Alginate stabilized gold nanoparticle as multidrug carrier: Evaluation of cellular interactions and hemolytic potential

Article abstract of DOI:10.1016/j.carbpol.2015.09.016

This work delineates the synthesis of curcumin (Ccm) and methotrexate (MTX) conjugated biopolymer stabilized AuNPs (MP@Alg-Ccm AuNPs). The dual drug conjugated nano-vector was characterized by FTIR, ¹H NMR and UV-vis spectroscopic techniques. H

Full text of DOI:10.1016/j.carbpol.2015.09.016

Carbohydrate Polymers 136 (2016) 71–80
Contents lists available at ScienceDirect
Carbohydrate Polymers
journal homepage: www.elsevier.com/locate/carbpol
Alginate stabilized gold nanoparticle as multidrug carrier: Evaluation
of cellular interactions and hemolytic potential
a
b
b
a,∗
Soma Dey , M. Caroline Diana Sherly , M.R. Rekha , K. Sreenivasan
a
Laboratory for Polymer Analysis, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojapura,
Trivandrum 695012, India
Division of Biosurface Technology, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Poojappura,
b
Trivandrum 695012, Kerala, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 2 March 2015
Received in revised form 27 August 2015
Accepted 7 September 2015
Available online 9 September 2015
This work delineates the synthesis of curcumin (Ccm) and methotrexate (MTX) conjugated biopoly-
mer stabilized AuNPs (MP@Alg–Ccm AuNPs). The dual drug conjugated nano-vector was characterized
by FTIR, H NMR and UV–vis spectroscopic techniques. Hydrodynamic diameter and surface charge
1
of the AuNPs were determined by DLS analysis and the spherical particles were visualized by TEM.
MP@Alg–Ccm AuNPs exhibited improved cytotoxic potential against C6 glioma and MCF-7 cancer cell
lines and was found to be highly hemocompatible. MP@Alg–Ccm AuNPs also exhibited active targeting
efficiency against MCF-7 cancer cells due to the presence of “antifolate” drug MTX. Thus MP@Alg–Ccm
AuNPs may find potential application in targeted combination chemotherapy for the treatment of cancer.
The study is also interesting from the synthetic point of view because, here generation of AuNPs was done
using “green chemical” alginate and dual drug conjugated AuNPs were created in two simple reaction
steps using “green solvent” water.
Keywords:
Curcumin
Alginate
Polymer–drug conjugates
Methotrexate
Gold nanoparticles
Confocal laser scanning microscopy
© 2015 Elsevier Ltd. All rights reserved.
1
. Introduction
Ccm can also down regulate the levels of Pgp, ABCG2 and MRP-1
(three major ABC drug transporters that are mainly responsible for
Curcumin (Ccm) is a polyphenolic compound extracted from the
the development of multi drug resistance [MDR] in cancer cells)
and thus it has significant role in controlling MDR (Misra & Sahoo,
2011). However, Application of Ccm as a chemotherapeutic agent
is restricted due to its extremely low aqueous solubility, instabil-
ity and consequent poor bioavailability (Anand, Kunnumakkara,
Newman, & Aggarwal, 2007). In order to redress these problems,
several approaches have been proposed and among them conjuga-
tion of Ccm to hydrophilic polymers is a wise approach. Conjugation
of hydrophobic drugs to suitable polymers augments aqueous sol-
ubility of the drug, ensures unhindered release and offers a chance
to alter drug pharmacokinetics and biodistribution which are basi-
cally useful for those drugs that exhibit rapid metabolism (e.g.
curcumin), faster clearance and/or off target toxicities (anticancer
drugs) (Larson & Ghandehari, 2012). It has been reported that Ccm
can boost the antitumor efficiency of several chemotherapeutic
agents like paclitaxel and doxorubicin (Duan et al., 2012; Ganta
& Amiji, 2009; Manju, Sharma, & Sreenivasan, 2011). A recent
report suggests that curcumin can significantly enhance the uptake
and cytotoxicity of methotrexate (MTX) in KG-1 leukemic cells
rhizome of the plant Curcuma longa (Indian spice turmeric). Within
the last couple of decades, extensive research work has unveiled
a wide range of striking pharmacological activities in Ccm such
as antioxidant, anti-inflammatory, antiproliferative and antiangio-
genic activities (Aggarwal, Kumar, & Bharti, 2003; Aggarwal & Sung,
2
009; Lantz, Chen, Solyom, Jolad, & Timmermann, 2005; Motterlini,
Foresti, Bassi, & Green, 2000; Shi et al., 2006). It has been reported
thatthetranscriptionalnuclearfactorkappabeta(NF␬␤) is thechief
controller of cell proliferation, inflammation, apoptosis and resis-
tance in cells and Ccm inhibits NF-␬␤ resulting in blockage of the
function of protein kinase C, receptor tyrosine kinase, Her-2 and
epidermal growth factor to induce apoptosis. Most interestingly,
development of resistance to Ccm is less likely as it induces apo-
ptosis through multiple cell signaling pathways (Ravindran, Prasad,
&
Aggarwal, 2009). In cancer therapy Ccm can exhibit pleiotropic
therapeutic effect because of its ability to inhibit multiple levels
of diverse cell signaling pathways (Maher et al., 2011). Moreover,
(Dhanasekaran, Biswal, Sumantran, & Verma, 2013).
