钙纳米粒子的制备及毒性研究
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摘要
近年来，人用疫苗佐剂的研究成为一个热点。目前，铝盐复合物是人用疫苗佐剂中使用最广泛的佐剂。铝盐是良好的体液免疫增强剂，但铝盐有许多副作用，包括：严重的局部组织刺激、注射部位的长时间持续的炎症反应、几乎不能诱导细胞免疫反应以及不期出现的IgE抗体应答等。
纳米粒技术(Nanoparticle technology)是目前药物载体输送系统研究的热点之一。纳米粒载体可以增加药物的口服吸收、靶向药物、控释或缓释药物、穿透生理屏障等，从而增加药物疗效，减轻药物副作用。将纳米粒子作为肿瘤、AIDS等的疫苗佐剂，国外已有少量报道。纳米粒子用作疫苗佐剂可以增强疫苗的免疫保护作用，延长疫苗的免疫保护时间。此外，使用纳米载体还可以改变疫苗接种方式。
以往研究表明，日本血吸虫抗独特型抗体NP30是一个极有潜力的血吸虫病疫苗候选分子。钙纳米粒子能明显提高NP30对小鼠的免疫保护作用，钙纳米粒子-NP30结合物既能增强小鼠的体液免疫反应又能增强细胞免疫反应，提示钙纳米粒子可作为血吸虫抗独特型抗体疫苗NP30的佐剂。本文制备了钙纳米粒子，并观察了其对小鼠的急性毒性和对大鼠的长期毒性作用，以评价钙纳米粒子用于人体的安全性。
研究目的
1．制备钙纳米粒子并进行表征鉴定。
2．观察小鼠一日内给予大剂量钙纳米粒子的急性中毒及死亡情况，得出半数致死量(LD50)或最大耐受量(MTD)。
3．观察连续重复给予钙纳米粒子对大鼠所产生的毒副作用情
南京医科大学硕卜学位论文
况，为拟定人用安全剂量提供参考。
研究方法
1.采用液相沉淀法，在不同温度、缓冲液、液体混合方式下制
备钙纳米粒子。
2.采用透射电镜(TEM)、粒度分布仪、X射线能谱仪(X Ps)等测
试手段对钙纳米粒子的结构、大小、形状和成分等进行表征鉴定。
3.急性毒性试验:ICR小鼠(18一229)随机分为2组，雌雄各半，
灌胃给药组以钙纳米粒子2.76吵g灌胃，一日内分两次给药;腹腔注
射给药组以钙纳米粒子1.159众g腹腔注射，单次给药。给药后连续
7d观察小鼠的毒性反应及死亡数。
4.长期毒性试验:SD大鼠(70一909)60只，雌雄各半，随机分为
3组。设两个给药组(loomg众g、smg众g)和一个对照组(等体积0.5%
发甲基纤维素钠)，腹腔注射给药，0.sml/l 009体重，每周2次，连续给
药3m，停药后继续饲养Zw作为恢复期观察。给药期及恢复期每日
观察一般性状，每周测体重和摄食量一次，3m后每组随机处死大鼠
10只(雌雄各半)，检测血常规、血生化、脏器系数，心、肝、脾、
肺、’肾、脑、’肾上腺、胸腺、胃、淋巴结、前列腺、争丸、子宫、卵
巢等主要脏器切片作病理学检查，心、肝、脾、肺、肾、脑作电镜观
察及TUNEL法检测细胞凋亡。余动物停药Zw后处死，处理同给药
期动物。
研究结果
1.低温下向柠檬酸钠中匀速缓慢加入等量氯化钙和磷酸二氢钠
并搅拌，可得到钙纳米粒子。TEM显示该纳米粒子为颗粒状。粒度
分布仪显示钙纳米粒子平均粒径260nm，粒子直径99%在900nm以
内。XPS结果显示其化学成分为磷酸钙。
2.急性毒性试验期间小鼠未出现明显的毒性反应，全部小鼠均
未死亡。钙纳米粒子对小鼠的一日内灌胃的最大药量大于2.769瓜g，
南京医科人学了吹卜学位论文
腹腔注射的最大药量大于1.159/kg。
3.大鼠长期毒性试验结果显示钙纳米粒子对大鼠的体重、活动
情况、摄食量、外表体征、血像、血液生化、脏器系数、组织学结构、
超微结构、细胞凋亡等指标均无明显影响。
结论
1.通过液相沉淀法，制备了大小、形态较为理想的钙纳米粒子。
2.急性毒性试验结果表明，钙纳米粒子对小鼠是安全的。
3.长期毒性试验结果表明，钙纳米粒子在本试验的剂量和疗程
下对大鼠是安全的。
Vaccine adjuvants for human have been the focus of attention in recently years. Aluminium compounds are the most extensively used adjuvants in licensed vaccines for humans. Although they effectively enhance humoral immune responses, there are several disadvantages associated with their use. The disadvantages of alum-based adjuvants include the severity of local tissue irritation, the longer duration of the inflammatory reaction at the injection site, minimal induction of cell-mediated immunity, and a propensity to elicit undesirable immunoglobulin E (IgE) responses.
