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Materials Science & Engineering C
journal homepage: www.elsevier.com/locate/msec
Synthesis and evaluation of the antiproliferative efficacy of BRM270
phytocomposite nanoparticles against human hepatoma cancer cell lines
Meeta Geraa, Nameun Kima, Mrinmoy Ghoshb, Neelesh Sharmac, Do Luong Huynha,
Nisansala Chandimalia, Hyebin Koha, Jiao Jiao Zhanga, Tae Yoon Kanga, Yang Ho Parkd,
Taeho Kwona, Dong Kee Jeonga,⁎
a Laboratory of Animal Genetic Engineering and Stem Cell Biology, Department of Animal Biotechnology, Jeju National University, Jeju, Jeju-Do 690-756, Republic of
Korea
bDepartment of Biotechnology, Division of Research and Development, Lovely Professional University, Punjab 144411, India
c Division of Veterinary Medicine, Faculty of Veterinary Science and Animal Husbandry, Sher-e-Kashmir University of Agricultural Sciences and Technology of Jammu, R.S.
Pura, Jammu 181102, India
d BRM Institute, Seoul 06111, Republic of Korea
A R T I C L E I N F O
Keywords:
Anticancer activity
BRM270 nanoparticles
Human hepatoma cancer cells
Mechanical-milling method
Phyto-composite
A B S T R A C T
BRM270 is the most leading phytochemical extract that possesses potent anticancer properties. A major chal-
lenge associated with this drug is its low bioavailability and thus requires high dosages for cancer treatment.
Here, we report the novel nano-synthesis of phyto-composite, BRM270 for the first time by mechanical milling
method with specific modifications for enhanced cytotoxicity against HepG2 human hepatoma cancer cells.
Unlike free BRM270 and other phytomedicines, BRM270 nanoparticles (BRM270 NPs) are well-dispersed and
small sized (23 to 70 nm) which is believed to greatly enhanced cellular uptake. Furthermore, the acidic tumor
microenvironment attracts BRM270 NPs enhancing targeted therapy while leaving normal cells less affected.
The comparative cytotoxicity analysis using MTT assay among the three treatment groups, such as free BRM270,
BRM270 NPs, and doxorubicin demonstrated that BRM270 NPs induced greater cytotoxicity against HepG2 cells
with an effective drug concentration of 12 μg/ml. From FACS analysis, we observed an apoptotic cell death of
44.4% at BRM270 NPs treated cells while only 12.5% found in the free BRM270 treated cells. Further, the
comparative relative expression profiling of the candidate genes were showed significant (p 0.5 million annual
mortalities, making it the third leading cause of cancer-related deaths
[1]. Several types of chemotherapeutic drugs are available to treat
hepatic cancer [2,3]. However, these drugs usually have some major
drawbacks of cancer resistance due to multidrug resistance (MDR)
[4,5], low apoptotic proteins, and high toxicity associated with the
current treatment modalities, which are a big challenge [6,7]. There-
fore, the last decade has witnessed a renewed interest in biologically
safe and active naturally occurring compounds isolated from a plethora
of plant sources for targeting cancer cells [8–10].
Phytochemicals have attracted researchers worldwide for various
therapeutic purposes because of their minimal toxicity and maximum
efficacy [11]; however, phytotherapeutics needs an investigative
methodology to deliver the components in a sustained manner to in-
crease cellular efficacy and avoid bulk drug administration [12].
https://doi.org/10.1016/j.msec.2018.11.055
Received 3 May 2018; Received in revised form 17 October 2018; Accepted 27 November 2018
⁎ Corresponding author at: Department of Animal Biotechnology, Faculty of Biotechnology, Jeju National University, Ara-1 Dong, Jeju-Do 690-756, Republic of
Korea.
E-mail address: newdkjeong@gmail.com (D.K. Jeong).
Materials Science & Engineering C 97 (2019) 166–176
Available online 28 November 2018
0928-4931/ © 2018 Published by Elsevier B.V.
