Breast Cancer

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ORIGINAL RESEARCH
Functional design of pH-responsive folate-

targeted polymer-coated gold nanoparticles for
International Journal of Nanomedicine downloaded from https://www.dovepress.com/ by 188.68.0.75 on 25-Oct-2019
drug delivery and in vivo therapy in breast cancer

This article was published in the following Dove Press journal:
International Journal of Nanomedicine
Sneha Mahalunkar 1 Background: Curcumin has been widely used owing to its various medicinal properties
Amit Singh Yadav 2 including antitumor effects. However, its clinical application is limited by its instability, poor
Mahadeo Gorain 2 solubility and low bioavailability. Folic acid (FA)-functionalized nanoformulations may
Vinay Pawar 3,4 enhance the sustained release of an anticancer drug (curcumin) by tumor-specific targeting
For personal use only.
to improve therapeutic benefit. This study aims to design a nanoconjugate (NC) comprised of
Ranveig Braathen 5,6
folate–curcumin-loaded gold–polyvinylpyrrolidone nanoparticles (FA–CurAu-PVP NPs) for
Siegfried Weiss 3
targeted delivery in breast cancer model systems.
Bjarne Bogen 5,6
Methods: We developed curcumin-loaded FA-functionalized Au-PVP NCs by layer-by-
Suresh W Gosavi 7 layer assembly. The folic acid–curcumin Au-PVP NCs (FA–CurAu-PVP NCs) were char-
Gopal C Kundu 2 acterized by ultraviolet–visible spectra, Fourier transform infrared spectroscopy, X-ray
1
School of Basic Medical Science, powder diffraction and thermogravimetric analysis. In vitro anticancer and antimigratory
Savitribai Phule Pune University, Pune effects of NCs were examined by performing MTT and wound migration assays. The in vivo
411007, Maharashtra, India; 2Laboratory
of Tumor Biology, Angiogenesis and antitumor efficacy of NCs was investigated using a preclinical breast cancer orthotopic
Nanomedicine Research, National mouse model.
Centre for Cell Science (NCCS), Pune
Results: Curcumin (40 µg/mL) was loaded along with conjugation of folate onto Au-PVP
411007, India; 3Institute of Immunology,
Hannover Medical School, Hannover, NPs to form FA–CurAu-PVP NCs. The size and charge of the NCs were increased gradually
Germany; 4Department of Molecular through layer-by-layer assembly and showed 80% release of curcumin at acidic pH. The NC
Bacteriology, Helmholtz Centre for
Infection Research, Braunschweig,
did not show aggregation when incubated with human serum and mimicked an intrinsic
Germany; 5K.G. Jebsen Centre for peroxidase-like property in the presence of 3,3ʹ,5,5ʹ-tetramethylbenzidine substrate. The
Influenza Vaccines Research, Institute of MTT data using these NCs showed efficient anticancer activity at lower doses in estrogen/
Clinical Medicine, University of Oslo,
Oslo, Norway; 6Oslo University Hospital, progesterone receptor (ER/PR)-negative cells compared with ER/PR-positive cells.
Oslo 0027, Norway; 7Department of Furthermore, the NCs did not show cytotoxicity at the investigated concentration in human
Physics, Savitribai Phule Pune University, breast epithelial and mouse fibroblast cell lines. They showed inhibitory effects on cell
Pune 411007, Maharashtra, India
migration and high antitumor efficacy in in vivo analysis.
Conclusion: These results suggest that folate-based tumor targeting using CurAu-PVP NCs
Correspondence: Gopal C Kundu is a promising approach for tumor-specific therapy of breast cancer without harming normal
Laboratory of Tumor Biology, cells.
Angiogenesis and Nanomedicine
Research, National Centre for Cell Keywords: curcumin, gold nanoconjugate, folic acid, breast cancer cell line, cytotoxic
Science (NCCS), Pune 411007, India activity
Tel +91 202 570 8104
Fax +91 202 569 2259
Email [email protected]
Suresh W Gosavi Introduction

