Chapter 1 Introduction
1.6 Research Framework
In this study, the first task was to successfully synthesize phenol-modified glycol chitosan, and then verified the synthesis by 1H-NMR and UV spectroscopy analysis.
Then, the hydrogels were fabricated by photochemical crosslinking method. Secondly, the main characteristic of this tissue adhesive is tissue adhesion with animal tissue; thus, the experiment of tissue adhesion was done first before other experiments. After the experiment of tissue adhesion, the best-modified hydrogels, which performed maximum of tissue adhesion, could be selected to be the research object. The selected hydrogels would be tested via various experiments of material evaluation, including rheological analysis, hydration, degradation and cell viability, to observe material
property. Subsequently, the experiments of functional evaluation, such as in vitro drug release, antibacterial activities and in vivo hemostatic ability, would be done to verify whether this hydrogels could serve as tissue adhesive, drug release system, antibacterial material and hemostat, and be efficacious in pragmatic application.
Figure 1-1 Experimental scheme of this research Synthesis of phenol-modified glycol chitosan
Fabrication of photochemically crosslinked hydrogels
Tissue adhesion
Rheological analysis
Hydration Degradation Cell viability
In vitro drug release Antibacterial ability In vivo hemostatic ability
Chapter 2
Materials and Methods
2.1 Chemicals
2.1.1 Synthesis of HPP-GC
1. Glycol-chitosan (≧60% (titration), crystalline): Cat. # G7753, Sigma-Aldrich, USA
2. N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC): Cat. # 03450, Fluka, United Kingdom
3. N-Hydroxysuccinimide (NHS): Cat. # H7377, Sigma-Aldrich, USA
4. 3-(4-Hydroxyphenyl)propionic acid (phloretic acid, PA): Cat. # A14567, Alfa Aesar, Great Britain
5. 2-(N-Morpholino)ethanesulfonic acid hydrate (MES hydrate): Cat. # M2933, Sigma, USA
2.1.2Preparation of photo-crosslinked adhesives
1. Tris(2,2′-bipyridyl)dichlororuthenium(II) hexahydrate (Ru(bpy)3Cl2): Cat. # 224758, Aldrich, USA
2. Sodium persulphate (SPS): Cat. # 13457, Sigma-Aldrich, USA
2.1.3Swelling and degradation
1. Sodium chloride (NaCl): Cat. #4058-01, J. T. Baker, USA
2. Lysozyme from chicken egg white: Cat. # L6876, Sigma-Aldrich, USA
2.1.4MTS assay
1. CellTiter 96® AQueous One Solution Reagent: Cat. # G358A, Promega, USA 2. Fetal bovine serum (FBS): Cat. # 04-001-1A, BI, USA
3. Gentamicin solution: Cat. # SV30080.01, GE, USA
4. Minimum essential medium alpha medium (alpha-MEM): Cat. # 11900-024, Gibco, USA
5. 2-Mercaptoehanol: Cat. # M3148, Sigma-Aldrich, USA 6. Penicillin Streptomycin (P/S): Cat. # 15140122, Gibco, USA 7. Phenol: Cat. # P1037, Sigma-Aldrich, USA
8. Sodium bicarbonate: Cat. # S7277, Sigma, USA
9. Sodium chloride (NaCl): Cat. #4058-01, J. T. Baker, USA
10. Tris(hydroxymethyl)aminomethane (Tris): Cat. # 4109-02, J.T.Baker, USA 11. Trypan blue: Cat. # T8154, Sigma, USA
12. Trypsin-EDTA solution: Cat. # T4174, Sigma, USA
2.1.5 In vitro drug release
1. Amoxicillin: Cat. # A8523 Sigma-Aldrich, USA
2.1.6 Antibacterial ability
1. LB Broth, Miller (Luria-Bertani): Cat. # 244620, Difco, USA
2. Agar Granulated: Cat. # 214530, Difco, USA
3. Gentamicin sulfate salt: Cat. # G1264, Sigma-Aldrich, USA
2.2 Experimental Instruments and Materials
2.2.1 Experimental instruments
