Chapter 2

MATERIALS AND METHODS

Materials and Methods Chapter 2

2.1 MATERIALS

2.1.1 Chemicals and Consumables

Hennotannic acid was procured from M/s Sigma Aldrich and the ER stress

kit was procured from M/s Cell Signaling Technology. iScript™ cDNA Synthesis

Kit and iProof HF Master Mix were purchased from Bio-Rad Laboratories. All the

remaining unspecified chemicals were also procured from M/s Sigma Aldrich

without undergoing further purification.

LIST OF INSTRUMENTS UTILISED FOR THIS STUDY

S. No. Name of the instruments Manufacturer

1. Thermal Cycler Eppendorf
2. Digital balance Sartorius
3. Magnetic stirrer Tarsons Spinot Digital
4. Cooling centrifuge Hermle Centrifuge
5. Lyophilizer Scanvac – Coolsafe
6. Nanopure Water Barnstead, Merck Millipore
7. Sonicator Sonics
8. UV- Visible Spectrophotometer Jasco
Fourier Transform Infra-Red
9. Perkin Elmer, Jasco
Spectrophotometer
10. Scanning Electron Microscope Quanta 200 FEG
11. CO2 Incubator Binder
12. Fluorescent Microscope Leica DMIRB, DMi8
13. BD FACS Calibur Flow Cytometry Becton Dickinson
14. Gel – Doc System Bio-Rad
15. ELISA plate reader Bio-Rad
16. Nanodrop Thermo Fischer Scientific
◦
17. Ultra-low Temperature Freezer (- 80 C) Haier Biomedical
◦
18. Deep Freezer (-40 C) Haier Biomedical

Table 5 List of instruments utilised for this study

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2.1.2 CELL CULTURE AND MAINTENANCE

S. No. Cell Line Media Used for Culture

Cancer cell line A549
1. ((Human epithelial cell line derived HAM'S F-12 medium
from a lung carcinoma tissue)
Minimum Essential Medium
2. PA-1(Human ovarian teratocarcinoma)
Eagle medium
SK-OV-3 (Human Ovarian Cancer Cell
3. McCoy's 5A (modified) Medium
Line)
Minimum Essential Medium
4. MG-63 (Osteosarcoma cell line)
Eagle medium
Dulbecco′s Modified Eagle′s
5. 293T (Human embryonic kidney)
Medium - low glucose
Dulbecco’s Modified Eagle
6. Hepa1-6 (Murine hepatoma)
Medium (DMEM),
Normal cell line L-132 (Human Dulbecco’s Modified Eagle
7.
embryonic lung) Medium (DMEM)
CHO (Epithelial cell line derived from Minimum Essential Medium
8.
the ovary) Eagle medium
Dulbecco’s Modified Eagle
9. BRL-3A (Rat liver fibroblast)
Medium (DMEM)
Minimum Essential Medium
10. A-498 (Human kidney cancer cell line)
Eagle medium

Table 6: Enumerates the cell lines, delineating the respective culture media
employed for their cultivation

The cell lines were supplemented with 10% (v/v) fetal bovine serum

(Sigma), 100 units/ml penicillin/streptomycin/ Gentamicin / Ampicillin (Sigma) and

maintained at 37°C in a CO2 incubator with a humidified atmosphere of 5% CO2 and

95% air.

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2.2 BIOCHEMICAL ASSAYS

2.2.1 MITOCHONDRIAL DEHYDROGENASE ASSAY

The impact of the samples on cytotoxicity was assessed by evaluating

mitochondrial dehydrogenaseactivityhroughtheuseof3-(4,5-dimethylthiazolyl-2)-2,5-

diphenyltetrazoliumbromide (MTT) salt. Cultures containing around 15,000 cells per

well were exposed to various concentrations of the specimens and incubated for 24

hours in a CO2 environment. Subsequently, the cells underwent treatment with an MTT

solution (0.5 mg/mL) for 4 hours at 37°C. Following this exposure, the resultant

blue/purple formazan crystals were dissolved in DMSO. The absorbance of the solution

was then gauged at 570 nm using a Bio-Rad Elisa plate reader.(Ghasemi et al., 2021)

2.2.2 APOPTOSIS ASSAY

A live/dead assay was employed to assess cell viability in both normal and

cancer cell lines. Around 40,000 cells were plated on a culture dish and incubated in a

CO2 incubator at 37°C for 24 hours. The next day, the cells were subjected to varying

concentrations of samples and further incubated in a CO2 incubator at 37°C for an

additional 24 hours. Following this incubation period, the cells were treated with

acridine orange (4μM) and propidium iodide (3μM) dyes. Fluorescent images of the

cells were captured using a Leica fluorescent microscope to examine the presence of

viable and non-viable cells.(Chan et al., 2016; Darzynkiewicz et al., 1992)

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2.2.3 ESTIMATION OF REACTIVE OXYGEN SPECIES (ROS) GENERATION

Cells were subjected to varying concentrations of the samples for 24 hours.

