Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in

May 23, 2016 - Nontransfected cells do not show red membrane fluorescence around the blue nuclei (Figure 4D). It is worth noting that within transfect...
0 downloads 0 Views 6MB Size
Download PDF
Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells Giulia Vindigni,†,# Sofia Raniolo,†,# Alessio Ottaviani,‡ Mattia Falconi,‡ Oskar Franch,§,⊥ Birgitta R. Knudsen,§,⊥ Alessandro Desideri,*,‡ and Silvia Biocca*,† †

Department of Systems Medicine, University of Rome Tor Vergata, Via Montpellier 1, 00133, Rome, Italy Department of Biology, Interuniversity Consortium, National Institute Biostructure and Biosystem (INBB), University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy § Department of Molecular Biology and Genetics and ⊥Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Aarhus 8000, Denmark ‡

S Supporting Information *

ABSTRACT: DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor “oxidized lowdensity lipoprotein receptor-1” (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin−streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells. KEYWORDS: DNA nanotechnology, DNA nanocage stability, cell uptake, drug delivery, LOX-1, scavenger receptors

D

Translation to biomedical applications requires high stability in biological environments and efficient cell binding and internalization of the assembled DNA nanostructures. Evidence has indicated that stability in tissue culture is very different for linear and 3D DNA nanostructures, and it is likely to depend on the geometrical arrangement. Hence, in the presence of 10% fetal bovine serum (FBS) the decay time constants in vitro vary from 0.8 h for a linear DNA to over 42 h for a tetrahedral structure and reach 48 h inside cells.18,19 In general, nucleic acids do not cross the plasma membrane due to their negative charge. Assembly of DNA oligonucleotides into more compact three-dimensional structures, such as

NA is an extremely suitable polymer for the generation of nanocapsules to be used for biomedical applications for its intrinsic properties of high stability, biocompatibility, and versatility. In the last years DNA nanotechnology has been extensively used to build an impressive variety of DNA cage structures including tetrahedra,1,2 cubes,3 truncated octahedra,4,5 trigonal bipyramids,6 dodecahedra,7 icosahedra,8 and truncated icosahedra as well as hybrid structures with synthetic linkers and/or large DNA origami-based structures.9 Several groups have been involved in the encapsulation of molecular cargo in DNA-based cages for their release in vivo.10−12 Attachment or entrapment of proteins or other functional molecules is indeed an important goal required for possible drug delivery purposes.13,14 DNA structures have been shown to be suitable for a slower release or selective targeting toward intracellular organelles of DNA intercalating drugs, such as doxorubicin.15−17 © 2016 American Chemical Society

Received: February 25, 2016 Accepted: May 23, 2016 Published: May 23, 2016 5971

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

www.acsnano.org

Article

ACS Nano

streptavidin−biotin assays. The nanocage stability in human serum has been studied together with its ability to enter normal and LOX-1-overexpressing cells. Efficient uptake of the octahedral cages into cells is observed only in LOX-1expressing cells, demonstrating a receptor-mediated uptake mechanism and conceiving the possibility to target specific receptors for a selective cellular uptake of DNA nanocages.

nanotubes and nanocapsules, enhances their intracellular uptake without the need of transfection reagents.19,20 However, whether this effect is due to the presence of cellular scavenger receptors has not yet been investigated. Interestingly, by decorating DNA nanostructures with folate, MUC 1 aptamer, or nucleolin, able to recognize specific receptors, a selective targeting to specific cell types has been obtained and improved by engineering shape-changing nanostructures,21−27 indicating receptor-mediated endocytosis as a mechanism for cell entry of DNA nanocapsules. Scavenger receptors, originally identified for their ability to recognize lipoproteins, are known to bind and internalize a variety of different macromolecules. The scavenger oxidized low-density lipoprotein receptor-1 (LOX-1) is the major receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells and plays a crucial role in cardiovascular diseases.28,29 It is up-regulated in pathological conditions affecting the cardiovascular system (i.e., hypertension, diabetes). Its altered expression has been associated with atherosclerosis, obesity, inflammation, and, more recently, tumor development.30−32 Of note, a meta-analysis of gene expression profiles reveals that the orl1 gene coding for the LOX-1 receptor is upregulated in many cancer cells, including bladder, cervix, mammary gland, lung, and colon/rectal.32 LOX-1 is a transmembrane receptor member of the C-type lectin-like protein family29,33 that binds the substrate ox-LDL through electrostatic interactions due to the presence of a spine formed by positively charged amino acids.34−38 The globular form and the negative charge of truncated octahedral DNA cage structures, used in this work, are similar to the natural substrate of LOX-1. The DNA nanocage dimensions are on the same order of magnitude as ox-LDL, as shown in Figure 1, envisaging a possible role of this scavenger receptor in DNA nanoparticle recognition and intracellular uptake.

RESULTS AND DISCUSSION Stability of DNA Nanocages under Physiological Conditions. To investigate the mechanism of entry of DNA nanocages in cells, we have assembled a truncated DNA octahedral cage with a biotin molecule on one edge of the structure, as depicted in Figure 2A and Supporting Information

Figure 2. Assembly of octahedral biotinylated DNA nanocages. (A) Scheme of the truncated octahedral cage with a biotin covalently bound to one strand of a double helix. (B) Gel electrophoretic result of a ladder experiment where an increasing number of DNA oligonucleotides that form the cages have been annealed and ligated before analysis. Lanes 1−7 show the result of subjecting assembly reactions containing 1−2, 1−3, 1−4, 1−5, 1−6, 1−7, and 1−8 oligonucleotides, while lane M contains DNA markers.