1. Absorbance microplate readers: ELx800, BioTek, USA 2. Analytical balances: AB104-S, Mettler Toledo, USA 3. Autoclave: TM-326, Tomin, ROC
4. Centrifuge: 5804R, Eppendorf, Germany
5. Constant temperature water bath: WB212-B2, Kansin, Taiwan 6. Digital force gauge: FGP-5, NIDEC-SHIMPO, Japan
7. Incubator: Class-100 HEPA, Thermo Scientific, USA 8. Laminar flow hood
9. LED high power lamp (~8 W, 440–460 nm): PAR20, VITALUX, ROC 10. Motorized test stand: FGS-50VB-H, NIDEC-SHIMPO, Japan
11. Nuclear magnetic resonance (NMR): AVIII-500MHz FT-NMR, Bruker, USA 12. Orbital Shaker Incubator, DENG YNG, ROC
13. Oven, HSIANGTAI, ROC
14. pH electrodes, TN-TXW600-GB, TNI, ROC
15. Phase contrast optical microscopy: TS-100, Nikon, Japan
16. Modular compact rheometer: MCR-102, Anton Paar, Canada 17. Stirrer/hot plate: Model PC-420, Corning, USA
18. UV-visible spectrophotometer: Cary 300, Agilent, USA 19. Vortex-genie 2: G560, Scientific Industries, Inc., USA
20. Vacuum dry oven, DOV40, DENG YNG, ROC
2.2.2 Experimental materials
1. Dialysis membranes (MWCO 12,000~14,000), Cat. # 1230-45, Cellu Sep, USA 2. 75 mm bottle top filter, Cat. # 595-3320, Thermo Scientific, USA
3. 96-well cell culture microplate, Cat. # 267313, Nunc, Denmark 4. 100 mm TC-treated culture dish, Cat. # 430167, Corning, USA 5. 15 mL PP centrifuge tube, Cat. # 430791, Corning, USA 6. 50 mL PP centrifuge tube, Cat. # 430829, Corning, USA 7. 1.5 mL microcentrifuge tubes, Cat. # 1260-00, SSI, USA
8. 2 mL microcentrifuge tubes, Cat. # 1310-00, SSI, USA
2.3 Solution Formula
1. MES buffer, pH 5.0
19.52 g MES hydrate is dissolved in 800 mL of deionized water. The pH is adjusted to 5.0 with NaOH, and deionized water is added to a total volume of 1 L. MES buffer is sterilized by filtration through a 0.2 µm filter and stored at 4 °C.
2. Minimum Essential Medium Alpha Medium
A bag of alpha-MEM power and 2.2 g sodium bicarbonate are dissolved in 800 mL deionized water. Then, 400 μL 2-Mercaptoehanol, 1 mL gentamycin solution, 10 mL P/S, and 100 mL FBS are added. The pH is adjusted with HCl and NaOH to 7.4, and deionized water is added to a total volume of 1 L. The solution is sterilized by filtration through a 0.2 µm filter and stored at 4 °C.
3. Phosphate buffered saline solution (PBS)
PBS consisted of 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.245 g KH2PO4 in 1 liter deionized water with its pH value of 7.4. The pH value was adjusted to pH 7.4
by 0.1 N HCl and 0.1 N NaOH.
2.4 Methods of Material Evaluation
2.4.1 Synthesis of HPP-GC
The synthesis of HPP-GC was modified from the previous research [30] (Figure 2-1).
Briefly, 0.2 g glycol chitosan was dissolved in 50 ml pH 5.0 MES buffer; the solution was put into Erlenmeyer flasks with magnetic stirring bar continuously stirring for 1 hour until glycol chitosan was totally dissolved. Secondly, 0.03 g of HPP were dissolved in 50 ml pH 5.0 MES buffer. HPP solution was avoided from light and then wait for 1 hour until HPP was totally dissolved. Afterwards, two solution mentioned above were mixed, and then 0.22 g EDC and 0.12 g NHS were added into the flask. The solution
was stirred at room temperature overnight. After reaction completed, the product was purified by dialysis against deionized water through a seamless cellulose tube (MWCO:
12,000~14,000, Cellu Sep, USA) for 2 days. Last, the frozen solution lyophilized for three days to obtain HPP-GC sample.
