Biotin Decorated Gold Nanoparticles for Targeted Delivery of a Smart-Linked Anticancer Active Copper Complex: In Vitro and in Vivo Studies

Article abstract of DOI:10.1021/acs.bioconjchem.6b00537

The synthesis and anticancer activity of a copper(II) diacetyl-bis(N4-methylthiosemicarbazone) complex and its nanoconjugates are reported. The copper(II) complex is connected to a carboxylic acid group through a cleavable disulfide link to enable smart delivery. The copper complex is tethered to highly water-soluble 20 nm gold nanoparticles (AuNPs), stabilized by amine terminated lipoic acid-polyethylene glycol (PEG). The gold nanoparticle carrier was further decorated with biotin to achieve targeted action. The copper complex and the conjugates with and without biotin, were tested against HeLa and HaCaT cells. They show very good anticancer activity against HeLa cells, a cell line derived from cervical cancer and are less active against HaCaT cells. Slow and sustained release of the complex from conjugates is demonstrated through cleavage of disulfide linker in the presence of glutathione (GSH), a reducing agent intrinsically present in high concentrations within cancer cells. Biotin appended conjugates do not show greater activity than conjugates without biotin against HeLa cells. This is consistent with drug uptake studies, which suggests similar uptake profiles for both conjugates in vitro. However, in vivo studies using a HeLa cell xenograft tumor model shows 3.8-fold reduction in tumor volume for the biotin conjugated nanoparticle compared to the control whereas the conjugate without biotin shows only 2.3-fold reduction in the tumor volume suggesting significant targeting.

Full text of DOI:10.1021/acs.bioconjchem.6b00537

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Article
Biotin Decorated Gold Nanoparticles for Targeted Delivery of a Smart-
linked Anticancer Active Copper Complex: In Vitro and In Vivo Studies
Anup Kumar Pramanik, * Siddikuzzaman, Duraippandi Palanimuthu,
Kumaravel Somasundaram, and Ashoka Gnanadoss Samuelson
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00537 • Publication Date (Web): 21 Nov 2016
Downloaded from http://pubs.acs.org on November 28, 2016
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Biotin Decorated Gold Nanoparticles for Targeted Delivery of a
Smart-linked Anticancer Active Copper Complex: In Vitro and In
Vivo Studies
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Anup K. Pramanik†, Siddikuzzaman‡, Duraippandi Palanimuthu†, Kumaravel Somasundaram*‡
and Ashoka G. Samuelson*†
†Department of Inorganic and Physical Chemistry, ‡Department of Microbiology and Cell
Biology, Indian Institute of Science, Bangalore- 560012, India
Abstract: The synthesis and anticancer activity of a copper(II) diacetylꢀbis(N4ꢀ
methylthiosemicarbazone) complex and its nanoconjugates are reported. The copper(II) complex
is connected to a carboxylic acid group through a cleavable disulfide link to enable smart
delivery. The copper complex is tethered to highly water soluble 20 nm gold nanoparticles
(AuNPs), stabilized by amine terminated lipoic acidꢀpolyethylene glycol (PEG). The gold
nanoparticle carrier was further decorated with biotin to achieve targeted action. The copper
complex and the conjugates with and without biotin, were tested against HeLa and HaCaT cells.
They show very good anticancer activity against HeLa cells, a cell line derived from cervical
cancer and are less active against HaCaT cells. Slow and sustained release of the complex from
conjugates is demonstrated through cleavage of disulphide linker in the presence of glutathione
(GSH), a reducing agent intrinsically present in high concentrations within cancer cells. Biotin
appended conjugates do not show greater activity than conjugates without biotin against HeLa
cells. This is consistent with drug uptake studies which suggests similar uptake profiles for both
conjugates in vitro. However, in vivo studies using a HeLa cell xenograft tumor model shows 3.8
fold reduction in tumor volume for the biotin conjugated nanoparticle compared to the control
whereas the conjugate without biotin shows only 2.3 fold reduction in the tumor volume
suggesting significant targeting.
Key words
Copper bis(thiosemicarbazone), gold nanoparticles, smartꢀlinked drug, targeted delivery, HeLa
cell xenograft.
*Ashoka G. Samuelson Department of Inorganic and Physical Chemistry, Indian Institute of
Science, Bangalore- 560012, India Email: ashoka@ipc.iisc.ernet.in
Kumaravel Somasundaram Department of Microbiology and Cell Biology, Indian Institute of
Science, Bangalore- 560012, India Email: skumar@mcbl.iisc.ernet.in
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Introduction
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Cancer is one of the foremost causes for premature death.1 Although chemotherapy increases the
lifespan of cancer patients, the quality of life deteriorates significantly due to the nonꢀspecific
cytotoxicity and inefficient delivery of anticancer drugs to the diseased tissue resulting in serious
side effects.2ꢀ4 A major challenge then, is to achieve selective delivery of the drug within cancer
cells so that side effects are minimized.5 In addition, if the drug is effective only in cancer cells
and not in normal cells, one would have the magic bullet envisaged by Paul Ehrlich nearly a
century ago.6 Selective entry of the drugs to cancer cells requires them to be linked to a suitable
targeting agent. Targeted drugs would then recognize cancer cells via receptors over expressed
for the target on the surface of cancer cells and leave normal cells relatively unaffected.7
Although conceptually simple, applying this paradigm to fight cancer is fraught with difficulties
and so research in this area remains a major challenge.8, 9
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In its simplest form, the targeting agent and the drug are both linked to a suitable delivery vehicle
such as a simple polymer like polyethylene glycol, or to a more complex system like a
dendrimer,10 vesicle,11 liposome,12 nanoparticle,13 or a nanotube.14 Large delivery agents have an
additional advantage in having many copies of the drug and the targeting agent on a single entity,
increasing the probability of targeting cancer cells and killing them. If the large molecule is
nanosized, they are also “passively targeted” as they leave the blood circulation system only in
the vicinity of solid tumors due to the leaky vasculature characteristic of rapidly proliferating
cancer cells in solid tumors.15 Reꢀentry into the blood vessel is also less probable, leading to
what is known as the Enhanced Permeation and Retention (EPR) effect.16 Suitable decoration of
the nanoparticle can also improve solubility, in vivo stability, prolonged blood circulation time
and selective biodistribution.17 For the drug to be effective, it has to be released from the delivery
agent which, either requires the presence of a cleavable linkage or binding of the drug to the
carrier via weak nonꢀcovalent interactions.18, 19 This provides an opportunity to have another
level of control in the release and activation of cytotoxic agents in side cancer cells. Smart
release of the attached drugs from the nanocarrier can be triggered by cancer specific conditions
such as higher levels of GSH in the cell, acidic pH or even by local application of heat or light on
the affected tissue.20ꢀ23 Nanomedicine has immense potential with wide ranging applications