Figure S1. The assembly of the cages is not perturbed by the presence of biotin, as shown by the preparative gel ladder showing the predominately one well-defined product with higher molecular weight resulting from annealing and ligating an increasing number of oligonucleotides from 1 to 8 (Figure 2B). The assembly efficiency of the fully annealed cages was estimated to be approximately 40%, which is in agreement with previous reports of cage assembly without the addition of a biotin.4,5 The presence of biotin inserted in the assembled cages allows the detection of cages after gel running and transfer to a membrane, through a streptavidin (HRP)−biotin reaction in DNA blots. Due to the very low streptavidin−biotin dissociation constant (Kd 10−14 M), the system has a sensitivity up to 100 times higher than the ethidium bromide staining, as we have calculated in a comparative test. The result of a typical DNA blot of 10 ng of purified biotinylated octahedral DNA nanocages is visualized in Figure 3A (lane 1). Importantly, the size of the DNA cages was shifted after incubation in the presence of FBS (Figure 3A, lane 2). Incubation with proteinase K restored the correct mobility, as shown by the gel electrophoretic mobility of the band resulting from the cages as a function of digestion time with the protease (Figure 3A, lanes 3−6). This result demonstrates that serum proteins bind and surround the surface of nanocages and that this binding can be totally eliminated by 2 h proteinase K digestion (Figure 3A, lane 6). This effect, called protein corona, happens

Figure 1. 3D model of LOX-1 receptor interactions with truncated octahedral DNA cage (on the left) and its natural substrate ox-LDL (on the right).

Starting from this hypothesis, in this work, we present an investigation of the mechanism of cellular uptake of pristine DNA nanocages. For this purpose, we have used a fibroblast cell line overexpressing LOX-1, and we have prepared truncated octahedral DNA nanocages functionalized with a single biotin molecule to permit DNA nanocage detection through 5972

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano

Figure 3. Stability of biotinylated DNA nanocages in biological fluids. (A) Time-course digestion with proteinase K of DNA nanocages incubated with serum proteins. Lanes 1 and 2 show gel-purified DNA nanocages and cages incubated with 10% FBS at 37 °C for 1 h, respectively. DNA nanocages incubated in 10% FBS at 37 °C for 1 h and digested with proteinase K (100 μg/mL) for 15, 30, 60, and 120 min are shown in lanes 3−6. (B) DNA blot analysis of nanocages incubated with 10% FBS (lanes 2−7), 10% HS (lanes 9−14), and 30% HS (lanes 15−17). Purified DNA nanocages (20 ng) before incubation with serum proteins (input) are shown in lanes 1 and 8. Incubation times are indicated under each gel. (C) Densitometric analysis of four different experiments. The intensity of the bands was calculated by using ImageJ software and corresponds to the normalized amount of DNA nanocages (y-axis) over time (x-axis). Values are expressed as an average ± SEM.

in a very short time (30 s) upon contact with biological fluids and has been reported for nanoparticles of various origin.39 The stability of DNA nanostructures in biological fluids at the physiological temperature of 37 °C is an important prerequisite for their use in biomedical applications. We have analyzed the stability of octahedral DNA nanocages in tissue culture medium supplemented with FBS or in the presence of human serum (HS). This was done by incubating 20 ng of biotinylated cages in FBS (10%) or HS (10% and 30%) at 4 and 37 °C for different time intervals (1, 3, 5, 24, 48, and 72 h). After incubation, each sample was treated with proteinase K (100 μg/mL) for 2 h at 37 °C, for removing the bound proteins. Samples were run in 4% polyacrylamide gels and analyzed by the streptavidin (HRP)−biotin reaction in DNA blots. At 4 °C DNA nanocages are fully stable up to 72 h (Supporting Information, Figure S2). On the contrary, at 37 °C DNA nanocages are stable in FBS for 5 h (Figure 3B, lanes 2− 4), and then they are degraded as a function of time. After 24 and 48 h in FBS the cages did not appear as a single band in the gel but rather as a low molecular weight smear below the intact cage band (Figure 3B, lanes 5 and 6). At 72 h no bands were visible in the gel, indicating that the cages are completely degraded (Figure 3B, lane 7). The relative intensity of each band, quantified using ImageJ software, was normalized to the intensity of the band corresponding to the input cage and reported in the graph (Figure 3C). The mean lifetime and the half-life of octahedral DNA cages in FBS are respectively 38.4 and 26.6 h and have been calculated following the first-order exponential decay equation reported in Supporting Information Table S3. Interestingly, DNA cages were much more stable in HS and migrated as a single band with an intensity similar to the input band up to 72 h at 10% HS (Figure 3B, lanes 9−14) and up to 48 h at 30% concentration HS (Figure 3B, lanes 15,

16). At 72 h at 30% HS the band started to appear less intense (Figure 3B, lane 17). The higher stability of the DNA cages in HS compared to FBS, as found for other DNA structures,40 is probably due to different nucleases present in the sera. Receptor-Mediated DNA Nanocage Entry into LOX-1Expressing Cells. Binding and uptake of the biotin-labeled octahedral DNA nanocages in mammalian cells were monitored in COS fibroblasts transiently transfected with a plasmid encoding for the full-length LOX-1 receptor containing a V5tag at the C-terminus (LOX-1-V5). This is a well-established cell system that allows a high transfection efficiency (about 40%) (Supporting Information, Figure S4) and a very high expression level of functional LOX-1 receptors.14 Since nontransfected COS cells do not express endogenous LOX-1, the system allows studying the specificity of the interaction between LOX-1 receptors and DNA cages within the same cell population. COS cells were transfected and, after 24 h, incubated with the biotinylated DNA nanocages at 4 °C to investigate the cell−cage binding, as described in the Experimental Methods. Cells were stained with anti-V5 antibodies for visualizing LOX1 receptors or with streptavidin−FITC for visualizing biotinylated cages and with DAPI for localizing the nuclei. In Figure 4A LOX-1 receptors are represented by red fluorescent intense dots, indicating that the receptors localize into the plasma membrane. Nontransfected cells do not show red membrane fluorescence around the blue nuclei (Figure 4D). It is worth noting that within transfected LOX-1 cells some cells do not express LOX-1 according to the transfection efficiency (see white arrows in Figure 4A). Figure 4B shows in green the pattern of the DNA nanocages bound to the cells. The dot-like fluorescence is almost identical to that observed for LOX-1 receptors, indicative of specific 5973

