Preparation Of Colon-Specific Delivery Systems Based On Chitosan/Sodium Alginate And Their Application As Anti-inflammatory Agents In Vivo

Oral colon-specific drug delivery systems (OCDDS), which could directly release drug in colon for treatment of colon diseases such as inflammatory bowel disease and colorectal cancer,had drawn greater attention in recent years. Various OCDDS with different drug release mechanism have been developed for treatment of colonic disease. However, safety, accuracy for colon-specific delivery, and therapeutic effect in vivo of various OCDDS remains a challenge for their application. In order to sovle this problem, natural polysaccharides chitosan (CS) and sodium alginate (SA) were selected as carrier materials to develop a series OCDDS including microparticles and microspheres in this thesis. The physicochemical properties of these developed OCDDS were evaluated by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), differential scanning calorimeters (DSC), and energy dispersive spectrometer(EDS). In vitro releases of these OCDDS were investigated in different medium simulating the conditions of different parts of a real gastrointestinal tract (GIT). Furthermore, the in vivo therapeutic efficacy of these OCDDS were assessed in colitis rat models induced by 2, 4,6-trinitro-benzene-sulfonic acid (TNBS). The main research contents and results were shown as follows:1. The thiolated chitosan (CS-NAC) was synthesized by condensation reaction between N-acetyl-L-cysteine and chitosan. The thiolated chitosan/sodium alginate (CS-NAC/SA)composite microparticles were prepared by ionic gelation and the polyelectrolyte complexation,and coated with Eudragit S-100. The physicochemical properties of CS-NAC/SA composite microparticles were characterized using Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). Thiol group content on CS-NAC/SA composite microparticles was determined using the Ellman's reagent. The mucoadhesive performance of the CS-NAC/SA composite microparticles was also assessed, ex vivo, on colon mucosa using everted sac technique. Furthermore, the release preporties of 5-ASA and CUR from uncoated and coated composite microparticles were performed in a pH progression medium simulating the conditions of different parts of a real (GIT). Finally, the therapeutic efficacy of uncoated and coated composite microparticles was evaluated in colitis rat models induced by TNBS. The results indicated that CS-NAC/SA composite microparticles have irregular shape and rough outer surface.After coated with Eudragit S-100, the composite microparticles became spherical and smooth-face in shape. The CS-NAC/SA composite microparticles contained free thiol onto its surface, and exhibited excellent mucoadhesive properties for the colonic mucosa of rats. In vitro releases results showed that Eudragit S-100 coated CS-NAC/SA composite microparticles had a pH-sensitive release property, which can avoid drug been released at upper of GIT. Finally,Eudragit S-100 coated CS-NAC/SA composite microparticles could markedly alleviate the colon inflammation of colitis rats induced by TNBS. Therefore, the enteric CS-NAC/SA microparticulates systems that combined the properties of pH-sensitive delivery, mucoadhesion,and control release, could be an available tool for the treatment of human inflammatory bowel disease (IBD).2. Based on our work in the previous chapter, the sodium alginate-curcumin conjugation(SA-CUR) was synthesized. The genipin (GNP) cross-linked CS-NAC/SA-CUR composite microparticles were prepared by chemical cross-linking and the polyelectrolyte complexation for further improving drug loading efficiency and decreasing drug release in the upper GIT.Physicochemical characterization of GNP cross-linked CS-NAC/SA-CUR composite microparticles was assessed using Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and optical microscopy. The drug encapsulation efficiency (EE) and loading efficiency (LE) were determined by ultraviolet-visible spectrophotometry. Ellman's reagent was used to determine the thiol group content on CS-NAC/SA-CUR composite microparticles. Also, an everted sac technique was employed to study mucoadhesive properties of the CS-NAC/SA-CUR composite microparticles. Furthermore, the in vitro release properties of CUR from composite microparticles were investigated in a pH progression medium simulating the conditions of different parts of a real GIT. Finally, the visible observation colon, the determination of clinical score and C/B ratio, and the pathological observation of colon were as assessment criterias to investigate the therapeutic efficacy of CS-NAC/SA-CUR composite microparticles in colitis rat models induced by TNBS. The results indicated that GNP cross-linked CS-NAC/SA-CUR composite microparticles could increase the EE and LE of drug by chemical cross-linking between GNP and CS-NAC, and the CS-NAC/SA-CUR composite microparticles exhibited an irregular shape and rough outer surface. FTIR results indicated that the three-dimensional network structure was formed by cross-linking reaction between the amino of CS-NAC and the carboxyl of SA-CUR, the amino of CS-NAC and GNP. Moreover, GNP cross-linked CS-NAC/SA-CUR composite microparticles exhibited excellent mucoadhesive