∗ Corresponding author.
E-mail address: sreeni@sctimst.ac.in (K. Sreenivasan).
MTX, an antimetabolite, is structurally analogous to folic acid
(FA). The difference in structure between FA and MTX is due to
http://dx.doi.org/10.1016/j.carbpol.2015.09.016
0
144-8617/© 2015 Elsevier Ltd. All rights reserved.

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2
S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
the functional group at 4th position of pteridine ring (FA has OH
and MTX has NH2 group). This structural feature of MTX allows
it to bind tightly to dihydrofolate reductase (DHFR) which is a vital
enzyme in folic acid cycle catalyzing the conversion of dihydrofo-
late to active tetrahydrofolate. Thus MTX interrupts DNA and RNA
synthesis by inhibiting DHFR and induces cellular apoptosis. Hence
the antifolate drug can play the role of a chemotherapeutic agent.
In addition to this, being a FA analog MTX has been established
to be an efficient ligand for active targeting (Dhar, Liu, Thomale,
Dai, & Lippard, 2008; Kohler, Sun, Wang, & Zhang, 2005). The main
drawback associated with MTX is its cellular efflux and consequent
resistance to the drug developed in the targeted cells. In order to
offset this fact, MTX conjugates with suitable polymers such as
poly(ethyleneglycol), hyaluronic acid and poly (glutamic acid) have
been developed (Piper, McCaleb, & Montgomery, 1983; Riebeseel
et al., 2002).
The conventional drug delivery systems (DDS) possess several
limitations like poor bioavailability, non-specific biodistribution,
lack of targeting, side effects and low therapeutic indices. Nan-
otherapeutics has the potential to surmount these limitations
to a large extent (Malam, Loizidou, & Seifalian, 2009; Moorthi,
Manavalan, & Kathiresan, 2011). Among various nanotherapeutics,
gold nanoparticles (AuNPs) have been widely used as the potential
drug delivery vehicle (Boisselier & Astruc, 2009). Facile synthetic
procedure, convenient surface modification, excellent stability and
low cytotoxicity are some of the distinctive attributes which have
made AuNPs a subject of intense research (Daniel & Astruc, 2004;
Giljohann et al., 2010). Polymeric nanoparticles (micelles, vesi-
cles), liposomes are also widely studied for delivery of hydrophobic
drugs. Compared to those nanoparticles, functionalized AuNPs with
much smaller size are advantageous for passive tumor targeting
via enhanced permeation and retention (EPR) effect along with
reduced clearance through reticuloendothelial system (RES) (Gref
et al., 1994; Maeda, Wu, Sawa, Matsumura, & Hori, 2000). Thus
AuNPs functionalized with suitable targeting ligands can serve as
excellent vehicles for hydrophobic drugs and other bioactive agents
with augmented longevity in blood stream and improved uptake by
cells due to both EPR effect and targeting ligand mediated endocy-
tosis (Manju & Sreenivasan, 2012). However, in most of the cases
AuNPs containing manifold functionalities (e.g., various drugs and
targeting ligands) are developed following multiple synthetic steps
and using hazardous organic solvents. Often rigorous purification
steps are employed in designing such type of AuNPs based nano-
vectors. All these can have adverse effect on the stability of the
nano-vectors.
