【作者】 刘丽芳；

【导师】 张阳德；

【作者基本信息】 中南大学 ， 生物医学工程， 2011， 博士

【摘要】 研究目的本研究利用纳米生物技术,以传统中药外用复方矾冰液为研究模型,研制矾冰纳米乳,达到优化剂型、提高载药量、减小给药剂量、降低毒副作用和提高疗效的目的。研究方法一、矾冰纳米乳制剂研究1.空白纳米乳处方筛选。筛选油相,根据冰片在油相中的溶解性,确定矾冰纳米乳剂的油相为油酸乙酯。固定油相,以聚氧乙烯山梨醇脂肪酸-80(Tween80)、聚氧乙烯醚(40)氢化蓖麻油(RH40)、聚氧乙烯辛基苯基醚(OP-10)、蓖麻油聚氧乙烯(EL)等为表面活性剂,以丙二醇、甘油、正辛醇、乙二醇乙醚、聚甘油酯为助表面活性剂,与油相混合,考察其乳化能力。2、矾冰纳米乳的制备。用伪三元相图进行处方优选,分别将冰片、白矾加入RH40/油酸乙酯／水纳米乳中制备矾冰纳米乳。建立矾冰纳米乳中冰片(龙脑)含量检测的气相色谱分析方法,并考察其载药量和包封率；以络合滴定法测定白矾(含水硫酸铝钾)的含量；利用透射电子显微镜、激光粒度分布仪对其形态、粒径进行考察；通过稳定性参数测定、热压灭菌试验、常温试验、冷冻-加热循环试验等考察其稳定性。3、矾冰纳米乳的体外释放、透皮试验。采用Franz扩散池进行体外经皮渗透试验,气相色谱法测定皮肤接收液中矾冰纳米乳的药物浓度,并与矾冰液比较,考察矾冰纳米乳的透皮特性。二、安全性评价1、家兔皮肤急性毒性试验。家兔36只,随机分为6组：完整皮肤对照组、完整皮肤矾冰纳米乳低剂量组、完整皮肤矾冰纳米乳高剂量组、破损皮肤对照组、破损皮肤矾冰纳米乳低剂量组、破损皮肤矾冰纳米乳高剂量组。每天局部给药4次,连续14天。每天观察给受试物后动物的全身中毒表现和死亡情况。2、长期毒性试验。48只家兔,随机分为8组,即完整皮肤对照组(基质组)、完整皮肤矾冰纳米乳低、中、高剂量组,破损皮肤对照组、破损皮肤矾冰纳米乳低、中、高剂量组,每组各6只。各组连续皮肤涂药13周后进行常规观察、血液学、血液生化、系统尸检、组织病理学检查。3、细胞毒性试验。体外培养L-929细胞作为体外细胞模型,MTT法测定矾冰纳米乳对皮肤成纤维细胞的细胞毒性结果。4、皮肤过敏性试验。30只白色雄性豚鼠,随机分为3组,包括阴性对照组、阳性对照组和受试物组。致敏接触和激发接触空白纳米乳、阳性对照物2,4-二硝基氯苯和矾冰纳米乳后,观察皮肤过敏性反应。5、皮肤刺激性试验。家兔6只,将背部皮肤脱毛区分为完整皮肤对照区、完整皮肤给药区、破损皮肤对照区、破损皮肤给药区4个区域,给予矾冰纳米乳、空白纳米乳,每天1次,连续给药7天,观察皮肤刺激性。三、药效学研究1、矾冰纳米乳对大鼠深Ⅱ度烫伤模型的影响。140只SD大鼠,造成深Ⅱ度烫伤模型,随机分为7组,即空白对照组、基质组、阳性对照组、矾冰纳米乳低、中、高剂量组、矾冰液组,采用相应药物局部涂抹,连续给药21d。每天观察创面愈合情况,记录创面愈合时间,分别于第7、14及21天计算创面愈合率,第7天取创面组织测定皮肤羟脯氨酸含量并进行病理组织学检验。2、体外抗菌试验。分别采用琼脂扩散法、体外杀菌试验及试管稀释法,测定矾冰纳米乳对临床常见病原菌的体外抑菌、杀菌效果及最低抑菌浓度(MIC)3、抗炎作用。50只KM小鼠,随机分为5组,即基质组、醋酸地塞米松软膏组、矾冰纳米乳低、中、高剂量组,用二甲苯诱导小鼠右耳肿胀,左耳对照,观察两耳肿胀度。60只SD大鼠,随机分为6组,即基质组、醋酸地塞米松组、矾冰纳米乳低、中、高剂量组、矾冰液组,用蛋清致炎制作大鼠蛋清性足肿胀模型,于致炎前后用毛细管放大法测定右后足跖容积,观察致炎前后肿胀度。4、镇痛作用。50只KM小鼠,随机分为5组,即基质组、阳性药物对照组、矾冰纳米乳低、中、高剂量组,连续3d分别将各组药物均匀涂于小鼠腹部,第4d末给药30分钟后,每只小鼠腹腔注射醋酸溶液,记录小鼠15分钟内的扭体次数,比较药物组与对照组的扭体次数差异。取预选痛阈值合格的雌性小鼠50只,随机分为5组(分组方法同上),记录用药前后小鼠放入预热烧杯至出现舔后足所需时间(s)。5、抗痒痛作用。60只KM小鼠,随机分为6组：基质组、矾冰液组、醋酸地塞米松软膏组、矾冰纳米乳低、中、高剂量组,甲醛注射制作小鼠痒痛模型,记录不同时间小鼠舔咬右后足或抓咬阴器的次数,比较各组之间的差异。60只豚鼠,随机分为6组,分组方法同上,以磷酸组胺溶液制作局部瘙痒模型,记录并比较各组的致痒阈。研究结果一、矾冰纳米乳研究伪三元相图显示RH-40与油酸乙酯形成的纳米区域最大,并确定RH40与OP-10为表面活性剂及其和油相的比例。纳米乳处方筛选结果显示：纳米乳的表面活性剂为聚氧乙烯醚(40)氢化蓖麻油,助表面活性剂为甘油,汕相为汕酸乙酯；制备的空白纳米乳为澄清、透明、略带淡蓝色乳光的液体,电镜下呈球形,粒径为28.15nm,亚甲基蓝染料在其中的扩散速度明显大于苏丹红Ⅲ的扩散速度,为水包油型。经伪三元相图及稳定性试验得到了矾冰纳米乳中油酸乙酯的最佳用量为0.6%(g/g),油酸乙酯与RH-40的比例为1：3.5。建立了气相色谱法检测冰片中龙脑含量,龙脑在0.1015 mg.mL-1～1.015 mg.mL-1范围内线性关系良好,平均回收率为100.59%(RSD为2.72%),龙脑的平均含量为1.48mg.mL-1。络合滴定法测定矾冰纳米乳中含水硫酸铝钾平均含量为13.70 mg.mL-1,平均回收率为98.26%(RSD为0.92%)。经考察连续制备的三批聚氧乙烯醚(40)氢化蓖麻油/油酸乙酯/白矾、冰片纳米乳为澄清、透明、略带淡蓝色乳光的液体,电镜下呈球形,平均粒径为26.1 nm,Zeta电位为-0.51±1.47 mV,载药量为1.35mg.mL-1,包封率大于80%,矾冰纳米乳的稳定性参数为4.48±0.63。矾冰纳米乳24h的累积释放量为(2083.00±431.17)μg·cm-2,其平均透皮速率为84.07μg·cm-2·h-1,结果显示矾冰纳米乳的透皮能力优于对照组。二、安全性评价1、家兔连续给予矾冰纳米乳14天后未见全身中毒表现和死亡情况,动物体重增加,对呼吸、循环、中枢神经系统、四肢活动等无影响。破损皮肤个别动物有轻微红斑而无水肿,停药后24h即消失,用药区无药物残留斑点。2、矾冰纳米乳各剂量组动物的外观行为、体重、脏器系数、血液学和血液生化学指标与对照组比较无显著性差异,组织病理学检查未见明显异常,停药后也未见药物延迟性毒性反应。3、纳米矾冰乳对L929培养细胞无明显毒性作用,细胞形态良好,细胞毒性属于1级。4、受试豚鼠的局部皮肤未出现红斑、水肿等过敏反应。5、家兔单次和多次给药后,完整皮肤和破损皮肤均未见红斑、水肿等皮肤刺激反应。三、药效学试验1、矾冰纳米乳各剂量组创面于19-25d基本愈合；低剂量、中高剂量组与空白对照组比较,能缩短创面愈合时间(P<0.05；P<0.01),创面愈合百分率明显增高(P<0.05；P<0.01)；各实验组羟脯氨酸含量在烫伤后7d高于空白对照组,但差异无显著性；创面组织病理形态学结果提示矾冰纳米乳具有明显的促进烫伤创面愈合的作用。