Preparation Of Hydroxybutyl Chitosan And Its Sensitive Hydrogel (Temperature And PH) And Biocompatibility

Chitosan (CS) is a widespread amino polysaccharide in nature, and is the product ofchitin after deacetylation. CS has many unique properties, such as biodegradability,non-toxicity, good biocompatibility, and antibacterial activity. In the biomedical field, CS iswidely used for drug delivery carriers, tissue engineering scaffolds, wound dressings, and soon. However, there are many amino and hydroxyl groups in the macromolecular chain of theCS, and those groups can form intramolecular and intermolecularhydrogen bonds. Thisreason led to be higher crystallinity and poor soluble of CS, which greatly limits itsdevelopment and application. Preparation for water soluble CS derivatives by chemicalmodification will be one important way of improving the performance and broadening itsapplication range. In this thesis we prepare a good water-soluble CS derivative–hydroxybutyl chitosan (HBCS) by etherifying modification method, which introducehydroxybutyl groups into the CS molecular chain. Then for this modified product we researchthe physical and chemical properties, gelation properties, bioactivities and biocompatibilities,and all of these will have great significance for the development of the CS products on thepotential values.In this study, we use the ring-opening product of1,2-epoxy butane in the alkalinecondition as the etherification agent to modify the CS. We research the main reactionconditions, such as the reaction medium, the dose of1,2-epoxy butane, the temperature andtime, all of which would have an influence on the substitution of product, the intrinsicviscosity and water-solubility. The results show that the optimum condition for thepreparation of the HBCS is as follows: CS1g,10ml50%NaOH solution, under N2protection, reacting for24h, then squeezing out the excess lye, and dispersing in the mixtureof water and isopropanol (isopropanol: water (v: v)=1:1), then adding1,2-epoxy butane20ml, and at60℃reacting for24h. By the elemental analysis, we know the substitution of theproduct under this condition is1.91. By FTIR and solid-state13C NMR, we character themolecular structure of the product, and demonstrate the successful introduction of thehydroxybutyl groups into the CS molecular, confirm the substituted position is C6-OH andC2-NH2. We further determine the physical and chemical properties of the synthesis product, and find HBCS has good water-solubility, pH wide adaptation, strongly hygroscopic andmoisture capacity and other advantages, comparing with the raw material of CS.We research the inhibition of cell migration, immune activity and antibacterial activity ofHBCS, respectively. Using Scratch Test, we study HBCS play the role in L929cell migration.There were two phases in this experiment, the pretreatment and cell migration, and both of thephases affect the role of HBCS impacting on the cell migration. After pretreatment for24h,HBCS show the strong inhibition of L929cell migration. During the cell migration phase, weuse HBCS (0.25mg/mL) to stimulate cells, after24h scratching, and respectively at differenttimes observe the situation of cell migration. The results show that for L929cells thestrongest inhibiting effect of HBCS is after6h, and the relative mobility is not relevant to thecell migration time. In addition, we also examine different concentration of HBCS in the roleof cell migration, the higher concentration bring the stronger inhibition of cell migration.Compared with the control group, when HBCS is0.25mg/mL, the maximum inhibiting cellmigration speed could reach48%. The results of the effects of HBCS on the phagocytosis ofmacrophage and the proliferation of lymphocyte in normal mice show that HBCS canenhance the phagocytosis function of monocyte and peritoneal macrophages, and as the doseincrease, the phagocytosis capacity enhance. Similarly, HBCS can stimulate the proliferationof lymphocyte in mice spleen, and also promote ConA, LPS-induced T, B lymphocyteproliferation. By turbidity method, we determine the antibacterial activity of the HBCS, at theconcentration>0.01%, HBCS show inhibiting the growth of Escherichia coli andStaphylococcus aureus, and this inhibiting capacity has the dependence of the concentrationof the HBCS, increasing with the concentration.The HBCS hydrogel is prepared through intramolecular and intermolecular hydrogenbonds without any cross-linking agent. The HBCS hydrogel is temperature-sensitive, it isclear and transparent solution in an ice bath and become to be turbidity when the