Download Gold nanoparticle-mediated gene delivery induces immunity genes

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SHORT COMMUNICATION
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Gold nanoparticle-mediated gene delivery induces
widespread changes in the expression of innate
immunity genes
E-Y Kim1, R Schulz2, P Swantek1, K Kunstman1, MH Malim3 and SM Wolinsky1
The unique properties of oligonucleotide (and small interfering RNA)-modified gold nanoparticle conjugates make them
promising intracellular gene regulation agents. We found that gold nanoparticles stably functionalized with covalently attached
oligonucleotides activate immune-related genes and pathways in human peripheral blood mononuclear cells, but not an
immortalized, lineage-restricted cell line. These findings have strong implications for the application of oligonucleotide-modified
gold nanoparticle conjugates in translational research and in the development of therapeutics and gene delivery systems.
Gene Therapy (2011) 0, 000–000. doi:10.1038/gt.2011.95
Keywords: gold nanoparticle–oligonucleotide complexes; gene delivery; gene expression; signaling pathways; immune activation
INTRODUCTION
New nanotechnologies that deliver oligonucleotides or small interfering RNA into cells to regulate gene transcript and protein
abundance are emerging. By conjugating antisense constructs to the
surface of gold nanoparticles, for example, polynucleotide stability,
binding properties and cellular uptake can be enhanced, and
ostensibly gene expression can be suppressed.1 This extent of regulation mediated by a nanoparticulate system could provide an effective
strategy to decipher gene pathways and function, and improve
the efficacy of gene therapy. Nevertheless, targeted delivery of nucleic
acids into immune cells by engineered nanoparticles may trigger
effector functions and elicit untoward molecular and biological
effects.2–6
Most cells have natural sensors of pathogen-associated molecular
patterns, other pathogen-associated molecules, cellular damage and
environmental irritants. Pattern recognition receptors (PRRs) of the
innate immune system, including the transmembrane Toll-like receptors (TLRs) and C-type lectin receptors as well as the cytoplasmic
RIG-I-like receptors and NOD-like receptors, recognize these structures and initiate signaling pathways that lead to the production of
type-1 interferons, proinflammatory cytokines and chemokines.7
These inducible gene products regulate the sensitivity and intensity
of the innate immune response to infection and injury. Though
oligonucleotide-modified gold nanoparticle conjugates are purportedly nontoxic for lineage-specific cell types, their effects on signaling
pathways that respond to activating stimuli in primary immune
cells, which are differentially activated depending upon the cell
type involved, has not been studied. Here, we show that human
peripheral blood mononuclear cells (PBMCs) exposed to gold
nanoparticles stably functionalized with covalently attached oligonucleotides lead to widespread transcriptional activation of innate
immune responses.
RESULTS AND DISCUSSION
Because pathogen-associated molecular patterns or other non-microbial
structures trigger signaling pathways and effector mechanisms of
innate immunity, we determined whether gold nanoparticle–oligonucleotide complexes impact gene expression with a functional genomics
approach. A phosphorothioate-modified non-targeting antisense
DNA sequence complementary to enhanced green fluorescent protein
(EGFP) that inhibits green fluorescent protein expression was
conjugated to the gold nanoparticle (13±1 nm) surface with a
mono-thiol group.1 In experiments meant to visualize their uptake
in the cell, the gold nanoparticle–oligonucleotide complexes were
functionalized with a 5¢ Cy5.5-label; for experiments aimed at determining the effect on cell signaling and gene expression, they were not
labeled with the fluorophore. The size and morphology of the gold
nanoparticle–oligonucleotide complexes were well characterized as
described.8 (Supplementary Figures S1 and S2). Real-time quantitative
PCR (RT-qPCR) analysis of the 16S ribosomal RNA gene and limulus
ameobocyte lysate analysis of bacterial endotoxin ruled out the
possibility for contamination of the gold nanoparticle–oligonucleotide
complex preparations by microbial products.9,10
The gold nanoparticle–oligonucleotide complexes or a commercially available cationic lipid transfectant (Oligofectamine, Invitrogen,
Carlsbad, CA, USA) with an equivalent amount of antisense EGFP
oligonucleotide were introduced onto PBMCs or 293T cells. There
were no differences in cell morphology in relation to the treated cells
or cell viability under the conditions of our experiments as assessed by
Trypan blue staining. As shown in Figure 1, there was efficient
internalization of gold nanoparticle–oligonucleotide complexes in
almost all PBMCs, regardless of cell type (Figure 1a). The amount
of gold nanoparticle–oligonucleotide complex uptake in the
cytoplasm increased in a time (and dose)-dependent manner
(Figure 1b), but did not further enter the nucleus.
