Ultrafast synthesis of stabilized gold nanoparticles using aqueous fruit extract of Limonia acidissima L. and conjugated epirubicin: Targeted drug delivery for treatment of breast cancer

Article abstract of DOI:10.1039/c6ra01482h

In this study, a green chemistry approach was used for the quick synthesis (within 30 seconds) of gold nanoparticles (AuNPs) by using the fruit extract of Limonia acidissima L. The study focused on the formation of L. acidissima L. stabilized AuNPs withou

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Ultrafast synthesis of stabilized gold nanoparticles using aqueous
fruit extract of Limonia acidissima L and conjugated epirubicin:
Targeted drug delivery for treatment of breast cancer
Received 00th January 20xx,
Accepted 00th January 20xx
a
c
a
b
a,b,*
C.Senthil Kumar , Ayyavu Mahesh , M.Gover Antoniraj , S.Vaidevi and K.Ruckmani
DOI: 10.1039/x0xx00000x
www.rsc.org/
In this study, the green chemistry approach was used for quick synthesis (within 30 seconds) of gold
nanoparticles (AuNPs) by using the fruit extract of Limonia acidissima L. The study focused on the formation
of L. acidissima L stabilized AuNPs without using any catalytic agent. The synthesis of the AuNPs was
confirmed by observation of the surface plasmon resonance (SPR) band at 537 nm. On the surface of these
capped AuNPs, epirubicin (EPI) was conjugated along with activated folic acid (FA) for targeted drug
delivery. The EPIꢀFAꢀAuNPs complex was characterized using FTIR and UVꢀvisible spectrophotometer and
the AuNPs were characterized by HRꢀTEM, particle size analyzer, and zeta potential measurement. In vitro
stability revealed that the AuNPs were stable in physiological conditions. The in vitro cytotoxicity of free EPI
and EPIꢀFAꢀAuNPs was investigated against MCFꢀ7 cells, and the results showed that 50% of EPIꢀFAꢀ
AuNPs was enough to achieve inhibition of growth (IC ) and that the amount was lower than that of free EPI.
50
2
Flow cytometry analysis showed significant reduction in G /M cells after treatment with EPIꢀFAꢀAuNPs, and
molecular levels of apoptosis were studied using western blotting. Over all, the results revealed that the EPIꢀ
FAꢀAuNPs have better regression activity on tumor cells than free EPI.
of the conventional anticancer drugs in order to meet a good
1
. Introduction
1
therapeutic index . To overcome this problem, we have established
a method to synthesize gold nanoparticles (AuNPs) functionalized
While chemotherapy drugs are effective, they, unfortunately, have a
lot of side effects, namely: (i) the conventional cancer drugs are
lethal and cause death of healthy cells as well as cancerous cells; (ii)
the drugs have a short lifetime in the body and up to 90 % of
intravenously delivered drugs may be gathered up by macrophages
within the first 5 min of their entering the body; and (iii) the drugs
have low solubility, and hence, it is necessary to deliver a larger dose
with folic acid (FA) for targeted delivery of epirubicin (EPI), an
2
anthracycline chemotherapy agent . EPI has been clinically used to
treat various types of cancer such as breast cancer, lymphomas,
sarcomas in softꢀtissue, pancreatic cancer, gastric cancer, smallꢀcell
lung cancer, and acute leukemia. EPI shows less hematologic or
3
myocardial toxicity at comparable doses . AuNPs are identified as
favorable candidates for drug delivery applications due to their
unique dimensions, tunable surface functionalities, nontoxicity, and
4
controlled drug release capability . Green chemistryꢀbased ecoꢀ
a. Department of Pharmaceutical Technology, Anna University BIT campus,
Tiruchirappalli-620024, Tamilnadu, India. E-mail:rmani48@gmail.com, : Fax: +91- friendly methods are predominantly used for the synthesis of AuNPs
431-2407333: Tel : +91-431-2459922.