properties for the colonic mucosa of rats. In vitro releases results showed that the GNP cross-linked CS-NAC/SA-CUR composite microparticles had a pH-sensitive release property,which could reduce drug release in simulated gastric juice and simulated intestinal juice, and sustained release drug in simulated colon juice. In vivo study results indicated that GNP cross-linked CS-NAC/SA-CUR composite microparticles had obvious treatment effect for colitis rats, and could significantly relieve the colon inflammation of colitis rats. Thus, the developed GNP cross-linked CS-NAC/SA-CUR composite microparticles had great potential application in treatment of IBD.3. N-succinyl chitosan (NSC) was synthesized by acylation reaction between chitosan and succinic anhydride. Zn2+ cross-linked N-succinyl chitosan/sodium alginate (NSC/SA)micorspheres were developed using emulsification/ionic cross-linking method. The physicochemical properties of Zn2+ cross-linked NSC/SA micorspheres including morphology,size, chemical structures and elemental component, were characterized using SEM, FTIR and EDS.The in vitro drug release properties of 5-ASA from Zn2+ cross-linked NSC/SA micorspheres were investigated in different mediums simulating the conditions of different parts of a real GIT.Futhremore, the treatment effect of Zn2+ cross-linked NSC/SA microspheres in TNBS induced-colitis rat models after oral administration were investigated by the visible observation of colon, the determination of C/B ratio and clinical score, and the pathological observation of colon.Finally, the in vivo toxicity of Zn2+ cross-linked NSC/SA micorspheres were evaluated in rats after oral administration by the determination of blood indicators and serum biochemical indicators, and the observation of pathological changes of main organs. The results indicated that the microspheres had spherical shapes, smooth outer surface with a few porous regions. The mean particles size of Zn2+ cross-linked microspheres ranged from 5?m to 10?m. The EDS results indicated the presence of zinc element in Zn2+ cross-linked micorspheres, and increased Zn2+concentration could result in an increasing of zinc element content in Zn2+ cross-linked micorspheres. In vitro drug release results showed that the release of 5-ASA from Zn2+cross-linked micorspheres was pH-sensitive, which could release a small amount of 5-ASA in HCl solution at pH 1.2 and in PBS at pH 4.5, and release more 5-ASA in simulated colon (pH 7.4 PBS).In addition, Zn2+ cross-linked NSC/SA micorspheres markedly alleviated the colonic inflammation of colitis rats induced by TNBS. Finally, Zn2+ cross-linked NSC/SA micorspheres exhibited excellent security and biocompatibility. Therefore, Zn2+ cross-linked NSC/SA micorspheres might be employed as safe and effective carriers for clinical treatment of IBD.4. Based on our work in the previous chapter, GNP and Zn2+ dual cross-linked NSC/SA microspheres were prepared with ionic/chemical cross-linking reaction for further improving the stability of microspheres and decreasing the drug release in the upper GIT. SEM, FTIR, EDS, and DSC were used to characterize the physicochemical properties of GNP and Zn2+ dual cross-linked NSC/SA micorspheres including morphology, size, chemical structures, and elemental component and thermostability. The in vitro release properties of berberine (BBR) from GNP and Zn2+ dual cross-linked microspheres were investigated in different medium simulating the conditions of different parts of GIT. Drug distribution in GIT and contents of GIT was determined to evaluate in vivo colon-specific delivery role after oral administration for rats. Finally, the treatment effect of GNP and Zn2+ dual cross-linked microspheres was evaluated after oral administration on induced-colitis rat models by the visible observation, the determination of C/B ratio and clinical score, the myeloperoxidase activity, and the pathological observation of colon. The results indicated that the GNP and Zn2+ dual cross-linked microspheres exhibited spherical shapes with smooth surface. The mean particles size of dual cross-linked NSC/SA microspheres was?5?m,and was smaller than that of Zn2+ cross-linked NSC/SA micorspheres. DSC study showed that dual cross-linked NSC/SA microspheres had better thermalstability compared to Zn2+ cross-linked NSC/SA micorspheres. The EDS results indicated the presence of zinc element in GNP and Zn2+dual cross-linked micorspheres. In vitro drug release study indicated that GNP and Zn2+ dual cross-linked micorspheres had an excellent stability in upper GIT and pH-sensitive release properties. The cumulative release of drug from GNP and Zn2+ dual cross-linked microspheres was less than that of Zn2+ cross-linked micorspheres at the same conditions. In vivo drug distribution results confirmed the ability of GNP and Zn2+ dual cross-linked microspheres to colon-specific deliver drug for treatment of IBD. Finally, GNP and Zn2+ dual cross-linked microspheres showed a superior in vivo therapeutic efficacy in alleviating inflammation of colitis rats by reduction of the C/B ratio, clinical score and myeloperoxidase activity of colon. Therefore,GNP and Zn2+ dual cross-linked NSC/SA microspheres might be suitable and effective tool vehicles for IBD treatment.