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1Division of Infectious Diseases, The Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; 2Department of Medical and Molecular Genetics, King’s College
London School of Medicine, London, UK and 3Department of Infectious Diseases, King’s College London School of Medicine, London, UK
Correspondence: Dr SM Wolinsky, Division of Infectious Diseases, Northwestern University Feinberg School of Medicine, 303 East Superior Street, 645 North Michigan Avenue
Suite 900, Chicago, IL 60611, USA.
E-mail: [email protected]
Received 14 February 2011; revised 27 April 2011; accepted 28 April 2011
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Figure 1 Gold nanoparticle–oligonucleotide complexes are localized within membrane-bound organelles with electron-dense cores. (a) PBMCs were treated
with a 10 nM concentration of gold nanoparticle–oligonucleotide complexes with a 5¢ Cy5.5-label for 24 h. Representative cells from independent healthy
blood donors are shown (Cy5.5 fluorescence is red, actin is green and DNA is blue). A deconvoluted magnified image (100) with stitched panels (left) and
a single panel (right). Images were acquired with constant microscope settings. The uptake of the gold nanoparticle–oligonucleotide complexes is not specific
to a particular cell type. Almost every mononuclear cell or granulocyte incorporated the gold nanoparticle–oligonucleotide complexes in its cytoplasm.
(b) PBMCs were treated with 1, 6 and 10 nM concentrations of gold nanoparticle–oligonucleotide complexes with a 5¢ Cy5.5-label for 6 24 or 48 h. Uptake
was measured by flow cytometry. The data represent five independent experiments. (c) A representative primary human PBMC treated with the gold
nanoparticle–oligonucleotide complexes without a 5¢ Cy5.5-label (10 nM concentration) for 24 h is shown (2900, bar¼2 mm (left); and 11 000,
bar¼500 nm, with 12 000 inset magnification (right)). The distribution of discrete, highly electron-dense particles (alone or in aggregate) is confined
exclusively to cytoplasmic membrane-bound organelles.
To understand where the gold nanoparticle–oligonucleotide complexes are localized in the cytoplasmic compartment, we imaged
transverse sections of individual cells treated with a 10 nM concentration of the same gold nanoparticle–oligonucleotide complexes with
the use of transmission electron microscopy. For a given cell, discrete
gold nanoparticles (singly or in aggregate) were located mainly in
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B0.5-mm-diameter membrane-bound organelles with electron-dense
cores (Figure 1c), and not free in the cytosol or in the nucleus.
Electron microscopic morphological studies of trafficking of vesicles in
the endolysosomal system suggest that this cell structure is formed by
direct fusion of late endosomes and lysosomes to create a hybrid
organelle for the digestion of endocytosed macromolecules.11–14 Q4
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Figure 2 Principal component analysis of the gene expression profiles and the distribution of the percent explained variance across the first five principal
components. (a) Projection of all transcription profiles onto the plane spanned by the first two principal components, that is, the two orthogonal dimensions
along which the data exhibit the largest variance (the two eigenvectors whose eigen values have the largest variances). The first principal component explains
72% of the variance in the data and corresponds to a separation of the sample set into PBMCs (left) and 293T cells (right), that is, most of the variance in
gene expression across all arrays was caused by the difference in cell types. The second principal component explains 11% of the overall variance and
represents the difference between treatment with gold nanoparticle–oligonucleotide complexes and oligofectamine or no treatment of PBMCs. The 293T cell
samples were not separated along the second principal component axis. Accordingly, their gene expression hardly varied in response to treatment. (b) The
transcription profiles are shown in the space spanned by the first and fourth principal components for the PBMC samples only, explaining 42% and 8% of
the variance, respectively. Along the first principal component, the samples treated with gold nanoparticle–oligonucleotide complexes were widely separated
from the other samples, that is, most of the changes in PBMC gene expression occurred between these two sample groups. The fourth principal component
represented the variance introduced by the two different durations (24 h versus 48 h) of treatment with gold nanoparticle–oligonucleotide complexes.