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano

Figure 4. Confocal analysis of DNA nanocages bound and internalized in cells. Membrane LOX-1 receptors were visualized with Mab anti-V5 IgG as primary antibody (A and D). LOX-1-transfected and nontransfected COS cells (COS nt) were incubated with biotinylated cages (B and E) and with the single biotinylated oligonucleotide (OL8BIO) for 1 h at 4 °C (C and F). (G−L) Transfected COS and COS nt incubated with biotinylated cages at 37 °C for 2, 5, and 24 h. Biotinylated cages and oligonucleotide OL8BIO were detected with streptavidin−FITC, and nuclei are blue stained with DAPI. White arrows in A and B indicate nuclei of nontransfected cells. Scale bar: 20 μm.

show DNA cage internalization in nontransfected cells. The cages appear in many small fluorescent dots in the cytoplasm after 2 h of incubation, which become fewer and larger after 5 h of incubation (Figure 4G and H), indicating that they are internalized in LOX-1-expressing cells in vesicles that fuse together. The fluorescence in larger vesicles remains visible, although less intense, for at least 24 h (Figure 4I). The observed uptake and traffic mechanism of DNA cages in LOX1-expressing cells is identical to that observed for the natural substrate ox-LDL. Thus, in COS cells transfected with LOX-1, the fluorescently labeled ox-LDL is found in vesicles dispersed in the cytoplasm after 1 h of incubation and, at longer time of incubation, is found inside cells in larger endosomes.41 It is worth mentioning that nontransfected cells do not show any detectable fluorescent dots at 2 or 5 h of incubation time (Figure 4J and K), and only a very weak fluorescence is detectable after 24 h of DNA cage incubation with cells (Figure 4L), indicating that a small amount of DNA cages is internalized in cells by a LOX-1-independent mechanism. Moreover, no fluorescence was detectable inside the cells when uptake experiments were done with the biotinylated oligonucleotide OL8BIO (Supporting Information, Figure S5), further confirming a LOX-1-dependent cellular uptake of the fully assembled DNA nanocages. The experiment of co-localization of DNA nanocages and LOX-1 receptors at the cell surface was unsuccessful. This negative result is actually an additional demonstration that the

DNA nanocage binding to discrete regions of plasma membranes.41 Green fluorescent cells are about 40% of the total cells, indicating a direct correlation between LOX-1 positive and DNA nanocage positive cells (Supporting Information, Figure S4). Remarkably, nontransfected cells do not show any detectable fluorescent dots on the cell surface (Figure 4E). Of note, no fluorescence was detectable at the cell surface or inside cells when binding (Figure 4C and F), and internalization experiments (Supporting Information, Figure S5) were done with the single biotinylated oligonucleotide OL8BIO in nontransfected and LOX-1-transfected cells, further confirming a LOX-1-dependent cellular binding of the fully assembled octahedral DNA nanocages. In order to verify whether the proteins surrounding the cages (protein corona) play an important role in LOX-1 receptor recognition or the receptor directly interacts with the DNA strands, we have performed a binding experiment at 4 °C by adding the pristine cages to a cell culture previously depleted of serum proteins. Of note, DNA nanocages efficiently bind to LOX-1 receptors either in the absence or in the presence of serum proteins, indicating that the protein corona does not affect the interaction (Supporting Information, Figure S6). To investigate the intracellular uptake and traffic, DNA nanocages were incubated with LOX-1-expressing cells for increasing time intervals at 37 °C. Panels G, H, and I of Figure 4 show DNA cage internalization in LOX-1-transfected COS cells at 2, 5, and 24 h, respectively, while panels J, K, and L 5974

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano

Figure 5. Co-detection analysis of LOX-1 and DNA cages in cells. Double fluorescence of LOX-1-V5-expressing COS cells incubated with DNA cages at 37 °C for 2 h. Intracellular LOX-1 receptors were visualized using mouse monoclonal anti-V5 antibody as primary antibody and Rhodamine Red-X-conjugated donkey anti-mouse IgG as the secondary antibody (red), and intracellular biotinylated cages were detected by using streptavidin−FITC (green). The nuclei were stained with DAPI. Scale bar: 10 μm.

Figure 6. LOX-1-mediated binding and uptake of DNA nanocages in cells. (A) Representative DNA blotting of cell extracts obtained from nontransfected (nt) and LOX-1-transfected COS cells cultured in 48 wells/plate and incubated with biotinylated DNA cages (2 μg/mL) at 4 °C for 1 h or at 37 °C for 2 and 24 h. DNA cages were detected with streptavidin-HRP. (B) Histogram shows the amount of DNA nanocages bound in 1 h at 4 °C or internalized in 2 h at 37 °C, detected in cell extracts of nontransfected and LOX-1-transfected cells, as indicated and expressed in ng/106 cells. For image processing and densitometric analysis, photographic films were digitized by scanning. Bands were analyzed by using ImageJ software. Values were normalized for the total number of LOX-1-transfected cells and expressed as a mean ± SEM.