5
instead of chemical synthesis . Plant extracts are used as reducing
b. National Facility for Drug Development for academia, Pharmaceutical and Allied
Industries (NFDD), Anna University, BIT campus, Tiruchirappalli-620024, Tamilnadu,
India.
and stabilizing agents to synthesise the nanoparticles. Plant extracts
contain different concentrations and combinations of organic
reducing agents, which influence the characteristics of the
c. School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, India
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nanoparticles produced . Flavonoids, the plant metabolites, contain Activation and attachment of FA to AuNPs
various functional groups capable of triggering nanoparticle Activation of FA has been reported earlier by Pandey and coworkers
1
5.
formation. It has been suggested that the tautomeric transformation
It was carried out by dissolving 0.25 g of FA in 20 mL of
of flavonoids from the enolꢀform to the ketoꢀform releases a reactive dimethyl sulfoxide (DMSO), and the mixture was then subjected to
7
hydrogen atom that can reduce the metal ions into nanoparticles . sonication for 45 minutes. The carboxylate group of FA was
Fruits of L. acidissima L, contain higher quantities of flavonoids, activated by the addition of 0.225 gm of nꢀhydroxy succinamide
tyramines,
tannins,
phytosterols,
saponins,
glycosides, (NHS) and 0.125 gm of dicarboxy aminocarbodiimide (EDC). The
carbohydrates, vitamins, coumarins, triterpenoids, and amino acids reaction was allowed to take place in an inert environment created
8
o
as their chemical constituents . The targeted drug delivery system by argon gas at 28 C for 12 hours (FA/NHS/EDC molar ratio
uses the anticancer drug for treatment of cancer cells alone, which 2: 2: 1). The resultant mixture was filtered through Whatman filter
reduces the effects of the drug on noncancerous cells and paper and was used for further characterization. Attachment of FA
9
simultaneously increases its efficacy on cancer cells . The main aim was carried out by adding 9 ml of AuNPs to 1 ml of activated FA.
of using the targeted drug delivery system is to deliver the anticancer The solution was purged continuously for 4 hours under N2
1
0,11
drug to the cancerous cells without loss of the drug’s efficacy
.
atmosphere and stirred constantly using a magnetic stirrer. After 4 h,
The folate receptor (FR) is a meticulously studied ligand for the both inlet and outlet valves were sealed, and the solution was
1
2
selective delivery of anticancer drugs on FRꢀpositive tumor cells
.
allowed to rest for 24 h. After 24 h the compound was filtered using
o
In general, the FRs are highly upꢀregulated on the surface of Whatman filter paper and stored at 20 C. Free FA was removed
1
3
different types of malignant cells . In this study, the zebrafish using a 3000 KDa dialysis bag against phosphate buffered saline (pH
embryo has been used as an in vivo prominent vertebrate model for 7.2). The postꢀdialyzed samples were centrifuged at 6000 RPM for
o
assessing the toxicity of AuNPs. The zebrafish offers several 15 min at 20 C. The pellet was redialyzed with deionized water for
advantages as a model for in vivo highꢀthroughput drug screening. It 24 h under constant stirring. The samples were subjected to UVꢀVis
is low cost, transparent, and the human genome and the zebrafish spectroscopy at systematic intervals for examination.
genome are highly comparable in terms of tissue types, fertilization, Synthesis of AuNPs-FA-EPI complex
and the development of different systems and their active functions EPI was conjugated to the AuNPsꢀFA complex for the destruction of
14
.
This article deals with the rapid green chemistryꢀbased synthesis cancer cells. Equimolar concentrations of EPI (0.25mM) and
of AuNPs that can be used as a carrier for EPI, a drug used to treat AuNPsꢀFA (0.25mM) were subjected to reduction with
breast cancer. Zebrafish embryos were used to examine the trimethylamine, 0.5mM (TEA) using DMSO as a solvent. The molar
developmental toxicity of both normal and drug coupled AuNPs. ratio of AuNPs–FA/EPI/TEA was 1: 1: 2. The mixture was purged
o
The efficacy of both EPI and EPIꢀFAꢀAuNPs were tested on the using argon gas with continuous stirring at 50 C for 4 h. The
MCFꢀ7 cell line (breast cancer).
resultant EPIꢀFAꢀAuNPs conjugate was refined using dialysis
against nanoꢀpure water for three days with a dialysis tube (MW cutꢀ
off of 3000 Da) to remove the excess amount of unbound EPI
molecules and DMSO. The water was changed at 6ꢀhour intervals.