To regulate gene transcript and protein abundance, the covalently
attached antisense polynucleotides or the engineered gold nanoparticle constructs per se must transverse this membrane-bound cellular
compartment to penetrate into the cytosol.15
mRNA levels were quantified in PBMCs under five conditions and
293T cells under four conditions for B47 000 gene transcripts with
the Affymetrix U133 Plus 2.0 expression array (Affymetrix, Santa
Clara, CA, USA; Supplementary Table S1). To match other studies of
the HeLa cell response to these gold nanoparticle–oligonucleotide
complexes, we performed genome-wide expression profiling of
PBMCs and 293T cells treated with gold nanoparticle–oligonucleotide
complexes.12,16 Three of the five PBMC conditions (24 and 48 h after
treatment with the gold nanoparticle–oligonucleotide complexes and
the negative control, respectively) were assayed independently a
second and a third time (technical and biological replicates, respectively) to control for experimental variability (15 microarrays
hybridized in total). A different batch of these same gold nanoparticle–oligonucleotide complexes was used in the generation of the four
293T cell samples and the three biologically replicate PBMC samples,
and the microarrays for these samples with calibration controls
included were processed separately.
The gene expression profiles of the replicate PBMC samples were
highly correlated (r240.92), indicating high experimental reproducibility. This was true for the 293T cell samples irrespective of the
treatment condition (r240.98), consistent with the finding that gold
nanoparticle–oligonucleotide complexes have little effect on gene
expression in immortalized lineage-restricted cell types, such as
293T cells and HeLa cells.12,16 In contrast, the correlation between
PBMCs and 293T cell samples was low (r2o0.46), indicating that
primary human cells in the blood and 293T cells have very distinct
transcription profiles. Unsupervised hierarchical clustering of the
transcription profiles confirmed and refined these observations.
We quantified the contributions of cell type and treatment with
gold nanoparticle–oligonucleotide complexes to the overall variance in
the microarray data using principal components analysis. We first
performed this analysis with the entire data set. The projection of the
transcription profiles onto the plane spanned by the leading principal
components showed that 72% of the variance in gene expression
across all samples was due to the difference between the PBMC and
293T cell types (Figure 2a). The second largest source of variance
(11%) was the difference between treatment of PBMCs with gold
nanoparticle–oligonucleotide complexes and the EGFP oligonucleotide cationic lipid transfectant control or no treatment (Figure 2a).
This difference had little effect on gene expression in 293T cells.
To further examine the contribution of treatment to changes in
the gene expression profile, we restricted the analysis to PBMCs
only (Figures 2b). For these samples, the largest source of
gene expression variance (42%) was the treatment with gold
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Figure 3 The most overrepresented GO terms and KEGG pathways in PBMCs treated with gold nanoparticle–oligonucleotide complexes. Shown are the –
log10-transformed multiple testing-corrected P-values of the (a) 19 most overrepresented GO term and (b) the 13 most overrepresented KEGG pathways for
two significance thresholds of differential gene expression (multiple testing-corrected SAM P-value o0.01 (blue) versus P-value o0.0025 (orange)). DAVID,
Database for Annotation, Visualization and Integrated Discovery; MHC, major histocompatibility complex.
nanoparticle–oligonucleotide complexes versus the EGFP oligonucleotide cationic lipid transfectant control or no treatments. The second
and third principal components (26% and 14% of variance, respectively) represent the differences between medium-only-negative
Q5 PBMC controls and the other controls, as well as nonbiological
differences between groups of samples. This batch effect explains
B20% of the variance in the PBMC data. After adjusting for batch
effects or other types of systematic bias in the microarray data using
principal component analysis, we found that the source of nonbiological variation did not correlate with the differences in gene expression
between PBMCs treated with gold nanoparticle–oligonucleotide
complexes and PBMCs not treated or treated otherwise. Thus, any
technical bias in the microarray data did not convolve true biological
differences. The fourth principal component (8% of variance) partitioned the four samples treated with gold nanoparticle–oligonucleotide
complexes according to the length of treatment (24 h versus 48 h).