DNA cages bound to the cell surface. In parallel, for monitoring cage stability during the internalization process, nontransfected or LOX-1-V5-expressing cells were incubated with the biotinylated DNA cages (2 μg/mL) for 2 or 24 h at 37 °C. The DNA cages were then purified as described in the Experimental Methods. Samples were analyzed in 4% polyacrylamide gels, transferred onto Zeta Probe membranes, and visualized by using streptavidin-HRP (Figure 6A). Lane 1 shows the band of DNA cages (10 ng) before incubation with the cells. The DNA cages were purified after binding to the plasma membrane at 4 °C run as a single product in the gel with a mobility comparable to the input but with an intensity much higher in transfected than in nontransfected cells (Figure 6A, lanes 2 and 3). A similar result was obtained when the cages were purified from cells after 2 h of incubation at 37 °C in the two populations of cells (Figure 6A, lanes 4 and 5), indicating that they are intact and correctly assembled inside cells. Lower molecular weight biotinylated DNA products were detected when cells were harvested after 24 h incubation (Figure 6A, lanes 6 and 7), indicating that the cages start to be degraded at these conditions. The amount of bound and internalized cages in nontransfected cells is very weak (Figure 6A, lanes 2, 4, and 6). Densitometric analysis allowed us to calculate the difference, expressed in ng/106 cells, of DNA cages bound at 4 °C and internalized in 2 h at 37 °C into the two populations of cells (Figure 6B). The amount of bound cages in LOX-1-expressing cells is about 6−7-fold higher than that bound to nontransfected cells, as normalized to the total number of

cages bind to LOX-1 receptors on the cell surface. In fact, the anti-V5 antibody cannot bind to the membrane receptors if they are already involved in binding the nanocages. In order to detect LOX-1 and DNA nanocages in the same cell, we took advantage of the fact that LOX-1, at the steady state, is always present in two distinct pools, one membrane bound and one intracellular. Thus, LOX-1 receptors distribute in the endoplasmic reticulum and Golgi apparatus during its biosynthesis before trafficking to the plasma membrane, as described.42 The biotinylated DNA cages were incubated at 37 °C for 2 h for their internalization in LOX-1-transfected cells and analyzed by confocal microscopy after permeabilization to allow the detection of either internalized DNA nanocages or intracellular LOX-1. Figure 5 shows a representative field in which the LOX-1-expressing cell (red fluorescence) has internalized DNA nanocages (green fluorescence and merge image) at variance with cells not expressing LOX-1. Intracellular Stability of DNA Nanocages. For drug delivery or other biomedical purposes, it is important to understand the cellular stability of DNA nanocages in order to achieve, for example, controlled or prolonged drug release. Confocal analysis does not allow verifying whether DNA nanocages are partially degraded or maintain their structural integrity inside cells. We evaluated the integrity of the cellinternalized octahedral DNA nanocages, monitoring their electrophoretic mobility. The biotinylated DNA cages (2 μg/ mL) were incubated with nontransfected or LOX-1-transfected COS cells for 1 h at 4 °C, for evaluating the amount of intact 5975

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano

Figure 7. Co-localization analysis of DNA nanocages and lysosomes. Double immunofluorescence of LOX-1-V5-expressing COS cells incubated with DNA cages at 37 °C for 2 h (A−F) and 5 h (G−L). Biotinylated cages were detected by using streptavidin−FITC (A and G), and lysosomes were visualized using mouse monoclonal anti-LAMP-1 antibody as primary antibody and Rhodamine Red-X-conjugated donkey anti-mouse IgG as the secondary antibody (B and H). The nuclei were stained with DAPI. Higher magnification of the merged images (C and I) is shown in panels E and K. Panels F and L represent XZ stack images. Co-localization analysis was performed using IMARIS software, and scatter plots are reported in panels D and J. Scale bar: 20 μm.

incubation with fibroblast cell extracts at least up to 24 h (Supporting Information, Figure S7). It is worth noting that in the confocal analysis the signal of bound cages in nontransfected cells was undetectable (Figure 4B (white arrows) and E), while in DNA blots a weak but still detectable band was seen when the cages were purified from nontransfected cells (Figure 6A, lane 2). This is probably due to the higher sensitivity of the blot analysis. The weak binding in nontransfected cells could be a LOX-1-independent mechanism of entry due to the presence of other scavenger

transfected cells (see Experimental Methods). Remarkably, the amount of internalized cages after 2 h at 37 °C is more than 30fold higher in LOX-1-expressing cells. These findings indicate a very high efficiency of LOX-1 receptors in selectively binding the DNA cages at 4 °C and, more importantly, its higher efficiency in internalizing the cages in living cells at the physiological temperature of 37 °C. At 24 h the total amount of internalized cages increased, but, since the cages were partly degraded (Figure 6A, lanes 6 and 7), the quantification was not done. Importantly, the DNA nanocages are fully stable after 5976

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano

exploiting this receptor for delivering therapeutic drugs encapsulated in pristine DNA nanocages.

receptors or plasma membrane molecules interacting with DNA cages. Intracellular Trafficking of DNA Nanocages. Guided by our observation that DNA nanocages are taken up by a receptor-mediated mechanism and that the scavenger receptor LOX-1 is responsible for their internalization, we studied whether these nanostructures, once endocyted in vesicles, traffic toward lysosomes. This is important information in case DNA cages are to be used as a delivery system, since many drugs (such as anticancer intercalating drugs) are released from double-stranded DNA by acidic pH, typical of endocytic vesicles and lysosomes. To study the intracellular traffic of the DNA cages, we incubated LOX-1-transfected COS cells with biotinylated DNA cages for different time intervals and performed double immunofluorescence analysis with streptavidin−FITC for visualizing biotinylated cages and antibodies directed against the lysosomal-associated membrane protein 1 (LAMP-1) for detecting lysosomes. Figure 7 shows representative confocal images of cells incubated for 2 and 5 h with 2.6 μg/mL DNA cage. After 2 h the fluorescent dots representing the DNA cages (Figure 7A) appeared dispersed in the cytoplasm of transfected cells, and only some of them colocalize with lysosomes (Figure 7B) as seen in the merged images (Figure 7C, higher magnification in E, and XZ stack in F). In panel D is shown the scatter plot corresponding to the co-localization events. In the case of complete co-localization the dots in the scatter plot should appear centered on a line.43 In our analysis dots form a cloud, and from the slope of the fitted line the Pearson coefficient can be calculated as 0.37. A similar analysis is shown for cells incubated for 5 h with DNA cages (see merged image in panel H and higher magnification and Z stack shown in I and J). Interestingly, the Pearson coefficient in this case was higher (0.51), indicating an increase of co-localized dots. This finding indicates that the internalized DNA nanocages reached lysosomes in a time-dependent way. It is noteworthy that the endocytic internalization pathway and subsequent intracellular traffic strongly depend on the specific targeted receptor. Here we report the case of pristine DNA nanocages recognized by the scavenger receptor LOX-1, but DNA nanostructures can be functionalized with targeting ligands specific for selected overexpressed receptors, for altering their cellular interaction and their traffic to subcellular environments.44−46

