Study On Two Kinds Of Self-aggregated Nanoparticles Of Hydrophobically Modified Glycol Chitosan Used In Drug Delivery System

Glycol chitosan is a novel chitosan derivative,and can be soluble in water at all pH values.It has chitosan-based structure and physicochemical properties,leading to excellent biocompatibility,biodegradability,low immunogenicity and biological activities.In the present study,a series of novel amphiphilic graft glycol chitosan were synthesized by introducing different hydrophobical groups.These polymeric amphiphiles can form self-aggregated nanoparticles via the intra- and/or intermolecular interactions of hydrophobic segments in aqueous media.These nanoparticles exhibit unique core-shell architecture composed of hydrophobic segments as internal core and hydrophilic segments as surrounding corona in aqueous media.The hydrophobic core serves as a reservoir for water-insoluble drugs.Hence,these nanoparticles can be used as carriers for hydrophobic drugs.For the synthesis of cholesterol-modified glycol chitosan(CHGC) conjugates,a carboxyl group was initially introduced to cholesterol molecule using succinic anhydride,and then covalently coupled with the primary amino group of glycol chitosan in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxyl succinimide(NHS).These conjuages were characterized by FTIR,1^H NMR and elemental analysis.Three kinds of CHGC conjugates were prepared by controlling the feed ratio of cholesteryl hemisuccinate to glycol chitosan.The degree of substitution(DS),defined as the number of cholesterol groups per 100 sugar residues of glycol chitosan The physicochemical properties of the self-aggregated nanoparticles were studied using dynamic light scattering(DLS),transmission electron microscopy (TEM) and fluorescence spectroscopy.CHGC conjugates were well-dispersed in aqueous media,and formed self-aggregated nanoparticles by probe sonication. Fluorescence measurement using pyrene as fluorescent probe was adopted to determine the critical aggregation concentration(CAC) of amphiphilic copolymers.As the DS of the conjugates increased,the CAC decreased.The CHGC self-aggregated nanoparticles were almost spherical in shape,and the mean diameters of these nanoparticles were in the range of 228-353 nm.Indomethacin(IND) was chosen as a model drug to assess the potential of CHGC self-aggregated nanoparticles as a drug carrier.IND was physically entrapped into the CHGC nanoparticles by a dialysis method.IND-loaded CHGC nanoparticles were almost spherical in shape,and their sizes were larger than blank CHGC nanoparticles.The in vitro release behavior of IND-CHGC nanoparticles was well fitted by Weibull equation and presented a biphasic drug release pattern.Stability experiments revealed that IND-loaded CHGC nanoparticles should be stored at low temperature or in freeze-dried state.Because the hydrophobic core of CHGC nanoparticles can provide a loading space for water-insoluble drugs,doxorubicin(DOX)-loaded CHGC nanoparticles were prepared.The drug-loaded nanoparticles were hoped to be stealthy nanoparticles, prolong circulation time in plasma and improve antitumor efficacy by enhanced permeability and retention(EPR) effect.The DOX-loaded CHGC1(the DS of CHGC1 was 6.7%) nanoparticles were prepared by a dialysis method.As the weight ratio of feed DOX to CHGC1 nanoparticles increased,the DOX-loading content increased and the DOX entrapment efficiency decreased.The DOX-loaded CHGC1 nanoparticles(DCN) were roughly spherical in shape determined by TEM observation.As the drug-loading content increased,the size of these nanoparticles increased.DOX in the polymer matrix was dispersed in an amorphous or molecular state,which was investigated by differential scanning calorimetry(DSC) and fluorescent quenching studies.The zeta potential of DCN series increased as the pH values of PBS solution decreased.DOX release from DCN in vitro was much faster in PBS at pH 5.5 than in PBS at pH 6.5 and 7.4.As the drug loading content of these nanoparticles increased,the release rate of DOX was much slower under the same condition.A notably slower degradation rate was observed for the encapsulated DOX,while free