Herein we report the fabrication of a double drug containing
biopolymer stabilized AuNPs via green synthetic route and fol-
lowing only two simple reaction steps. Recently we developed
alginate–curcumin (Alg–Ccm) conjugate (Dey & Sreenivasan, 2014)
in our laboratory. The study showed that Alg–Ccm enhances aque-
ous solubility, stability and therapeutic efficacy of Ccm. In the
present study we simultaneously generated and stabilized AuNPs
using the Alg–Ccm conjugate in aqueous medium under thermal
activation. Here we hypothesized that alginate possessing sec-
ondary hydroxyl groups can reduce Au(III) to Au(0). Besides, the
structure of alginate being oxygen rich, it can effectively cap the
new born AuNPs and protect them from aggregation. With an aim
to redress the efflux of MTX from cancer cells, we covalently con-
jugated MTX to bis(aminopropyl) terminated poly(ethylene glycol)
in aqueous buffer solution (of pH 7.4) to get MP conjugate which
was subsequently conjugated onto the Alg–Ccm AuNPs in aque-
ous medium to get the hybrid nano-structured DDS MP@Alg–Ccm
AuNPs. Alginate, present in brown algae and bacteria, is consid-
ered as a “green chemical” (Yang, Ren, & Xie, 2011). The entire
synthetic work was done using “green solvent” water and dou-
ble drug conjugated biopolymer stabilized AuNPs were designed
through two facile reaction steps. The cytotoxic potential of the dual
drug conjugated DDS was evaluated against C6 glioma (from rat
brain tumor) and MCF-7 (human breast cancer) cells. Facile uptake
of MP@Alg–Ccm AuNPs by the cancer cell lines was confirmed by
both fluorescence microscopic and confocal laser scanning micro-
scopic imaging. Percentage of hemolysis caused by the DDS was
also studied to assess the blood compatibility of the DDS.
2. Experimental
2.1. Materials
Tetrachloroauric acid(III) trihydrate (HAuCl ·3H O), 1,3-
4
2
dicyclohexylcarbodiimide
(DCC),
4-dimethylaminopyridine
(
DMAP),
hydrochloride
aminopropyl) terminated polyethyleneglycol (Bis(aminopropyl)
N-(3-dimethylaminopropyl)-N-ethylcarbodiimide
(EDC), N-hydroxysuccinimide (NHS), Bis
(
terminated PEG), Methotrexate hydrate (MTX) were purchased
from Sigma–Aldrich (Bangalore, India). Curcumin (95% total cur-
cuminoid content) from turmeric rhizome was obtained from Alfa
Aesar (Bangalore, India). Sodium alginate was purchased from SD
fine chemicals (Mumbai, India). The average molecular weight of
5
Sodium alginate used in this study was found to be 4 × 10 g/mol
by GPC analysis. Sodium poly(guluronate) (G-block) was isolated
according to the procedure by Bouhadir, Hausman and Mooney
(1999) (the procedure is mentioned in brief in Supplementary
Information). GPC analysis of the G-block resulted in polymer
with significantly lower molecular weight of 10,281 g/mol. The
molecular weight of guluronic acid and mannuronic acid in its
deprotonated form (C H7O ) is 176.1 g/mol. Hence there are
6
6
almost (10,281/176.1) 58 units of guluronic acids in the G-block
polymer isolated from sodium alginate.
Dimethyl sulfoxide (DMSO) and ethanol (EtOH) were obtained
from Merck (Mumbai, India). Ultra pure water (18.2 mꢀ resistivity)
was obtained from a Mili-Q water purification system. Deionized
water was used all through the reaction and purification steps in
this study.
C6 (Glial cells from rat Glioma) and MCF-7 (human breast can-
cer cells) cell lines were obtained from the National Centre for
Cell Sciences (NCCS), Pune, India. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyl tetrazolium bromide (MTT reagent), fetal bovine serum
(FBS), minimum essential medium (MEM), Dulbecco’s Modified
Eagle’s Medium (DMEM)/Nutrient F-12 Ham and Trypsin/EDTA
were purchased from Sigma-Aldrich (Bangalore, India). RPMI 1640
medium, Hoechst 33342 and Fluorescence isothiocyanate (FITC)
were purchased from Invitrogen (Bangalore, India).
2.2. Synthesis of Alg–Ccm conjugate and generation of Alg–Ccm
AuNPs
The Alg–Ccm conjugate was prepared according to our previous
report (Dey & Sreenivasan, 2014). In brief, hydrophobic drug cur-
cumin was directly covalently conjugated to the C-6 carboxylate
functional group of biopolymer alginate via esterification reac-
tion using DCC/DMAP in H O-DMSO medium. The conjugate was
2
purified by dialysis (using a dialysis membrane of MWCO 3500)
against DMSO for one day and against H O for three days to remove
2
any unreacted molecules. Alg–Ccm conjugate was lyophilized and
stored in the refrigerator for further applications.