2.4.2 Characterization of HPP-GC
1H-NMR and UV spectroscopy analysis were used to verify the successful
derivatization and determine the degree of substitutions (DS) of phenol groups in HPP-GC samples. For 1H-NMR, 10 mg samples were dissolved in 0.5 ml deuterium oxide and analyzed by using Nuclear magnetic resonance (AVIII-500MHz FT-NMR, Bruker, USA). The DS of phenol groups in HPP-GC polymer was quantified using UV-Visible Spectrophotometer (Cary 300, Agilent, USA). The glycol chitosan and HPP-GC samples were dissolved in deionized water and the absorbance was measured at 276 nm. Phenolic substitution was calculated from a standard curve prepared using various concentrations (200, 100, 50, 25, 12.5 µg HPP / ml ultra-pure water) of HPP dissolved in ultra-pure water.
2.4.3 Preparation of photo-crosslinked HPP-GC adhesives
All hydrogel precursor solutions were prepared in deionized water and the concentration of solutions are 3% (wt. %). The solution was mixed with light-activated metal catalyst RuII(bpy)3Cl2 (1mM) and SPS (20 mM). Then, irradiating the solutions
for 30 s with a LED high power lamp (8 W, 440–460 nm: PAR20, VITALUX, ROC) from a distance of 15 cm. The crosslinking mechanism was illustrated in Figure 2-2 and 2-3.
2.4.4 Tissue adhesion
Tissue adhesion was examined using a digital force gauge (FGP-5, NIDEC-SHIMPO, Japan) shown in Figure 2-4 (A) with a 50 N digital force gauge. Two pieces of egg membrane was attached to glass cylindrical vials (diameter: 10 mm) by using O-ring.
90 μl HPP-GC solution was applied on the top of the egg membrane, and then make the upper glass cylinder move downward to attach to the hydrogel. LED high power lamp was turned up, and the hydrogel was exposed to the light for 30 s. After turning off the lamp, wait 30 secs until hydrogel was stable. Afterwards, the detachment stress was tested by making the upper glass cylinder move upward to pull the hydrogel apart. The maximum of detachment stress was recorded. The procedure is shown in Figure 2-4 (B). This experiment mainly aims at testing the tissue adhesion of hydrogel with animal tissue (egg membrane). The reason that using egg membrane is its low cost, easiness to operate and thinness that can let blue light penetrate appropriately.
2.4.5 Rheological analysis
The rheological properties were evaluated by using a Modular compact rheometer (MCR-102, Anton Paar, Canada). Parallel plate geometry (20 mm diameter) was
performed at 37℃ to monitor the elastic modulus values (G’) and the viscous modulus
values (G’’). A constant strain 1 % was used for the measurements. For the frequency sweep measurements, the sweep of the frequency varied from 0.1 Hz to 1 Hz, and each frequency sweep took 10 points to be completed. Through rheological analysis, the mechanical strength and elastic property can be measured to ensure the hydrogel is able to serve as adhesives that would not break or be destroyed easily.
2.4.6 Hydration
After the fabrication of HPP-GC hydrogels, hydrogels were lyophilized and the dry weights of hydrogels (Wdry) were measured. Afterwards, the dry samples were immersed in pH 7.4 PBS. Then, at specific time interval, water on the surface of hydrogels was removed by kimwipes, and the wet weights of hydrogels (Wwet) were recorded. Hydration values of HPP-GC hydrogels with different degree of substitutions were calculated by the following equation. Hydration is highly correlated with morphology and stability of hydrogel, this test can help us analyze other characteristics such as degradation and drug release of this hydrogel.
Hydration (%) = (𝑊𝑤𝑒𝑡− 𝑊𝑑𝑟𝑦)
𝑊𝑑𝑟𝑦 × 100%
2.4.7 Degradation
Hydrogel samples (200 mg) were prepared in vials accurately weighted (Wi).
