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Supplementary Data
Poly(amido)amine (PAMAM) dendrimer-cisplatin complexes for chemotherapy of ovarian
cancer: Efficacy evaluation in vitro
Venkata K Yellepeddi, Kiran Kumar Vangara, Srinath Palakurthi*
1. Synthesis of Biotin-PAMAM NH2 dendrimers
Figure S1: Scheme of synthesis of PAMAM NH2-biotin. sulfo-NHS-LC-biotin and PAMAM
NH2 dendrimers were reacted in pH 9.0 buffer for 4 hrs at room temperature. (Only few of
available 64 amine surface groups are shown for convenience).
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2. Synthesis of Biotin PAMAM COOH dendrimers
Figure S2: Scheme of synthesis of PAMAM COOH–biotin. Biotin-LC-hydrazide and PAMAM
G3.5 COOH were reacted in MES buffer pH 4.7 overnight at room temperature. (Only few of
available 64 carboxylate surface groups are shown for convenience).
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3.
1H
NMR spectra of biotinylated PAMAM NH2 dendrimers
Figure S3: 1H NMR spectra of biotinylated PAMAM NH2 dendrimers. Dendrimers were
dissolved in D20 at 5 mg/ml concentration. (a) PAMAM NH2 and (b) Biotinylated NH2
dendrimer.
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4.
1H
NMR spectra of biotinylated PAMAM COOH dendrimers
Figure S4: 1H NMR spectra of biotinylated PAMAM COOH dendrimers. Dendrimers were
dissolved in D20 at 5 mg/ml concentration. (a) PAMAM COOH and (b) Biotinylated COOH
dendrimer.
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5. MALDI-TOF spectra of biotinylated PAMAM NH2 dendrimers
Figure S5. MALDI-TOF spectra of biotinylated PAMAM NH2 dendrimers. PAMAM G4 NH2
(Top) and Biotin PAMAM G4 NH2 (Bottom). Dendrimers were dissolved in methanol
(0.1mg/ml) and spectra were recorded using 2,5 – dihydroxybenzoic acid as matrix.
6. MALDI-TOF spectra of biotinylated PAMAM COOH dendrimers
Figure S6. MALDI-TOF spectra of biotinylated PAMAM G3.5 COOH dendrimers. PAMAM
G3.5 COOH (Top) and Biotin PAMAM G3.5 COOH (Bottom). Dendrimers were dissolved in
methanol (0.1mg/ml) and spectra were recorded using 2,5 – dihydroxybenzoic acid as matrix.
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7. Analytical method development for estimation of cisplatin
Analytical methods for quantification of cisplatin were developed using UV, HPLC and
ICP-MS techniques. Various parameters such as ease of sample preparation, sensitivity and
robustness of method were considered when choosing the most suitable analytical method for
platinum estimation. Optimizations of the parameters for all the analytical methods investigated
are given in Table 1. Based on the data, ICP-MS was considered to be the most sensitive and
robust method with very simple sample preparation technique. UV and HPLC methods not only
involved cumbersome derivatization in sample preparation methods but also were not sensitive.
Moreover, the HPLC and UV methods were not robust as the results were not consistent when
the pH of the sample was altered.
Table S1: Analytical method development parameters for measuring cisplatin.
Analytical
Method
Conditions
Derivatization
Lower Limit of
Quantification
(Sensitivity)
Linearity
Range
Robustness of
Method
Ultraviolet
Absorption
Absorbance
measured at
254 nm using
a double beam
spectrophotometer.
Samples needed
to be derivatized
with DDTC
(diethyldithiocar
bamate)
0.1 µg/mL
0.1 to
10µg/mL
Sensitivity
reduced by 100
times with
change in pH
of the sample.
High
Performance
Liquid
Chromatograph
y (HPLC)
Samples were
eluted using a
C18 column
with
1.5mL/min
rate with a
mobile phase
A [methanol:
water (30:40)]
and B
[Acetonitrile]
in 80:20 ratio.
The peaks
were detected
using a UV
detector at
254nm.
Samples needed
to be derivatized
with DDTC
(diethyldithiocar
bamate) prior to
elution.
0.1µg/mL
0.1 to
100µg/mL
Split peaks
were observed
with change in
pH of sample.
Inductively
Coupled Plasma
Mass
Spectroscopy
(ICP-MS)
Samples
dissolved in
1% HNO3 and
introduced
into ICP-MS
in 2Xi screen
mode.
No
derivatization
required.
100 pg/mL
100pg/mL
to
10ng/mL
No change in
sensitivity with
changes in pH.