Following exposure, the cells underwent a rinse and were suspended in a phosphate-

buffered solution. Subsequently, the cells were treated with 5 µM of dichloro

dihydrofluorescein diacetate (DCFH-DA) in PBS for 30 minutes under dark conditions.

After the incubation period, cellular images were captured using a Leica fluorescent

microscope equipped with a blue filter (495 nm excitation and 523 nm emission). The

green colour observed in the images represented the visualized levels of reactive oxygen

species (ROS) in the cells. (Ghosh & Roy, 2023; Matsumoto et al., 2021)

2.2.4 MITOCHONDRIAL MEMBRANE POTENTIAL (ΔΨM) ASSAY

To evaluate the mitochondrial membrane potential (Δψm), cells were treated

with various concentrations of samples over 24 hours. After exposure, the cells

underwent washing and suspension in a phosphate-buffered solution. Subsequently,

they were incubated with 5 µg/mL of JC-1 for 30 minutes at 37°C. Following this

incubation, the cells were rinsed with PBS and suspended in 0.5 mL of sterile PBS.

The fluorescence emitted by the cells was then examined using a Leica fluorescent

microscope to assess alterations in mitochondrial membrane potential.

2.2.5 DETECTION OF APOPTOSIS

The impact of the compound on the initiation of programmed cell death was

assessed through DAPI staining. Lung cancer cells (A549) and a normal cell line (L-

132) were cultured in 24-well plates and exposed to the samples for 24 hours.

Following treatment, the cells underwent staining with DAPI. The nuclear

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morphology of the stained cells was subsequently scrutinized using fluorescence

microscopy to identify any alterations indicative of programmed cell death. Antony

et al., 2021; Saadat et al., 2015; Surti et al., 2022)

2.2.6 INTRACELLULAR Ca2+ MEASUREMENT

Intracellular calcium levels were quantified employing FLUO-8 AM. Both the

cancer cell line (A549) and the normal cell line (L-132) were subjected to the samples

for 24 hours. The subsequent day, the cells were exposed to FLUO-8 AM in the absence

of light for 30 minutes. (Harada, 2008; Lee et al., 2019) After a thorough washing step,

the cells were observed and analyzed using a Leica DMi8 fluorescent microscope

equipped with a FITC filter, enabling the visualization and assessment of intracellular

calcium levels in the cells.(Saadat et al., 2015; Surti et al., 2022)

2.2.7 WOUND HEALING/SCRATCH MIGRATION ASSAY

A wound-healing assay was conducted on the cells, involving the creation of

a wound by scraping a sterile pipette tip across the cell monolayer. Subsequently, the

cells were exposed to varying concentrations of Hennotannic acid. To eliminate cell

debris, the cells underwent a wash with sterile phosphate-buffered saline (PBS) and

were then maintained in a CO2 incubator for 24 hours. Following this incubation, the

cells were fixed by incubating them with 80% ethanol for 10 minutes at room

temperature. Post-fixation, the cells were stained with crystal violet. Using a Leica

DMi8 microscope, images of the cells were captured, enabling the visualization and

analysis of the wound healing process. (Bahar & Yoon, 2021)

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2.2.8 IN VITRO MIGRATION AND INVASION ASSAY

The migration and invasion capabilities of the cancer cell line (A549) were

evaluated using a customized 6.5 mm trans-well chamber featuring polycarbonate

membranes with an 8.0-mm pore size. In the migration assay, 5×10^4 cells were

suspended in serum-free HAM'S F-12 medium and placed in the upper chamber, while

a chemoattractant medium containing 10% FBS was introduced into the lower chamber.

Following a 24-hour incubation at 37 °C, the cells on the lower surface of the membrane

were stained, photographed, and quantified using a microscope. For the invasion assay,

1×10^5 cells were similarly prepared and positioned in the upper chamber, with the

chemoattractant medium containing 10% FBS in the lower chamber. After 24 hours of

incubation at 37 °C, the cells that invaded the membrane and migrated to the lower

surface were stained, photographed, and counted under a microscope. (Justus et al.,

2023; Kramer et al., 2013; Pijuan et al., 2019; Yarrow et al., 2004)

2.2.9 CHICK EMBRYO CHORIOALLANTOIC MEMBRANE (CAM) ASSAY

Fertile eggs underwent an incubation period of four to six days at 37°C in an

atmosphere of 97% air before being subjected to the test compounds. These

compounds, prepared as a liquid suspension with a final concentration of 1 mg/mL,

were applied to the chorioallantoic membrane (CAM) of 5- to 6-day-old embryos

using small-dose beads. Each dose bead contained 10 to 30 µg of the test compound,

administered in a 5 µL volume. Following a 48-hour incubation period, the impact

of the samples on blood vessel formation was assessed by observing the number of

newly formed blood vessels around the dose beads. Each concentration underwent

testing in a minimum of two separate experiments, with each experiment involving 5

