2‐1 Materials and reagents
1. Chitosan MW = 210000, degrees of deacetylation = 91.3 %, C&B company.
2. Isopropanol MW = 60.10, ACS grade, TEDIA.
3. Sodium hydroxide MW = 40.00, SHOWA.
4. Chloroacetic acid MW = 94.50, J.T. Baker.
5. Methanol MW = 32.04, ACS grade, TEDIA.
6. Haxanoic anhydride MW = 214.31, 99 % purity, ACROS.
7. Ethanol MW = 46.07, ACS grade, TEDIA.
8. Dialysis tubing cellulose membrane avg. flat width 33 mm (1.3 in.), Retains > 90 % of cytochrome C (MW = 12400) in solution over a 10 hour period, SIGMA.
9. Sodium alginate from brown algae, viscosity of 2 % solution at 25℃ ~ 250 cps, SIGMA
10. Glycerol MW = 92.09, purity = 99 %, Riedel‐de Haën.
11. Calcium chloride Mw = 110.98, SHOWA.
12. Olive oil viscosity at 20℃ ~ 84 cps, SIGMA.
13. Trypan blue 0.4 % solution, SIGMA.
14. L‐(+)‐ascorbic acid (vitamin C), MW = 176.12, J.T. Baker.
15. Retinoic acid (vitamin A), MW = 300.43, SIGMA.
16. WS1 (human fetal skin fibroblast cell lines, derice from BCRC; BCRC number: 60300) 17. Simulated body fluid (SBF), 1.5 times concentration, prepared by myself,
composition shows in Table 2‐1.
18. Phosphate buffered saline (PBS), UniRegion Bio‐tech.
19. Minimum essential medium (MEM), 20. Fetal bovine serum (FBS), Sigma.
21. Non‐essential amino acids 10mM, Gibco.
22. Sodium pyruvate 100mM, Gibco.
23. Trypsin EDTA Sigma.
24. 3‐(2,5‐dimethylthiazol‐w‐yl)‐2,5‐diphenylterazolium bromide (MTT reagent) 25. New Zealand white rabbits (animal study)
Component Na+ K+ Mg2+ Ca2+ Cl‐ HCO3‐ HPO42‐ SO2‐
Concentration 213.0 7.5 2.3 3.8 281.7 6.3 1.5 0.8 Unit: (mM) Table 2‐1 1.5 times concentration of simulated body fluid (SBF)
2‐2 Apparatus
1. Magnetic stirrer (MS‐211, Fargo Instruments Co.) 2. Electronic balance (Precisa, XS225A, Swiss Made) 3. Freeze dry system (FreeZone 4.5 Liter, LABCONCO) 4. Cooling centrifuges (Z326K, Hermle Labortechnik GmbH) 5. UV‐vis spectrometer (Evolution 300, Thermo scientific) 6. Rheometer (Rheological Scientific, ARES instrument) 7. Incubator (NUAIRE)
8. Autoclave (TOMIN) 9. ELISA (DV990BV4, GDV)
10. Fluorescence Microscopy (D‐eclipise C1, Nikon)
2‐3 Synthesis of CHC/SAL composite hydrogels
2‐3‐1 Synthesis of amphiphilic chitosan (CHC) polymer
Amphiphilic chemically‐modified chitosan, named carboxymethyl‐hexanoyl chitosan (CHC), has been synthesized in previous studies6, 7. Briefly, 5 g of chitosan was suspended in 50 ml isopropanol and stirred for 30 min at room temperature. Then the well‐suspended chitosan suspension was slowly mixed by instilling total 12.5 ml of 13.3 N sodium hydroxide solutions. To acquire a hydrophilic chitosan with a high degree of carboxymethyl substitution, named carboxymethyl chitosan (CC), 25 g chloroacetic acid was added into the prepared chitosan suspension at least five times in batches and stirred for 30 min to make sure all chloroacetic acid dissolved. The resulting suspension was heated to 60℃ under an oil bath for 4 hours and then followed by suction filtration method washing by 500 ml dilute methanol (90 % v/v). The resulting muddy solid was dried at 60℃ for one day. Taking 2 g of the dried CC sample dissolved in 50 ml distilled water one day and mixed with 50 ml methanol after CC samples completely dissolved. To gain the hydrophobic long carbon chains, 1.4 ml hexanoyl anhydride was added for a final concentration of 0.5 M. After 12 hours reaction process, the mixture solution was dialyzed with a dialysis membrane against dilute ethanol (75 % v/v) for one day and pure ethanol for another one day. The dialyzed samples were dried by oven at 60℃ for one day and the dried samples were amphiphilic chitosan (CHC) for the following using.