Nanoparticle technology has been one of the focuses in drug carrier systems. Nanoparticles carriers could be used to increase the availability of drug for oral uptake, targeted release drugs, controlled or sustained release drugs, across the physical barriers and so on. Nanoparticles carriers were used for enhanced therapeutic efficacy or reduced toxicity. There are several studies reported nanoparticles used as vaccine adjuvant for tumors or AIDS in foreign countries. Nanoparticles, used as vaccine adjuvant, could enhance the protective immunity of vaccine, prolong the protective immunity and alter the route of vaccination.
Previous studies have demonstrated that anti-idiotypic antibody NP30 may represent a promising vaccine candidate for schistosomiasis japonica. Calcium(Ca) nanoparticles could enhance significantly the protective immunity of NP30 against infection of Schistosomajaponicum, CA-NP30 conjugate enhanced both humoral and cellular immune
-4 -
responses in mice. Ca nanoparticle could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. In this study, Ca nanoparitcles were prepared and the acute and long-term toxicity of Ca nanoparitcles was also investigated to evaluate the safety of Ca nanoparitcles for humans. Objectives
1. To prepare and identify Ca nanoparitcles.
2. To observe the acute toxicity and death of the mice to which calcium nanoparticles were given in maximal dose and obtain the lethal dose 50(LD50) or the maximal tolerance dose (MTD).
3. To investigate the toxic effects of the rats to which calcium nanoparticles were given twice per week, in order to offer reference of the safe dose for humans.
Methods
1. The Ca nanoparitcles were prepared by precipitation method at different temperatures, buffers or mixing orders.
2. The structure, particle size, morphology and components of resulting nanoiparticles were characterized by transmission electron microscope (TEM), particle meter and X-ray photoelectron spectrometer (XPS).
3. The acute toxicity test: Both sexes of ICR mice (weight 18-22g) were randomly divided into 2 groups. For one group, Ca nanoparitcles were administered by oral gavage to the mice at a total dose of 2.76g/kg (2 individual doses of 1.38g/kg), separated by 6-hour intervals. For the other, Ca nanoparitcles were administered by intraperitoneal injection in a single total dose of 1.15g/kg.