from simple therapy, to detection, diagnosis and theranostics.24
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AuNPs are being extensively investigated for targeted drug delivery of drugs by tethering them
to homing agents like antibodies, folic acid, biotin, suitable peptides, or monoclonal
antibodies.25ꢀ28 The synthesis, functionalization and loading of drugs is easier in the case of
AuNPs in comparison to other nano particles. Studies on AuNPs have shown that capping them
with polyethylene glycol improves their lifetime in the circulatory system and hence the
bioavailability of their payload improves significantly at the desired site.29, 30 AuNPs targeted in
this fashion can home in on cancer cells and deliver their payload to the cells by receptor
mediated endocytosis (active targeting) and these large nanoconjugates (less than 200 nm) exit
only through the leaky blood vessels near the tumor tissue (passive targeting) which in turn
improves the pharmacokinetics.31ꢀ33
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Among the various metal complexes studied for anticancer activity, transition metal complexes,
especially copper containing bis(thiosemicarbazone) ligands are extensively studied.34, 35 Some
of these complexes show cytotoxicity similar to cisplatin, the drug widely used in clinics.36
Based on the mechanistic studies carried out on several copper(II) bis(thiosemicarbazone)
complexes, particularly derived from glyoxal, it is generally believed that their cytotoxicity is a
result of reactive oxygen species (ROS) they generate, whereas other complexes activate
multiple pathways such as inhibition of DNA and RNA synthesis and disruption of ATP
production resulting in apoptosis and growth inhibition.37, 38 Although these complexes, like
most copper(II) complexes, suffer from low water solubility and specificity, a few of them
including CuATSM and amine terminated CuATSM (CuATSMꢀA) show selectivity towards
hypoxic cells, again a characteristic feature of solid tumors, which results in accumulation of
toxic copper in cancer cells.39ꢀ41
We hypothesized that conjugation of these molecules to a water soluble nanocarrier decorated
with biotin as a targeting agent, and its delivery to the desired site by capitalizing on the EPR
effect might improve their chance of being used as drugs.42 Furthermore, if the link between the
nano delivery agent and the copper complex is cleaved by GSH, the complex would be released
inside cancer cells.43 To this end we have synthesised a Cu(II) bis(thiosemicarbazone) complex,
a close analog of CuATSM, by linking it to a PEG decorated gold nanoparticle through a redox
active linkerꢀ a disulfide bond. We have examined its cytotoxicity against HeLa and HaCaT cells
in vitro and its effectiveness in growth inhibition of a tumor expressed in nude mice using a
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HeLa cell xenograft model. The biotin targeted vehicle was more effective in inhibiting the
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tumor growth in vivo than the simple gold nanoparticle.
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Results and Discussion
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Synthesis and characterization of
a
smart-linked cytotoxic agent: Copper(II)
bis(thiosemicarbazone) complex (CuATSM-SS-COOH)
In order to make smart delivery of the copper bis(thiosemicarbazone) complex possible, a
disulfide linker with a carboxylic acid group to link it to the delivery agent was attached to it
(Scheme 1). The amine terminated biacetylꢀbis(4ꢀmethylꢀ3ꢀthiosemicarbazone) ligand, ATSMꢀ
A, was prepared following the literature procedure and the structure was confirmed by 1H NMR
spectroscopy and ESIꢀHRMS spectrometry.44 ATSMꢀA was conjugated with 3,3´ꢀ
dithiobis(propionic acid) in the presence of 1.1 equivalents of OꢀbenzotriazoleꢀN,N,N',N'ꢀ
tetramethylꢀuroniumꢀhexafluoroꢀphosphate (HBTU) and N,Nꢀdiisopropylethylamine (DIPEA).
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1H NMR and C NMR spectroscopy and ESIꢀHRMS confirmed the successful synthesis of the
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ligand, ATSMꢀSSꢀCOOH (Figure S1-S3). Two singlets at 2.20 and 2.23 ppm in the H NMR
spectrum were assigned to the six methyl protons of the biacetyl unit, whereas a doublet at 3.01ꢀ
3.03 ppm was due to the methyl group on the nitrogen split by the NH proton. Two multiplets at
2.57ꢀ2.65 ppm and 2.89ꢀ2.96 ppm correspond to the methylene protons of the disulfide linker.
The ratios of the peaks assigned to ATSMꢀA to the disulphide linker corresponded to the
formation of ATSMꢀSSꢀCOOH. The corresponding copper complex, CuATSMꢀSSꢀCOOH, was
prepared by reaction of the ligand with copper acetate and was characterized by ESIꢀHRMS
spectrometry and elemental analysis (Figure S4 and S5).
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Scheme 1: Synthetic scheme for the preparation of CuATSM-SS-COOH.
Synthesis and characterization of lipoic acid functionalized polyethylene glycol
The use of lipoic acid, a cyclic disulphide, to anchor PEG to the gold nanoparticle has shown
greater stability in the biological milieu compared to the use of monoꢀthiols.45 Hence a capping
agent, was prepared by reacting PEGꢀbis(amine) with Nꢀhydroxysuccinimide activated lipoic
acid (LAꢀNHS) in the presence of a weak base, NaHCO3 following the literature procedure to
give lipoic acid conjugated PEG (LAꢀPEG) (Scheme 2).46, 47 The monoꢀfunctionalized PEG was
purified by size exclusion chromatography (SEC) using lipophilic Sephadex LHꢀ20 with
methanol as eluent. Its structure was confirmed using 1H NMR spectroscopy (Figure S11) prior
to its use as the capping agent for stabilizing the gold nanoparticles.
Synthesis and characterization of PEGylated AuNP and nanoconjugates
Colloidal gold nanoparticles were prepared by the TurkevichꢀFrens method using trisodium
citrate as the reducing agent in water,48, 49 followed by ligand exchange with LAꢀPEG resulting
in highly stabilized AuNPs (Scheme 2). AuNPꢀ1 prepared in this fashion was purified by
dialysis against MilliꢀQ water using a dialysis tube (MWCO: 12 kDa) and solid AuNPꢀ1 was
obtained after lyophilisation of the purified solution. A surface plasmon resonance band (SPR) at
526 nm (λmax) confirmed the formation of PEGylated AuNPs (Figure 1). TEM images reveal
aspherical AuNPs without any agglomeration of the nanoparticles (Figure 2). The core size of
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nanoparticles was calculated by taking the average size of a large number of particles from TEM
images. The histogram of the particle size shows a narrow distribution with an average diameter
of 19.4 ± 1.9 nm. Further, the average height (17.7 ± 1.7 nm) of the nanoparticles in the AFM
images is reasonably close to the size estimated from TEM measurements (Figure 3). The
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stabilization of nanoparticles by the PEG linker was confirmed by H NMR spectroscopy and
further supported by FTIR spectroscopy by the presence of bands at 2880 cmꢀ1 (ꢀCH stretching),
1655 cmꢀ1 (C=O stretching), 1541 cmꢀ1 (NꢀH bending).50 The extent of the surface
functionalization by LAꢀPEG was estimated from thermogravimetric (TGA) analysis (Figure
S13). Thermal decomposition pattern of AuNPꢀ1 was similar to that of LAꢀPEG for which
decomposition started at 300°C and ended at 420°C, with a weight loss of 89% suggesting a gold
core accounting for the remaining 11% coated with LAꢀPEG. (Table-S1).