We interrogated the gold nanoparticle–oligonucleotide complex
regulatory effects in PBMCs by exploring the functional composition
of significantly differentially expressed genes. At a significance level of
0.01 (multiple testing-corrected P-value o0.01), 2919 microarray
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probe sets detected a change in gene expression between the treated
and the untreated (both the EGFP oligonucleotide cationic lipid transfectant control or no treatment) PBMCs (Supplementary Figure S3 and
Supplementary Table S2). Thus, the non-targeting EGFP oligonucleotide
by itself was not responsible for the cell response. We used RT-qPCR to
confirm the microarray expression changes for 12 genes—evaluating the
TLR signaling, T-cell activation, endocytosis and apoptosis pathways.
Genes were selected based on P-values o0.05 and fold change 42 in
PBMCs. RT-qPCR analysis of the representative genes experimentally
confirmed the microarray data (Supplementary Table S3). This indicates
that the gold nanoparticle–oligonucleotide complexes per se can have a
significant impact on gene expression in PBMCs.
To discover dynamic changes of cellular processes, we determined
the categories of functionally related genes, using gene ontology (GO)
term and Kyoto Encyclopedia of Genes and Genomes (KEGG)
pathway analysis, which contained a significant number of genes
with altered expression levels in response to treatment with gold
nanoparticle–oligonucleotide complexes. A GO analysis revealed an
overrepresentation of differentially expressed genes linked to an innate
immune response (Figure 3a and Supplementary Table S4). The same
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result could be produced at a significance level of 0.0025 (multiple
testing-corrected P-value o0.0025) for 1341 probe sets, indicating
that the functional composition of the differentially expressed genes
was invariant with respect to arbitrary changes of the significance level,
and therefore robust. A similar pattern of enrichment was observed for
the KEGG pathways that reflected the identified GO terms involved in
innate immunity (Figure 3b and Supplementary Table S5; multiple
testing-corrected P-value o0.001; Figure 3 and Supplementary
Figure S4). Most consistently overrepresented were the immune
response, inflammatory response, response to wounding and defense
response categories (multiple testing-corrected P-value o105).
We did not identify any significant changes in the gene expression
profile for 293T cells treated with gold nanoparticle–oligonucleotide
complexes for at least 24 h after correction for multiple testing
(Supplementary Table S6). Nevertheless, we performed GO and
KEGG pathway analyses for 293T cells using the genes that exhibited
significant differential expression before multiple test correction to
determine whether there was any overlap with respect to overrepresented gene categories (Supplementary Tables S7 and S8). We found
no overlap between 293T cells and PBMCs for the raw P-value
thresholds examined (P-value o0.05: 2349 probe sets, P-value
o0.0125: 628 probe sets; Supplementary Figure S5). Consequently,
the extent of functional gene category sharing across the gold
nanoparticle–oligonucleotide complex-treated PBMC and 293T cell
types is insignificant.
Although innate immune signaling receptors evolved to recognize
specific microbial products, it appears that these same receptors can
detect a broad range of other activating stimuli. The changes in the
signaling pathways activated in response to the gold nanoparticle–
oligonucleotide complexes were highly complex, suggesting that
multiple PRRs were responsible for recognizing these exogenous
molecules. Because the oligonucleotide-modified gold nanoparticle
conjugates activate signaling cascades that lead to the induction of
genes involved in cytokine signaling and immune regulation, it is not
surprising that there is a direct effect on the physiological state of
PBMCs and not cell lines. The engineered nanoparticles presumably
interact with PRRs outside the cell and in endosomes and lysosomes
to produce these signaling events in innate immune cells, such as
dendritic cells, macrophages and neutrophils, among others, through
mechanisms that are currently not clear. Activation of PRRs by these
non-microbial-activating signals induces the synthesis and secretion of
regulatory molecules known to mediate cell–cell communication that
can strongly amplify the innate immune response to cellular stress.
