Available via license: CC BY 4.0
Content may be subject to copyright.
Supplementary information
Folate Receptor-targeted Aminoglycoside-derived
Polymers for Transgene Expression in Cancer Cells
Sudhakar Godeshalaa, Rajeshwar Nitiyanandanb, Brian Thompsona, Sheba
Goklanya, David R. Nielsena, and Kaushal Regea (*)
aChemical Engineering
bBiological Design Program
Arizona State University, Tempe, AZ 85287-6106, USA.
(*) To Whom All Correspondence is to be addressed
Kaushal Rege
Chemical Engineering
501 E. Tyler Mall, ECG 303
Arizona State University
Tempe, AZ 85287-6106
Email: rege@asu.edu
Phone: 480-727-8616
Fax: 480-727-9321
Primer sequences
TRAIL -- EcoRI - F
ATA GAA TTC GCC GCC ACC ATG GCT ATG ATG GAG GTC CAG GG
TRAIL -- NotI - R
ATT GCG GCC GCT TAG CCA ACT AAA AAG GCC CCA AAA AAA CTG
p EF - T R AI L
5141 bp
A MP
p B R 3 2 2
h TR A I L
P -E F
R b G lob P A Te r m i na to r
Complete pEF-TRAIL sequence ( portions confirmed by sequencing )
GTCGACATTGATTATTGACTAGATCATCGCGTGAGGCTCCGGTGCCCGTCAGTGGGC
AGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAA
CCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGG
CTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTG
AACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGT
TCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCA
CGCCCCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGG
GAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCC
TGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTC
GCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTT
TTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGG
TTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCG
AGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGG
CCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGC
AAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCC
CTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGA
GTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTC
CACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTAC
GTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTG
GGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGC
CCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTT
TTTCTTCCATTTCAGGTGTCGTGAGGAATTCGCCGCCACCATGGCTATGATGGAGGT
CCAGGGGGGACCCAGCCTGGGACAGACCTGCGTGCTGATCGTGATCTTCACAGTGCT
CCTGCAGTCTCTCTGTGTGGCTGTAACTTACGTGTACTTTACCAACGAGCTGAAGCA
GATGCAGGACAAGTACTCCAAAAGTGGCATTGCTTGTTTCTTAAAAGAAGATGACA
GTTATTGGGACCCCAATGACGAAGAGAGTATGAACAGCCCCTGCTGGCAAGTCAAG
TGGCAACTCCGTCAGCTCGTTAGAAAGATGATTTTGAGAACCTCTGAGGAAACCATT
TCTACAGTTCAAGAAAAGCAACAAAATATTTCTCCCCTAGTGAGAGAAAGAGGTCC
TCAGAGAGTAGCAGCTCACATAACTGGGACCAGAGGAAGAAGCAACACATTGTCTT
CTCCAAACTCCAAGAATGAAAAGGCTCTGGGCCGCAAAATAAACTCCTGGGAATCA
TCAAGGAGTGGGCATTCATTCCTGAGCAACTTGCACTTGAGGAATGGTGAACTGGTC
ATCCATGAAAAAGGGTTTTACTACATCTATTCCCAAACATACTTTCGATTTCAGGAG
GAAATAAAAGAAAACACAAAGAACGACAAACAAATGGTCCAATATATTTACAAATA
CACAAGTTATCCTGACCCTATATTGTTGATGAAAAGTGCTAGAAATAGTTGTTGGTC
TAAAGATGCAGAATATGGACTCTATTCCATCTATCAAGGGGGAATATTTGAGCTTAA
GGAAAATGACAGAATTTTTGTTTCTGTAACAAATGAGCACTTGATAGACATGGACCA
TGAAGCCAGTTTTTTTGGGGCCTTTTTAGTTGGCTAAGCGGCCGCACTCCTCAGGTGC
AGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACC
ACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGC
ATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTT
TTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAA
TGAGTATTTGGTTTAGAGTTTGGCAACATATGCCATATGCTGGCTGCCATGAACAAA
GGTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTAT
TCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTT
TTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCT
CTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGAAGATCCCTCGACCTG
CAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCC
GCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTG
CCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTC
GGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCG
CCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCC
ATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCT
ATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAAC
TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACA
AATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTAT
CTTATCATGTCTGGATCCGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCG
GTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGT
TCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAG
AATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAG
GAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGA
GCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA
GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCC
GCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGC
TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTG
CACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAG
TCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGAT
TAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT
ACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT
TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGT
GGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGA
TCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGG
GATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAA
ATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCA
ATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTT
GCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCC
AGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATA
AACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTC
CATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAG
TTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGT
ATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATG
TTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTG
GCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC
CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT
AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGC
CACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAA
CTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCC
AACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGA
AGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCA
TACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGG
ATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCC
CCGAAAAGTGCCACCTG
Figure S1. Primer sequence and complete pEF-TRAIL DNA sequences.