The entire compound was characterized using UVꢀVis
spectrophotometry and FTIR.
2
. Experimental methods
Material
L. acidissima L fruit was used for this study. EPI HCl was obtained
from SRL Limited, Mumbai (India). Hydrochloroauric acid
(
HAuCl4), FA, 1ꢀ(3ꢀdimethylaminopropyl)ꢀ3ꢀethylcarbodiimide
Characterization of AuNPs and EPI-FA-AuNPs
hydrochloride (EDC), and 3ꢀ(4, 5ꢀdimethylthiazolylꢀ2)ꢀ2, 5ꢀ
diphenyltetrazolium bromide (MTT) were obtained from Sigmaꢀ
Aldrich Chemicals (USA). MCFꢀ7 cell lines were obtained from
National Center for Cell Science (NCCS), Pune, (India). The
antibody to actin, Bclꢀ2, caspasesꢀ3 and caspasesꢀ8, Fas, FasL, and
FADD bax were purchased from Santa Cruz Biotechnology, CA,
USA and Neo Markers, USA. Millipore milli Q water was used for
all the experiments. All other chemicals and reagents were of
analytical grade.
Preliminary characterization of AuNPs and EPIꢀFAꢀAuNPs was
carried out by FTIR, XRD, HRꢀTEM, particle size and zeta potential
analysis method. LC–MS analysis of the extract was also been
carried out. The detailed procedures are given as supplementary
information.
In vitro stability studies
1
ml of AuNPs was incubated with 0.5 ml each of 0.9 % NaCl saline
and phosphate buffer saline (PBS, pH 1.2, 4.5, 6.8 and 7.4),
2
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respectively, at 37 °C for 48 hours and were analyzed as described in another paper . The detailed methodology
spectrophotometrically by measuring at 480 nm.
descriptions are given in the Supplementary information.
In vivo toxicity study of AuNPs in zebrafish embryos
High content imaging
Fertilized eggs of zebrafish were obtained from the natural mating of Localization of EPIꢀFAꢀAuNPs and the cytotoxic effect were tested
adult zebrafish, and embryos were collected within 2 hours of on MCFꢀ7 cell line. Briefly, 2×104 cells/well of MCFꢀ7 cancer cell
spawning. Six healthy embryos from fertilized eggs, approximately 2 line were seeded on 96 well CellCarrier microplates (PerkinElmer,
hours post fertilization (hpf), were transferred to each well of a 24ꢀ US).When the cells reached 80% confluence, the medium was
well plate containing 1 mL of the E3 medium. The embryos were changed, and then the cells were treated with AuNPs, EPI, and EPIꢀ
exposed to different concentrations of AuNPs for 4 days. Duplicates FAꢀAuNPs, and the plate was incubated for 24 hours in a humidified
were maintained without the AuNPs as control. Toxicity was incubator at 37 °C with 5% CO . The cells were washed twice with
2
assessed by studying the hatching rate, percentage survival rate, and iceꢀcooled PBS, and the drug was localized in live cells using the
morphology changes in the embryos.
Operetta High Content Imaging System (PerkinElmer, US).
Loading efficiency
3
. Results and discussion
From the total concentration of EPI used for loading onto AuNPs,
the unloaded drug was removed by separating the supernatant after
centrifugation (Eppendorf centrifuge 5430R) at 15,000 × g for 15
min. Free EPI present in the supernatant was determined by UVꢀVis
spectrophotometry by measuring at 480 nm.
Characterization of EPI-FA-AuNPs
In the present study, we explain the reducing, stabilizing, and
biocompatible properties of L. acidissima L extract for synthesis of
AuNPs. L.acidissima
L has higher quantity of polyphenolic
compounds that can actively chelate and reduce metal ions to
nanoparticles. It was assumed that the tautomeric conversions of
flavonoids from the enolꢀform to the ketoꢀform may release an
The percentage of drug loading efficiency was calculated
using the following formula (1)
In vitro drug release characteristics
EPIꢀconjugated AuNPs (equivalent to 1000 µg of EPI) were dialyzed
against 100 ml of sodium phosphate buffer (pH 5.7 and 7.4), at 37
°C with continuous stirring at 100 rpm. One milliliter of the sample
was withdrawn at specific time intervals and analyzed
spectrophotometrically. The sink condition was maintained by
replacing equal volumes of the buffer. The release studies were
performed in triplicate, and the average was taken. The percentage of
drug release was calculated by the using following formula (2):
In vitro cytotoxicity studies
Cell viability was measured by MTT assay described by Mossman
16
and coworkers . The detailed procedure is given as Supplementary
information.