Our results highlight the need to study the potential harmful
interactions and system effects caused by engineered nanoparticle
structures with a relevant biological system and the appropriate
discovery tools that can identify perturbations of genes important
for cell signaling and innate immunity. Our data furthermore question
the mechanism by which gold nanoparticles stably functionalized with
covalently attached oligonucleotides could regulate gene transcript
and protein abundance.1 In light of the cell-type-specific differences in
cell signaling and effector responses, our data imply that assessments
of the toxic potential for engineered nanoparticles in immortalized
lineage-restricted cell lines may not predict their phenotypic effects in
relevant biological systems and should be interpreted with caution.
MATERIALS AND METHODS
Cell culture and DNA transfection
PBMCs derived from healthy donors were isolated on Ficoll-Paque gradients
(MP Biomedicals, Santa Ana, CA, USA). 293T cells were obtained from the
American Type Culture Collection. Cells were grown in 5% CO2 at 37 1C in
RPMI1640 medium or Dulbecco’s modied Eagle’s medium (Invitrogen) with
10% fetal bovine serum supplemented with 100 U ml1 penicillin, 100 ı̀g ml1
streptomycin and 200 mM L-glutamine. Cells were seeded at 50% confluent in
24-well plates, grown for 24 h, washed with 1 phosphate-buffered saline
(PBS) and fresh media was added. Antisense EGFP oligonucleotide was
delivered onto the freshly washed cells using cationic lipid transfection
(Oligofectamine, Invitrogen) according to the manufacturer’s direction. Gold
nanoparticle–oligonucleotide complexes were filtered (0.2 mm) and added onto
the freshly washed cells. After 6, 24 or 48 h, the cells were washed in 1 PBS
and collected for analysis. The gold nanoparticle–oligonucleotide complexes
had no shift in color from red to purple associated with their aggregation, and
therefore remained stable under the cell culture conditions we used. Cell
viability was assessed by the Trypan blue exclusion method (Sigma-Aldrich,
St Louis, MO, USA).
Limulus amoebocyte lysate assay analysis
Because nanoparticles often interfere with limulus amoebocyte lysate assays
owing to their intrinsic physicochemical properties, endotoxin contamination
was assessed with the Lonza gel-clot limulus amoebocyte lysate assay format
(sensitivity of 0.03 EU ml1) using certified reagents and supplies according to
the manufacturer’s instructions (Lonza BioWitthaker, Walkersville, MD, USA).
A United States Pharmacopeia (USP)-certified Escherichia coli 055:B5 endotoxin
standard spiked into endotoxin-free water was used to generate a standard
curve and allow calculation of the input amount of endotoxin. A gold
nanoparticle–oligonucleotide complex control and inhibition/enhancement
control (USP-certified endotoxin standard at the standard curve midpoint
concentration added to the gold nanoparticle–oligonucleotide complexes) were
used to screen for potential false-positives or false-negatives.9,10 Controls were
used within 30 min of their preparation. Replicate samples were tested at four
different dilutions in triplicate. The parameters for the gel-clot limulus
amoebocyte lysate assay met the validity criteria for evaluation of endotoxin
contamination as described.9
Bacterial culture
Gold nanoparticle–oligonucleotide complexes were inoculated into sterile
Luria–Bertani broth without antibiotics and grown in a shaking incubator
(225 r.p.m.) at 37 1C. After overnight culture, the tubes were screened for visible
bacteria growth and then centrifuged (6000 g, 15 min, at 4 1C) to concentrate
any bacteria for RT-qPCR analysis for bacterial 16S ribosomal DNA.