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fertile eggs per test group. The average percentage response was then determined for

each group. (Ribatti, 2017)

2.2.10 AORTIC RING ASSAY

Aortas were harvested from 9th-day fertilized chick egg embryos and

promptly placed in ice-cold PBS buffer upon extraction. The arteries were then

roughly sectioned into lengths of approximately 1-1.4 mm. Following this, the

sections underwent multiple washes with PBS (Chen et al., 2021) Subsequently, the

aortic sections were immersed in a collagen gel, and various concentrations of

Hennotannic acid were applied. The impact of Hennotannic acid on endothelial

sprouting from the aortic sections was assessed through microscopic examination

and compared with the control group. (Baker et al., 2012; Bellacen & Lewis, 2009;

Fuente et al., 2016; Lokman et al., 2012)

2.2.11 TUMOR SPHEROID GROWTH INHIBITION

The potential anti-cancer therapeutic properties of Hennotannic acid were

assessed using a 3D tumour spheroid model. In this model, cancer cells were

suspended in a 2% agarose solution at a cell density of 10^7 cells/100μL. The

agarose mixture solidified at 37°C for 10 minutes, forming agarose molds. (Jubelin

et al., 2022)These molds were then transferred to 96-well culture plates containing

the appropriate medium for the specific cell line, and the plates were placed in an

incubator with 5% CO2 at 37°C. As a control, cell aggregates consisting of 10^7

cells were deposited at the bottom of each tube after centrifugation, and they were

cultured under the same conditions as the experimental group. Once the tumour

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spheroids formed within the tubes, they were treated with Hennotannic acid. The

impact of Hennotannic acid on the 3D cancer cell spheroid was monitored using

microscopic imaging, enabling the observation and analysis of its therapeutic effects

on the tumour spheroids in a 3D environment.(Jubelin et al., 2022)

2.3. MOLECULAR BIOLOGICAL ASSAYS

2.3.1 GENE EXPRESSION STUDIES

RNA ISOLATION

Total RNA extraction was carried out using TRIzol reagent following the

manufacturer's guidelines (Ambion, Life Technologies). After treating the cells with

the samples for varying time intervals, cell harvesting was performed by

homogenizing them with 1 mL of TRIzol reagent to create a homogenate. To this

homogenate, 0.2 mL of chloroform was added, vigorously shaken for 30 seconds,

and then incubated for 10 minutes at room temperature. The contents underwent

centrifugation at 12,000 x g for 15 minutes at 4 °C, and the resulting aqueous phase,

containing the RNA, was carefully transferred to a fresh sterile tube. (“TRI

Reagent,” 2008)

RNA in the aqueous phase was precipitated by adding 0.5 mL of 100%

isopropanol (v/v) per mL of TRIzol reagent used in the initial homogenization. The

mixture was incubated at room temperature for 10 minutes, followed by

centrifugation at 12,000 x g for 10 minutes at 4 °C. The resulting RNA pellet was

washed with 1 mL of 75% ethanol per mL of TRIzol reagent used in the initial

homogenization. The samples were vortexed and then centrifuged at 7,500 x g for 5

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minutes at 4 °C. The washed RNA pellet was air-dried and dissolved in

diethylpyrocarbonate (DEPC)-treated water. The isolated RNA was subsequently

stored at -80 °C until further analysis. (Thermo Fisher Scientific, 2016)

2.3.2 RNA QUANTIFICATION AND COMPLEMENTARY DNA

CONVERSION

The assessment of RNA yield and purity was conducted using NanoDrop 2000

(Thermo Fisher Scientific, USA). Subsequently, the samples were normalized and

subjected to cDNA conversion utilizing the iScript™ cDNA Synthesis Kit from Bio-

Rad Laboratories, Hercules California, United States, in a thermal cycler (Eppendorf

Master Cycler Gradient). (Thermo Fisher Scientific, 2016) To achieve this, the RNA

concentration was normalized, and an equivalent concentration of about 2 µg of total

RNA was mixed with 10 µL of 2X Reverse Transcriptase master mix on ice. This mix

comprised 2.0 µL of 10X RT Buffer, 0.8 µL of 25X dNTP Mix (100 mM), 2.0 µL of

10X Reverse Transcriptase Random Primers, 1 µL of Multi Scribe™ Reverse

Transcriptase, 1 µL of RNase Inhibitor, and 3.2 µL of Nuclease-free H2O. The reaction

mix was then briefly spun and underwent incubation in a thermal cycler with the

following running conditions: an initial 10-minute reaction at 25 °C for RNA

denaturation, followed by incubation at 37 °C for 2 hours for cDNA synthesis. Finally,

the reverse transcriptase enzyme was deactivated by heating the reaction mixture to 85

°C for 5 minutes. The resulting cDNA was diluted and stored at -80 °C, serving as a

template for studying gene expression in the samples.

