METHODOLOGY

4.1 Procedure of Protein Extraction

4.1.1 Biomass Sampling

Material
Airtight container ( Ziploc)
Mortar and Pestle

Equipment
Freezer ( 20 C)
Freezer ( 110 C)

Gracilaria gracilis will be collected (Cabid-an, Sorogon) and about 3 Kg of wet

biomass will be sampled respectively. Next, the algal biomass will be washed with
distilled water and their epiphytes will be removed. The fresh seaweed will be placed
in a freezer (20C) immediately after collection. After that, the cleaned seaweed will
be freeze-dried at 110 C for 3 days and then ground to fine powder and stored in
airtight containers at 20C.

4.1.2 Phycobiliprotein Extraction and Analysis

Reagent
Alumina
1M acetic acid-sodium acetate
Sodium azide

Material
Centrifuge glass tube

Equipment
Potter Homogeniser (any type
of blender)
UV-Vis Spectrophotometer

An amount of 0.5 g of fine powder sample will be mixed with 0.5 g of alumina. The
mixture will be suspended in 10 mL of 1 M acetic acidsodium acetate buffer (pH 5.5)
with 0.01% of sodium azide for 30 min in the dark. After the incubation with buffer,
samples will be ground for 5 min using a Potter homogeniser (Marconi, model

MA099) or any type of blender. The mixture will be then transferred to a centrifuge
glass tube and will be centrifuged at 5 C, 15,000 rpm per g for 20 min. Supernatant
will be collected and the pellet will be extracted three times again with buffer as
described. Supernatants will be combined and the final volume of the extract will be
collected

for

about

40.0

mL.

Then,

the

Phycobiliproteins

(identified

as

R-

phycoerythrin R-PE, phycocyanin PC and allophycocyanin APC) will be quantified by

UV-Vis spectrophotometer according to Kursar et al.

4.1.3 Purification of Phycobiliproteins

Purification of Phycobiliproteins will be achieved in less than 24 h as
follows: The algae will be homogenized, substituting a buffer of 100 mM NaPi (pH
5.5), and centrifugation process will take place. The supernatant will be precipitated
with (NH4)2SO4 and the precipitate will be dialyzed against 1.0 mM NaPi, (pH 5.5)
containing 0.20 M NaCl. (Kursar, Van Der Meer, & Albert, 1983)

Reagent

Material

Napi buffer ( 100mM at ph 5.5)

Centrifuge tubes

(NH4)SO4

Equipment
Homogenizer(alternative blender)
Centrifuge Machine

1.0 mM NaPi, (pH 5.5)

containing 0.20 M NaCl.

4.2 In- vitro Biocompatibility testing through MTT Assay

4.2.1 Cytotoxicity Test: (measure cell viability of cultured cells)

Samples (Phycobiliproteins) will be incubated in 5mL RPMI media
with 10% (v/v) of fetal bovine serum (FBS) and 1% penicillin/streptomycin antibiotics
for 3 days at 37oC, while shaking at 100 rpm in an incubator to obtain an extract
solution. Fibroblast cells, 1 x 104 cells/100 L, will be seeded in 96-well culture plates

Equipment
RPMI Media
10% v/v fetal bovine serum (FBS)
1% penicillin/streptomycin antibiotics
DMSO
Dimethyl Sulfoxide

the

Reagent
Incubator

n
inc
uba

ted for 24 h at 37oC, 5% CO2. Then, extract solutions will be filtered with 0.20 m
filters and diluted to the following concentrations relative to fresh media: 25, 50 and
100%. Next, the diluted extract solutions will be added to the cell-seeded tissue
culture plates and incubated for another 3 days at 37oC and 5% CO2. Cell-seeded wells
without an extract solution (0%) will be also prepared as a control. MTT solution will
be added to the wells after 3 days and re-incubated for 4 h. Media will be discarded
from the wells and replaced with 200 L of DMSO to dissolve the formazan salts. The
cell viability will be quantified by measuring the absorbance at 595 nm using an
ELISA

reader

(Turner

Biosystems

CE,

Promega

Corporation,

USA).

These

experiments will be conducted in three replicates and cell viability will be calculated
as percentage relative to the cell-seeded wells containing media without the extract
solutions.

4.2.2 Test for Cell Proliferation

Fibroblast cells, 1 x 104 cells/ml, will be seeded on the electrospun PCL/PLGA
membranes that are 15 mm in diameter, and Phycobiliprotein samples will be
incubated for 1, 3 and 5 days in 24-well culture plates. Media will be replaced every
other day.

After each incubation time, media will be discarded and MTT solution will

be added and re-incubated for another 4 h. DMSO will be then added and plates were
shaken for 1 h and the absorbance will be read at 595 nm using an ELISA plate reader.
The cell viability will be calculated as the ratio of absorbance to the control after 1 day
of incubation and expressed as a percentage.

Reagent:
Polycaprolactone/Polyclacto-co-glycolic acid (PCL/PLGA)