Surface modified PAMAM dendrimers with gallic acid inhibit, cell proliferation, cell migration and inflammatory response to augment apoptotic cell death in human colon carcinoma cells

Article abstract of DOI:10.1080/07391102.2020.1802344

To overcome the obstacle of anti-cancer therapy significant attention has been drawn for improving drug delivery system. Since recent past, different approaches were applied using synthetic or natural derivatives for improving efficacy of anti-cancer drugs in cancer therapeutics. Gallic acid (GA) is a natural polyphenol, which exhibits a broad spectrum of biological activities, but its therapeutic application was limited due to poor bioavailability and toxicity. In the present study, we had conjugated the GA with PAMAM dendrimers and proposed the insights of molecular mechanism on inhibition of cell proliferation and programmed cell death through apoptotic pathway in human colon carcinoma cells. GA was chemically conjugated with 4.0 G PAMAM dendrimer at outer surface and characterized by different biophysical methods. We further examined its bioavailability, anti-cancer activity and explored the molecular mechanism of programmed cell death signaling in HCT116 cells. The results show that PAMAM-GA conjugate inhibits cell proliferation of different origin of cancer cells, improves cellular uptake of GA, inhibits colonogenic ability, restricts cancer cell migration by down regulating the expression of MMP-9, inhibits NF-kB activation and release of pro-inflammatory cytokines to manifest apoptotic cell death in HCT 116 cells rather than necrosis. On other hand, PAMAM-GA conjugate showed negligible cytotoxic response as compared to the free Gallic acid to the normal cells. In conclusion, findings of this study revealed that PAMAM-GA conjugate improves the bioavailability of GA and specificity towards cancer cellsto manifests apoptotic cell death. This indispensable approach may be beneficial for the revolution of anti-cancer therapy. Communicated by Ramaswamy H. Sarma.

Full text of DOI:10.1080/07391102.2020.1802344

Journal of Biomolecular Structure and Dynamics
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tbsd20
Surface modified PAMAM dendrimers with gallic
acid inhibit, cell proliferation, cell migration and
inflammatory response to augment apoptotic cell
death in human colon carcinoma cells
Khushbu Priyadarshi , Kavita Shirsath , N. Bhargav Waghela , Anupama
Sharma , Ajay Kumar & Chandramani Pathak
To cite this article: Khushbu Priyadarshi , Kavita Shirsath , N. Bhargav Waghela , Anupama
Sharma , Ajay Kumar & Chandramani Pathak (2020): Surface modified PAMAM dendrimers with
gallic acid inhibit, cell proliferation, cell migration and inflammatory response to augment apoptotic
cell death in human colon carcinoma cells, Journal of Biomolecular Structure and Dynamics, DOI:
10.1080/07391102.2020.1802344
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JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
https://doi.org/10.1080/07391102.2020.1802344
Surface modified PAMAM dendrimers with gallic acid inhibit, cell proliferation,
cell migration and inflammatory response to augment apoptotic cell death
in human colon carcinoma cells
a#
a,b#
a
a
c
Khushbu Priyadarshi , Kavita Shirsath , N. Bhargav Waghela , Anupama Sharma , Ajay Kumar and
a
Chandramani Pathak
a
Cell Biology Laboratory, School of Biological Sciences & Biotechnology, Indian Institute of Advanced Research, Gandhinagar, India;
Department of Zoology, The Maharaja Sayajirao University of Baroda, Vadodara, India; Department of Zoology, Banaras Hindu University,
b
c
Varanasi, India
Communicated by Ramaswamy H. Sarma
ABSTRACT
ARTICLE HISTORY
Received 17 April 2020
Accepted 23 July 2020
To overcome the obstacle of anti-cancer therapy significant attention has been drawn for improving
drug delivery system. Since recent past, different approaches were applied using synthetic or natural
derivatives for improving efficacy of anti-cancer drugs in cancer therapeutics. Gallic acid (GA) is a nat-
ural polyphenol, which exhibits a broad spectrum of biological activities, but its therapeutic applica-
tion was limited due to poor bioavailability and toxicity. In the present study, we had conjugated the
GA with PAMAM dendrimers and proposed the insights of molecular mechanism on inhibition of cell
proliferation and programmed cell death through apoptotic pathway in human colon carcinoma cells.
GA was chemically conjugated with 4.0 G PAMAM dendrimer at outer surface and characterized by dif-
ferent biophysical methods. We further examined its bioavailability, anti-cancer activity and explored
the molecular mechanism of programmed cell death signaling in HCT116 cells. The results show that
PAMAM-GA conjugate inhibits cell proliferation of different origin of cancer cells, improves cellular
uptake of GA, inhibits colonogenic ability, restricts cancer cell migration by down regulating the
expression of MMP-9, inhibits NF-kB activation and release of pro-inflammatory cytokines to manifest
apoptotic cell death in HCT 116 cells rather than necrosis. On other hand, PAMAM-GA conjugate
showed negligible cytotoxic response as compared to the free Gallic acid to the normal cells. In con-
clusion, findings of this study revealed that PAMAM-GA conjugate improves the bioavailability of GA
and specificity towards cancer cellsto manifests apoptotic cell death. This indispensable approach may
be beneficial for the revolution of anti-cancer therapy.
KEYWORDS
Cancer; apoptosis; gallic
acid; PAMAM dendrimers;
PAMAM-GA conjugate
CONTACT Chandramani Pathak
cmpathak@iiar.res.in
Supplemental data for this article can be accessed online at https://doi.org/10.1080/07391102.2020.1802344.
First and second authors have an equal contribution.
#
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
K. PRIYADARSHI ET AL.
Gallic acid (GA) is conjugated with 4.0 G PAMAM dendrimers via an amide linkage to form
PAMAM-GA conjugate. The treatment of PAMAM-GA conjugate to cancer cells may induces loss
of mitochondrial membrane potential with subsequent release of cytochrome c from mitochon-
drial intermembrane space into the cytosol where it forms apoptosome complex in association
with Apaf-1 and Procaspase-9. This event leads to the proteolytic activation of caspase-3 and
ultimately cells undergo toward apoptotic cell death.
Introduction
highly branched structure and suitable core moiety for
entrapment or attachment of the drug. Among the other
nanocarriers, dendrimers have distinctive features and
emerging applications in nanomedicine (Kesharwani et al.,
Colorectal cancer (CRC) is the third most commonly diag-
nosed cancer and fourth leading cause of cancer-related
death worldwide. Biological and epidemiological studies of
2
014). Among all the dendrimers, PAMAM (Poly(amidoamine)
colorectal cancer indicate
a clear association between
has drawn the attention for the application of the drug deliv-
ery system due to unique and versatile attractive features.
Polyamidoamine (PAMAM) dendrimers possess free amine
groups on their surface, which contribute to their enhanced
cytoplasmic delivery via the proton-sponge mechanism.
Besides these unique characteristics, their nanosize and
highly aqueous nature confers excellent drug delivery prop-
erties (Esfand & Tomalia, 2001). PAMAM dendrimers are the
first synthesized and commercialized dendrimers family. They
are considered as ideal carriers for the delivery of therapeutic
agents including anticancer drugs because of their small size,
high aqueous solubility and a large number of chemically
chronic inflammation. Generally, chemotherapy is the first
preferred anti-cancer therapy in many types of cancer, but
within a short span of treatment exhibits adverse side effects
along with systemic toxicities and immune surveillance that
may causes failure of anti-cancer therapy. In the recent past,
several strategies have been employed for the drug delivery
system to improve cancer therapy. Since the recent past
application of nanocarrier has drawn considerable attention
to improve drug delivery system.
Dendrimers are emerging biopolymers, which have been
considered as the versatile nanocarriers for drug delivery
owing to their unique structure and presence of a multi-
functional group on their surface (Sherje et al., 2018). versatile surface groups (Medina
&
El-Sayed, 2009).
Dendrimers have distinctive features, which may include the Interestingly, controlled synthesis of PAMAM dendrimers pro-
capability to improve the solubility, bioavailability, and per- vides a suitable nanocarrier for drug delivery. Typically,
meability of drugs or desired molecules. They are unique bio- PAMAM dendrimers have a size of 2.3 nm in generation 2.0
degradable and non-immunogenic polymer. They have a (G 2.0) to 5.3 nm in G 5.0 (Svenson & Tomalia, 2012).