Apoptosis study
The influence of EPIꢀFAꢀAuNPs inducing apoptosis in the breast
1
7
cancer cells was confirmed using staining methodology . The
detailed methodology is given as Supplementary information.
Flow cytometric analysis and Western blotting
To investigate the effect of the drug on the cell cycle distribution
Fig. 1 . LC–ESI–MS/MS analysis of an optimised aqous extract of Limonia
acidissima L fruit.
1
8
analysis described by Krishnan , Western blotting was carried out
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Scheme 1: Schematic representation for the generation of EPI-FA-AuNPs
activated hydrogen atom that can reduce metal ions to form
nanoparticles (yellow color converted to wine red color).
Figure 2: (A) Optimization of AuNPs by changing various concentrations of (L.
acidissima L extract) while keeping the gold solution constant. (B) FT-IR study of:
(
i) L. acidissima L extract (ii) AuNPs (iii) FA (iv) EPI (v) EPI-FA-AuNPs.
The Photochemical profiling of the extract was done by LCꢀMS
analysis which revealed the presence of phytocompounds such as (i)
kinetin, (ii) esculin, (iii) 3,4,2’,4’,6’ꢀpentamethoxychalcone, (iv) Nꢀ
desmethylpheniramine. The identification of compounds was
verified by mass fragmentation analysis, and the LCꢀMS spectra of
the compounds are shown in Figure 1. The combined action of these
chemical components and others may be responsible for the
observed reduction of the metal ions to form nanoparticles. The
AuNPs were formed by simple mixing without applying any external
energy (heating, sun light, or microwaving). The resultant mixture
produced a significant color change (to wine red) within a few
seconds after the addition. Further, our study focused on using the
synthesized AuNPs to enable conjugation of biomolecules for
However, in the FTIR spectrum of AuNPs Figure 2 B (ii), the peak
−1
at 3420 cm becomes comparatively narrow, which could be
attributed to the phenolic hydroxyls (OꢀH bond) confirming that the
phenolic hydroxyls react with the AuNPs resulting in the partial
destruction of the hydrogen bonds in the molecules of the L.
−1
acidissima L extract. A shift in the peak from 1048 cm to 1017
−1
cm also indicates that L. acidissima L extract interacts with AuNPs
through its adjacent phenolic hydroxyls and/or formed quinones. The
comparison of FTIR spectra of bare AuNPs, FA, EPI, and EPIꢀFAꢀ
AuNPs shows that the spectrum of FAꢀAuNPs exhibits the
characteristic IR absorption peaks of FA Figure 2B (iii), showing a
–1
ꢀ1
peak at 1697cm (amide I stretching), 1603 cm (amide II
different applications in drug delivery (Scheme 1)
.
–1
stretching), and 1481cm (heteroꢀring, conjugated double bond).
3+
We observed that the solution containing gold ions (Au ) and L.
acidissima L turned into wine red (537 nm) within 30 seconds (The
synthesis of AuNPs is given in the video file).We found that 75µL of
L. acidissima L extract Figure 2 A was sufficient to reduce 1mM
FTIR spectra of EPI and EPIꢀFAꢀAuNPs are shown in Figure 2B (iv)
and (v). FTIR spectra of EPI clearly confirm the basic structural
units such as OH, CH, and carbonyl groups carboxyl groups by the
–1
–1
–1
peaks that appear at 3300 cm , 2940 cm , and 1620 cm ,
respectively. The FTIR spectrum of EPIꢀFAꢀAuNPs shows the peaks
4
HAuCl . FTIR spectrum of L. acidissima L extract Figure 1B (i))
−1
shows apeak at 3448 cm , which could be attributed to the phenolic
–1
–1,
at 3300 cm and 2940 cm demonstrating the OH and CH content
hydroxyls (OꢀH bond) in extracts, the absorption peaks at 1048 and
of EPIꢀFAꢀAuNPs. Moreover, the disappearance of the peak at 1730
−
1
1
612 cm representing costretching functional present in the extract.