Subsequently, 5 mL of PBS solutions containing lysozyme (1 mg/mL) were added into
the vials to cover the hydrogels and then incubated in 37 ℃ at 50 rpm Orbital Shaker Incubator (DENG YNG, ROC). At specific time intervals (1, 4, 7, 14 days), the buffer solution was removed from the samples and the hydrogels were weighted (Wt) after water on the surface of hydrogels was removed by kimwipes. The remaining mass ratios were determined by the following equation. Degradation time can demonstrate that whether the hydrogel can sustain for enough time to serve as adhesives, wound dressing in wound healing process.
Remaining mass ratios (%) = 𝑊𝑡
𝑊𝑖 × 100%
2.4.8 MTS assay
Cytotoxicity of hydrogel was studied using L929 cells according to ISO10993 standard test. The hydrogel was prepared in a 2 mL centrifuge tube from 0.2 mL (3 wt. %) HPP-GC solution. Each tube of hydrogels was extracted using 1 mL of serum-free medium and incubated for 48 hours at 37°C shaker (50 rpm). Followed by incubation, the final extract solutions containing 10% fetal bovine serum were diluted using culture medium to 100%, 50%, and 10% for culturing L929 cells. L929 cells were seeded in 96-well plate at a density of 2 × 104 cells per well, and cultured in 100 µL of culture medium for 1 day in 37 ℃ incubator. Afterward, the culture medium was replaced with 100 µL of prepared dilution, and the plate was incubated for 1 day in 37 ℃ incubator.
Subsequently, 20μl of CellTiter 96® AQueous One Solution Reagent (MTS) in 100μl
of culture medium replaced the solution of each well, and the plate was incubated for 3 hours. Finally, absorbance was measured at 490 nm using an Elisa reader (ELx800, BioTek, USA). The results were compared with cells treated using culture medium and 0.64% phenol solution in culture medium. This test is significant for this biomaterial since this hydrogel will directly adhere to human tissue; thus, the cytotoxicity should
be seriously considered.
2.5 Methods of Functional Evaluation
2.5.1 In vitro drug release
Amoxicillin solution (0.2 wt. %) was prepared, and then HPP-GC samples were dissolved in this solution (3 wt. %). After crosslinked by irradiation of blue light, the hydrogels were incubated in 10 ml pH 7.4 PBS, and 0.5 ml solution would be taken out from the vials for Uv-visible test; 0.5 ml fresh pH 7.4 PBS was then added to the vial.
The solution would be taken out in different time intervals including 10, 20, 30, 45, 60, 90, 120, 180, 300 minutes. Amoxicillin could be detected at 229 nm in Uv-visible spectrophotometer (Cary 300, Agilent, USA), and then the release ratios of antibiotics were also determined from the calibration curves.
2.5.2 Antibacterial ability
Antibacterial activities were tested by inhibition of zone. E. coli as an example of Gram-negative bacteria and Staphylococcus epidermidis as an example of Gram-positive
bacteria were used as target organisms. Concretely, each bacterium was incubated in sterile Luria-Bertani (LB) liquid culture medium at 37 ℃, with a 200 rpm shaking rate.
Afterward, the bacteria solutions were diluted to a 0.01 optical density at 600 nm with fresh LB broth. LB agar medium was prepared and sterilized in an autoclave. Then the agar medium was poured into the sterile petri plates when the medium was in a warm molten state. After the medium solidified, 10 μl of bacterial solution was transferred into sterile petri plates and daubed the surface uniformly. Then, hydrogels (containing 0.005%, 0.01%, 0.02% and 0.1% gentamycin) was tailored into circle with the diameter of 8 mm and placed on the LB agar medium. The agar plates were then placed in an incubating oven at 37 °C and were left for 14 hours to assess the zone of inhibition for each of the hydrogel. The image of zone of inhibition was photographed and percentage of the inhibition was estimated through the following equation. By this antibacterial experiment, the fact that whether the hydrogels can serve as ideal drug release system and whether the gentamycin entrapped hydrogels by photochemically crosslinked method can effectually against bacterial can be verify.