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2.3.3 PRIMER DESIGNING AND SYNTHESIS

Novel primers targeting genes, including AMPK, SIRT1, SIRT3, ACC,

COX-2, p53, PGC-1α, mTOR, Caspase 8, caspase 3, BAD, CHOP, and the

housekeeping gene β-Actin, were developed using the NCBI Primer-Blast tool.

Subsequently, the designed primers underwent a purity assessment through Net

Primer online software to detect secondary structures such as hairpins, self-dimers,

cross-dimers, palindromes, repeats, and runs. Moreover, the designed primers

underwent sequence similarity analysis with the NCBI nucleotide database for

validation. The appropriate primers (as indicated in Table 7) were then custom

synthesized and procured at a scale of 5 OD from Priority Imperial Life Science

(India). Post-reconstitution in sterile Tris EDTA (10 mM Tris, 1 mM EDTA) buffer,

the primers were stored at -40 °C for subsequent use.

2.3.4 PCR ANALYSIS

PCR analysis was conducted to explore the expression patterns of genes,

including AMPK, SIRT1, SIRT3, ACC, COX-2, p53, PGC-1α, mTORC1, Caspase

8, Caspase 3, and BAD, in addition to the housekeeping gene β-Actin, in cells

subjected to the samples. The gene expression study was performed using a thermal

cycler (Eppendorf Master Cycler Gradient). The PCR conditions consisted of an

initial denaturation step at 95 °C for 2 minutes, followed by denaturation at 95 °C

for 45 seconds, annealing at the specific primer's optimized annealing temperature

(Ta) for 25 seconds, extension at 72 °C for 45 seconds, and final elongation at 72 °C

for 2 minutes. The primer sequences and their respective annealing temperatures are

detailed in Table 7. After amplification, the resulting PCR amplicons were separated

by electrophoresis on a 1.8% agarose gel, alongside a 100 bp DNA ladder. The gel

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was stained with ethidium bromide, and the resolved gel was visualized under UV

illumination and photographed using a GelDoc XR documentation system from Bio-

Rad. The expression levels of the target genes were analyzed by comparing the

treated cells to the control (untreated) cells. An endogenous control, likely the

housekeeping gene β-Actin, was employed for normalization and quantification.

Amplicon
S.
Gene Primer Sequence Ta°C Length
No.
(bp)
β Forward GGTGCTGACTATCCAGTTGA
1 58 145
catenin Reverse TCCATTTGTATTGTTACTCCTAAAG
Forward GTACCAGGTCATCAGTACAC
2. AMPK 47 117
Reverse ACTACTCCAGGTACATCAG
Forward TGCCGGAAACAATACCTCCA
3. SIRT1 59 179
Reverse AGACACCCCAGCTCCAGTTA
Forward TCCGGAGGACTCCTTGGACTG
4 SIRT3 58 100
Reverse GCCTCGACCCGTTCAACTAC
Forward GGTCTGGTGCCTGGTCTGATGATG
5 COX-2 58 724
Reverse GTCCTTTCAAGGAGGAGAATGGTGC
Forward GCTAGGTGCATTGACATACAACA
6 mTOR 59 200
Reverse AGTGCTAGTTCACAGATAATGGC
Forward CAGCAGTTCCTCCACGCAGG
7. ACC 60 672
Reverse CACTGGCACATAGTGATCTGC
Forward TCCCGATCACCATATTCC
8 PGC-1α 58 465
Reverse TTCAAGAGCAGCAAAAGC
Forward CCTGGATTGGCCAGACTGC
9. P53 60 121
Reverse TTTTCAGGAAGTAGTTTCCATAGGT
Caspase- Forward TTTGGAACCAAAGATCATACATGG
10 61 186
3 Reverse GTTTCCCTGAGGTTTGCTGC
Caspase- Forward TGGTTGCCAGGACGAATTGA
11 51 183
8 Reverse AAGATACGAGATCCCGCTGC
Forward ACTGAGGTCCTGAGCCGACA
12. BID 58 224
Reverse CCAGGCTAACTCTGGGATCTG
Forward TCGCCGAGCTCTGATTGAC
13 CHOP 58
Reverse CCCTGCGTATGTGGGATTGAG 163

Table 7 Enumerates the primers employed in the scope of this research

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2.3.5 ENZYME-LINKED IMMUNOSORBENT ASSAY

To evaluate endoplasmic reticulum (ER) stress, an Enzyme-Linked

Immunosorbent Assay (ELISA) was conducted using cell lysates obtained from

A549 cells treated with various concentrations of the samples. The protein

concentration in the lysates was determined using the BCA protein estimation assay.