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8. ICP-MS Analysis of Cisplatin
The ICP-MS system was comprised of an X Series ICP-MS (Thermo Electron
Corporation, Madison, WI, USA) equipped with a Cetac 500 auto sampler (Cetac
Technologies, Omaha, NB, USA). Analysis was performed in X-series default mode using 2.Xi
+ screen. The results were analyzed using PlasmaLab software (Thermo Electron Corporation.
Details of ICP-MS method and operation conditions are provided in supplementary data Table1. In vitro release of cisplatin from dendrimer-cisplatin complexes was determined using ICPMS. Major isotopes of platinum and iridium were monitored at m/z 195 and 193, respectively.
Sample nebulization was performed using a concentric nebulizer and detection modes for both
isotopes were ‘scanning’. Details of ICP-MS operating conditions are given in Table 1.
Quantification was based on the mean (n=3) intensity ratios for platinum and iridium against a
calibration curve using linear regression analysis. All standards and samples were prepared in 1
% OmniTrace® nitric acid. A standard platinum calibration curve was prepared with
concentrations from 0.1 to 1000 ng/mL. In all standard solutions iridium was added to get a
final concentration of 10 ng/mL.
Table S2: ICP-MS instrument settings
Flow Conditions (L min-1)
Torch
(mm)
Plasma flow
Sampling
130
Auxillary flow
Sheath gas
Nebulizer flow
18.0
0.90
0.25
0.95
alignment
Ion optics (volts)
depth Lens 1
0.28
Lens 2
-29.02
Lens 3
-195.29
Pole Bias
0.51
-3.02
Hexapole Bias
Extraction
-235.29
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9. Quantification of gene expression
Gene expression analysis was performed by relative quantitation using the comparative
threshold cycle (CT) method. The housekeeping gene glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) was used as an internal control and untreated A2780 and CP70 cells
were used as calibrators. The reaction mixture for each assay containing 2 µL of cDNA, 12.5
µL of Power SYBR Green PCR Master Mix (2X; Applied Biosystems, Carlsbad, CA, USA),
and 2.5 µL of each primer (Bax - 5 µM, Bcl-2 - 500 nM, p53 – 5 µM, and GAPDH – 5 µM) was
prepared on ice and final volume was made upto 25 µL with water. The reaction conditions
included an initial step at 95° C for 10 min (AmpliTaq Gold DNA Polymerase activation),
followed by 40 cycles at 95° C for 15 s (melt) and 60°C for 1 min (anneal/extend). Each sample
was repeated in triplicate in the PCR reaction in order to estimate the reproducibility of data. A
dissociation curve analysis followed the amplification reaction to distinguish specific from
nonspecific products and/or primer dimers (data not shown).
Table S3: Oligonucleotide sequence of primers used for real-time RT-PCR
Gene quantified
(GeneBank acc #)
Forward (F) and reverse (R)
Human Bax
Size of PCR
product (bp)
F: 5’- CAGGCTTGAGTGCAATGGCATGAT -3’
108
(AY217036)
R: 5’- TGCACACCCATAATCCCAGCTACT - 3’
Human Bcl-2
F: 5’- GCTAAAGACCAATGGGCCAAAGCA - 3’
(AY220759)
R: 5’- TGGTCTCAAACTCCTGGGCTCAAT - 3’
Human p53
F: 5’- TTGGTCGGTGGGTTGGTAGTTTCT - 3’
(AB082923)
R: 5’- ACCAAGAGGTTGTCAGACAGGGTT - 3’
GAPDH
F: 5’- TCCTGGTTGCAGGAATAGCTGAGT- 3’
(AY340484)
R: 5’- TTCACAATGACCACCCAGAGCAGA-3’
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104
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10. Quantification of caspases, FAM-FLICA method
Briefly, 0.5 X 106 treated cells in 300 µL were mixed with 10 µL of FAM-FLICATM
reagent for each of the caspases (3, 8 and 9) and incubated for 1 hour at 37°C under 5% CO2
protecting the cells from light. To distinguish the cells undergoing apoptosis from necrotic, dead,
and membrane-compromised, cells were also stained with 2 µL of propidium iodide before
analysis. After FAM-FLICATM binds to caspases inside cell the fluorescence of
carboxyfluorescein will be excited using 15 mW argon laser at 488 nm. The histograms were
generated and analyzed using Cell Quest Pro software (BD Bioscience, San Jose, CA, USA). The
green fluorescence was measured on the FL1 channel and red fluorescence of PI was measured
on FL3 channel. A dot plot with a log FL1 (X-axis) versus log FL3 (Y-axis) was generated and
the quadrant of the plot containing living positive cells which are carboxyfluorescein positive
and propidium iodide negative were selected for quantification of caspases.