temperatureincrease.2.5wt%of the HBCS hydrogel is translucent and has a certain intensity, which canbe clamped. The main factors affect the gel process are investigated by systematic rheologicalmethod, and the results are as follows:1. The gelation time decrease with increasing CSconcentration and temperature, the gelation time is <90s under different circumstances,belonging to a rapidly responsive hydrogel.2. The concentration, pH and solvent types ofHBCS all affect the gelation temperature. The gelation temperature is decrease with increasing the HBCS concentration. The solvent pH also affect the gelation temperature of theHBCS hydrogel.5wt%HBCS solution in distilled water has a gelation temperature of16.5℃, in PBS7.4is15.5℃, in pH4HCl is17.5℃and in pH10NaOH can not be a gel. Theions in the solvent also affect the gelation temperature, when the CaCl2, MgCl2is added to thesystem, the HBCS show gel characteristics at0℃. The Na+, when the concentration is0.1mol/L, the gelation temperature is3.5℃.3. HBCS hydrogel is heat reversible exchange withsurroundings temperature and can occur sol-gel-sol transformation continuously with theconversion of ambient temperature. The internal morphology of the hydrogel was observed bySEM and the results show that it is porous network structure, with smooth and dense porewalls. The pore size of the hydrogel decreased with the increasing content of HBCS. Theresults of the swelling ratio test certify the hydrogel could reach swelling equilibrium within60min, belonging to a fast swelling hydrogel.To study the biocompatibilities of the HBCS hydrogel, the cytotoxicity test, hemolysistest, acute systemic toxicity test, muscle and subcutaneous implantation experiments areconducted according to the "Chinese Ministry of Health, Biological evaluation of biomaterialsand medical devices". The cytotoxicity test is performed using fetal fibroblast cells as targetcells and the cell proliferation degree is detected. The result show that in the group of HBCSsolution, the HBCS demonstrate weak cytotoxicity when its concentration>0.5mg/mL.While in the extract group, the fetal mouse fibroblast cells relative growth rate is higher than75%, independent of the concentration and the stimulus time, indicating the goodcyto-compatibility. The hemolysis test of CS and HBCS is detected by direct contact methodand the results show that the1mg/mL of CS solution has a higher hemolytic rate with15.33%. HBCS could improve the hemolytic properties of CS, when the m (CS): m (HBCS)≦1:1and the concentration is1mg/ml, the hemolysis rate is <5%. The hemolysis rates are alsodifferent when the solvents of the HBCS are different, the solution with a0.9%NaCl assolvent has a2.24%hemolysis rate, while solution with a2%acetic acid as solvent has a2.67%hemolysis rate. The results of the acute systemic toxicity show that the HBCShydrogel extracts has not toxic effects in mice. The weight gain of the mice is normal, noadverse reactions are found, and sections of the main organs in mice are normal too. When theHBCS solution is embedded in the back muscles and subcutaneous tissue of mice, it can forma gel structure in situ, the tests are conducted at different time after implantation. There are no significant surrounding tissue inflammation, tissue degeneration and necrosis afterimplantation. In the early stages after implantation, the fibrous capsule can be seen around thehydrogel. With the time extend, the fibrous capsule become thinner until disappearance. TheHBCS hydrogel could be slowly depredated within the muscle tissue and subcutaneous tissue.All results prove that the HBCS hydrogel has no cytotoxicity in vitro, no acute hemolytic, noacute toxicity in vivo, and good tissue compatibility.We use HBCS as the carrier material and BSA as a model drug to prepare HBCShydrogel delivery systems which is loaded BSA. The release behaivor in vitro is studied. Therelease of BSA resulted from HBCS hydrogel is affected by the concentration of HBCS,environmental temperature and pH values. As the increase of the HBCS concentration, theBSA release speed becomes slower and the release ratio decrease. Compared with the releasebehavior at25℃, the BSA release speed becomes faster at37℃. The pH also affects theBSA release behavior, at pH=6.0, HBCS begins to be dissolute and all of the BSA is releasedout in1h. Compared with at pH=9.0, the BSA release is faster at pH=6.0. These resultssuggest that HBCS hydrogel is an ideal delivery system, which can be adjusted by theconcentration of HBCS, the release of environmental including the release temperature andpH to control the release behavior.