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Therefore, recently, nanotechnology-based strategies are being aggres-
sively explored worldwide because of their ability to transport specific
anticancer drugs to the tumor site with enhanced phytodrug bioavail-
ability [13–15]. In addition, nanomedicine offers various additional
benefits, such as improved cellular uptake, targeted drug delivery, ex-
cellent stability, and controlled release functions, leading to enhance-
ment of its pharmacological activities [16,17]. Herein, BRM270 is a
phytocomposite extract formulated from seven plants (Saururus chi-
nensis, Citrus unshiu markovich, Scutellaria baicalensis, Portulaca oleracea,
Prunella vulgaris, Aloe vera, Arnebia euchroma) used as traditional Asian
medicine that inhibits the proliferation of many types of cancer cells
[18]. It is one of the most promising anticancer medicinal plant extract
and widely distributed in Northeast Asia mainly China, Korea and
Japan [19]. Among different anticancer drugs derived from natural
resources available, a phyto-composite, BRM270 shows tremendous
cytotoxic activity against different cancer cell lines might due to its
versatile chemical nature of different plant extract composite or the
presence of free hydroxyl groups.
The present context of this research investigates the nanoformula-
tions of a phytocomposite, BRM270, using an ingenious synthesis ap-
proach. Their physicochemical characterization and its evaluation of
anticancer activity against HepG2 cell lines. The uniquely prepared
BRM270 NPs are highly stable and eco-friendly with increased bioa-
vailability and exert significant inhibitory effects towards cancer cells.
Earlier studies on free BRM270 have suggested that it plays an im-
portant role in the inhibition of the NF-ĸB signaling cascade in MDR-
induced stem-like cancer-initiating cells (SLCICs) and promotes pro-
grammed cell death [20]. It has also been shown in previous studies
that the BRM270 phytodrug was capable of inhibiting lipocalin-2-in-
duced epithelial-mesenchymal transition (EMT) process in CD133 in
A549 cells, the tumor-initiating cancer stem-like cell xenograft, by in-
hibiting the NF-κB pathway [21].
The present study encompasses the preparation and characteriza-
tion of nano-range particles of the phytodrug BRM270 for the effective
antiproliferative activity against HepG2 cell lines. This nanoformulated
phytodrug has the potential for effective intracellular delivery of a drug
into cancer cells. It can be a suitable alternative to other chemother-
apeutic modes of treatment against cancer cells.
2. Material and methods
2.1. Chemicals and reagents
The BRM270 phytocomposite drug was procured from the
Biological Response Modifier International Health Town Corp., Korea.
The organic solvents used for the extraction were of analytical grade
from Fisher Scientific (UK). Acetonitrile (HPLC grade, Fisher Scientific,
UK) and phosphoric acid were of analytical grade (BHD, England).
Ultrapure water from Milli-Qsystem (Millipore, Bedford, MA, USA) was
used for the mobile phase preparation. Dimethylsulfoxide (DMSO) was
purchased from Junsei Chemical Co. Ltd. (South Korea). The reagents
used in the cell culture assay were of molecular biology grade. The
reagent and culture media Dulbecco's modified eagle's medium
(DMEM) were purchased from GIBCO®, Invitrogen Corporation
(Carlsbad, CA, USA). The glutaraldehyde was purchased from Sigma-
Aldrich Chemie GmbH (Munich, Germany). All the other reagents used
in the present study were of analytical grade.
2.2. Preparation of BRM270 nanoparticles
The total of 1ml solution was used from the standard BRM270 pack
and sprayed dropwise into 50ml boiling water with a flow rate of
0.2 ml/min for 5min under constant stirring at 200 to 800 rpm at room
temperature for 30min. After continuous stirring, the solution was kept
in an ultrasonicator for 2 h with an ultrasonic power of 100W and a
frequency of 30 kHz. After sonication, a clear pale yellow-orange co-
lored solution was obtained. The solution was further concentrated up
to 5ml under reduced pressure at 40 °C using a rotavapor and then
freeze-dried to obtain an orange-brown powder using lyophilizer
without using any cryoprotectant and stored at −20 °C for further use
(Fig. 1).