4. The long-term toxicity test: Both sexes of sixty SD rats (weight
70-90g) were randomly divided into 3 groups. Ca nanoparitcles were administered, by intraperitoneal injection in 100 mg/kg, 5mg/kg twice a week for three monthes, exposure to 2 separate groups (each containing 10 male and 10 female animals). A third group served as an control group administered by intraperitoneal injection with 0.5% sodium carboxymethylcellulose (CMC-Na). During the period of observation, the following indexes were concerned: exercise, signs per day, body weight food-eating quantity per week. After 3 months 10 rats( both sexes) of each group were sacrificed, hemogrotm, blodbiochemistry, organs facter were examined, heart liver, spleen, lungs, kidneys, brain, adrenal glands thymus glands stomach lymph nodes prostate, testicles, uterus and ovaries were extracted for pathologic examination, heart, liver, spleen lungs kidneys and brain for electron microscope examination and apoptosis study. Results
1. It was indicated that granular nanop
纳米粒技术(Nanoparticle technology)是目前药物载体输送系统研究的热点之一。纳米粒载体可以增加药物的口服吸收、靶向药物、控释或缓释药物、穿透生理屏障等，从而增加药物疗效，减轻药物副作用。将纳米粒子作为肿瘤、AIDS等的疫苗佐剂，国外已有少量报道。纳米粒子用作疫苗佐剂可以增强疫苗的免疫保护作用，延长疫苗的免疫保护时间。此外，使用纳米载体还可以改变疫苗接种方式。
以往研究表明，日本血吸虫抗独特型抗体NP30是一个极有潜力的血吸虫病疫苗候选分子。钙纳米粒子能明显提高NP30对小鼠的免疫保护作用，钙纳米粒子-NP30结合物既能增强小鼠的体液免疫反应又能增强细胞免疫反应，提示钙纳米粒子可作为血吸虫抗独特型抗体疫苗NP30的佐剂。本文制备了钙纳米粒子，并观察了其对小鼠的急性毒性和对大鼠的长期毒性作用，以评价钙纳米粒子用于人体的安全性。
研究目的
1．制备钙纳米粒子并进行表征鉴定。
2．观察小鼠一日内给予大剂量钙纳米粒子的急性中毒及死亡情况，得出半数致死量(LD50)或最大耐受量(MTD)。
3．观察连续重复给予钙纳米粒子对大鼠所产生的毒副作用情
南京医科大学硕卜学位论文
况，为拟定人用安全剂量提供参考。
研究方法
1.采用液相沉淀法，在不同温度、缓冲液、液体混合方式下制
备钙纳米粒子。
2.采用透射电镜(TEM)、粒度分布仪、X射线能谱仪(X Ps)等测
试手段对钙纳米粒子的结构、大小、形状和成分等进行表征鉴定。
3.急性毒性试验:ICR小鼠(18一229)随机分为2组，雌雄各半，
灌胃给药组以钙纳米粒子2.76吵g灌胃，一日内分两次给药;腹腔注
射给药组以钙纳米粒子1.159众g腹腔注射，单次给药。给药后连续
7d观察小鼠的毒性反应及死亡数。
4.长期毒性试验:SD大鼠(70一909)60只，雌雄各半，随机分为
3组。设两个给药组(loomg众g、smg众g)和一个对照组(等体积0.5%
发甲基纤维素钠)，腹腔注射给药，0.sml/l 009体重，每周2次，连续给
药3m，停药后继续饲养Zw作为恢复期观察。给药期及恢复期每日
观察一般性状，每周测体重和摄食量一次，3m后每组随机处死大鼠
10只(雌雄各半)，检测血常规、血生化、脏器系数，心、肝、脾、
肺、’肾、脑、’肾上腺、胸腺、胃、淋巴结、前列腺、争丸、子宫、卵
巢等主要脏器切片作病理学检查，心、肝、脾、肺、肾、脑作电镜观
察及TUNEL法检测细胞凋亡。余动物停药Zw后处死，处理同给药
期动物。
研究结果
1.低温下向柠檬酸钠中匀速缓慢加入等量氯化钙和磷酸二氢钠
并搅拌，可得到钙纳米粒子。TEM显示该纳米粒子为颗粒状。粒度
分布仪显示钙纳米粒子平均粒径260nm，粒子直径99%在900nm以
内。XPS结果显示其化学成分为磷酸钙。
2.急性毒性试验期间小鼠未出现明显的毒性反应，全部小鼠均
未死亡。钙纳米粒子对小鼠的一日内灌胃的最大药量大于2.769瓜g，
南京医科人学了吹卜学位论文
腹腔注射的最大药量大于1.159/kg。
3.大鼠长期毒性试验结果显示钙纳米粒子对大鼠的体重、活动
情况、摄食量、外表体征、血像、血液生化、脏器系数、组织学结构、
超微结构、细胞凋亡等指标均无明显影响。
结论
1.通过液相沉淀法，制备了大小、形态较为理想的钙纳米粒子。
2.急性毒性试验结果表明，钙纳米粒子对小鼠是安全的。
3.长期毒性试验结果表明，钙纳米粒子在本试验的剂量和疗程
下对大鼠是安全的。
Vaccine adjuvants for human have been the focus of attention in recently years. Aluminium compounds are the most extensively used adjuvants in licensed vaccines for humans. Although they effectively enhance humoral immune responses, there are several disadvantages associated with their use. The disadvantages of alum-based adjuvants include the severity of local tissue irritation, the longer duration of the inflammatory reaction at the injection site, minimal induction of cell-mediated immunity, and a propensity to elicit undesirable immunoglobulin E (IgE) responses.
