List of Supplementary information:
Methods:
Agarose gel analysis of siRNA-dendrimer complexes. The dendrimers were serially
diluted to an appropriate concentration in 50 mM Tris-HCl buffer (pH 7.4), with all
solutions stored at -20°C. The 27 mer Tat/rev siRNA was diluted with H2O. Both
solutions were mixed at various N/P (= [total end amines in cationic dendrimer] /
[phosphates in siRNA]) ratios and incubated at 37°C for 30 min in Tris-HCl buffer. The
final concentration of siRNA was adjusted to 25 ng/L (100 ng per well). After
incubation the binding complexes were loaded on a 1.2% agarose gel for electrophoresis.
The siRNA bands were stained by ethidium bromide and then detected by a Herolab
EASY CCD camera (Type 429K) (Herolab, Wiesloch, Germany).
Stability of siRNA-dendrimer complexes against RNase A. An aliquot of 27 mer
Tat/rev siRNA (1 g) and the indicated amounts of dendrimers were kept at 37°C for 30
min. The complexes were then incubated in the presence of 0.05 g/L RNase A for
different times as indicated in the gel at 37°C. Aliquots (4 L) of the corresponding
solution were withdrawn, added to 1.5 L 1% SDS solution on ice, and the subjected to
electrophoresis in 1% agarose gel in standard TAE buffer. The siRNA bands were stained
by ethidium bromide and then detected by a Herolab EASY CCD camera (Type 429K)
(Herolab, Wiesloch, Germany).
Transmission electron microscopy (TEM) analysis. Studies were performed with a
JEM-2000FX transmission electron microscopy. A solution (10 L) of 27 mer Tat/rev
siRNA (5 ng/L) was mixed with a solution (10 L) of G5 dendrimer in Opti-MEM
transfection medium. After equilibration (30 min), this mixture (4 L) was dropped on a
standard carbon-coated copper TEM grid and allowed to evaporate (1 h at 30°C, ambient
pressure). In the case of siRNA-dendrimer solutions, the samples were premixed and
allowed to equilibrate for 30 min before placing on the grid. The grid was then stained
with uranyl acetate (2% in 50% alcoholic solution) for 3 min. Imaging was performed
after air-drying for 20 min.
Cellular uptake study of dendrimer-siRNA complexes: To evaluate cellular uptake
and subsequent intracellular routing of complexes, the allStars non-silencing siRNA
modified with 3’-Alexa488 conjugated by an aminohexyl linker was obtained from
Qiagen (Courtaboeuf, France). One day before use, HeLa cells were seeded at a density
of 5×104 cells/chamber in 8-well glass chamber slides (Labtek, Nunc, USA). Preparation
of the Alexa 488-labeled siRNA/dendrimer complexes was performed as described
previously. The complex containing Alexa 488-labeled siRNA was added to the cells in
Opti-MEM transfection medium.
Confocal microscopy: After incubation for 4 hour at 37 ºC, the cells were washed with
PBS and staining with Hoechst 34580. Zeiss LSM 510 Meta laser scanning confocal
microscope equipped with inverted Zeiss Axiovert 200M stand (Carl Zeiss GmbH, Jena,
Germany) was used for visualization. Images were acquired using LSM 510 software
(Carl Zeiss GmbH, Jena, Germany). For examination of actin rearrangement, after
incubation for 15 min at 37 ºC with siRNA/dendrimer nanaoparticles, cells were fixed in
4% paraformaldehyde, permeabilized with 0.1% triton, incubated with 1% bovine serum
albumin (BSA) and stained with Alexa-Fluor 647 phalloidin to label actin fibers, then
with DAPI to label the nuclei.
Flow cytometry: The uptake mechanism of the siRNA/G5 nanoparticles was examined
by means of specific inhibitors of different endocytic pathways. For inhibition
experiments, the cells were seeded at a density of 5×105 cells in 3.5-cm dishes (Nunc,
USA) one day before, then they were incubated with one of following inhibitors:
cytochalasin D (to inhibit macropinocytosis), genistein (to inhibit caveolae-mediated
endocytosis), or chlorpromazine (to inhibit clathrin-mediated endocytosis) for 1 hour in
completed medium before the G5 dendrimer-siRNA complexes were added. Inhibitors
were used at concentrations in which they were not cytotoxic for HeLa cells. After
incubation for 15 minutes at 37 ºC with the G5 dendrimer-Alexa 488-labeled siRNA
complex, the cell uptake efficiency was measured using flow cytometry analysis
(Beckman Coulter Epics Elite, Beckman Inc., Miami, FL). Each assay was performed in
triplicate.