AuNPs were generated and stabilized by the Alg–Ccm conjugate
in aqueous medium under thermal activation. An aqueous solution
of HAuCl , 3H O (1 mM) was slowly added to the aqueous solution
4
2
of Alg–Ccm conjugate (0.41 mg/mL) to maintain a final concen-
−
2
tration of 10 M of chloroauric acid in the solution. After proper
mixing the solution was heated gradually in a water bath and the

S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
73
generation of AuNPs was indicated by a color change of the solu-
tion from yellow to pinkish red. The AuNPs solution was cooled to
room temperature and dialyzed against deionized water (using a
dialysis membrane of MWCO 3500) for 1 day. Finally the solution
was lyophilized and stored in refrigerator.
was collected. Using this supernatant solution, total Ccm content
was determined by UV–vis spectroscopy (at ꢂmax = 428 nm).
2.6. Evaluation of cytotoxicity
The quantitative cytotoxic potential of MP@Alg–Ccm AuNPs
was determined using two cancer cell lines: (i) C6 cancer cell
and (ii) MCF-7 cancer cells. For the study against C6 cells, cells
were maintained in 50:50 mixture of Dulbecco’s Modified Eagle’s
Medium/Nutrient F-12 Ham and MEM supplemented with 10% FBS.
Then 80% confluent cells were trypsinized and seeded in 96 well
2
.3. Synthesis of MTX conjugated Alg–Ccm AuNPs (MP@Alg–Ccm
AuNPs)
In order to develop MTX conjugated Alg–Ccm AuNPs, at
first MTX was conjugated to Bis (aminopropyl) PEG to generate
MTX–PEG conjugate (MP conjugate). For MP conjugate synthesis,
MTX was dissolved in PBS (pH 7.4) and was activated using EDC and
NHS (MTX:EDC:NHS = 1:1.3:1.1 molar ratio) at room temperature
for one hour under the blanket of N2 (g) in dark. Then solution of
bis(aminopropyl) PEG (MTX:PEG = 1:1 molar ratio) in PBS (pH 7.4)
was gradually added to the activated MTX solution and the mixture
was allowed to stir at room temperature for around four hours in
dark. Next, the MP conjugate was dialyzed (using dialysis mem-
brane of MWCO 1000) against PBS (pH 7.4) for one day and against
3
plates (1 × 10 cells/well) and incubated for 24 h. Then the cells
were exposed to a series of doses of MP@Alg–Ccm AuNPs, free MTX
◦
and free Ccm in a CO2 (5%) incubator at 37 C. After 24 h incuba-
tion, the medium containing sample and free drug was removed
from respective wells and 200 ␮L of freshly prepared MTT solution
(0.5 mg/mL) in culture medium was added into each well. After 4 h
incubation, MTT solution was carefully removed. DMSO (200 ␮L)
was then added into each well and the plate was gently shaken for
10 min at room temperature to dissolve all precipitates formed. The
absorbance of individual wells at 570 nm was then detected by a
microplate reader (Tecan Infinite M200, Switzerland). Cell viability
was expressed as the mean percentage of sample absorbance rel-
ative to untreated cells (control) as shown in the equation below
(where As is the absorbance of sample and Ac is the absorbance of
control). Here each reported value is the mean of three replicates.
distilled H O for one day and was dried by lyophilization.
2
In the second step, MP conjugate was conjugated to the Alg–Ccm
AuNPs utilizing the EDC chemistry. The free carboxyl functional-
ity on Alg–Ccm AuNPs (15 mg) was activated with EDC/NHS (in
aqueous carbonate/bicarbonate buffer medium with pH 8.2) for one
◦
hour at 25 C followed by the addition of aqueous solution of MP
conjugate (25 mg) and the reaction mixture was moderately stirred
overnight at 25 C. The reaction mixture was then dialyzed (MWCO
As
◦
Cell viability (%) =
× 100
Ac
3
500) and lyophilized to get MP@Alg–Ccm AuNPs.
For the study with MCF-7 cells, the cells were maintained in
RPMI Medium. Then 80% confluent cells were trypsinized and
2
.4. Physicochemical characterizations of Alg–Ccm AuNPs and
3
MP@Alg–Ccm AuNPs
seeded in 96 well plates (1 × 10 cells/well) and incubated for 24 h.
Then similar procedure was adopted as described for C6 cell lines.