Scheme 2: Schematic illustration for the synthesis of PEG stabilized gold nanoparticles and
nano-conjugates.
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Figure 1: UV-visible spectra of PEG stabilized gold nanoparticles and nanoconjugates in
Milli-Q water.
Synthesis and characterization of biotin containing nanoconjugates
Biotin was attached to AuNPꢀ1 using the HBTU/DIPEA amide coupling protocol to link the
carboxylic acid groups of biotin and the amine groups on the AuNPꢀ1 surface thus creating
AuNPꢀ2. There was no shift in the SPR band of AuNPꢀ2 after conjugation of biotin indicating
stability of the nanoconjugate. Excess reagents were removed from AuNPꢀ2 by extensive
dialysis against MilliꢀQ water and dry AuNPꢀ2 was obtained from the purified solution by freeze
drying. Biotin conjugation on AuNPꢀ2 was confirmed by the presence of two sets of doublet of
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doublets between 4.38ꢀ4.41 and 4.58ꢀ4.60 ppm in the H NMR spectrum of the conjugate
corresponding to two protons on the ring of biotin.51 Particle size was measured from TEM and
AFM images (Figure 2 and 3, Table 1) and the PEG content was estimated from TGA analysis
(Figure S13, Table S1). The extent of biotin loading on the nanoparticles was estimated using
the biochemical HABA assay to be 71.4 ± 3.3 nmol/mg of nanoconjugates present in AuNPꢀ2
(Table S2).
Synthesis and characterization of drug and biotin containing nanoconjugates
The copper complex, CuATSMꢀSSꢀCOOH, containing a carboxylic acid end group was
conjugated to the amine group of AuNPꢀ1 following the same amide coupling procedure leading
to AuNPꢀ3. To make the targeted nanocarrier, biotin was added to AuNPꢀ3 using the same
HBTU/DIPEA coupling agent resulting in AuNPꢀ4 which was purified by the same techniques
described earlier and the solid nanoconjugate was obtained by freeze drying. The average size of
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AuNPꢀ3, and AuNPꢀ4, estimated from TEM and AFM images presented in the Table 1. These
values suggest that there is no appreciable change in the size of the nanoconjugates after
conjugation of the drug and/or biotin. More importantly, TEM and AFM images reveal there is
no agglomeration or collapse of nanoconjugates which is also confirmed by no shift in the SPR
band of AuNPꢀ3 and AuNPꢀ4 (Figure 2 and 3, Table 1). The hydrodynamic radius for the
nanoconjugates were obtained from dynamic light scattering (DLS) measurements (Table-1)
which showed no change in hydrodynamic radius after conjugation of biotin or CuATSMꢀSSꢀ
COOH to PEGylated AuNPs signifying the stability of the conjugates.
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The drug loading was estimated from the copper content in the nanoconjugates by inductively
coupled plasma mass spectrometry (ICPꢀMS) and found to be 1.11 ± 0.04 and 1.30 ± 0.20% in
AuNPꢀ3 and AuNPꢀ4 respectively (Table S3). The amount of biotin, the targeting agent,
attached to AuNPꢀ4 was estimated using the HABA assay and found to be 25.1 ± 0.8 nmol/mg of
nanoconjugates (Table S2). All the freeze dried nanoconjugates were stored at 4°C. 52
Figure 2: Transmission electron microscopy images and particle size distributions of
AuNP-1 (top left), AuNP-2 (top right), AuNP-3 (bottom left), and AuNP-4 (bottom right).
HRTEM is shown on the right-top of each image and the histogram (inset) shows the
particle size distribution.
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Table 1: Summary of the particle size of AuNPs from TEM, AFM and DLS measurements
± standard deviation (SD).
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Hydrodynamic
Nanoparticle
Size from TEM(nm)
Size from AFM(nm)
radius(nm)
30.8 ± 1.0
29.1 ± 1.8
29.9 ± 1.6
28.7 ± 1.8
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AuNPꢀ1
AuNPꢀ2
AuNPꢀ3
AuNPꢀ4
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19.2 ± 1.6
19.0 ± 1.8
17.7 ± 1.7
19.2 ± 1.9
18.7 ± 1.8
19.5 ± 2.2
Colloidal stability of nanoconjugates
Evaluation of colloidal stability of nanoconjugates under physiological conditions is essential
before one can use nanoparticles in biomedical applications as they are exposed to different
electrolyte and pH conditions on its journey from the point of administration to the tumor site.
The pH in the blood stream is 7.3ꢀ7.5 whereas pH in endosomes (pH 5.0ꢀ6.5) and lysosomes (pH
4.5ꢀ5.0) are very low.53 So, the nanoconjugate should be stable in a wide range of pH. As the
SPR is very sensitive to aggregation, the stability of gold nanoparticles can be monitored by
following the UVꢀvisible profiles at different pH. No shift was observed in the SPR bands for
AuNPꢀ1 and AuNPꢀ4 at different pH, moreover, there was no new peak appearing in the spectra
(Figure S15 and S16). Thus, it was concluded that the nanoconjugates used in the current study
were stable in a wide range of pH and suitable for biological applications. AuNPs under
investigation showed no discernible change in the intensity or position of the absorbance band at
526 nm at different concentrations of NaCl (Figures S17 and S18) ensuring their stability under
different electrolyte concentrations.54
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Figure 3: AFM images and height distribution of AuNP-1(top left), AuNP-2 (top right),
AuNP-3 (bottom left), and AuNP-4 (bottom right).
GSH mediated drug release kinetics
Glutathione (GSH), an important redox active biomolecule reduces the disulfide bond.55 The
concentration of GSH in the extracellular medium is low, whereas within the cell it is relatively
high.56 Additionally, some tumor tissues have up to 7ꢀfold higher concentration of GSH due to
the highly reducing and hypoxic condition in them compared to normal tissues.57, 58 The release
kinetics of the drug from the nanoconjugates in presence of GSH was estimated to understand its
activity. Cleavage of the disulfide bond caused by GSH at 5 mM concentration and release of the
conjugated drug was followed by analysis of the copper content of dialyzed solutions at different
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time points for 48h using ICPꢀMS (Figure 4). The release profile indicated a slow and partial
release (about 60%) after 48h whereas in the absence of GSH as a trigger, there was less than
10% release of the conjugated drug in the same time period. This trend is in line with previous
results where others have also observed slow and incomplete release of the conjugated drugs
linked through a disulfide in the presence of GSH. Huo et al. reported 82% and 52% release of
paclitaxel from a redoxꢀsensitive polyethene glycol−paclitaxel prodrug over a period of 48h in
the presence of 20mM and 10mM of GSH respectively.59 The controlled and slow release of the
drug could be advantageous to maintain an effective drug concentration in cancer cells which
will increase the therapeutic efficacy of the drug.60 The fact that in vitro release increases more
than 80% with excess GSH suggests that the release is controlled by an equilibrium and would
be controlled by the concentration of GSH in the cell being tested. Hence it is unlikely that
inaccessibility of the SꢀS bond for cleavage is a reason for incomplete release.