In the ELISA procedure, 100 µL of protein-normalized cell lysates was coated onto

ELISA plates and left to incubate overnight at 4°C. To minimize nonspecific

binding, the coated plates were blocked with a 1% BSA (bovine serum albumin)

solution. (Lin, 2015; Trakunram et al., 2019)

Primary antibodies, including BiP (Rabbit monoclonal IgG), calnexin

(Rabbit monoclonal IgG), Ero1-Lα (Rabbit monoclonal), IRE1α (Rabbit monoclonal

IgG), PDI (Rabbit monoclonal), CHOP (Mouse monoclonal IgG2a), and PERK

(Rabbit monoclonal IgG), were diluted at 1:500 and allowed to react in the BSA-

blocked wells of the ELISA plate for 1 hour. Subsequently, the wells were washed,

and secondary antibodies conjugated with Horseradish peroxidase (HRP),

specifically goat anti-rabbit IgG and horse anti-mouse IgG (for CHOP), were added

to the wells and incubated for 1 hour. Following another round of washing with

PBST, the wells were incubated with 3,3’,5,5’-tetramethylbenzidine (TMB)

substrate solution. The colour developed was measured at 405 nm using a Bio-Rad

ELISA plate reader. (Iswardy et al., 2017)

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2.3.6 IMMUNOFLUORESCENCE ASSAY

To investigate the relative expression of the CHOP protein post-treatment

with Hennotannic acid, an immunofluorescence assay was performed. A549 cells

subjected to varying concentrations of Hennotannic acid were washed three times

with PBS (phosphate-buffered saline) and fixed with 4% paraformaldehyde for 30

minutes. After fixation, the cells were permeabilized using 0.1% Triton X-100 for 10

minutes at room temperature. (Iswardy et al., 2017)

To prevent nonspecific binding, the cells were then incubated with

phosphate-buffered saline containing 5% bovine serum albumin for 2 hours.

Subsequently, after blocking, the cells were incubated overnight at 4°C with the

primary CHOP antibody. On the following day, the A549 cells were washed with

PBST and treated with a FITC (fluorescein isothiocyanate)-conjugated secondary

antibody for 3 hours at room temperature. For visualization of cellular components,

the cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI).

Immunofluorescence images were captured using a Leica DMi8 microscope.

2.4 ISOLATION OF COLLAGEN

The isolation of acid-soluble collagen was conducted using the salt

precipitation method. The procedures were carried out with approval from the

Institutional Animal Ethical Committee (sanction numbers IAEC No. Rev. 03/2017

and IAEC No. 07/2021). Tail tendons from 6-month-old Wistar rats were carefully

extracted, and any lipid content was removed by treating them with a 1:1 ratio of

diethyl ether to chloroform (v/v). Following this, the tendons were allowed to swell

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in 0.5 M acetic acid overnight, ground, and centrifuged to collect the supernatant.

For salt precipitation, 5% NaCl was gently added to the supernatant, and the

resulting mixture was centrifuged to collect the crude collagen. (Rittié, 2017a)The

crude collagen was subsequently dialyzed against 0.05 M acetic acid to eliminate

excess salts, yielding a purified collagen solution. This collagen solution underwent

freeze-drying using a lyophilizer and was stored at -20 °C. To evaluate the isolated

collagen protein, Sodium Dodecyl Sulfate Poly-Acrylamide Gel Electrophoresis

(SDS-PAGE) analysis was performed. SDS-PAGE, with a 4% separating gel and an

8% resolving gel, was carried out to determine the subunit composition of the

extracted collagen protein. A protein ladder of known molecular weight was

included for comparison with the isolated protein. (Xiong et al., 2009a) The collagen

samples were denatured with sample solubilizing buffer and loaded onto the

electrophoretic gel. After electrophoresis, the gel was stained with Coomassie

brilliant blue (CBB R-250) dye, and the stained protein bands were visualized using

a gel documentation system. (Chen, 2019; Rittié, 2017b; Xiong et al., 2009b)

The SDS-PAGE Gel composition is given below

Stacking Gel (4%) (5 mL)

• 30% Acrylamide: Bisacrylamide : 0.67 mL

• Tris pH 6.8 : 1.3 mL

• 10% SDS : 0.05 mL

• Ammonium Per-sulfate : 0.05 mL

• TEMED : 0.005 mL

• Water : made up of 5 mL

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Resolving Gel (8%) (9mL)

• 30% Acrylamide: Bisacrylamide : 2.4 mL

• Tris pH 8.9 : 2.25 mL

• 10% SDS : 0.09 mL

• Ammonium Per sulfate : 0.09 mL

• TEMED : 0.009 mL

• Water : made up with 9 mL

Lammelli Sample Buffer (4X)

• 500 mM Tris-HCl (pH: 6.8)

• 8% SDS

• 0.06 % Bromophenol Blue

• 30% glycerol

• 200 mM β Mercapto ethanol (add just before use)

SDS-PAGE Running Buffer (10X)

• 25 mM Tris Base

• 193 mM Glycine

• 1% SDS

Staining solution

• 0.25% Coomassie Brilliant Blue

• 25% Methanol

• 10% Acetic acid

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De-staining Solution

• 25% Methanol

• 10% Acetic acid

2.4.1 KINETICS STUDY OF COLLAGEN FIBRIL SELF-ASSEMBLY

Collagen fibril formation, a self-organizing process leading to the

development of helical collagen fibrils, was evaluated through a collagen fibrillation

assay. The experiment involved treating and incubating 3 mg/mL collagen, along

with Hennotannic and glutaraldehyde-treated collagen (used as a reference group).