%inhibition = 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 𝑧𝑜𝑛𝑒 𝑜𝑓 𝑖𝑛ℎ𝑖𝑏𝑖𝑡𝑖𝑜𝑛 (𝑚𝑚)
𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑙 (𝑚𝑚) × 100%
2.5.3 In vivo hemostatic ability
To evaluate the hemostatic potential of the HPP-GC hydrogels, a hemorrhaging liver mice model was employed (22–25 g, 5 weeks, male). All animal studies were performed
in compliance with guidelines set by national regulations and were approved by the local animal experiments ethical committee. Briefly, a mice was anesthetized using 400~600 μl 2% xylocaine mixed with water (vol.: vol. = 1:3) and fixed on a surgical corkboard. The liver of the mouse was exposed by abdominal incision, and serous fluid around the liver was carefully removed to prevent inaccuracies in the estimation of the blood weight obtained by the filter paper. A pre-weighted filter paper on a paraffin film was placed beneath the liver. Bleeding from the liver was induced using a 28 G needle and 100 μl of hydrogel was immediately applied to the bleeding site and expose the gel to blue light 10s (solution: 3 wt.% of HPP-GC). After bleeding for 2 min, the weight of
the filter paper with absorbed blood was measured and compared with a control group.
2.6 Statistic Analysis
The data was presented as means ± standard error of the mean (SEM). The statistical analyses between different groups were determined with Student- Newman-Keuls Multiple Comparisons Test. A value of p ≤ 0.05 was considered statistically significant difference.
Figure 2-1 Synthesis of phenol groups modified glycol chitosan via EDC & NHS method.
Figure 2-2 Fabrication of HPP-GC hydrogel.
Figure 2-3 Mechanism of photochemical crosslinking [35].
Figure 2-4 (A) Digital force gauge. (B) Procedure of testing the detachment stress of hydrogel by using egg membrane.
Egg membrane
HPP-GC hydrogel
(A)
(B)
Chapter 3
Results and Discussion
3.1 Synthesis and Characterization of HPP-GC
HPP-GC was synthesized by the reaction of glycol chitosan (GC) with 3-(4-Hydroxyphenyl) propionic acid (HPP). The successful derivatization was verified by
1H NMR (Figure 3-1). Quantitative Uv-visible analysis (Figure 3-2) was used to
determine the degree of substitution (DS) of phenol groups in HPP-GC samples.
The successful synthesis was verified by 1H NMR. Figure 3-1 shows 1H NMR spectra of glycol chitosan and HPP-GC. By comparison with the spectrum of glycol chitosan, new signals were observed at δ = 6.8 ppm and δ = 7.2 ppm in the spectrum of HPP-GC. The signals of new peaks corresponding to aromatic protons confirmed the successful graft of phenol groups in the HPP-GC polymer. The reduction of the signal of hydrogen on amino groups at δ = 2.7 ppm after the reaction confirmed that the reaction completed correctly.
The degree of substitution (DS) was determined by Uv-visible spectrophotometer.
According to the Figure 3-2, the phenol groups on HPP-GC could be detected at 276 nm wavelength. Among all the samples produced, the DS arranged from 0.200 to 0.600 μmoles of phenol groups per mg of HPP-GC. The DS could be controlled by the amount of HPP. After the actual DS was determined, the following experiments of different
characteristics could be analyzed and explained.
3.2 Fabrication of Photocrosslinked HPP-GC Hydrogel
After mixed with light-activated metal catalyst RuII(bpy)3Cl2 and SPS, the solution become orange ready to be crosslinked. This injectable solution could be crosslinked whenever the user wants and could be made into any shape (Figure 3-3). In the photo crosslinking process, the solution could be crosslinked rapidly within 5 seconds, yet waiting for 30 seconds to make the hydrogel crosslinked completely in our procedure.
These advantages make applying hydrogel to irregular wound possible and the moment for crosslinking could be controlled easily. Compared with other crosslinking methods, widely used enzymatically crosslinked hydrogels always need special device to fabricate hydrogel such as double syringes [30, 34], that is an additional expense and hard to operate. Besides, after mixing with HRP and H2O2, the gelation will occur immediately and the moment is hard to control that would definitely cause inconvenience in certain condition. Moreover, some hybrid crosslinking methods are under restraint like temperature and pH of environment [33]. This photochemically crosslinked hydrogel can conquer those disadvantages with the merits mentioned above.