Department of Physics, Savitribai Phule Chemotherapy, although a major treatment, is associated with various side effects
Pune University, Pune 411007,
Maharashtra, India as well as poor compliance, and this has boosted significant efforts to find a better
Tel +91 202 569 2678 anticancer drug modality that uses natural herbal compounds.1,2 As a result,
Fax +91 202 569 1684
Email [email protected] attempts made by the National Cancer Institute have led to the identification of
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Mahalunkar et al Dovepress
natural compounds that exhibit chemopreventive activity.3 Chemically engineered nanocarriers made up of various
Curcumin, a polyphenol compound isolated from Curcuma materials can be formulated and tuned to act as efficient
longa Linn, can sensitize tumor cells by passing through drug-delivery vehicles. To achieve better targeting and drug
their lipid membrane.4 Extensive experimental research release ability, gold nanoparticles (AuNPs) have predomi-
has clearly shown that native curcumin can modulate nantly been used as nanocarriers.19 A literature survey
molecular targets, thereby inducing cell apoptosis in showed effective cytotoxic activity of curcumin when it is
human tumor-derived cell lines.5–10 Therefore, we have attached to one of the side chains of soluble polymers such
selected curcumin as a model compound to inform readers as PEG, polyvinylpyrrolidone (PVP) and chitosan, while
about advanced drug-delivery therapies and assist them in the other chain is attached to an AuNP, which also tackles
exploring other untouched platforms in chemotherapeutic its problems of poor solubility and non-specificity.20 We
prevention. have explored FA conjugation and loading of curcumin
The major hurdles in assigning natural compounds onto PVP-functionalized AuNPs to target breast cancer
such as curcumin as chemopreventives are their low bioa- cells, which thereby were proven to impart high cell-killing
vailability, in vivo stability, non-specific biodistribution ability. Therefore, a state-of-the-art layer-by-layer (LBL)
and non-specific targeting properties. To overcome such assembly method for designing nanoconjugates on a solid
issues, various target specific drug-delivery systems have surface of AuNPs by adsorbing anticancer drug with an
been designed, consisting of nanoparticles loaded with oppositely charged polymer such as PVP, involving steric
drugs and specific biomarkers, and this is the major area stabilization, is attracting much attention.21 The LBL
of interest in this particular study.11,12 Nanocarrier-based assembly can be further enhanced by loading activated
delivery systems enable passive targeting due to the leaky folate onto the above-formed conjugate using carbodiimide
vasculature of tumors because of differences in their pore coupling chemistry. The cross-linking agents that help in
size which enable nanocarriers to target the tumor and forming an amide bond via coupling reactions are 1-ethyl-3-
accumulate at the tumor site, thereby blunting their effect [3-dimethylaminopropyl]carbodiimide (EDC) and dicyclo-
on normal tissues. Under many circumstances, cancer hexylcarbodiimide (DCC). Manju and Sreenivasan
patients with few other pathophysiological conditions demonstrated conjugation of hyaluronic acid with curcumin
show a non-specific distribution of nanoconjugates to mul- using DCC/DMAP as a catalyst and coupling agent.21 The
tiple sites which, to some extent, affects the targeting carboxyl group of EDC/N-hydroxysuccinimide (NHS)-
specificity to the tumor zone.13,14 For this reason, nano- functionalized folate can be conjugated with amine-functio-
carriers conjugated with drugs can be designed for active nalized AuNPs on which the anticancer drug curcumin is
targeting using targeting ligands such as folic acid (FA), loaded, as shown in Scheme 1.22
which can recognize specific receptors overexpressed on With this background, we hypothesize that we can
certain cancer cell types. remove the barriers and limitations associated with classical
FA is a well-known vitamin for cell proliferation as drug-delivery protocols by encasing an anticancer drug such
well as targeting specific biomarkers. FA-functionalized as curcumin with properties of a multi-drug-resistant mod-
formulations may enhance sustained release of an antic- ulator and a chemosensitizer on Au-PVP NPs. A target-
ancer drug (curcumin) at the cellular targets to improve the specific strategy is warranted to enhance the therapeutic
therapeutic benefits. Folate-conjugated nanomaterials have efficacy of drug-loaded nanoparticles by conjugating them
elevated interest in the field of cancer biology, mainly with extensively investigated epithelial cancer markers such
focusing on diagnosis and targeted drug-delivery as folate. Thus, our study aims to develop curcumin-loaded
protocols.15–17 Salmaso et al formulated targeted delivery FA-functionalized Au-PVP NCs by LBL assembly, which
by covalently conjugating polyethylene glycol (PEG) to can augment the therapeutic effects and improve the clinical
FA at one end of a polymeric carrier which has a cyclo- outcomes of curcumin in breast cancer therapy.
dextrin–curcumin complex at the other end. Even though
such nanoconjugate complexes entered the clathrin-inde-
pendent pathway leading to cell endocytosis through tar- Materials and methods
geting overexpressed folate receptors, they showed limited Materials
cellular uptake; hence, further investigation is required in The chemical and biological compounds used in this
terms of drug release efficacy (DRE).18 research are as follows: folic acid (FA) (C19H19N7O6),
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Dovepress Mahalunkar et al
Scheme 1 Schematic representation of folicacid–curcumin–gold–polyvinylpyrrolidone nanoparticles (FA–CurAu-PVP NCs) with layer-by-layer assembly.
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydro- Synthesis of bare AuNPs and PVP-coated

chloride (EDC.HCl) (C8H17N3.HCl), N-hydroxysuccini- AuNPs
mide (NHS) (C4H5NO3) and 3,3ʹ,5,5ʹ-tetramethylbenzidine Several methods of gold synthesis exist, which produce
(TMB) were obtained from Sigma-Aldrich (Bangalore, diverse characteristics in the final product. The method
India). Curcumin crystalline (C21H20O6) was purchased devised by Brust et al23 showed extensive use of organic
from Loba Chemie (Mumbai, India); acetone, dimethyl phase synthesis concerning a double- or a single-phase
sulfoxide (DMSO) and H2O2 from Thomas Baker. process, based on gold salt reduction by sodium citrate
Chloroauric acid (HAuCl4) and trisodium citrate as reported by Turkevitch et al,24,25 which was refined by
(Na3C6H5O7) were purchased from SRL and were used as Frens,26 who produced spherical size-tunable nanoparti-
received. Polyvinylpyrrolidone [(C6H9NO)n] was collected cles. The purified AuNPs thus obtained were treated with
from CDH. Trypsin/EDTA, penicillin, streptomycin, MTT PVP polymer, which forms a thin covering around the
reagent, propidium iodide, RNase, FBS, DMEM and L-15 AuNP and can now be used to conjugate with the activated
media were obtained from Sigma-Aldrich (Bangalore, ligands or with another nanoparticle or anticancer agents.
India). All chemicals were of reagent grade and were used
without further purification.
Activation of FA and its conjugation with
CurAu NPs to form FA–CurAu-PVP NCs
Maintenance of cell lines FA was converted into activated folate to achieve success-
Human breast adenocarcinoma (MDA-MB-231 and ful conjugation with the amine terminated nanoparticles.
MCF-7), mouse fibroblast (L929) and human breast The activation of FA was achieved by the previously
epithelial (MCF 10A) cell lines were purchased from reported method with few modifications, as follows.27–29
American Type Culture Collection (ATCC, Manassas, In brief, 2 mg FA was dissolved in 20 mL DMSO to obtain
VA, USA). Mouse mammary carcinoma cell line (4T1) a clear solution. To the above solution, EDC/NHS (0.4 M/
was purchased from Caliper Life Sciences (USA). Cell 0.1 M) mixture was added, which resulted in activation of
lines were maintained in L-15 (MDA-MB-231) and COOH groups in FA (Fa/EDC/NHS 2:1:1) and the result-
DMEM (MCF-7, T47D, 4T1, MCF 10A and L929) sup- ing solution was continuously stirred for about 1 h.
plemented with 10% FBS, 100 units penicillin and Similarly, curcumin (1 mg/mL) was dissolved in acetone
100 μg/mL streptomycin. Cells were subcultured at and was added in the amine terminated Au-PVP NP solu-
70–80% confluence and incubated at 37°C in an incubation in a dropwise manner with stirring and heating con-
tor supplied with 5% CO2. tinuously for 3 h, followed by washing the pellet several
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International Journal of Nanomedicine 2019:14 8287
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times to remove the traces of unattached curcumin. The by UV–visible spectroscopy. The amount of drug released
activated folate mixture prepared earlier was then added to and cumulative release percentage were calculated using
5 mL of amine terminated CurAu-PVP NPs solution. The the following formula:
conjugated FA–CurAu-PVP NPs were washed multiple
DRE ¼ ðDrug released at time t = Total drugÞ 100
times with deionized water followed by suspending the
pellet in PBS. The NC thus formed was stored at 4°C until
further use. Colorimetric response of FA–CurAu-
PVP NCs
Physicochemical characterization of drug- To investigate the peroxidase-mimetic activity of
loaded NPs FA–CurAu-PVP NCs, a 1:1 ratio of TMB and H2O2 was
The functionalized AuNPs were characterized by ultra- mixed with 100 µL of 1 µg/mL NC and 0.01 M HNO3
violet (UV)–visible spectra (Jasco V-670 spectrophot- solution. Next, the mixture of solution was kept at 50°C in
ometer) recorded at 200–800 nm, and Fourier-transform a water bath for 30 min and then cooled to room tempera-
infrared (FTIR) spectra (Jasco FT/IR 6100) were also ture. Subsequently, color change photographs were
recorded. The X-ray diffraction (XRD) patterns of curcu-
acquired immediately and UV–visible readings were
min and FA–CurAu-PVP NCs were obtained using a
recorded at 655 nm.
Bruker D8 Advance X-ray diffractometer equipped
with Cu Kα radiation source from 10 to 80°C.
Thermogravimetric analysis (TGA) was carried out Protein adsorption studies