To initiate collagen fibrillation, specific components were added to achieve a total

reaction volume of 1000 µL. The components included: collagen (with or without

cross-linkers) - 750 µL, phosphate buffer - 100 µL, 2 M NaCl - 75 µL, distilled H2O

- 15 µL. The pH of the mixture was adjusted to 7.2±0.2 using 1% NaOH (60 µL).

The resulting mixture was promptly transferred into a quartz cuvette with a 10 mm

path length. To monitor the progression of fibril formation, turbidity measurements

were recorded using a Jasco UV-visible spectrophotometer. The spectrophotometer

was set at 313 nm using time drive scanning mode, and measurements were taken

over 100 minutes at 32°C. The rate of fibril formation was determined based on the

t1/2 value, representing the time taken for the turbidity to reach half of the

logarithmic phase of the fibrillation curve. The t1/2 values were obtained by plotting

optical density measurements against time in minutes, and the point at which the

optical density reached half of the maximum turbidity was identified. The t1/2 value

serves as a measure of the rate of fibril formation during the experiment.

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2.4.2 DEGREE OF CROSS-LINKING

To assess the cross-linking efficiency of Hennotannic Acid on collagen

scaffolds, the quantity of free amino groups in collagen after cross-linking was

estimated using the 2,4,6-trinitrobenzene sulfonic acid (TNBS) assay. Cross-linked

collagen samples and native collagen samples ranging from 1 to 3 mg were

incubated in a 4% (w/v) solution of NaHCO3 for 30 minutes. After incubation, a

freshly prepared solution of TNBS (0.5 wt %) in 4% (w/v) NaHCO3 was added to

the mixture. The reaction was allowed to proceed for 2 hours at a temperature of

60°C. Following the 2-hour incubation, HCl (6 M) was introduced to the TNBS-

treated samples (3 mL) to solubilize the solid components in the reaction mixture.

The temperature was maintained at 40°C to facilitate solubilization. The resulting

samples were then diluted with distilled water, and the absorbance was measured at

345 nm using a Perkin Elmer UV-visible spectrophotometer. The absorbance at 345

nm provides information about the amount of free amino groups remaining after

cross-linking, allowing for an assessment of the cross-linking efficiency of

Hennotannic acid on the collagen scaffolds.

2.4.3 BIOPHYSICAL ANALYSIS: TENSILE STRENGTH

The tensile strength of the collagen films was evaluated using an Instron

tensile tester instrument. To prepare the samples, the collagen films were cut to

achieve a uniform thickness and dimensions of 5 cm in length and 1 cm in width.

Before conducting the tensile strength test, the thickness of each sample was

measured using a digital vernier. For the tensile strength test, elongation at break,

and modulus assessment, a load of 2N was applied to the collagen film samples. The

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measuring speed of the instrument was set to 5 mm/min. Throughout the

experiment, the testing environment was maintained at a relative humidity of 65%

and a temperature of 20°C. The tensile strength, elongation at break, and modulus of

the films were determined by applying controlled force and measuring the response

of the collagen films. (Bittner et al., 2021)

2.4.4 SWELLING RATIO ANALYSIS

The swelling behaviour of the scaffold materials was investigated to assess

their fluid uptake capacity. For this evaluation, a consistent amount of collagen

scaffold, weighing 8 mg, was placed in 1 mL of 1x PBS. After specific time intervals

of 12 and 24 hours, the scaffolds were removed from the PBS solution. Any excess

adhered water was meticulously blotted using sterile tissue paper to ensure precise

measurements. The weights of the collagen scaffold sheets were then recorded after

removing the excess water. The degree of swelling of the scaffold materials in PBS

was determined by comparing the weights before and after each time interval. This

measurement provided valuable insights into the fluid uptake capacity and swelling

behaviour of the native collagen and Hennotannic Acid-reinforced collagen

scaffolds. (Liu et al., 2019)

2.4.5 HEMOCOMPATIBILITY ASSAY

The hemocompatible behaviour of both the native collagen and Hennotannic

Acid-reinforced collagen scaffold was assessed using heparinized blood. Initially,

red blood cells (RBCs) were subjected to multiple washes with a 150 mM NaCl

solution. Subsequently, the blood was diluted in a 1:50 ratio with phosphate-buffered

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saline (PBS), pH 7.4. A total of 200 μL of the diluted RBCs was incubated with the

native collagen and Hennotannic acid-reinforced collagen scaffold at a temperature

of 37°C for 1 hour. Following the incubation period, intact red blood cells were

separated by centrifugation, and the supernatant was collected. (Liu et al., 2019)The

optical density of the supernatant was measured at a wavelength of 540 nm using a

spectrophotometer. This measurement enabled the assessment of any hemolysis or

release of haemoglobin resulting from the interaction between the blood and the

scaffold materials. A higher optical density at 540 nm would indicate greater

hemolysis in the samples, while a lower optical density would suggest a more

hemocompatible behaviour of the scaffolds. (Kirkness & Forde, 2018)

