贝尼地平对成骨细胞功能影响及其纳米载药系统的生物应用
|
摘要
高血压是中老年人群中的常见疾病，有调查显示中国成年人中高血压患病率接近20%，这一比例随着我国老龄化社会的进程而不断提高。口腔种植及修复的患者当中，中老年人所占比例高，因此高血压患者的比重就更大。钙拮抗剂是目前治疗高血压疾病的常用药物，包括使用历史最长、最普遍的硝苯地平（心痛定），以及氨氯地平（络活喜）等。然而此类药物对于常年用药患者的骨质有何影响，是否会减低骨密度，进而影响患者接受口腔种植及修复治疗的疗效；或是能够促进骨质的生成，对骨质疏松具有防治作用，这些都是值得关注并探究的问题。
贝尼地平为第二代二氢吡啶类钙拮抗药，常用于治疗高血压和心绞痛。有研究显示贝尼地平能够促进成骨细胞的分化，但未见对其促进成骨作用的系统性研究。本研究通过一系列体外实验评价贝尼地平对于成骨细胞功能的影响，以期望发现贝尼地平除常规降压作用外，还具有促进骨质生成这一新的临床药用价值。同时本实验制备了硅酸镁纳米球，并首次以其作为药物载体，建立载贝尼地平药物的硅酸镁纳米载药系统，以提升贝尼地平促进成骨的作用效果，并增强其作为局部治疗药物的稳定性，为贝尼地平促进成骨作用的临床应用提供理论依据。
此外本实验中还首次以硅酸镁纳米球作为化疗药物载体，制备载阿霉素药物的硅酸镁纳米载药系统，评价其载药性能及药物释放特性，并通过体外及体内实验探究其抑制肿瘤的作用效果，为该纳米载药系统的临床应用提供理论基础。
1.贝尼地平对成骨细胞功能的影响
目的：采用MC3T3-E1Subclone14小鼠前成骨细胞系作为成骨细胞模型，系统性评价贝尼地平在体外对成骨细胞功能的影响。
方法：配制浓度为10-10~10-4mol/L的贝尼地平（benidipine，BD）梯度药物溶液，通过MTT法检测BD对MC3T3-E1细胞增殖活性的影响，筛选促进该细胞增殖的药物浓度区间进行后续实验。通过ALP活性水平及茜素红染色检测BD对MC3T3-E1细胞分化活性及矿化能力的影响，并通过RT-PCR及Western Blot检测BD对于MC3T3-E1细胞BMP2、OCN及Runx2mRNA和蛋白表达水平的影响。
结果：BD浓度为10-5~10-4mol/L时对细胞增殖具有抑制作用，在较低浓度10-10mol/L时对细胞的增殖无显著影响，而浓度区间于10-9~10-6mol/L时该药物能够显著促进细胞的增殖，因此筛选此浓度区间进行后续实验。浓度区间于10-9~10-6mol/L的BD能够提高MC3T3-E1细胞ALP的活性水平及矿化能力，其中浓度为10-7mol/L时作用效果最为显著。同时此浓度区间的BD也能够上调MC3T3-E1细胞BMP2、OCN及Runx2mRNA和蛋白的表达水平。
结论：浓度为10-9~10-6mol/L的BD能够促进MC3T3-E1细胞的增殖、分化及矿化，并能上调该细胞BMP2、OCN及Runx2mRNA和蛋白的表达水平。其中浓度为10-7mol/L的BD体外促进成骨的作用效果最为显著。
2.硅酸镁纳米球的制备及其生物相容性的研究
目的：制备硅酸镁纳米球（magnesium silicate nanospheres，MSNs），检测该纳米材料的形貌结构特征，并通过体外及体内各项实验评价其生物相容性，为后续实验中MSNs载药系统的建立及应用提供理论基础。
方法：采用经典St ber法合成二氧化硅胶体球，并以此材料为前体制备MSNs。采用扫描电镜、透射电镜观察MSNs的形态结构，EDS分析MSNs元素组成，XRD分析MSNs衍射图谱，获取材料的结构信息。判断MSNs表面孔隙特点，并计算出其比表面积及外壳表面的孔径尺寸。细胞形态观察、MTT法及溶血实验检测MSNs的体外毒性，并将MSNs通过尾静脉注入小鼠体内，通过对小鼠30天内体重和行为的观察，组织学及血液学等检测手段评价MSNs的体内毒性。
结果：本实验所合成的MSNs为大小均一，直径约400nm的空心球体结构，比表面积为498m2/g。其表面具有介孔结构，平均直径为4.32nm。体外实验表明，浓度为200μg/ml的MSNs对细胞形态及活性均无显著影响，且溶血率小于1%。体内实验中，MSNs在小鼠脾脏及肝脏累积最多。100mg MSNs/kg的剂量条件下，小鼠的体重、行为、血液学及主要脏器的组织学检测均显示MSNs没有显著毒性。
结论：本实验所制备的空心MSNs具有较高的生物相容性及较低的毒性，可以作为一种安全、高效的纳米药物载体。该纳米材料在纳米载药系统的应用中有着巨大的潜力。
3.载贝尼地平硅酸镁纳米球对成骨细胞功能的影响
目的：制备载贝尼地平药物的硅酸镁纳米球（BD-MSNs），探讨该纳米载药系统对于成骨细胞增殖、分化及矿化的作用，为其日后促进成骨作用的相关临床应用提供理论基础。
方法：制备BD-MSNs纳米载药系统并测定其载药量。以含有BD浓度为10-7mol/L的BD-MSNs为实验浓度，观测该浓度纳米载药系统对于MC3T3-E1细胞形态的影响，并通过MTT法、ALP活性水平及茜素红染色检测BD-MSNs对MC3T3-E1细胞增殖、分化及矿化水平的影响，并以相同浓度BD做为对照，比较该纳米载药系统促进成骨作用的优越性。
结果：所制备的BD-MSNs载药量为450mg/g，即当BD浓度为10-7mol/L时，所含MSNs浓度为12.04μg/ml，且该浓度下BD-MSNs对于MC3T3-E1细胞形态无显著影响。与相同浓度单纯BD药物相比，BD-MSNs能够显著促进MC3T3-E1细胞的增殖、分化及矿化。
结论：本实验所制备的BD-MSNs具有良好的生物安全性，体外实验证实BD-MSNs纳米载药系统较单纯BD药物促进成骨的作用更为显著。
4.载阿霉素硅酸镁纳米球在抗肿瘤治疗中的应用
目的：以硅酸镁纳米球（MSNs）作为药物载体，制备载阿霉素（DOX）的纳米载药系统（DOX-MSNs），测定其载药性能及药物释放特性，通过体外及体内实验检测其抑制肿瘤的作用效果。
方法：分别将不同浓度的DOX药物溶解于pH为5~8的溶液中，每份样品中加入MSNs10mg，搅拌以制备不同载药量的DOX-MSNs，并检测载药量及药物释放特性。选择HepG2细胞作为肿瘤细胞模型，细胞内吞实验检测MSNs在HepG2细胞中的聚集情况，通过MTT法检测该纳米载药系统于体外对肿瘤细胞的抑制效果。建立荷瘤小鼠的动物模型，通过观测小鼠肿瘤的体积变化及TUNEL试验检测肿瘤细胞凋亡情况，评价DOX-MSNs于动物体内抗肿瘤的作用效果。
结果：本实验中制备的DOX-MSNs在DOX浓度为500μg/ml，pH为8的条件下载药量最高，达到2140mg/g。24h内DOX-MSNs在pH为5.0条件下药物释放量显著高于pH为7.4条件下的释放量。细胞内吞实验显示MSNs能够进入肿瘤细胞内部并大量聚集于溶酶体中。MTT结果显示DOX-MSNs抑制肿瘤细胞增殖的效果较相同浓度单纯DOX药物的作用效果更为显著。体内实验对于肿瘤体积的观测及TUNEL试验检测肿瘤细胞凋亡的结果也显示DOX-MSNs能够显著抑制肿瘤的生长。
结论：本实验制备的DOX-MSNs载药量高，且药物释放具有pH敏感性。MSNs纳米药物载体能够大量聚集于胞内溶酶体中，有利于提高所载化疗药物的作用效果。DOX-MSNs较单纯DOX药物抑制肿瘤生长的作用效果更为显著。
Hypertension is a common disease in the elderly, the prevalence rate of this desease isclose to20%in our country. Most patients of dental restoration and implantation are middleaged and elderly people, so the proportion of patients with high blood pressure is larger.Calcium antagonist is one of the most commonly used medicine in the treatment ofhypertensive disease, including nifedipine, amlodipine, etc. Whether the calcium antagonistwould cause osteoporosis and further affect oral treatment, or the drug could promote boneformation and prevent osteoporosis. These issues are worthy of attention and exploration.
Benidipine (BD) is the second generation of dihydropyridine calcium antagonist, whichis often used for hypertension and angina pectoris. Previous studies have demonstrated thatBD has a positive effect on bone metabolism. Inspired by this promoting phenomenon, thepresent study investigated the effects of BD on osteoblast function in vitro. We preparedmagnesium silicate nanospheres (MSNs) and used it as nanocarriers to make BD-MSNs drugdelivery system for promoting the function of osteoblasts. The preparation of BD-MSNs drugdelivery system improved the stability of the drug, and also provided a theoretical basis forthe further clinical application.