with a Mettler Toledo TGA/DSC 1 Stare System. The UV–visible and FTIR spectra, particle size and zeta
FA–CurAu-PVP NCs were also measured by the dynamic potential of NPs can vary after serum protein adsorption or
light scattering (DLS) technique using a Zetasizer Nano interaction. To evaluate this phenomenon, human serum
ZS90 instrument (Malvern) with an He-Ne laser beam at (HS) was mixed with FA–CurAu-PVP NCs in a 1:1 ratio
a wavelength of 633.8 nm. Transmission electron micro- with a pipette tip to obtain a homogeneous mixture. The
scopy (TEM) was used to examine the morphology of the mixture was incubated for 22 h at 4°C followed by several
nanosystems (Tecnai G22). steps of washing with deionized distilled water before
measurement.
Determination of drug loading efficiency
The drug loading efficiency (DLE) of curcumin was deter-
Cell viability assay
mined. Curcumin solution was mixed with Au-PVP NPs in
The effect of drug-loaded FA–CurAu-PVP NCs on cell
a 1:1 ratio and sealed in porous dialysis tube to be dialyzed
viability in breast cancer was evaluated by the MTT
against PBS (1×, pH 7.4) at room temperature for 24 h.
assay. In brief, human breast cancer cells (MCF-7 and
The released drug concentration was monitored by record-
MDA-MB-231) and mouse breast cancer cells (4T1)
ing UV–visible spectra. The drug loading efficiency was
were seeded (2×104) in a 96-well tissue culture plate.
calculated as follows:
Cells were treated with FA–CurAu-PVP NCs (10, 20, 50,
DLE ¼ ðDrug loaded at time t = Total drugÞ 100 100 and 200 µg/mL) for 24 h. After 24 h incubation under
experimental conditions, the derivatives in the medium
In vitro release of curcumin were removed and MTT (0.5 mg/mL) was added and
Two volumes of 5.0 mL of FA–CurAu-PVP NCs were incubated for 4 h. Thereafter, the formazan crystals were
enclosed in two dialysis bags with a molecular cut-off of dissolved in 200 µL of isopropanol and the absorbance
10 kDa and placed separately in two beakers, one contain- was recorded immediately at 570 nm using an automated
ing 150 mL of PBS (1×, blood pH level 7.4) and another microplate reader (EPOCH2; BioTek Instruments,
with acetate buffer (1×, acidic pH 5.3), with continuous Highland Park, VT, USA). Cell viability was analyzed
stirring at 100 rpm. At predetermined time intervals, using SigmaPlot 10.0 software. In separate experiments,
100 µL of sample was collected and replaced with an Human mammary epithelial cells, MCF 10A, and mouse
equivalent volume of release buffer solution. The released fibroblast cells (L929) were grown in 96-well plates and
concentration of drug as a function of time was analyzed the MTT assay was performed.

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h i
Wound healing assay V ¼ π=6 ðlxbÞ3=2
The effect of FA–CurAu-PVP on breast cancer cell migra-
tion was examined by a wound healing assay. In brief, After the required time period, mice were killed and
monolayers of MDA-MB-231 and 4T1 cells were grown tumors were removed, photographed, weighed and pre-
in 12-well plates and wounds of uniform size were made sented graphically.
with a sterile 10 µL pipette tip. Then, cells were treated
with different concentrations of NCs (5–20 µg/mL for Statistical analysis
MDA-MB-231 and 2.5–10 µg/mL for 4T1 cells) in low- The data are presented as mean ± SEM using SigmaPlot
serum media with 4% FBS. Wound photographs were 10.0 software. The level of significance was calculated
captured at T=0 and T=18 h for MDA-MB-231 cells and using the unpaired Student’s t-test. A p value less than
at T=0 and T=6 h for 4T1 cells using a phase-contrast 0.05 (p<0.05) was considered statistically significant.
microscope (Nikon). The area migrated was quantified
(Image-Pro Plus software), analyzed and represented as a
Results
bar graph (SigmaPlot 10.0 software).
Physiochemical characterization of FA–
Determination of cell cycle by flow CurAu-PVP NCs
At the start, AuNPs were synthesized by the Turkevitch
cytometry method and analyzed by UV–visible spectroscopy. AuNPs
The effect of FA–CurAu-PVP on cell cycle progression in were coated with PVP, followed by loading of curcumin
MDA-MB-231 cells was analyzed by fluorescence-acti- on Au-PVP NPs and activation of FA into folate, thereby
vated cell sorting. In brief, MDA-MB-231 cells were achieving functionalization of nanoparticles with the EDC
seeded in a 60 mm dish at a density of 2×105 cells/dish coupling reaction, and then conjugating it with activated
and treated with NCs (20 and 50 µg/mL) for 24 h. After folate to form FA–CurAu-PVP NCs. The NP thus formed
treatment, cells were fixed overnight in 95% ethanol and was analyzed and confirmed by UV–visible spectroscopy
washed twice in chilled PBS, then the pellet was resus- as well as by FTIR, as shown in Figure 1.
pended in 50 μL of RNase A (0.5 mg/mL) and incubated The unmodified AuNPs displayed characteristic sur-
for 20 min at 37°C. After that, 450 μL of propidium iodide face plasmon absorption at 533 nm; when they were
(50 μg/mL) was added. The proportion of cells in different coated with a polymer PVP there was a shift in the peak
phases of the cell cycle was monitored by a BD position (532 nm), suggesting that the AuNPs had been
FACSCalibur instrument (BD Biosciences). successfully coated with PVP (Figure 1A). The UV–visi-
ble spectra for pure FA showed one broad absorption band
In vivo antitumor efficacy around 285 nm, corresponding to the л-л* transition of the
All animal experiments were conducted as approved by C–C bond, and another broad shoulder around 364 nm,
the Institutional Animal Care and Use Committee corresponding to the n-л* transition of the C–O bond in
(IACUC) of the National Centre for Cell Science the enone moiety of FA. The spectrum of functionalized
(NCCS, Pune, India), in compliance with the ‘Committee FA also showed two broad peaks but with a bathochromic
for the Purpose of Control and Supervision of Experiments shift from 285 to 312 nm and 364 to 376 nm. Thus, the
on Animals’ (CPCSEA) guidelines. 4T1 (1×105) cells in a shift in the spectra of FA before and after functionalization
mixture with Matrigel (1:1) (BD Biosciences) were suggests the successful conversion of FA to activated
injected into the mammary fat pads of 6-week-old female folate (Figure S1). Curcumin-conjugated AuNPs showed
Balb/c mice. Once tumors had formed, mice were ran- a surface plasmon band around 223 nm and 532 nm,
domly divided into three groups. The first group was left suggesting loading of curcumin onto Au-PVP NPs
untreated as a control, while the second and third groups (Figure 1A). This new red peak indicates that curcumin
were treated intratumorally with curcumin and FA–CurAu- interacted with Au-PVP NPs. Two absorption maxima
PVP NCs, respectively (10 mg/kg body weight) for 2 peaks, at 285 nm and 364 nm, were observed in the
weeks. Tumor length and breadth were measured at reg- UV–visible spectrum of folate-conjugated nanomaterials
ular intervals using Vernier calipers. Tumor volume was elsewhere, and the same data can be used to confirm
calculated using the formula: covalent attachment of the folate with AuNPs. Thus, we