Bacterial 16S ribosomal DNA
We used RT-qPCR to determine the presence of bacterial 16S ribosomal DNA
in the gold nanoparticle–oligonucleotide complex preparations directly and
from the concentrated overnight cultures. Samples were amplified with Taqman
Universal PCR master mix reagent (Applied Biosystems, Foster City, CA, USA)
as per manufacture’s protocol. Primer sequences for the bacterial 16S ribosomal
gene were forward (8F: 5¢-AGTTTGATCCTGGCTCAG-3¢), reverse (515R: 5¢-GW
ATTACCGCGGCKGCTG-3¢) and probe (338P: 5¢-FAM-GCTGCCTCCCGTAG
GAGT-BHQ1-3¢). After Taq polymerase activation at 94 1C for 10 min, the
cycling was performed with 45 cycles of denaturation at 94 1C for 15 s and
primer–probe annealing at 60 1C for 1 min with the ABI 7900 Sequence
Detection System. Data were analyzed with the system’s SDS software Q6
(v2.1).17 Serial dilutions of a standard and negative controls were included to
generate a standard curve and allow calculation of the input amount of
bacterial 16S ribosomal DNA. Reactions were performed in triplicate and
results for the reaction plate were confirmed by gel electrophoresis.
Cell imaging studies
Cells were grown in a 24-well tissue culture plate. The gold nanoparticle–
oligonucleotide complexes were then added to the culture plate wells. After 6,
24, or 48 h of incubation, the cells were washed with PBS, immobilized onto
chambered glass coverslip treated with Cell Tak (BD Bioscience, Bedford, MA,
USA), and then fixed with 3.7% formaldehyde (Polysciences, Warrington, PA,
USA) in 0.1 M PIPES buffer (pH 6.8). Cells were stained with fluorescein Q7
isothiocyanate-labeled anti-actin mouse monoclonal antibody (Cytoskeleton
Inc., Denver, CO, USA) to access the cytosol and Hoechst dye to access nuclear
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E-Y Kim et al
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morphology, washed and mounted on glass slides for imaging. Images were
collected with a digital camera (CoolSnap HQ; Photometrics, Tucson, AZ,
USA) using a DeltaVision Deconvolution Microscope equipped with a 100
1.4 NA objective lens (Applied Precision, Issaquah, WA, USA).
Electron microscopy
Cells treated with the gold nanoparticle–oligonucleotide complexes were
washed, fixed with 2% paraformaldehyde/2.5% glutaraldehyde in cacaodylate
buffer (0.1 M), and then embedded in 2% agar. After additional fixation, cells
dehydrated through a graded ethanol series, and infiltrated and embedded in
LX112 resin at 60 1C for 24 h. Ultrathin sections (60 nm) were cut from the
embedded blocks, mounted onto a 200-mesh copper grid, and then stained
with uranyl acetate and lead citrate by standard methods. Stained grids
were examined using a FEI Tecnai Spirit G2 120 kV transmission electron
microscope and imaged with a FEI Eagle 4k CCD camera (FEI, Hillsboro, OR,
USA). The gold nanoparticle–oligonucleotide complex preparations were
added directly onto a 3-mm diameter copper grid coated with carbon film.
The particle size distribution was obtained from the transmission electron
microscope images.
Ultraviolet–visible absorption spectrum
The ultraviolet–visible absorption spectrum of the gold nanoparticle–oligonucleotide complexes was recorded on a SpectraMax M2 spectrometer (Molecular
Devices, Sunnyvale, CA, USA) with a spectral resolution of 1 nm.18
Microarray studies
Total RNA was extracted from cell cultures using an RNeasy Mini Kit (Qiagen,
Valencia, CA, USA). RNA quantity (concentration greater than 0.5 mg ml1) and
purity (OD260/OD280 ratio above 1.8) were verified in the extracted samples.
The microarray workflow was monitored at the cDNA production, in vitro
transcription, complementary RNA fragmentation and labeling, and target
hybridization stages. Internal hybridization controls were included. Biotinylated
complementary RNA probes prepared according to standard protocols were
hybridized to Affymetrix Human HG-U133 Plus 2.0 arrays (B52 000 probe
sets). The arrays were scanned with the Affymetrix GeneChip Scanner 3000
(Affymetrix). Microarray data probe-level analysis and filtering were performed
using the xps v1.6.2 BioConductor package implementations of the FARMS19
and I/NI methods.20 Differential gene expression was ascertained using the
siggenes v1.18.0 and multitest v2.2.0 BioConductor package implementations
of the SAM and Benjamini–Hochberg multiple testing correction methods.21,22
The Matlab r2007a platform was used for correlation, hierarchical clustering
and principal component analyses. The Database for Annotation, Visualization
and Integrated Discovery (DAVID) tool v6.7 (http://david.abcc.ncifcrf.