2‐3‐2 Preparation of CHC/SAL combination solutions
A 2 wt% CHC solution was prepared in a vial, using sodium hydroxide solution to adjust the pH value of the CHC solution to slightly alkaline (pH = 7.5 ‐ 8.5). CHC/SAL combination solutions with different compositions were formed by mixing CHC solution containing 0, 5, or 10 % glycerol) with an equal volume SAL solution, having a concentration of 2, 3, or 4 wt%.
2‐3‐3 Formation of CHC/SAL composite hydrogels
Composite hydrogels were formed in the presence of various calcium ions concentrations by submerging 2 ml of CHC/SAL combination solution in a glass Petri dish into 50 ml of 1, 2, or 3 wt% calcium chloride solutions at room temperature.
2‐4 Characterization of CHC/SAL composite hydrogels
In this section, the fundamental physical properties such as gelation time, equilibrium swelling degree, and water retention percentage were investigated to characterize CHC/SAL composite hydrogels under various components and concentrations of gelation medium.
2‐4‐1 Gelation time measurement
The time to form a gel (designated as gelation time) was determined using a vial tilting method, where no flow within 1 min of inverting the vial was criterion for gel state42, 43. Main
gelation factors such as sodium alginate (1, 1.5, and 2 wt%) and calcium chloride (1, 2, and 3 wt%) in gelation medium were controlled in this study.
2‐4‐2 Equilibrium swelling degree test
To determine the equilibrium swelling, samples of about 2 g of a gel made from CHC/SAL were lyophilized by Freeze dry system (FreeZone 4.5 Liter, LABCONCO) and weighted (Wd). The dried hydrogels were immersed in di‐water, medium (α‐MEM+10% FBS), or SBF for 1 day until equilibrium swelling state had been attained. After removal of water from the surface of swollen hydrogels, the samples were weighted (Ws). The equilibrium
swelling degree (ESD) was calculated using the following equation:
ESD ⁄ (2‐1)
2‐4‐3 Water retention test
Equilibrium swollen gels, were subsequently dried at 25℃ and 54 % relative humidity.
At predetermined time points, the samples were weighted (Wr) and the water retention ratio
(WR) was calculated using the following equation:
WR ⁄ (2‐2)
2‐5 Rheological properties of CHC/SAL composite hydrogels
Rheological characterization of the CHC/SAL composite hydrogels was performed on a strain‐controlled rheometer (Rheological Scientific, ARES instrument) using parallel‐plate fixture. Olive oil was used to cover the surface of the composite hydrogels in order to avoid water evaporation during the analysis. The gap at the apex of the parallel‐plate was set to be 2 mm and samples were placed between the parallel‐plate and the platform.
2‐5‐1 Strain sweep test
The dynamic mechanical properties of CHC/SAL composite hydrogels were characterized by strain sweep test (γ 0.01%~100%) using a fixed frequency (ω 10 ⁄ ) and a temperature of 37℃.
2‐5‐2 Small deformation test
The difference of dynamic mechanical properties under various concentrations of glycerol and CaCl2 in gelation medium studied by small deformation test (γ 0.015%~7%) using a fixed frequency (ω 10 ⁄ ) and a temperature of 37℃.
2‐5‐3 Strain step test
To investigate the recovery properties of the samples after exposure to high shear strain, the following program was applied: γ= 0.1% (100s) →γ= 100% (100s) →γ= 0.1% (200s) →γ=
100% (200s) →γ= 0.1% (300s).
2‐5‐4 Self‐healing test
To investigate the self‐healing capability of the composite hydrogels, two types of samples were prepared, one was colored by Trypan blue and the other was a pure hydrogel.
The samples were cut into the size of 3cm 1cm 0.5cm and the freshly produced surfaces of two samples with different color were brought together within one minute. After allowing healing to proceed for one, the healed composite hydrogel bridge was suspended horizontally and vertically.
2‐6 Biomedical properties of CHC/SAL composite hydrogels
2‐6‐1 Protection of easily degradable drugs inside the composite hydrogels
To determine the protective action of our composite hydrogels, the degradation of drug loaded inside the hydrogels was compared with the degradation of drug in water solution. As a model drug the easily degradable Vitamin C (L‐ascorbate) was chosen. Vitamin C was separately dissolved (1 mg/ml) in water and 2 % CHC solution. To form a gel film, we designed a mold to produce 5cm 3cm 0.1mm thin films and prepared a pure gel film as background in the UV‐analysis. At predetermined time points the concentration of remaining vitamin C was determined through the absorbance at 270 nm, using a spectrophotometer (Evolution 300, Thermo scientific).