ꢀ1
cm describes the amide bond formation between the carboxyl
Further, carboxyl stretching vibration peak formed at 1730 cm−1
functional of EPI and amine functional of FA. These spectra
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external of the AuNPs at pH (1.2). The results from these in vitro
stability studies established that the AuNPs were intact and
Figure 3: (A) The EDAX spectrum, (B) X-ray diffraction (XRD) spectrum, (C) SAED
pattern of L. acidissima L fruit extract reduced AuNPs.
Figure 4: HRTEM image of (A) AuNPs (B) EPI-FA-AuNPs, (C)The UV–Vis spectra
showing the in vitro stability of EPI-FA-AuNPs in the pH range 1.2–7.4 and PBS,
(
D) AuNPs conjugated EPI-FA-AuNPs.
confirm the formation of EPIꢀFAꢀAuNPs. Energy dispersive Xꢀray
(
EDX) spectrum analysis of AuNPs showed the presence of AuNPs demonstrated excellent in vitro stability in biological fluids at
2
0
in the sample. In Figure 3 (A) reveals a strong and typical optical various pH . EPI binding onto the AuNPs was further confirmed by
absorption peak at approximately 2.2 keV, which could be attributed the shift of the SPR band in Figure 4 (D) towards a higher
to the SPR of the metallic Au nanocrystals. The crystalline structure wavelength (from 537 nm to 543nm). The percentage of EPI loaded
of nanoparticles was determined based on Xꢀray diffraction (XRD) onto the AuNPs was determined based on EPI content in the
analysis. The crystalline peaks that were identified as AuNPs based obtained pellet, and it was found that 52 ± 4% of the drug could be
on XRD analysis showed that the intense peaks of reflected radiation loaded.
Inductively
coupled
plasma
optical
emission
(Bragg peaks) were at the points (1 1 1), (2 0 0), (2 2 0), and (3 1 1) spectrophotometer (ICPꢀOES) was performed to quantify the amount
and were the diffraction lines of the faceꢀcenteredꢀcubic (fcc) gold of gold in the aqueous solutions of AuNPs, and the same was used in
are shown in Figure 3 (B). The selected area of electron diffraction further studies.
(
SAED) outline of the AuNPs showing that the rings designated 1, Particle size and zeta potential measurements
, 3, and 4 arise because of the reflections from (1 1 1), (2 0 0), The hydrodynamic particle size of the AuNPs was found to be 134 ±
2 2 0), and (3 1 1) are shown in Figure 3 (C). The morphological 4 nm with a polydispersity index of 0.219 are shown in Figure 5 (i).
2
(
results (HRTEM) images Figure 4 (A) reveals that the AuNPs and The average hydrodynamic diameter of EPIꢀFAꢀAuNPs is 139 ± 3
EPIꢀFAꢀAuNPs appear to be nearly spherical in shape as seen in nm with a polydispersity index of 0.249 shown in Figure 5 (ii). A
Figure 4 (B). The stability of EPIꢀFAꢀAuNPs in different buffers low polydispersity index shows that the particle size distribution of
was examined in Figure 4 (C), and only slight changes (< 15%) were the AuNPs and EPIꢀFAꢀAuNPs is uniform. Zeta potential is
observed in the SPR band at 537 nm when the AuNPs were exposed considered an essential parameter to study the surface charge of the
to different buffers for 48 hours. However, at pH 1.2, about 20% nanoparticle surface and predict the longꢀterm stability of the
21
decline in the absorbance at 537 nm was observed compared with nanoparticles . The zeta potential of the AuNPs was found to be
the absorbance of EPIꢀFAꢀAuNPs at pH (7.4). The observed −27.9 mV Figure 5 (iii) indicating that the AuNPs were properly
reduction in the absorption of EPIꢀFAꢀAuNPs in extremely acidic capped with anionic (carboxyl group) FA. EPIꢀFAꢀAuNPs were
conditions could be attributed to the limited aggregation of EPIꢀFAꢀ isolated by centrifugation and then suspended in an aqueous solution
AuNPs as a result of the screening of the negative charge on the to quantify the electrophoretic mobility. The calculated zeta potential
of EPIꢀFAꢀAuNPs was −16.0 mV as seen in Figure 5 (iv). Though
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the surface charge is reduced to some extent, EPIꢀFAꢀAuNPs has AuNPs displays the slow and controlled release of the drug, with the
found to be stable without aggregation when analysed after sixꢀ release rate calculated showing negligible release when compared to
month storage period. Hence, it is clear that EPIꢀFAꢀAuNPs are that of free EPI in acidic and neutral atmospheres, respectively.