2.2.1. Physico-chemical characterization of BRM270 NPs
The synthesized BRM270 NPs were characterized using the
LAMBDA25 ultraviolet (UV)-visible spectroscopy (Perkin Elmer,
Waltham, MA, USA). The aqueous solution of BRM270 NPs was
scanned within a wavelength ranging from 200 to 800 nm and the
characteristic peaks were detected. The comparative analysis of surface
Fig. 1. Schematic preparation of BRM270 nanoparticles using mechanical milling method.
M. Gera et al. Materials Science & Engineering C 97 (2019) 166–176
167
morphology and the shape of the free BRM270 vs. BRM270 NPs was
performed using the JSM-6700F field emission scanning electron mi-
croscope (FE-SEM) (JEOL Ltd., Tokyo, Japan). The sample was pre-
pared by spreading the aqueous dispersion of free BRM270 and
BRM270 NPs separately over the carbon tape and drying it under a
nitrogen stream. The sample was then coated with the OPC80 T osmium
plasma sputter coated with a platinum layer 200 Å thick under vacuum
conditions. The JEOL JEM 1200EX II high-resolution transmission
electron microscope (HR-TEM) (JEOL Ltd., Tokyo, Japan) was used to
examine the powdered BRM270 NPs and to record the exact NP size
and shape. The samples for HR-TEM analysis were prepared by ultra-
sonically dissolving the powdered free BRM270 and BRM270 NPs in
double-distilled water. A drop of this solution was subsequently de-
posited onto a carbon-coated copper grid and allowed to evaporate
under ambient conditions. The vibrational spectra of BRM270 were
recorded on a Nicolet 6700 Fourier transform infrared (FTIR) spectro-
photometer (Thermo Electron Corporation, Pasadena, TX, USA). The
dried NP sample was ground with KBr pellets and measured at a wa-
velength ranging from 600 to 4000 cm−1. The FTIR analysis was per-
formed to study the functional groups of the BRM270 molecules. The
size distribution or average diameter of synthesized BRM270 NPs was
further confirmed by Dynamic light scattering (DLS, Zetasizer Nano ZS,
Malvern Instruments, UK).
2.2.2. Physico-chemical stability studies
The stability analysis of BRM270 NPs was explored over time in
phosphate-buffered saline (PBS; 0.01M) at different pH values of
pH 4.5, pH 5.5 and pH 7.5. The BRM270 NPs were stored at 4 °C tem-
perature. After every 24 h of incubation, the particle size of the
BRM270 NPs was analyzed for evaluating the physicochemical stability.
2.2.3. In vitro drug-release study
The BRM270 NPs release was investigated in a time-dependent
manner in different buffers at pH 7.4 and pH 5.0 for 12 h at 37 °C.
Accordingly, 1 mg/ml of the BRM270 NPs solution was transferred to
the dialysis membrane tube (3.5 kDa). The released drug in the different
pH buffers was analyzed at different time intervals by using the
UV–visible spectrometer (Perkin Elmer, Waltham, MA, USA).
2.2.4. High-performance liquid chromatography (HPLC) analysis
HPLC analysis was conducted on an Agilent 1200 liquid chroma-
tography system (Agilent Technologies, Palo Alto, CA, USA), equipped
with a double pump and a diode array detector. The separations
were carried out on an Agilent Eclipse ODS C18 column
(4.6 mm×250.0 mm×5.0 μm). The column temperature was set at
25 °C. The mobile phase consisted of 0.4% aqueous phosphoric acid
(A) and acetonitrile (B). The gradient concentration was as follows:
15% B (v/v) at 0–16min, 15–25% B at 16–30min, 25% B at
30–32min, 25–30% B at 32–35min, 30% B at 35–37min, 30–33% B at
37–40min, 33% B at 40–45min, 33–48% B at 45–55min, 48–55% B at
55–75min, 55–80% B at 75–82min, and 80% B at 82–88min, and the
re-equilibration time of gradient elution was 15min. The flow rate
was 0.8 ml/min and the injection volume was 10 μl. The detection was
carried out at a set wavelength of 280 nm. The absorption spectra of
the compounds were recorded between 200 and 800 nm.