2、矾冰纳米乳对金黄色葡萄球菌、表皮葡萄球菌、大肠埃希菌、铜绿假单胞菌、白假丝酵母菌的抑制及杀灭活性均明显强于矾冰液(p<0.05)；对金黄色葡萄球菌、铜绿假单胞菌、大肠埃希菌临床菌株MIC9o值分别为1.02、2.04和2.04 mg. mL-1,均明显低于矾冰液的MIC90值(p<0.05)。3、矾冰纳米乳各剂量组对二甲苯诱导的小鼠耳肿胀均有不同程度的抑制作用,中、高剂量组与基质组比较,差异具有显著性意义(P<0.05)；低、中、高剂量组于致炎后1-6h,可不同程度的抑制大鼠蛋清性足跖肿胀,与基质组比较差异有显著性：(P<0.05,P<0.01)。4、矾冰纳米乳各剂量组具有抑制醋酸诱发的小鼠扭体反应,但与对照组比较差异无显著性意义(P>0.05)；与对照组比较均有延长小鼠热板致痛反应痛阈值的趋势,但差异无显著性意义(P>0.05)。5、矾冰纳米乳各剂量组能减少小鼠舔咬后足或抓咬阴器次数,与基质组比较差异有统计学意义(P<0.01)；中、高剂量组能明显提高豚鼠的致痒阈(P<0.05,P<0.01)。结论1、筛选的空白纳米乳符合纳米乳的要求。2、通过优选处方、工艺制得的矾冰纳米乳为O/W型复合纳米乳,为澄清、透明、略带淡蓝色乳光的液体,平均粒径为26.1 nm,质量稳定,符合2010年版中国药典乳剂项下的要求。3、矾冰纳米乳具有良好的透皮能力,其透皮作用的机制与冰片和乳剂的纳米尺寸有关。4、矾冰纳米乳对动物正常皮肤及破损皮肤长期给药无明显毒性反应,对L929细胞无明显毒性作用,对动物皮肤无致敏性,对正常皮肤和破损皮肤均无刺激性。5、与传统药物矾冰液比,矾冰纳米乳具有明显的促进烫伤创面愈合、抗炎、抗痒痛作用。更多还原

【Abstract】 AimsThe aims of this study was to develop Alum-Borneol nanoemulsion based on the traditional Alum-Borneol liquid. Nano biotechnology was used to optimize the formulation and technology to improve drug loading, reduce the dose, decrease side effects and improve efficacy.Methods1. The preparation study of Alum-Borneol nanoemulsion1.1 Blank nanoemulsion formulation screening.The oil phase of the Alum-Borneol nanoemulsion has been determined on the basis of solubility of the borneol in oil phase. The oil phase has been set firstly. Tween80, RH40, OP-10, and EL were selected as surfactant, while propylene glycol, glycerin, octanol, ethylene glycol ether and polyethylene glycerol were selected as co-surfactant, and then all of those were mixed with oil phase to investigate the emulsification.1.2 The preparation of Alum-Borneol nanoemulsionThe optimized prescription was screened by pseudo-ternary phase method. The borneol and alum were added into RH40, ethyl oleate or water nanoemulsion to produce Alum-Borneol nanoemulsion. The analysis method of gas chromatography was established to detect the content of borneol in the Alum-Borneol nanoemulsion, and its drug loading and encapsulation efficiency. The content of alum (hydrated aluminum potassium sulfate) was detected by complexometric titration. The particle morphology and particle diameter were assayed by transmission electron microscopy and laser particle size distribution analyzer, respectively. Stability parameters determination, hot sterilized test, temperature test, freezing-heating cycle test were used to study its stability.1.3 In vitro release and penetration tests of Alum-Borneol nanoemulsionFranz diffusion cell was used to do percutaneous penetration test, while the gas chromatography was used to detect the drug concentration of Alum-Borneol nanoemulsion from the skin received liquid and compared with Alum-Borneol and then the transdermal properties of Alum-Borneol nanoemulsion were investigated.2. Safety evaluation of Alum-Borneol nanoemulsion2.1 Rabbit skin acute toxicity trial36 rabbits were randomly divided