用于胰岛素口服给药和肿瘤靶向的壳聚糖衍生物接枝纳米粒

【作者】 崔福英；

【导师】 印春华；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 为研究生物大分子药物口服给药系统,分别制备了壳聚糖盐酸盐接枝纳米粒（CHNPs）、壳聚糖季铵盐接枝纳米粒（TMCNPs）、羧化壳聚糖接枝纳米粒（CCNPs）和羧甲基壳聚糖接枝纳米粒（CMCNPs）,考察其理化性质。以胰岛素为模型药物,研究纳米粒的载药特性、体外释药规律、体内作用及吸收机理。以香豆素（6-coumarin）标记的CCNPs为载体,研究其在肿瘤靶向中的应用。1壳聚糖衍生物接枝纳米粒的制备和表征分别以壳聚糖盐酸盐（CH）、壳聚糖季铵盐（TMC）、羧化壳聚糖（CC）和羧甲基壳聚糖（CMC）为亲水大分子单体,甲基丙烯酸甲酯（MMA）为疏水单体,经接枝共聚形成两亲性大分子,水溶液中自组装形成核壳型的CHNPs、TMCNPs、CCNPs和CMCNPs。考察纳米粒的结构、粒径、Zeta电势、形态和稳定性等。结果表明,制得的纳米粒粒径为100～300nm,粒径分布均匀;CHNPs和TMCNPs的Zeta电势为正值,CCNPs和CMCNPs的Zeta电势为负值;CCNPs在胃液中聚集,肠液中分散成纳米粒;CCNPs和CMCNPs冻干和室温贮存稳定性良好。2载胰岛素纳米粒研究考察载胰岛素纳米粒的形态、粒径、Zeta电势、包封率、载药量及体外释放性质。采用内源荧光光谱、外源荧光光谱、圆二色谱和双荧光标记法研究胰岛素在纳米粒中的载药方式。结果表明,TMCNPs和CCNPs的包封率大于80%,载药量高于10%;CHNPs和CMCNPs的包封率和载药量较低;载胰岛素前后CCNPs的粒径和形态未发生明显变化;Zeta电势随胰岛素浓度增加其绝对值减小;胰岛素可通过氢键、静电作用及范德华力包载至纳米粒;载胰岛素纳米粒的体外释放具pH敏感性。研究胰岛素-CCNPs在正常大鼠、糖尿病大鼠及Beagle犬体内的口服降血糖效果。与胰岛素溶液相比,正常大鼠皮下注射胰岛素-CCNPs后血糖最低下降至初始值的41%。正常大鼠口服胰岛素溶液及不同剂量胰岛素-CCNPs,100u·kg^-1时,2～12h内血糖降为初始值的80%～67%;15u·kg^-1时,降血糖效果不明显;对照组胰岛素溶液无降血糖效果;胰岛素-CCNPs(25u·kg^-1)的口服药理生物利用度为9.7%。糖尿病大鼠口服胰岛素-CCNPs,6～16h内血糖浓度降低为初始值的80%以下,16h达到最低值,为初始值的35%。Beagle犬口服胰岛素-CCNPs(16.5u·kg^-1),4h内血糖降低为初始值的60～80%。考察CCNPs体外抑酶作用和对肠道紧密连接的影响,并研究香豆素标记的CCNPs在Peyer’s结处的吸收。结果表明,CCNPs可使胰蛋白酶活力下降75%,胃蛋白酶活力下降80%;CCNPs可打开肠道紧密连接,并在肠道Peyer’s结处被吸收。选择FITC-胰岛素和藻蓝蛋白包载至纳米粒,进一步研究胰岛素口服吸收机理。测定FITC-胰岛素与胰蛋白酶作用后的荧光光谱;考察SD大鼠口服FITC-胰岛素和载FITC-胰岛素的CCNPs后的体内分布;分别测定藻蓝蛋白与胰蛋白酶和胰凝乳蛋白酶作用后的紫外光谱和荧光光谱。结果表明,FITC-胰岛素与胰蛋白酶作用后,荧光强度增强;大鼠口服FITC-胰岛素和载FITC-胰岛素的CCNPs6h后,肝、脾中荧光均较强;藻蓝蛋白与胰蛋白酶和胰凝乳蛋白酶作用后,紫外吸收和荧光强度均减小,蛋白浓度与紫外吸收及荧光强度呈正相关。3载黏膜黏附性纳米粒双层膜口服给药载体以Na₂-EDTA为交联剂制备壳聚糖水凝胶,加入CCNPs冻干粉,以乙基纤维素膜为背衬层制备载纳米粒壳聚糖水凝胶双层膜(NP-Film^CH)。对壳聚糖水凝胶膜的形态、粘度、流变行为和体外黏附力等进行表征,并考察CCNPs在不同介质中的体外释放特性。壳聚糖水凝胶具有典型的网络结构,纳米粒可分散其中。壳聚糖水凝胶膜的黏膜黏附力为壳聚糖盐酸盐膜的2～8倍,在回肠处的黏膜黏附力较大。pH 1.2释放介质中,纳米粒从水凝胶膜中释放较慢;pH 7.4 PBS释放介质中,纳米粒释放较快。将CCNPs冻干粉载入海藻酸钠水凝胶中并以疏水性乙基纤维素膜为背衬层制备双层膜(NP-Film^SA)。荧光显微镜和扫描电镜观察纳米粒在双层膜中形态,考察水凝胶的溶胀性、黏膜黏附性和纳米粒释放特性,以钙黄绿素作为荧光模型多肽,观察载药纳米粒在水凝胶膜中的形态。离子强度较低时,海藻酸钠水凝胶膜溶胀率较高;离子强度较高时,其溶胀率较低;pH 5.8和pH 8.0时,其溶胀率较高,pH 1.7时,其溶胀率较低。海藻酸钠水凝胶膜的体外黏膜黏附力达6000N·m^-2。0.01 mol·l^-1HCl中,CCNPs释放较慢,2h累计释放量约为10%;pH 6.5和pH 7.4 PBS中,CCNPs快速释放。海藻酸钠水凝胶膜含较多孔隙,大小均一的纳米粒可均匀分散在孔隙中。4香豆素标记CCNPs在肿瘤细胞中的分布以亲脂性荧光染料香豆素标记CCNPs,比较香豆素标记CCNPs在H1299肺癌和HEK293胚肾细胞中的分布情况,研究其组织分布、活体光学成像及体外巨噬细胞摄取。结果表明,香豆素标记CCNPs可被H1299细胞摄取,而不被HEK293胚肾细胞摄取。纳米粒定向聚集于肿瘤部位,肺、肝、肾和脾脏的摄取较少,具有一定的靶向性。5生物相容性研究以溶血试验、动态凝血时间测定、血小板黏附和形态学观察等方法研究纳米粒的血液相容性,小肠生化损伤、肌肉植入和组织损伤等试验研究材料的组织相容性。CCNPs、CMCNPs和TMCNPs溶血率均低于5%,符合生物材料溶血率要求。与硅化玻璃相比,CCNPs的动态凝血时间较长;CCNPs和CMCNPs的血浆复钙时间延长达40%。CHNPs、CCNPs、CHNPs和壳聚糖水凝胶膜与血小板作用后,血小板未发生变形。CCNPs和NP-Film^CH的乳酸脱氢酶（LDH）泄漏值与PBS测定结果相近,表明材料对肠细胞没有生化损伤。壳聚糖水凝胶、CCNPs和CMCNPs对肌肉组织没有损伤。更多还原

【Abstract】 Chitosan derivative graft nanoparticles including chitosan hydrochloride graft nanoparticles（CHNPs）,trimethylated chitosan graft nanoparticles（TMCNPs）, carboxylation chitosan graft nanoparticles（CCNPs） and carboxymethyl chitosan graft nanoparticles（CMCNPs） were synthesized and investigated as peroral delivery systems for biomacromolecular drugs.With insulin as a model drug,drug loading,in vitro release,in vivo hypoglycemic effect and absorption mechanism in the GI treact were studied.6-coumarin labeled CCNPs were also evaluated as a potential candidate in tumor targeting.1 Preparation and characterization of chitosan derivative graft nanoparticlesNanoparticles were prepared by graft polymerization of methyl methacrylate （MMA） and chitosan derivatives including chitosan hydrochloride（CH）, trimethylated chitosan（TMC）,carboxylation chitosan（CC） and carboxymethyl chitosan（CMC） in