2.3. Cell culture and treatment conditions
The human liver cancer cell lines, HepG2 was maintained in DMEM
supplemented with 10.0% fetal bovine serum (FBS) (Hyclone, South
Logan, UT, USA), penicillin (100.0 U/ml), and streptomycin (100mg/
ml) were then incubated under standardized conditions at humidified
37 °C and 5% CO2 atmosphere. The human bone marrow cells (hBMCs)
were purchased from Korean Cell Line Bank, Seoul, South Korea (KCLB
No. 10246). The potential hBMCs were further cultured in DMEM high
glucose supplied with 10% FBS, 1% antibiotic-antimycotic, and 10 nM
E9644-Sigma epithelial growth factor (Sigma-Aldrich, St. Louis, MO,
USA).
2.3.1. Intracellular distribution of BRM270 NPs
To investigate the dynamic intracellular distribution of BRM270
NPs, 6.0× 105 cells were seeded in each culture dish and allowed to
adhere and acclimate for 1 day. BRM270 NPs at a concentration of
12 μg/ml were then exposed to each culture dish for 12 h. The cells then
were harvested by trypsinization, fixed in ice-cold 2.5% glutaraldehyde,
and preserved at 4 °C for further analysis. The cells were then post-fixed
in 1% osmium tetraoxide in 2.5% glutaraldehyde, dehydrated in graded
alcohol, embedded in Epon 812, sectioned with an ultramicrotome, and
stained with uranyl acetate and lead citrate. The sections were ex-
amined under the HR-TEM operating at 200 kV, with a point resolution
of 0.23 nm and C3=1.0mm.
2.3.2. Evaluation of cytotoxicity assay
Cytotoxicity of free BRM270, BRM270 NPs and doxorubicin was
assessed by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay against normal HBM and cancerous HepG2 cells. Both
types of cells were seeded at a density of 5× 103 cells/well in a 96-well
plate (Nunc™, Wiesbaden, Germany) in 100 μl of DMEM medium. The
cells were incubated for 24 h to adhere properly, then the cells were
treated with both free BRM270 and BRM270 NPs at concentrations
ranging from 0.0 to 12 μg/ml and Doxorubicin (Dox) at 0 to 5 μM
concentration. The DMEM medium itself was used as a solvent to dis-
solve various drugs for the exposure in cells. After the further incuba-
tion of 24 h, the drug-treated cells were washed twice with phosphate-
buffered saline (1× PBS) and incubated for another 4 h with 0.5mg/ml
MTT solution in the culture medium. In the cells, dehydrogenase, and
reductase would reduce this solution into formazan, a purple color of
artificial chromogenic products. After 4 h of incubation, the cells were
lysed into 100 μl dimethyl sulfoxide (DMSO) and the absorbance was
measured at the wavelength of 570 nm using a bio-assay reader (Bio-
Rad, Berkeley, CA, USA). Cell viability was determined using the fol-
lowing equation:
= ×Number of viable cells Absorbance of sample Absorbance of control( / ) 100
Final results of the assay were reported as the mean value ±
standard error of the mean (SEM) of triplicate independent experi-
ments.
2.3.3. Apoptosis assayby flow cytometry
The cells (1× 105) suspended in 2ml fresh DMEM media were
seeded in each well of a 6-well flat-bottomed microtiter plate and in-
cubated overnight. After the overnight incubation, the free BRM270
and BRM270 NPs have added an optimized concentration of 12 μg/ml
in the media. After 24 h, cells were harvested and washed twice with
pre-cold PBS followed by re-suspension in 1× Annexin V binding buffer
(BD Biosciences, Franklin Lakes, NJ, USA) at a concentration of
1×106 cells/ml. Approximately 100 μl of such solution (1× 105 cells)
was mixed with 5 μl of Annexin V-FITC and 5 μl of propidium iodide
(PI, BD Biosciences, San Jose, CA, USA) according to the manufacturer's
instructions. The mixed solution was incubated at room temperature
(25 °C) in the dark for 15min. Then 400 μl of 1× dilution buffer was
added to each tube. The analysis was performed by Beckman Coulter
FC500 Flow Cytometry System with CXP Software (Beckman Coulter,
Fullerton, CA, USA) within 1 h of the treatment of cells with dilution
buffer.