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
3
Importantly, PAMAM dendrimers have the size in the range 2, PARP, procaspase-9, procaspase-3, MMP-9, p53, p65, GAPDH,
of 1 to 10 nm, which can diffuse directly into tumor cells COX-IV, Histone H3, b-actin, and anti-rabbit HRP linked second-
(
Sinek et al., 2004). These formulations tend to accumulate in ary antibodies were purchased from Cell Signaling Technology
solid tumors via the enhanced permeability and retention (Danvers, MA). Rabbit polyclonal antibody against Apaf-1 was
purchased from BD Biosciences (New Jersey, USA). All other
chemicals used were of analytical grade and purchased from
Merck (Darmstadt, Germany).
(
EPR) effect, thus avoiding accumulation in noncancerous tis-
sues (Medina & El-Sayed, 2009).
Many chemotherapeutic drugs are derivatives of natural
polyphenols or flavonoids. The major obstacles include their
toxicity or poor bioavailability. Gallic acid (3,4,5-trihydroxy
benzoic acid) is a natural polyhydroxy phenolic compound,
Synthesis of 4.0 G PAMAM dendrimers
generally found in various plants, fruits and foods 4.0 G EDA core PAMAM dendrimer was synthesized using
Kahkeshani et al., 2019). In recent past, GA has been shown Tomalia’s divergent growth method (Tomalia et al., 1986).
(
chemopreventive and anti-proliferative activities against dif- The Michael addition of primary amine (EDA in the first step)
ferent types of human cancers (Verma et al., 2013). GA is to the double bond of methyl acrylate (MA) resulted in the
formation of ester terminated half-generation dendrimers,
designated as G (n 0.5). This was followed by amidation of
the carbomethoxy intermediate with a large excess of EDA in
methanol to produce amine-terminated full generation den-
drimers referred to as G (n). The reiteration of these two
steps led to the formation of the next higher generation
dendrimers. 4.0 G PAMAM dendrimer was synthesized using
the above approach shown in Figure 1.
The Michael addition of EDA to methyl acrylate in metha-
nol yields half-generation dendrimer denoted as G 0.5
whereas its further amidation with EDA in methanol yields a
full generation dendrimer (G 1.0). The repetition of these two
reactions yields a G 4.0 PAMAM dendrimer.
known to have has a broad spectrum of biological activities
including antioxidant, anti-inflammatory, anti-microbial anti-
viral, anti-allergic, anti-melanogenic, and anti-mutagenic
activities etc. (Badhani et al., 2015). Similar to other natural
polyphenolic derivatives, GA also has poor bioavailability,
limited tissue distribution and rapid degradation, which limit
its clinical application to consider as drug candidate (Brglez
Mojzer et al., 2016; Manach et al., 2005). In the recent past,
GA was conjugated with PAMAM dendrimes and shown anti-
proliferative response, but not explored the molecular mech-
anism of cell death (Sharma et al., 2011). In the present
study, we chemically conjugated the GA with 4.0 G PAMAM
dendrimers to extend the bioavailability and explored the
molecular mechanism of inhibition of cell proliferation and
induction of cell death in HCT116 cells.
Synthesis of PAMAM-GA conjugate
The PAMAM-GA conjugate was synthesized by coupling reac-
tion (Sharma et al., 2011). Gallic acid was covalently linked to
Materials & methods
4
.0 G PAMAM dendrimer using EDAC as a cross-linking agent.
Materials
An aqueous solution of EDAC (2.24 mM) was mixed with
The molecular biology grade chemicals were purchased com-
ethanolic (1:1) solution of GA (2.24 mM) with constant stir-
mercially. Gallic acid (GA), ethylenediamine (EDA), methyl acryl- ring. Subsequently, 4.0 G PAMAM dendrimer (0.070 mM) was
ate (MA), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide added dropwise with constant stirring in the dark. The stir-
hydrochloride (EDAC), Poly-L-lysine, (3-(4,5-dimethylthiazol-2-yl)- ring was continued for another 24 h at room temperature.
0
2
,5-diphenyltetrazolium bromide (MTT), Trypan blue, 4 ,6-diami- Thereafter, the reaction mixture was placed for dialysis under
dino-2-phenylindole (DAPI), bicinchoninic acid (BCA) protein esti- distilled water to remove the byproducts and/or the
mation kit, dialysis tubing cellulose membrane and anti-mouse unreacted reagents. The scheme of synthesis of PAMAM-GA
horseradish peroxidase (HRP) conjugated secondary antibody conjugate has been shown in Figure 2.
were purchased from Sigma-Aldrich (St. Louis, MO, USA). RPMI-
1
640, DMEM, Dulbecco’s Phosphate buffer saline (DPBS),
Characterizations of PAMAM-GA conjugate
.0 G of PAMAM dendrimer and PAMAM-GA conjugate were
Penicillin, Streptomycin, Neomycin (PSN) antibiotic cocktail, Fetal
bovine serum (FBS), EnzChekCaspase-3 Assay Kit, Opti-MEM,
Lipofectamine LTX, LysoTracker Red DND-99, Hoechst 33342 dye
and antibody against cytochrome c, human IL1b and IL-6 ELISA
4
characterized by UV- Visible spectrophotometry, Fourier
1
transform infrared spectroscopy (FT-IR) and H NMR spectros-
kits were purchased from Invitrogen (Life Technologies, USA). copy. Briefly, 0.01% (w/v) 4.0 G PAMAM dendrimer and
0
0
Annexin V-FITC/Propidium Iodide (PI) staining kit, 5,5 ,6,6 -tetra- PAMAM-GA conjugate were prepared in distilled water and
0
0
chloro-1,1 ,3,3 -tetraethylbenzimidazolylcarbocyanine iodide (JC- scanned in the range of 200–400 nm using Multimode micro-
) dye and Protein A-Sepharose beads were purchased from plate reader (SpectraMax M2e, Molecular Devices, USA).
1
Biovision Inc. (California, USA). LDH cytotoxicity assay kit was Further, FT-IR Analysis was carried out by using Perkin Elmer,
purchased from Takara Bio Inc. (Shiga, Japan). The complete G-FTIR. Various peaks were interpreted for different groups.
EDTA-free Protease inhibitor cocktail (PIC) was purchased from Surface group modification on dendrimer was studied
Roche Applied Science (Penzberg, Germany). 0.2 lm PVDF mem- through IR spectra. To confirm the synthesis of a conjugate,
1
brane, iScript cDNA Synthesis Kit and ECL substrate were pur-
H NMR spectra of 4.0 G PAMAM dendrimer and PAMAM-GA
chased from Bio-Rad (Philadelphia, USA). Antibodies against Bcl- conjugate were recorded on Bruker Avance II (500 MHz) Bru

4
K. PRIYADARSHI ET AL.
Figure 1. Schematic representation illustrating the synthesis of 4.0 G PAMAM dendrimer.
obtained from National Centre for Cell Science (NCCS), Pune,
India. HCT 116and HT29 cells were maintained in RPMI-1640
and , MCF7, and NIH 3T3 cells were maintained in DMEM cul-
ture media, supplemented with 2 mmol/L L-Glutamine, 10%
FBS and an antibiotic cocktail containing 5 mg/ml penicillin,
5
mg/ml streptomycin and 10 mg/ml neomycin (Gibco, Life
Technologies, USA). The cells were maintained under humidi-
ꢀ
fied atmosphere with 5% CO at 37 C. Exponentially grow-
2
ing cells were used for all the experiments.
Treatment to cells with GA and PAMAM-GA conjugate
Stock solutions of PAMAM-GA conjugate was prepared in
ultrapure RNase-free distilled water at a concentration of
1
00 mM and 60 mM respectively. The PAMAM-GA conjugate
was filtered through a 0.2 mm sterilized filter before use.
Exponentially cultured cells were treated with different con-
centrations of GA and PAMAM-GA conjugate (2.5 mM, 5 mM,
and 10 mM) for mentioned time points in the figure legends.
Figure 2. Scheme of the conjugation of GA with 4.0 G PAMAM dendrimer. GA
was conjugated with PAMAM dendrimer using EDAC as a linker. This occurs via
2
amide linkage between the –COOH group of GA and surface –NH group of
PAMAM dendrimer.
Determination of IC50
spectrometer. The samples were solubilized in methanol. The IC50 of GA and PAMAM-GA conjugate was determined
4
Chemical shifts were reported as d (ppm) relative to by MTT assay. Briefly, 1 ꢁ 10 HCT 1116 and NIH 3T3 cells
Tetramethylsilane (TMS) as a standard. Various shifts in the were seeded in a 96 well plate and incubated for 24 h. Cells
peaks were interpreted for different groups present in the were treated with GA (0–20 mM) and PAMAM-GA conjugate
conjugated system. Moreover, the particle of the size of 4.0 G (0–20 mM) for 24 h. After completion of incubation, cells
PAMAM dendrimer and PAMAM-GA conjugate was analyzed were washed using DPBS (pH 7.4) and 0.5 mg/ml of MTT
by Dynamic Light Scattering (DLS) particle size analyzer.
solution was added to each well and allowed incubation for
ꢀ
4
h at 37 C in a CO incubator. Residual MTT was discarded
2
and DMSO was added to dissolve the formazan crystals.
Absorbance was recorded at a wavelength of 570 nm with a
Cell lines and cell culture
The human colorectal carcinoma (HCT 116), human breast reference of 650 nm using a Multimode microplate reader
carcinoma (MCF7), human colorectal adenocarcinoma (HT29), (SpectraMax M2e, Molecular Devices, USA). The results were
and mouse embryonic fibroblast (NIH 3T3) cell lines were represented in terms of percentage inhibition of cell