2.4.6 PROTEOLYTIC SUSCEPTIBILITY ASSAY

To evaluate the susceptibility of both native collagen and Hennotannic Acid-

reinforced collagen scaffolds to proteolytic enzymes, a degradation assay using

collagenase enzyme was conducted. Equal amounts of scaffolds were weighed and

incubated in PBS containing 0.1% type 1 A collagenase at a temperature of 37°C. At

regular intervals, the scaffolds were removed from the enzyme solution by

centrifugation, and their weights were measured. The greater the observed loss in

weight, the higher the susceptibility of the scaffolds to collagenase enzyme.

(Kirkness & Forde, 2018)The experiment indicated the degradation susceptibility of

the native collagen and Hennotannic Acid-reinforced collagen scaffolds by

proteolytic enzymes, as monitored by the weight loss over time.

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2.5 CONFORMATIONAL ANALYSIS

2.5.1 FTIR MEASUREMENTS

FTIR spectra of native collagen, Hennotannic acid cross-linked collagen, and

glutaraldehyde cross-linked collagen sponges (utilized as a positive control) were

recorded using a Spectrum Two Perkin-Elmer spectrophotometer. The transmission

mode was employed for spectral analysis. To prepare the samples for analysis,

native collagen, Hennotannic acid cross-linked collagen, and glutaraldehyde cross-

linked collagen were ground with KBr in a ratio of 1:100. The ground samples were

then compressed into pellets using an Atlas Manual 15T Hydraulic Press pellet

maker. Each sample underwent spectral scanning from 800 to 4000 cm-1 with 8

scans per sample and a resolution of 1 cm-1. Subsequently, the FTIR spectra were

baseline-corrected and normalized using the solvent spectrum, which consisted of 50

mM acetic acid. The FTIR analysis allowed for the examination of the characteristic

infrared absorption patterns of the samples, providing valuable information about

the structural features and chemical composition of native collagen, Hennotannic

acid cross-linked collagen, and glutaraldehyde cross-linked collagen sponges.

(Cavalu et al., 2019; Li et al., 2018)

2.5.2 SEM ANALYSIS OF COLLAGEN SCAFFOLDS

A lyophilized collagen sample weighing 0.3 mg was measured and dissolved

in 1 mL of 50 mM acetic acid. The resulting collagen solution was then treated with

the redox modulator, Hennotannic Acid, and freeze-dried. The biomatrix was

subsequently cut into small pieces measuring 2 mm in size. Scanning Electron

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Microscope (SEM) analysis was conducted to examine the structural organization of

collagen. Both the collagen biomatrix treated with Hennotannic Acid and the

untreated control collagen were subjected to SEM examination. This analysis

facilitated the visualization and assessment of the 3D structural morphology of the

biomatrices at the microscopic level.(Kesavan et al., 2022)

2.5.3 CELL VIABILITY ON COLLAGEN BIOMATRIX CROSS-LINKED

WITH HENNOTANNIC ACID

Collagen reconstituted with various concentrations of Hennotannic Acid,

ranging from 100 µM to 300 µM, was subjected to cell viability testing using the

MTT assay. After gelation, the collagen was allowed to air dry in 48 well tissue

culture plates, resulting in a monolayer of collagen sheets. The collagen sheets were

extensively washed with distilled water to remove acetic acid and other salts. The

culture plates were surface sterilized by exposure to UV light. Approximately 12,000

A549 and L132 cells were seeded into each culture well containing the cross-linked

and non-cross-linked collagen scaffolds. The culture plates were then incubated in a

CO2 incubator. After various time intervals of incubation, the culture medium was

removed, and the cells were treated with 0.5 mg/mL of 3-(4,5-dimethylthiazolyl-2)-

2,5-diphenyltetrazolium bromide (MTT) salt in PBS. The cells were then incubated

in the dark at 37°C. Following a 4-hour incubation period, the blue/purple formazan

crystals formed by the viable cells were solubilized using dimethyl sulfoxide

(DMSO). The absorbance of the solution was measured at 570 nm using a Bio-Rad

ELISA plate reader. The MTT assay allowed the quantification of cell viability

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within the collagen scaffolds treated with different concentrations of Hennotannic

Acid. The absorbance readings at 570 nm provided insight into the metabolic

activity of the cells and their viability within the collagen scaffold environments.