Cellular uptake studies (Fluorescent Microscopy analysis). 2105 CEM cells per well
were seeded in 24-well tissue culture plates in 300 L fresh complete medium containing
10% FBS. CEM cells were transfected with 50 nM Cy3-labeled Tat/Rev Site I dsiRNA
(27 mer) using dendrimers G4 and G5 as above described. 24 hours of post-transfection,
the images were collected using a Nikon Eclipse TE2000-S fluorescent microscopy
system at 20× magnification.
Flow cytometry: Downregulation of CD4 levels by G5-siRNA nanoparticles in CEM
cells detected by flow cytometry. 2105 CEM cells per well were seeded in a 24-well
tissue culture plate in 300 L fresh complete medium containing 10% FBS. Before
transfection, complexes of dsiRNA/dendrimer reagents were prepared. The desired
amount of dsiRNA and dendrimer reagent was diluted in 50 L of serum-free medium
(Opti-MED), respectively. The dendrimer solution was mixed gently and incubated for
10 min at room temperature. After the 10-minute incubation, the diluted dsiRNAs and the
dendrimer reagent were mixed gently and incubated for 30 minutes at room temperature.
The 100 L of dsiRNA/dendrimer complex was added to each well containing cells and
medium and mixed gently by rocking the plate back and forth. A commercial transfection
reagent TransIT-TKO (Mirus, Madison) was used as control according to the
manufacturer’s instructions.
The cells were incubated at 37°C in a CO2 incubator for 48 hours for further assays. The
cells were harvested, washed once with PBS and resuspended in 100 µL of ice cold PBS,
0.5 % BSA, 0.2 % Sodium Azide. Primary labelled antibody: 0.1~10 µg/mL (10 µL of
CD4 antibody [EDU-2] (FITC) (Abcam) is sufficient for labeling of 1*106 cells). After
incubation at least for 30 min at RT, reaction solutions were centrifuged to remove the
supernatants and washed twice with 320 µL of cold PBS, 0.5 % BSA, 0.2 % Sodium
Azide at RT, and finally resuspended in 350 µL of cold PBS, 0.5 % BSA, 0.2 % Sodium
Azide at 25°C and spun down at 1500 rpm for 4 min at 25°C. Cells were immediately
assayed using flow cytometry keeping the cells in the dark on ice.
5’-RACE PCR assay. Total RNA (5 g) from HEK 293 cells transfected with 50 nM
experimental RNAs (TNPO3 25/27-mer siRNA or scrambled siRNA) in the presence of
the G5 dendrimer was isolated using Trizol per the manufacture’s protocol. Residual
DNA was digested using the DNA-free kit per the manufacturer’s instructions (Ambion).
Subsequently, total RNA was ligated to a GeneRacer adaptor (Invitrogen, Carlsbad, CA)
without prior treatment. Ligated RNA was reversed transcribed using a gene specific
primer 1 (GSP-Rev 1: 5’- TCC CGT AAA GAG GCA TGA GAG TCT GT -3’). To
detect cleavage products, PCR was preformed using primers complementary to the RNA
adaptor (5’-cDNA primer) and gene-specific primer 2 (GSP-Rev 2: 5’- CCG GAT CTG
TAA CAA CTG GTC TGA GA -3’). Amplification products were resolved by agarose
gel electrophoresis and visualized by ethidium bromide staining. The specific PCR
products were recovered using a QIAquick Gel purification Kit and then were cloned into
TOPO TA cloning vector pCR 2.1-TOPO vector (Invitrogen). Individual clones were
identified by DNA sequencing.
Northern Blot analyses of CEM cells and PBMCs. Cells were transfected with 50 nM
Tat/Rev Site I dsiRNA (27 mer) using dendrimers G4 and G5 as described above. After 48
hours post-transfection, the total RNAs were harvested for analysis with STAT-60 (TELTEST “B”, Friendswood, TX) according to the manufacturer’s instructions. Five
micrograms of total RNAs were electrophoresed in a 15% polyacrylamide-8 M urea gel
and then transferred to a Hybond N+ membrane (Amersham pharmacia Biotech, USA).