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AuNP-4 in the presence of GSH
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Time (h)
Figure 4: GSH triggered drug release profile of AuNP-4 in PBS solution at 370 C.
In Vitro Cytotoxicity:
Copper(II) bis(thiosemicarbazone) complexes have been shown to be active against several cell
61ꢀ63
lines
of which the cervical tumor cell line, HeLa is one of the most studied. It has been
shown that Hela cells overexpress biotinꢀspecific receptors.64 Dong et al. have shown enhanced
cellular uptake of the biotinylated gold nanoparticles by HeLa cells compared to A549 (lung
cancer), MG63 (osteosarcoma) or NIH3T3 (normal fibroblast) cells.65 The higher uptake of
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biotinylated polyamidoamine (PAMAM) dendrimer with respect to the conjugate without biotin
has been shown to be energy dependent.66 So in the current study, the targeting efficacy of
biotinylated AuNP carriers of the drug has been probed using the HeLa cell line. The
unconjugated disulfide containing the copper complex, CuATSMꢀSSꢀCOOH, showed very good
cytotoxicity with IC50 = 6.9 ± 0.2 µM which is the concentration required to kill 50% of the cells
(Figure 5a). A model compound with disulfide linker between two bis(thiosemicarbazone)
moiety, CuATSMꢀSSꢀCuATSM, was also checked for cytotoxicity and was found to have a
similar activity, IC50 = 7.2 ± 0.8 µM (Figure 5a). The nanoparticle, AuNPꢀ1, and the
biotinylated nanoparticle, AuNPꢀ2 demonstrated no cytotoxicity up to 50 µg/ml in HeLa cells
(Figure S19). Hence, AuNPꢀ1 and AuNPꢀ2 were considered safe for the delivery of the
cytotoxic agents specifically to the tumor cells. Unlike AuNPꢀ1 and AuNPꢀ2, AuNPꢀ3, a
CuATSMꢀSSꢀCOOH tethered AuNPꢀ1, and AuNPꢀ4, a biotinylated AuNPꢀ3, were found to have
IC50 values of 15.2 ± 0.3 µM and 17.7 ± 1.0 µM respectively in HeLa cells (Figure 5a and Table
2). The cell viability was also checked against nonꢀtumorigenic immortalized keratinocyte cells
(HaCaT) and the data is compared with the cytotoxicity values obtained with HeLa cells (Table
2). The IC50 of model copper complexes are significantly higher against HaCaT cells thus
implying that the complexes are less cytotoxic to normal cells (Figure 5b and Table 2). In good
correlation, AuNPꢀ3 and AuNPꢀ4, are also found to be less cytotoxic against HaCaT cells than
against HeLa cells as well (Figure 5b and Table 2).
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Figure 5: Cell viability of (a) HeLa cells and (b) HaCaT cells on treatment with CuATSM-
SS-COOH, CuATSM-SS-CuATSM, AuNP-3 and AuNP-4 assessed by MTT assay at 48 h
incubation. Data are presented as the mean of triplicate measurements ± SD.
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Table 2: Summary of the cell viability of HeLa and HaCaT cells on treatment with
CuATSM-SS-COOH, CuATSM-SS-CuATSM, AuNP-3 and AuNP-4.
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IC50 (µM)
Compound
HeLa
HaCaT
CuATSMꢀSSꢀCOOH
CuATSMꢀSSꢀCuATSM
AuNPꢀ3
6.9 ± 0.2
7.2 ± 0.8
15.2 ± 0.3
17.7 ± 1.0
19.6 ± 1.7
11.9 ± 1.1
30.0 ± 2.0
> 30
AuNPꢀ4
The reduced cytotoxicity observed for the drug nanoconjugate compared to the free drug is in
keeping with observations made by others. The delivery of cisplatin through PEGylated PRINT
hydrogel against A549, SKOVꢀ3 (ovarian cancer) and MDAꢀMBꢀ468 (breast cancer) showed
less cytotoxicity compared to cisplatin due to slow and incomplete release of the drug which was
approximately 60% after 72h.67 Yu et al. demonstrated reduced activity of doxorubicin (DOX)
on magnetic DOXꢀanchored nanogel (MDANꢀgel) against HeLa cells and the result was
attributed to the partial release of the drug from the nanoconjugate.68 In this study also, the
reduced activity of AuNPꢀ3 and AuNPꢀ4 compared to the parent drug could be ascribed to the
slow and partial release of the active component. The expected increase in cytotoxicity of the
biotinylated nanoconjugate was not observed in vitro, however, in vivo results proved more
encouraging (vide infra). The reason for this apparent lack of targeting in the in vitro experiment
is not obvious. However, the reduced cytotoxicity of the complexes and the nanoꢀconjugates
towards normal cells (HaCaT) could prove advantageous for in vivo applications.
Quantification of in vitro drug uptake by ICP-MS
For an anticancer agent to act, it has to first enter the cancer cell and activate pathways leading to
cell death. So the activity or the cytotoxicity should be directly correlated to the amount of drug
internalized by the cells. Drug internalization was estimated by ICPꢀMS by measuring copper
content in the whole cell following a 6h incubation. Copper content nearly doubled from 0.67±
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0.03 to 1.44 ± 0.33 in the case of AuNPꢀ3 incubated cells. Biotin decorated AuNPꢀ4 also showed
a similar increase. Although the expected enhanced uptake with biotinylated AuNPꢀ4 was not
observed in HeLa cells, the similar drug uptake of AuNPꢀ3 and AuNPꢀ4 is consistent with the in
vitro MTT assay result (Figure 6). As an excess of biotin was being used in the functionalization
step, further increase in the extent of biotinylation of the gold nanoparticle was not possible with
the current protocol. Decoration of the nanoparticle with more biotin is probably inhibited by the
presence of copper bis(thiosemicarbazone) complex.
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Figure 6: Drug uptake estimated from copper content estimated by ICP-MS in Hela cells
6h post treatment with AuNP-3 and AuNP-4.