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
5
proliferation and IC50 values were determined from the dose- RPMI 1640 complete cell culture medium for 8–10 days for
response curve using Sigma Plot 12.0.
colony formation. The visible colonies were fixed with metha-
nol and stained with 0.2% crystal violet stain. The data were
represented as percentage plating efficiency (PE), defined as
Cell proliferation and cell viability assay
%
PE ¼ (Number of colonies formed/Number of cells seeded)
The effect of GA and PAMAM-GA conjugate on cell prolifer- ꢁ 100.
ation was evaluated by MTT assay as described above.
Subsequently, cell viability was carried out by trypan blue
Wound healing assay
exclusion assay. HCT 116, HT 29, MCF 7 and NIH 3T3 cells
were treated with 2.5 mM, 5 mM and 10 mM of GA and
PAMAM-GA conjugate for 6 h, 12 h, and 24 h respectively.
Cells were harvested, washed and resuspended in DPBS (pH
The effect of PAMAM-GA conjugate on cell migration was
monitored by in vitro wound-healing assay as described ear-
lier (Vaidya et al., 2019). Briefly, 5 ꢁ 10 HCT 116 cells were
5
7.4). The cell suspension was mixed with an equal amount of
0.4% trypan blue solution and allowed to incubate for
2–3 min. Live and dead cells were counted and the percent-
seeded in a 6 well plate and allowed to reach confluency. A
scratch was created in the confluent monolayer using a ster-
ile tip. The cells were washed with DPBS to remove debris
and subsequently, treated with different concentrations of
PAMAM-GA conjugate in a serum-free medium as described
in the figure legend. After completion of incubation, the
medium was replaced with fresh serum-free medium and
cells were allowed to migrate for 72 h. The images of
scratches were captured under the DIC filter of the inverted
microscope (DP71, Olympus, Japan). The distance covered by
the migrating cells was calculated by comparing the same
field at 0 h and 72 h using Image-Pro MC 6.1 (Bethesda,
MD, USA).
age of cell viability was calculated using formula, % cell
death ¼ Number of dead cells/Total number of cells ꢁ 100.
The results are represented in terms of the percentage of
cell death relative to untreated control.
In vitro release and cellular uptake assay
In vitro release of Gallic acid (GA) from PAMAM-GA conjugate
was monitored by the dialysis method. A known quantity
(
1.0 mg/ml) of GA and PAMAM-GA conjugate were filled in a
dialysis tube (10 KDa). The dialysis tube was suspended in
1
0 ml of RPMI-1640 medium supplemented with 10% FBS
ꢀ
and 1% PSN. The entire system was maintained at 37 ± 0.5 C
at the constant stirring of 200 ± 2 rpm. 1 ml of the medium
was withdrawn at respective time points and replaced with
fresh media. The absorbance of GA was recorded at 227 nm
using a Multimode microplate reader (SpectraMax M2e,
Molecular Devices, USA). Further, the concentration of drug
release was quantified using the standard curve of GA. In
addition, cellular uptake of PAMAM-GA conjugate in the cells
was examined by detecting the presence of GA in the cell
Analysis of apoptotic cell death
The apoptotic potential of PAMAM-GA conjugate was exam-
ined by Annexin V/PI staining using Annexin V-FITC/PI apop-
tosis assay kit (Biovision Inc, California) as described
previously (Vaidya et al., 2019). More than 100 cells from ran-
dom fields were examined for apoptotic cell death. All the
images were acquired by fluorescence inverted microscope
using Image-Pro MC 6.1 software (Bethesda, MD, USA) and
analyzed by Image J software(NIH, USA). Further, to confirm
changes in mitochondrial membrane potential (MMP) was
monitored by using a lipophilic fluorescent probe JC-1. For
qualitative analysis, cells were treated with 2.5 mM, 5 mM
and 10 mM of PAMAM-GA conjugate for different time points
mentioned in the figure legend. Thereafter, cells were
5
lysate. In brief, 1 ꢁ 10 HCT-116 cells were seeded in a 24
well plate and allowed to grow for 24 h. Thereafter, the cells
were treated with different concentrations of PAMAM-GA
conjugate for 6 h and 12 h. The cells were washed with DPBS
(
pH 7.4) twice and lysed with 0.1% Triton-X 100 (in DPBS).
GA concentration in supernatants was measured at a wave-
length of 290 nm using a Multimode microplate reader
(
lation of GA was normalized to total protein content. The
protein concentration was determined by using a BCA pro-
tein assay kit (Sigma-Aldrich, USA) according to the manufac-
turer’s instructions.
ꢀ
stained with JC-1 dye (5 mg/ml) for 20 min in dark at 37 C.
Subsequently, the cells were counterstained with DAPI for
SpectraMax M2e, Molecular Devices, USA). Cellular accumu-
5
–7 min in dark at room temperature and observed under a
fluorescent microscope (DP-71, IX81, Olympus, Japan). More
than 100 cells from three random fields were examined for
the change in MMP. All the images were acquired by Image-
Pro MC 6.1 (Bethesda, MD, USA) and analyzed by Image J
software (NIH, USA). In addition, for quantitative analysis, the
treated cells were stained with JC-1 dye (5 lg/ml) for 20 min
Clonogenic ability assay
The in vitro cell survival assay was performed to examine the
effect of PAMAM-GA conjugate on colony-forming ability of
HCT 116 cells. Briefly, 4 ꢁ 10 cells were seeded in a 48-well
ꢀ
in the dark at 37 C. Subsequently, the fluorescence was
4
quantified using a Multimode microplate reader at the exci-
plate. Cells were treated with 2.5 mM, 5 mM and 10 mM of tation of 485 nm and the emission at 527 nm (green fluores-
PAMAM-GA conjugate for 6, 12 and 24 h. After completion of cence) and 590 nm (red fluorescence). The ratio of red
incubation, cells were trypsinized and 1,000 viable cells were fluorescence to green fluorescence was considered as a
seeded in a 6-well plate. The cells were allowed to grow change in the Mitochondrial Membrane Potential (DWm).