Scheme S1. Schematic showing synthesis of the paromomycin/neomycin-resorcinol/glycerol
diglycidyl ether parent polymers and synthesis of the folic acid conjugated polymers carried out
in presence of EDCi/NHS. This illustration is meant to indicate the general reactions that take
place and not the exact structure of the resulting product formed.
Figure S2. 1H-NMR spectra of parent polymers (A) NR, (B) PR, (C) NG, and (D) PG using
DMSO-d6 as the solvent. Nomenclature of the different polymers is described in the
Experimental section.
Figure S3. 1H-NMR spectra of folic acid conjugated polymers (A) NRFA, (B) PRFA, (C)
NGFA, and (D) PGFA using DMSO-d6 as the solvent. Proton peaks corresponding to folic acid
shown in blue circle. Nomenclature of the different polymers is described in the Experimental
section.
Figure S4. FT-IR spectra of folic acid conjugated polymers (A) NRFA, (B) PRFA, (C) NGFA,
and (D) PGFA.
0
100000
200000
300000
400000
500000
MDA-MB-231 48h
RLU/mg
20:1
25:1
*
*
0
100000
200000
300000
400000
500000
UMUC3 48h
RLU/mg
20:1
25:1
*
*
AB
Figure S5. Luciferase transgene expression (RLU/mg) with plasmid pGL3.0 observed with FA-
conjugated polymers, their corresponding parental polymers and Lipofectamine-3000 in (A)
MDA-MB-231 (48h), (B) UMUC3 (48h), cells. Transgene expression was determined for
polymer: pDNA weight ratios for 20:1 and 25:1. * = p-value < 0.05 using Student’s t-test; p-
values were obtained by comparing RLU/mg values of each FA-conjugated polymer or the
parental polymer with Lipofectamine-3000 under corresponding conditions. UT: untreated
control. Data represent mean ± one standard deviation of three independent experiments (n=3).
0
20000
40000
60000
80000
100000
120000
MDA-MB-231 48h
RLU/mg
0.01µL
0.05µL
0.15µL
0.3µL
0.5µL
*
*
1.0µL
A
T24 Positive controlates folate expressionbe converted into some concentration units if available / possibile.rch Initiative (FURI) award and wT24 negative control
Figure S6. Luciferase expression in MDA-MB-231 human breast cancer and UMUC3 human
bladder cancer cells following delivery of pGL3.0 plasmid using different doses of
Lipofectamine-3000 and P3000 reagent as per vendor’s protocol. The legend indicates volume of
Lipofectamine-3000 used. (n=3).
Figure S7. Confocal images of T24 and UMUC3 human bladder cancer cells following
immunostaining with folate receptor antibody alpha. Cell morphology can be visualized in
0
20000
40000
60000
80000
100000
120000
UMUC3 48h
RLU/mg
0.01µL
0.05µL
0.15µL
0.3µL
0.5µL
*
*
1.0µL
B
phase-contrast images and the green fluorescence emission of Alexa fluor-488 indicates folate
expression (top); the overlay of these two can be seen in images (bottom). T24 cells stained with
primary antibody against FR- and FITC-tagged anti-rabbit antibody (secondary) were used as a
positive control and FITC-tagged anti-rabbit antibody only as the negative control.
Figure S8. Western blot analyses showing TRAIL protein expression following polymer-
mediated delivery of the pEF-TRAIL plasmid to UMUC3 cells; a plasmid expressing green
fluorescent protein, pEF-GFP, was used as the control. Lane information is provided in the
legend table in which the numbers in the first column indicate lane numbers in the gels and
entries in the second column indicate corresponding polymer-plasmid complexes delivered.
Folic acid conjugated polymer Calculated Folic acid/polymer mole ratio
PRFA 1.12
NRFA 0.85
PGFA 0.72
NGFA 0.83
Table S1. Extent of modification for folic acid conjugated polymers from parental polymers.
The folic acid: polymer mole ratio was calculated using NMR.
Polymer Concentration (mM) of amines by Ninhydrin assay Molar ratio
3.5 kDa / 10kDa
After 3.5 kDa dialysis tubing After 10 kDa dialysis tubing
PR 3.087 0.32 9.65
NR 2.853 0.18 15.85
Table S2. Amine content analysis (Ninhydrin assay) of solution left behind followling dialysis of
NR and PR polymers first through 3.5 kDa and then through 10kDa MWCO dialysis membranes
indicates that most of the polymers ranged from 3.5-10 kDa in molecular weight, since almost all
polymer was lost following dialysis through a 10 kDa membrane.