EXPERIMENTAL METHODS Preparation of Biotinylated DNA Octahedra. Octahedral cages were prepared as described, 4 with some modifications. All oligonucleotides were HPLC purified and purchased from DNA Technology with the exception of the biotinylated oligonucleotide (OL8BIO), which was purchased from Sigma-Aldrich. The sequences of the oligonucleotides are reported in Table S1. Briefly, cages were assembled by combining equimolar amounts of each strand at a concentration of 0.8 μM in TAM buffer (40 mM Tris-acetic acid pH 7.0, 12.6 mM magnesium acetate). Samples were heated at 95 °C for 10 min, then 80 °C for 5 min, cooled to 60 °C (4 min/1 °C), and finally slowly cooled to 4 °C (6 min/1 °C).25 Cages were then incubated for 2 h at 25 °C with T4 DNA ligase (New England Biolabs) to covalently close all nicks and analyzed on native 6% polyacrylamide gels in TAEM buffer (40 mM Tris-acetic acid pH 7.0, 1 mM EDTA, 12.6 mM magnesium acetate) following standard procedures. The 50 bp ladder was purchased from Tecnochimica Moderna. After staining with ethidium bromide, the band of correctly assembled cages was cut out of the gel and eluted by incubating it in diffusion buffer (500 mM NH4 acetic acid, 10 mM magnesium acetate, 0.1 mM EDTA, 0.1% SDS) overnight at 30 °C. The samples were then concentrated by 2-propanol precipitation. DNA Construct. For the expression in mammalian cells, human LOX-1 was subcloned into pEF/V5-His vectors (Invitrogen) as previously described.47 Cell Cultures and Transfection. COS cells were grown in DMEM (Dulbecco’s modified Eagle’s medium) (Biowest) supplemented with 10% fetal bovine serum (Gibco), 1 mM L-glutamine (Sigma-Aldrich), 1 mM sodium pyruvate (Biowest), and 100 U/mL penicillin−streptomycin (Euroclone). COS cells were transiently transfected with JetPEI (Polyplus Transfection), following the manufacturer’s instructions, with a DNA/transfectant reagent ratio (w/v) of 1:2. Human Serum Preparation. Whole human blood was collected from healthy volunteers into BD Vacutainer SSTTM gel separator tubes, allowed to clot for 1 h at room temperature, and centrifuged at 3000 rpm for 15 min. Human serum samples were filtered, dialyzed overnight against phosphate-buffered saline (PBS) pH 7.4, and stored at −20 °C.48 DNA Octahedra Stability. Biotinylated cages were incubated in FBS or HS at different concentrations and temperatures for different times (1, 3, 5, 24, 48, and 72 h). Each sample was then treated with proteinase K (100 μg/mL) for 2 h at 37 °C, and protein digestion was stopped by adding phenylmethylsulfonyl fluoride (PMSF) to a final concentration of 5 mM. Samples mixed with loading buffer (Tris-Cl 500 mM pH 6.8; glycerol 20%; SDS 4%; Bromophenol Blue 0.02%) were run in 4% polyacrylamide gels in TBE (Tris-Cl 89 mM pH 8, boric acid 89 mM, EDTA 2 mM) at 100 V for 1 h and blotted. Purification and Blotting of DNA Nanocages. Nontransfected and LOX-1-transfected COS cells, cultured in 48 wells/plate, were incubated with 60 μL of biotinylated DNA cages (2 μg/mL) for 1 h at 4 °C or 2 and 24 h at 37 °C. After incubation, cells were washed three times with PBS and lysed with lysis buffer (20 mM Tris/Cl pH 7.8, 10 mM EDTA, 100 mM NaCl, 0.5% NP40, 0.5% NAD) for 20 min on ice. Lysates were centrifuged at 12 000 rpm for 20 min, and supernatants were digested with proteinase K (100 μg/mL) for 2 h at 37 °C. The reaction was stopped by adding PMSF to a final concentration of 5 mM. Samples were electrophoresed on 4% polyacrylamide gels and transferred to a positively charged nylon membrane (Zeta Probe, BioRad) for 40 min at 23 V (SemiDry transfer cell). The membrane was then cross-linked for 15 min at 80 °C and blocked with Tris-buffered saline pH 7.8/BSA 6%. Biotin detection was carried out using streptavidin-HRP (Abcam) and visualized by enhanced chemiluminescence (ECL Turbo Euroclone). For image processing and densitometric analysis, photographic films were digitized by scanning. Bands were analyzed by ImageJ software.

CONCLUSIONS We have demonstrated that pristine octahedral DNA nanocages are efficiently bound by the scavenger receptor LOX-1 and are internalized through a receptor-mediated mechanism. The here-described DNA-based nanocages have a controllable structure, are easy to assemble, and have an enhanced stability over single-stranded aptamers in biological fluids and in cells. The amount of internalized cages is 30 times higher in LOX-1expressing cells than in normal fibroblasts, demonstrating the occurrence of an efficient receptor-mediated uptake of pristine DNA nanocages that can be pursued for a selective cellular internalization. The nanoparticles are internalized in vesicular structures, which fuse to lysosomes in a time-dependent way, suggesting that DNA nanocages enter the cells through the endolysosomal pathway. The typical low pH of endocytic vesicles should help the therapeutic payload release from nanocages for selective delivery toward LOX-1-expressing cells. Importantly, LOX-1 is up-regulated in many pathological conditions, including tumors, which opens possibilities for 5977