6
K. PRIYADARSHI ET AL.
Caspase-3 activity assay
(Waghela et al., 2015). In brief, After completion of treatment,
cells were harvested and lysed in lysis buffer containing
The activity of caspase-3 was examined by using the EnzChek
Caspase-3 Assay Kit (Invitrogen, Life Technologies, USA)
2
50 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na HPO ,
2 4
1
.4 mM KH PO 100 lM PMSF and 1X protease inhibitor cock-
6
2
4,
according to the manufacturer’s protocol. Briefly, 1 ꢁ 10 HCT
tail (Roche, Germany) for 5 min on ice. The lysate was centri-
1
16 cells were treated with 2.5 mM, 5 mM and 10 mM of
ꢀ
fuged at 1,000 ꢁ g for 5 min at 4 C and the resulting
PAMAM-GA conjugate for different time points as shown in
the figure legend. Untreated cells were used as control. After
completion of incubation, cells were harvested and washed
twice with DPBS. The pellet was resuspended in 1X cell lysis
supernatant was collected as the cytosolic fraction. The pellet
was resuspended in mitochondrial lysis buffer containing
5
0 mM Tris-HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 2 mM
EGTA, 0.2% (v/v) Triton X-100, 0.3% NP-40, 100 lM PMSF and
X protease inhibitor cocktail for 5 min on the ice. Pellet was
ꢀ
buffer for 30 min and centrifuged at 5000 rpm for 5 min at 4 C.
1
The amount of protein in the resultant supernatant was esti-
mated using the BCA protein estimation kit. The 100 mg pro-
tein was mixed with a 2X substrate solution containing 50 mM
Z-DEVD-R110 substrate and incubated at room temperature
for 30 min in the dark. The fluorescence was measured at an
excitation and emission wavelength of 496 nm and 520 nm
respectively using multimode plate reader (SpectraMax M2e,
Molecular Devices, USA).
disrupted in cell disruptor and centrifuged at 10,000 ꢁ g for
ꢀ
5
min at 4 C and the supernatant was collected as a mitochon-
drial fraction to examine the expression of cytochrome c and
COX-IV. The whole cell lysate was prepared using RIPA buffer
containing a protease inhibitor cocktail (Roche, USA). Cells
were kept on the ice for 30 min followed by disruption and
ꢀ
centrifugation at 8,000 rpm for 10 min at 4 C and supernatant
was collected. For collection of nuclear fractions, cells were
lysed in hypotonic buffer (10mM HEPES-Kþ pH 7.5, 10mM KCl,
1
1
.5mM MgCl2, 0.1mM DTT, 0.5% Triton X-100, 1mM PMSF and
Necrotic cell death assay
ꢀ
x PIC) for 5 min on ice and spun at 3000 rpm for 2 min at 4 C.
The Necrotic cell death (cytotoxic effect) of GA and PAMAM-
GA conjugate was examined by release of LDH using LDH
activity assay kit (Takara Bio Inc., Shiga, Japan) according to
the manufacturer’s instruction. Briefly, HCT 116 cells and NIH
The pellets were washed with wash buffer (10mM HEPES-Kþ
pH 7.5, 10mM KCl, 1.5mM MgCl2, 0.1mM DTT, 1mM PMSF and
1
1
x PIC) and subsequently lysed with lysis buffer (150mM NaCl,
% Triton X-100, 0.5% Sodium deoxycholate, 0.1% SDS, 50mM
3
T3 cells were treated with 2.5 mM, 5 mM and 10 mM of
Tris pH 8.0, 1mM PMSF, 1mM NaV and 1x PIC) for 25 min on
ice and spun at 10,000 rpm for 15 min. The supernatant was
collected as nuclear fraction. Protein concentration was deter-
mined by using a BCA protein estimation kit (Sigma-Aldrich,
USA) according to the manufacturer’s protocol. 50 lg protein
was resolved on 10% SDS-PAGE and transferred to PVDF mem-
PAMAM-GA conjugate for 6, 12 and 24 h. The assay was per-
formed as mentioned previously (Ranjan & Pathak, 2016).
Microscopic assessment of cytotoxicity was carried out using
Hoechst and propidium iodide staining as described previ-
ously (Vaidya et al., 2019). Cells were analyzed under a fluor-
escence inverted microscope (DP71, Olympus, Japan) and
images were analyzed using ImageJ software (NIH,
Bethesda). Hoechst-stained nuclei were counted and judged
as either viable (lack of propidium iodide staining), apoptotic
ꢀ
brane by wet electro-blotting method at 4 C. The membrane
was blocked with 5% non-fat milk in TBST (10 mM Tris-HCl (pH
7
.4), 0.9% NaCl and 0.05% Tween 20) for 4 h at room tempera-
ture followed by overnight incubation with primary antibody
against p65 (1:1000), cytochrome c (1:1000), procaspase-3
(
Hoechst-stained condensed nuclei with propidium iodide-
stained spots), or necrotic (large nuclei intensely stained with
Hoechst and propidium iodide-stained).
(
1:1000), PARP (1:1000), Bcl-2 (1:250), MMP-9 (1:250), Histone
H3 (1:1000), COX-IV (1:1000), b-actin (1:10,000) and GAPDH
ꢀ
(1:1000) at 4 C. After washing with TBST, the membrane was
probed with HRP conjugated secondary antibodies (1:10,000).
Expression of immune reactive protein was detected by using
Lysosomal integrity assay
The effect of GA and PAMAM-GA conjugate on lysosomal mem- Clarity Western ECL substrate kit according to the man-
brane integrity was examined by using LysoTracker Red DND- ufacturer’s instruction and developed on Kodak X-Omat blue
9
9 dye (Invitrogen, Life Technologies, USA). HCT 116 cells were film (NEN Life Sciences, Inc., Boston, MA) in the dark.
treated with GA and PAMAM-GA conjugate shown in the figure
ꢀ
legend and further stained with Lysotracker red for 1 h at 37 C,
Co-immunoprecipitation assay
followed by counterstaining with DAPI (1 mg/ml) for 5–7 min in
the dark. More than 100 cells from random fields were exam-
ined under a fluorescence inverted microscope (DP-71, IX81,
Olympus, Japan). All the images were acquired by Image-Pro
MC 6.1 (Bethesda, MD, USA) and analyzed by Image J
To analyze the formation of the apoptosome complex, a co-
immunoprecipitation assay was carried out as described ear-
6
lier (Ranjan & Pathak, 2016). In brief, 1 ꢁ 10 HCT 116 cells
were seeded in 60 mm dishes and treated with PAMAM-GA
conjugate for 12 h. The cells were lysed using lysis buffer
(
NIH, USA).
(20 mM Tris-Cl (pH 7.4), 150 mM NaCl, 10% glycerol, 1%
Triton-X-100) containing 1X protease inhibitor cocktail
Sub-cellular fractionation and western blotting
ꢀ
(
Roche, USA) for 30 min at 4 C, followed by disruption and
ꢀ
The expression of cell death regulatory proteins was examined centrifugation at 15,000 ꢁ g for 15 min at 4 C. The clarified
from mitochondrial and cytosolic fractions as described earlier supernatant was collected and its protein concentration was

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
7
Figure 3. Characterization of PAMAM-GA conjugate by FT-IR Spectroscopy. FT-IR spectra of (A) 4.0 G PAMAM dendrimer (B) PAMAM-GA conjugate.
estimated using the BCA protein estimation kit (Sigma- HCT 116 cells were transfected with pEGFP-p65 (gifted by Dr.
Aldrich, USA) according to the manufacturer’s protocol. Johannes Schmid, Med. Uni. Vienna) using Lipofectamine LTX
2
00 lg of protein was incubated with 20 ll Protein A- with Plus reagent (Life Technologies, USA). 24 h post-trans-
ꢀ
Sepharose beads (BioVision, USA) for 1 h at 4 C with gentle fection, the cells were treated with 2.5 mM, 5 mM and 10 mM
shaking. The mixture was centrifuged at 3000 rpm for 5 min PAMAM-GA conjugate for 6 h followed by treament of TNF-a
ꢀ
at 4 C and the supernatant was further incubated with 1 lg for 1 h. Thereafter, the cells were washed with DPBS and
anti-Apaf-1 antibody and 20 ll Protein A-Sepharose beads at analyzed under fluorescence inverted microscope. Untreated
ꢀ
4
C with gentle shaking for overnight. The immunoprecipi- cells were used as control. More than 100 cells from random
tated complexes were washed thrice with RIPA buffer, dena- fields were examined for translocation of GFP tagged p65
tured in 6X Laemmli sample buffer and subjected to from cytosol to nucleus. All the images were acquired under
immunoblotting using the antibody against pro-caspase-9 inverted microscope (DP71, Olympus Japan) using Image-Pro
(
1:1000), cytochrome-c (1:500) and Apaf-1 (1:250) as MC 6.1 software (Bethesda, MD, USA) and analyzed by Image
described above.
J software (NIH, USA).
p65 translocation assay
Quantitative gene expression by real-time PCR
To examine the NF-kB activation the translocation of p65 The mRNA expression of IL-1b and IL-6 was analyzed in
from cytoplasm to nucleus was monitored using fluorescent HCT116 cells upon treatment of PAMAM-GA conjugate for
microscope as described earlier (Vaidya et al., 2019). In brief, 24 h followed by exposure of TNF-a (10 ng/mL) for 1 h. Total