2.3.4. DNA fragmentation using Hoechst 33258 staining
The cells (5× 104) were seeded in sterile six-well microtitre plates
(Nunc Nunclon™ Delta). After overnight growth, the cells were treated
with free BRM270 and its NPs. The cells were later washed with 1×
PBS (Gibco Life Technologies™, USA), fixed with 4% paraformaldehyde
for 10min, and incubated with 50 μM Hoechst 33258 staining solution
M. Gera et al. Materials Science & Engineering C 97 (2019) 166–176
168
for 5min. After three washes with cold PBS, the cells were viewed
under a fluorescence microscope (Olympus, Tokyo, Japan) to analyze
the apoptotic morphological changes in the nuclear chromatin of both
the treated and non-treated cancer cells.
2.3.5. Annotation of Gene Ontology and pathway analysis
The biological processes and pathway analysis of the candidate
genes, i.e., IL6, CASP 9, BAX, BCL2, p53, MMP9; which are associated
with the anticancer activity were investigated. To define the molecular
mechanism of tumorigenesis in liver mediated by nanoparticles of
BRM270, specific cancer-associated protein-protein interaction network
was constructed. All protein-encoding genes were grouped and orga-
nized through protein-protein associations using the web-based Search
Tool for Retrieval of Interacting Genes/Proteins (STRING) software
(v.9.1) (www.string-db.org/). The pathway mapping was performed
using the Kyoto Encyclopedia of Genes and Genomes (KEGG) (www.
genome.jp/kegg) tool. It is an integrated resource with associated
software consisting of three types of databases for genomic, chemical
and network information. The web-based ToppCluster (www.
toppcluster.cchmc.org/) tool was used to visualize the shared and
specific features associated with any number of genes involved in bio-
logical processes.
2.3.6. Determination of relative expression of candidate genes by qRT-PCR
Cells were seeded at a concentration of 1× 105 in sterile six-well
plates and incubated overnight under standard culture conditions.
After incubation, the cells were treated with free BRM270 and
BRM270 NPs and incubated for 24 h. The cells were then detached
with 0.5% trypsin-EDTA (Gibco) and total RNA was extracted with
easy Blue (Intron Biotech, Seongnam-si, Gyeonggi-do, Korea). The
purified RNA was quantified using a photometer (Bio-Rad Hercules,
CA, and the USA). The reverse transcription was performed using a
Superscript III first-strand cDNA synthesis kit and oligo (DT) primers
(Invitrogen, Waltham, MA, USA). The cDNA was subjected to qRT-
PCR for the quantification of the relative transcript levels of the in-
flammatory cytokine IL6, pro-apoptotic/anti-apoptotic gene CASP 9,
BAX, p53, BCL2, and MMP9 using the specific primers. The oligonu-
cleotide primers for the targeted genes and β-actin as an endogenous
gene were designed based on sequences available from the NCBI da-
tabase using online program Primer-3 (https://www.ncbi.nlm.nih.
gov/tools/primer-blast/) (Table 1).
RT-PCR was conducted using 2.5 μl of cDNA with an Eppendorf
Master Cycler gradient instrument. The thermal cycling reaction was
conducted as follows: an initial 5 min at 94 °C; 35 cycles of 30 s at 94 °C,
30 s at 60 °C, and 1min at 72 °C; and a single extension period of 10min
at 72 °C. Differential expressions of candidate genes were evaluated
using real-time qRT-PCR using the Step-One Real-Time PCR system
(Applied Biosystems). Each sample was quantified in triplicate using
the dye Eva Green (Biotium, USA) under the following amplification
conditions: 95 °C for 10min, followed by 40 cycles of 95 °C for 30 s and
60 °C for 60 s. The efficiency of gene amplification was normalized
using β-actin with the mean cycle threshold value (Ct), and the 2−ΔΔCT
method was used to measure the relative gene expression.