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano Confocal Analysis and Lysosome Co-localization. Membrane immunofluorescence was performed in transfected COS cells seeded onto the poly- L-lysine-coated glass cover-slides as previously described.49 For binding experiments, cells were incubated with 2.6 μg/mL of biotinylated cages in DMEM 10% FBS for 1 h at 4 °C, washed in PBS, fixed in 4% paraformaldehyde, and neutralized with NaBH4. For uptake experiments, cells were incubated at 37 °C for 2, 5, and 24 h, fixed, and permeabilized with Tris HCl 0.1 M pH 7.7/Triton 0.1% for 4 min. For co-detection of DNA nanocages and LOX-1, cells were incubated at 37 °C for 2 h with 2.6 μg/mL of biotinylated cages, fixed, and permeabilized. LOX-1 was visualized using Mab anti-V5 as the primary antibody (Invitrogen) and Rhodamine Red-X-conjugated AffiniPure donkey anti-mouse IgG as the secondary antibody (Jackson). Biotinylated cages were detected by using streptavidin− FITC (Jackson). The nuclei were stained with DAPI (Invitrogen). For lysosome co-localization COS cells transfected with LOX-1-V5 were cultured for 48 h, incubated with 2.6 μg/mL of biotinylated cages in DMEM/10% FBS for 2 and 5 h at 37 °C, and then processed for confocal imaging (Olympus FV1000). Lysosomes were visualized using mouse monoclonal anti-LAMP-1 antibody (Abcam) as primary antibody and Rhodamine Red-X-conjugated donkey anti-mouse IgG as the secondary antibody. Images were obtained with a laser confocal fluorescent microscope Olympus FV1000 at 60× magnification, and the fluorescence signal was evaluated with IMARIS software.

S.; Veigaard, C.; Koch, J.; Rubinstein, J. L.; Guldbrandtsen, B.; Hede, M. S.; Karlsson, G.; Andersen, A. H.; Pedersen, J. S.; Knudsen, B. R. Assembly and Structural Analysis of a Covalently Closed Nano-Scale DNA Cage. Nucleic Acids Res. 2008, 36, 1113−1119. (5) Oliveira, C. L. P.; Juul, S.; Jørgensen, H. L.; Knudsen, B.; Tordrup, D.; Oteri, F.; Falconi, M.; Koch, J.; Desideri, A.; Pedersen, J. S.; Andersen, F. F.; Knudsen, B. R. Structure of Nanoscale Truncated Octahedral DNA Cages: Variation of Single-Stranded Linker Regions and Influence on Assembly Yields. ACS Nano 2010, 4, 1367−1376. (6) Erben, C. M.; Goodman, R. P.; Turberfield, A. J. A SelfAssembled DNA Bipyramid. J. Am. Chem. Soc. 2007, 129, 6992−6993. (7) Zimmermann, J.; Cebulla, M. P. J.; Mönninghoff, S.; von Kiedrowski, G. Self-Assembly of a DNA Dodecahedron from 20 Trisoligonucleotides with C(3h) Linkers. Angew. Chem., Int. Ed. 2008, 47, 3626−3630. (8) Bhatia, D.; Mehtab, S.; Krishnan, R.; Indi, S. S.; Basu, A.; Krishnan, Y. Icosahedral DNA Nanocapsules by Modular Assembly. Angew. Chem., Int. Ed. 2009, 48, 4134−4137. (9) Benson, E.; Mohammed, A.; Gardell, J.; Masich, S.; Czeizler, E.; Orponen, P.; Högberg, B. DNA Rendering of Polyhedral Meshes at the Nanoscale. Nature 2015, 523, 441−444. (10) Banerjee, A.; Bhatia, D.; Saminathan, A.; Chakraborty, S.; Kar, S.; Krishnan, Y. Controlled Release of Encapsulated Cargo from a DNA Icosahedron Using a Chemical Trigger. Angew. Chem., Int. Ed. 2013, 52, 6854−6857. (11) Bhatia, D.; Surana, S.; Chakraborty, S.; Koushika, S. P.; Krishnan, Y. A. Synthetic Icosahedral DNA-Based Host-Cargo Complex for Functional. In Vivo Imaging. Nat. Commun. 2011, 2, 339. (12) Juul, S.; Iacovelli, F.; Falconi, M.; Kragh, S. L.; Christensen, B.; Frøhlich, R. F.; Franch, O.; Kristoffersen, E. L.; Stougaard, M.; Leong, K. W.; Ho, Y. P.; Sørensen, E. S.; Birkedal, V.; Desideri, A.; Knudsen, B. R. Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage. ACS Nano 2013, 7, 9724−9734. (13) De Vries, J. W.; Zhang, F.; Herrmann, A. Drug Delivery Systems Based on Nucleic Acid Nanostructures. J. Controlled Release 2013, 172, 467−483. (14) Biocca, S.; Desideri, A. The Potential of Nucleic Acid-Based Nanoparticles for Biomedical Application. Nano LIFE 2015, 05, 1541004. (15) Kumar, V.; Bayda, S.; Hadla, M.; Caligiuri, I.; Russo, S.; Palazzolo, S.; Kempter, S.; Corona, G.; Toffoli, G.; Rizzolio, F. Enhanced Chemotherapeutic Behavior of Open-Caged DNA@ Doxorubicin Nanostructures for Cancer Cells. J. Cell. Physiol. 2016, 231, 106−110. (16) Zhao, Y. X.; Shaw, A.; Zeng, X.; Benson, E.; Nyström, A. M.; Högberg, B. DNA Origami Delivery System for Cancer Therapy with Tunable Release Properties. ACS Nano 2012, 6, 8684−8691. (17) Chan, M. S.; Tam, D. Y.; Dai, Z.; Liu, L. S.; Ho, J. W.; Chan, M. L.; Xu, D.; Wong, M. S.; Tin, C.; Lo, P. K. Mitochondrial Delivery of Therapeutic Agents by Amphiphilic DNA Nanocarriers. Small 2016, 12, 770−781. (18) Keum, J. W.; Bermudez, H. Enhanced Resistance of DNA Nanostructures to Enzymatic Digestion. Chem. Commun. 2009, 45, 7036−7038. (19) Walsh, A. S.; Yin, H.; Erben, C. M.; Wood, M. J.; Turberfield, A. J. DNA Cage Delivery to Mammalian Cells. ACS Nano 2011, 5, 5427− 5432. (20) Hamblin, G. D.; Carneiro, K. M.; Fakhoury, J. F.; Bujold, K. E.; Sleiman, H. F. Rolling Circle Amplification-Templated DNA Nanotubes Show Increased Stability and Cell Penetration Ability. J. Am. Chem. Soc. 2012, 134, 2888−2891. (21) Chang, M.; Yang, C. S.; Huang, D. M. Aptamer-Conjugated DNA Icosahedral Nanoparticles as a Carrier of Doxorubicin for Cancer Therapy. ACS Nano 2011, 5, 6156−6163. (22) Watanabe, T.; Hirano, K.; Takahashi, A.; Yamaguchi, K.; Beppu, M.; Fujiki, H.; Suganuma, M. Nucleolin on the Cell Surface as a New Molecular Target for Gastric Cancer Treatment. Biol. Pharm. Bull. 2010, 33, 796−803.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b01402. Oligo-DNA sequences, additional stability data, analysis of DNA nanocage lifetime and half-life, details on transfection efficiency, uptake experiments with OL8BIO, effect of protein corona on DNA nanocage recognition (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail (A. Desideri): [email protected]. *E-mail (S. Biocca): [email protected]. Author Contributions #