Pre-hybridization and hybridization were carried out using PerfectHyb Plus
Hybridization buffer (Sigma, USA) at 37°C with 3 pmol of a 27-mer DNA
oligonucleotide probe end-labeled with T4 polynucleotide kinase and γ-32P-ATP. Filters
were washed three times at 37°C for 15 min, prior to autoradiography. We also probed
for human U6 snRNA as an internal RNA loading standard. In this case, the same siRNA
was electroporated into CEM cells as system control following the manufacturer’s
instructions (Amaxa, MD).
Interferon
assays
(qRT-PCR
analysis).
CEM
cells
were
treated
with
dsiRNA/dendrimer complex or IFN-alpha (100 U/mL). After 48 h, total RNAs were
isolated with STAT-60 (TEL-TEST “B”, Friendswood, TX). Expression of human
mRNAs encoding p56(CDKL2) and OAS1 were analyzed by quantitative RT-PCR using
2X iQ SyberGreen Mastermix (BIO-RAD) as described above and specific primer sets
for these genes at final concentrations of 400 nM. Primers were as follows: P56 (CDKL2)
forward, 5’-GCC TCC TTG GGT TCG TCT ATA A - 3’; P56 (CDKL2) reverse, 5’-CTC
AGG GCC CGC TCA TAG TA - 3’; OAS 1 forward, 5’ –GGA GGT TGC AGT GCC
AAC GAA G - 3’; OAS 1 reverse, 5’-TGG AAG GGA GGC AGG GCA TAA C - 3’.
GAPDH expression was used for normalization of the qPCR data.
MTT Cytotoxicity Assay. The cell viability of siRNAs plus dendrimers on CEM or
PBMCs was determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium
bromide (MTT) assay (Sigma-Aldrich, USA). Cells were seeded in each well of 96-well
microtiter plates and then treated with dendrimer-siRNA complexes. After 48 hour of
post-transfection with dendrimer-siRNA, cells pellets were collect by centrifugation and
placed into fresh medium. 10 L MTT solution (5 mg/mL in PBS) was added to each
well and then incubated with cells for 2 h in the 37°C. After careful removal of the
supernatant the cells were frozen for at least 1 h at -80°C. To each well was added 100
L of DMSO to dissolve the crystals properly, the absorbance was measured with a
Wallack plate reader using the Kamings protocol at 595 nm. The viability was expressed
as the ratio of absorbance obtained from transfected cells to non-transfected cells (n=6).
In vivo distribution and cryosection observation of G5 dendrimer-Cy5 siRNA
complexes
Cy5-labeled siRNA was provided by Suzhou Ribo Life Science Co. Ltd. The sequence
was as follows: sense strand: 5’- UUCUCCGAACGUGUCACGUTT -3’, antisense
strand: 5’- ACGUGACACGUUCGGAGAATT -3’. Optimal cutting temperature (OCT)
compound was purchased from Sakura Finetek USA, Inc. (Torracne, CA 90501, USA).
DAPI
(4',
6-diamidino-2-phenylindole,
for
staining
nuclei)
and
Fluorescein
Isothiocyanate Labeled Phalloidin (for staining F actin) were purchased from Zhongshan
Goldenbridge Biotechnology Co. Ltd., Beijing, China and Sigam-Aldrich, USA,
respectively.
Animals were maintained in the Peking University Laboratory Animal Center (an
AAALAC accredited experimental animal facility). All procedures involving
experimental animals were performed in accordance with protocols approved by
Institutional Animal Care and Use Committee (IACUC) of Peking University. Male
C57BL/6 mice at the age of 5-7 weeks, weighing in the range of 18-22 gram, were
purchased from the Academy of Military Medical Sciences of China. All animals were
subjected to hair-clipping of the abdominal region a day before administration to
facilitate observation. Three mice were administered with the following solutions via tailvein injection: (1) 200 μL saline; (2) naked Cy5-labeled siRNA in 200 μL saline at the
dosage of 0.3 mg/kg; (3) dendrimer G5/Cy5-labeled siRNA complex (N/P = 10:1) in 200
μL saline at the dosage of 0.3 mg/kg, respectively. Cyanine Cy5’s fluorescence signal
from the mice whole body imaging was detected using a Kodak in vivo imaging system
(Kodak In-Vivo Imaging System FX Pro, Carestream Health, USA) at the given time
points. At the last time point, the mice were sacrificed and the major tissues were isolated
and examined again. These analyses were carried out independently three times The
tissues were placed on omnisette tissue cassettes, embedded in optimal cutting
temperature (OCT) compound, and frozen in a prechilled Dewar flask containing a liquid
nitrogen/dry ice slurry for ~1 minute until the OCT turned white and opaque.