into 6 groups:the complete skin control group, intact skin of ice nanoemulsion low dose alum group, intact skin of ice nanoemulsion high dose alum group, skin and the control group, skin and Alum-Borneol nanoemulsion low dose group, skin and Alum-Borneol nanoemulsion high dose group. The drugs were administered 4 times a day for 14 days to observe the performance of systemic poisoning of animals and death situation of subjects on a daily basis.2.2 The long-term toxicity tests48 rabbits were randomly divided into 8 groups, including intact skin the control group (matrix group), alum skin, full of ice nanoemulsion low, medium and high dose group, skin and the control group, skin and Alum-Borneol nanoemulsion low, medium, high-dose group with 6 rabbits, respectively. Routine observations of hematology, blood biochemistry, systems autopsy and histopathologic examination were conducted after 13 weeks of continuous skin application in every group.2.3 Cytotoxicity testL-929 cells were cultivated in vitro cell as model, while MTT method was used to test the cytotoxicity of Alum-Borneol nanoemulsion to skin fibroblast cell.2.4 Skin allergy test30 white male guinea pigs were randomly divided into 3 groups, including the negative control group, positive control and test substance groups. The skin allergy was observed after the allergy exposure and stimulation in negative control group with blank nanoemulsion, positive control group with 2,4-dinitrochlorobenzene and the test substance group with Alum-Borneol nanoemulsion.2.5 Skin irritation testThere were in total 6 rabbits whose back skin hair was removed. They were divided into completed control areas of skin, completed areas of skin for drug administration, broken skin control area, and broken skin for drug administration. Those 4 areas were given Alum-Borneol nanoemulsion and blank nanoemulsion on a daily basis for 7 days and then the skin irritation was observed.3. Pharmacodynamics3.1 Deep II degree burn model in rats.140 SD rats with deepⅡdegree burn were randomly divided into 7 groups, which were control group, matrix group, positive control group, low, medium and high dose alumina nano-ice milk group and alum icing fluid group. After appropriate medication, continuous administration was carried out in each group for 21 days. The situation of wound healing was observed everyday and the wound healing time were recorded respectively. The calculation of wound healing rate was calculated on the seventh, fourteenth and twenty-first days. The wound tissue was collected on the seventh day to do the skin hydroxyproline determination test and histopathological examination.3.2 In vitro antibacterial test Agar diffusion method, in vitro bactericidal test and tube dilution method were used to test. The effects of alumina ice nanoemulsion on clinical common pathogens in vitro antibacterial, bactericidal effects and minimum