The generation and stabilization of AuNPs using Alg–Ccm
conjugate and its functionalization with MP conjugate to pro-
duce MP@Alg–Ccm AuNPs were characterized by analyzing the
surface plasmon resonance (SPR) absorption spectra of the respec-
tive AuNPs using Ultraviolet–Visible (UV–Vis) spectrophotometer
2.7. Cellular uptake study
Cellular images were acquired with a fluorescence microscope
(Leica DM IRB, Germany) using C6 and MCF-7 cell lines. Cells were
seeded and incubated on a 4-well plate at 37 C for 24 h. Then both
◦
(
Carry model 100 bio UV–Vis spectrophotometer, Melbourne,
Australia). Generation of Alg–Ccm AuNPs was verified by compar-
ing its X-ray diffraction pattern (Bruker D8 Advance; equipped with
Cu K␣ radiation source) with that of Alg–Ccm conjugate. The Fourier
transform infrared spectra were recorded using a Nicolet 5700 FTIR
C6 and MCF-7 cells were exposed to FITC tagged MP@Alg–Ccm
AuNPs. After incubation for 2.5 h, Hoechst 33342 solution (15 ␮L
of concentration 10 ␮M) was added to each well for staining the
nucleus. Then, after 30 min of incubation, the medium containing
AuNPs sample and Hoechst was removed from each well and the
cells were washed with PBS (twice) to remove any non-specific
binding. After fixing the cells, uptake was detected in fluorescence
microscope.
−
1
spectrometer in the range of 4000–500 cm
modification. H NMR spectra of Alg–Ccm AuNPs and MP@Alg–Ccm
AuNPs were analyzed by 500 MHz spectrometer (Brucker Avance
after each step of
1
DPX 500) using D O as the solvent. The hydrodynamic diameter of
2
the AuNPs was determined by dynamic light scattering instrument
In order to confirm the targeting efficiency of MTX, FITC was
tagged at the same density on MP@Alg–Ccm AuNPs and Alg–Ccm
AuNPs. MCF-7 cells were treated with same concentration of FITC
labeled MP@Alg–Ccm AuNPs and FITC labeled Alg–Ccm AuNPs. The
cellular uptake in each case was studied following the same proce-
dure as mentioned above.
(
DLS, Malvern Zetasizer Nano ZS, UK) with a He–Ne laser beam at
a wavelength of 633.8 nm. To get an idea about the stability of the
◦
functionalized AuNPs, zeta potential (ꢁ, at 25 C and pH 7.4) was
determined using the same instrument. The spherical morphology
of the AuNPs was visualized by Transmission Electron microscopy
(
TEM; Hitachi H-7650; Tokyo, Japan). Sample for TEM analysis was
Cellular uptake in both C6 glioma and MCF-7 cell lines was con-
firmed by confocal laser scanning microscopic images recorded in
Nicon A1R.
prepared by depositing a drop of nanoparticle suspension on a 200
mesh copper TEM grid with formvar film and air dried at room
temperature.
2.8. Evaluation of hemolytic toxicity
2.5. Determination of drug content in MP@Alg–Ccm AuNPs
The hemolytic toxicity of MP@Alg–Ccm AuNPs was determined
The total MTX content in MP@Alg–Ccm AuNPs was determined
by evaluating the percentage of hemolysis. Blood was drawn from a
healthy unmedicated human donor and collected in anticoagulant
ACD. MP@Alg–Ccm AuNPs was placed in polystyrene culture plates
and agitated with PBS before they are exposed to blood. To each
plate 10 mL blood was added (to maintain sample concentration of
0.5 mg/mL). 5 mL blood was immediately taken for initial analysis
by UV–vis spectroscopy (at ꢂmax = 369 nm) in PBS (pH 7.4). In order
to determine the total Ccm content in MP@Alg–Ccm AuNPs, a
known amount of fridge-dried sample was dispersed in EtOH and
◦
incubated for 24 h (at 37 C and 120 rpm). After that the dispersion
was centrifuged (at 14,000 rpm for 15 min) and the supernatant

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4
S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
and remaining 5 mL blood (with sample) was incubated for 30 min
AuNPs were further fabricated with another anticancer drug MTX
to generate MP@Alg–Ccm AuNPs. Most interestingly, the dual drug
conjugated polymer functionalized AuNPs were developed follow-
ing a green synthetic route as depicted in Scheme 1 without using
any hazardous organic solvent. We have recently developed the
water soluble Alg–Ccm conjugate (Dey & Sreenivasan, 2014). AuNPs
were generated using the aqueous solution of Alg–Ccm conju-
gate under thermal activation. Here alginate played the role of
both reducing and stabilizing agent. In order to develop a dual
drug delivery vehicle with improved targeting ability, antifolate
drug MTX was also covalently conjugated to the Alg–Ccm AuNPs.