In addition we used MSNs as the chemotherapy drug carrier for the first time to preparethe drug delivery system of doxorubicin (DOX), evaluated its drug loading capacity and drugrelease property. We also explored the effects of DOX-MSNs drug delivery system on theinhibition of tumor in vitro and in vivo, which provided a theoretical basis for the clinicalapplication of DOX-MSNs drug delivery system.
1. Effects of benidipine on osteoblast function in vitro
Objective: To investigate the effects of benidipine (BD) on osteoblast function in vitro.
Methods: A solution of BD was prepared by dissolving solid BD in DMSO solvent.Experiments were designed and performed, including an MTT assay, reverse transcription- polymerase chain reaction, western blot analysis, alkaline phosphatase activity measurementsand alizarin red staining.
Results: BD promoted proliferation of MC3T3-E1cells at concentrations of10-9~10-6mol/L. The higher concentrations of BD inhibited cell proliferation whereas no significantdifference from the control was observed when the lower concentrations of BD were applied.So the concentrations of10-9~10-6mol/L was chosen for further experiments. Followingtreatment of the drug, BMP2, OCN and Runx2mRNA and protein levels were markedlyupregulated compared with those in the control group. ALP activity was enhancedsignificantly in a time dependent manner when osteoblasts were treated with BD. BD alsopromoted the formation of mineralized matrix nodules in the MC3T3-E1cells.
Conclusion: BD promoted cell proliferation and osteogenic differentiation atconcentrations of10-9~10-6mol/L by upregulating BMP2, OCN and Runx2gene expressionlevels. BD promoted osteogenesis most markedly at concentrations of10-7mol/L.
2. The preparation of magnesium silicate nanospheres and evaluation of theirbiocompatibility
Objective: To investigate morphological and structural features of magnesium silicatenanospheres (MSNs) and evaluate their biocompatibility in vitro and in vivo.
Methods: Monodispersed silica colloidal spheres were synthesized in accordance withthe St ber method and then were used for the preparation of MSNs. Detailed morphologicaland structural features of MSNs were examined via scanning electron microscopy (SEM) andtransmission electron microscopy (TEM). Energy dispersive spectroscopy (EDS) analysisconfirmed the elements of our product. The N2adsorption and desorption analysis wasintroduced to investigate the pore properties of MSNs. Cellular modality, hemolysis assay andMTT assay were used to evaluate the cytotoxicity of MSNs in vitro. Body weightmeasurements, histology analysis, hematology analysis and blood biochemical assay wereused to detect the biocompatibility of MSNs in vivo.
Results: The MSNs were uniform hollow spheres, the average diameter was nearly400nm and the specific surface area was498m2/g. MSNs were mesoporous materials, an averagediameter width of pores was4.32nm. In vitro experiment no obvious differences in the cell morphology and cell viability for the cells treated with MSNs were observed at theconcentration of200μg/ml. No hemolysis of RBCs could be detected upon the maximalconcentration of200μg/ml. There was nearly no difference in body weight measurements,histology analysis, hematology analysis and blood biochemical assay between test andcontrol groups in vivo.
Conclusion: The MSNs had good biocompatibility and low biological toxicity, thus itcould be used as a practical and powerful nanocarrier for drugs. Nanocarriers based on MSNscould serve meeting the criteria as a high performance biomedical material.
3. Effects of benidipine-loaded magnesium silicate nanospheres on osteoblast function invitro
Objective: To investigate the effects of benidipine-loaded magnesium silicatenanospheres (BD-MSNs) on osteoblast function in vitro.
Methods: Prepared BD-MSNs and evaluated the drug loading capacity. Theconcentration of BD was10-7mol/L in this experiment. Cell morphology was observed, MTTassay, alkaline phosphatase activity measurements and alizarin red staining were used toevaluate the effect of BD-MSNs on cell proliferation and osteogenic differentiation.
Results: The BD loading capacity of BD-MSNs was450mg/g. No obvious differencesin the cell morphology for the cells treated with BD-MSNs were observed at the experimentalconcentration. BD-MSNs had a better effect on proliferation and osteogenic differentiation ofMC3T3-E1cells compared to BD at the concentration of10-7mol/L.
Conclusion: BD-MSNs prepared in our experiment had good biocompatibility. Thisdrug delivery system had a better effect on proliferation and osteogenic differentiation ofMC3T3-E1cells compared to BD at the concentration of10-7mol/L.
4. Uniform magnesium silicate nanospheres as high drug-loading nanocarriers forcancer therapy
Objective: To evaluate drug loading capacity and drug release property of drug deliverysystem of doxorubicin (DOX-MSNs), and to explore the effects of DOX-MSNs on theinhibition of tumor in vitro and in vivo.
Methods: MSNs (200μg/mL) at different pH values ranging from5to8was dispersed in various concentrations of DOX solution and stirred overnight to get the DOX-MSNs. TheUV-vis absorbance method was used to determine the loading and releasing amount of DOX.Endocytosis study was used to detect the accumulation of MSNs in tumer cells. MTT assay,tumer size measurement and TUNEL assay were used to evaluate the effects of DOX-MSNson the inhibition of tumor.
Results: The drug-loading capacity was2140mg/g upon optimum conditions (pH8anda DOX concentration of500μg/ml). Approximately70%of the drug was released within24hat pH5.0, whereas at pH7.4, about32%of the drug was released. Most DOX-MSNsmolecules accumulated in lysosomes of tumer cells. DOX-MSNs had a better effect oninhibiting tumor cell proliferation in vitro compared to DOX at the same concentration.Tumer size measurement and TUNEL assay showed the same effect of DOX-MSNs in vivo.
Conclusion：MSNs exhibited a significantly high storage capacity of DOX (2140mg/g)and held a sustained release property, showing more potential for cancer therapy. MSNs weremostly accumulated in lysosome, indicating their pH-dependent release character.DOX-MSNs could effectively suppress tumor growth and induce cancer cell destruction.
贝尼地平为第二代二氢吡啶类钙拮抗药，常用于治疗高血压和心绞痛。有研究显示贝尼地平能够促进成骨细胞的分化，但未见对其促进成骨作用的系统性研究。本研究通过一系列体外实验评价贝尼地平对于成骨细胞功能的影响，以期望发现贝尼地平除常规降压作用外，还具有促进骨质生成这一新的临床药用价值。同时本实验制备了硅酸镁纳米球，并首次以其作为药物载体，建立载贝尼地平药物的硅酸镁纳米载药系统，以提升贝尼地平促进成骨的作用效果，并增强其作为局部治疗药物的稳定性，为贝尼地平促进成骨作用的临床应用提供理论依据。
此外本实验中还首次以硅酸镁纳米球作为化疗药物载体，制备载阿霉素药物的硅酸镁纳米载药系统，评价其载药性能及药物释放特性，并通过体外及体内实验探究其抑制肿瘤的作用效果，为该纳米载药系统的临床应用提供理论基础。
1.贝尼地平对成骨细胞功能的影响
目的：采用MC3T3-E1Subclone14小鼠前成骨细胞系作为成骨细胞模型，系统性评价贝尼地平在体外对成骨细胞功能的影响。
方法：配制浓度为10-10~10-4mol/L的贝尼地平（benidipine，BD）梯度药物溶液，通过MTT法检测BD对MC3T3-E1细胞增殖活性的影响，筛选促进该细胞增殖的药物浓度区间进行后续实验。通过ALP活性水平及茜素红染色检测BD对MC3T3-E1细胞分化活性及矿化能力的影响，并通过RT-PCR及Western Blot检测BD对于MC3T3-E1细胞BMP2、OCN及Runx2mRNA和蛋白表达水平的影响。
结果：BD浓度为10-5~10-4mol/L时对细胞增殖具有抑制作用，在较低浓度10-10mol/L时对细胞的增殖无显著影响，而浓度区间于10-9~10-6mol/L时该药物能够显著促进细胞的增殖，因此筛选此浓度区间进行后续实验。浓度区间于10-9~10-6mol/L的BD能够提高MC3T3-E1细胞ALP的活性水平及矿化能力，其中浓度为10-7mol/L时作用效果最为显著。同时此浓度区间的BD也能够上调MC3T3-E1细胞BMP2、OCN及Runx2mRNA和蛋白的表达水平。
结论：浓度为10-9~10-6mol/L的BD能够促进MC3T3-E1细胞的增殖、分化及矿化，并能上调该细胞BMP2、OCN及Runx2mRNA和蛋白的表达水平。其中浓度为10-7mol/L的BD体外促进成骨的作用效果最为显著。
2.硅酸镁纳米球的制备及其生物相容性的研究
目的：制备硅酸镁纳米球（magnesium silicate nanospheres，MSNs），检测该纳米材料的形貌结构特征，并通过体外及体内各项实验评价其生物相容性，为后续实验中MSNs载药系统的建立及应用提供理论基础。
方法：采用经典St ber法合成二氧化硅胶体球，并以此材料为前体制备MSNs。采用扫描电镜、透射电镜观察MSNs的形态结构，EDS分析MSNs元素组成，XRD分析MSNs衍射图谱，获取材料的结构信息。判断MSNs表面孔隙特点，并计算出其比表面积及外壳表面的孔径尺寸。细胞形态观察、MTT法及溶血实验检测MSNs的体外毒性，并将MSNs通过尾静脉注入小鼠体内，通过对小鼠30天内体重和行为的观察，组织学及血液学等检测手段评价MSNs的体内毒性。