2‐6‐2 Drug release behavior
Retinoic acid released from different preparations of CHC/SAL composite hydrogels was determined in various release environments. A stock solution of retinoic acid was prepared by dissolving 100 mg of retinoic acid in 50 ml isopropanol (IPA). CHC solution with drug was prepared by diluting the stock solution to achieve a final retinoic concentration 100 ⁄ , subsequently CHC nanoparticles were added to reach a concentration of 2 wt% CHC. Using the process mentioned in section 2‐3‐3, composite hydrogels with different compositions were prepared. To investigate the release profiles for the drug‐loaded CHC/SAL composite
hydrogels, samples were submerged in 3 ml di‐water or SBF. At predetermined times 1 ml samples were extracted and centrifuged at 12000 rpm for 5 min. Subsequently, the drug concentration in the supernatant was determined from the absorbance at 340 nm using a UV‐Vis spectrophotometer (Evolution 300, Thermo scientific). The extracted volume was replaced with an equal volume of fresh dissolution medium, which was accounted for in the release calculations.
2‐6‐3 Cell culture – cytotoxicity assay
WS1 human fetal skin fibroblast cell lines (BCRC number: 60300) were grown in minimum essential medium (Gibco) (MEM) with 10 % FBS, 0.1 mM non‐essential amino acids, and 1.0 mM sodium pyruvate. The cells were incubated at 37℃, in a 5 % CO2 humidified atmosphere. The culture medium was changed every two to three days. For all experiments, cells were harvested from sub‐confluent cultures using trypsin and were re‐suspended in fresh complete medium before plating. To investigate the in vitro cytotoxicity of the CHC/SAL composite gel, viability of human WS1 fetal skin fibroblasts was analyzed with the MTT assay.
CHC/SAL gels were formed from 0.5ml combination solution with the weight ratios CHC/SAL/glycerol = 1/1.5/0 prepared by exposure to 2 wt% CaCl2. Two kinds of samples, one loaded with 100 ⁄ retinoic acid and the other without drug (pure gel), were prepared in the 24‐well plates. Besides, pure sodium alginate gels (1.5 wt%) prepared by exposure to 2
wt% CaCl2 were also determined to compare with CHC/SAL composite gels. The control group was determined without any gels and set as 100 % survival ratio. Briefly, 3×104 cells were plated in 24‐well plates to allow the cells to attach at 37℃ in an atmosphere with 5 % CO2. After 1 or 2 days incubation, 100 μl of volume ratios MTT/medium = 1/9 combination solution was added and incubation was continued for another 4 hours. Then, DMSO was added to solve the precipitate, which formed from the reaction between MTT reagents and live cells, and the solution was transferred to a 96‐well plate. The result solution absorbance values were determined at 595 nm using a Sunrise absorbance microplate reader (DV990/BV4, GDV Programmable MPT Reader).
2‐6‐4 Animal study – skin irritation test
The Draize model and its modification such as UNI EN ISO10993‐10:1996 and USP Biological Tests are generally used to examine the degree of skin primary irritation utilizing healthy rabbits44‐46. Following the Draize model, the back of healthy male New Zealand White rabbits were narrowly clipped free of fur with an electric clipper 4 hours before application of samples. Each rabbit received six parallel epidermal abrasions with a sterile needle (26G 1 2 0.45mm⁄ 13mm) at one test site while the skin at the opposite site remained intact. Samples were prepared by coating a total of 0.5 ml gel (weight ratios CHC/SAL/glycerol = 1/0.5/0 and 1/1.5/0 prepared by exposure to 2 wt% CaCl2) on fixed size
with a non‐reactive tape and the entire test site was swathed with a non‐occlusive bandage.
After a 24 hours treatment, the bandage and gauze sample were removed. The test sites were swabbed with physiological saline solution to remove any remaining test article residue.
The procedure adopted in the U.S. Federal Hazardous Substance Act (FHSA)47 is described in Table 2‐2.
At 24, 48, and 72 hours after sample application, the test sites were evaluated for dermal reactions, defined as erythema and edema, with Draize – FHSA scoring system (Table 2‐3). The score of primary irritation of the test was calculated for various dosages. The Primary Irritation Index was calculated as the arithmetical mean and the evaluation of PII was performed according to the categories showed in Table 2‐4. The following formula was
used to calculate the primary irritation index45, 46:
PII ∑ , , ∑ , , (2‐3)
Number of animals 6 rabbits (clipped)
Test sites 2 1 inch sites on dorsum
One site intact, the other abraded Test materials Applied undiluted to both test sites
Liquids: 0.5ml; Solids/semisolids: 0.5g.
Occlusion 1 surgical gauze over each test sites Rubberized cloth over entire trunk
Occlusion period 24 hours
Assessment Visual scoring system after 24, 48, and 72hours
Reaction Score
Severe edema (raised more than 1mm and extending beyond the area of exposure)
Fig. 2‐1 Primary irritation test on the back of healthy rabbits.