inhibitory concentration (MIC).3.3 Anti-inflammatory effect50 KM mice were randomly divided into 5 groups, including matrix group, dexamethasone acetate ointment group, low, medium and high dose Alum-Borneol nanoemulsion groups, with the xylene to induce right ear swelling, and left ear as control to observe the two swelling ears.60 SD rats were randomly divided into 6 groups, including matrix group, dexamethasone acetate group, low, medium and high Alum-Borneol nanoemulsion dose groups, alum icing fluid group. Making white rat inflamed paw swelling model using egg white. Capillary enlarge method was used to test the volume of right hind paw and the swelling was observed before and after the inflammation.3.4 Analgesic effect50 KM mice were randomly divided into 5 groups, including matrix groups, positive control group, low, medium and high Alum-Borneol nanoemulsion dose groups. Each drug was applied to each group evenly to the abdomen of mice.30 minutes after the end of administration on the fourth day, each mouse was injected with acetic acid solution, and then the mice’s writhing within 15 minutes was recorded, the differences in the times of writhing between the control group and drug group were compared.50 female mice which were selected according to pre-qualified pain threshold were randomly divided into 5 groups (groups as above). The time between the mice been put in to pre-heated beaker and the time they licked the back legs were recorded before and after the treatment with drugs.3.5 Anti-itching effect60 KM mice were randomly divided into 6 groups, including matrix group, alum icing fluid group, dexamethasone acetate ointment group, Alum-Borneol nanoemulsion low, medium and high dose groups. The mice tickle model was established by injecting mice with formaldehyde. The different time when the mice licking the right hind bite or scratch and bite genitals were recorded to compare the differences among groups.60 guinea pigs were randomly divided into 6 groups described as above in order to make local itching histamine phosphate solution model. The itching threshold of each group was recorded and compared among each group.Results1.The preparation of Alum-Borneol nanoemulsionPseudo-ternary phase diagram showed the nano region formed by the RH-40 and the oil ethyl was the biggest, and the ratio of surfactant and oil phase were determined. Nanoemulsion formulation screening results showed that the surfactant of nanoemulsion was 40-polyoxyethylene castor oil, the co-surfactant was glycerol and oil phase was ethyl oleate. Blank nanoemulsion was clear, transparent, slightly opalescent pale blue liquid. The particles were spherical under electron microscope with diameter of 28.15 nm. The diffusion rate of methylene blue was significantly higher than that of Sudan III for oil in water type which is in line with the requirements of nanoemulsion.The pseudo-ternary