G. Vindigni and S. Raniolo contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We thank E. Romano from the Centre of Advanced Microscopy “Patrizia Albertano” for skillful assistance in confocal analysis, F. Iacovelli for useful support with figure preparation, and M. Piacentini for the anti-LAMP-1 antibody. The work was supported by the Dagmar Marshalls Foundation. REFERENCES (1) Goodman, R. P.; Schaap, I. A. T.; Tardin, C. F.; Erben, C. M.; Berry, R. M.; Schmidt, C. F.; Turberfield, A. J. Rapid Chiral Assembly of Rigid DNA Building Blocks for Molecular Nanofabrication. Science 2005, 310, 1661−1665. (2) Erben, C. M.; Goodman, R. P.; Turberfield, A. J. Single-Molecule Protein Encapsulation in a Rigid DNA Cage. Angew. Chem., Int. Ed. 2006, 45, 7414−7417. (3) Edwardson, T. G. W.; Carneiro, K. M. M.; McLaughlin, C. K.; Serpell, C. J.; Sleiman, H. F. Site-Specific Positioning of Dendritic Alkyl Chains on DNA Cages Enables Their Geometry-Dependent Self-Assembly. Nat. Chem. 2013, 5, 868−875. (4) Andersen, F. F.; Knudsen, B.; Oliveira, C. L. P.; Frøhlich, R. F.; Krüger, D.; Bungert, J.; Agbandje-McKenna, M.; McKenna, R.; Juul, 5978

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979

Article

ACS Nano (23) Koutsioumpa, M.; Papadimitriou, E. Cell Surface Nucleolin as a Target for Anti-Cancer Therapies. Recent Pat. Anti-Cancer Drug Discovery 2014, 9, 137−152. (24) Lee, H.; Lytton-Jean, A. K.; Chen, Y.; Love, K. T.; Park, A. I.; Karagiannis, E. D.; Sehgal, A.; Querbes, W.; Zurenko, C. S.; Jayaraman, M.; Peng, C. G.; Charisse, K.; Borodovsky, A.; Manoharan, M.; Donahoe, J. S.; Truelove, J.; Nahrendorf, M.; Langer, R.; Anderson, D. G. Molecularly Self-Assembled Nucleic Acid Nanoparticles for Targeted In Vivo Sirna Delivery. Nat. Nanotechnol. 2012, 7, 389−393. (25) Fakhoury, J. J.; McLaughlin, C. K.; Edwardson, T. W.; Conway, J. W.; Sleiman, H. F. Development and Characterization of Gene Silencing DNA Cages. Biomacromolecules 2014, 15, 276−282. (26) Kim, K. R.; Kim, D. R.; Lee, T.; Yhee, J. Y.; Kim, B. S.; Kwon, I. C.; Ahn, D. R. Drug Delivery by a Self-Assembled DNA Tetrahedron for Overcoming Drug Resistance in Breast Cancer Cells. Chem. Commun. 2013, 49, 2010−2012. (27) Ohta, S.; Glancy, D.; Chan, W. C. W. DNA-Controlled Dynamic Colloidal Nanoparticle Systems for Mediating Cellular Interaction. Science 2016, 351, 841−845. (28) Sawamura, T.; Kume, N.; Aoyama, T.; Moriwaki, H.; Hoshikawa, H.; Aiba, Y.; Tanaka, T.; Miwa, S.; Katsura, Y.; Kita, T.; Masaki, T. An endothelial receptor for oxidized low-density lipoprotein. Nature 1997, 386, 73−77. (29) Mehta, J. L.; Chen, J.; Hermonat, P. L.; Romeo, F.; Novelli, G. Lectin-Like, Oxidized Low-Density Lipoprotein Receptor-1 LOX-1: a Critical Player in the Development of Atherosclerosis and Related Disorders. Cardiovasc. Res. 2006, 69, 36−45. (30) Hirsch, H. A.; Iliopoulos, D.; Joshi, A.; Zhang, Y.; Jaeger, S. A.; Bulyk, M.; Tsichlis, P. N.; Shirley Liu, X.; Struhl, K. A. Transcriptional Signature and Common Gene Networks Link Cancer with Lipid Metabolism and Diverse Human Diseases. Cancer Cell 2010, 13, 348− 361. (31) Khaidakov, M.; Mitra, S.; Kang, B. Y.; Wang, X.; Kadlubar, S.; Novelli, G.; Raj, V.; Winters, M.; Carter, W. C.; Mehta, J. L. Oxidized LDL Receptor 1(ORL1) as a Possible Link Between Obesity Dyslipidemia and Cancer. PLoS One 2011, 6, e20277. (32) Murdocca, M.; Mango, M.; Pucci, S.; Biocca, S.; Testa, B.; Capuano, R.; Paolesse, R.; Sanchez, M.; Orlandi, A.; Di Natale, C.; Novelli, G.; Sangiuolo, F. The Lectin-Like Oxidized LDL Receptor-1: a New Potential Molecular Target in Colorectal Cancer. Oncotarget 2016, DOI: 10.18632/oncotarget.7430. (33) Xu, S.; Ogura, S.; Chen, J.; Little, P. J.; Moss, J.; Liu, P. LOX-1 in Atherosclerosis: Biological Functions and Pharmacological Modifiers. Cell. Mol. Life Sci. 2013, 70, 2859−2872. (34) Ohki, I.; Ishigaki, T.; Oyama, T.; Matsunaga, S.; Xie, Q.; Ohnishi-Kameyama, M.; Murata, T.; Tsuchiya, D.; Machida, S.; Morikawa, K.; Tate, S. Crystal Structure of Human Lectin-Like, Oxidized Low-Density Lipoprotein Receptor 1 Ligand Binding Domain and its Ligand Recognition Mode to OxLDL. Structure 2005, 13, 905−917. (35) Park, H.; Adsit, F. G.; Boyington, J. C. The 1.4 Angstrom Crystal Structure of the Human Oxidized Low Density Lipoprotein Receptor LOX-1. J. Biol. Chem. 2005, 280, 13593−13599. (36) Falconi, M.; Biocca, S.; Novelli, G.; Desideri, A. Molecular Dynamics Simulation of Human LOX-1 Provides an Explanation for the Lack of OxLDL Binding to the Trp150Ala Mutant. BMC Struct. Biol. 2007, 7, 73. (37) Biocca, S.; Falconi, M.; Filesi, I.; Baldini, F.; Vecchione, L.; Mango, R.; Romeo, F.; Federici, G.; Desideri, A.; Novelli, G. Functional Analysis and Molecular Dynamics Simulation of LOX-1 K167N Polymorphism Reveal Alteration of Receptor Activity. PLoS One 2009, 4, e4648. (38) Biocca, S.; Arcangeli, T.; Tagliaferri, E.; Testa, B.; Vindigni, G.; Oteri, F.; Giorgi, A.; Iacovelli, F.; Novelli, G.; Desideri, A.; Falconi, M. Simulative and Experimental Investigation on the Cleavage Site that Generates the Soluble Human LOX-1. Arch. Biochem. Biophys. 2013, 540, 9−18. (39) Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.;