2.4. Statistical analysis
Independent experiments were repeated at least three times, and
data are presented as mean ± SEM for all duplicates within an in-
dividual experiment. The relative quantitative expressions of the genes
by qRT-PCR were analyzed using analysis of variance (ANOVA). The
significant differences and multiple comparisons between the mean
differential expressions of candidate genes in different treated groups at
phttps://www.ncbi.nlm.nih.gov/tools/primer-blast/
nitro group, and NeH bending in primary amines respectively (Fig. 2f).
Moreover, the physicochemical stability analysis of BRM270 NPs re-
vealed that the particle size gradually increases over time. The BRM270
NPs in pH 7.5 showed a rapid increase in their size as compared to the
acidic pH conditions such as pH 4.5 and pH 5.5 (Fig. 2g). The size
distribution histogram of DLS indicated that the average size was
46.5 nm (Fig. 2h).
3.1.1. Drug release study of BRM270 nanoparticles
BRM270 NPs released in different pH levels at 37 °C in different pH
buffered solution at pH 5.0 and pH 7.4 (Fig. 2i). Maintaining the tem-
perature at 37 °C mimics the normal physiological temperature. Low
drug release was observed at pH 7.4 at 12 h but at pH 5, the drug release
was nearly 80%. The release study suggested that BRM270 NPs was
completely acidic pH-responsive thus releasing at the lower pH condi-
tions.
3.1.2. HPLC fingerprints for BRM270 drug
The HPLC chromatograms were detected at 280 nm for free
BRM270 vs. BRM270 NPs (1mg/ml) solution showed major peaks at
retention times 38.8, 65, and 67.5min, respectively (Fig. 2j). The spe-
cific phytochemical compounds were identified by comparing their
retention times and the UV–visible spectra with earlier reported study
[16]. Thus, the purity of each chromatographic peak was well identified
with their characteristic retention times at 280 nm wavelength. The
peaks were observed and determined to be quercetin, dihydroguaiaretic
acid and sauchinone.
Fig. 2. Characterization of BRM270 NPs
formulation (a) FE-SEM image of free
BRM270 particles (scale bar 20 μm). (b) FE-
SEM image of BRM270 NPs (scale bar 1 μm).
(c) HR-TEM analysis of free BRM270 parti-
cles (scale bar 20 μm). (d) HR-TEM analysis
of BRM270 NPs (scale bar 2 μm). (e)
Absorption spectra of the BRM270 NPs were
analyzed using UV–visible spectroscopy. (f)
Fourier transform infrared spectrum of the
BRM270 NPs. (g) Physico-chemical stability
analysis of BRM270 NPs over time in phos-
phate buffer saline at pH 4.5, pH 5.5 and
pH 7.5 levels at 4 °C (h) DLS histogram
showing particle size distribution of synthe-
sized silver nanoparticles (i) pH dependent
release study of BRM270 NPs by calculating
the amount of BRM270 NPs in acetate
(pH 5.0) and phosphate (pH 7.4) buffer ana-
lysis filtrate at different time intervals. (j)
HPLC chromatograms of BRM270 methanol
extract and BRM270 NPs. Quercetin, dihy-
droguaiaretic acid, sauchinone.
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3.1.3. Distribution and internalization mechanisms of BRM270 NPs in
cancer cells
The HR-TEM images of HepG2 cells treated with BRM270 NPs de-
monstrated the distribution and internalization of the NPs into the
HepG2 cells (Fig. 3). The control group of cells without any treatment
was shown in Fig. 3a. Several dispersed BRM270 NPs appeared in the
cell cytoplasm (Fig. 3b). More importantly, Fig. 3c & d shows the NPs
that were residing completely within the nucleus. These results re-
garding the detailed BRM270 NP location at the subcellular level have
not been previously reported. BRM270 NPs were rapidly internalized
into the cell after the NPs were added. TEM images showed that after
1 h of BRM270 NP treatment, the NPs reached the surrounding areas of
the nucleus, and the cells continued to uptake the BRM270 NPs. The
results showed that the intracellular distribution among the cells was
the same regardless of the size of the particle. The presence of BRM270
NPs was found on the membrane while entering inside the cell (Fig. 3d
& e). A typical endosome-like structure was found near the cell
membrane containing two groups of BRM270 NPs, which later moved
inside the cell (Fig. 3f). Cellular internalization was more efficient with
the endocytosis process because of the smaller size of the NPs; there-
fore, a greater accumulation of BRM270 NPs was observed in the cell
membrane and cytoplasm. The presence of BRM270 NPs was found
inside the nuclear membrane (Fig. 3g & h). All of these images indicate
that endocytosis is the mechanism behind the internalization of
BRM270 NPs in the HepG2 cells.