Biotinylated gold nanoparticle, AuNP-4 is an efficient inhibitor of tumor growth
Biotin containing drug delivery systems have been shown to target tumor sites in animal
models.69ꢀ72 Biotin tagged polymeric nanoparticles encapsulated with paclitaxel demonstrated
significant inhibition of tumor growth in a mouse model compared to the drug bearing
nanoparticles without the biotin tag.70 Biotin conjugated human serum albumin nanoparticles
carrying methotrexate (MTX) was able to inhibit tumor growth of a breast tumor model in mice
with enhanced therapeutic effect and reduced side effects compared to the nonꢀtargeted
nanoparticle.73 Hence, we decided to take up an in vivo study of AuNPꢀ3 and AuNPꢀ4. A solid
tumor cervical xenograft model induced in nude mice with HeLa cells was used for this purpose
and has been described in detail earlier.74 Tumor volumes were compared in four groups of
animals. The first group (Control) had saline treated mice whose tumors increased rapidly, the
second group consisted of AuNPꢀ2 treated mice which did not have significant inhibition
compared with the control group (saline treated) as expected (Figure 7). In contrast, AuNPꢀ3
treated mice (third group) showed 2.3ꢀfold tumor growth inhibition at the end of 18 days.
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Furthermore, AuNPꢀ4 treated mice (fourth group) showed higher tumor growth inhibition than
the third group with the reduction in tumor volume reaching a 3.8ꢀfold compared with the AuNPꢀ
2 treated group. The percentage tumor growth was plotted for these four groups as a function of
time (Figure 7). A twoꢀway ANOVA test showed the comparisons to be very significant (p <
0.001). Although, in vitro studies showed that AuNPꢀ3 and AuNPꢀ4 exhibited similar activity
with no enhancement from biotin conjugation (Figure 5a), in vivo tumor growth inhibitory effect
of biotin conjugated nanoparticle (AuNPꢀ4) was significantly better compared with AuNPꢀ3
treated mice (Figure 7 and Figure S20).
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Figure 7: In vivo anticancer activity of AuNPs in mice bearing HeLa xenografts. Mice
received a dosage of 7.5 mg/kg/d nanoconjugates, AuNP-2, AuNP-3 and AuNP-4 and
vehicle (PBS) daily for 7 days by ip injection. Arrows at the bottom of the figure indicates
the treatment days. Tumor volumes were measured at regular intervals. Tumor volumes
are expressed with ± standard error. Statistical analysis was made between different
groups by 2-way ANOVA with the Bonferroni test for post hoc comparisons. ns refers to no
significance, *** indicates p < 0.001.
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Mice in all the four groups had no systematic weight loss during the experiment suggesting
absence of systematic toxicity of the targeted or nonꢀtargeted AuNPs (Figure 8).75 Thus, drug
conjugation to gold nanoparticles increased drug efficacy without increasing toxicity.
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Figure 8: Plot of body weight of treated mice during treatment. Data are presented as the
mean ± SD.
Conclusions
We have demonstrated the first smart delivery of a copper bis(thiosemicarbazone) complex,
CuATSMꢀSSꢀCOOH, using AuNPs stabilized by PEGylation. In vivo targeted delivery of these
gold nanoparticles by biotin decoration has also been established. Unlike the copper complex,
CuꢀATSM and its close analogs, the AuNP conjugates have very good aqueous solubility and
stability in a wide range of pH and salt concentrations. Substantial release of the cytotoxic agent
with cellular reducing agents, such as GSH could be achieved, and demonstrated ex-vivo.
Although, this smart linking reduced the cytotoxicity of AuNPꢀ3 and AuNPꢀ4 relative to the
parent copper complex, in vivo studies demonstrated the effectiveness of these nanoparticle
carriers as suitable delivery vehicles as they exhibited efficient reduction of tumor volume
without significant loss in body weight. Moreover, we also found that the biotin conjugated gold
nanoparticle, AuNPꢀ4, was more efficient in inhibiting the tumor growth compared to the gold
nanoparticle without biotin, AuNPꢀ3. This smart linking strategy can be extended to other
cytotoxic complexes that suffer from nonꢀspecificity, low aqueous solubility and toxicity. Our
current efforts are directed towards finding better complexes to link and improving biotin
loading.
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Experimental Section
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Materials and Methods
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Nꢀhydroxy succinimide, polyethylene glycol (MW 3000), (±)ꢀαꢀlipoic acid, Sephadex LHꢀ20, 3ꢀ
mercaptopropionic acid, and dialysis tubing (12 kDa MWCO) were purchased from Sigma
Aldrich. Dialysis tubing (3.5 kDa MWCO) was purchased from ThermoFischer Scientific.
HAuCl4, citrate were obtained from SRL chemicals (India). N, N'ꢀDicyclohexylcarbidoimide
(DCC) was purchased from Spectrochem Pvt. Ltd. (India). OꢀBenzotriazoleꢀN,N,N',N'ꢀ
tetramethylꢀuroniumꢀhexafluoroꢀphosphate (HBTU), N,Nꢀdiisopropylethylamine (DIPEA) were
obtained from Avra Synthesis Pvt. Ltd. (India). Water was purified using Biocel MilliꢀQ water
purification system (18.2 Mꢁ cmꢀ1) and used for all experiments. All the solvents were dried and
distilled prior to use, following standard procedures. Standards for ICPꢀMS analysis were
purchased from Fluka Analytical.
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1H and 13C NMR spectra were recorded in Bruker AMX 400 spectrometer (operating at 400 and
100 MHz respectively). UVꢀvis spectra were recorded using Perkin Elmer Lambda 35 UVꢀ
visible spectrometer. IR spectra in KBr phase were obtained from Perkin Elmer Spectrum One
FTꢀIR spectrometer. TEM images were obtained from samples placed on a carbon coated copper
grid using a JEOL 2100F instrument operated with an accelerating voltage of 200 kV. AFM
images were acquired on Multimode Scanning Probe Microscope using a NanoScope IV A
controller using the tapping mode on the cleaved mica surface. TGA analysis of the samples
were done on a NETZSCH TG 209 F1 thermogravimetric analyzer. The copper content of the
nanoꢀconjugates was estimated using a Thermo Scientific XSeries 2 ICPꢀMS. Mass spectra were
recorded using Agilent 6538 ultraꢀhigh definition (UHD) accurateꢀmass QꢀTOF (LCꢀHRMS)
instrument. Brookhaven ZetaPALS instrument was used to determine hydrodynamic radius of
the nanoconjugates. Copper content in nanoconjugates and in cells was estimated using a
Thermo Scientific XSeries 2 inductively coupled plasma mass spectroscopy (ICPꢀMS).