MTX serves not only as a chemotherapeutic agent; being an ana-
log of folic acid it also plays the role of a targeting ligand. In
this study MTX was first conjugated to bis(aminopropyl) termi-
nated PEG to produce MTX-PEG conjugate (MP conjugate; see
the inset in Scheme 1) in aqueous buffer solution. The MP con-
jugate was eventually conjugated to Alg–Ccm AuNPs following
EDC chemistry to generate MP@Alg–Ccm AuNPs (as shown in
Scheme 1).
under agitation at 70 ± 5 rpm using Environ shaker thermostated at
◦
3
5 ± 2 C. The total hemoglobin (Hb) in the initial blood sample was
measured using automatic hematology analyzer (Sysmex-K4500).
The free Hb liberated into the plasma after exposure to the samples
was estimated in each sample by measuring absorbance of diluted
plasma with diode array spectrophotometer (HP 8453, Hewlett-
Packard GmbH/Germany). Percentage of hemolysis was calculated
from the following formula:
Free Hb
Total Hb
%
Hemolysis =
× 100
Here reported value is the mean of three replicates.
3
. Results and discussion
3.1. Synthesis and physicochemical characterization of Alg–Ccm
AuNPs and MP@Alg–Ccm AuNPs
Generation and stabilization of AuNPs by Alg–Ccm conjugate
was proved by the distinctive SPR absorption of Alg–Ccm AuNPs
at ∼ 525 nm (Fig. 1A). It is clear from Fig. 1A that the charac-
teristic absorbance of curucmin (at around 427 nm in Alg–Ccm)
was masked by the strong SPR band of Alg–Ccm AuNPs. How-
ever, the sharp peak at 280 nm evidently indicated the capping of
AuNPs by Alg–Ccm conjugate. In Fig. 1B the X-ray diffractogram of
both Alg–Ccm conjugate and Alg–Ccm AuNPs are shown. The X-ray
diffractogram of the conjugate indicated almost amorphous or dis-
ordered crystalline phase. However, the XRD pattern of Alg–Ccm
Among the nanotherapeutics, suitably functionalized AuNPs are
widely used for drug delivery applications. Different types of reduc-
tants and capping agents have been used for the generation and
stabilization of AuNPs with desired size and shape. Most of the
studies describe multiple steps for the generation of drug conju-
gated polymer functionalized AuNPs. Besides, hazardous organic
solvents are usually employed for conjugating hydrophobic drugs
onto the surface of functionalized AuNPs. In this study water soluble
curcumin conjugated biopolymer stabilized AuNPs were fabri-
cated in a single step using Alg–Ccm conjugate and the Alg–Ccm
Scheme 1. Schematic presentation for the fabrication of MP@Alg–Ccm AuNPs via green route (inset: synthetic route to develop MP conjugate) (For interpretation of the
references to colour in this scheme legend, the reader is referred to the web version of this article.).

S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
75
Fig. 1. (A) SPR absorption of Alg–Ccm AuNPs (inset: picture of the same in the visible range) and (B) XRD patterns of Alg–Ccm conjugate and Alg–Ccm AuNPs.
AuNPs showed distinct Bragg reflections corresponding to (1 1 1),
2 0 0), (2 2 0) and (3 1 1) Miller indices. Thus the diffractogram con-
firmed face centered cubic (FCC) crystalline geometry of the AuNPs
formed using Alg–Ccm conjugate.
is due to the ester
spectrum of MP@Alg–Ccm AuNPs (in Fig. 3B), the appearance of
new peak at around 1409 cm clearly indicates the presence of
MTX and the new peak at 1550 cm (due to combination of N
C O vibration in the conjugate. In the FTIR
(
−
1
−
1
H
The formation of MP conjugate was confirmed by FTIR and
UV–vis spectroscopic analyses and the results are detailed in the
Supplementary Information (Fig. S1 and S2).
stretch and N H bending) supports the formation of amide link-
age. Formation of both Alg–Ccm AuNPs and MP@Alg–Ccm AuNPs
was confirmed by H NMR spectra of both the systems as shown in
1
Shift in the SPR absorption of AuNPs is an indication of surface
modification of the NPs (Liang et al., 2010). In MP@Alg–Ccm AuNPs
the SPR band was red-shifted to 539 nm (Fig. 2). Chemical adsorp-
tion of MP conjugate onto the surface of Alg–Ccm AuNPs caused
perturbation in the dielectric constant of the medium surrounding
the NPs resulting in a red-shift of the SPR band. In the absorption
spectrum of MP@Alg–Ccm AuNPs a distinct peak at around 369 nm
Fig. 4.