highly stable over a reasonable period of time.
Figure 5: (i) The particle size distribution of AuNPs (ii) EPI-FA-AuNPs (iii) The zeta
potential distribution of AuNPs in an aqueous medium. (iv)The zeta potential of
distribution EPI-FA-AuNPs in an aqueous medium.
Toxicity of AuNPs in zebrafish embryos
Zebrafish embryos, which are ideal organisms, were used to
22
investigate the developmental toxicity of AuNPs without EPIꢀFA
The survival rate was better for the embryos that had hatched within
6 hours post fertilization, as compared with the control group as seen
.
9
in Figure 6 A (i). The mortality level was indicated by the dead
embryos 96 hours post fertilization as compared to the control group
in Figure 6 A (ii).The survival rate and mortality rate examined
shows no apparent toxicity at various concentrations ranging from 50
23
to 450 µg/mL . The AuNPsꢀtreated embryos were observed using a
compound stereo microscope to find abnormalities such as pericardial
edema, pigmentation, deformity of the pericardial sac of the larvae,
Figure 6: (A) (i) Graph representing the toxicity of AuNPs in terms of survival rate
%) of larvae, (ii) Mortality rate (%) analysis of AuNPs toxicity, (iii) Examples of
(
zebrafish larvae exposed to control and various concentrations of AuNPs.
Significant differences compared to control calculated with the one-way ANOVA
and deformity of the tail. Exposure of zebrafish egg embryos to test and are indicated by ***p < 0.001. (B) In vitro release profiles of EPI from
the EPI-FA-AuNPs and free EPI in phosphate buffer solution (pH5.7 and 7.4) at
AuNPs did not produce any seeming toxicity in the development of
o
7 C.
3
the zebrafish are shown in Figure 6 A (iii). Hence, this investigation
of results clearly indicated that the AuNPs might be carriers for drug
delivery applications.
Furthermore, the release efficiency of the drug from the EPIꢀFAꢀ
AuNPs was rapid, higher at pH (5.7) than at pH (7.4) EPI from EPIꢀ
FAꢀAuNPs in pH (7.4) will help to reduce toxicity of EPI to the
normal tissue since the physiological pH of the body is maintained at
Release profile of EPI-FA-AuNPs
The in vitro study of pHꢀcontrolled EPI release from the EPIꢀFAꢀ
25
pH (7.4)
.
AuNPs was performed in PBS (pH = 7.4) and acetate buffer (pH =
o
In vitro cytotoxicity studies
5
.7) at 37 C, pH (7.4) and pH (5.7) being near the physiological and
To determine the cytotoxic effect of EPI and EPIꢀFAꢀAuNPs, cell
viability study was done by a standard MTTꢀreduction assay with
endosomal pH conditions, respectively, of a cancer cell. The results
are presented in Figure 6 (B),The pHꢀdependent drug release is also
2
6
2
4
slight modifications . The results of the MTT assay for free and EPIꢀ
considered to be an essential factor in cancer therapy . The EPIꢀFAꢀ
6
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FAꢀAuNPs on MCFꢀ7 cell lines are shown in Figure 7 (A). The IC50 Effect of EPI-FA-AuNPs on cell cycle analysis
value for EPIꢀFAꢀAuNPs was found to be around 2 µg/mL, while that Generally, the flow cytometry study was performed to investigate
whether apotosis and the cell cycle arrest are closely related. In our
Figure 7: (A) In vitro cytotoxicity studies of drug EPI and EPI-FA-AuNPs in MCF-7
cell lines. All assays were performed in triplicate, and the mean ± standard
deviations are shown. (B) Images of MCF-7 cell lines visualized under inverted
microscope. (i) Control, (ii) Blank, (iii) EPI, and (iv) EPI-FA-AuNPs.