3.2. Cellular viability in HBM and HepG2 cells
To understand the effect of free BRM270 vs. BRM270 NPs with
Doxorubicin in both HepG2 and HBM cell proliferation, and to de-
termine an effective concentration of BRM270 NPs, we performed an
MTT assay following the treatment at a range of doses from 0 to 12 μg/
ml. BRM270 NPs caused a dose-dependent decrease in HepG2 cells and
inhibited their proliferation (Fig. 4a). The results revealed that the
Fig. 3. Intracellular distribution of BRM270 NPs in HepG2 cells. (a) Control cells without BRM270 NPs treatment. (b) Group of BRM270 NPs located inside the
cytoplasm. (c, d) BRM270 NPs residing on and inside the nucleus. (e) BRM270 NPs entering inside the cytoplasm through the membrane. (f) Spherical nanoparticles
are found in the cytoplasm. (g, h) BRM270 NPs residing inside and on the nuclear membrane (scale bar= 500 nm to 2 μm).
M. Gera et al. Materials Science & Engineering C 97 (2019) 166–176
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inhibitory effect of BRM270 NPs on the cancer cells was much stronger
as compared to the free BRM270 particles and the standard drug dox-
orubicin, the positive control at the same concentration at 24 h. How-
ever, it was found that free BRM270, BRM270 NPs, and doxorubicin
showed less or no effect on the growth of normal HBM cells.
3.2.1. Apoptosis assay by Annexin-V-FITC/PI staining
The apoptotic cell death was measured with fluorescein iso-
thiocyanate-conjugated Annexin V and PI staining of HepG2 cells after
treatment with the optimized concentration of free BRM270 and
BRM270 NPs. The results showed that the number of apoptotic cells
(Annexin V-positive cells) increased in the BRM270 NP-treated cells
after 24 h exposure. The Annexin V-FITC assay showed 12.5% of the
free BRM270 treated cells and 44.4% of the BRM270 NPs treated cells
within 24 h underwent apoptosis. No significant (papoptosis. Caspase 9
binds to Apaf-1 leading to the activation of the protease which then
cleaves and activates caspase 3. It also promotes DNA damage-induced
apoptosis in an ABL1/c-Abl-dependent manner and proteolytically,
cleaves poly (ADP-ribose) polymerase (PARP). MMP9 plays an essential
role in local proteolysis of the extracellular matrix and in leukocyte
migration.
The graphical network among the candidate genes was viewed
using the “abstracted” option of ToppCluster, excluding the genes from
the network and the “gene level” option. To provide a dissected gene
level view, we choose the GO of the biological process shared among
the genes expressed (Fig. 5b). From the abstracted network analysis, the
distinct functional separation was observed. ToppCluster allows se-
lecting the terms of interest to be included in the network. Notably, the
pathway analysis revealed close associations of the variety of biological
processes including transcriptional misregulation in cancer
(p=0.00029) (IL6, p53, MMP9), PI3K-Akt signaling pathway
(p=3.75E−05) (IL6, CASP9, BCL2, p53), HIF-1 (p=0.00485) (IL6,
Fig. 4. Cell culture assay (a) cellular viability of HepG2 and HBM cells treated with BRM270, BRM270 NPs, and doxorubicin. Percentage of viable cells was evaluated
using MTT assay. Each value represents the mean ± SE. (b) Apoptosis assay for the HepG2 cells treated with (a) free BRM270 and (b) BRM270 nanoparticles. (c)
Confirmation of the DNA fragmentation apoptosis, condensation, and nuclei of early apoptotic cells show lobular or fragmented and shrunk blue bodies in the
BRM270 NPs treated a group of cells. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
M. Gera et al. Materials Science & Engineering C 97 (2019) 166–176
172
BCL2), TNF-signaling pathways (p=0.00491) (IL6, MMP9), apoptosis
(p=2.26E−07) and p53 signaling pathway (p=2.61E−05) (CASP9,
BAX, BCL2, p53), thyroid hormone signaling pathway (p=0.00532)
(CASP9, p53), neurotrophin signaling pathway (p=8.92E−05) (BAX,
BCL2, p53), proteoglycan signaling pathway (p=0.0143) (p53,
MMP9).