8
K. PRIYADARSHI ET AL.
Figure 5. Release of GA from PAMAM-GA conjugate & uptake. (A) A known
quantity (1 mg/ml) of GA and PAMAM-GA conjugate was placed in a dialysis
ꢀ
tube and suspended in RPMI 1640 medium with 10% FBS at 37 C. At men-
tioned time points, 1 ml of medium was withdrawn and the absorbance of GA
was measured at 430 nm. The released amount of GA was quantified using its
standard curve. The plot represents the amount of GA released at mentioned
time points. (B) HCT 116 cells treated with PAMAM-GA conjugate for 6 h and
1
Figure 4. Characterization of PAMAM-GA conjugate by NMR. H NMR spectra
of (A) 4.0 G PAMAM dendrimer (B) PAMAM-GA conjugate.
1
2 h, were harvested and lysed with 0.1% Triton-X100. The absorbance of GA
was recorded in cell lysate at 290 nm using a Multimode microplate reader. The
RNA was isolated with Trizol reagent (Invitrogen, UK) accord- graphs represent intracellular concentration of GA against the concentration of
PAMAM-GA conjugate. Error bars represent mean ± SEM of three independent
ing to manufacturer’s protocol. The concentration of total
experiments. The difference is significant between GA and PAMAM-GA treat-
RNA was determined by taking the absorbance at 260/
80 nm under UV-visible spectrophotometer (PG Instrument,
ꢂ
ꢂꢂ
ꢂꢂꢂ
ment groups at p ꢃ 0.05, p ꢃ 0.01 and p ꢃ 0.001.
2
USA, T90); 1 lg of total RNA was reversely transcribed, and
cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-
Rad Laboratories Inc., Hercules, CA, USA). A real-time PCR
was performed using the StepOne plus real-time PCR detec-
tion system (Applied Biosystems, Carlsbad, California). The
0
0
AGGCACAGAACCA-3 and reverse, 5 -ACTCCTTAA AGCTGCGC
0
0
AGAA-3 ,18S rRNA Forward,5 -AGAAACGGCTA CCAC ATCCAA-
0
0
0
3
and reverse, 5 -TGTCACTACCTCCCCGTGTCA-3 . Each assay
was normalized by using the difference in critical thresholds
between target genes and 18SrRNA.
reaction was conducted with 20 lL final reaction volume
TM
containing 0.2 lL (100 pMolar) of cDNA, 10 lL iQ
SYBRVR
Green Supermix (Bio-Rad, USA), and 2 lL (1 lM) of each pri-
mer and remaining 5.8 lL of nuclease free water. All reac-
tions were performed in MicroAmp fast optical 96well PCR
plates (Applied Biosystem) and sealed with optical adhesive
covers (Applied Biosystem). 18SrRNA was used as the
endogenous control. Thermal cycler conditions were as fol-
ELISA (enzyme-linked immunosorbent assay)
To examine the release of pro-inflammatory cytokines, ELISA
5
was carried out. 1 ꢁ 10 HCT116 cells were treated with
PAMAM-GA conjugate for 24 h followed by exposure of TNF-a
(10ng/mL) for 1h. The media was collected, and the ELISA was
ꢀ
performed. ELISA plate was coated with 100 ml/well of captured
lows: After denaturing at 95 C for 10 min, PCR was per-
antibody in Coating buffer and the plate was sealed and incu-
bated overnight at 4 C. Next, buffer was aspirated from each
Afterwards, final PCR products were heated to 72 C for 30 s, well and washed 3 times with 250 ml/well wash buffer for 1 min
and the expected size products were confirmed by melting each. The surface of wells were blocked with 200 mL/well of 1x
curve analysis. These following sets of PCR primers were assay diluent solution and incubated at room temperature for 1
formed for 40 cycles, each of which consisted of denaturing
ꢀ
ꢀ
ꢀ
at 95 C for 15 s, annealing/extending at 65 C for 1 min.
ꢀ
0
0
used. IL-1b forward, 5 -GACAACGAGGCGTACGTTCA-3 and h . 100 ml/well of the sample was added to the appropriate well
reverse, 5 - CGATTTCTGTTGACTATCCCGTAA-3 , IL-6 for- and plate was sealed and incubated at room temperature for 2
0
0
0
ward,5 -GCTGC
h. Subsequently, each well was washed with wash buffer and

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
9
Figure 6. Effect of GA and PAMAM-GA conjugate on cell proliferation. IC50 values of (A) GA and (B) PAMAM-GA conjugate on HCT-116 and NIH-3T3 cells by MTT
assay. Cells were treated with a series of concentrations of GA or PAMAM-GA conjugate for 24 h and MTT assay was carried out to determine the inhibition in cell
proliferation. IC50 values were calculated from the dose-response curve using SigmaPlot 13.0
Figure 7. Effect of GA and PAMAM-GA conjugate on cell proliferation. (A) HCT 116, (B) HT-29, (C) MCF-7, and (D) NIH 3T3 cells were treated with 2.5 mM, 5 mM
and 10 mM GA and PAMAM-GA conjugate and MTT assay was carried out to determine their effect on cell proliferation. Error bars represent mean ± SEM of three
ꢂ
ꢂꢂ
ꢂꢂꢂ
p ꢃ 0.001.
independent experiments. The difference is significant between GA and PAMAM-GA treatment groups at p ꢃ 0.05, p ꢃ 0.01 and

1
0
K. PRIYADARSHI ET AL.
Differences were considered statistically significant at
ꢂ
ꢂꢂ
ꢂꢂꢂ
p ꢃ 0.05, p ꢃ 0.01,
p ꢃ 0.001.
Results
Characterization of 4.0 G PAMAM dendrimer and
PAMAM-GA conjugate
Gallic acid was conjugated with 4.0 G PAMAM dendrimer
shown in the schematic representation (Figure 2). The conju-
gation was confirmed by UV-Vis spectroscopy, FT-IR spectral
analysis, NMR spectroscopy, and particle size analysis. The
kmax of 4.0 G PAMAM dendrimer was recorded at 300 nm,
whereas kmax of PAMAM-GA conjugate was shifted to
2
90 nm that was examined by UV-Vis spectroscopy (Figure
S1). Further, FT-IR analysis revealed the appearance of the
ꢄ1
peak of the amide linkage at 1643 cm and aromatic link-
ꢄ1
age (C ¼ C) at 1465 cm
that denotes the conjugation of
4
.0 G PAMAM dendrimer with GA (Figure 3). The NMR spectra
also revealed the formation of PAMAM-GA conjugate with
significant changes in integral values and shifts of the sec-
ondary –CH2 group that was observed upon conjugation.
The important shifts obtained in NMR spectra that were for;
4
.0 G PAMAM Dendrimer: 1H NMR (MeOD) d 2.54 (–NCH2
CH N–); d 2.53 (–NCH CH CO–); d 2.37 (–NCH CH CO–); d
2
2
2
2
2
3
.29 (–CONHCH CH N–); d 2.55 (–CONHCH CH N–); d 3.47
2 2 2 2
Figure 8. Effect of PAMAM-GA conjugate on cell survival. The effect of (–CONHCH CH NH );
d
2.57 (–CONHCH CH NH ); 4.0 G
2 2 2
2
2
2
PAMAM-GA conjugate on cell survival was monitored by colony formation
assay. HCT 116 cells were treated with indicated concentrations of PAMAM-GA
PAMAM- GA conjugate: 1H NMR (MeOD) d 3.13 (–NCH CH
2
2
conjugate for 6 h, 12 h, and 24 h. Subsequently, the cells were harvested, 1000 N–); d 3.31 (–NCH CH CO–); d 2.88 (–NCH CH CO–); d 3.59
2
2
2
2
viable cells were seeded in a 6 well plate and allowed to grow for 8–10 days.
The colonies were fixed with methanol and stained with crystal violet. The colo-
nies were counted and plating efficiency was determined from it. (A)
(
(
–CONHCH CH N–);
–NHCO-GA); d 8.07–8.10 (Ar–H). The appearance of newer
d
3.45 (–CONHCH CH N–);
d
8.53
2
2
2
2
Representative images of the colonies formed. (B) The graph represents the peaks of amide linkage [–NHCO-GA] at 8.53 ppm and Ar–H at
relative plating efficiency of the cells at indicated time points and the concen-
tration of PAMAM-GA conjugate treatment. Error bars represent mean ± SEM of
8
.07–8.10 ppm, confirms the formation of PAMAM-GA conjugate
ꢂ
ꢂꢂ
ꢂꢂꢂ
three independent experiments. p ꢃ 0.05, p ꢃ 0.01 and
p ꢃ 0.001 com- (Figure 4). PAMAM dendrimer and PAMAM-GA conjugate
were further subjected to analysis of particle size distribution.
The particle size of 4.0G PAMAM dendrimer and PAMAM-GA
pared to untreated control cells.
after that 100 mL/well detection antibody was added. The plate
was sealed and incubates at room temperature for one hour
followed by repeated washing. 100 mL/well of Avidin-HRP was
added and plate was sealed and incubated at room tempera-
ture for 30 min followed by repeated washing. 100 mL/well of
substrate solution was added to each well and the plate was
incubated at room temperature for 15 min. Finally, 50 mL/well of
stop solution was added to each well and absorbance was
conjugate was found ꢅ 5.2 nm. However, the conjugation of
GA with PAMAM dendrimer did not show a major alteration
in the particle size. Minor changes in effective diameter were
observed. The average effective diameter of PAMAM dendri-
mer and PAMAM-GA conjugate was obtained as 198.0 and
211.7 nm respectively (Figure S2).
recorded at a wavelength of 450 nm and 570 nm respectively. PAMAM-GA conjugate exhibits sustained release and
The value of absorbance of 570 nm was substracted from the cellular uptake of GA from PAMAM-GA conjugate
absorbance of 450 nm for further analysis. The concentration of
secreted IL-6 and IL-1b was determined from its standard curve.
conjugate in vitro at various time points. We found that
We examined the release study of Gallic acid from PAMAM-GA
PAMAM-GA conjugate exhibits slow release than the free
Gallic acid (Figure 5(A)). Thus, the results illustrate that conju-
gation of Gallic acid with PAMAM dendrimer shows improved
Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA)
followed by Student Newman Keuls (SNK) test for multiple
comparisons using Sigma Plot 13.0 statistical analysis soft-
stability and sustained release that may enhance permeable
retention effects and cellular uptake. Further, the uptake of GA
from PAMAM-GA conjugate on HCT 116 was examined. The
ware. The data were tested for normality by the Shapiro-Wilk results showed that the amount of GA in the cell increased in
test prior to ANOVA. Values were expressed as mean ± SEM a dose-dependent manner at 6 h. The highest amount of GA
from three independent experiments. For the experiments concentration was found on the cells treated with 10mM
involving only two groups, a Student t-test was used. PAMAM-GA conjugate at
6 h as compared to other