As the Figure 3-4 shown, different DS have significant influence on the mechanical properties, structure and appearance of hydrogels. High DS (> 0.500 µmole/mg
HPP-GC) would make the hydrogel shrink, and the high crosslinking density would squeeze out the water of hydrogels. Sometimes the hydrogel may be stiff and crack after
crosslinked.
3.3 Strong Tissue Adhesion
Strong tissue adhesion is the main advantage of this photochemically crosslinked HPP-GC hydrogel. In previous study, it puts forth the fact that by irradiation of blue light, the phenol groups can not only form intermolecular crosslinking but bind the amino and thiol groups on animals’ tissue as well [35, 41]. Tissue adhesion could be measured by utilizing digital force gauge to measure the detachment stress at the break point of hydrogels when they were pulled apart. The results indicates that different DS would critically affect the tissue adhesion of hydrogels. Figure 3-5 shows that the detachment stress of the least DS (around 0.250 µmole/mg HPP-GC) is 15.99 ± 9.29 kpa, the moderate DS (around 0.370 µmole/mg HPP-GC) is 77.96 ± 12.10 kpa, the higher DS (larger than 0.534 µmole/mg HPP-GC) is lower than 10.40 ± 7.90 kpa. This photochemically crosslinked HPP-GC hydrogel possess approximately same or larger adhesive strength than other crosslinking method or polymer [31, 33, 64]. Especially, compared with the commercial fibrin sealant fabricated by same photochemical method [46], the adhesive strength of this HPP-GC hydrogel is much larger.
From Figure 3-5, we could also find out that low DS (< 0.300 µmole/mg HPP-GC)
would not provide enough phenol groups to crosslink that the hydrogel would not have adequate crosslinking density; therefore, it would lose the cohesive ability to become hydrogel and could not form enough bonds with animal tissue for adhesion as well. As for hydrogels with high DS, they tend to have too high crosslinking density and that would cause the hydrogel have less elasticity, shrink and sometimes crack into pieces [41]. This tendency would severely destroy the structure of hydrogel and finally fail to bind animal tissue. After finding out the proper DS of hydrogels, which possess the strongest tissue adhesion, this most promising hydrogel would serve as the major object of this research.
In addition, as Figure 3-6 shown, different concentration also have crucial effect on tissue adhesion. Higher concentration (3%) possessed better tissue adhesion and moderate concentration (2%) just slightly lower than the higher one (3%); nevertheless, low concentration (1%) could not become strong hydrogel and it still had a lot of water which had not been crosslinked by irradiation of blue light. Therefore, it is ideal to
fabricate hydrogel between 2% to 3%.
3.4 Rheological Analysis and Mechanical Property
The rheological analysis can characterize the elastic and viscous property of this photocrosslinked HPP-GC hydrogel. As Figure 3-7 shown, the storage modulus of 3%
HPP-GC hydrogel is approximately 6663 ± 65 Pa, which indicates the fact that this
hydrogel is highly elastic and it has great mechanical strength [65]. Previous studies have shown that photo cross-linking 2% glycol chitosan solution has storage modulus of 1800 Pa [66]. Similarly, crosslinking 1.5% glycol chitosan solution with various concentrations of benzaldehyde functionalized PEG analogues has storage modulus ranging from 210~1400 Pa [30]. Hence, we can say that this ruthenium-based photochemical crosslinking process make HPP-GC hydrogel have much higher elastic and mechanical strength. The high storage modulus can also verify the fact that this photochemical crosslinking method can make hydrogel form strong intermolecular covalent bond between polymers. The outstanding elastic and viscosity of HPP-GC hydrogels is significant for tissue adhesion, which can better bear load and dissipate
elastic energy, and can be applicable to flexible soft tissue [67].
3.5 Hydration of Different DS HPP-GC Hydrogels
Swelling and hydration ability of taking up liquid play important part in wound dressing.
Swelling and hydration ability of taking up liquid play important part in wound dressing.