Landfester, K.; Schild, H.; Maskos, M.; Knauer, S. K.; Stauber, R. H. Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol. 2013, 10, 772−781. (40) Goltry, S.; Hallstrom, N.; Clark, T.; Kuang, W.; Lee, J.; Jorcyk, C.; Knowlton, W. B.; Yurke, B.; Hughes, W. L.; Graugnard, E. DNA Topology Influences Molecular Machine Lifetime in Human Serum. Nanoscale 2015, 7, 10382−10390. (41) Matarazzo, S.; Quitadamo, M.; Mango, R.; Ciccone, S.; Novelli, G.; Biocca, S. Cholesterol-Lowering Drugs Inhibit Lectin-Like Oxidized Low-Density Lipoprotein-1 Receptor Function by Membrane Raft Disruption. Mol. Pharmacol. 2012, 82, 246−254. (42) Mango, R.; Biocca, S.; del Vecchio, F.; Clementi, F.; Sangiuolo, F.; Amati, F.; Filareto, A.; Grelli, S.; Spitalieri, P.; Filesi, I.; Favalli, C.; Lauro, R.; Mehta, J. L.; Romeo, F.; Novelli, G. In Vivo and In Vitro Studies Support that a New Splicing Isoform of OLR1 Gene Is Protective against Acute Myocardial Infarction. Circ. Res. 2005, 97, 152−158. (43) Bolte, S.; Cordelières, F. P. A Guided Tour into Subcellular Colocalization Analysis in Light Microscopy. J. Microsc. 2006, 224, 213−232. (44) Modi, S.; Nizak, C.; Surana, S.; Halder, S.; Krishnan, Y. Two DNA Nanomachines Map pH Changes Along Intersecting Endocytic Pathways inside the Same Cell. Nat. Nanotechnol. 2013, 8, 459−467. (45) Li, J.; Pei, H.; Zhu, B.; Liang, L.; Wei, M.; He, Y.; Chen, N.; Li, D.; Huang, Q.; Fan, C. Self-Assembled Multivalent DNA Nanostructures for Noninvasive Intracellular Delivery of Immunostimulatory CpG Oligonucleotides. ACS Nano 2011, 5, 8783−8789. (46) Liang, L.; Li, J.; Li, Q.; Huang, Q.; Shi, J.; Yan, H.; Fan, C. Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells. Angew. Chem., Int. Ed. 2014, 53, 7745−7750. (47) Biocca, S.; Filesi, I.; Mango, R.; Maggiore, L.; Baldini, F.; Vecchione, L.; Viola, A.; Citro, G.; Federici, G.; Romeo, F.; Novelli, G. The Splice Variant LOXIN Inhibits LOX-1 Receptor Function Through Hetero-Oligomerization. J. Mol. Cell. Cardiol. 2008, 44, 561−570. (48) Magee, L. S. LabNotes 2005, 15, 1−4. (49) Cardinale, A.; Filesi, I.; Vetrugno, V.; Pocchiari, M.; Sy, M. S.; Biocca, S. Trapping Prion Protein in the Endoplasmic Reticulum Impairs PrPC Maturation and Prevents PrPSc Accumulation. J. Biol. Chem. 2005, 280, 685−694.

5979

DOI: 10.1021/acsnano.6b01402 ACS Nano 2016, 10, 5971−5979