Synthesis of ATSM-SS-COOH:
Bis(thiosemicarbazone) ligand with a disulfide linker was synthesized as follows. Initially, the
ATSMꢀA was prepared according to the previously reported procedures (Scheme 1).44, 76
A
solution of 3,3'ꢀdithiobis(propionic acid) and HBTU in dry N,Nꢀdimethyl formamide (DMF) was
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stirred for 1h at 0°C in an iceꢀbath. After 1h, diacetylꢀ2ꢀ(4ꢀNꢀmethylꢀ3ꢀthiosemicarbazone)ꢀ3ꢀ(4ꢀ
Nꢀaminoꢀ3ꢀthiosemicarbazone) (ATSMꢀA) dissolved in dry DMF was added dropwise to the
above mentioned iceꢀcold solution over a period of 15 min. Next, N,Nꢀdiisopropylethyl amine
(DIPEA) was added to the reaction mixture. The resulting reaction mixture was then brought to
room temperature and stirred for 24h. The reaction mixture was concentrated under vacuum at
65°C and subsequently added to a large excess of water which resulted in precipitation of a
yellow solid. The precipitate was washed with copious amounts of water and dried in vacuo.
Yield – 75%. 1H NMR (400 MHz; DMSOꢀd6): δ 2.20 (3H, s, CH3), δ 2.23 (3H, s, CH3), δ 2.57ꢀ
2.65 (4H, m, CH2ꢀC=O), δ 2.89ꢀ2.96 (4H, m, CH2ꢀS), δ 3.01ꢀ3.03 (3H, d, NꢀCH3), δ 8.40ꢀ8.41
(1H, d, NH), δ 8.40ꢀ8.41 (1H, d, NH), δ 9.98 (1H, s, NH), δ 10.16 (1H, s, NH), δ 10.25 (1H, s,
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NH), δ 10.61 (1H, s, NH), δ 12.34 (1H, s, COOH). C NMR (100 MHz; DMSOꢀd6): δ 12.5, δ
12.7 (CH3), δ 32.1 (NCH3), δ 34.0, δ 34.2, δ 34.3, δ 34.6 (CH2), δ 148.7, δ 150.6 (C=N), δ 170.0
(COOH), δ 173.6 (C=O), δ 179.4, δ 180.1(C=S), ESIꢀHRMS calculated [MꢀH+]-: m/z –
452.0667, found m/z – 452.0716 (100%). Elemental analysis (%): calculated for
C13H23N7O3S4.H2O: C – 33.1, H – 5.3, N – 20.8, S – 27.2, found (C13H23N7O3S4.H2O): C – 32.2,
H – 5.5, N – 20.7, S – 26.8.
Synthesis of CuATSM-SS-COOH:
ATSMꢀSSꢀCOOH (0.20 g, 0.44 mmol) was suspended in dry ethanol (10 mL) and copper acetate
(0.09 g, 0.45 mmol) was added to the above mixture. The reaction mixture was refluxed for 6h at
80°C. The product was filtered and washed thoroughly with ethanol and dried in vacuo. Yield –
90%. ESIꢀHRMS calculated [MꢀH+]-: m/z – 512.9812, found m/z – 512.9817(100%), calculated
[M+H+]+: m/z – 514.9957, found m/z – 514.9934(100%). IR data (cmꢀ1): 3365 (m, COOH), 3245
(m, NH), 2925 (m), 1670 (m, C=O), 1507 (s, C=N), 1395 (vs, amide), 1225 (vs, thioamide), 832
(w, C=S). UV−visible in DMSO [λmax, nm (ε, M−1 cm−1)]: 312 (21750) and 475 (7320).
Elemental analysis (%): calculated for CuC13H21N7O3S4: C – 30.3, H – 4.1, N – 19.0, S – 24.9,
found (CuC13H21N7O3S4): C – 29.9, H – 4.1, N – 18.9, S – 24.6.
Synthesis of ATSM–SS–ATSM:
HBTU (0.50 g, 1.3 mmol) in DMF (3 mL) was added to DMF (3 mL) solution of 3,3′ꢀ
dithiobis(propionic acid) (0.13 g, 0.62 mmol) and stirred for 1 h at 0 ˚C. In another flask,
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ATSM/A (0.3 g, 1.2 mmol) was dissolved in DMF (10 mL). The ATSM/A solution was added
slowly to the activated 3,3′ꢀdithiobis(propionic acid) over a period of 15 min and then DIPEA
(0.2 g, 1.6 mmol) was added. The resulting mixture was stirred for 6 h at room temperature
under nitrogen. The reaction mixture was concentrated to about 2 mL by applying vacuum at 65
˚C and the product precipitated out by the addition of water. The precipitate formed was washed
with adequate amount of water to obtain ATSM–SS–ATSM as a brownishꢀyellow solid. Yield:
75% (0.3 g). 1H NMR (400 MHz; DMSOꢀd6): δ 2.20 (6H, s, CH3 ), δ 2.23 (6H, s, CH3 ), δ 2.59ꢀ
2.63 (4H, t, CH2 –C=O), δ 2.95ꢀ2.99 (4H, t, CH2 –S), δ 3.02ꢀ3.03 (3H, d, CH3), δ 8.39ꢀ8.40 (1H,
q, NHꢀCH3), δ 9.98 (1H, s, NHꢀC=O), δ 10.17 (1H, s, NH), δ 10.22 (1H, s, NH), δ 10.60 (1H, s,
NH ). 13C NMR (100 MHz; DMSOꢀd6): δ 12.6 (CH3 ), δ 12.8 (CH3 ), δ 32.1 (CH3N), δ 34.2
(CH2), δ 148.8, δ 150.6 (C=N), δ 170.1 (C=O), δ 179.4 δ 180.1 (C=S). ESIꢀHRMS calculated
[M+H+]+: m/z – 697.1543, found m/z – 697.1553.
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Synthesis of CuATSM–SS–CuATSM:
To a suspension of ATSM–SS–ATSM (0.12 g, 0.17 mmol) in ethanol (10 mL), copper acetate
(0.069 g, 0.34 mmol) was added and refluxed for 4 h. The solution was cooled to room
temperature and filtered, the precipitate washed with ethanol and dried in vacuo to give
CuATSM–SS–CuATSM as a darkꢀbrown solid (0.12 g). Yield: 86%. ESIꢀHRMS calculated
[M+H+]+: m/z – 820.9801, found m/z – 820.9836. IR data (cmꢀ1): 3164 (m, NH), 1679 (m, C=O),
1484 (vs, C=N), 1224 (s, thioamide), 828 (w, C=S). UV−visible in DMF [λmax, nm (ε, M−1
cm−1)]: 312 (31 200) and 480 (10300). Elemental analysis (%): calculated for
Cu2C20H32N14O2S6.2H2O:
C
–
26.9,
H
–
4.5,
N
–
21.9,
S
–
21.5, found
(Cu2C20H32N14O2S6.2H2O): C – 25.9, H – 3.7, N – 19.4, S – 20.4.
Synthesis of LA-NHS:
A
solution of Nꢀhydrosuccinamide (NHS) (0.40 g, 4.31 mmol) and N,N'ꢀ
Dicyclohexylcarbidoimide (DCC) (1.5 g, 4.3 mmol) in dry DCM (60 mL) and DMF (2ml) was
cooled to 0o C in an ice bath. To this, (±)ꢀαꢀlipoic acid (0.80 g, 3.9 mmol) in dry DCM (60 mL)
was added. The reaction mixture was warmed up to room temperature and stirred for 48h in the
dark. After 48h, the precipitated dicyclohexylurea (DCU) was filtered off and the solvent was
evaporated under reduced pressure. Isopropanol was added to dissolve the crude product and
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scratching the walls of the beaker resulted in the formation of a pale yellow solid which was
filtered and washed with hexane. The product was air dried. Yield (0.64g, 55.0%). 1H NMR (400
MHz, CDCl3): 1.51ꢀ1.60 (2H, m, ꢀCHCH2), 1.69ꢀ1.81 (4H, m, ꢀCOCH2), 1.88ꢀ1.96 (1H, m, ꢀ
CH), 2.43ꢀ2.51 (1H, m, ꢀCH), 2.61ꢀ2.64 (2H, t, ꢀCH2), 2.83ꢀ2.84 (4H, d, ꢀNCH2), 3.10ꢀ3.19 (2H,
m, ꢀSCH2), 3.54ꢀ3.61 (1H, q, ꢀSCH).
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Synthesis of LA-PEG-NH2:
Diaminoꢀpolyethylene glycol (PEG, MW 3000) was synthesized following the literature
procedure.77, 78 The capping agent, LAꢀPEGꢀNH2, was prepared and purified in the following
way. To a solution of diamineꢀPEG (8.05 g, 2.68 mmol) and sodium hydrogencarbonate (0.23 g,
2.70 mmol) in DMF/water (10 mL/10 mL) at 0°C, a solution of LAꢀNHS (0.49 g, 1.60 mmol) in
DMF (3mL) was added dropwise. The solution was then brought to room temperature and stirred
for 24h at RT. The product was extracted with CHCl3 (3x50 mL). The organic fraction was
washed with water (3x30 mL) and dried over anhydrous Na2SO4. The crude product was
obtained by evaporation of the solvent and was purified by passing through a Sephadex (LHꢀ20)
1
column using methanol as eluent. Yield (3.8 g, 45 %). H NMR (400 MHz, CDCl3): 1.41ꢀ1.47
(m, ꢀCOCH2ꢀ, 2H), 1.62ꢀ1.69 (m, ꢀCH2, 4H), 1.86ꢀ1.97 (m, ꢀSCH2CH2, 1H), 2.15ꢀ2.19 (t, ꢀCH2ꢀ
CONHꢀ, 2H), 2.42ꢀ2.49 (m, ꢀSCH2CH2, 1H), 2.89ꢀ2.92 (t, NH2ꢀCH2, 2H), 3.43ꢀ3.80 (m, ꢀ
O(CH2)2, 264H).
Synthesis of AuNP-1:
HAuCl4 0.03% in 400 mL of MilliꢀQ water was heated to boiling with vigorous stirring and a
preheated (60°C) solution of trisodium citrate (0.18 g in 18 ml of MilliꢀQ water) was added.
Within a few minutes a gradual color change from yellow to pink red was observed. The mixture
was kept for another 15 min at the same temperature and then allowed to cool to room
temperature naturally. LAꢀPEGꢀNH2 (1.68 g) dissolved in 4 mL of MilliꢀQ water was added to
the above colloidal solution and stirred overnight at RT. The LAꢀPEGꢀNH2 stabilized
nanoparticle solution was concentrated and dialyzed (12 kDa MWCO) against MilliꢀQ water for
1
48h. Yield (0.50 g). H NMR (400 MHz, D2O): 1.17ꢀ1.21 (t, ꢀCH2, 2H), 1.35ꢀ1.47 (m, ꢀ
COCH2CH2, 4H), 1.97ꢀ2.04 (m, ꢀCH2CHꢀ, 1H), 2.25ꢀ2.29 (t, ꢀCH2CH2, 2H), 2.44ꢀ2.51 (m, ꢀ
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CH2CH, 1H), 2.92 (br, NH2ꢀCH2, 2H), 3.20ꢀ3.25 (m, ꢀSCH2, 2H), 3.39ꢀ3.89 (m, ꢀO(CH2)2,
264H).
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Biotin (1.9 mg, 7.8 ꢂmol) dissolved in DMF (3 mL) was added dropwise at 0°C to a solution of
HBTU (2.9 mg, 7.6 ꢂmol) in DMF (2 mL). The solution was allowed to stir at room temperature
for 1h. AuNPꢀ1 (90 mg) in DMF (3 mL) was added drop wise to the solution at 0°C. After the
addition, the mixture was allowed to stir for 15 min. To this DIPEA (7 mg, 0.05 mmol) was
added and stirred overnight at room temperature. The reaction mixture was then dialyzed (12
kDa MWCO) against MilliꢀQ water for 48h (water changed every 6h), and finally lyophilized to
get solid nanoparticles of AuNPꢀ2. Yield ꢀ 65 mg.
Synthesis of AuNP-3:
CuATSMꢀSSꢀCOOH (20 mg, 39.0 ꢂmol) dissolved in DMF (3 mL) was added drop wise at 0°C
to a solution of HBTU (16 mg, 42.2 ꢂmol) in DMF (2 mL). The solution was allowed to stir at
room temperature for 1h. AuNPꢀ1 (235 mg) in DMF (3 ml) was added drop wise to the solution
at 0°C. After the addition, the mixture was allowed to stir for 15 min. To this DIPEA (37 mg,
0.285 mmol) was added and stirred overnight at room temperature. The reaction mixture was
then dialyzed (12 kDa MWCO) against MilliꢀQ water for 48h (water changed every 6h), and
finally lyophilized to get solid nanoparticles. Yield ꢀ 180 mg.
Synthesis of AuNP-4:
Biotin was conjugated to AuNPꢀ3 following the same procedure as that described for preparation
of AuNPꢀ2. To a solution of HBTU (3.8 mg, 10.0 ꢂmol) in DMF (2 mL), biotin (2.4 mg, 9.8
ꢂmol) in DMF (3 mL) was added drop wise at 0°C. The solution was allowed to stir at room
temperature for 1h. AuNPꢀ3 (115 mg) in DMF (3 mL) was added drop wise to the solution at
0°C. After the addition, the mixture was allowed to stir for 15 min. To this DIPEA (15 mg, 0.115
mmol) was added and stirred overnight at room temperature. Yield ꢀ 75 mg.