结果：本实验所合成的MSNs为大小均一，直径约400nm的空心球体结构，比表面积为498m2/g。其表面具有介孔结构，平均直径为4.32nm。体外实验表明，浓度为200μg/ml的MSNs对细胞形态及活性均无显著影响，且溶血率小于1%。体内实验中，MSNs在小鼠脾脏及肝脏累积最多。100mg MSNs/kg的剂量条件下，小鼠的体重、行为、血液学及主要脏器的组织学检测均显示MSNs没有显著毒性。
结论：本实验所制备的空心MSNs具有较高的生物相容性及较低的毒性，可以作为一种安全、高效的纳米药物载体。该纳米材料在纳米载药系统的应用中有着巨大的潜力。
3.载贝尼地平硅酸镁纳米球对成骨细胞功能的影响
目的：制备载贝尼地平药物的硅酸镁纳米球（BD-MSNs），探讨该纳米载药系统对于成骨细胞增殖、分化及矿化的作用，为其日后促进成骨作用的相关临床应用提供理论基础。
方法：制备BD-MSNs纳米载药系统并测定其载药量。以含有BD浓度为10-7mol/L的BD-MSNs为实验浓度，观测该浓度纳米载药系统对于MC3T3-E1细胞形态的影响，并通过MTT法、ALP活性水平及茜素红染色检测BD-MSNs对MC3T3-E1细胞增殖、分化及矿化水平的影响，并以相同浓度BD做为对照，比较该纳米载药系统促进成骨作用的优越性。
结果：所制备的BD-MSNs载药量为450mg/g，即当BD浓度为10-7mol/L时，所含MSNs浓度为12.04μg/ml，且该浓度下BD-MSNs对于MC3T3-E1细胞形态无显著影响。与相同浓度单纯BD药物相比，BD-MSNs能够显著促进MC3T3-E1细胞的增殖、分化及矿化。
结论：本实验所制备的BD-MSNs具有良好的生物安全性，体外实验证实BD-MSNs纳米载药系统较单纯BD药物促进成骨的作用更为显著。
4.载阿霉素硅酸镁纳米球在抗肿瘤治疗中的应用
目的：以硅酸镁纳米球（MSNs）作为药物载体，制备载阿霉素（DOX）的纳米载药系统（DOX-MSNs），测定其载药性能及药物释放特性，通过体外及体内实验检测其抑制肿瘤的作用效果。
方法：分别将不同浓度的DOX药物溶解于pH为5~8的溶液中，每份样品中加入MSNs10mg，搅拌以制备不同载药量的DOX-MSNs，并检测载药量及药物释放特性。选择HepG2细胞作为肿瘤细胞模型，细胞内吞实验检测MSNs在HepG2细胞中的聚集情况，通过MTT法检测该纳米载药系统于体外对肿瘤细胞的抑制效果。建立荷瘤小鼠的动物模型，通过观测小鼠肿瘤的体积变化及TUNEL试验检测肿瘤细胞凋亡情况，评价DOX-MSNs于动物体内抗肿瘤的作用效果。
结果：本实验中制备的DOX-MSNs在DOX浓度为500μg/ml，pH为8的条件下载药量最高，达到2140mg/g。24h内DOX-MSNs在pH为5.0条件下药物释放量显著高于pH为7.4条件下的释放量。细胞内吞实验显示MSNs能够进入肿瘤细胞内部并大量聚集于溶酶体中。MTT结果显示DOX-MSNs抑制肿瘤细胞增殖的效果较相同浓度单纯DOX药物的作用效果更为显著。体内实验对于肿瘤体积的观测及TUNEL试验检测肿瘤细胞凋亡的结果也显示DOX-MSNs能够显著抑制肿瘤的生长。
结论：本实验制备的DOX-MSNs载药量高，且药物释放具有pH敏感性。MSNs纳米药物载体能够大量聚集于胞内溶酶体中，有利于提高所载化疗药物的作用效果。DOX-MSNs较单纯DOX药物抑制肿瘤生长的作用效果更为显著。
Hypertension is a common disease in the elderly, the prevalence rate of this desease isclose to20%in our country. Most patients of dental restoration and implantation are middleaged and elderly people, so the proportion of patients with high blood pressure is larger.Calcium antagonist is one of the most commonly used medicine in the treatment ofhypertensive disease, including nifedipine, amlodipine, etc. Whether the calcium antagonistwould cause osteoporosis and further affect oral treatment, or the drug could promote boneformation and prevent osteoporosis. These issues are worthy of attention and exploration.
Benidipine (BD) is the second generation of dihydropyridine calcium antagonist, whichis often used for hypertension and angina pectoris. Previous studies have demonstrated thatBD has a positive effect on bone metabolism. Inspired by this promoting phenomenon, thepresent study investigated the effects of BD on osteoblast function in vitro. We preparedmagnesium silicate nanospheres (MSNs) and used it as nanocarriers to make BD-MSNs drugdelivery system for promoting the function of osteoblasts. The preparation of BD-MSNs drugdelivery system improved the stability of the drug, and also provided a theoretical basis forthe further clinical application.
In addition we used MSNs as the chemotherapy drug carrier for the first time to preparethe drug delivery system of doxorubicin (DOX), evaluated its drug loading capacity and drugrelease property. We also explored the effects of DOX-MSNs drug delivery system on theinhibition of tumor in vitro and in vivo, which provided a theoretical basis for the clinicalapplication of DOX-MSNs drug delivery system.
1. Effects of benidipine on osteoblast function in vitro
Objective: To investigate the effects of benidipine (BD) on osteoblast function in vitro.
Methods: A solution of BD was prepared by dissolving solid BD in DMSO solvent.Experiments were designed and performed, including an MTT assay, reverse transcription- polymerase chain reaction, western blot analysis, alkaline phosphatase activity measurementsand alizarin red staining.
Results: BD promoted proliferation of MC3T3-E1cells at concentrations of10-9~10-6mol/L. The higher concentrations of BD inhibited cell proliferation whereas no significantdifference from the control was observed when the lower concentrations of BD were applied.So the concentrations of10-9~10-6mol/L was chosen for further experiments. Followingtreatment of the drug, BMP2, OCN and Runx2mRNA and protein levels were markedlyupregulated compared with those in the control group. ALP activity was enhancedsignificantly in a time dependent manner when osteoblasts were treated with BD. BD alsopromoted the formation of mineralized matrix nodules in the MC3T3-E1cells.
Conclusion: BD promoted cell proliferation and osteogenic differentiation atconcentrations of10-9~10-6mol/L by upregulating BMP2, OCN and Runx2gene expressionlevels. BD promoted osteogenesis most markedly at concentrations of10-7mol/L.
2. The preparation of magnesium silicate nanospheres and evaluation of theirbiocompatibility
Objective: To investigate morphological and structural features of magnesium silicatenanospheres (MSNs) and evaluate their biocompatibility in vitro and in vivo.
Methods: Monodispersed silica colloidal spheres were synthesized in accordance withthe St ber method and then were used for the preparation of MSNs. Detailed morphologicaland structural features of MSNs were examined via scanning electron microscopy (SEM) andtransmission electron microscopy (TEM). Energy dispersive spectroscopy (EDS) analysisconfirmed the elements of our product. The N2adsorption and desorption analysis wasintroduced to investigate the pore properties of MSNs. Cellular modality, hemolysis assay andMTT assay were used to evaluate the cytotoxicity of MSNs in vitro. Body weightmeasurements, histology analysis, hematology analysis and blood biochemical assay wereused to detect the biocompatibility of MSNs in vivo.
Results: The MSNs were uniform hollow spheres, the average diameter was nearly400nm and the specific surface area was498m2/g. MSNs were mesoporous materials, an averagediameter width of pores was4.32nm. In vitro experiment no obvious differences in the cell morphology and cell viability for the cells treated with MSNs were observed at theconcentration of200μg/ml. No hemolysis of RBCs could be detected upon the maximalconcentration of200μg/ml. There was nearly no difference in body weight measurements,histology analysis, hematology analysis and blood biochemical assay between test andcontrol groups in vivo.
Conclusion: The MSNs had good biocompatibility and low biological toxicity, thus itcould be used as a practical and powerful nanocarrier for drugs. Nanocarriers based on MSNscould serve meeting the criteria as a high performance biomedical material.
3. Effects of benidipine-loaded magnesium silicate nanospheres on osteoblast function invitro
Objective: To investigate the effects of benidipine-loaded magnesium silicatenanospheres (BD-MSNs) on osteoblast function in vitro.
Methods: Prepared BD-MSNs and evaluated the drug loading capacity. Theconcentration of BD was10-7mol/L in this experiment. Cell morphology was observed, MTTassay, alkaline phosphatase activity measurements and alizarin red staining were used toevaluate the effect of BD-MSNs on cell proliferation and osteogenic differentiation.
Results: The BD loading capacity of BD-MSNs was450mg/g. No obvious differencesin the cell morphology for the cells treated with BD-MSNs were observed at the experimentalconcentration. BD-MSNs had a better effect on proliferation and osteogenic differentiation ofMC3T3-E1cells compared to BD at the concentration of10-7mol/L.
Conclusion: BD-MSNs prepared in our experiment had good biocompatibility. Thisdrug delivery system had a better effect on proliferation and osteogenic differentiation ofMC3T3-E1cells compared to BD at the concentration of10-7mol/L.