Nanoparticle technology has been one of the focuses in drug carrier systems. Nanoparticles carriers could be used to increase the availability of drug for oral uptake, targeted release drugs, controlled or sustained release drugs, across the physical barriers and so on. Nanoparticles carriers were used for enhanced therapeutic efficacy or reduced toxicity. There are several studies reported nanoparticles used as vaccine adjuvant for tumors or AIDS in foreign countries. Nanoparticles, used as vaccine adjuvant, could enhance the protective immunity of vaccine, prolong the protective immunity and alter the route of vaccination.
Previous studies have demonstrated that anti-idiotypic antibody NP30 may represent a promising vaccine candidate for schistosomiasis japonica. Calcium(Ca) nanoparticles could enhance significantly the protective immunity of NP30 against infection of Schistosomajaponicum, CA-NP30 conjugate enhanced both humoral and cellular immune
-4 -
responses in mice. Ca nanoparticle could act as a vaccine adjuvant of anti-idiotypic antibody NP30 against schistosomiasis. In this study, Ca nanoparitcles were prepared and the acute and long-term toxicity of Ca nanoparitcles was also investigated to evaluate the safety of Ca nanoparitcles for humans. Objectives
1. To prepare and identify Ca nanoparitcles.
2. To observe the acute toxicity and death of the mice to which calcium nanoparticles were given in maximal dose and obtain the lethal dose 50(LD50) or the maximal tolerance dose (MTD).
3. To investigate the toxic effects of the rats to which calcium nanoparticles were given twice per week, in order to offer reference of the safe dose for humans.
Methods
1. The Ca nanoparitcles were prepared by precipitation method at different temperatures, buffers or mixing orders.
2. The structure, particle size, morphology and components of resulting nanoiparticles were characterized by transmission electron microscope (TEM), particle meter and X-ray photoelectron spectrometer (XPS).
3. The acute toxicity test: Both sexes of ICR mice (weight 18-22g) were randomly divided into 2 groups. For one group, Ca nanoparitcles were administered by oral gavage to the mice at a total dose of 2.76g/kg (2 individual doses of 1.38g/kg), separated by 6-hour intervals. For the other, Ca nanoparitcles were administered by intraperitoneal injection in a single total dose of 1.15g/kg.
4. The long-term toxicity test: Both sexes of sixty SD rats (weight
70-90g) were randomly divided into 3 groups. Ca nanoparitcles were administered, by intraperitoneal injection in 100 mg/kg, 5mg/kg twice a week for three monthes, exposure to 2 separate groups (each containing 10 male and 10 female animals). A third group served as an control group administered by intraperitoneal injection with 0.5% sodium carboxymethylcellulose (CMC-Na). During the period of observation, the following indexes were concerned: exercise, signs per day, body weight food-eating quantity per week. After 3 months 10 rats( both sexes) of each group were sacrificed, hemogrotm, blodbiochemistry, organs facter were examined, heart liver, spleen, lungs, kidneys, brain, adrenal glands thymus glands stomach lymph nodes prostate, testicles, uterus and ovaries were extracted for pathologic examination, heart, liver, spleen lungs kidneys and brain for electron microscope examination and apoptosis study. Results
1. It was indicated that granular nanop
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