The Preliminary Study Of Temperature-Responsive Chitosan Gel Combined With Drug-loaded Chitosan Microspheres

Objective Periodontal disease is one of the most common oral disease, have a higher prevalence rate in the world. The ultimate goal of the treatment of periodontal disease is regenerate periodontal tissue, forming new periodontal attachment. In recent years, people have realized the application of the growth factor plays an important role in the regeneration of periodontal tissue. Exogenous growth factors, however, easily lost, volatile live, and would not be long efficient release. In actual applications, the therapeutic effect of exogenous growth factors is not satisfactory. If we can use a suitable carrier load goal growth factor, let it play the potential application of the growth factor. The application of exogenous growth factors is expected to become close to the most effective means of clinical periodontal tissue regeneration.Chitosan thermosensitive gel has prepared under mild conditions, and has good biocompatibility, can be used as a sensitive protein drugs with short biological half-life drugs, oral unstable drugs and systemic side effects of the drugs and other drugs carrier.Chitosan gel system has directed to accomplish something in the biomedical field, but there are some shortcomings of its own, such as poor mechanical properties, not good enough for drug release effect of some small molecule. To solve this problem, the present study we put nanospheres particles in chitosan thermosensitive gel to form temperature-responsive chitosan gel combined with drug-loaded chitosan microspheres system, and study the performance of the system.Methods The present experiment prepared chitosan microspheres (CMS) using ionic gelation method. Namely, when adding tripolyphosphate (TPP) into chitosan solution, chitosan free amino and TPP anion happened intermolecular or intramolecular cross-linking reaction, thereby preparing the chitosan spherical gel. We studied surface morphology, particle size distribution and other physical and chemical properties of the prepared chitosan microspheres. Making bovine serum albumin as model drug, we studied the change of conditions of preparing microspheres (CS concentration, the concentration of BSA, TPP concentration) how to impact CMS encapsulation efficiency and drug loading. We studied the release effect of CMS as a protein drug delivery carrier in vitro.The present experiment prepared chitosan temperature-responsive gel (CS/β-GP system system). Under ice-cooling, added β-glycerophosphate to CS solution was until turbidity, after a few minutes in a37℃water bath incubator fromed chitosan thermosensitive gel. The change of factors such as the ratio of β-GP/CS, pH and temperature impacted the characteristics of the gel (gel state, the gelation time).We also studied the sustained release effect of CS/β-GP system as carrier of protein drugs(the BSA proteins) in vitro.Adding dried CMS to the prepared CS/β-GP system sol, mixing evenly, after a few minutes in the37℃water bath incubator, we got temperature-responsive chitosan gel combined with drug-loaded chitosan microspheres(CS/β-GP/CMS system). We studied the different impactive factors how to impact the nature of the hydrogel (gel state, gelation time), and studied the drug release effect of the hydrogel as a sustained release carrier of drugs in vitro.Results1. When CS concentration between0.6mg/ml and2.0mg/ml, TPP concentration between0.5mg/ml and1.0mg/ml, we can generate CMS. Through scanning electron microscope, we can see the CMS is well dispersed, the particle diameter is uniform; surface is smooth, no wrinkles; diameter of resulting microspheres hbetween10and500nm. Drug loading-CMS particle size has increased, and the edge become irregular. BSA protein encapsulation efficiency and drug loading increases with increasing CS concentration; with increasing concentration of BSA solution, BSA encapsulation efficiency is gradually reduced, the drug loading of BSA is increased;When the TPP concentration changes, BSA encapsulation efficiency has less affected. In vitro experiments, along with the increase of the concentration of CS, the amount of drug release also increase. In early6hour of drug release, drug release increased from45%to62%. In the late,CMS are showing signs of a slow-release, the amount of the drug release are over80% within48hours.2. Gelation time of CS/β-GP system and β-GP/CS ratio, pH and temperature has a close relationship.When the volume ratio of58%β-GP and2%CS is0.2, the hydrogel system is sustainable to maintain the liquid phase at room temperature, and gelling does not occur within30min. When the temperature was raised to37℃, the gelation time is2.5min. Therefore selected this ratio of CS/β-GP system for drug loading experiments to examine the performance of sustained release of the drug-loaded gel. The experimental results show that the CS/β-GP system for BSA protein has sustained effect, different drug concentrations (2,4,6mg/ml) of CS/β-GP system has little effect in vitro release of BSA protein effect. Different drug concentrations of CS/β-GP system has a similar drug release pattern,in the early20h, they release faster, respectively,47%,50%,54%; after100h basic release completely, eventually the cumulative release rates were75%,78%,81%. Increases with the concentration of the drug, the drug release rate is slightly accelerated, the cumulative release rate is slightly elevated.3. Dried CMS added into CS/β-GP system was formed CS/β-GP/CMS system. CS/β-GP/CMS system’s gelation time has shortened than the CS/β-GP system, from1.5min to2.5min, and the gel strength of the system has also been enhanced. CS/β-GP system and the CS/β-GP/CMS system there is a certain degree of sustained release of BSA, the CMS adding significantly slows down the rate of release of the BSA in the gel, the CS/β-GP/CMS system24h cumulative release rate was only16%, sustained release for up to14days, the cumulative release rate is75%;24h of CS/β-GP system cumulative release rate is70%,4d total cumulative release rate is82%.Conclusions1. The present experiment prepared CMS using ionic gelation method. When CS concentration of2mg/ml, BSA concentration of1.5mg/ml, TPP concentration of1mg/ml, the prepared CMS has uniform particle size, smooth surface not wrinkled, high encapsulation efficiency and drug loading.2. When58%β-GP/2%CS ratio is0.2, pH=6.9, prepared CS/β-GP system sustainable to maintain the liquid phase at room temperature, does not appear gelled within30min, when the temperature was raised to37℃, the gelation time2.5min.3. Gelation time of CS/β-GP/CMS system was shorter, the gel strength was higher than the CS/β-GP system, CS/β-GP/CMS system show significantly slower release rate of BSA protein in vitro.