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Figure 1 Step-by-step synthesis of folicacid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs). (A) Ultraviolet–visible spectra of AuNPs and
AuNPs coated with PVP. (B) Fourier transform infrared spectra of variation in forming different layers of NCs. (C) X-ray diffraction (XRD) spectra of FA–CurAu-PVP NC.
Inset: XRD spectra of curcumin. (D) DTG result of FA–CurAu-PVP NCs.
observed three major peaks in the FA–CurAu-PVP NC plotted at 1500–1600 cm−1 were the stretching vibrations
spectrum. Two of these three peaks were at 285 nm and of C=C determined for the aromatic ring backbone
364 nm, indicating folate covalent bonds, and the other structure.34 Comparing the infrared spectrum of FA with
absorption peak was at 435 nm, confirming Au-PVP NP that of FA–CurAu-PVP NCs, both of them have intensity
formation, thus suggesting successful bonding of the FA absorptions plotted at 1429 cm−1, 2911 cm−1 and 2999
ligand to CurAu-PVP NPs.30–32 FA–CurAu-PVP NCs cm−1. The peak around 1508 cm−1 is assigned to N–H
showed peaks at 273 nm, 285 nm, 364 nm and 435 nm, bending vibration of the free amine group of folate on
respectively. The results also indicate the increase in the FA–CurAu-PVP NCs. When FA was conjugated with
size of the sample after modification and conjugation. CurAu-PVP NPs, a shift in the peak position was noticed
Comparing the spectral data of FA and FA–CurAu- with respect to FA, CurAu-PVP NP and FA–CurAu-PVP
PVP NCs shows that both of them had the same unique NCs between 3000 cm−1 and 3600−1, which was ascribed
peaks at 285 nm and 364 nm, which is assigned to the to the hydroxyl (OH) stretching bands (Figure 1B). These
π→π* transition of the pterin ring of the FA molecule, results suggest successful conjugation of FA on CurAu-
indicating that FA was successfully conjugated with PVP NPs to form FA–CurAu-PVP NCs.34,35 Figure 1C
CurAu-PVP NPs (Figure 1A).33 To further testify the shows the XRD pattern of FA–CurAu-PVP NCs, which
occurrence and qualitatively analyze conjugation of FA reveals a typical amorphous pattern that may be due to
on CurAu-PVP NPs, the nanoconjugates were character- AuNPs and a few characteristic peaks of curcumin in the
ized by FTIR spectroscopy. In the pure FA and functiona- 2θ range of 10≤2θ≤30 degrees, which indicates the distinct
lized FA, the characteristic peak at 1651 cm−1 is accredited crystalline structure of a hydrophobic drug. Characteristic
to the stretching vibration of –C=O groups. The peaks peaks of curcumin appeared at 8.90, 12.25, 14.55 17.24,

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23.33, 24.60 and 25.52 degrees (Figure 1C, inset).

Furthermore, the diffraction 2θ peaks observed in the
range of 20–30 degrees for curcumin match those in ear-
lier reports.36–40
To study the change in sample weight as a function of
temperature and to analyze its thermal stability, a very
important technique, namely thermogravimetric analysis
(TGA), was used. As shown in the thermogram in

Figure 1D, CurAu-PVP NPs showed only one step of
weight % decrease, while FA–CurAu-PVP NCs showed
two steps of weight % decrease. The temperature equiva-
lent to the deep peak at each weight change step is more
prominently observed in the first derivative of the TGA
curve (DTG) with respect to temperature to obtain a DTG
thermogram. The temperature which gives the maximum
rate of weight % for a sample (seen as peaks in the DTG
thermogram) is considered as the degradation temperature
of a component in the material. DTG thermogram of
CurAu-PVP NPs showed one step degradation at 1250C

with 99% weight loss which might be the degradation
temperature (Td) of curcumin linked Au-PVP NPs
whereas after conjugating FA to form FA-CurAu-PVP
NCs, temperature at 1140C and 1450C showed 95% and
97% weight loss where the second Td might be ascribed
to the degradation temperature of folic acid conjugated
with CurAu-PVP NPs. The above data also indicate no
weight gain in the samples, suggesting that the conjugate
was intact without oxidation, and this may be due to the
Figure 2 Size distribution and transmission electron microscopy images of (A)
inert nitrogen gas atmosphere. We can thus conclude that curcumin–gold–polyvinylpyrrolidonenanoparticles (CurAu-PVP NPs), (B) folic acid–
the conjugates were successfully formed as FA–CurAu- curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs).
PVP NCs, which are slightly more stable than CurAu-PVP

NPs, and this may be due to layering of FA molecules.
(Figure 2A),3 whereas the hydrodynamic diameter of
To ascertain the formation of nanoconjugates, we per-
FA-grafted NCs (~358.7 nm) compared to CurAu-PVP
formed DLS and zeta potential measurements and TEM
NPs with a fairly low polydispersity is consistent with the
before and after conjugation with the drug curcumin as
NHS–folate grafting (Figure 2B).12 The significant increase
well as with a biomarker FA, as shown in Table S1. The
in the particle size of the NC formulation can be attributed
TEM images showed discrete spherical outlines and mono-
dispersed size distribution (~250 nm) (Figure 2A and B, to the increase in shell volume that resulted from FA incor-
inset) which were lower values compared with DLS mea- poration onto the CurAu-PVP NP core.12 The zeta potential
surements. The nanoconjugate of 250 nm will be ideal for obtained for bare AuNPs was −7.47 mV, which shows a
passive tumor targeting as most solid tumors show a vas- noticeable change after its successful coating with PVP
cular cut-off between 380 and 780 nm, as mentioned (−9.91 mV) (Table S1). PVP is a neutral and hydrophilic
earlier.4–11 The DLS result reveals the average hydrody- polymer which stabilizes Au-PVP through hydrophilic
namic diameter of AuNPs to be 36.85 nm and that of interaction and therefore is efficiently loaded with hydro-
AuPVP to be 62.50 nm. The increase in hydrodynamic phobic oxygen-rich curcumin to form CurAu-PVP NPs.5
size of CurAu-PVP NPs (72.56 nm) compared to Attachment of partly negatively charged NHS–folate to
PVP-coated AuNPs can be credited to partial cross-linking amine-functionalized CurAu-PVP NPs not only results in
between the particles during the conjugation process increasing negative potential value of FA–CurAu-PVP NCs

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but also ensures that the formulations will repel each and Cl may be due to polymer, folate and curcumin cap-
other to enable long-term storage without particle ping. The EDS spectra of FA–CurAu-PVP NPs reveal the
aggregation.3–10 Previous reports show that particles with elemental composition of curcumin and FA, with a higher
a positive charge favor opsonization followed by reticuloen- percentage of Ca, H, O and N along with C, Cl and Au
dothelialsystem clearance. Hence, a negative zeta potential proving the successful conjugation of FA and curcumin
of approximately −5 to −20 mV is an appropriate system to onto AuNPs (Figure S2).
arbitrate drug delivery at sites other than the reticuloen-
dothelial system.8,9 A step-by-step increase in the absolute In vitro drug loading and release studies
negative value of the zeta potential of AuNPs with FA– The DLE of curcumin was gradual with respect to time
CurAu-PVP NCs by increasing the polymer, drug and folate and was calculated to be 40 µg/mL of FA–CurAu-PVP
content indicates that all three components were solubilized NCs, and the DRE in the initial hours (2 h) was found to
not only on the AuNPs but also in the periphery of NCs. be 40–45% at blood pH level (pH 7.4) and 80% at acidic
The more negative charge of the NC could be also due to pH level (pH 5.3), which were better results than proposed
the carboxylate group of FA located on the surface of the previously (Figure 3A and B).1 The release profiles at pH
nanoparticle.13 The elemental composition from the energy- 5.3 and pH 7.4 of curcumin are shown in Figure 3C and D.
dispersive X-ray spectroscopy (EDS) spectra shows the The loaded curcumin was released within a period of 24 h;
maximum percentage of AuNPs and the remainder as C maximum release was seen within 8 h at pH 7.4 and 6 h at
Figure 3 Drug loading and release of curcumin. (A) Ultraviolet–visible spectra of drug loading onto the nanoconjugate. (B) Cumulative percentage of curcumin released
from folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) at pH 7.4 and pH 5.3. (C) Curcumin released from FA–CurAu-PVP NCs at pH
5.3. (D) Curcumin released from FA–CurAu-PVP NCs at pH 7.4.

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pH 5.3, followed by a gradual slow release. This clearly of curcumin show striking changes and this may be due to
implies that curcumin was loaded successfully onto the change in particle size because of protein binding.3,4
Au-PVP NPs. Thus, the goal of integrating an anticancer These changes indicate adsorption of serum proteins onto
drug (curcumin) onto the nanoparticle, along with FA, to the nanoconjugates. The addition of HS can promote the
enhance its application in targeted drug-delivery protocols movement of curcumin from a hydrophilic to a hydropho-
was achieved. bic environment, as the aryl groups of curcumin are hydro-
phobically bound to the hydrophobic pockets of HS.5
Serum binding studies Comparing the spectra of HS alone and when incubated
Analysis of FA–CurAu-PVP NCs in PBS before and after with FA–CurAu-PVP NCs gives information about differ-
incubation in HS by UV–visible spectra provided addi- ent types of secondary structures obtained after binding.4
tional evidence about the protein coating. The presence of The FTIR spectra of HS displayed a strong signal at
such a protein coating layer on the nanoconjugates is also 1645 cm−1, while the spectrum of FA–CurAu-PVP NCs
known to prevent aggregation of the nanoparticles.11 showed changes in the strength and position of the absorp-
Curcumin shows intense absorption from 200 to 450 nm tion peak between 1600 and 1700 cm−1 (Figure 4B). The
with maximum absorption close to 420 nm. Furthermore, characteristic amide bands at about 1652, 1542 and 1241
the UV–visible absorption spectra of curcumin bound to cm−1 correspond to C=O stretching, N–H in-plane bend-
HS have a shoulder peak around 450 nm, which is present ing, and C–H stretching as well as C–N and N–H in-plane
stretching, respectively (Figure 4B).9,10 The changes in the
in the spectra of FA–CurAu-PVP NC as well (Figure 4A).6

However, in the presence of HS, the curves of absorption band intensity and slight shifts in infrared bands after
Figure 4 Serum binding studies. (A) Ultraviolet–visible spectra of folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) before and after
incubation with human serum and (B) Fourier transform infrared spectra of naive human serum and FA–CurAu-PVP NCs before and after incubation with human serum. (C)
Zeta potential measurements of FA–CurAu-PVP NCs before and after incubation with human serum. (D) Dynamic light scattering measurements of FA–CurAu-PVP NCs
before and after incubation with human serum.