4. Uniform magnesium silicate nanospheres as high drug-loading nanocarriers forcancer therapy
Objective: To evaluate drug loading capacity and drug release property of drug deliverysystem of doxorubicin (DOX-MSNs), and to explore the effects of DOX-MSNs on theinhibition of tumor in vitro and in vivo.
Methods: MSNs (200μg/mL) at different pH values ranging from5to8was dispersed in various concentrations of DOX solution and stirred overnight to get the DOX-MSNs. TheUV-vis absorbance method was used to determine the loading and releasing amount of DOX.Endocytosis study was used to detect the accumulation of MSNs in tumer cells. MTT assay,tumer size measurement and TUNEL assay were used to evaluate the effects of DOX-MSNson the inhibition of tumor.
Results: The drug-loading capacity was2140mg/g upon optimum conditions (pH8anda DOX concentration of500μg/ml). Approximately70%of the drug was released within24hat pH5.0, whereas at pH7.4, about32%of the drug was released. Most DOX-MSNsmolecules accumulated in lysosomes of tumer cells. DOX-MSNs had a better effect oninhibiting tumor cell proliferation in vitro compared to DOX at the same concentration.Tumer size measurement and TUNEL assay showed the same effect of DOX-MSNs in vivo.
Conclusion：MSNs exhibited a significantly high storage capacity of DOX (2140mg/g)and held a sustained release property, showing more potential for cancer therapy. MSNs weremostly accumulated in lysosome, indicating their pH-dependent release character.DOX-MSNs could effectively suppress tumor growth and induce cancer cell destruction.
引文
[1] Yao K, Nagashima K, Miki H. Pharmacological, pharmacokinetic, and clinicalproperties of benidipine hydrochloride, a novel, long-acting calcium channel blocker[J]. J Pharmacol Sci,2006,100(4):243-261.
[2]黄婧,李玉珍.长效二氢吡啶类钙离子拮抗剂贝尼地平[J].临床药物治疗杂志,2005,(04):55-58.
[3] Ishii A, Toyama J. Binding properties of (+/-)[3H] benidipine hydrochloride to ratheart membranes [J]. J Cardiovasc Pharmacol,1993,21(2):191-196.
[4] Ishii A, Nishida K, Oka T, et al. Slow dissociation of the new slow-onset andlong-acting calcium antagonist benidipine hydrochloride from3H-nitrendipine bindingsites [J]. Arzneimittelforschung,1988,38(11A):1681-1683.
[5] Catterall WA, Striessnig J. Receptor sites for Ca2+channel antagonists [J]. TrendsPharmacol Sci,1992,13(6):256-262.
[6] Fleckenstein A. History of calcium antagonists [J]. Circ Res,1983,52:13-16.
[7] Furukawa T, Yamakawa T, Midera T, et al. Selectivities of dihydropyridine derivativesin blocking Ca2+channel subtypes expressed in Xenopus oocytes [J]. J PharmacolExp Ther,1999,291(2):464-473.
[8] Furukawa T, Nukada T, Miura R, et al. Differential blocking action of dihydropyridineCa2+antagonists on a T-type Ca2+channel (alpha1G) expressed in Xenopus oocytes[J]. J Cardiovasc Pharmacol,2005,45(3):241-246.
[9] Moriyama T, Karasawa A. Cardiovascular effects of benidipine and amlodipine inisolated tissues and anesthetized dogs [J]. Biol Pharm Bull,1994,17(11):1468-1471.
[10]张维忠.新型钙拮抗剂贝尼地平基础与临床研究进展[J].高血压杂志,2005,(04):196-197.
[11] Kobayashi H, Ohishi T, Nishiie H, et al. Absorption, distribution and excretion afteroral administration of14C-benidipine hydrochloride in rats and dogs [J].Arzneimittelforschung,1988,38(11A):1742-1746.
[12] Kobayashi H, Kobayashi S, Dalrymple PD, et al. Absorption, metabolism andexcretion after oral administration of a new Ca antagonist, benidipine hydrochloride toman [J]. Xenobiotica,1997,27(6):597-608.
[13] Yamada S, Nakajima M, Kusaka T, et al. In vivo receptor binding of benidipine andamlodipine in mesenteric arteries and other tissues of spontaneously hypertensive rats[J]. Life Sci,2002,70(17):1999-2011.
[14] Grossman E, Messerli FH. Effect of calcium antagonists on plasma norepinephrinelevels, heart rate, and blood pressure [J]. Am J Cardiol,1997,80(11):1453-1458.
[15] Karasawa A, Nomura H, Nito M, et al. Effects of benidipine hydrochloride (Coniel) onblood pressure, heart rate and plasma norepinephrine concentration in spontaneouslyhypertensive rats [J]. Nihon Yakurigaku Zasshi,1999,113(5):317-326.
[16] Sugimoto K, Gotoh E, Takasaki I, et al. AT1receptor antagonist, TCV116, does notprevent cardiac hypertrophy in salt-loaded Dahl salt-sensitive rats [J]. Clin ExpPharmacol Physiol,1996,23(4):282-286.
[17] Namba M, Kim S, Zhan Y, et al. Combination treatment with a calcium channelblocker and an angiotensin blocker in a rat systolic heart failure model withhypertension [J]. Hypertens Res,2002,25(3):461-466.
[18] Kusaka H, Nomura H, Karasawa A. Effects of benidipine on renal function inanesthetized spontaneously hypertensive rats [J]. Jpn J Pharmacol,1994,66(1):81-86.
[19] Hayashi K, Ozawa Y, Fujiwara K, et al. Role of actions of calcium antagonists onefferent arterioles--with special references to glomerular hypertension [J]. Am JNephrol,2003,23(4):229-244.
[20] Kawata T, Hashimoto S, Koike T. Diversity in the renal hemodynamic effects ofdihydropyridine calcium blockers in spontaneously hypertensive rats [J]. J CardiovascPharmacol,1997,30(4):431-436.
[21] Yue W, Kimura S, Fujisawa Y, et al. Benidipine dilates both pre-and post-glomerulararteriole in the canine kidney [J]. Hypertens Res,2001,24(4):429-436.
[22]胡景云.贝尼地平对高血压病患者的降压作用及β_2微球蛋白的影响[J].医学临床研究,2004,(10):1167-1168.
[23] Ito A, Fukumoto Y, Shimokawa H. Changing characteristics of patients withvasospastic angina in the era of new calcium channel blockers [J]. J CardiovascPharmacol,2004,44(4):480-485.
[24]陈晶,刘宁,张毅杰.血管内皮细胞功能障碍与原发性高血压[J].中国实用医药,2009,(23):241-242.
[25]林忠伟,许香广,方卫华.原发性高血压与血管内皮细胞功能障碍[J].岭南心血管病杂志,2006,(01):69-72.
[26] Dohi Y, Kojima M, Sato K. Benidipine improves endothelial function in renalresistance arteries of hypertensive rats [J]. Hypertension,1996,28(1):58-63.
[27] Yamashita T, Kawashima S, Ozaki M, et al. A calcium channel blocker, benidipine,inhibits intimal thickening in the carotid artery of mice by increasing nitric oxideproduction [J]. J Hypertens,2001,19(3):451-458.
[28] Karasawa A, Kubo K, Shuto K, et al. Vasodilating effects of the new calciumantagonist benidipine hydrochloride in anesthetized dogs and cats [J].Arzneimittelforschung,1988,38(11A):1707-1712.
[29] Kitayama J, Kitazono T, Ooboshi H, et al. Long-term effects of benidipine on cerebralvasoreactivity in hypertensive rats [J]. Eur J Pharmacol,2002,438(3):153-158.
[30] Shirakura S, Karasawa A, Kubo K. Effect of benidipine hydrochloride (KW-3049), oncerebral ischemia induced by bilateral occlusion of the common carotid arteries in rats[J]. Biol Pharm Bull,1993,16(5):475-479.
[31] Higashiura K, Ura N, Takada T, et al. Alteration of muscle fiber composition linking toinsulin resistance and hypertension in fructose-fed rats [J]. Am J Hypertens,1999,12(6):596-602.
[32] Suzuki M, Kanazawa A, Hasegawa M, et al. Improvement of insulin resistance inessential hypertension by long-acting Ca antagonist benidipine [J]. Clin Exp Hypertens,1999,21(8):1327-1344.
[33] Kosaka N, Uchii M. Effect of benidipine hydrochloride, a dihydropyridine-typecalcium antagonist, on the function of mouse osteoblastic cells [J]. Calcif Tissue Int,1998,62(6):554-556.
[34] Nishiya Y, Kosaka N, Uchii M, et al. A potent1,4-dihydropyridine L-type calciumchannel blocker, benidipine, promotes osteoblast differentiation [J]. Calcif Tissue Int,2002,70(1):30-39.
[35] Nishiya Y, Sugimoto S. Effects of various antihypertensive drugs on the function ofosteoblast [J]. Biol Pharm Bull,2001,24(6):628-633.
[36] Tanaka H, Shoji Y, Ogami T, et al. The effect of benidipine hydrochloride on the bonesof male spontaneously hypertensive rats [J]. J New Remed Clin,1993,42:2298-2306.
[37] Teofilo JM, Brentegani LG, Carvalho TL. A histometric study in rats of the effect ofthe calcium antagonist amlodipine on bone healing after tooth extraction [J]. Arch OralBiol,2001,46(4):375-379.
[38] Moraes RB, Correa L, Luz JG. Adverse effects of the amlodipine on bone healing ofthe mandibular fracture: an experimental study in rats [J]. Oral Maxillofac Surg,2011,15(2):93-101.