aqueous solution.During polymerzation,amphiphilic graft polymers assembled in the solution to form core-shell type nanoparticles,which possessed hydrophobic cores and hydrophilic shell layers on their surfaces,resulting in an excellent aqueous dispersion.Structure of nanoparticles was characterized by dissolubility,FT-IR,¹H-NMR and X-ray.Particle size and zeta potential were determined;morphology was investigated and stability of nanoparticles in the GI tract and during storage and freeze drying process was evaluated.The nanoparticles varied from 100 to 300 nm in size.CHNPs and TMCNPs bore positive zeta potentials while CCNPs and CMCNPs showed negative zeta potentials.The CCNPs assembled in the gastric fluid while was well dispersed in the intestinal fluid.The CCNPs and CMCNPs were stable in the GI tract and during storage or freeze-drying.2 Insulin loaded nanopartielesThe encapsulation efficiencies,loading capacities,in vitro release,particle diameters,zeta potentials and morphologies of insulin loaded nanoparticles were investigated.Endogenous fluorescence,extrinsic fluorescence,circular dichroism（CD） and double labeled fluorescence were used to characterize the conformational structure of insulin when loaded in nanoparticles.Results demonstrated that the encapsulation efficiencies of TMCNPs and CCNPs were above 80%and their loading capacities surpassed 10%.Insulin was mainly located in the shell of the nanoparticles via hydrogen bonding,electrostatic interaction and Vanderwolf force.Morphologies, diameters and zeta potentials did not significantly alter after insulin loading.Insulin release from these nanoparticles exhibited pH-sensitivity.Hypoglycemic effects following oral administration of insulin-CCNPs in normal rats,diabetic rats and Beagle dogs were investigated.At 100 u·kg^-1 and 50 u·kg^-1,oral administration of insulin-CCNPs led to a significant blood glucose depression of 20-40%within 12 h,achieving a relative pharmacological bioavailability of 9.7%(25 u·kg^-1).Blood glucose level of diabetic rats decreased to below 80%following oral administration of CCNPs within 6-16 h,and reached the maximum hypoglycemic effect at 16 h（35%of the initial level）.Oral administration of insulin-CCNPs in Beagle dogs at a dose of 16.5 u·kg^-1 also exhibited remarkable hypoglycemic effect.The potential of the CCNPs on enzyme inhibition,opening of the epithelial tight junctions,and absorption enhancement at the Peyer’s patches were evaluated. Results showed that the activity of trypsin and pepsin decreased to 25%and 20%in the presence of the CCNPs,respectively.Transient and reversible opening of the tight juntions was achieved following treatment of the CCNPs and uptake of 6-coumarin labeled CCNPs at Peyer’s patch was enhanced correspondingly.Nanoparticles loaded with FITC-insulin or phycocyanin（PC） were used for the assessment of oral absorption mechanism.The fluorescence spectrum of trypsin treated FITC-insulin was monitored and the in vivo distributions of FITC-insulin and FITC-insulin loaded CCNPs following oral administration in SD rats were investigated.The ultraviolet and fluorescence spectra of trypsin treated PC and chymotrypsin PC were measured.Results showed that fluorescence of FITC-insulin was intensified following exposure to trypsin.Strong fluorescence was detected in the liver and spleen following oral administration of FITC-insulin and FITC-insulin loaded CCNPs.After incubation with trypsin and chymotrypsin,ultraviolet