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
11
Figure 9. Effect of PAMAM-GA conjugate on cell migration. The migration ability of HCT 116 cells was determined by wound scratch assay and western blotting
after treating the cells with mentioned concentrations of PAMAM-GA conjugate for 6 h, 12 h, and 24 h. (A) The graph represents the % migration of the cells. (B)
PAMAM-GA conjugated treated HCT 116 cells were harvested for analyzing the expression of Pro-MMP-9. b-actin was used as a loading control. Error bars represent
ꢂ
ꢂꢂ
ꢂꢂꢂ
p ꢃ 0.001 compared to untreated control cells.
mean ± SEM of three independent experiments. p ꢃ 0.05, p ꢃ 0.01 and
concentrations (Figure 5(B)). This result indicates that PAMAM- excessive cell death at a lower concentration in both HCT 116
GA conjugate gets internalized inside the cells within 6 h.
and NIH 3T3 cells, whereas PAMAM-GA conjugate showed sig-
nificant cell death to cancer origin cells, but not to the normal
NIH 3T3 cells (Figure S3). We found, similar response on cell
death examined by trypan blue exclusion assay in HCT-116 cell
Determination of IC50 of GA and PAMAM-GA conjugate
The half-maximal inhibitory concentration (IC ) of GA and (Figure S3). Next, we examined the effect of PAMAM-GA conju-
5
0
PAMAM-GA conjugate was determined from the dose- gate on cell survival by examination of the clonogenic ability of
response curve on HCT 116 and NIH 3T3 cells. These cells the cells. The results showed that PAMAM-GA conjugate inhibits
were treated with different concentrations of GA and colony formation in a dose and time-dependent manner on
PAMAM-GA conjugate for 24 h. The IC50 value for GA and HCT-116 cells (Figure 8(A, B)).
PAMAM-GA conjugate was found 69 mM and 3.68 mM
respectively in HCT 116 cells. On the other hand, the IC50
value for NIH 3T3 cells was found 260 mM with the treatment
of GA and 6.5 mM with the treatment of PAMAM-GA conju-
PAMAM-GA conjugate inhibits cell migration and
downregulates MMP-9 expression
gate. Indeed, IC50 of PAMAM-GA conjugate was significantly
higher in NIH 3T3 as compared to the HCT 116 cells (Figure
The effect of PAMAM-GA conjugate on cellular migration
was analyzed by wound healing assay and expression of
MMP-9, which is associated with cell migration and invasive-
ness. We found that PAMAM-GA conjugate profoundly inhib-
its the migration of HCT 116 cells in a dose and time-
dependent manner as compared to the control (Figure 9(A)).
Cell migration is associated with upregulated expression of
matrix metalloproteinases. The result show HCT116 cells
treated with PAMAM-GA conjugate downregulating the
expression of Pro form of MMP-9 (Figure 9(A, B)). The results
suggest that PAMAM-GA conjugate inhibits colony formation
and migration ability of HCT 116 cells for cell survival.
6
). These results indicate that PAMAM-GA is effective at low
concentration to cancer cells and less toxic to normal NIH
T3 cells. Moreover, GA showing inhibition in cell prolifer-
3
ation at lower concentration in HCT 116 and NIH 3T3 cells,
but the conjugation of GA with PAMAM dendrimers was
showing more efficacious and promising response as com-
pared to free GA. Thus, these results demonstrate that conju-
gation with PAMAM dendrimer reduces the toxicity of GA.
PAMAM-GA conjugate inhibits cell proliferation
specifically in cancer cells
We examined the anti-proliferative effect of PAMAM-GA conju-
gate on the different origins of cancer cells (HCT-116, HT-29,
MCF-7) using MTT assay. The results show that PAMAM-GA con-
PAMAM-GA conjugate induces cell death via activating
apoptosis signaling
jugate significantly inhibits cell proliferation of HCT-116, HT-29 PAMAM-GA conjugate inhibits cell proliferation of cancer
and MCF-7 (Figure 7). Although, free GA was found to induce cells. Therefore, it was important to unravel the molecular

1
2
K. PRIYADARSHI ET AL.
Figure 10. PAMAM-GA conjugate induces apoptotic cell death. (A) HCT 116 cells were treated with mentioned concentrations of PAMAM-GA conjugate for 6 h,
2 h, and 24 h, followed by staining with Annexin V-FITC and PI. The cells were analyzed using a fluorescence microscope. Scale bar: 10 lm. (B) After treating with
1
mentioned concentrations of PAMAM-GA conjugate for the indicated time, HCT 116 cells were harvested for analyzing the expression of Bcl-2, Procaspase-3, and
PARP cleavage. To monitor the release of cytochrome c from mitochondria to cytosol, the PAMAM-GA conjugate treated cells were subjected to subcellular frac-
tionation, followed by western blotting for cytochrome c. GAPDH and COX-IV represent loading control. (C) HCT 116 cells were treated with mentioned concentra-
tions of PAMAM-GA conjugate for 12 h and subsequently, harvested and subjected to immunoprecipitation using an anti-Apaf-1 antibody. The immunoprecipitated
complexes were subjected to western blotting for cytochrome c and Procaspase-9 to monitor the formation of the apoptosome complex. (D) After treating HCT
1
16 cells with mentioned concentrations of PAMAM-GA conjugate for 6 h, 12 h, and 24 h, the cells were harvested and the cell lysate was allowed to react with Z-
DEVD-R110 substrate, followed by reading the fluorescence at an excitation and emission wavelength of 496 nm and 520 nm, respectively. The plot represents a
fold increase in the caspase-3 activity compared to untreated control of the respective time points. Error bars represent mean ± SEM from three independent
ꢂ
ꢂꢂ
ꢂꢂꢂ
p ꢃ 0.001 compared to untreated control cells.
experiments. p ꢃ 0.05, p ꢃ 0.01 and
mechanism of cell death. First, we examined the apoptotic found an increased activity of caspase-3 on PAMAM-GA con-
cell death by Annexin V/Propidium Iodide staining. We jugate treated HCT116 cells as compared to the control
noticed that HCT 116 cells treated with different concentra- (Figure 10(D)).
tions of PAMAM-GA conjugate showed Annexin V and PI
positive cells at 6, 12 and 24 h. The higher number of
Annexin V and PI positive stained cells were found at 12 h. PAMAM-GA conjugate losses of mitochondrial
Further, we examined the expression of cell death regulatory membrane potential (MMP)
proteins i.e. Bcl-2, cytochrome c, procaspase-3 and PARP in
Loss of mitochondrial membrane potential (MMP) is another
HCT 116 cells by western blotting. We observed that
indication of apoptosis. Here, we examined the loss of MMP
PAMAM-GA conjugate alleviates the expression of the anti-
using a mitochondria specific lipophilic fluorescent probe JC-
apoptotic protein, Bcl-2 as compared to the control and
1
. When cells losses the MMP during apoptosis dye retains in
reduces levels of mitochondrial cytochrome c with a con-
comitant increase of the cytosolic cytochrome c, downregula-
tion of pro-caspase3 and cleavage of PARP was found on
shown time points (Figure 10(B)). The formation of the apop-
tosome complex was confirmed by co-immunoprecipitation
the cytoplasm and fluoresces green, while in healthy cells,
dye aggregates in the mitochondria and show red fluores-
cence. The quantitative analysis show that PAMAM-GA conju-
gate treated HCT-116 cells decrease in the ratio of red/green
(
co-IP) assay. PAMAM-GA conjugate augments the interaction fluorescence to the control (untreated cells), which indicates
of pro-caspase-9, cytochrome c, and Apaf- 1 in a dose- HCT-116 cells treated with PAMAM-GA conjugate loss mito-
dependent manner, which indicating apoptosome mediated chondrial membrane potential (Figure 11(A)). In addition,
activation of caspase-9 and caspase-3. In consistent, we microscopic analysis of the cells stained with JC-1 dye