TEM and AFM characterization:
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TEM samples were prepared by dissolving solid AuNPs in HPLC grade methanol (~ 0.5 mg/ml)
and drop coating on 400 mesh carbon coated copper grid. For AFM, solutions of the samples in
HPLC grade methanol were spin coated at 2000 RPM for 1 min on a cleaved mica surface and
dried under vacuum.
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GSH triggered release of copper complex:
AuNPꢀ4 (5 mg) in 1mL PBS buffer (pH 7.4) was suspended in the dialysis tube (3.5 kDa
MWCO). To this solution, GSH (3 mg, 9.77 ꢂmol) in 1 ml PBS buffer (pH 7.4) was added. The
dialysis tube was then immersed in a beaker having 160 mL of PBS buffer at 37°C for 48 h. A
control experiment was also carried without GSH. In both the cases, at time intervals 0.5 h, 1 h, 2
h, 6 h, 12 h, 24 h, 32 h and 48 h; 2 ml of the outer solution was taken out and 2 ml of fresh PBS
buffer (pH 7.4) was added to maintain the same volume. The copper content of the collected
samples at different time intervals was estimated by ICPꢀMS analysis and the % of drug released
plotted as a function of time.
Copper estimation by ICP- MS:
Three sets of HeLa cells (∼10 million cells each) were treated with 25 ꢂM of AuNPꢀ3, AuNPꢀ4
and one set of cells without any treatment were kept for 6 h at 37°C in a humidified 5% CO2
incubator. After 6h, the media was removed to exclude unabsorbed nanoconjugates and the cells
were washed with 5 ml of PBS, trypsinised, and collected in 5 ml of PBS. The scraped cells were
spun down, by centrifugation at 2000 RPM for 5 min. The supernatant was discarded and the
cells were once again washed with PBS. The cell pellet so obtained was suspended again and the
number of cells were counted. Cells were lysed in 1M NaOH (1 mL) and diluted with 2% (v/v)
HNO3 (9 mL) for determining whole cell copper content. The instrument was calibrated using
standard solutions containing 10, 50, 100, and 500 ppb copper.
Biotin estimation by HABA assay:
The degree of biotinylation in the biotinylated AuNPs was determined by HABA assay.79
Briefly, the avidin/HABA reagent was prepared according to the manufacturer’s instructions by
adding 10 ml of MilliꢀQ water to the orange colored solid. The final solution contains 0.3 mM
HABA, 0.45 mg/mL avidin, 0.3 M NaCl, 0.01 M HEPES, 0.01 M MgCl2, 0.02% NaN3 (as a
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preservative). To a 1 mL cuvette 900 ꢂL of HABA/Avidin solution was added. This was
followed by 100 ꢂL of biotin or, biotinylated AuNPs dissolved in PBS to make the final volume
of 1 mL. As a control, 100 ꢂL of PBS was added to the reference. For colored AuNPs samples, a
separate blank was made with 900 ꢂL water and 100 ꢂL of conjugate. The absorbance was
measured at 500 nm using a Perkin Elmer Lambda 35 UVꢀvisible spectrometer. The calculations
were done using the equation: ꢃA500 = 0.9(AHABA/Avidin) +Asample blank ꢀ AHABA/Avidin + sample), 0.9 =
dilution factor and ꢂmole biotin/ml = (ꢃA500/34)×10 where 34 = mM extinction coefficient at
500 nm and 10 = dilution factor. All measurements were done in triplicate.
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In vitro cytotoxicity analysis by MTT assay:
The MTT assay was carried out to measure cell viability as described earlier.33 Two thousand
cells in 100 ꢂL of growth media (DMEM) were seeded in a 96ꢀwell plate and kept in CO2
incubator. After 24 h, 100 ꢂL of various concentrations of copper bisꢀ(thiosemicarbazone) and
nanoconjugate solutions were added and incubated for 48 h at 37°C in a CO2 incubator. At the
45th hour after incubation, MTT (20 ꢂL of 5 mg/ml) was added to the wells. After removing the
media, the formazan crystals formed were dissolved in 200 ꢂL of DMSO, and the absorbance
was measured at 570 nm in a microplate reader (Molecular Devices, Spectramax M5e). The
cytotoxic effects of complexes and conjugates were quantified by calculating the drug
concentration inhibiting tumor cell growth by 50% (IC50).
In vivo xenograft model:
The anticancer activity of nanoconjugates was examined in a cervical xenograft model using
HeLa cells as previously described.64 Briefly, 10 million viable HeLa cells in 100 µL PBS were
injected subcutaneously into the right posterior flank of each female nude mouse aged 4−5
weeks. After the tumor size reached approximately 200 mm3, mice were divided into four groups
with four animals in the first two groups (PBS treated and AuNPꢀ2 treated) and five animals in
the other two groups (AuNPꢀ3 and AuNPꢀ4 treated). The nanoconjugate (7.5 mg/kg body
weight) dissolved in PBS was injected intraperitoneally (ip), and the control group was treated
with an equal volume of the vehicle for seven consecutive days. The tumor volumes were
measured at regular intervals. The tumor volume (Tv) was calculated using the formula: π/6 ×
(larger diameter) × (smaller diameter)2. The tumor volume and mice body weight was monitored
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for 18 days. The tumor growth was estimated using the formula: % tumor growth= [(Vnꢀ
V0)/Vn]×100, where Vn is tumor volume on nth day of treatment and V0 is the tumor volume on
the first day of treatment. Tumor volume was measured using Vernier Calipers. Statistical
analysis of the tumor volumes was made between different groups by 2ꢀway ANOVA with the
Bonferroni test for post hoc comparisons using GraphPad Prism 5 software. All experiments
used in our animal studies were approved by the ethics committee of Indian Institute of Science
for animal care and usage.
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Author Information
Corresponding Author Phone: (+91) 80ꢀ2293ꢀ2973. Fax: (+91) 80ꢀ2360ꢀ2697. Eꢀmail:
skumar@mcbl.iisc.ernet.in (K.S.). Phone: (+91) 80ꢀ2293ꢀ2663. Fax: (+91) 80ꢀ2360ꢀ1552. Eꢀ
mail: ashoka@ipc.iisc.ernet.in (A.G.S.).
Notes: The authors declare no competing financial interest.
Acknowledgement
A.K.P gratefully acknowledges Indian Institute of Science for a Senior Research Fellowship.
K.S. and A.G.S. thank the Department of Science and Technology (New Delhi) and Department
of Biotechnology (New Delhi) for the award of a research grant. K.S. is a J. C. Bose fellow
(DST). We thank the Central Animal Facility, Indian Institute of Science (IISc), for providing
the nude mice.
Supporting Information: Characterization data (NMR, ESIꢀMS, IR, TGA, and powder XRD
spectra), AuNP stability study by UVꢀvis, data for estimation of biotin, ICPꢀMS data, images of
tumor bearing mice. This material is available free of charge via the Internet at
http://pubs.acs.org.
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