Subsequently, the specimens were cut into 10 micron histology slices with a cryostat.
Each section was placed on a glass slide, stained by DAPI (Zhongshan Goldenbridge
Biotechnology Co. Ltd., Beijing, China) and by Phalloidin (Fluorescein Isothiocyanate
Labeled, Sigma-Aldrich, USA) to visualize actin and display the rough cellular outlines.
Frozen sections were observed under a confocal microscope (LSM 700, Carl Zeiss,
Germany).
Histopathology analysis: Toxicology studies of Dendrimer-siRNA treated, HIV
infected, humanized mice. Tissues from five mice labeled: J991, J990, J986, J983,
J984, and J987 were fixed in 10 %neutral buffered formalin for histological evaluation.
The tissues were routinely processed and sectioned at 4-5 micron thick sections.
HISTOPATHOLOGY: 24 H&E stained slides containing liver, spleen, lung, and kidney
cross sections were histologically evaluated using the criteria detailed below.
Standardized toxicological pathology criteria and nomenclature for the mouse were used
to categorize microscopic tissue changes. The tissues were evaluated without knowledge
of the specific pharmacologic activity or formulation of the test articles. The following
criteria were evaluated in at least three high power microscopic fields in each section of
liver provided avoiding processing and microtome artifacts.
Cellular swelling:
0 when there is no visible cellular swelling noticed
A score of 1 is given when less than 25% of hepatocytes or renal tubules are swollen
2 is scored when 25-50% of hepatocytes or renal tubules are involved
3 is scored when 50-75% of hepatocytes or renal tubules are involved
4 is scored when greater than 75% of hepatocytes or renal tubules are swollen
Necrosis:
0 where is no discernible necrosis noticed
1 when individual hepatocyte necrosis (piecemeal or minimal) or individual renal
epithelial necrosis is observed.
2 when there is a focal area of necrosis
3 when there are multifocal areas or locally extensive areas of necrosis
4 when there is massive necrosis
Hypertrophy:
0 when there is no visible hepatocellular or renal epithelial hypertrophy
A score of 1 is given when less than 25% of hepatocytes or renal tubules are hypertrophic
2 is given when 25-50% of hepatocytes or renal tubules are hypertrophic
3 is given when 50-75% of hepatocytes or renal tubules are hypertrophic
4 is given when greater than 75% of hepatocytes or renal tubules are hypertrophic
Hyperplasia:
0 when no mitoses are detected
1 when one mitotic figure/high power microscopic field (HPMF) is detected in enlarged
cells
2 When 1-2 mitotic figures/HPMF are detected in enlarged cells
3 when more than two mitotic figures /HPMF are detected in enlarged cells
4 when there are more than two mitotic figures per each HPMF in enlarged cells
Inflammation:
0 when there are no visible aggregates of inflammatory cells seen
1 when aggregates of less than 10 inflammatory cells are seen /HPMF
2 When aggregates of 10-20 inflammatory cells are seen /HPMF
3 when aggregates of 20-50 inflammatory cells are seen /HPMF
4 when aggregates of 50-100 inflammatory cells are seen /HPMF
Bile duct hyperplasia (only applies to livers):
0 when there in no bile duct hyperplasia detected
1 when there are 2-3 newly formed bile ductules in one portal triad
2 when there are 3-5 newly formed bile ductules in one or more portal triads
3 when there are 5-10 newly formed bile ductules in one or more portal triads
4 when there are greater than 10 bile ductules in one or more portal triads
Mineralization:
0 when there is no visible hepatocyte mineralization
1 when individual hepatocytes are obliterated by basophilic mineral deposits
2 when 2-3 hepatocytes/ HPMF are obliterated by basophilic mineral deposits
3 when 3-5 hepatocytes /HPMF are obliterated by basophilic mineral deposits
4 when more than 5 hepatocytes /HPMF are obliterated by basophilic mineral deposits
Total score:
Minimal if total score is less than 5
Mild if the total score is 5-10
Moderate if the total score is 10-15
Marked if the total if the score is 15-20