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interaction with HS confirm the association of HS proteins 655 nm (Figure 5A). The catalytic activity of NCs is also
to FA–CurAu-PVP NCs. This suggests that by interacting dependent on the pH of the reaction buffer and incubation
with FA–CurAu-PVP NCs, changes in the secondary temperature. Hence, the pH of the solution (1.0–3.0) and
structure of HS protein have occurred. temperature of the reaction system (40–60°C) were opti-
To gain an insight into the HS-induced variation in mized in this study. As shown in Figure 5C, the catalytic
net surface charge and size of Au-PVP NPs, CurAu- activity reduced with increasing pH conditions, in the pH
PVP NPs and FA–CurAu-PVP NCs, zeta potential and range 1–3. Reduced activity was observed in neutral or basic
DLS measurements were carried out under standard solutions compared to acidic solutions. The best result for a
experimental conditions. Before incubation of these par- color change from yellow to greenish blue was obtained at
ticles with HS, they were negatively charged. Upon pH 1.2 and temperature 50°C, with an HNO3 concentration
incubation, the mean particle surface charge increased; of approximately 220–230 µL. TMB has a structure similar
that is, it became less negative, from −9.91 mV to −3.82 to diamine, which leads to a lower solubility in a weak basic
mV for Au-PVP NPs, from −10.1 mV to −5.09 mV for medium. Such pH-dependent catalytic activity is mainly
CurAu-PVP NPs and from −12.5 mV to −3.74 mV for associated with the substrates and not with the NCs. On the
FA–CurAu-PVP NPs (Figure 4C and Table S2). HS other hand, the activity elevates with increasing incubation
protein binding to the FA–CurAu-PVP NCs is due to temperature. The experiments showed that the absorbance of
electrostatic effects. Thus, the measured zeta potential of FA–CurAu-PVP NCs in the TMB–H2O2 system was much
the NC was highly negative before serum incubation higher at pH 1.2, and the same system at pH 1.0 and pH 2.2
and reached neutrality when bound with serum proteins. showed slight absorbance at 655 nm (Figure 5C). The ele-
It can be estimated that there is sequential binding of vated absorbance at 655 nm is considered to be the gradual
cationic proteins initially, which may be covering the increase in the oxidation product of TMB.3 This result
negative charges on the particles, followed by opsoniza- demonstrated that both FA–CurAu-PVP NCs and H2O2
tion of negatively charged proteins to the positively were required to catalyze TMB oxidation at standard pH
charged protein coat.3–8 In this study, we also observed 1.2. Thus, the result also confirmed that FA–CurAu-PVP
an increase in the size of AuNPs when incubated with NCs exhibited an intrinsic peroxidase-mimetic activity,
HS. The adsorbed serum proteins decreased the absolute mainly due to the presence of AuNPs in the nanoconjugate.
charge on the particles but increased the size and kept The inset in Figure 5C compares the formation of reaction
them stable. This stability may be due to Coulombic products against different pH within a time interval of 30 min
repulsion between charged particles or to steric hin- after the addition of the catalysts (FA–CurAu-PVP NCs and
drance caused by the protein coating, as stated HNO3) for the color change of TMB. The color change was
elsewhere.10 As illustrated in Figure 4D, the size of very fast as soon as the pH was adjusted to 1.2, and after the
nanoparticles almost doubles after their incubation with addition of 0.01 M HNO3 the change from colorless to
HS, whereas the polydispersity index remains between greenish blue was observed. The maximum catalytic activity
0.2 and 0.6, which demonstrates that the nanoparticles occurred at pH 1.2 and temperature 50°C in the presence of
are suitable for DLS (Table S2). Thus, DLS was able to TMB–H2O2–FA–CurAu-PVP NCs in the ratio 1:1:1.
detect the increase in size due to enhanced serum pro- Therefore, FA–CurAu-PVP NCs show superior peroxidase-
tein attachment with the particles.1 According to pre- like catalytic performance to the peroxidase substrate TMB
vious investigations and the literature, degradation of in the presence of H2O2 and under standard pH conditions.1
curcumin has shown that HS acts as an efficient trans- Each component separately treated with NC does not give
porter for the curcumin molecule.7 absorbance at 655 nm but the NC with all three components
(TMB, H2O2 and HNO3) shows the color change (Figure 5B
Colorimetric assay and D). The AuNPs can catalyze the TMB–H2O2 reaction,
To analyze the peroxidase-mimetic activity of FA–CurAu- where H2O2 is adsorbed on the surface of AuNPs and the O–
PVP NCs, the catalytic oxidation of peroxidase substrate O bond of H2O2 may be broken up into the double HO˙
TMB in the presence of a catalyst, H2O2, was tested. As radical.4,5 Thus, the catalytic ability of AuNP is attributed to
shown in Figure 5, the AuNPs catalyze TMB substrate oxi- the OH radical thus generated. The catalytic efficiency of the
dation in the presence of H2O2 to produce a greenish blue NC is strongly dependent on the pH and temperature of the
color complex. The maximum absorption peak appeared at reaction. The 0.01 M HNO3 was used in the reaction media to

