Supporting Information
Aggregation of gold nanoparticles followed by methotrexate release enables Raman
imaging of drug delivery into cancer cells.
C.V. Durgadas1, C. P. Sharma 1, W. Paul1, M. R. Rekha1, K. Sreenivasan2*
1
Biosurface Technology Division, 2Laboratory for Polymer Analysis,
Biomedical Technology Wing,
Sree Chitra Tirunal Institute for Medical Sciences and Technology,
Trivandrum, Kerala, India 695 012
*
Address for correspondence. sreeni@sctimst.ac.in
Phone. 0914712520248 Fax. 0914712341814
Scheme S1: Schematic view of conjugation of MTX to DAPEG.
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Figure S2: Stability of synthesized gold nanoparticles obtained from TEM analysis.
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H- NMR and MALDI- TOF characterization of conjugates.
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H- NMR of DAPEG MTX
Where n=30 and m=x+y, (x=y=3)
d
2
g
f
d
e c
Figure S3: 1H-NMR spectra of DAPEG MTX
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a
b
Figure S4: Expanded NMR of DAPEG MTX from 6.5ppm to 9ppm
Figure S5 MALDI-TOF of DAPEG MTX.
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Preparation of Dansylated DAPEG and Fluorescence Microscopy.
Modification of GNPs by fluorophore to impart fluorescence to GNPs has been reported earlier1, 2. The
highly fluorescent DAPEG DANS was prepared by reacting DAPEG in basic condition with dansyl chloride
prepared in acetone. Thus obtained DAPEG DANS was isolated and characterized. The DAPEG DANS
showed a PL at 508 nm on 365 nm excitation (figure S6). AuNPs were prepared by reducing gold chloride in
a 1:1 mixture of the DAPEG MTX and DAPEG DANS. The nanoparticles formed here were assumed to be
of equal surface coverage by both the moieties. Up on incubation with the HePG2 cells, very bright green
fluorescence from the cytoplasmic region of the cells was observed when viewed through fluorescent
microscope (Figure S6 B). This clearly indicates cellular internalization of the particles.
Figure S6. PL spectrum of the DAPEG DANS (A) and the fluorescent microscopic image of the HepG2 cells
after incubation (B).
Cytotoxicity by MTT assay
Cytotoxicity by MTT assay was performed in the HEP G2 cells. In order to evaluate the cytotoxic property
of DAPEG MTX AuNPs, all the samples were separately tested. Repeated assay performed to confirm the
toxic effect of the attached moieties. Based on MTT assay result a concentration of 50µg/mL was found to
be enough to test the toxicities of the samples except for DAPEG MTX – DAPEG DNS GNPs (100µg/mL
for 1:1 synthesis) and the same concentration was used in cellular internalization studies. As the fluorescence
intensity was less in the case of GNPs synthesized from 1:3 mixture of DAPEG DNS and DAPEG MTX
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respectively (but it on incubation resulted only 20.41% of the cell viability), only GNPs synthesized using
1:1 ratio were incubated for fluorescence detection of cellular uptake.
We evaluated the toxic effect of DAPEG Au, DAPEG DNS, DAPEG MTX, free MTX and drug conjugated
nanoparticles. From the repeated experiments, it was evident that except drug containing moieties all others
entities caused minimal toxicity to cells. About 93% of the cells were viable when DAPEG Au was used and
about 94% cells were viable when DAPEG DNS was incubated.
Cell viability from MTT assay
Percentage of cell viability
100
93.75
94.57
80
60
40
20
13.95
18.80
20.41
23.90
E
F
0
A
B
C
D
Samples in MTT assay
Percentage was plotted assuming medium as control with the full cell viability. Sample A, B, C, D, E, F were
DAPEG Au, DAPEG DNS, free MTX, DAPEG MTX conjugate, Au nanoparticle synthesized by 1:3 of
DAPEG DNS to DAPEG MTX and Au nanoparticle synthesized by 1:1 of DAPEG to DAPEG MTX
respectively.
The cell viability of free MTX was 13.95% and DAPEG MTX conjugate showed a viability of 18.80%. But
the nanoparticle synthesized from the 1:1 mixture of the DAPEG DNS - DAPEG MTX, when incubated with
a concentration of 100µg/mL the cell viability was 23.99%. It can be concluded that on conjugating to
DAPEG and then to Au core resulted in 10% decrease in the toxicity compared to free MTX. The decrease in
toxicity can be attributed to the conjugation of the drug3.
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Calculation of number of ligands per nanoparticles
(a) Calculation of number of atoms per nanoparticles:
Assumed that all particles are completely spherical,
We’ve
Vcluster = NVatom
4/3П(Rcluster)3 = N 4/3П (Ratom)3
Rcluster = N1/3Ratom
Where V is the cluster or atom volume, R is the cluster or atomic radii and N is the total number of atoms in
the cluster.
Since from TEM radii of GNP formed were found to be 2.28 nm, 1.925nm and 1.78 nm when DAPEG alone,
dansylated DAPEG and DAPEG MTX respectively were used.
So, number of gold atoms per nanoparticles is
N = (Rcluster/Ratom)3
N DAPEG alone = 4609 (Rcluster= 2.28 nm)
NDNS-DAPEG = 2795.8 ~ 2796
(Rcluster= 1.93 nm)
NDRUG-DNS-DAPEG = 2193.363~ 2193 (Rcluster = 1.78 nm)………. (1)
(b). Calculation of Number of Fluorophore (dansylated DAPEG) per nanoparticles.
For determining the concentration of the nanoparticle formed in the stock solution, it was assumed that all
the gold chlorides were completely reduced by NaBH4 solution.
Amount of Nanoparticle in stock, Nstock = 5.83 × 1016
Concentration of the fluorophore used for the synthesis = 3mM
Concentration of unbound fluorophore = 0.3 mM
So the concentration of the fluorophore attached to GNPs = 3mM – 0.3 mM
= 2.7 mM
The number of moles of attached fluorophore = 1.26 × 10-6
Hence number of fluorophores attached to nanoparticles = 1.24 × 10 -6 NA
= 7.62 × 10 17
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So the number of fluorophores per nanopartilce = Number of attached fluorophore/ Number of nanoparticles
in stock solution
= 7.62 × 10 17/ 5.83 × 1016
= 13.07 ~ 13.
Approximately 13 fluorophore (DAPEG DNS) per nanoparticles.
Quantification of Drug loaded and calculation of the number of DAPEG MTX molecules per nanoparticle in
1:1 ratio synthesis: Calculation was based on the assumption that the presence of drug will not make any
remarkable change in the size of gold nanoparticles and hence the number of ligands bound to GNPs will be
in the range of 13 per nanoparticles.
Concentration of the HAuCL4. 3H2O used for the reaction = 0.6mM
As per above method the concentration of the nanoparticles formed in the stock
Solution,
N DRG DAPEG= 1.29 × 1017
Quantity of Drug used in initial conjugation to DAPEG = 4.4 mM
Amount of MTX in DAPEG conjugate = 4.12 mM
Quantity of DAPEG MTX used to synthesize gold nanoparticles = 4.12 mM
Amount of unconjugated DAPEG MTX = 0.37 mM
Concentration of DAPEG MTX conjugated to GNP = 3.75 mM (91.02%)
Number of moles of DAPEG MTX in 3.75 mM DRG DAPEG = 1.60 × 10-6
Number of DAPEG MTX molecule = 1.60 × 10 -6 NA
= 9.65 × 1017
Hence Number of drug molecules per nanoparticles = 9.57 × 10 17/ 1.29 × 1017
= 7.48 ~ 7-8 molecules of DAPEG MTX per nanoparticles.
So the rest of the nanoparticle surface will be occupied by the Fluorophore molecules (approximately 5-6
molecules).
References
1) K G Thomas and Prashant VK 2003 Chromophore functionalized gold nanoparticles Acc. Chem. Res. 36
888-98.
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2) K G Thomas and Prashant VK 2000 Making gold nanoparticles glow: enhanced emission from a surfacebound fluoroprobe J. Am. Chem. Soc. 122 2655-56.
3) Sezen Gurdag,Jayant Khandare, Sarah Stapels,Larry H. Matherly,and Rangaramanujam M. Kannan;
Activity of Dendrimer-Methotrexate Conjugates on Methotrexate Sensitive and -Resistant Cell Lines.
Bioconjugate Chem. 2006, 17, 275-283.
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