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
13
Figure 11. PAMAM-GA conjugate disrupts the mitochondrial membrane potential. HCT 116 cells were treated with 2.5 mM, 5 mM and 10 mM PAMAM-GA conju-
gate for indicated time and stained with JC-1 dye, followed by counterstaining with DAPI. The cells were analyzed under a fluorescence microscope. (A)
Representative images of cells (B) The plots represent a quantitative change in the Mitochondrial Membrane Potential (DW
mean ± SEM from three independent experiments. p ꢃ 0.05, p ꢃ 0.01 and p ꢃ 0.001 compared to untreated control cells.
m
). Scale bar: 20 lm. Error bars represent
ꢂ
ꢂꢂ ꢂꢂꢂ
showed similar results (Figure 11(B)). These results suggest PAMAM-GA conjugate inhibits the activity of NF-jB
that GA conjugated with PAMAM dendrimer causes changes (p65) and release of pro-inflammatory cytokines IL-1b
in mitochondrial membrane potential and executes mito- and IL-6
chondrial-dependent apoptosis in HCT-116 cells.
Nuclear factor kappa B (NF-jB) is a transcription factor that reg-
ulates expression of different sets of genes including prolifer-
ation, anti-apoptotic, inflammatory response for cell survival
Conjugation of GA with PAMAM dendrimer restricts
necrotic cell death
(Hayden & Ghosh, 2004) We examined the NF-jB activation
through cytosol to nuclear translocation of GFP tagged p65.
First, we examined the toxic response from necrotic cell The results show that PAMAM-GA conjugate inhibits the nuclear
death of PAMAM-GA conjugate in HCT116 and NIH3T3 cells. translocation of GFP tagged p65 (Figure 13(A)). This was further
We found neligible cytotoxicity (release of LDH) on PAMAM- confirmed by western blotting analysis of p65 subunit. We
GA conjugate treated HCT116 and NIH3T3 cells as compared observed a prominent cytosolic retention of p65 after treatment
to the free GA (Figure 12(A,B)). Next, we confirmed from of PAMAM-GA conjugate for 6 h (Figure 13(B)). Thus, PAMAM-
qualitative analysis using Hoechst and PI staining. Results GA conjugate efficiently inhibits NF-jB activity by restricting the
show that HCT 116 cells treated with GA for 6 h and 12 h cytosolic to nuclear translocation of p65 in HCT 116 cells. An
showed intensed stained nuclei with both Hoechst and PI, elevated level of inflammatory cytokines is associated with the
which indicate necrotic cell death. However, HCT-116 cells progression of malignancy (Landskron et al., 2014). PAMAM-GA
treated with PAMAM-GA conjugate showed Hoechst-stained treated HCT116 cells were tested for affect TNF-a induced secre-
condensed nuclei with inadequate PI positive stained cells tion of the pro-inflammatory cytokines. The level of cytokines
(Figure 12(D)). Lysosomal rupture is an another indicative examined by ELISA indicates that PAMAM-GA conjugate effi-
feature of necrotic cell death (Artal-Sanz et al., 2006). We ciently reduces the secretion of IL-1b and IL-6 (Figure 13(C, D)).
monitored the lysosomal membrane integrity using In addition, expression of pro-inflammatory cytokines at tran-
Lysotracker red DND-99 dye counter stained with DAPI stain. scriptional level was examined by real time PCR. The results
We observed a progressive decrease in the intensity of red show that PAMAM-GA conjugate treated cells downregulates
fluorescence of the dye in HCT 116 cells treated with free GA the gene expression of IL-1b and IL-6 mRNA in a dose-depend-
for 6 h and 12 h as compared to the control (untreated cells). ent manner as compared the control (Figure 13(E, F)).
Whereas, the cells treated with PAMAM-GA conjugate
showed intense red fluorescence (Figure 12(C)). Collectively,
these results suggest that at GA may induce cell death asso-
Discussion
ciated with necrosis whereas its conjugation with PAMAM Polymer-drug conjugate is an emerging interest for drug
dendrimer restricts cell death by necrosis and instigates delivery and manifests the efficacy of the drug with various
apoptotic cell death in HCT 116 cells.
advantages including improving the aqueous solubility,

1
4
K. PRIYADARSHI ET AL.
Figure 12. Effect of conjugation with PAMAM dendrimer on the cytotoxicity of GA. The percent of cytotoxicity was determined by measuring the intracellular LDH
left after treatment of (A) HCT 116 and (B) NIH 3T3 cells with different concentrations of GA or PAMAM-GA conjugate for mentioned time points. (C) The lysosomal
integrity was monitored in HCT 116 cells by staining the cells with Lysotracker Red DND-99 dye after treatment with indicated concentrations of GA and PAMAM-
GA conjugate for 6 h and 12 h. The cells were counterstained with DAPI and analyzed under a fluorescence microscope. (D) Qualitative analysis of cytotoxicity was
carried out in HCT 116 cells. The cells were treated with indicated concentrations of GA and PAMAM-GA conjugate for mentioned time points and subsequently
stained with Hoechst 33342 dye and propidium iodide, followed by analysis under a fluorescence microscope. Scale bar: 20 lm. Error bars represent mean ± SEM
ꢂ
ꢂꢂ
ꢂꢂꢂ
p ꢃ 0.001.
from three independent experiments. The difference is significant between GA and PAMAM-GA treatment groups at p ꢃ 0.05, p ꢃ 0.01 and
tissue distribution, sustained release and safety with reduced permeability and retention (EPR) effect. In this study, Gallic
toxicity. Dendrimers are one of the unique, versatile and syn- acid (GA) was conjugated with 4.0 GPAMAM dendrimers by
thetic nanocarriers, which have an emerging interest in drug the surface modification by taking into consideration that
delivery due to flexible structural and functional properties the conjugation of GA with the free amine groups of
(
Akbarzadeh et al., 2018). The cellular uptake of dendrimers PAMAM dendrimer may reduce the cytotoxicity of dendrimer
and dendrimer-based drug delivery systems is significantly as well as enhance the bioavailability of GA.
greater than the linear polymeric carriers like polyethylene
The mechanistic conjugation of desired molecules or
glycol (PEG), making them superior drug carriers (Khandare drugs with nanocarrier provides sustained release of the
et al., 2006). It has been shown that dendrimers have a drug in a spatiotemporal manner to reduce its cytotoxicity
hydrophobic interior and hydrophilic chain, which allows and can improve the efficacy of drug (Lee & Yeo, 2015).
solubilizing the hydrophobic drugs in the aqueous medium Here, we examined the sustained release of GA from
(
Twyman et al., 1999). PAMAM dendrimers have emerged as PAMAM-GA conjugate at physiological pH. We noticed that
excellent drug carriers because of large number of cationic the slow release of GA from PAMAM-GA conjugate was
groups on their surface provide multiple attachment sites found for an extended time as compared to the free GA. In
and facilitate their uptake into the cells via ‘proton sponge’ addition, we examined the uptake of PAMAM-GA conjugate
mechanism (Sonawane et al., 2003). Moreover, they show to monitor the intracellular accumulation of GA. We
great potential for in vivo targeting of tumors for therapeutic observed an increase concentration of GA in the cell lysates
and diagnostic applications, as they exhibit enhanced in a dose-dependent manner. Thus, this result suggests that

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
15
Figure 13. PAMAM-GA conjugate inhibits the NF-jB (p65) activation and subsequent release of Pro-inflammatory cytokines. HCT 116 cells were treated with
.5 mM, 5 mM and 10 mM PAMAM-GA conjugate for 6 h followed by TNF-a treatment for 1 h and analyzed for p65 translocation. (A) Microscopic analysis of GFP-
2
p65 translocation under a fluorescence microscope. Scale bar: 20 lm. (B) The western blots represent p65 expression in cytosol and nucleus. Actin and Histone H3
represent loading control. ELISA of released inflammatory cytokines (C) IL-1b and (D) IL-6. RT-qPCR of inflammatory cytokines (E) IL-1b and (F) IL-6. Error bars repre-
ꢂ
ꢂꢂꢂ
p ꢃ 0.001 compared to untreated control cells.
sent mean ± SEM from three independent experiments. p ꢃ 0.05 and
PAMAM dendrimers may act as a carrier for the delivery of inhibits cell proliferation of different origin of cancer cells
GA and intracellular internalization in HCT116 cells. First, we (HCT 116, HT-29, MCF-7). Interestingly, the inhibition of cell
examined the IC50 value of GA and PAMAM-GA conjugate in proliferation was negligible to the normal NIH3T3 cells,
cancer and normal cells using HCT 116 and NIH 3T3 cell which indicates that conjugation of GA with PAMAM den-
lines. We found higher toxicity of free GA to HCT116 and drimers manifest specificity towards cancer cells. Further, we
NIH3T3 cells as compared to PAMAM-GA conjugate. Next, we confirmed the inhibition of cell survival by inhibiting the col-
observed the inhibition of cell proliferation on different ori- ony formation ability of HCT 116 cells. We observed that
gin of cancer cells. We found that PAMAM-GA conjugate PAMAM-GA conjugate restricts colony formation ability of