3.2.3. Validation of the relative expression of candidate genes using qRT-
PCR
The relative gene expression levels of selected genes (IL6, Caspase 9,
BAX, BCL2, p53, and MMP9) have been assessed by normalizing ex-
pression levels of the samples from the different treated groups against
the transcript levels of an endogenous reference GAPDH (Fig. 5c).
The expression of multifunctional cytokine, IL6 showed significant
(pbecause of
their significant physicochemical properties within the nano-range,
which makes it highly efficacious. In support of these findings, one of
the previous studies conducted by Mongre et al. studied free BRM270
particles and showed cytotoxicity with relatively high concentrations of
125 μg/ml in Cal72 and SaOS-2 SLCICs [18]. This study concluded that
for free BRM270 particles, there must be a need to expose the cancer
cells to a high concentration of the drug because of its larger size and
irregular shape, which restricts its cellular permeability and lowers
bioavailability and the distribution of the cells; therefore, our prime
aim was to systematically design a BRM270 within a nano-range for
providing a minimum effective dose, which is critical for clinical trials
in future experiments.
In addition, BRM270 NPs were found to be more effective compared
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incubation period. This demonstrates that BRM270 showed better
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Mrinmoy Ghosh is working on genetic engineering, NGS
technology and stem cell research.
Neelesh Sharma is working on stem cell research and
mastitis in dairy animals with emphasis on use of nano
particles and herbal drugs for its management.
Do Luong Huynh is working on cancer stem cell research
in phytochemicals.
Nisansala Chandimali is working on cancer stem cell re-
search in phytochemicals.
Hyebin Koh is working on cancer stem cell research in
phytochemicals.
Jiao Jiao Zhang is working on cancer stem cell research in
phytochemicals.
Tae Yoon Kang is working on cancer stem cell research in
phytochemicals.
Yang Ho Park is from Jeju National University Cancer
Stem Cell and System Biology. He has published more than
80 papers in reputed journals.
Taeho Kwon is working on cancer stem cell research in
phytochemicals.
Dong Kee Jeong is from Jeju National University Cancer
Stem Cell and System Biology. He has published more than
80 papers in reputed journals.
M. Gera et al. Materials Science & Engineering C 97 (2019) 166–176
176
	Synthesis and evaluation of the antiproliferative efficacy of BRM270 phytocomposite nanoparticles against human hepatoma cancer cell lines
	Introduction
	Material and methods
	Chemicals and reagents
	Preparation of BRM270 nanoparticles
	Physico-chemical characterization of BRM270 NPs
	Physico-chemical stability studies
	In vitro drug-release study
	High-performance liquid chromatography (HPLC) analysis
	Cell culture and treatment conditions
	Intracellular distribution of BRM270 NPs
	Evaluation of cytotoxicity assay
	Apoptosis assay by flow cytometry
	DNA fragmentation using Hoechst 33258 staining
	Annotation of Gene Ontology and pathway analysis
	Determination of relative expression of candidate genes by qRT-PCR
	Statistical analysis
	Results
	Preparation and characterizationof BRM270 NPs
	Drug release study of BRM270 nanoparticles
	HPLC fingerprints for BRM270 drug
	Distribution and internalization mechanisms of BRM270 NPs in cancer cells
	Cellular viability in HBM and HepG2 cells
	Apoptosis assay by Annexin-V-FITC/PI staining
	Clustering of candidate genes and annotation of pathways
	Validation of the relative expression of candidate genes using qRT-PCR
	Discussion
	Conclusion
	Disclosure
	Acknowledgments
	References