[39] Halici Z, Borekci B, Ozdemir Y, et al. Protective effects of amlodipine and lacidipineon ovariectomy-induced bone loss in rats [J]. Eur J Pharmacol,2008,579(1-3):241-245.
[40] Ushijima K, Liu Y, Maekawa T, et al. Protective effect of amlodipine againstosteoporosis in stroke-prone spontaneously hypertensive rats [J]. Eur J Pharmacol,2010,635(1-3):227-230.
[41]周雅轩.纳米载药系统在医药领域中的应用进展[J].天津药学,2012,(01):47-49.
[42]吉顺莉,张春燕,戈延茹.纳米载药系统的研究进展[J].中国药业,2010,(14):82-83.
[43]郑延波,徐克.纳米载药系统靶向治疗肝细胞癌的研究进展[J].中国介入影像与治疗学,2008,(04):320-323.
[44]周蓉,艾立,余日跃,等.纳米载药系统及其在中药中的应用[J].亚太传统医药,2007,(08):41-44.
[45]蒋正立,朱萍.纳米脂质体研究新进展[J].海峡药学,2008,(11):5-7.
[46] Ali KA, Mudassir J, Mohtar N, et al. Advanced drug delivery to the lymphatic system:lipid-based nanoformulations [J]. Int J Nanomedicine,2013,8:2733-2744.
[47] Mehnert W, Mader K. Solid lipid nanoparticles: production, characterization andapplications [J]. Adv Drug Deliv Rev,2001,47(2-3):165-196.
[48]李泳雪,王春龙,李杰.纳米技术在现代中药制剂中的应用[J].中草药,2002,(08):3-5.
[49]杨雪峰,赵坤,姜金庆.纳米乳给药系统的应用[J].广东农业科学,2011,(07):125-127.
[50] Cheng L, Anglin E, Cunin F, et al. Intravitreal properties of porous silicon photoniccrystals: a potential self-reporting intraocular drug-delivery vehicle [J]. Br JOphthalmol,2008,92(5):705-711.
[51] Michel C, Aprahamian M, Defontaine L, et al. The effect of site of administration inthe gastrointestinal tract on the absorption of insulin from nanocapsules in diabetic rats[J]. J Pharm Pharmacol,1991,43(1):1-5.
[52] Babu A, Templeton AK, Munshi A, et al. Nanodrug Delivery Systems: A PromisingTechnology for Detection, Diagnosis, and Treatment of Cancer [J]. AAPS Pharm SciTech,2014.
[53] Sanna V, Pala N, Sechi M. Targeted therapy using nanotechnology: focus on cancer [J].Int J Nanomedicine,2014,9:467-483.
[54]高洁,李博华.靶向抗肿瘤纳米药物研究进展[J].中国医药生物技术,2008,(02):143-145.
[55] Peng F, Su Y, Wei X, et al. Silicon-nanowire-based nanocarriers with ultrahighdrug-loading capacity for in vitro and in vivo cancer therapy [J]. Angewandte Chemie(International ed. in English),2013,52(5):1457-1461.
[56]马宝花,王少华,王宏,等.5-氟尿嘧啶纳米级给药系统研究进展[J].中国药业,2009,(17):1-3.
[57]乔明艳.紫杉醇纳米级给药系统研究概述[J].黑龙江医药,2010,(05):759-761.
[58] Lu J, Li Z, Zink JI, et al. In vivo tumor suppression efficacy of mesoporous silicananoparticles-based drug-delivery system: enhanced efficacy by folate modification [J].Nanomedicine,2012,8(2):212-220.
[59] Wang B, Meng W, Bi M, et al. Uniform magnesium silicate hollow spheres as drug-loading nanocarriers for cancer therapy with low systemic toxicity [J]. Daltontransactions,2013,42(24):8918-8925.
[60]胡振夏,符旭东.纳米载药系统逆转肿瘤多药耐药的研究进展[J].中国医院药学杂志,2011,(20):1724-1727.
[61] Kalota A, Shetzline SE, Gewirtz AM. Progress in the development of nucleic acidtherapeutics for cancer [J]. Cancer Biol Ther,2004,3(1):4-12.
[62]林华型.靶向性纳米载药系统对中枢神经系统疾病作用的研究进展[J].西北药学杂志,2008,(04):256-258.
[63]郭志雷,车晓明.中枢神经纳米载药系统的研究进展[J].中国临床神经科学,2009,(03):318-323.
[64] Wohlfart S, Gelperina S, Kreuter J. Transport of drugs across the blood-brain barrierby nanoparticles [J]. J Control Release,2012,161(2):264-273.
[65] Roney C, Kulkarni P, Arora V, et al. Targeted nanoparticles for drug delivery throughthe blood-brain barrier for Alzheimer's disease [J]. J Control Release,2005,108(2-3):193-214.
[66] Yang S, Zhu J, Lu Y, et al. Body distribution of camptothecin solid lipid nanoparticlesafter oral administration [J]. Pharm Res,1999,16(5):751-757.
[67] DK Kim YZ, J Kehr TK, Bjelke B. Characterization and MRI study of surfactant-coated superparamagnetic nanoparticles administered into the rat brain [J]. JMagnetism and Magnetic Materials,2001,225(1):256-261.
[68]王蓉,原永芳.纳米载药系统在静脉注射药物中的应用[J].中国现代应用药学,2010,(03):206-209.
[69] Wang Y, Li Y, Wang Q, et al. Pharmacokinetics and biodistribution ofpaclitaxel-loaded pluronic P105/L101mixed polymeric micelles [J]. Yakugaku zasshi:Journal of the Pharmaceutical Society of Japan,2008,128(6):941-50.
[70] Nahar M, Mishra D, Dubey V, et al. Development, characterization, and toxicityevaluation of amphotericin B-loaded gelatin nanoparticles [J]. Nanomedicine,2008,4(3):252-261.
[71] Brusa P, Dosio F, Pacchioni D, et al. Pharmacokinetics of an antibody-ricin conjugateadministered intraperitoneally to mice [J]. J Pharm Sci,1994,83(4):514-519.
[72] Alexiou C, Arnold W, Klein RJ, et al. Locoregional cancer treatment with magneticdrug targeting [J]. Cancer Res,2000,60(23):6641-6648.
[73]何训贵,高河勇,吕伟,等.喜树碱及其衍生物的前药研究进展[J].药学进展,2005,(04):145-150.
[74] Chen G, Deng C, Li YP. TGF-beta and BMP signaling in osteoblast differentiation andbone formation [J]. Int J Biol Sci,2012,8(2):272-288.
[75] Matsumoto A. The effect of cell environment on osteoblastic function [J]. Nihonyakurigaku zasshi. Folia pharmacologica Japonica,1995,105(5):273-283.
[76] Sudo H, Kodama HA, Amagai Y, et al. In vitro differentiation and calcification in anew clonal osteogenic cell line derived from newborn mouse calvaria [J]. J Cell Biol,1983,96(1):191-198.
[77] Long F. Building strong bones: molecular regulation of the osteoblast lineage [J]. NatRev Mol Cell Biol,2012,13(1):27-38.
[78]刘爱旗,夏璐. CCK-8法与MTT法检测兔成纤维细胞活性的比较研究[J].中国医学创新,2013,(02):12-13.
[79]赵承彦,靖志安,牛青霞. MTT显色反应实验条件分析[J].河南医学研究,2000,(02):107-111+116.
[80] Teti A. Bone development: overview of bone cells and signaling [J]. Curr Rep,2011,9(4):264-273.
[81] Liu D, Zhang J, Wang G, et al. The dual-effects of LaCl3on the proliferation,osteogenic differentiation, and mineralization of MC3T3-E1cells [J]. Biol Trace ElemRes,2012,150(1-3):433-440.
[82] Neve A, Corrado A, Cantatore FP. Osteocalcin: skeletal and extra-skeletal effects [J]. JCell Physiol,2013,228(6):1149-1153.
[83] Tsuji K, Bandyopadhyay A, Harfe BD, et al. BMP2activity, although dispensable forbone formation, is required for the initiation of fracture healing [J]. Nat Genet,2006,38(12):1424-1429.
[84] Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfa1: a transcriptional activator ofosteoblast differentiation [J]. Cell,1997,89(5):747-754.
[85] Ducy P, Starbuck M, Priemel M, et al. A Cbfa1-dependent genetic pathway controlsbone formation beyond embryonic development [J]. Genes Dev,1999,13(8):1025-1036.
[86] Nakashima K, Zhou X, Kunkel G, et al. The novel zinc finger-containing transcriptionfactor osterix is required for osteoblast differentiation and bone formation [J]. Cell,2002,108(1):17-29.
[87] Phimphilai M, Zhao Z, Boules H, et al. BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype [J]. J Bone Miner Res,2006,21(4):637-646.
[88] Komori T. Regulation of bone development and extracellular matrix protein genes byRUNX2[J]. Cell Tissue Res,2010,339(1):189-195.
[89] Fink WS. Controlled growth of monodisperse silica spheres in the micron size range[J]. Journal of Colloid and Interface Science,1968,26(1):62-69.
[90] Hu L, Chen Q. Hollow/porous nanostructures derived from nanoscale metal-organicframeworks towards high performance anodes for lithium-ion batteries [J]. Nanoscale,2014,6(3):1236-1257.