DOX was subject to rapid decomposition in PBS(pH 7.4) at 37℃.Stability tests indicated that DCN should be freeze-dried in store. Reversed-phase high performance liquid chromatography(HPLC) was developed to quantify the content of DOX in rate plasma and mice tissues.Intraveneous pharmacokinetic behaviors in rats of DCN-16(drug loading content:9.36%) and free DOX were investigared.DCN-16 could prolong circulation time in rat plasma.In comparison with free DOX,DCN-16 has an increased MRT(P＜0.01) and decreased CL(P＜0.01).The AUC_0-∞ of DCN-16 was 6.61 times higher than that of free DOX(P＜0.01).Moreover,DCN-16 displayed a much greater systemic circulation time in S180 mice than free DOX.In addition,compared with free DOX,DCN-16 also produced significantly increased AUC in liver(P＜0.05),spleen(P＜0.05),and tumors(P＜0.05).DCN-16 can passively accumulate into the tumor tissues by the long systemic retention in blood circulation and the EPR effect.The AUC_0-∞ of DCN-16 was lower in heart(P＜0.05),lung(P＜0.05) and kidney(P＜0.05) than that of free DOX,which indicated DCN-16 could show longer circulation time and less toxic effect in S180-bearing mice.The relative tumor tissue exposure(R_e) and the ratio of peak concentration(C_e) were 2.56±0.30 and 1.49±0.24,respectively.It demonstrated that the tumor affinity of DCN-16 was higher than free DOX.Cytotoxicity of blank CHGC1 nanoparticles and DCN-16 nanoparticles against MCF-7 and HepG2 cells was evaluated by MTT assay.The IC₅₀ value of blank CHGC1 nanoparticles on MCF-7 and HepG2 cells was 0.226μg/mL and 0.351μg/mL, respectively.These results indicated that CHGC1 nanoparticles have relatively low cytotoxicity against both MCF-7 and HepG2 cells.Concentration-dependent cytotoxicity on MCF-7 and HepG2 cells was observed for both free DOX and DCN-16. DCN-16 showed a lower cytotoxicity against MCF-7 and HepG2 cells than free DOX. Confocal laser scanning microscopy(CLSM) demonstrated that cellular uptake of DCN-16 was in an endocytosis manner but that of free DOX was in a passive diffusion way.HepG2 ceils incubated with DCN-16 emitted lower fluorescent intensity than the cells incubated with free DOX after 0.5 or 4 h.This result was consistent with confocal microscopic observation and in vitro DOX release behavior.Compared with free DOX, DCN-16 exhibited more efficient tumor growth suppression,and simultaneously showed less toxicity effect in S 180-bearing mice.Low-molecular-weight glycol chitosan was obtained by enzymatic degradation of glycol chitosan and membrane separation.A series of linoleic acid-modified low-molecular-weight glycol chitosan(LALGC) conjugates were synthesized in the presence of EDC.The chemical structure of LALGC copolymers was characterized by FTIR,1^H NMR and colloidal titration method.The degree of substitution(DS) defined as the number of LA groups per 100 glucosamine units of low-molecular-weight glycol glycol chitosan was 4.5-13.4%.The LALGC conjugates can form mono-dispersed nanoparticles by probe sonication.These nanoaggregates were almost spherical in shape, and the mean diameters of these nanoparticles determined by dynamic light scattering (DLS) were in the range of 204-289 nm.The CAC decreased as the DS of LALGC conjugates increased.Paclitaxel(PTX),as a model drug,was physically entrapped into the LALGC nanoparticles by a dialysis method.The drug loading content and encapsulation efficiency of PTX-loaded LALGC(PTX-LALGC) nanoparticles increased with an increasing ratio of the hydrophobic LA to hydrophilic glycol chitosan in the conjugates.The PTX-LALGC nanoparticles were almost spherical in shape and their size was ranged from 238 to 307 nm.In vitro release revealed that release rate of PTX from the nanoparticles was slower as the drug-loading content increased.PTX appeared to be released in a biphasic way,which characterized by an initial rapid release period followed by a step of slower release.The results of cytotoxicity in vitro showed that PTX-LALGC1 nanoparticles exhibited comparable activity in inhibiting MCF-7 cell proliferation as free PTX.Therefore,these results indicated that LALGC self-aggregated nanoparticles had a potential as a drug carrier.