The cell viability was calculated by the following formula

% of Cell viability = [(OD of treated culture)/ (OD of control)]*100

2.5.4 REACTIVE OXYGEN SPECIES ASSAY ON COLLAGEN

BIOMETRICS

The ROS-specific fluorescent probe, 2ʹ,7ʹ-Dichlorofluorescein diacetate

(DCFH-DA), was utilized in L-132 and A549 cells that were grown on a

Hennotannic Acid-stabilized collagen biomatrix for determination of ROS levels in

the cells after interaction with the biomatrix. The cells were treated with 5 μM

DCFH-DA and incubated for 30 minutes at 37°C. DCFH-DA is a non-fluorescent

compound that can be converted into a fluorescent compound, DCF (2ʹ,7ʹ-

Dichlorofluorescein), in the presence of ROS. After the incubation period, the cells

were analyzed for the intensity of green fluorescence, indicating the presence of

ROS, using a Leica DMi8 microscope. The DCF fluorescence is proportionate to the

level of ROS present within the cells. This analysis provided insight into the extent

of ROS production within the L-132 and A549 cells grown on the Hennotannic

Acid-stabilized collagen biomatrix, highlighting the potential role of the biomatrix

in influencing ROS levels.

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2.5.5 ACRIDINE ORANGE /PROPIDIUM IODIDE NUCLEAR STAINING

AND FLUORESCENCE MICROSCOPY

A live/dead assay was conducted to determine the number of viable and dead

cells in control collagen and Hennotannic Acid cross-linked collagen biomatrix in

both a normal cell line (L-132) and a cancer cell line (A549). For the assay,

approximately 30,000 cells were seeded onto the native collagen and Hennotannic

Acid cross-linked collagen biomatrix. The cells were then incubated in a CO2

incubator at 37°C for 24 hours. Following the incubation period, the cells were treated

with acridine orange (4μM) and propidium iodide (3μM) dyes. Acridine orange stains

viable cells green, while propidium iodide stains non-viable cells red. Fluorescence

images of the cells were captured using a Leica fluorescent microscope to evaluate the

presence of viable and non-viable cells on the collagen biomatrix.

2.6 ANGIOGENIC ASSAY

2.6.1 AORTIC ARCH ASSAY FOR ANGIOGENESIS STUDIES

The aorta from an 8th-day fertilized egg embryo was delicately extracted and

rinsed with PBS (pH 7.4). Subsequently, it was placed in serum-free Dulbecco's

Modified Eagle's Medium with high glucose (DMEM-HG). The aortic arches were

then trimmed and cut into 1 mm pieces, which were further placed into a 24-well

tissue culture plate containing both native collagen scaffolds and Hennotannic Acid-

reinforced collagen scaffolds. This tissue culture plate, housing the aortic arches and

collagen scaffolds, was maintained in a CO2 incubator at 37°C. The sprouting of

endothelial cells from the aortic arches was documented every two days using a

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Leica DMi8 microscope. This assay facilitated the observation of the sprouting

process, providing insights into the growth and development of endothelial cells on

the collagen scaffolds over time.

2.6.2 CELL MIGRATION ASSAY IN CANCER CELL

The cells were seeded and allowed to grow until they reached confluency,

forming a monolayer. A sterile 100µl micropipette tip was gently used to scratch

across the cell layer to create a scratch or wound within the monolayer. The cells

were rinsed with DMEM (Dulbecco's Modified Eagle's Medium) to remove any cell

debris. Subsequently, the cells were incubated with fresh DMEM medium. The

migration of cells towards the wounded area was then observed and monitored at

different time intervals using an inverted Leica DMi8 microscope. (Ding et al.,

2018) This assay allowed the assessment of cell migration and the closure of the

scratch over time, indicating the ability of the cells to migrate and heal the wound-

like gap in the monolayer.

2.7 3D SPHEROID TUMOUR MODELS ASSAY

The 3D tumor spheroid assay was conducted to investigate the therapeutic

efficacy and tumor-penetrating capabilities of anti-cancer therapeutics. In this assay,

a three-dimensional (3D) in vitro culture model was established by embedding

cancer cells within collagen I, mimicking the architecture of a solid tumour. To

create the collagen gel, a solution was prepared and allowed to polymerize through

thermal cross-linking in a humidity box at 37°C for 2 hours. Cancer cells, including

A549, MG-63, 293T, SK-OV-3, PA-1, and A498, were trypsinized and resuspended

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at a concentration of 2 × 10^6 cells per mL. These cells were then used to generate

tumour spheroids, allowing them to grow for approximately six days. (Dhiman et al.,

2021)Photographs were taken on alternate days to document the formation and

growth of the spheroids. Following spheroid formation, they were treated with

Hennotannic acid, and the impact of Hennotannic acid on the 3D cancer cell

spheroids was monitored using microscopic imaging. This assay facilitated the

assessment of the therapeutic effects and influence of Hennotannic acid on the

spheroids within a 3D cellular environment.

2.8 STATISTICAL ANALYSIS

The experiments were performed in triplicate, and the provided data are

expressed as mean ± standard deviation (S.D). Statistical analysis was carried out

utilizing SPSS 22.0 software, with a significance threshold set at P<0.05 to

determine statistical significance.

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