Q8 gov:8080/) was used for in silico discovery of GO term and KEGG biochemical
pathways.23,24 Non-informative probe sets (I/NI P-value 40.6) did not enter
subsequent analyses.
RT-qPCR gene expression analysis
RT-qPCR analysis for selected genes (Supplementary Table S1) was performed
using Taqman RNA assays (Applied Biosystems), according to standard
protocols. Total RNA of 5 ng was reverse transcribed with random hexamers
Q9 using a Superscript III First strand cDNA synthesis kit (Invitrogen). Messenger
RNA expression was quantified by real-time PCR with an ABI 7900HT RealTime PCR machine (Applied Biosystems). Primer sequences were designed and
optimized by ABI Assays-on-Demand for human IL1B (Hs01555410_m1), JUN
(Hs99999141_s1), TLR1 (Hs00413978_m1), NFKBIA (Hs00153283_m1), IL2RA
(Hs00907779_m1), EHD1 (Hs00199030_m1), TLR4 (Hs00152939_m1), CLTA
(Hs00747351_mH), NFKB2 (Hs00174517_m1), CTLA4 (Hs03044418_m1), FYN
(Hs00176628_m1), MAP2K7 (Hs00178198_m1), GAPDH (Hs99999905_m1)
and â-actin (Hs99999903_m1). Reactions were performed in triplicate and
analyzed with the ABI SDS software (v2.1). Results for the duplicate samples
were averaged. Values were then normalized by the amount of â-actin and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in each sample by means
of the 2–DDCT method.
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Statistical analysis
Statistical analyses compared results from gold nanoparticle–oligonucleotide
complex-treated and -untreated controls in primary immune cells microarray,
flow cytometry and RT-qPCR analyses by using each analysis software and
GraphPad Prism 4.1. (GraphPad Prism, San Diego, CA, USA).
CONFLICT OF INTEREST
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The National Institutes of Health, the National Science Foundation, the United
Kingdom Medical Research Council and the International Institute for
Nanotechnology within Northwestern University supported this study. Data
were presented at the 2009 meeting of the International Institute for
Nanotechnology, Nanoscale Science and Engineering Center for Integrated
Nanopatterning and Detection Technologies, Northwestern University. EK is
the recipient of a Baxter Award from the Institute for BioNanotechnology in
Medicine. RS is a Research Councils UK Academic Fellow. All patient samples
were collected with Northwestern University Institutional Review Board (IRB)
consent. We thank David Giljohann and Chad Mirkin for generously providing
the gold nanoparticles stably functionalized with covalently attached oligonucleotides; Edward Campbell and Kelly Fahrbach for assistance with fluorescent microscopy; Robert Goldman and Lennell Raynolds for assistance with
transmission electron microscopy; Carlos Nahas and Maurice O’Gorman for
assistance with FACS analysis; and Jaejung Kim for assistance with the human
microarrays. The microarray data have been deposited in the National Center
for Biotechnology Information Gene Expression Omnibus (www.ncbi.nlm.nih.
gov/geo) and are accessible through GSE 20677 Series accession numbers
GSM518597 to GSM518609 and GSM535541 to GSM535543.
AUTHOR CONTRIBUTIONS
E-YK, SMW and MHM planned and designed the project. E-YK., PS and KK
performed the experiments and E-YK., RS, SMW analyzed the data. E-YK. and PS
performed experiments with PBMCs and the lineage-restricted 293T cell line, E-YK
performed fluorescent microscopy and transmission electron microscopy, E-YK and
PS performed the FACS analysis, E-YK and KK performed the RT-qPCR experiments, E.-YK and PSperformed human microarray experiments and RS performed
the human microarray data analysis. E-YK, RS, MHM and SMW interpreted the
results and drafted the manuscript. All the authors participated in critically
reviewing the text and approved the final version of the manuscript.
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Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)
Gene Therapy