[91] De Angelis F, Malerba M, Patrini M, et al.3D hollow nanostructures as buildingblocks for multifunctional plasmonics [J]. Nano Lett,2013,13(8):3553-3558.
[92] You L, Wang T, Ge J. When mesoporous silica meets the alkaline polyelectrolyte: acontrollable synthesis of functional and hollow nanostructures with a porous shell [J].Chemistry,2013,19(6):2142-2149.
[93] Wang Z, Zhou L, Lou XW. Metal oxide hollow nanostructures for lithium-ion batteries[J]. Advanced materials,2012,24(14):1903-1911.
[94] Williamson PA, Blower PJ, Green MA. Synthesis of porous hollow silicananostructures using hydroxyapatite nanoparticle templates [J]. Chemicalcommunications,2011,47(5):1568-1570.
[95] Ambrogio MW, Thomas CR, Zhao YL, et al. Mechanized silica nanoparticles: a newfrontier in theranostic nanomedicine [J]. Acc Chem Res,2011,44(10):903-913.
[96] Xue M, Zhong X, Shaposhnik Z, et al. pH-Operated mechanized porous siliconnanoparticles [J]. J Am Chem Soc,2011,133(23):8798-8801.
[97] Anglin EJ, Cheng L, Freeman WR, et al. Porous silicon in drug delivery devices andmaterials [J]. Adv Drug Deliv Rev,2008,60(11):1266-1277.
[98] Chen Y, Chen H, Guo L, et al. Hollow/rattle-type mesoporous nanostructures by astructural difference-based selective etching strategy [J]. ACS Nano,2010,4(1):529-539.
[99] Wang Y, Wang G, Wang H, et al. Chemical-template synthesis of micro/nanoscalemagnesium silicate hollow spheres for waste-water treatment [J]. Chemistry,2010,16(11):3497-3503.
[100]潘卫松,周永标,张讷敏,等.氨甲苯酸注射液引起溶血反应原因的实验研究[J].中国临床药理学与治疗学,2012,(08):878-883.
[101] Cheng L, Yang K, Shao M, et al. In vivo pharmacokinetics, long-term biodistributionand toxicology study of functionalized upconversion nanoparticles in mice [J].Nanomedicine (London, England),2011,6(8):1327-1340.
[102]刘颖,秦继勇,李文辉,等.土当归对辐射损伤小鼠体重与血常规的影响[J].江苏中医药,2013,(01):71-72.
[103]陈陵,赵学成,邓琼,等.纳米氧化铈急性染毒对雄性小鼠体重和脏器系数及血常规的影响[J].环境与健康杂志,2010,(10):899-902.
[104]马慧萍,焦阳,高荣敏,等.大苞雪莲乙醇提取物对模拟高原缺氧小鼠生化指标的影响[J].中药材,2014,(01):103-107.
[105] Zhou YT, Yu LS, Zeng S, et al. Pharmacokinetic drug-drug interactions between1,4-dihydropyridine calcium channel blockers and statins: factors determininginteraction strength and relevant clinical risk management [J]. Ther Clin Risk Manag,2014,10:17-26.
[106]薛梅,扶玲,李楚云,等.盐酸贝尼地平片溶出度的测定[J].中国药房,2010,(25):2384-2385.
[107] Nishiya Y, Kosaka N, Uchii M, et al. A potent1,4-dihydropyridine L-type calciumchannel blocker, benidipine, promotes osteoblast differentiation [J]. Calcif Tissue Int,2002,70(1):30-39.
[108] Redlich K, Pietschmann P, Stulc T, et al. Comparative study on the effect of calciumchannel blockers on basal and parathyroid hormone-induced bone resorption in vitro[J]. Pharmacol Toxicol,1997,80(6):262-265.
[109] Carvalho C, Santos RX, Cardoso S, et al. Doxorubicin: the good, the bad and the uglyeffect [J]. Curr Med Chem,2009,16(25):3267-85.
[110] Climent E, Bernardos A, Martinez-Manez R, et al. Controlled delivery systems usingantibody-capped mesoporous nanocontainers [J]. J Am Chem Soc,2009,131(39):14075-14080.
[111] Liu T, Li L, Teng X, et al. Single and repeated dose toxicity of mesoporous hollowsilica nanoparticles in intravenously exposed mice [J]. Biomaterials,2011,32(6):1657-1668.
[112] Yang P, Gai S, Lin J. Functionalized mesoporous silica materials for controlled drugdelivery [J]. Chem Soc Rev,2012,41(9):3679-3698.
[113] He X, Li J, An S, et al. pH-sensitive drug-delivery systems for tumor targeting [J].Ther Deliv,2013,4(12):1499-1510.
[114] Xiao Z, Ji C, Shi J, et al. DNA self-assembly of targeted near-infrared-responsive goldnanoparticles for cancer thermo-chemotherapy [J]. Angewandte Chemie,2012,51(47):11853-11857.
[115] Peng F, Su Y, Wei X, et al. Silicon-nanowire-based nanocarriers with ultrahigh drug-loading capacity for in vitro and in vivo cancer therapy [J]. Angewandte Chemie,2013,52(5):1457-1461.
[116] Loo DT. In situ detection of apoptosis by the TUNEL assay: an overview of techniques[J]. Methods in molecular biology,2011,82:3-13.
[117] Stadelmann C, Lassmann H. Detection of apoptosis in tissue sections [J]. Cell TissueRes,2000,301(1):19-31.
[118] Sgonc R, Gruber J. Apoptosis detection: an overview [J]. Exp Gerontol,1998,33(6):525-533.
[2]黄婧,李玉珍.长效二氢吡啶类钙离子拮抗剂贝尼地平[J].临床药物治疗杂志,2005,(04):55-58.
[3] Ishii A, Toyama J. Binding properties of (+/-)[3H] benidipine hydrochloride to ratheart membranes [J]. J Cardiovasc Pharmacol,1993,21(2):191-196.
[4] Ishii A, Nishida K, Oka T, et al. Slow dissociation of the new slow-onset andlong-acting calcium antagonist benidipine hydrochloride from3H-nitrendipine bindingsites [J]. Arzneimittelforschung,1988,38(11A):1681-1683.
[5] Catterall WA, Striessnig J. Receptor sites for Ca2+channel antagonists [J]. TrendsPharmacol Sci,1992,13(6):256-262.
[6] Fleckenstein A. History of calcium antagonists [J]. Circ Res,1983,52:13-16.
[7] Furukawa T, Yamakawa T, Midera T, et al. Selectivities of dihydropyridine derivativesin blocking Ca2+channel subtypes expressed in Xenopus oocytes [J]. J PharmacolExp Ther,1999,291(2):464-473.
[8] Furukawa T, Nukada T, Miura R, et al. Differential blocking action of dihydropyridineCa2+antagonists on a T-type Ca2+channel (alpha1G) expressed in Xenopus oocytes[J]. J Cardiovasc Pharmacol,2005,45(3):241-246.
[9] Moriyama T, Karasawa A. Cardiovascular effects of benidipine and amlodipine inisolated tissues and anesthetized dogs [J]. Biol Pharm Bull,1994,17(11):1468-1471.
[10]张维忠.新型钙拮抗剂贝尼地平基础与临床研究进展[J].高血压杂志,2005,(04):196-197.
[11] Kobayashi H, Ohishi T, Nishiie H, et al. Absorption, distribution and excretion afteroral administration of14C-benidipine hydrochloride in rats and dogs [J].Arzneimittelforschung,1988,38(11A):1742-1746.
[12] Kobayashi H, Kobayashi S, Dalrymple PD, et al. Absorption, metabolism andexcretion after oral administration of a new Ca antagonist, benidipine hydrochloride toman [J]. Xenobiotica,1997,27(6):597-608.
[13] Yamada S, Nakajima M, Kusaka T, et al. In vivo receptor binding of benidipine andamlodipine in mesenteric arteries and other tissues of spontaneously hypertensive rats[J]. Life Sci,2002,70(17):1999-2011.
[14] Grossman E, Messerli FH. Effect of calcium antagonists on plasma norepinephrinelevels, heart rate, and blood pressure [J]. Am J Cardiol,1997,80(11):1453-1458.
[15] Karasawa A, Nomura H, Nito M, et al. Effects of benidipine hydrochloride (Coniel) onblood pressure, heart rate and plasma norepinephrine concentration in spontaneouslyhypertensive rats [J]. Nihon Yakurigaku Zasshi,1999,113(5):317-326.
[16] Sugimoto K, Gotoh E, Takasaki I, et al. AT1receptor antagonist, TCV116, does notprevent cardiac hypertrophy in salt-loaded Dahl salt-sensitive rats [J]. Clin ExpPharmacol Physiol,1996,23(4):282-286.
[17] Namba M, Kim S, Zhan Y, et al. Combination treatment with a calcium channelblocker and an angiotensin blocker in a rat systolic heart failure model withhypertension [J]. Hypertens Res,2002,25(3):461-466.
[18] Kusaka H, Nomura H, Karasawa A. Effects of benidipine on renal function inanesthetized spontaneously hypertensive rats [J]. Jpn J Pharmacol,1994,66(1):81-86.
[19] Hayashi K, Ozawa Y, Fujiwara K, et al. Role of actions of calcium antagonists onefferent arterioles--with special references to glomerular hypertension [J]. Am JNephrol,2003,23(4):229-244.