as well as fluorescent intensities of PC were diminished,and positive correlation was determined between protein concentrations and ultraviolet absorption as well as fluorescent intensity.3 CCNPs loaded bilaminated film as oral delivery vehiclesA novel smart drug delivery system(NP-Film^CH) consisting of CCNPs and bilaminated films was developed,the films composed of the mucoadhesive chitosan-EDTA hydrogel layer（CH-EDTA） and the hydrophobic ethylcellulose layer.The CH-EDTA was characterized by morphology,viscosity,rheology and in vitro muco-adhesive force.In vitro release in different media was also performed. Results showed that the chitosan-EDTA hydrogel possessed typical network structure, where nanoparticles could be well-dispersed.Chitosan-EDTA hydrogel film showed a 2-8 fold increase in the muco-adhesive force as compared to the chitosan chloride film,and it was especially pronounced in the ileum.Nanoparticles were slowly released from the film at pH 1.2 while easily released at pH 7.4.Another novel polymeric composite carrier consisting of CCNPs and bilaminated films which were composed of the mucoadhesive alginate-Ca²⁺ hydrogel and the hydrophobic ethylcellulose layer was developed,which might be a promising drug carrier.Morphology of nanoparticles in the bilaminated films was examined using fluorescence microscopy and scanning electronic microscopy.Swelling, muco-adhesion and nanoparticle release was studied,and morphology of calcein loaded nanoparticles in the films was also examined.At low ionic strength,the alginate hydrogel film possessed high swelling ratios and vice-versa at high ionic strength.Swelling ratios were high at pH 5.8 and 8.0,while low at pH 1.7.In vitro muco-adhesive force of the alginate film was 6000 N-m^-2.Slow release of the CCNPs was achived in 0.01 mol·l^-1 HCl with a total release amount of 10%within 2h.Fast dissolution was observed in pH 6.5 and pH 7.4 PBS.Large quantities of pores were present in the alginate film,where nanoparticles could be well distributed.4 Distribution of coumarin-labeled nanoparticles in tumorsCCNPs were labeled with lipophilic fluorescent 6-coumarin,and their distributions in H1299 and HEK293 cells were compared.Besides,tissue distribution, in vivo optical imaging and in vitro macrophage uptake was investigated.Results showed that coumarin-labeled CCNPs could be uptaken by H1299 cells rather than HEK293 cells.Nanoparticles were concentrated in tumor tissues with low distribution in lungs,livers,kidneys and spleen,which indicated tumor-targeting properties.5 BiocompatibilityBlood compatibility of the nanoparticles was evaluated in terms of hemolysis, kinetic thrombus time,platelet adhesion.Tissue compatibility was assessed with respect to intestinal biochemical damage,in vivo implantation and tissue damage. Hemolysis ratios of the CCNPs,CMCNPs and TMCNPS were below 5%,which met the requirements of biomedical materials.The CCNPs showed prolonged thrombus time compared to silicated glass;CCNPs and CMCNPs exhibited prolonged recalcification time of 40%.Morphological deformation and activation of the platelets were not detected upon treatment with CHNPs,CCNPs,CHNPs,and chitosan hydrogel films.Lactate dehydrogenase leakage in the small intesetine of SD rats was minimal following exposure to CCNPs and NP-film^CH,indicating integrity of the intestinal epithelia.No damage towards the muscle was detected after implantation of chitosan-EDTA hydrogel film,CCNPs or CMCNPs,either.更多还原