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Figure 5 Colorimetric assay. (A) Ultraviolet (UV)–visible peaks showing the effect of different concentrations of HNO3. (B) Images illustrating color change after addition of
different concentrations of HNO3: (i) 20 μL, (ii) 30 μL, (iii) 50 μL, (iv) 100 μL, (v) 200 μL, (vi) 300 μL, (vii) 220 μL, (viii) 230 μL, (ix) 240 μL, (x) 260 μL, (xi) 280 μL, (xii)
300 μL. (C) UV-visible spectra depicting pH standardization to obtain color change. Inset: Color change at (i) pH 1.0 colorless, (ii) pH 1.2 greenish blue and (iii) pH 2.2
yellow. (D) Absorbance at 655 nm of different reaction components: (i) 3,3ʹ,5,5ʹ-tetramethylbenzidine nanoconjugates (TMB-NC), (ii) H2O2-NC, (iii) TMB-H2O2 and (iv)
folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs), TMB and H2O2.
achieve optimum pH conditions. Thus, the results showed of FA–CurAu-PVP NCs (IC50: 50 µg/mL) in both human
that the catalytic activity was faster in acidic pH solution and and mouse mammary cancer cells (Figure 6A–C).
this may be due to the presence of enough positive charges on Moreover, incubation of normal cell lines (L929 and
the NCs only in acidic conditions. As tumor tissues have a MCF 10A) with FA–CurAu-PVP NCs demonstrated low
more acidic environment than normal tissues, this peroxi- cytotoxicity up to 50 µg/mL dose compared to cancer cells
dase-mimetic activity of the NC could be beneficial for (Figure 6D and E). However, higher doses (100 and
intelligent cancer treatments, which would not only kill 200 µg/mL) showed significant cytotoxicity in both can-
tumor cells by producing OH radicals but also relieve oxida- cerous and normal cells. Hence, the data suggest that NCs
tive stress in normal cells by reducing the H2O2 level.6 can exhibit anticancer activity at low doses without harm-
ing normal cells. NCs exhibited a higher inhibitory effect
In vitro anticancer activity in estrogen receptor (ER)/progesterone receptor (PR)-
To examine the in vitro anticancer activity of NCs, the negative breast cancer cells (MDA-MB-231 and 4T1)
MTT assay was performed as described previously.41 compared to ER/PR-positive breast cancer cells (MCF-7).
Incubation of cancer cells (MDA-MB-231, MCF-7 and To further confirm our findings, we performed cell-cycle
4T1) with the NCs showed the efficient antitumor effect analysis in MDA-MB-231 cells using flow cytometry. The

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Figure 6 In vitro antitumor effect of folicacid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) in breast cancer. (A–C) MCF-7, MDA-MB-
231 and 4T1 cells were seeded into 96-well plates and treated with increasing concentrations of FA–CurAu-PVP NCs (0–200 µg/mL) for 24 h. Cell death induced by
NCs was analyzed by MTT assay. The percentage inhibition of cell viability is represented graphically. Values are presented as mean ± SEM of three independent
experiments, (D, E) Mouse fibroblast cells (L929) and Human mammary epithelial (MCF 10A) were seeded into 96-well plates and treated with increasing
concentrations of FA–CurAu-PVP NCs (0–200 µg/mL) for 24 h. Cell death induced by NCs was analyzed by MTT assay. The percentage inhibition of cell viability
is presented graphically.
data showed an increase in the sub-G0 population in NC- PVP NCs (10 mg/kg body weight) and the effect on
treated cells compared to controls, suggesting that NC tumor growth was compared with untreated controls.
treatment induced cell death (Figure S3). Tumor volumes were measured twice a week by
Vernier calipers (Figure 8A). At the end of the experi-
In vitro antimigratory potential of FA– ment, mice were killed; tumors were removed, photo-
graphed and weighed (Figure 8A–D). The data
CurAu-PVP NCs
demonstrated that FA–CurAu-PVP NCs significantly
We further examined the antimigratory property of the
inhibited the 4T1 breast tumor growth compared to
NCs in MDA-MB-231 and 4T1 cells. Cells grown in
controls. However, at the same dose curcumin did not
monolayers were wounded and then treated with different
lead to any significant reduction in tumor growth com-
concentrations of NCs; they were then observed until a
pared to controls. This may be due to low water solu-
specific time-point at which cell death did not occur. As
bility, non-specific targeting and early clearance of
illustrated in Figure 7, the NCs significantly inhibited
curcumin from the body. The above findings suggest
migration of 4T1 and MDA-MB-231 cells even at very
that FA–CurAu-PVP NCs enhanced the efficacy of cur-
low doses (Figure 7A and B), proving their antimetastatic
cumin at a dose as low as 10 mg/kg body weight.
potential.
Discussion
In vivo antitumor efficacy of FA–CurAu- Nanotechnology has immense potential to produce major
PVP in breast cancer advances in areas of enzyme immobilization, energy, food,
To examine the antitumor effect of FA–CurAu-PVP NCs agriculture and medicine.42 Nanotechnology-based deliv-
under in vivo conditions, 4T1 mouse mammary tumors ery systems can overcome shortcomings such as drug
were generated in Balb/c mice. After tumor generation, solubility, permeability and early clearance issues asso-
mice were treated with free curcumin and FA–CurAu- ciated with small molecules and drugs, and can provide

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Figure 7 Folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) inhibit migration of breast cancer cells. (A) Confluent monolayer of MDA-
MB-231 cells wounded with constant width and treated with FA–CurAu-PVP NCs (0–20 µg/mL) for 18 h. Photographs of wounds were taken at T=0 and 18 h. (B)
Confluent monolayer of 4T1 cells wounded with constant width and treated with FA–CurAu-PVP NCs (0–10 µg/mL) for 6 h. Photographs of wounds were taken at T=0 and
6 h. Migrated distances were measured using Image-Pro Plus software and analyzed statistically and presented graphically using SigmaPlot software. Values are presented as
mean ± SEM of three independent experiments.
smart therapeutic solutions such as tissue-specific che- degradation of curcumin was observed to be reduced in
motherapy and phototherapy.43–46 the presence of a serum protein in a buffer solution, which
In this study, an innovative targeted drug-delivery vehi- also proves that HS acts as an efficient transporter for the
cle (FA–CurAu-PVP NC) with a pH-responsive drug- curcumin molecule. FA–CurAu-PVP NCs showed
release system, favorable cytocompatibility and folate enhanced peroxidase-like activity in the presence of perox-
receptor-mediated targeting is formulated via a simple cova- idase substrate TMB with H2O2 as a catalyst under a
lent cross-linking method for cancer therapy. In the present standard pH condition of 1.2.
study, folate-targeted nanoconjugates of CurAu-PVP NPs The MTT assay and flow cytometry studies indicated the
were synthesized successfully for the delivery of curcumin. higher potential of folate-modified NCs in the inhibition of
Curcumin-loaded optimized FA–CurAu-PVP NCs were breast cancer cell proliferation and migration in vitro. Further,
spherical, with a mean particle size of approximately 250 the data demonstrated a higher cytotoxicity toward ER/PR-
nm estimated by DLS and TEM, a zeta potential of −12.5, a negative compared to positive cells. This may be due to the
narrow size distribution of 0.6 and curcumin DLE of differential expression of FA receptors on ER/PR-negative and
40 µg/mL. The NCs showed a sustained release behavior positive cells. It has been reported that there is a higher
during 48 h in two different pH conditions. In this study, we expression of folate receptors in triple-negative breast cancer,
also observed an increase in the size of AuNPs when justifying the higher anticancer activity of FA–CurAu-PVP in
incubated with HS. The adsorbed serum proteins decreased triple-negative breast cancer cells.47,48 These findings thereby
the absolute charge on the particles and kept them stable, as establish the fact that the synthesized FA–CurAu-PVP NCs
proven by zeta potential and UV–visible spectra analysis. showed prominent cytotoxicity in FA receptor-expressing
According to previous investigations and the literature, breast cancer cells at doses significantly lower than the doses