Fig. 4A shows the 1H NMR spectrum of Alg–Ccm AuNPs in
which the methine protons of hexuronic acid residues of algi-
nate appeared at ı ≈ 3.5–5 ppm and the peaks at ı 6.9 and 7.9 are
due to the aromatic protons of Ccm. However, the chemical shift
due to the characteristic OCH3 protons of Ccm probably merged
with the chemical shift of C-5 methine proton of mannuronic
acid (M) unit of alginate (ı ≈ 3.81 ppm). In Fig. 4B, the multiple
peaks at ı ≈ 1.7–3.8 ppm were observed due to the protons from
bis(aminopropyl) terminated PEG unit. Some of the peaks merged
with that of alginate protons. However, the peaks that appeared
in the range of ı ≈ 6.9–8.7 ppm can be attributed to the aromatic
protons of both Ccm and MTX.
(
see the inset of Fig. 2) further indicated the presence of MTX in
MP@Alg–Ccm AuNPs.
In order to thoroughly characterize the chemical structure of
drug conjugated AuNPs, FTIR spectra of the AuNPs were recorded
after each stage of modification. Fig. 3A and B represents the spectra
of Alg–Ccm AuNPs and MP@Alg–Ccm AuNPs respectively. In Fig. 3A,
−
1
the peak at 3437 cm is assigned to the OH stretching vibration
The sizes and zeta potential values for the aqueous suspension
of Alg–Ccm AuNPs and MP@Alg–Ccm AuNPs determined by DLS are
tabulated in Table 1. The hydrodynamic diameter of MP@Alg–Ccm
AuNPs being small enough (∼187 nm), the DDS can be considered
as a suitable candidate for entering into the cancer cells exploi-
ting the EPR effect. Stability of Alg–Ccm AuNPs and MP@Alg–Ccm
AuNPs in aqueous suspension was evaluated by the zeta poten-
−1
−1
and peaks at around 1650 cm and 1647 cm are attributed to
the stretching vibration of O functionality in Alg–Ccm AuNPs.
According to our hypothesis, alginate reduced Au(III) to Au(0) and
the OH functional group of alginate was oxidized to O func-
tionality and this O stretching vibration appeared as a small
peak at around 1701 cm (Fig. 3A). The small peak at 1211.2 cm
C
C
C
−1
−1
tial measurement. The sufficiently high negative zeta potential
◦
(
ꢃ = −39.5 ± 0.02 mV at pH 7.4 and 25 C) for Alg–Ccm AuNPs indi-
cates potential stability of the nano-system in aqueous medium.
However, the ꢃ-potential value dropped to −25.8 ± 0.05 mV upon
chemisorptions of MP conjugate onto the surface of Alg–Ccm
AuNPs. This decrease in surface charge can be attributed to the
presence of PEG chains (possessing stealth potential) onto the sur-
face of MP@Alg–Ccm AuNPs (Manson, Kumar, Meenan, & Dixon,
2
011). However; reduced negative surface charge was adequate to
impart stability to the NPs as evident from the TEM images of the
well-dispersed particles shown in Fig. 5.
Table 1
DLS data of Alg–Ccm AuNPs and MP@Alg–Ccm AuNPs.
Material
Hydrodynamic diameter
nm)
Zeta potential
(ꢃ, mV)
(
Alg–Ccm AuNPs
MP@Alg–Ccm AuNPs
132 ± 2 (PDI: 0.60)
187 ± 4 (PDI: 0.43)
−39.5 ± 0.02
−25.8 ± 0.05
Fig. 2. SPR absorption of MP@Alg–Ccm AuNPs (inset: picture of the same from 300
to 800 nm range emphasizing the peak at 369 nm).

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S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
Fig. 3. FTIR spectra of (A) Alg–Ccm AuNPs and (B) MP@Alg–Ccm AuNPs.
Fig. 4. 1H NMR spectra of (A) Alg–Ccm AuNPs and (B) MP@Alg–Ccm AuNPs.

S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
77
Fig. 5. TEM images of (A) Alg–Ccm AuNPs and (B) MP@Alg–Ccm AuNPs.