for free EPI ranged from 28 µg/mL on the MCFꢀ7 cells. These results
show that the EPIꢀFAꢀAuNPs exhibited better cytotoxic activity than
free EPI. This could possibly be due to the result of the variations in
the cellular uptake profile leading to better activity of EPIꢀFAꢀAuNPs
2
7
as suggested by Ormrod and coworkers . The results of this study
suggests that FA functionalized AuNPs can effectively deliver the
drug to MCFꢀ7 cancer cells by means of active targeting.
Apoptosis study
Figure 8: (A) Effects of EPI-FA-AuNPs on cell cycle analysis. MCF-7 non-small cell
breast cancer cells were cultured for 36 hours (i) Control (ii) EPI (iii) EPI-FA-
AuNPs. (B) Western blot, illustrating the different protein expressions in
apoptosis after treatment with EPI and EPI-FA-AuNPs. Lane 1: Control, Lane 2:
Apoptotic changes, cytotoxic effect caused by EPIꢀFAꢀAuNPs
occurred in MCFꢀ7 cells when treated using acridine AuNPs, Lane 3: EPI, Lane 4: EPI-FA-AuNPs.
orange/ethidium bromide differential staining method. The stained
study, the effect of EPI and EPIꢀFAꢀAuNPsꢀon cell cycle (MCFꢀ7
cells characterized to EPIꢀFAꢀAuNPs caused more effective cell
death than free EPI. The number of nonviable cells increased
dramatically after treatment with EPIꢀFAꢀAuNPs in MCFꢀ7 cells are
shown in Figure 7 B (iv). The red color is due to the nonviable cells
cells) was studied. Figure 8 (A) shows the cell cycle analysis of (i)
control, (ii) EPI and (iii) EPIꢀFAꢀAuNPs. Control cells after 36
2
hours had 20.3 % the proportion of cells in the G /M phase in Figure
8
(A) (i), and the EPI and EPIꢀFAꢀAuNPs produced a dramatic
(
dead cells), and it was revealed that EPIꢀFAꢀAuNPs treatment
increase of 28.95 % in Figure 8 (A) (ii) and 43.21% are shown in
resulted in a significant increase in apoptosis compared to EPI
treatment. The control and blank cells in Figure 7 B (i) and (ii), the
condensed nuclei, as well as the membrane blebbing fluoresced
uniformly bright green indicating (early apoptotic) that they did not
undergo any apoptotic changes.
Figure 8 (A) (iii) in the G /M population, respectively. These data
2
2
clearly prove that the cell cycle is arrested significantly at the G /M
2
8
phase in EPIꢀFAꢀAuNPs induced MCFꢀ7 cells . The antiꢀcancer
therapy completely eradicates the cancer cells by triggering different
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caspaseꢀmediated cell death pathways , which confirms the FAꢀAuNPsꢀtreated cells showed maximum localization of EPIꢀFAꢀ
activation of the apoptotic pathway after EPIꢀFAꢀAuNPs’ exposure.
Western blot analysis
AuNPs into cells compared to EPIꢀtreated cells.
The Western blot results showed mechanisms of EPIꢀFAꢀAuNPsꢀ
mediated cell death; the apoptotic regulators have been measured
using mRNA and protein expression patterns. Cyclin D1 regulates
the proliferation process by adjusting its expression levels and,
3
0
accordingly, regulates the cell cycle control machinery . EPI
3
1
controls the reduction of Cyclin D1 levels in tumors . The results
as seen in Figure 8 (B) showed that EPIꢀFAꢀAuNPs inhibited the
function of Cyclin D1, and its expression was dramatically
diminished. Cyclin E showed a high intense band when AuNPs and
native EPIꢀtreated cells were compared. The functions of Cyclinꢀ
dependent kinase CDK4, CDK6, and CDK2 mediated
phosphorylation of the retinoblastoma (Rb) family of tumor
32
suppressor proteins during apoptosis were also observed . EPIꢀFAꢀ
AuNPs induced apoptosis in cancer cells was evident from the
reduced expression of antiꢀapoptotic CDK6, CDK4, and CDK2 in
MCFꢀ7 cells. In this study on the efficacy of CDK inhibition as a
means to arrest the proliferation of HCC, βꢀactin was used as a
loading control, and it showed similar expression in all the lanes.
