2.1. and ethylene glycol obtained from Chem-Lab, Belgium.

2.1. Materials

L-lactide (LLA) and Glycolide (GA) were prepared from 90% L-lactic
acid and glycolic acid solutions, (Merck Inc., Darmstadt, Germany) according to
our previous study. These monomers were purified by multiple
re-crystallizations from ethyl acetate and eluted by the diethyl ether to
remove residual impurities and finally, dried under vacuum at room temperature
for 24 hr 20,22. Stannous octoate (Sn(Oct)2)
(Sigma, St. Louis, USA), was purified by vacuum distillation, if necessary 22. Sodium periodate, 1-hydroxybenzotriazole hydrate (HOBt), hyaluronidase,
polyvinyl alcohol (PVA) (MW=85,000-124,000) and Dulbecco’s modified Eagle’s
medium (DMEM) were all purchased from Sigma-Aldrich. Dexamethasone Sodium
Phosphate (DEX) with molecular
weight: 516.40 gr/mol, was purchased from Caspian Tamin Co, Iran. Dichloromethane (DCM)
and 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) were purchased from Merck, Germany. Hyaluronic acid (HA) was
purchased from Bloomage Freda Biopharm Co.,Ltd.,
China (MW 1.2 MDa), Genipin (Gp) was provided from
Challenge Bioproducts Co., Ltd, Taiwan. Ethylene Diamine (ED) and ethylene glycol obtained from Chem-Lab, Belgium.

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2.2. Synthesis
of the polylactide-co-glycolide (PLGA)

on our previous studies, calculated amounts of L-lactide and glycolide mixture,
in a LLA:GA = 85:15 mol% ratio were mixed together and poured in glass tubes
with Sn(Oct)2 (mole ratio of reacting monomer/catalyst kept at 5000)
were placed into
a polymerization tube and kept under vacuum at 50 °C for 2-3 h, until all
volatiles were removed. Then, the polymerization tubes were sealed under vacuum
and immersed in an oil bath at 120 °C for three days. At the end of the
reaction, the tubes were broken, and the contents were dissolved in chloroform,
filtered, and finally precipitated in cold methanol. The purified products were
finally dried under vacuum for 24 h 20,22,26. The schematic of
synthesis reaction of PLGA is presented in Fig 1.

2.3. Preparation
of PLGADEX nanoparticles

The PLGADEX nanoparticles were synthesized using the emulsification-diffusion method with few
modifications as reported by Davachi et al 26. At the first
step, polyvinyl alcohol (PVA) with conc.
5% (w/v) was completely dissolved in distilled water to form an emulsification phase and act as a stabilizer during the nanoparticle
preparation 26,27. Then, 300 mg
of DEX was dissolved in 8 ml distilled water and prepared DEX solution was
added to PVA solution (solution 1). After the preparation of solution 1, synthesized
PLGA was dissolved in chloroform (solution 2). Two mixtures were mixed using a magnetic stirrer for 10 min at 300 rpm and were
further mixed using an overhead stirrer for 20 min at 1200 rpm. The prepared
oil in water mixture was then sonicated twice, each time for 10 min while being
stirred. The organic solvent was eliminated from the nanoparticle suspension by
the vacuum steam distillation at 35°C and 100 rpm under controlled pressure at
70 mm-Hg using a two-way glass valve. The nanoparticle suspensions were centrifuged
using an ultracentrifuge Sigma 3-30k at 20000 rpm for 20 min and were frozen at -70°C for 12 h. Finally, they
were freeze-dried using an Alpha 1-2 Lo Plus (Christ, Germany) freeze-drier at
0.074 mb and -53°C for 24 h 26. In the
current work, for preparation of PLGADEX nanoparticles, 80 mL of chloroform as
an organic solvent, 8 gr of PVA and 160
mL of distilled water was used and DEX
used in three different concentrations 10, 20 and 40% with respect to the PLGA
weight. Finally, to preserve the morphology of the nanoparticles after freeze
drying samples were grinded in liquid nitrogen.

2.4. Synthesis of
double-crosslinked hyaluronic acid hydrogel

2.4.1. Synthesis of
amino-Hyaluronic acid (amino-HA)

(HA-NH2) was synthesized in aqueous conditions. Briefly, 0.5 g HA
was dissolved in 100 ml distilled water
to form a 5 mg/ml solution. Afterwards, this solution was added to a ethylenediamine
excess solution. The pH of the solution was controlled by adding HCl to be 6.8.
Then, 0.8 g EDC and 0.7 g HOBt were dissolved in DMSO: H2O (1:1 v/v,
5 ml each) and added to the reaction mixture. The reaction was stirred for 24 h
at room temperature and then was exhaustively dialyzed (MWCO 10 000,
Spectra/Por membrane, Rancho Dominguez, CA, USA) against distilled water for 3 days. NaCl was then added
to produce a 5% w/v solution and the HA-NH2 was precipitated in ethanol.
The precipitate was resolved in water and dialyzed for 3 days to remove the
salt. The purified product was freeze-dried at ?50 ?C (Freezone 4.5, Labconco,
USA) and kept at 4°C. The percentage of substitution in the HA-NH2
was quantified as 53% using a ninhydrin assay 9.

2.4.2. Synthesis of
aldehyde-Hyaluronic acid (aldehyde-HA)

the synthesis of Aldehyde-HA (HA-CHO), 1.0
g HA was dissolved in 100 ml distilled
water at a concentration of 10 mg/ml. An aqueous solution of sodium periodate
(0.5 M, 5 ml) was added dropwise and the reaction was stirred for 2 h at room
temperature in the dark. Ethylene glycol (1 ml) was then added to inactivate
any unreacted periodate. The reaction was stirred for 1 h in an ambient
temperature and the solution was purified by exhaustive dialysis against water
for 3 days, and the dry product was obtained by freeze-drying. The percentage
oxidation of HA-CHO was
quantified by measuring the number of aldehydes in the polymer, using t-butyl carbazate. Determination of the actual aldehyde
content of HA-CHO revealed an extent of oxidation of 47% 9.

2.4.3. Double crosslinked
HA hydrogels fabrication

for preparation of
the HA double-crosslinked hydrogels (HA-NH2/HA-CHO with
genipin) (XHA), genipin was dissolved in the HA-CHO solution at the desired
concentration. HA double crosslinked hydrogels were formed by mixing HA-NH2 and HA-CHO/genipin solutions in various volume
ratios of 2:8, 4:6, 5: 5, 6:4 and 8:2 at room temperature. The genipin concentration
was 4mM according to Tan et al as this concentration was the optimum amount to
be used 9. The chemical reaction of XHA
hydrogels fabrication is shown in Fig 2.


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