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Figure 8 Folic acid–curcumin–gold–polyvinylpyrrolidone nanoconjugates (FA–CurAu-PVP NCs) suppress breast cancer growth under in vivo conditions. (A, B) 4T1 mouse
mammary cells were injected orthotopically into Balb/c mice and then 10 mg/kg body weight of curcumin and FA–CurAu-PVP NCs were injected intratumorally twice a
week for 2 weeks. Tumor volumes were measured twice a week using Vernier calipers, analyzed statistically and represented graphically (mean ± SEM, n=5; *p<0.006
compared to control tumor, **p<0.05 compared to curcumin-treated tumor). (C, D) Tumors were excised, photographed, weighed and analyzed statistically. The bar graph
represents mean tumor weights (mean ± SD, n=5; *p<0.05 compared to control).
at which they developed toxicity in normal cells (MCF 10A cancer.49 During the development of most human cancers,
and L929). The cytotoxic nature of these NCs can also be primary cells move out and invade the neighboring tissues
explained by their enhanced cellular uptake via FA-mediated and ultimately travel to distant sites to make new colonies,
endocytosis.5 The ability of cancer cells to undergo migration leading to 90% of human cancer deaths. Hence, targeting
and invasion has been designated as one of the hallmarks of cancer cell migration and invasion is an important aspect of

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cancer chemotherapy.41,50 Our data showed that FA–CurAu- 6. Anand P, Kunnumakkara AB, Newman RA, Aggarwal BB.
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We would like to acknowledge the Department of Science 17. Das M, Mohanty C, Sahoo SK. Ligand-based targeted therapy for
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37. Patel A, Hu Y, Tiwari JK, Velikov KP. Synthesis and characterization Med. 2013;138:890–895. doi:10.5858/arpa.2013-0309-OA
of zein-curcumin colloidal particles. Soft Matter. 2010;6:6192–6199. 49. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation.
doi:10.1039/c0sm00800a Cell. 2011;144:646–674. doi:10.1016/j.cell.2011.02.013
38. Dey S, Sreenivasan K. Conjugating curcumin to water soluble poly- 50. Yadav AS, Pandey PR, Butti R, et al. The biology and therapeutic
mer stabilized gold nanoparticles via pH responsive succinate linker. implications of tumor dormancy and reactivation. Front Oncol.
J Mater Chem. 2015;3:824–833. doi:10.1039/C4TB01731E 2018;8:72. doi:10.3389/fonc.2018.00072

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Supplementary materials
Figure S1 UV-Visible spectra representing activation of folic acid in o folate.

A) B)
Figure S2 (A) EDS graph of AuNP, (B) EDS graph of FaCur @AuNC.
Figure S3 Effect FA@CurAu-PVP treatment on cell cycle in MDA-MB-231 cells was analyzed by flow cytometry. Briefly, 2×105 cells were seeded in 60 mm dish and treated
with 20 and 50 µg/ml of FA@CurAu-PVP for 24 hrs. Cells were stained with propidium iodide and analyzed by FACSCalibur (BD Bioscieces).

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Table S1 Hydrodynamic size (Dh), polydispersity index (PDI) and zeta potential of gold nanoparticles (AuNPs) and successively
functionalized AuNPs, curcumin–gold polyvinylpyrrolidone nanoparticles (CurAu-PVP NPs) and folic acid–curcumin gold–polyvinyl-
pyrrolidone nanoparticles (FA–CurAu-PVP NPs) at physiological pH
Sr. no. Formulation Dh (nm) PDI Zeta potential (mV)
1 AuNP 36.85 0.296 −7.47

2 AuPvP 62.50 0.338 −9.91
3 CurAu-PVP NP 72.56 0.582 −10
4 FA–CurAu-PVP NC 358.7 0.6 −12.5
Table S2 Summary of size (dynamic light scattering), polydispersity index (PDI) and charge of gold–polyvinylpyrrolidone (Au-PVP)
functionalized nanoparticles (NPs), curcumin–gold (CurAu NPs) and folic acid–curcumin gold (FA–CurAu) NPs before and after
incubation with human serum
Formulation DLS: hydrodynamic diameter (nm) PDI Zeta potential (mV)
Au-PVP NPs before incubation 62.5 0.338 −9.91

Au-PVP NPs after incubation 559 0.239 −3.82
CurAu NPs before incubation 72.5 0.582 −10.1
CurAu NPs after incubation 320 0.393 −5.09
FA–CurAu NPs before incubation 358 0.6 −12.5

FA–CurAu NPs after incubation 657 0.393 −3.74
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Open Access Full Text Article

Functional design of pH-responsive folate-

drug delivery and in vivo therapy in breast cancer

Suresh W Gosavi Introduction

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N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydro- Synthesis of bare AuNPs and PVP-coated

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Thermogravimetric analysis (TGA) was carried out Protein adsorption studies

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23.33, 24.60 and 25.52 degrees (Figure 1C, inset).

(TGA), was used. As shown in the thermogram in

CurAu-PVP NPs showed one step degradation at 1250C

PVP NCs, which are slightly more stable than CurAu-PVP

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in the spectra of FA–CurAu-PVP NC as well (Figure 4A).6

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targets in cancer chemoprevention by curcumin. Aaps J. 2006;8:

14. Siﬂinger-Birnboim A, Del Vecchio PJ, Cooper JA, Blumenstock FA,

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cancer cells. Bioorg Med Chem. 2010;18:5528–5534. doi:10.1016/j. doi:10.1002/adfm.v29.26

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Figure S1 UV-Visible spectra representing activation of folic acid in o folate.

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1 AuNP 36.85 0.296 −7.47

4 FA–CurAu-PVP NC 358.7 0.6 −12.5

Au-PVP NPs before incubation 62.5 0.338 −9.91

FA–CurAu NPs before incubation 358 0.6 −12.5

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