It is well studied that NPs with positive surface charge or
hydrophobic exteriors are prone to plasma protein adsorption and
hence are susceptible to reticuloendothelial system (RES) clear-
ance. As here the negatively charged AuNPs are protected with
hydrophilic, non-immunogenic biopolymer “alginate” and biocom-
patible polymer PEG, hence the particles might escape unwanted
protein adsorption and RES clearance resulting in longevity in sys-
temic circulation and enhanced EPR effect (Aryal, Grailer, Pilla,
Steeberb, & Gong, 2009).
attributed to several factors like the synergistic effect exerted by
the co-administration of MTX and Ccm, pleiotropic effect of Ccm
and improved uptake of the nano-sized drug delivery system (DDS)
by cancer cells. Here it can be reasoned that as MTX was conjugated
with bis(aminopropyl) terminated PEG and then conjugated with
Alg–Ccm AuNPs, hence the efflux of the drug from cancer cells was
probably reduced considerably resulting in improved cytotoxicity
of the DDS than that of the equivalent concentrations of free drugs.
Improvement in the internalization of the DDS may be expected
due to the size in the nm scale and presence of MTX as one of the
drug.
3.2. Drug content and cytotoxic potential of MP@Alg–Ccm AuNPs
From UV–vis spectroscopic study it was revealed that 100 mg
3.3. Cellular uptake study
of MP@Alg–Ccm AuNPs contained 1.05 ± 0.01 mg of Ccm and
1
.26 ± 0.05 mg of MTX. In order to assess the cytotoxic potential
The nano-sized DDS was easily uptaken by both C6 and MCF-7
cancer cells. The bright green intracellular fluorescence observed in
both the cells indicated the facile uptake. MTX uptake is known to
take place through at least two different carrier systems such as (i)
the folate receptors (FR) and (ii) the reduced folate carriers (Kohler
et al., 2005). Here both C6 and MCF-7 cells possess folate receptors
and in addition, MCF-7 contains reduced folate carriers to which
MTX has superior affinity than that of folate receptor (Trippett et al.,
2001; Wang, Zhao, & Goldman, 2004). In Fig. 7(A)–(F) the bright
green intracellular fluorescence from C6 and MCF-7 cells indicating
potential uptake of the DDS by the cancer cells.
of the double drug containing nano-vector, cell viability (%) was
checked against C6 glioma and MCF-7 cancer cell lines after inter-
action with different concentration of MP@Alg–Ccm AuNPs and
thereafter compared with that of each of the free drug of equivalent
concentration.
Fig. 6 shows the cytotoxic activity of MP@Alg–Ccm AuNPs after
interaction with the cancer cells in comparison with free drugs of
equivalent concentrations. For each cell line, MP@Alg–Ccm AuNPs
exhibited improved cytotoxicity compared to the individual drug
(
MTX/Ccm) at all tested concentrations. This observation can be
Fig. 6. Cell viability of (A) glioma cells and (B) MCF-7 cells after being exposed to different concentrations of MP@Alg–Ccm AuNPs in comparison with equivalent amount
of free drugs, where I: 20.5 ␮M MTX + 21 ␮M Ccm in DDS & equivalent free drugs and II: 41 ␮M MTX + 42 ␮M Ccm in DDS & equivalent free drugs. The error bars indicate
mean ± standard deviations and here n = 3.

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S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
Fig. 7. Fluorescence microscopic images after incubation with FITC labeled MP@Alg–Ccm AuNPs for 3 h; glioma cells: (A) fluorescent image, (B) Hoechst 33342 stained
nucleus and (C) merged fluorescent image; MCF-7 cells: (D) fluorescent image, (E) Hoechst 33342 stained nucleus and (F) merged fluorescent image; (G) and (H): merged
fluorescent images of MCF-7 cells after being exposed to FITC labeled MP@Alg–Ccm AuNPs and FITC labeled Alg–Ccm AuNPs, respectively.
In a study by Chen et al. (2013), it has been shown by microscopic
images that a multifunctional fluorescent nanogel based DDS was
uptaken in a higher extent by FR positive MDA-MB-231 cells when
it was decorated with FA and uptake was lower for the DDS without
any FA decoration. Similarly in this study, MCF-7 cells were exposed
to MP@Alg–Ccm AuNPs and Alg–Ccm AuNPs both of which was
tagged with same density of FITC. Here MCF-7 cell line was chosen
for this study as MTX has higher affinity to reduced folate carriers
Fig. 8. Confocal laser scanning microscopic images after incubation with FITC labeled MP@Alg–Ccm AuNPs for 3 h; (I) C6 glioma cells and (II) MCF-7 cells: (A) fluorescent
image, (B) Hoechst 33342 stained nucleus, (C) merged fluorescent image in dark field and (D) merged fluorescent image in bright field.

S. Dey et al. / Carbohydrate Polymers 136 (2016) 71–80
79
that are over expressed in MCF-7 cells. Comparing the merged fluo-
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. Conclusions
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