[20] Kawata T, Hashimoto S, Koike T. Diversity in the renal hemodynamic effects ofdihydropyridine calcium blockers in spontaneously hypertensive rats [J]. J CardiovascPharmacol,1997,30(4):431-436.
[21] Yue W, Kimura S, Fujisawa Y, et al. Benidipine dilates both pre-and post-glomerulararteriole in the canine kidney [J]. Hypertens Res,2001,24(4):429-436.
[22]胡景云.贝尼地平对高血压病患者的降压作用及β_2微球蛋白的影响[J].医学临床研究,2004,(10):1167-1168.
[23] Ito A, Fukumoto Y, Shimokawa H. Changing characteristics of patients withvasospastic angina in the era of new calcium channel blockers [J]. J CardiovascPharmacol,2004,44(4):480-485.
[24]陈晶,刘宁,张毅杰.血管内皮细胞功能障碍与原发性高血压[J].中国实用医药,2009,(23):241-242.
[25]林忠伟,许香广,方卫华.原发性高血压与血管内皮细胞功能障碍[J].岭南心血管病杂志,2006,(01):69-72.
[26] Dohi Y, Kojima M, Sato K. Benidipine improves endothelial function in renalresistance arteries of hypertensive rats [J]. Hypertension,1996,28(1):58-63.
[27] Yamashita T, Kawashima S, Ozaki M, et al. A calcium channel blocker, benidipine,inhibits intimal thickening in the carotid artery of mice by increasing nitric oxideproduction [J]. J Hypertens,2001,19(3):451-458.
[28] Karasawa A, Kubo K, Shuto K, et al. Vasodilating effects of the new calciumantagonist benidipine hydrochloride in anesthetized dogs and cats [J].Arzneimittelforschung,1988,38(11A):1707-1712.
[29] Kitayama J, Kitazono T, Ooboshi H, et al. Long-term effects of benidipine on cerebralvasoreactivity in hypertensive rats [J]. Eur J Pharmacol,2002,438(3):153-158.
[30] Shirakura S, Karasawa A, Kubo K. Effect of benidipine hydrochloride (KW-3049), oncerebral ischemia induced by bilateral occlusion of the common carotid arteries in rats[J]. Biol Pharm Bull,1993,16(5):475-479.
[31] Higashiura K, Ura N, Takada T, et al. Alteration of muscle fiber composition linking toinsulin resistance and hypertension in fructose-fed rats [J]. Am J Hypertens,1999,12(6):596-602.
[32] Suzuki M, Kanazawa A, Hasegawa M, et al. Improvement of insulin resistance inessential hypertension by long-acting Ca antagonist benidipine [J]. Clin Exp Hypertens,1999,21(8):1327-1344.
[33] Kosaka N, Uchii M. Effect of benidipine hydrochloride, a dihydropyridine-typecalcium antagonist, on the function of mouse osteoblastic cells [J]. Calcif Tissue Int,1998,62(6):554-556.
[34] Nishiya Y, Kosaka N, Uchii M, et al. A potent1,4-dihydropyridine L-type calciumchannel blocker, benidipine, promotes osteoblast differentiation [J]. Calcif Tissue Int,2002,70(1):30-39.
[35] Nishiya Y, Sugimoto S. Effects of various antihypertensive drugs on the function ofosteoblast [J]. Biol Pharm Bull,2001,24(6):628-633.
[36] Tanaka H, Shoji Y, Ogami T, et al. The effect of benidipine hydrochloride on the bonesof male spontaneously hypertensive rats [J]. J New Remed Clin,1993,42:2298-2306.
[37] Teofilo JM, Brentegani LG, Carvalho TL. A histometric study in rats of the effect ofthe calcium antagonist amlodipine on bone healing after tooth extraction [J]. Arch OralBiol,2001,46(4):375-379.
[38] Moraes RB, Correa L, Luz JG. Adverse effects of the amlodipine on bone healing ofthe mandibular fracture: an experimental study in rats [J]. Oral Maxillofac Surg,2011,15(2):93-101.
[39] Halici Z, Borekci B, Ozdemir Y, et al. Protective effects of amlodipine and lacidipineon ovariectomy-induced bone loss in rats [J]. Eur J Pharmacol,2008,579(1-3):241-245.
[40] Ushijima K, Liu Y, Maekawa T, et al. Protective effect of amlodipine againstosteoporosis in stroke-prone spontaneously hypertensive rats [J]. Eur J Pharmacol,2010,635(1-3):227-230.
[41]周雅轩.纳米载药系统在医药领域中的应用进展[J].天津药学,2012,(01):47-49.
[42]吉顺莉,张春燕,戈延茹.纳米载药系统的研究进展[J].中国药业,2010,(14):82-83.
[43]郑延波,徐克.纳米载药系统靶向治疗肝细胞癌的研究进展[J].中国介入影像与治疗学,2008,(04):320-323.
[44]周蓉,艾立,余日跃,等.纳米载药系统及其在中药中的应用[J].亚太传统医药,2007,(08):41-44.
[45]蒋正立,朱萍.纳米脂质体研究新进展[J].海峡药学,2008,(11):5-7.
[46] Ali KA, Mudassir J, Mohtar N, et al. Advanced drug delivery to the lymphatic system:lipid-based nanoformulations [J]. Int J Nanomedicine,2013,8:2733-2744.
[47] Mehnert W, Mader K. Solid lipid nanoparticles: production, characterization andapplications [J]. Adv Drug Deliv Rev,2001,47(2-3):165-196.
[48]李泳雪,王春龙,李杰.纳米技术在现代中药制剂中的应用[J].中草药,2002,(08):3-5.
[49]杨雪峰,赵坤,姜金庆.纳米乳给药系统的应用[J].广东农业科学,2011,(07):125-127.
[50] Cheng L, Anglin E, Cunin F, et al. Intravitreal properties of porous silicon photoniccrystals: a potential self-reporting intraocular drug-delivery vehicle [J]. Br JOphthalmol,2008,92(5):705-711.
[51] Michel C, Aprahamian M, Defontaine L, et al. The effect of site of administration inthe gastrointestinal tract on the absorption of insulin from nanocapsules in diabetic rats[J]. J Pharm Pharmacol,1991,43(1):1-5.
[52] Babu A, Templeton AK, Munshi A, et al. Nanodrug Delivery Systems: A PromisingTechnology for Detection, Diagnosis, and Treatment of Cancer [J]. AAPS Pharm SciTech,2014.
[53] Sanna V, Pala N, Sechi M. Targeted therapy using nanotechnology: focus on cancer [J].Int J Nanomedicine,2014,9:467-483.
[54]高洁,李博华.靶向抗肿瘤纳米药物研究进展[J].中国医药生物技术,2008,(02):143-145.
[55] Peng F, Su Y, Wei X, et al. Silicon-nanowire-based nanocarriers with ultrahighdrug-loading capacity for in vitro and in vivo cancer therapy [J]. Angewandte Chemie(International ed. in English),2013,52(5):1457-1461.
[56]马宝花,王少华,王宏,等.5-氟尿嘧啶纳米级给药系统研究进展[J].中国药业,2009,(17):1-3.
[57]乔明艳.紫杉醇纳米级给药系统研究概述[J].黑龙江医药,2010,(05):759-761.
[58] Lu J, Li Z, Zink JI, et al. In vivo tumor suppression efficacy of mesoporous silicananoparticles-based drug-delivery system: enhanced efficacy by folate modification [J].Nanomedicine,2012,8(2):212-220.
[59] Wang B, Meng W, Bi M, et al. Uniform magnesium silicate hollow spheres as drug-loading nanocarriers for cancer therapy with low systemic toxicity [J]. Daltontransactions,2013,42(24):8918-8925.
[60]胡振夏,符旭东.纳米载药系统逆转肿瘤多药耐药的研究进展[J].中国医院药学杂志,2011,(20):1724-1727.
[61] Kalota A, Shetzline SE, Gewirtz AM. Progress in the development of nucleic acidtherapeutics for cancer [J]. Cancer Biol Ther,2004,3(1):4-12.
[62]林华型.靶向性纳米载药系统对中枢神经系统疾病作用的研究进展[J].西北药学杂志,2008,(04):256-258.
[63]郭志雷,车晓明.中枢神经纳米载药系统的研究进展[J].中国临床神经科学,2009,(03):318-323.
[64] Wohlfart S, Gelperina S, Kreuter J. Transport of drugs across the blood-brain barrierby nanoparticles [J]. J Control Release,2012,161(2):264-273.
[65] Roney C, Kulkarni P, Arora V, et al. Targeted nanoparticles for drug delivery throughthe blood-brain barrier for Alzheimer's disease [J]. J Control Release,2005,108(2-3):193-214.
[66] Yang S, Zhu J, Lu Y, et al. Body distribution of camptothecin solid lipid nanoparticlesafter oral administration [J]. Pharm Res,1999,16(5):751-757.
[67] DK Kim YZ, J Kehr TK, Bjelke B. Characterization and MRI study of surfactant-coated superparamagnetic nanoparticles administered into the rat brain [J]. JMagnetism and Magnetic Materials,2001,225(1):256-261.
[68]王蓉,原永芳.纳米载药系统在静脉注射药物中的应用[J].中国现代应用药学,2010,(03):206-209.
[69] Wang Y, Li Y, Wang Q, et al. Pharmacokinetics and biodistribution ofpaclitaxel-loaded pluronic P105/L101mixed polymeric micelles [J]. Yakugaku zasshi:Journal of the Pharmaceutical Society of Japan,2008,128(6):941-50.