phase diagram and the stability test were used to establish oil Alum-Borneol nanoemulsion dosage of ethyl to be 0.6% (g /g), ethyl oleate and RH-40 ratio of 1:3.5. Gas chromatography was used to detect borneol content, the standard curve for borneol had a good linear relationship between 0.1015 mg.mL-1 to 1.015 mg.mL-1. The average content of borneol was 1.48 mg·mL-1 with the average recovery rate as 100.59% and the recovery rate of RSD was 2.72%. The average concentration of aluminum potassium sulfate was 13.70 mg·mL-1 in Alum-Borneol nanoemulsion which was detected by complexometric titration. Its average recovery was 98.26%, and the recovery rate of the RSD was 0.92%. The consecutive three batches of 40-polyoxyethylene castor oil/ethyl oleate/alum borneol nanoemulsion were clear, transparent and slightly opalescent pale blue liquid. It was spherical under electron microscope with average particle diameter of 26.1 nm and zeta potential wa -0.51±1.47 mV. Its drug loading was 1.35mg·mL-1 and had more than 80% encapsulation efficiency rate. The stability parameter of Alum-Borneol nanoemulsion was 4.48±0.63.The release rate of Alum-Borneol nanoemulsion after 24 h accumulation was (2083.00±431.17)μg·cm-2, which average penetration rate 84.07μg·cm-2·h-1. The results show that the transparency of Alum-Borneol nanoemulsion was better than the control group.2. Safety evaluation2.1 There was no systematic toxicity and death in the subjects of rabbits after giving Alum-Borneol nanoemulsion. The rabbits had gained weight and there was no harm to the rabbits’biological system including respiratory, circulatory, central nervous system, limbs and other activities. There was slight erythema but no individual animal edema in the very few subjects whose symptoms disappeared after 24 h with no drug residues spots in drug area.2.2 There was no significant difference in appearance of each dose group behavior of animals, body weight, organ coefficient, hematology and hematological indices in the Alum-Borneol nanoemulsion group when compared with the control group. There were no obvious abnormalities in histopathological examination and no delayed symptoms of toxicity after stopping the drugs.2.3 The Alum-Borneol nanoemulsion had no significant toxicity on L929 cells and the cell morphology was good with first class of cell toxicity.2.4 There was no local skin erythema and no other allergic reactions in tested guinea pigs.2.5 Neither of the intact skin or damaged skin of tested rabbits showed erythema, edema or other skin irritations after single and multiple dosing.3.pharmacodynamics3.1 The wound treated with different doses of Alum-Borneol nanoemulsion were healed between 19 and 25 days. The healing time of the low and high dose groups was significantly shorten than the blank control group (P<0.05; P<0.01). The wound healing rate of the former was significantly higher (P<0.05; P<0.01) than the later and the content of hydroxyproline in each experimental groups was higher than the control group 7 days after the burn, but the difference was not significant. The pathological results suggested that Alum-Borneol nanoemulsion had significantly better healing effects on burn wounds.3.2 The effects of Alum-Borneol nanoemulsion to staphylococcus aureus, staphylococcus epidermidis, escherichia coli, pseudomonas aeruginosa, candida albicans. The killing or inhibitory activity was significantly stronger than Alum-Borneol fluid (P<0.05); the MIC90 values of Alum-Borneol nanoemulsion on staphylococcus aureus, pseudomonas aeruginosa, clinical escherichia coli were 1.02,2.04 and 2.04 mg.mL-1 which were significantly lower than the MIC90 values of Alum-Borneol fluid (P<0.05).3.3 The Alum-Borneol nanoemulsion dose groups had different degrees of inhibition for the xylene-induced ear edema in mice. There was significant differences (P<0.05) between the medium, high dose groups and the base group. There was significant different degrees of inhibition of egg white rat paw edema when compared with low, medium and high dose groups in inflammation after 1-6 h with matrix groups (P <0.05, P<0.01).3.4 The Alum-Borneol nanoemulsion could significantly inhibit the acetic acid induced writhing in mice, but there was no significant difference when compared with the control group (P> 0.05). There was a trend of prolonging the hot plate pain threshold in mice when compared with the control group, but the difference was not significant (P> 0.05).3.5 The Alum-Borneol nanoemulsion could reduce the times of licking biting or scratching biting genitals in mice when compared with the matrix group and the difference was significant (P<0.01); in medium and high dose groups it could significantly improve the itching threshold of guinea pig(P<0.05, P<0.01). Conclusions1. The proposed blank nanoemulsion can meet the requirements of nanoemulsion.2.The Alum-Borneol nanoemulsion obtained through the optimization of formulation is O/W type, it was clear, transparent and slightly opalescent pale blue liquid with average particle diameter of 26.1 nm, which meets the requirements of Chinese Pharmacopoeia (2010 edition).3.The Alum-Borneol nanoemulsion has no significant toxic effect on animals’skin, normal skin and damage after long-term administration. There was no significant toxic effect on L929 cell, or on skin allergies as well as no irritation to broken skin or normal skin.4.The Alum-Borneol nanoemulsion obviously improves the burn wound healing. It has anti-inflammatory and anti-itching effects. It may have analgesic and antibacterial effects on a broad-spectrum of bacteria and fungus. The Alum-Borneol nanoemulsion is significantly more effective than the traditional drug of Alum-Borneol fluid.5.The Alum-Borneol nanoemulsion has a good penetration capacity through skin.更多还原