1
6
K. PRIYADARSHI ET AL.
HCT116 cells in a dose-dependent manner. Thus, the results integrity in HCT 116 cells. We found that the cells treated
indicate that the biological activity of GA and efficacy were with GA showed a prominent loss of lysosomal membrane
improved and PAMAM-GA conjugate inhibits the prolifer- integrity, but PAMAM-GA conjugate protect the lysosomal
ation of cancer cells. It is known that cancer cells have inva- integrity. In concordance, the morphological assessment of
siveness and migration properties to spread the tumor. Here, the nucleus revealed that the cells treated with GA exhibit
we tested the migration ability of HCT-116 cells by wound
healing scratch assay. We noticed that PAMAM-GA conjugate
inhibits migration of HCT116 cells. Previous report suggests
that secretion of MMP-9 enhances cell migration and angio-
genesis (Mira et al., 2004). We noticed that treatment of
PAMAM-GA conjugate diminished the expression of Pro-
MMP-9.
PAMAM-GA conjugate significantly induces cell death,
inhibits cell proliferation and migration of cancer cell. It was
important to confirm molecular mechanism of cell death
induced by PAMAM-GA conjugate. The results show that
HCT 116 cells treated with PAMAM-GA conjugate showing
Annexin V-FITC and PI positivity stained cells at lower con-
centrations at the earlier time points at 6–12 h. At 24 h, mas-
sive cell death was found. In continuation, we examined the
expression of apoptosis regulatory proteins and found that
decreased expression of anti-apoptotic protein Bcl-2 with
necrotic cell death. Whereas PAMAM-GA conjugate confers
cell death via the mode of apoptosis. Thus, these results col-
lectively indicate that the conjugation of GA with PAMAM
dendrimer surmounts it apoptotic effects. Importantly, activa-
tion of NF-kB and release of pro- inflammatory cytokines are
prominent characteristics of the cancer cells (Xia et al., 2014).
Here, we examined TNF-a primed activation NF-kB and
expression of pro-inflammatory cytokines IL-1b and IL-6. Our
results show that PAMAM-GA conjugate restricts cytoplasmic
to nuclear translocation and expression of p65. Moreover,
PAMAM-GA conjugate downregulated the expression of pro-
inflammatory cytokines IL-1b and IL-6 at protein and
mRNA level.
Conclusion
subsequent activation of executioner caspase-3 and cleavage Taken together, this study demonstrates that conjugation of
of PARP in PAMAM-GA conjugate treated HCT 116 cells. In GA with PAMAM dendrimers show sustained release of GA,
addition expression of cytochrome-c was found to be down- which might facilitate to attain a steady-state therapeutic
regulated on mitochondrial fraction and upregulated in cyto-
solic fractions. Upon release, cytochrome c from mitochon-
dria interacts with Apaf-1 and activates intrinsic signaling of
apoptosis via the formation of apoptosome complex (Bratton
concentration of GA for an extended time. PAMAM-GA con-
jugate inhibits cell proliferation, improves cellular uptake of
GA, inhibits colonogenic ability for cell survival, restricts can-
cer cell migration by down regulating the expression of
MMP-9, inhibits NF-kB activation and release of pro-inflam-
matory cytokines. PAMAM-GA conjugate augments mito-
chondria mediated apoptotic cell death in HCT 116 cells
rather than necrotic cell death. On other hand PAMAM-GA
conjugate showed negligible cytotoxic response as com-
pared to the free Gallic acid to the normal cells. In conclu-
sion, findings of this study revealed that PAMAM-GA
conjugate improved the bioavailability of GA and directs the
specificity toward cancer cells to manifests apoptotic cell
death. This indispensable approach may be helpful for inter-
vention of cancer therapy.
&
Salvesen, 2010). Immuno-precipitation and changes in
mitochondrial membrane potential assays revealed that
PAMAM-GA conjugate instigates apoptosome formation after
the release of cytochrome-c from the mitochondria and acti-
vates executioner caspases for initiation of apoptotic cell
death. Further, it was important to validate necrotic cell
death. Cell death as a result of necrosis and developing
resistance against apoptosis contributes to a major obstacle
in cancer therapy (Krysko et al., 2017). Necrotic cell death is
accompanied by the loss of plasma membrane integrity
resulting in the leakage of intracellular milieu to the external
environment and promotes metastasis in cancer (Lee et al.,
2
018). The previous report suggests that GA can induce cal-
pain dependent necrotic cell death in hepatic stellate cells
Hsieh et al., 2014). Therefore, it was important to examine
Acknowledgements
(
the effect of PAMAM-GA conjugate on necrotic cell death. FT-IR and NMR analysis were carried out at CSMCRI, Bhavanagar, Gujarat,
India. Authors acknowledge IIAR, Puri Foundation for Education in India
for providing junior Research Fellowship to KP, infrastructure facility and
support. Department of Biotechnology, Gov of India is gratefully
acknowledged for providing research grant to CP.
The release of LDH is one of the considerable markers of
necrotic cell death (Vaidya et al., 2019). We observed an ele-
vated level of LDH after the treatment of GA to HCT 116
cells, but it was negligible after the treatment of the
PAMAM-GA conjugate. In addition, we examined the effect
on normal mouse fibroblast NIH 3T3 cells, which showed
that the cytotoxicity of GA to the normal cells, but PAMAM-
Authors contribution
Chandramani Pathak (CP): Conceptualization; Kavita Shirsath (KS),
Khushbu Priyadarshi (KP), Bhargav Waghela (BW), Anupama Sharma (AS),
GA conjugate was showing negligible effect. Considering the
pivotal role of necrosis as a “calpain-cathepsin hypothesis”,
which postulates that the activation of calpain I instigates
lysosomal rupture, which is the key step of rapid cell necrosis
Ajay Kumar (AK), Chandramani Pathak (CP): Methodology
&
Experimentation; CP, KS, KP, BW, AS: Data curation Software; KS, KP &
CP: Writing and original draft preparation; KS, KP, BW & CP: Software
and data validation; CP: Visualization, Investigation Supervision; CP:
(
Yamashima & Oikawa, 2009). Hence, we also examined the
effect of GA and PAMAM-GA conjugate on the lysosomal Writing- Reviewing and Editing.

JOURNAL OF BIOMOLECULAR STRUCTURE AND DYNAMICS
17
Landskron, G., De la Fuente, M., Thuwajit, P., Thuwajit, C., & Hermoso,
M. A. (2014). Chronic inflammation and cytokines in the tumor micro-
environment. Journal of Immunology Research, 2014, 149185.
Lee, J. H., & Yeo, Y. (2015). Controlled drug release from pharmaceutical
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Lee, S. Y., Ju, M. K., Jeon, H. M., Jeong, E. K., Lee, Y. J., Kim, C. H., Park,
H. G., Han, S. I., & Kang, H. S. (2018). Regulation of tumor progression
by programmed necrosis. Oxidative Medicine and Cellular Longevity,
Disclosure statement
The authors declare no conflict of interest.
Funding
This work was financially supported by the Department of
Biotechnology, Ministry of Science & Technology, Government of India
under Nano Science & Nanotechnology scheme (BT/PR14259/NNT/28/
2018, 3537471. https://doi.org/10.1155/2018/3537471
4
85/2010) to CP.
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Bioavailability and bioefficacy of polyphenols in humans. I. Review of
97 bioavailability studies. The American Journal of Clinical Nutrition,
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ORCID
Medina, S. H., & El-Sayed, M. E. (2009). Dendrimers as carriers for delivery
of chemotherapeutic agents. Chemical Reviews, 109(7), 3141–3157.
https://doi.org/10.1021/cr900174j
Chandramani Pathak
http://orcid.org/0000-0002-9389-8096
Mira, E., Lacalle, R. A., Buesa, J. M., de Buitrago, G. G., Jim ꢀe nez-Baranda,
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