Intrinsic pathways of Western blot studies are shown in Figure 8 (B).
The present study also investigated EPIꢀFAꢀAuNPs induced upꢀ
regulation of Bax proteins and downꢀregulation of Bclꢀ2 proteins in
EPIꢀFAꢀAuNPs cells. p53 is a leading switch that coordinates stress
3
3
signals between apoptosis and cell cycle arrest is shown in Figure
(B). EPIꢀFAꢀAuNPs induced increase in the levels of p53, the
tumor suppressor protein. Caspases, a family of cysteine acid
8
Figure 9: Drug localization studies of control, EPI, AuNPs, and EPI-FA-NPs in
34
proteases, can be regarded as key factors in apoptosis . In Figure 8 MCF-7 cellline.
B) shows that EPIꢀFAꢀAuNPs treated cells induced cleavage of
(
Conclusion
caspase 3 and caspase 9. On the whole, these pH results clearly
indicate that the molecular level apoptosis was induced when MCFꢀ7
cells were treated with EPIꢀFAꢀAuNPs. Cell death was further
studied based on PARP cleavage after treatment with EPIꢀFAꢀ
AuNPs, as seen in Figure 8 (B). Our studies showed that PARP
proteins cleaved into fragments after treatment with EPIꢀFAꢀAuNPs.
High content imaging
To summarize the present study, a rapid and green chemistryꢀbased
method, which relies on the reduction of gold ions by L. acidissima
L extract acting as both reducing and capping agents, has been
established to synthesize AuNPs.The EPIꢀFAꢀAuNPs exhibits
prominent anticancer activity against MCFꢀ7 cells. Results of the
toxicity study in the zebrafish embryo model revealed that no
significant malformation occurred, ensuring that the AuNPs
nanoparticles are highly compatible for drugꢀdelivery applications.
Furthermore, maximum localization into cells compared with EPI in
the MCFꢀ7 cell line was observed. The EPIꢀFAꢀAuNPs could be
promising for targeting breast cancer with the enhanced therapeutic
activity of EPI.
The cellular uptake study was made in MCFꢀ7 cell line for EPI,
AuNPs, and EPIꢀFAꢀAuNPs using high content imaging methods,
and the results are shown in Figure 9. EPIꢀFAꢀAuNPs’ localization
into the cell was compared with EPI in which bright field and
fluorescent images were taken. The merged images help to calculate
the localization of EPIꢀFAꢀAuNPs. Control cells without the
35
exposure to EPI and AuNPs indicated no fluorescence . The EPIꢀ
8
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Acknowledgements
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17.S.Karthik, R.Sankar, K.Varunkumar,
The authors are thankful to the University Grants Commission
UGC), India for financial support through the project grant no. F1ꢀ
7.1/2011ꢀ12/RGNFꢀSCꢀTAMꢀ2228/ (SAꢀIII) to one of the authors,
V.Ravikumar,
(
Biomedicine & Pharmacotherapy, 2014,68, 327ꢀ334.
18. A.Krishnan, The Journal of Cell Biology,1975,66,188ꢀ193.
19. L.C.Tu, C.K.Chou, C.Y.Chen, Y.T.Chang, Y.C.Shen, S.F.Yeh,
Biochimica et Biophysica Acta, 2004, 1672, 148ꢀ156.
1
C. Senthil Kumar. We thank National Facility for Drug
Development for Academia, Pharmaceutical and Allied Industries
(
NFDD) for particle size analyser facility LCꢀMS and FTIR studies.
20. M.Ganeshkumar, M.Sathishkumar, T.Ponrasu, M.G.Dinesh,
L.Sugun, Colloids and Surfaces B: Biointerfaces, 2013,106,
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ꢀ
ꢀ
ꢀ
Green synthesis of AuNPs using extract of Limonia acidissima L as a reducing agent
Epirubicin was successfully loaded on Folic acid-AuNPs
This formulation revealed better site specific delivery of epirubicin to MCF-7 cells