[70] Nahar M, Mishra D, Dubey V, et al. Development, characterization, and toxicityevaluation of amphotericin B-loaded gelatin nanoparticles [J]. Nanomedicine,2008,4(3):252-261.
[71] Brusa P, Dosio F, Pacchioni D, et al. Pharmacokinetics of an antibody-ricin conjugateadministered intraperitoneally to mice [J]. J Pharm Sci,1994,83(4):514-519.
[72] Alexiou C, Arnold W, Klein RJ, et al. Locoregional cancer treatment with magneticdrug targeting [J]. Cancer Res,2000,60(23):6641-6648.
[73]何训贵,高河勇,吕伟,等.喜树碱及其衍生物的前药研究进展[J].药学进展,2005,(04):145-150.
[74] Chen G, Deng C, Li YP. TGF-beta and BMP signaling in osteoblast differentiation andbone formation [J]. Int J Biol Sci,2012,8(2):272-288.
[75] Matsumoto A. The effect of cell environment on osteoblastic function [J]. Nihonyakurigaku zasshi. Folia pharmacologica Japonica,1995,105(5):273-283.
[76] Sudo H, Kodama HA, Amagai Y, et al. In vitro differentiation and calcification in anew clonal osteogenic cell line derived from newborn mouse calvaria [J]. J Cell Biol,1983,96(1):191-198.
[77] Long F. Building strong bones: molecular regulation of the osteoblast lineage [J]. NatRev Mol Cell Biol,2012,13(1):27-38.
[78]刘爱旗,夏璐. CCK-8法与MTT法检测兔成纤维细胞活性的比较研究[J].中国医学创新,2013,(02):12-13.
[79]赵承彦,靖志安,牛青霞. MTT显色反应实验条件分析[J].河南医学研究,2000,(02):107-111+116.
[80] Teti A. Bone development: overview of bone cells and signaling [J]. Curr Rep,2011,9(4):264-273.
[81] Liu D, Zhang J, Wang G, et al. The dual-effects of LaCl3on the proliferation,osteogenic differentiation, and mineralization of MC3T3-E1cells [J]. Biol Trace ElemRes,2012,150(1-3):433-440.
[82] Neve A, Corrado A, Cantatore FP. Osteocalcin: skeletal and extra-skeletal effects [J]. JCell Physiol,2013,228(6):1149-1153.
[83] Tsuji K, Bandyopadhyay A, Harfe BD, et al. BMP2activity, although dispensable forbone formation, is required for the initiation of fracture healing [J]. Nat Genet,2006,38(12):1424-1429.
[84] Ducy P, Zhang R, Geoffroy V, et al. Osf2/Cbfa1: a transcriptional activator ofosteoblast differentiation [J]. Cell,1997,89(5):747-754.
[85] Ducy P, Starbuck M, Priemel M, et al. A Cbfa1-dependent genetic pathway controlsbone formation beyond embryonic development [J]. Genes Dev,1999,13(8):1025-1036.
[86] Nakashima K, Zhou X, Kunkel G, et al. The novel zinc finger-containing transcriptionfactor osterix is required for osteoblast differentiation and bone formation [J]. Cell,2002,108(1):17-29.
[87] Phimphilai M, Zhao Z, Boules H, et al. BMP signaling is required for RUNX2-dependent induction of the osteoblast phenotype [J]. J Bone Miner Res,2006,21(4):637-646.
[88] Komori T. Regulation of bone development and extracellular matrix protein genes byRUNX2[J]. Cell Tissue Res,2010,339(1):189-195.
[89] Fink WS. Controlled growth of monodisperse silica spheres in the micron size range[J]. Journal of Colloid and Interface Science,1968,26(1):62-69.
[90] Hu L, Chen Q. Hollow/porous nanostructures derived from nanoscale metal-organicframeworks towards high performance anodes for lithium-ion batteries [J]. Nanoscale,2014,6(3):1236-1257.
[91] De Angelis F, Malerba M, Patrini M, et al.3D hollow nanostructures as buildingblocks for multifunctional plasmonics [J]. Nano Lett,2013,13(8):3553-3558.
[92] You L, Wang T, Ge J. When mesoporous silica meets the alkaline polyelectrolyte: acontrollable synthesis of functional and hollow nanostructures with a porous shell [J].Chemistry,2013,19(6):2142-2149.
[93] Wang Z, Zhou L, Lou XW. Metal oxide hollow nanostructures for lithium-ion batteries[J]. Advanced materials,2012,24(14):1903-1911.
[94] Williamson PA, Blower PJ, Green MA. Synthesis of porous hollow silicananostructures using hydroxyapatite nanoparticle templates [J]. Chemicalcommunications,2011,47(5):1568-1570.
[95] Ambrogio MW, Thomas CR, Zhao YL, et al. Mechanized silica nanoparticles: a newfrontier in theranostic nanomedicine [J]. Acc Chem Res,2011,44(10):903-913.
[96] Xue M, Zhong X, Shaposhnik Z, et al. pH-Operated mechanized porous siliconnanoparticles [J]. J Am Chem Soc,2011,133(23):8798-8801.
[97] Anglin EJ, Cheng L, Freeman WR, et al. Porous silicon in drug delivery devices andmaterials [J]. Adv Drug Deliv Rev,2008,60(11):1266-1277.
[98] Chen Y, Chen H, Guo L, et al. Hollow/rattle-type mesoporous nanostructures by astructural difference-based selective etching strategy [J]. ACS Nano,2010,4(1):529-539.
[99] Wang Y, Wang G, Wang H, et al. Chemical-template synthesis of micro/nanoscalemagnesium silicate hollow spheres for waste-water treatment [J]. Chemistry,2010,16(11):3497-3503.
[100]潘卫松,周永标,张讷敏,等.氨甲苯酸注射液引起溶血反应原因的实验研究[J].中国临床药理学与治疗学,2012,(08):878-883.
[101] Cheng L, Yang K, Shao M, et al. In vivo pharmacokinetics, long-term biodistributionand toxicology study of functionalized upconversion nanoparticles in mice [J].Nanomedicine (London, England),2011,6(8):1327-1340.
[102]刘颖,秦继勇,李文辉,等.土当归对辐射损伤小鼠体重与血常规的影响[J].江苏中医药,2013,(01):71-72.
[103]陈陵,赵学成,邓琼,等.纳米氧化铈急性染毒对雄性小鼠体重和脏器系数及血常规的影响[J].环境与健康杂志,2010,(10):899-902.
[104]马慧萍,焦阳,高荣敏,等.大苞雪莲乙醇提取物对模拟高原缺氧小鼠生化指标的影响[J].中药材,2014,(01):103-107.
[105] Zhou YT, Yu LS, Zeng S, et al. Pharmacokinetic drug-drug interactions between1,4-dihydropyridine calcium channel blockers and statins: factors determininginteraction strength and relevant clinical risk management [J]. Ther Clin Risk Manag,2014,10:17-26.
[106]薛梅,扶玲,李楚云,等.盐酸贝尼地平片溶出度的测定[J].中国药房,2010,(25):2384-2385.
[107] Nishiya Y, Kosaka N, Uchii M, et al. A potent1,4-dihydropyridine L-type calciumchannel blocker, benidipine, promotes osteoblast differentiation [J]. Calcif Tissue Int,2002,70(1):30-39.
[108] Redlich K, Pietschmann P, Stulc T, et al. Comparative study on the effect of calciumchannel blockers on basal and parathyroid hormone-induced bone resorption in vitro[J]. Pharmacol Toxicol,1997,80(6):262-265.
[109] Carvalho C, Santos RX, Cardoso S, et al. Doxorubicin: the good, the bad and the uglyeffect [J]. Curr Med Chem,2009,16(25):3267-85.
[110] Climent E, Bernardos A, Martinez-Manez R, et al. Controlled delivery systems usingantibody-capped mesoporous nanocontainers [J]. J Am Chem Soc,2009,131(39):14075-14080.
[111] Liu T, Li L, Teng X, et al. Single and repeated dose toxicity of mesoporous hollowsilica nanoparticles in intravenously exposed mice [J]. Biomaterials,2011,32(6):1657-1668.
[112] Yang P, Gai S, Lin J. Functionalized mesoporous silica materials for controlled drugdelivery [J]. Chem Soc Rev,2012,41(9):3679-3698.
[113] He X, Li J, An S, et al. pH-sensitive drug-delivery systems for tumor targeting [J].Ther Deliv,2013,4(12):1499-1510.
[114] Xiao Z, Ji C, Shi J, et al. DNA self-assembly of targeted near-infrared-responsive goldnanoparticles for cancer thermo-chemotherapy [J]. Angewandte Chemie,2012,51(47):11853-11857.
[115] Peng F, Su Y, Wei X, et al. Silicon-nanowire-based nanocarriers with ultrahigh drug-loading capacity for in vitro and in vivo cancer therapy [J]. Angewandte Chemie,2013,52(5):1457-1461.
[116] Loo DT. In situ detection of apoptosis by the TUNEL assay: an overview of techniques[J]. Methods in molecular biology,2011,82:3-13.
[117] Stadelmann C, Lassmann H. Detection of apoptosis in tissue sections [J]. Cell TissueRes,2000,301(1):19-31.
[118] Sgonc R, Gruber J. Apoptosis detection: an overview [J]. Exp Gerontol,1998,33(6):525-533.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |