CN110475546A - 生理活性物质载体 - Google Patents
生理活性物质载体 Download PDFInfo
- Publication number
- CN110475546A CN110475546A CN201880022677.1A CN201880022677A CN110475546A CN 110475546 A CN110475546 A CN 110475546A CN 201880022677 A CN201880022677 A CN 201880022677A CN 110475546 A CN110475546 A CN 110475546A
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- Prior art keywords
- particle
- bioactive substance
- porous silica
- silicon particle
- silica silicon
- Prior art date
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- 229960000641 zorubicin Drugs 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
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Abstract
本发明涉及一种生物活性物质载体,其包括:生物活性物质;和负载生物活性物质且具有多个5nm至100nm直径的孔的多孔二氧化硅颗粒,其中所述多孔二氧化硅颗粒具有特定物理性质,可以以持续方式递送负载量的各种药物,并且可以肠胃外施用。
Description
技术领域
本发明涉及一种生理活性物质(“生物活性物质”)载体。
背景技术
药物递送系统是指可以有效递送所需量的药物,例如,蛋白质、核酸或其它低分子量化合物,同时最小化现存药物的副作用并且最大化其功效和效果的制药技术。可以减少开发新药所需的成本和时间的上述技术最近已成为高科技应用领域,其与纳米技术组合的同时在医学科学中创造新的附加价值。此外,自上世纪80年代后期,例如美国和日本等技术先进国家致力于药物递送系统以及与包括起关键作用的制药公司在内的企业合作的新药的开发。
迄今为止,病毒基因、重组蛋白、脂质体、阳离子聚合物和各种类型的纳米颗粒和纳米材料已用于向动物细胞的药物递送。然而,已发现许多阳离子脂质体和阳离子聚合物对细胞具有强毒性,使得它们不适合应用于临床实践。此外,为了核酸稳定的膜渗透,还尝试了化学修饰核酸中的主链的方法。然而,这样的方法是昂贵的,花费很长时间并且需要劳动密集型工艺,因此不适于临床应用。使用包括量子点、磁性颗粒或金纳米颗粒的各种类型的纳米颗粒来开发药物递送系统(DDS)是重大的尝试。例如,存在如“image diagnosticdrug delivery carrier using porous silicon particles and a manufacturingmethod thereof(韩国公开专利No.2010-0117433).”等的相关研究。然而,这样的颗粒具有对细胞的毒性、不易于引入例如核酸等生物分子的结构、和向细胞中的低导入效率的缺点。
为了研究细胞中生物活性物质的功能或细胞内递送,需要有效的递送系统。然而,可递送广泛的生物活性物质的用于一般用途的递送系统、用于接收和递送大量药物的系统、药物缓释系统等的开发仍是不完整的。
发明内容
发明要解决的问题
本发明的一个目的是提供用于一般用途的生物活性物质载体。
本发明的另一个目的是提供可以以持续方式递送各种生物活性物质的生物活性物质载体。
用于解决问题的方案
1.一种生物活性物质载体,其包括:生物活性物质;和
多孔二氧化硅颗粒,其负载所述生物活性物质,具有多个5nm至100nm直径的孔,并且具有在通过下式1测定的吸光度的比为1/2下的‘t’为24以上:
[式1]
At/A0
其中A0表示在将5ml的含有1mg/ml的多孔二氧化硅颗粒的悬浮液置于具有50kDa直径的孔的圆筒状渗透膜中后测定的多孔二氧化硅颗粒的吸光度,
其中15ml的与渗透膜接触并且与悬浮液实质上相同的溶剂存在于渗透膜的外部,在37℃和60rpm下水平搅拌渗透膜的内部和外部,
悬浮液的pH为7.4,和
其中At表示在测定A0后‘t’小时测定的多孔二氧化硅颗粒的吸光度。
2.根据上述1所述的生物活性物质载体,其中悬浮液是选自由磷酸盐缓冲盐水(PBS)和模拟体液(SBF)组成的组的一种或多种。
3.根据上述1所述的生物活性物质载体,其中式1中的At在其中每隔预定时间更换渗透膜外部的溶剂的环境中测定。
4.根据上述1所述的生物活性物质载体,其中‘t’的范围为24至120。
5.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒是生物降解性的。
6.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒具有在通过式1测定的吸光度的比为1/5下的“t”为70至120。
7.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒具有在通过式1测定的吸光度的比为1/20下的“t”为130至220。
8.根据上述1所述的生物活性物质载体,其中通过式1测定的吸光度的比和“t”之间的皮尔森相关系数为0.8以上。
9.根据上述1所述的生物活性物质载体,其中孔径的范围为7nm至30nm。
10.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒具有球状。
11.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒的平均直径的范围为150nm至1000nm。
12.根据上述1所述的生物活性物质载体,其中多孔二氧化硅的BET表面积的范围为200m2/g至700m2/g。
13.根据上述1所述的生物活性物质载体,其中多孔二氧化硅的BET表面积的范围为300m2/g至450m2/g。
14.根据上述1所述的生物活性物质载体,每克多孔二氧化硅颗粒的体积的范围为0.7ml至2.2ml。
15.根据上述1所述的生物活性物质载体,每克多孔二氧化硅颗粒的体积的范围为1.0ml至2.0ml。
16.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒在颗粒的外表面或孔的内部具有亲水性取代基或疏水性取代基。
17.根据上述1所述的生物活性物质载体,其中多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带正电或带负电。
18.根据上述1所述的生物活性物质载体,其中生物活性物质是难溶性的(non-soluble),并且多孔二氧化硅颗粒在颗粒的外表面或孔的内部具有疏水性取代基。
19.根据上述1所述的生物活性物质载体,其中生物活性物质是难溶性的,多孔二氧化硅颗粒在孔的内部具有疏水性取代基并且在颗粒的外表面具有亲水性取代基。
20.根据上述1所述的生物活性物质载体,其中生物活性物质在中性pH下带负电,并且多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带正电。
21.根据上述1所述的生物活性物质载体,其中生物活性物质在中性pH下带正电,并且多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带负电。
22.一种生物活性物质载体,其包括:生物活性物质;和球状多孔二氧化硅颗粒,其负载生物活性物质,粒径为150nm至500nm,并具有多个7nm至30nm直径的孔,并且
具有在通过下式2测定的吸光度的比为1/2下的‘t’为24至120的范围:
[式2]
At/A0
其中A0表示在将5ml的含有1mg/ml的多孔二氧化硅颗粒的悬浮液置于具有50kDa直径的孔的圆筒状渗透膜中后测定的多孔二氧化硅颗粒的吸光度,
其中15ml的与渗透膜接触并且与悬浮液实质上相同的溶剂存在于渗透膜的外部,在37℃和60rpm下水平搅拌渗透膜的内部和外部,
悬浮液为PBS或SBF并具有7.4的pH,和
其中At表示在测定A0后‘t’小时测定的多孔二氧化硅颗粒的吸光度。
23.根据上述22所述的生物活性物质载体,其中多孔二氧化硅颗粒的BET表面积的范围为300m2/g至450m2/g,并且每克的体积的范围为1.0ml至2.0ml。
24.根据上述22所述的生物活性物质载体,其中生物活性物质是难溶性的,并且多孔二氧化硅颗粒在颗粒的外表面或孔的内部具有疏水性取代基。
25.根据上述22所述的生物活性物质载体,其中生物活性物质是难溶性的,并且多孔二氧化硅颗粒在孔的内部具有疏水性取代基并且在颗粒的外表面具有亲水性取代基。
26.根据上述22所述的生物活性物质载体,其中生物活性物质在中性pH下带负电,并且多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带正电。
27.根据上述22所述的生物活性物质载体,其中生物活性物质在中性pH下带正电,并且多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带负电。
28.一种制造生物活性物质载体的方法,其包括在溶剂中将多孔二氧化硅颗粒与生物活性物质接触。
29.根据上述28所述的方法,其中溶剂为选自由以下组成的组的一种或多种:水、氯仿、二氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷、丙酮、甲基异丁酮、环己酮、苯、甲苯、二甲苯、N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮、甲醇、乙醇、丙醇、丁醇、PBS、SBF、硼酸盐缓冲生理盐水和tris缓冲生理盐水。
30.根据上述28所述的方法,其中多孔二氧化硅颗粒与生物活性物质按重量计的比为1:0.05至0.8。
31.一种递送生物活性物质的方法,其包括向个体受试者肠胃外施用根据上述1至27任一项所述的生物活性物质载体。
32.根据上述31所述的方法,其中肠胃外施用是眼眶内、眼内、输注、动脉内、关节内、心内、皮内、肌内、腹膜内、肺内、脊柱内、胸骨内、鞘内、子宫内、静脉内、蛛网膜下、被膜下、皮下、转化粘液质或跨器官(transorgan)施用。
发明的效果
本发明的生物活性物质载体可包括负载生物活性物质的多孔二氧化硅颗粒,其在体内缓慢降解,因此以持续方式递送药物。
本发明的生物活性物质载体可包括负载(即,携带)生物活性物质的多孔二氧化硅颗粒,其在体内完全降解,因此完全递送负载的生物活性物质至生物体中。
本发明的生物活性物质载体可为肠胃外施用。
本发明的生物活性物质载体可以以持续方式递送各种药物。
附图说明
图1是显示根据本发明的一个实施方案的多孔二氧化硅颗粒的显微照片。
图2是显示根据本发明的一个实施方案的多孔二氧化硅颗粒的显微照片。
图3是显示在生产根据本发明的一个实施方案的多孔二氧化硅颗粒的过程中产生的微多孔颗粒的显微照片。
图4是显示根据本发明的一个实施方案的微多孔颗粒的显微照片。
图5是显示根据本发明的一个实施方案的孔径的多孔二氧化硅颗粒的显微照片。
本文中,降解性递送运载体(Degradable Delivery Vehicle,DDV)是指说明性颗粒,其中括号中的数字表示粒径,下标中的数字表示孔径。例如DDV(200)10意为具有200nm粒径和10nm孔径的颗粒。
图6是说明根据本发明的一个实施方案的多孔二氧化硅颗粒的生物降解性的识别的显微照片。
图7说明了根据一个实例的设置有圆筒状渗透膜的管。
图8说明了根据本发明的一个实施方案的多孔二氧化硅颗粒的吸光度随时间的减少。
图9说明了根据本发明的一个实施方案的多孔二氧化硅颗粒的根据粒径的吸光度随时间的减少。
图10说明了根据本发明的一个实施方案的多孔二氧化硅颗粒的根据粒径的吸光度随时间的减少。
图11说明了根据本发明的一个实施方案的多孔二氧化硅颗粒的根据环境中的pH的吸光度随时间的减少。
图12说明了根据本发明的一个实施方案的多孔二氧化硅颗粒的吸光度随时间的减少。
图13至17分别说明了在根据本发明的一个实施方案的多孔二氧化硅颗粒上负载的生物活性物质随时间的释放曲线。
图18说明了用于确认根据一个实施例的生物活性物质的释放的管。
图19至25分别说明了在根据本发明的一个实施方案的多孔二氧化硅颗粒上负载的生物活性物质随时间的释放曲线。
图26是显示在根据本发明的一个实施方案的多孔二氧化硅颗粒上负载Cas9蛋白、接着递送其至细胞中的结果的显微照片。
图27是显示在根据本发明的一个实施方案的多孔二氧化硅颗粒上负载siRNA、接着在小鼠中检测siRNA释放的确认结果的显微照片。
具体实施方式
本发明的生物活性物质载体可包括:生物活性物质;和负载生物活性物质且具有多个5nm至100nm直径的孔的多孔二氧化硅颗粒。
生物活性物质
生物活性物质是负载在多孔二氧化硅颗粒上的并可递送至个体对象来显示活性的生理活性物质/生物功能控制物质(biofunction control substance),其可以起到可对人或动物有机体赋予直接、间接、治疗的、生理的和/或药理的效果的治疗活性剂的作用。
如上所述的这类治疗活性剂可包括,例如,一般药物、药剂、药或前药、靶组、或含有靶组的药或前药。
更具体地,治疗活性剂可包括,例如:心血管药物,具体地,抗高血压剂(例如,钙通道阻滞剂或钙拮抗剂)和抗心律失常药;充血性心力衰竭药;肌肉收缩剂;血管舒张药;ACE抑制剂;利尿剂;脱氧脱水酶抑制剂;强心苷;磷酸二酯酶抑制剂;阻滞剂;β-阻滞剂;钠通道阻滞剂;钾通道阻滞剂;β-肾上腺素激动剂;血小板抑制剂;血管紧张素II拮抗剂;抗凝血药;血栓溶解剂;出血的治疗药(bleeding drug);贫血治疗剂;凝血酶抑制剂;抗寄生虫药;抗菌剂;抗炎剂,具体地,非甾体抗炎剂(NSAIDs),更具体地,COX-2抑制剂;甾体抗炎药;预防性抗炎剂;抗青光眼剂;肥大细胞稳定剂;散瞳剂;影响呼吸系统的药物;过敏性鼻炎药;α-肾上腺素拮抗剂;皮质类固醇;慢性阻塞性肺病的丸剂;黄嘌呤氧化酶抑制剂;抗关节炎剂;痛风治疗剂;多能药物和多能药物拮抗剂;抗结核病药;抗真菌剂;抗原农药;驱虫药;抗病毒剂,具体地,针对呼吸道、疱疹、巨细胞病毒、人体免疫缺陷病毒和肝炎感染的抗病毒剂;卡波西氏肉瘤和白血病治疗剂;疼痛管理剂,具体地,麻醉剂和止痛药,包括阿片受体激动剂、阿片受体部分激动剂、阿片类拮抗剂、阿片受体混合激动剂-拮抗剂等的阿片类药物;安定药;拟交感神经药;肾上腺素拮抗剂;影响神经递质的吸收和排出的神经传递药;抗胆碱能刺激剂;抗痔疮治疗剂;具有化学疗法的治疗效果的放射或预防或治疗剂;脂肪形成剂(adipogenics);减脂剂;抗肥胖药如脂肪酶抑制剂;拟交感神经剂;胃溃疡和炎症治疗剂如质子泵抑制剂;前列腺素;VEGF抑制剂;抗血脂异常剂,具体地,他汀类药物;影响中枢神经系统(CNS)的药物,例如抗精神病药、抗癫痫药和抗惊厥药(抗惊厥剂)、精神活性剂、刺激剂、抗焦虑剂和安眠药;抗抑郁药;抗帕金森剂;激素或其片段如性激素;生长激素拮抗剂;促性腺激素释放激素及其类似物;类固醇激素及其拮抗剂;选择性雌性激素调节剂;生长因子;抗糖尿病剂如胰岛素、胰岛素片段、胰岛素类似物、胰高血糖素样肽和降血糖剂;H1、H2、H3和H4抗组胺剂;肽、蛋白质、多肽、核酸、和寡核苷酸药物;类似物、片段和变体如天然蛋白质、多肽、寡核苷酸和核酸;用于治疗偏头痛的药物;止喘药;胆碱能拮抗剂;糖皮质激素;雄激素;抗雄激素;肾上腺皮质激素生物合成抑制剂;骨质疏松治疗剂如双磷酸盐类;抗甲状腺药;防晒剂、防UV保护剂和过滤剂;细胞因子拮抗剂;抗肿瘤剂;抗阿尔兹海默症剂;HMGCoA还原酶抑制剂;贝特类;胆固醇吸收抑制剂;HDL胆固醇提升剂;甘油三酯还原剂;抗衰老或抗皱纹剂;蛋白质如胶原和弹性蛋白、抗微生物剂;抗痤疮剂;抗氧化剂;头发护理和皮肤美白剂;人载脂蛋白的变体;用于产生激素的前体分子;蛋白质及其肽;氨基酸;植物提取物如葡萄籽提取物;DHEA;异黄酮;营养素包括维生素、植物固醇和环烯醚萜苷、倍半碳酸盐内酯、萜烯、酚苷、三萜、对苯二酚衍生物、苯丙氨酸;抗氧化剂如视黄醇和其它类维生素A类包括维A酸(视黄酸)和辅酶Q10;Ω-3脂肪酸;葡萄糖胺;核酸、寡核苷酸、反义医学;酶;辅酶;细胞因子类似物;细胞因子激动剂;细胞因子拮抗剂;免疫球蛋白;抗体;抗体医学;基因治疗剂;脂蛋白;红细胞生成素;疫苗;用于治疗或预防例如以下的人和动物疾病的小分子治疗剂:过敏/哮喘、关节炎、癌症、糖尿病、生长障碍、心血管疾病、炎症、免疫功能障碍、脱发、疼痛、眼科疾病、癫痫、妇科病症、CNS病症、病毒感染、细菌感染、寄生虫感染、G1疾病、肥胖和血液病,等,但不限于这些。
治疗活性剂可为另外的活性剂,包括,例如,红细胞生成素(EPO),血小板生成素,细胞因子如白细胞介素(包括IL-1至IL-17)、胰岛素、胰岛素样生长因子(包括IGF-1和IGF-2)、表皮生长因子(EGF)、转化生长因子(包括TGF-α和TGF-β)、人生长激素、转铁蛋白、低密度脂蛋白、高密度脂蛋白、瘦素、VEGF、PDGF、睫状神经营养因子、催乳素、促肾上腺皮质激素(ACTH)、降钙素、人绒毛膜促性腺激素、皮质醇、雌二醇、促卵泡激素(FSH)、促甲状腺激素(TSH)、黄体化激素(LH),毒素包括黄体酮、睾酮、蓖麻毒素,等。
治疗活性剂可选自用于治疗肿瘤疾病和细胞或组织转化的药物组。合适的治疗剂型可包括抗肿瘤剂,例如:烷基磺酸盐例如白消安、英丙舒凡、哌泊舒凡、等,烷化剂包括苯佐替派、卡波醌、美妥替哌、氮丙啶例如乌瑞替派,等;六甲蜜胺、三亚乙基蜜胺、三亚乙基磷酰胺、三亚乙基硫代磷酰胺、氮丙啶例如三羟甲基三聚氰胺、和甲基三聚氰胺;苯丁酸氮芥、萘氮芥、环磷酰胺、雌莫司汀、异环磷酰胺、恩比兴、氧氮芥、氧氮芥盐酸盐、美法仑、新恩比兴、苯芥胆甾醇、泼尼莫司汀、曲磷胺、所谓的氮芥类如乌拉莫司汀;亚硝基脲化合物如卡莫司汀、氯脲霉素、福莫司汀、洛莫司汀、尼莫司汀、雷莫司汀;达卡巴嗪、甘露醇氮芥、二溴甘露醇(mitobranitol)、二溴卫矛醇;哌泊溴烷;索拉非尼(Sorafenib);阿霉素(Doxorubicin)和顺铂及其衍生物,和上述物质的任何组合和/或衍生物。
治疗活性剂可选自由抗病毒剂和抗菌剂组成的组,例如阿克拉霉素、放线菌素、安曲霉素、偶氮丝胺酸、博莱霉素、放线菌素、洋红霉素、嗜癌素、色霉素、更生霉素、柔红霉素、6-重氮-5-氧代-1-正亮氨酸、阿霉素、表阿霉素、丝裂霉素、mycophenolsaure、诺拉霉素、橄榄霉素、匹来霉素、光辉霉素、泊非霉素、嘌呤霉素、棕霉素、链脲菌素、杀结核菌素、乌苯美司、净司他丁、佐柔比星、氨基糖苷类、多烯类或大环内酯类抗体,以及上述物质的任何组合和/或衍生物。
治疗活性剂可选自:内皮抑素,血管抑素,干扰素,血小板因子4(PF4),血小板反应素,转化生长因子β,金属蛋白酶-1、-2和-3(TIMP-1、-2和-3)的组织抑制剂,TNP-470,马立马司他,新伐司他,BMS-275291,COL-3,AG3340,沙利度胺,角鲨胺,考布他汀,放射增敏剂如SU5416、SU6668、IFN-[α]、EMD121974、CAI、IL-12和IM-862,甾体或非甾体抗炎药,或用于血管新生的制剂,和上述物质的组合和/或衍生物。
治疗活性剂可选自包括核酸的组。在这方面,例如,为了赋予基因治疗或反义效果,术语“核酸”可包括含有通过共价键连接在一起的至少两个核苷酸的寡核苷酸。核酸优选具有磷酸二酯键。可选地,还包括了分别具有不同骨架的类似物。类似物可包括骨架,例如,磷酰胺、硫代磷酸酯、二硫代磷酸酯、O-甲基亚磷酰胺化合物和肽-核酸-骨架及其化合物。其它类似物可具有离子骨架、非离子骨架、或非核糖骨架。具有一个或多个碳环糖的核酸可合适作为本发明中使用的核酸。除了选择本领域已知的核酸或核酸类似物,还可应用天然产生的核酸和核酸类似物或核酸和核酸类似物的混合物的任何组合。
治疗活性剂可包括,例如,依维莫司、他克莫司、西罗莫司、山喜多(mycofenolate-mofetil)、雷帕霉素、紫杉醇、放线菌素D、兰瑞肽、巴马司他、雌二醇、VEGF、抑胃酶氨酸等,以及它们的衍生物和类似物,其可用作抗迁移、抗增殖、免疫抑制、抗炎或再内皮化剂。
治疗活性剂可包括,阿片类受体、激动剂和拮抗剂,显示激动剂/拮抗剂混合活性的化合物和显示部分激动剂活性的化合物,例如,吗啡、长效吗啡(depot morphine)、阿托品、二乙酰吗啡、双氢吗啡、氢羟吗啡酮、左啡诺、美沙酮、左美沙酮(levomethadyl)、杜冷丁、芬太尼、舒芬太尼、阿芬他尼、可待因、二氢可待因酮、羟可待酮、蒂巴因、地素吗啡、尼可吗啡、二丙酰吗啡、苄吗啡、乙基吗啡、哌替啶、美沙酮、曲马多、右旋达尔丰;纳洛酮和纳曲酮;叔丁啡、纳布啡、布托啡诺、喷他佐辛和乙基氯代环唑星。
治疗活性剂及其组合可选自,例如:肝素、合成的肝素类似物(例如,磺达肝癸钠)、水蛭素、抗凝血酶III、重组人活化蛋白C(drotrecogin alpha);纤维蛋白溶解剂如阿替普酶、纤维蛋白溶酶、溶解激酶、因子VIIa、前尿激酶、尿激酶、阿尼普酶、链激酶,等;血小板聚集抑制剂如乙酰水杨酸[阿司匹林]、噻氯匹定、氯吡格雷、阿昔单抗、葡聚糖,等;皮质类固醇如阿氯米松、安西奈德、增强的倍他米松、倍氯米松、倍他米松、布地奈德、可的松、氯倍他索、氯可托龙、地索奈德、去羟米松、地塞米松、氟西奈德、醋酸氟轻松、氟氢缩松、氟尼缩松、氟替卡松、哈西奈德、卤贝他索、氢化可的松、甲泼尼龙、莫美他松、泼尼卡酯、泼尼松、泼尼松龙、曲安西龙,等;所谓的非甾体抗炎药(NSAIDs)如双氯芬酸、二氟尼柳、依托度酸、菲诺洛芬、氟比洛芬、布洛芬、吲哚美辛、酮基布洛芬、酮咯酸、甲氯灭酸钠、甲灭酸、美洛昔康、萘丁美酮、萘普生、奥沙普秦、吡罗昔康、双水杨酸酯、舒林酸、托麦汀、塞来昔布、罗非昔布,等;细胞抑制剂,包括生物碱类如长春质碱、长春新碱等和鬼臼毒素,等;细胞毒素抗生素如柔红霉素、阿霉素、其它蒽环类药物和相关物质、博莱霉素、丝裂霉素等;抗代谢物,如叶酸类似物、嘌呤类似物或嘧啶类似物,等;紫杉醇、多西他赛、西罗莫司;铂化合物如卡铂、顺铂或奥沙利铂,等;安吖啶、伊立替康、伊马替尼、拓扑替康、干扰素-α2a、干扰素-α2b、羟基脲、米替福新、喷司他丁、卟菲尔、阿地白介素、贝沙罗汀、维A酸;抗雄激素和抗雌激素;抗心律不齐药,包括奎尼丁型抗心律不齐药,具体地,I型抗心律不齐药如奎尼丁、达舒平、阿义吗啉、重酒石酸普拉马林、重酒石酸地他义铵,等;利多卡因型抗心律不齐药,例如,利多卡因、美西律、苯妥英、妥卡尼,等;Ic型抗心律不齐药,例如,普罗帕酮、氟卡尼(醋酸),等;II类抗心律不齐药β-受体阻滞剂如美托洛尔、艾司洛尔、普萘洛尔、阿替洛尔、氧烯洛尔,等;III型抗心律不齐药如胺碘酮、索他洛尔,等;IV型抗心律不齐药如地尔硫卓、维拉帕米、戈洛帕米等;其它抗心律不齐药如腺苷、奥西那林、异丙托溴铵,等;用于促进心肌中血管新生的制剂如血管内皮生长因子(VEGF)、碱性成纤维生长因子(bFGF)、非病毒DNA、病毒DNA、内皮生长因子,等;FGF-1、FGF-2、VEGF、TGF;抗生素、单克隆抗体、抗运载蛋白;干细胞、内皮祖细胞s(EPC);洋地黄糖苷类如乙酰地高辛/甲基地高辛、洋地黄毒甙、地高辛,等;强心苷如哇吧因、海葱次苷,等;抗高血压剂,例如甲基多巴,咪唑啉受体激动剂如CNS活性抗肾上腺素能物质,等;二氢吡啶型钙通道阻滞剂如硝苯地平、尼群地平,等;ACE抑制剂;喹普利拉、西拉普利、莫西普利、群多普利、螺普利、咪达普利;血管紧张素II拮抗剂;坎地沙坦酯、缬沙坦、替米沙坦、奥美沙坦酯、依普罗沙坦;外周活化α-受体阻滞剂如哌唑嗪、乌拉地尔、多沙唑嗪、布纳唑嗪、特拉唑嗪、吲哚拉明,等;血管扩张药如双肼酞嗪、二氯醋酸二异丙胺、米诺地尔、硝普酸钠,等;其它抗高血压剂如吲达帕胺、甲磺酸双氢麦角碱、甲磺酸二氢麦角碱、西氯他宁、波生坦、氟氢可的松,等;磷酸二酯酶抑制剂如米力农、依诺昔酮和,具体地,抗高血压药如肾上腺素能和多巴胺能物质,例如,多巴酚丁胺、肾上腺素、依替福林、去甲苯福林、去甲肾上腺素、奥洛福林、多巴胺、米多君、福来君、阿美铵甲(ameziniummetil)等;部分肾上腺素能受体激动剂如双氢麦角胺;炎性细胞因子如纤连蛋白、聚赖氨酸、乙烯醋酸乙烯酯、TGFβ、PDGF、VEGF、bFGF、TNFα、NGF、GM-CSF、IGF-a、IL-1、IL-8、IL-6、生长激素,等;粘合物质如氰基丙烯酸酯、铍、二氧化硅,等;生长因子如红霉素,激素如促肾上腺皮质激素、促性腺激素、生长激素、促甲状腺激素、去氨加压素、特利加压素、pxytocin、醋酸西曲瑞克、可的瑞林、亮丙瑞林、曲普瑞林、戈那瑞林、醋酸加尼瑞克、布舍瑞林、那法瑞林、戈舍瑞林,等,和调节肽如生长抑素、奥曲肽,等;骨和软骨刺激肽,重组人BMP-2(rhBMP-2),重组BMP如磷酸双盐(例如,利塞膦酸、帕米膦酸、伊班膦酸、唑来膦酸、氯屈瞵酸、羟基乙叉二膦酸、阿仑膦酸、替鲁膦酸),骨形态发生蛋白(BMPs)包括氟化物如单氟磷酸钠、氟化钠,等;降钙素、双氢速甾醇;表皮生长因子(EGF)、血小板源性生长因子(PDGF)、成纤维细胞生长因子(FGFs)、转化生长因子-b(TGFs-b)、转化生长因子-a(TGFs-a)、红细胞生成素(EPO)、胰岛素样生长因子-I(IGF-I)、胰岛素样生长因子-II(IGF-II)、白细胞介素-1(IL-1)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-a(TNF-a)、肿瘤坏死因子-b(TNF-b)、干扰素-g(INF-g)、集落刺激因子(CSFs);单核细胞趋化蛋白、成纤维细胞刺激因子1、组胺、纤维蛋白或纤维蛋白原、内皮素-1、血管紧张素II、胶原、溴隐亭、美西麦角、甲氨蝶呤、四氯化碳、硫代乙酰胺和乙醇;此外,银(离子)、二氧化钛,具体地,例如,β-内酰胺酶敏感青霉素如苄青霉素(青霉素G)、苯氧甲基青霉素(青霉素V);例如,β-内酰胺酶抗性青霉素如阿莫西林、氨苄青霉素、巴氨西林,等;酰氨基青霉素如美洛西林、哌拉西林,等;羧基青霉素如头孢唑啉、头孢呋肟、头孢西丁、头孢替安、头孢克洛、头孢羟氨苄、头孢氨苄、氯碳头孢、头孢克肟、头孢呋辛酯、头孢布烯、头孢泊肟酯,等;氨曲南、厄他培南、美罗培南;β-内酰胺抑制剂如舒巴坦、对甲苯磺酸舒他西林,等;四环素如强力霉素、米诺环素、四环素、金霉素、土霉素,等;氨基糖苷类如庆大霉素、新霉素、链霉素、妥布霉素、阿米卡星、奈替米星、巴龙霉素、新霉素B、大观霉素,等;大环内酯类抗生素如阿奇霉素、克拉霉素、红霉素、罗红霉素、螺旋霉素、交沙霉素,等;林可胺类如克林霉素、林可霉素等;旋转酶抑制剂如氟喹诺酮类,例如,环丙沙星、氧氟沙星、莫西沙星、诺氟沙星、加替沙星、依诺沙星、氟罗沙星、左氧氟沙星,等;喹诺酮类如吡哌酸;磺胺、甲氧苄啶、磺胺嘧啶、磺胺林;糖肽类抗生素如万古霉素、替考拉宁,等;多肽类抗生素如抗敌素、多粘菌素,例如,多粘菌素-b,等,硝基咪唑类衍生物,例如,甲硝唑、替硝唑,等;氨基喹诺酮如氯喹、甲氟喹、硫酸羟基氯喹,等;双胍类如氯胍;奎宁生物碱类如乙胺嘧啶、和二氨基嘧啶;酰胺醇类如氯霉素;利福布汀、氨苯砜、夫西地酸、磷霉素、硝呋太尔、泰利霉素、夫沙芬净、喷他脒羟乙磺酸盐、利福平、牛磺罗定、阿托伐醌、利奈唑胺,等;病毒静力学药如阿昔洛韦、更昔洛韦、泛昔洛韦、膦甲酸、肌苷-(二甲氨丙醇-4-乙酰氨基苯甲酸酯)、缬更昔洛韦、伐昔洛韦、西多福韦、溴夫定,等;抗逆转录病毒活性成分核苷类似物逆转录酶抑制剂和衍生物如拉米夫定、扎西他滨、地达诺辛、齐多夫定、泰诺福韦、司他夫定、阿巴卡韦,等;非核苷类似物逆转录酶抑制剂;安普那韦、茚地那韦、沙奎那韦、洛匹那韦、利托那韦、甲磺酸萘非那韦;金刚烷胺、利巴韦林、扎那米韦、奥司他韦、拉米夫定,以及其的任何组合和其混合物。
治疗活性剂可包括,例如,抗抑郁、抗精神病或抗焦虑剂,例如阿普唑仑、阿莫沙平、苯他西泮、溴西泮、氯拉卓酸、氯巴占、氯噻西泮、地西泮、劳拉西泮、氟硝西泮、氟西泮、氯甲西泮、美达西泮、硝西泮、奥沙西泮、替马西泮、马普替林、米安舍林、去甲替林、利培酮、舍曲林、曲唑酮、氟哌啶醇、马来酸曲米帕明氟西汀、恩丹西酮、咪达唑仑、氯丙嗪、氟哌啶醇、三唑仑、氯氮平、氯丙嗪、癸氟奋乃静、氟阿尼酮、奋乃静、匹莫奇特、丙氯拉嗪、舒必利、硫利达嗪、帕罗西汀、西酞普兰、安非他酮、苯乙肼、奥氮平、双丙戊酸钠和文拉法辛。
治疗活性剂可包括,例如,阿片受体激动剂和拮抗剂,表现激动剂/拮抗剂组合活性的化合物,和表现部分活性的化合物,例如,吗啡、长效吗啡、乙烯啡、二乙酰吗啡、双氢吗啡、氢羟吗啡酮、左啡诺、美沙酮、左美沙酮、杜冷丁、芬太尼、舒芬太尼、阿芬他尼、可待因、二氢可待因酮、羟可待酮、tevine、地素吗啡、尼可吗啡、二丙酰吗啡、苄吗啡、乙基吗啡、哌替啶、曲马多、右旋达尔丰;纳洛酮和纳曲酮;叔丁啡、纳布啡、布托啡诺、喷他佐辛和乙基氯代环佐辛。
治疗活性剂可包括,例如,三环化合物,例如偶氮噻吩、阿米替林、法莫替丁、异丙嗪、帕罗西汀、奥卡西平和米氮平。
治疗活性剂可包括,例如,抗糖尿病药,例如醋磺环已脲、氯磺丙脲、格列本脲、格列齐特、格列吡嗪、甲福明、妥拉磺脲、格列本脲、格列美脲和甲苯磺丁脲。
治疗活性剂可包括,例如,抗癫痫剂,例如氯丙酰苄胺、卡马西平、加巴喷丁、噻加宾、氨己烯酸、托吡酯、氯安定、乙苯妥英、美芬妥因、甲琥胺、甲苯比妥、奧卡西平、甲乙双酮、苯乙酰脲、苯巴比妥、苯妥英、密朗丁、扑米酮、舒布硫胺、苯妥英钠、呋喃妥因一水合物、加巴喷丁、拉莫三嗪、唑尼沙胺、乙琥胺和丙戊酸。
治疗活性剂可包括,例如,催眠/镇静药和/或肌肉松弛剂,例如酒石酸唑吡坦、异戊巴比妥、巴比妥、丁巴比妥、戊巴比妥、溴替唑仑、乙溴酰脲、氯氮卓、氯美噻唑、瓦尔米、甲丙氨酯、美他沙酮、环苯扎林、环苯扎林、替扎尼定、巴氯芬、异丁烯丙巴比妥、佐匹克隆、阿曲库铵、筒箭毒碱和苯巴比妥。
治疗活性剂可包括,例如,抗真菌药、抗原虫药或抗寄生虫药,例如:两性霉素、硝酸布康唑、克霉唑、硝酸益康唑、氟康唑、氟胞嘧啶、灰黄霉素、伊曲康唑、酮康唑、咪康唑、纳他霉素、制霉菌素、硝酸硫康唑、曲康唑、噻康唑和十一烯酸;苄硝唑、氯碘羟喹、癸氧喹酯、双碘喹啉、糠酸二氯尼特、二硝托胺、呋喃唑酮、甲硝唑、尼莫拉唑、呋喃西林、奥硝唑、特比奈芬、克霉唑、氯喹、甲氟喹、乙胺嘧啶、吡喹酮、疟疾平、甲苯达唑和替硝唑。
治疗活性剂可包括,例如,抗高血压剂或心脏病药,例如坎地沙坦、肼屈嗪、可乐定、氨苯蝶啶、非洛地平、吉非罗齐、非诺贝特、硝苯地平、哌唑嗪、美加明、多沙唑嗪、多巴酚丁胺和坎地沙坦(cilexetil)。
治疗活性剂可包括,例如,抗偏头痛药,例如甲磺酸二氢麦角胺、酒石酸麦角胺、马来酸美西麦角、马来酸苯噻啶和琥珀酸舒马曲坦。
治疗活性剂可包括,例如,抗毒蕈碱剂,例如阿托品、苯海索、比哌立登、普罗吩胺、莨菪碱、溴美喷酯、奥昔布宁、盐酸奥西克利平和托吡卡胺。
治疗活性剂可包括,例如,抗肿瘤剂(或免疫抑制剂),例如氨鲁米特、安吖啶、硫唑嘌呤、白消安、苯丁酸氮芥、环孢素、达卡巴嗪、雌莫司汀、依托泊苷、洛莫司汀、美法仑、巯嘌呤、甲氨蝶呤、丝裂霉素、米托坦、米托蒽醌、甲基苄肼、枸橼酸他莫昔芬、睾内酯、他克莫司、巯嘌呤和西罗莫司。
治疗活性剂可包括,例如,抗帕金森剂,例如甲磺酸溴隐亭、左旋多巴、托卡朋、罗匹尼罗、溴隐亭,降血糖剂,例如磺酰脲类双胍、α-葡糖苷酶抑制剂、噻唑烷二酮、卡麦角林、卡比多巴和马来酸麦角乙脲。
治疗活性剂可包括,例如,抗甲状腺剂如比马唑和丙硫氧嘧啶。
治疗活性剂可包括,例如,强心药如氨力农、米力农、洋地黄毒甙、依诺昔酮、毛花苷C和甲地高辛。
治疗活化剂可包括,例如,低血脂症或高血脂症药如非诺贝特、安妥明、普罗布考、依折麦布和托彻普,等。
治疗活性剂可包括,例如,抗炎剂如美洛昔康、去炎松、色甘酸、奈多罗米、硫酸羟基氯喹、孟鲁司特、齐留通、和扎鲁司特。
治疗活性剂可包括,例如,抗组胺药如非索非那定、水合氯醛、羟嗪、异丙嗪、西替利嗪、西咪替丁、赛克利嗪、美克洛嗪、茶苯海明、氯雷他定、和尼扎替丁。
治疗活性剂可包括,例如,抗溃疡药如奧美拉唑、兰索拉唑、潘妥拉唑和雷尼替丁。
治疗活化剂可包括,例如,利尿剂如氢氯噻嗪、阿米洛利、乙酰唑胺、呋塞米和托拉塞米。
治疗活性剂可包括维甲酸类,例如:第一代维甲酸类如视黄醇、视黄醛、维A酸(视黄酸(retinoic acid),retin-A)、异维A酸和阿利维A酸;第二代维甲酸类如依曲替酯和阿维A作为其代谢产物;和第三代维甲酸类如他扎罗汀、贝沙罗汀和阿达帕林。
治疗活性剂可包括,例如,他汀类及其衍生物,如阿托伐他汀、氟伐他汀、洛伐他汀、制霉菌素、瑞舒伐他汀、普伐他汀、奧利司他和辛伐他汀。
治疗活性剂可包括,例如,刺激剂如安非他命、芬特明、酪胺、麻黄碱、间羟胺、去氧肾上腺素、右旋安非他命、右芬氟拉明、氟苯丙胺、尼古丁、咖啡因和马吲哚。
治疗活性剂可包括,例如,血管舒张药如卡维地洛、特拉唑嗪、酚妥拉明和薄荷醇。
治疗活性剂可包括,例如,抗阿尔茨海默病药如左乙拉西坦、左乙拉西坦和多奈哌齐。
治疗活性剂可包括,例如,ACE抑制剂如贝那普利、依那普利、雷米普利、福辛普利钠、赖诺普利、米诺地尔、异山梨酯、雷米普利和喹那普。
治疗活性剂可包括,例如,β-肾上腺素受体拮抗剂如阿替洛尔、噻吗洛尔、吲哚洛尔、盐酸普萘洛尔、比索洛尔、艾司洛尔、琥珀酸美托洛尔、美托洛尔和酒石酸美托洛尔。
治疗活性剂可包括,例如,血管紧张素II拮抗剂如氯沙坦。
治疗活性剂可包括,例如,血小板抑制剂如阿昔单抗、氯吡格雷、替罗非班和阿司匹林。
治疗活性剂可包括,例如,醇类或酚类,如曲马多、盐酸曲马多、别嘌醇、骨化三醇、西洛他唑、索他洛尔、熊去氧胆酸溴哌醇、氟哌利多、氟哌噻吨癸酸酯、沙丁胺醇、硫酸沙丁胺醇、卡利普多、氯倍他索、罗匹尼罗、拉贝洛尔和美索巴莫。
治疗活性剂可包括,例如,酮类或酯类如胺碘酮、氟替卡松、螺内酯、泼尼松、曲唑酮、去羟米松、甲基泼尼松、苯佐那酯萘丁美酮和丁螺环酮。
治疗活性剂可包括,例如,抗呕吐药如甲氧氯普胺。
治疗活性剂可包括,例如,眼科治疗药如多佐胺、溴莫尼定、奥洛他定、环喷托酯、匹罗卡品和碘依可酯。
治疗活性剂可包括,例如,抗凝血剂或抗血栓剂如华法林、依诺肝素、和来匹卢定。
治疗活性剂可包括,例如,痛风治疗药如丙磺舒和磺吡酮。
治疗活性剂可包括,例如,COPD或哮喘治疗药如异丙托溴铵。
治疗活性剂可包括,例如,骨质疏松症治疗药如雷洛昔芬、帕米膦酸钠和利塞膦酸。
治疗活性剂可包括,例如,用于化妆品的肽,如乙酰基六肽-3、乙酰基六肽-8、乙酰基八肽和1-肌肽。
治疗活性剂可包括,例如:包含类毒素(灭活的有毒化合物)的疫苗;蛋白质、蛋白质亚基和多肽;聚核苷酸如DNA和RNA;缀合物;包含皂苷、病毒体、无机和有机佐剂的疫苗,例如zostavax。
治疗活性剂可包括营养医学或美容医学活性物质,包括,例如:Q10(或泛醌)、泛醇或白藜芦醇;类胡萝卜素类如α、β或γ-胡萝卜素、番茄红素、叶黄素、玉米黄质和虾青素;植物养分如番茄红素、叶黄素、和硫代黄嘌呤;omega-3-脂肪酸包括亚油酸、缀合的亚油酸、二十二碳六烯酸(DHA)、二十碳五烯酸(EPA)和它们的甘油酯;油溶性维生素包括维生素D(D2、D3及其衍生物)、维生素E(α、β、γ、δ-生育酚或α、β、γ、δ-生育三稀酚)、维生素A(视黄醇、视黄醛、维A酸及其衍生物)、维生素K(K1、K2、K3、及其衍生物)、三癸酸/辛酸甘油酯、叶酸、铁、烟酸、单亚油酸甘油酯、omega-6脂肪酸、维生素F、硒、氰钴胺、芦荟、β-葡聚糖、红没药醇、山茶茶(绿茶)提取物、三癸酸/辛酸甘油酯、崩大碗(雷公根)提取物、橄榄油鲸蜡醇酯、叶绿素、甜橙油、椰油酰基脯氨酸、二辛基醚、月桂亚氨基二丙酸生育酚磷酸酯盐二钠(维生素E磷酸盐)、甘油、甘油单油酸酯、甘草提取物、榛子(金缕梅)提取物、乳酸、卵磷脂、叶黄素、澳洲坚果籽油、洋甘菊提取物、月见草油、橄榄叶提取物、米糠油、鳄梨油、何首乌根提取物、石榴固醇类、白藜芦醇、玫瑰油、檀香油、二氧化钛、维生素A棕榈酸酯、葡萄籽油、卤贝他索、腺苷、三磷酸腺苷、果酸、尿囊素、透明质酸及衍生物、isolutrol、传明酸、甘醇酸、精氨酸、抗坏血酸葡糖胺、抗坏血酸棕榈酸酯、水杨酸、鼠尾草酸、α-硫辛酸、γ-亚麻酸(GLA)、泛醇、维生素A丙酸酯、维生素A棕榈酸酯、呋喃甲基腺嘌呤、视黄醛、醣肽、艾地苯醌、二甲基乙醇胺(DMAE)、烟酰胺、β-葡聚糖、棕榈酰五肽-4、棕榈酰寡肽/四肽-7、依托考昔、神经酰胺、苯丙氨酸、葡醛内酯、左旋肉碱、羟磷灰石、棕榈酰三肽-3、磷胆碱(phoscholine)、氧化锌、α-红没药醇、丁香酚、水飞蓟宾、大豆异黄酮、梓醇、卡密松山金车源伪愈创木内酯、迷迭香酸、迷迭香酚、水杨酸类,例如,水杨甙、水杨醇和水杨酸,蒲公英甾醇、α-山莴苣醇、异山莴苣醇、蒲公英苷(taraxacoside)、神经酰胺、熊果苷、姜酚、姜烯酚、金丝桃素、弹力素、胶原及其的肽。
多孔二氧化硅颗粒
根据本发明的二氧化硅颗粒(多孔二氧化硅颗粒,pSP)是具有纳米级粒径的二氧化硅材料(SiO2)的颗粒。
根据本发明的二氧化硅纳米颗粒是具有纳米级孔的多孔颗粒。
根据本发明的多孔二氧化硅颗粒可在颗粒的外表面或孔的内部负载生物活性物质。
根据本发明的多孔二氧化硅颗粒是生物降解性的并且可在体内负载生物活性物质,并且当施用至体内时,可被生物降解来释放生物活性物质。
即,生物降解多孔二氧化硅颗粒来释放生物活性物质。具体地,根据本发明的多孔二氧化硅颗粒在体内逐步降解,使得负载的生物活性物质具有缓释特性。例如,在下式1中的吸光度的比达到1/2下的‘t’为24以上。
[式1]
At/A0
其中,A0表示在将5ml的含有1mg/ml的多孔二氧化硅颗粒的悬浮液置于具有50kDa直径的孔的圆筒状渗透膜中后测定的多孔二氧化硅颗粒的吸光度,
其中,15ml的与渗透膜接触并且与悬浮液实质上相同的溶液存在于渗透膜的外部,并且在37℃和60rpm下水平搅拌渗透膜的内部和外部,
悬浮液的pH为7.4,和
其中At表示在测定A0后‘t’小时测定的多孔二氧化硅颗粒的吸光度。
式1表示多孔二氧化硅颗粒在类似于体内生理条件的环境中降解的速率。
如图7中所示,可以通过将多孔二氧化硅颗粒和悬浮液置于圆筒状渗透膜中并且还将相同悬浮液注入渗透膜的外部,然后测定吸光度来确定式1中的吸光度A0和At。
本发明的多孔二氧化硅颗粒可为生物降解性的并且在悬浮液中缓慢降解。由于50kDa的直径近似对应于5nm,生物降解的多孔二氧化硅颗粒可穿过具有50kDa直径的渗透膜。在60rpm下水平搅拌圆筒状渗透膜使其均匀混合,同时降解的多孔二氧化硅颗粒可任选地通过渗透膜出来。
式1中的吸光度可以在,例如,用新的悬浮液替换渗透膜外部的悬浮液的环境中确定。可以在预定时间段内连续更换或替换悬浮液。在此,预定时间段可为定期或不定期的时间段。例如,可在以下范围中的预定间隔下替换悬浮液:1小时至1周、每小时、每2小时、每3小时、每6小时、每12小时、每24小时、每2天、3天、4天、7天等,但不限于这些。
吸光度的比达到1/2意味着‘t’时间后的吸光度变为初始吸光度的一半,并且因此表明已降解近似一半的多孔二氧化硅颗粒。
悬浮液可以是缓冲溶液,并且具体地,选自由磷酸盐缓冲生理盐水(PBS)和模拟体液(SBF)组成的组的至少一种或多种,并且更具体地,PBS。
根据本发明,其中在通过式1确定的吸光度的比达到1/2下的‘t’可为24以上,例如,‘t’的范围可为24至120,并且更具体地,在上述范围内的24至96、24至72、30至70、40至70、50至65等,但不限于此。
关于根据本发明的多孔二氧化硅颗粒,其中在通过式1确定的吸光度的比达到1/5下的‘t’,例如,范围可为70至140,并且更具体地,在上述范围内的80至140、80至120、80至110、70至140、70至120、70至110等,但不限于此。
关于根据本发明的多孔二氧化硅颗粒,其中在通过式1确定的吸光度的比达到1/20下的‘t’,例如,范围可为130至220,并且更具体地,在上述范围内的130至200、140至200、140至180、150至180等,但不限于此。
此外,关于根据本发明的多孔二氧化硅颗粒,其中在通过式1确定的吸光度的比变为0.01以下下的‘t’,例如,可为250以上,并且更具体地,300以上、350以上、400以上、500以上、1000以上等,并且可进一步具有2000的上限,但不限于此。
此外,关于根据本发明的多孔二氧化硅颗粒,其中通过式1确定的吸光度的比和‘t’具有高水平的正相关。例如,皮尔森相关系数可为0.8以上,例如,0.9以上、和0.95以上。
式1中的‘t’表示多孔二氧化硅颗粒在类似于体内生理条件的环境中降解的比率,并且可通过调节例如多孔二氧化硅颗粒的表面积、粒径和孔径、颗粒表面和/或孔内部的取代基、表面的致密性等来控制。
例如,可以通过增加颗粒的表面积来减少t或通过减少其来增加t。可通过调整颗粒的直径、或孔的直径来控制表面积。此外,取代基位于颗粒的表面和/或孔的内部可减少多孔二氧化硅颗粒对环境(溶剂等)的直接暴露,从而增加t。此外,可通过在多孔二氧化硅颗粒上负载生物活性物质和增加生物活性物质和多孔二氧化硅颗粒之间的亲和性来减少多孔二氧化硅颗粒对环境的直接暴露,从而增加‘t’。此外,可通过在颗粒制备期间更密集地形成颗粒的表面来增加‘t’。在上文中,已描述了控制式1中‘t’的各种实例,但不限于此。
根据本发明所述的多孔二氧化硅颗粒可为球状颗粒,但不限于此。
根据本发明所述的多孔二氧化硅颗粒可具有例如150nm至1000的平均直径,更具体地,在上述范围内的150nm至800nm、150nm至500nm、150nm至400nm、150nm至300nm或150nm至200,但不限于此。
根据本发明所述的多孔二氧化硅颗粒可具有例如1nm至100nm的平均孔径,更具体地,在上述范围内的5nm至100nm、7nm至100nm、7nm至50nm、10nm至50nm、10nm至30nm或7nm至30nm,但不限于此。如果具有如上所述的大直径,可负载大量的生物活性物质,甚至还可负载具有大尺寸的生物活性物质。
根据本发明所述的多孔二氧化硅颗粒可具有例如200m2/g至700m2/g的BET表面积。例如,BET表面积的范围可为在上述范围内的200m2/g至700m2/g、200m2/g至650m2/g、250m2/g至650m2/g、300m2/g至700m2/g、300m2/g至650m2/g、300m2/g至600m2/g、300m2/g至550m2/g、300m2/g至500m2/g、或300m2/g至450m2/g等,但不限于此。
根据本发明所述的多孔二氧化硅颗粒可具有例如0.7ml至2.2ml的每克的体积(体积/g)。例如,体积/g的范围可为上述范围内的0.7ml至2.0ml、0.8ml至2.2ml、0.8ml至2.0ml、0.9ml至2.0ml、1.0ml至2.0ml,但不限于此。如果体积/g过小,则降解率过高。此外,过大的颗粒是难以制备的或不具有完整形状。
根据本发明所述的多孔二氧化硅颗粒在颗粒的外表面和/或孔的内部可具有亲水性取代基和/或疏水性取代基。例如,仅亲水性取代基或仅疏水性取代基可存在于表面和孔的内部两者。另外,亲水性取代基或疏水性取代基可存在于颗粒的表面或孔的内部。可选地,亲水性取代基可存在于颗粒的表面上,同时疏水性取代基存在于孔的内部,反之亦然。
生物活性物质通常通过降解纳米颗粒来释放。因此,多孔二氧化硅颗粒与生物活性物质释放环境的相互作用可通过调整取代基来控制,其反过来可调整纳米颗粒自身的降解率,从而控制生物活性物质的释放率。此外,生物活性物质可从纳米颗粒中扩散并释放,其中生物活性物质对纳米颗粒的结合力可通过调整取代基来调节,从而控制生物活性物质的释放。
此外,为了改善对不溶性(疏水性)生物活性物质的结合力,考虑到易用性和制剂化,可以进行一些操作使得疏水性取代基存在于孔的内部同时在颗粒的表面赋予亲水性取代基。
亲水性取代基可包括,例如,羟基、羧基、氨基、羰基、巯基、磷酸酯基、硫醇基、铵基(ammonium)、酯基、酰亚胺基、硫代酰亚胺基、酮基、醚基、茚基、磺酰基、或聚乙二醇基等。此外,疏水性取代基可包括,例如,取代或未取代的C1至C30烷基、取代或未取代的C3至C30环烷基、取代或未取代的C6至C30芳基、取代或未取代的C2至C30杂芳基、卤素、C1至C30酯、含卤素的基团等。
此外,根据本发明所述的多孔二氧化硅颗粒可在颗粒的外表面和/或孔的内部带正电或带负电。例如,颗粒的外表面和孔的内部都可带正电或带负电。此外,颗粒的外表面或孔的内部可带正电或带负电。另外,颗粒的外表面可带正电,而孔的内部可带负电,并且反之亦然。
可通过阳离子取代基或阴离子取代基的存在来带电。
阳离子取代基可包括,例如,碱性基团如氨基或含氮基团,同时阴离子取代基可包括,例如,酸性基团如羧基(-COOH)、磺酸基(-SO3H)、巯基(-SH)等,但不限于此。
同样,多孔二氧化硅颗粒对生物活性物质释放环境的相互作用可通过带电来控制,其反过来可调节纳米颗粒的降解率,从而控制生物活性物质的释放率。可选地,生物活性物质可从纳米颗粒中扩散并释放,当调整取代基时,可调整生物活性物质对纳米颗粒的结合力,从而控制生物活性物质的释放。
此外,根据本发明的多孔二氧化硅颗粒可含有取代基,除上述目标外,所述取代基用于在颗粒的表面和/或孔的内部负载生物活性物质、将生物活性物质递送至靶细胞、负载用于其它用途的材料、或结合其它另外的取代基。另外,多孔二氧化硅颗粒可进一步包括与取代基结合的抗体、配体、细胞渗透肽或适体。
可例如通过表面修饰来添加上述取代基、电荷、结合材料等。
表面修饰可以例如通过将含有待引入的取代基的化合物与颗粒反应来进行,其中所述化合物可以是,例如,具有C1至C10烷氧基的烷氧基硅烷,但不限于此。烷氧基硅烷具有1个以上的烷氧基,例如,1至3个烷氧基。可选地,烷氧基硅烷可在烷氧基未键合的位点包括待引入的取代基、或由上述取代基取代的另一取代基。
多孔二氧化硅颗粒的制备
根据本发明所述的多孔二氧化硅颗粒可通过,例如,用于制备具有小孔的颗粒和用于孔扩张(pore expansion)的工艺来形成。必要时,可通过煅烧、和表面修饰等进一步的工艺来形成颗粒。当煅烧和表面修饰的工艺都已进行时,可在煅烧后表面修饰颗粒。
具有小孔的颗粒可以具有例如1至5nm的平均孔径。
具有小孔的颗粒可以通过添加表面活性剂和二氧化硅前体至溶剂中、接着搅拌并均质化来得到。
本文使用的溶剂可以是水和/或有机溶剂。有机溶剂可包括,例如:醚类(具体地,环醚类)如1,4-二噁烷等;卤代烃类如氯仿、氯甲烷、四氧化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷等;酮类如丙酮、甲基异丁酮、γ-丁内酯、1,3-二甲基-咪唑啉酮、甲基乙基酮、环己酮、环戊酮、4-羟基-4-甲基-2-戊酮等;芳香烃类如苯、甲苯、二甲苯、四甲基苯等;烷基酰胺类如N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等;醇类如甲醇、乙醇、丙醇、丁醇等;乙二醇醚类(溶纤剂)如乙二醇单乙醚、乙二醇单甲醚、乙二醇单丁醚、二乙二醇单乙醚、二乙二醇单甲醚、二乙二醇单丁醚、丙二醇单甲醚、丙二醇单乙醚、二丙二醇二乙醚、三乙二醇单乙醚等;其它化合物包括,例如,甲基乙酰胺(DMAc)、N,N-二乙基乙酰胺、二甲基甲酰胺(DMF)、二乙基甲酰胺(DEF)、N,N-二甲基乙酰胺(DMAc)、N-甲基吡咯烷酮(NMP)、N-乙基吡咯烷酮(NEP)、1,3-二甲基-2-咪唑啉酮、N,N-二甲基甲氧基乙酰胺、二甲基亚砜、吡啶、二甲基砜、六甲基磷酰胺、四甲基脲、N-甲基己内酰胺、四氢呋喃、间二噁烷、对二噁烷、或1,2-二甲氧基乙烷等。可优选使用醇,更优选地甲醇,但不限于此。
当使用水和有机溶剂的混合溶剂时,它们之间的相对比例可以是体积比1:0.7至1.5,例如,1:0.8至1.3,但不限于此。
表面活性剂可包括,例如,十六烷基三甲基溴化铵(CTAB)、十六烷基三甲基溴化铵(TMABr)、十六烷基三甲基氯化吡啶(TMPrCl)、或四甲基氯化铵(TMACl)等。优选地,使用CTAB。
表面活性剂可以以1至10g对1升溶剂的量加入,例如,在上述范围内的1至8g、2至8g、或3至8g等,但不限于此。
二氧化硅前体可在添加表面活性剂后添加至溶剂中并且将其搅拌。二氧化硅前体可包括,例如,正硅酸四甲酯(TMOS),但不限于此。
例如,搅拌可以进行10至30分钟,但不限于此。
二氧化硅前体可以以0.5至5ml对1升溶剂的量添加,例如,在上述范围内的0.5至4ml、0.5至3ml、0.5至2ml、或1至2ml等,但不限于此。
必要时,可进一步添加氢氧化钠作为催化剂。在这种情况下,可以在添加表面活性剂之后和在添加二氧化硅前体至溶剂中之前添加该化合物同时搅拌。
氢氧化钠可以以1M氢氧化钠溶液为基准以0.5至8ml对1升溶剂的量来添加,例如,在上述范围内的0.5至5ml、0.5至4ml、1至4ml、1至3ml、或2至3ml等,但不限于此。
在添加二氧化硅前体后,溶液可在搅拌下进行反应。搅拌可进行2至15小时,例如,在上述范围内的3至15小时、4至15小时、4至13小时、5至12小时、6至12小时、或6至10小时等,但不限于此。如果搅拌时间(反应时间)过短,可导致成核不充分。
搅拌后,可对溶液进行老化(aging)。老化可进行8至24小时,例如,在上述范围内的8至20小时、8至18小时、8至16小时、8至14小时、10至16小时、或10至14小时等,但不限于此。
其后,可以清洗并干燥反应产物来制备多孔二氧化硅颗粒。
必要时,可在清洗前去除未反应的材料。
例如,可通过离心分离上清液来进行这样的除去。
离心可在6,000至10,000rpm下进行3至60分钟,例如,在上述范围内的3至30分钟、4至30分钟、或5至30分钟等,但不限于此。
清洗可用水和/或有机溶剂进行。更具体地,由于不同类型的材料溶于不同溶剂中,水和有机溶剂可以轮流使用一次或多次。可选地,可单独使用水或有机溶剂清洗一次或多次。多次可以是2次以上且10次以下。优选地,3次以上且10次以下、4次以上且8次以下、或4次以上且6次以下等。
有机溶剂可包括,例如:醚类(具体地,环醚类)如1,4-二噁烷等;卤代烃类如氯仿、氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷等;酮类如丙酮、甲基异丁酮、γ-丁内酯、1,3-二甲基-咪唑啉酮、甲基乙基酮、环己酮、环戊酮、4-羟基-4-甲基-2-戊酮等;芳香烃类如苯、甲苯、二甲苯、四甲基苯等;烷基酰胺类如N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等;醇类如甲醇、乙醇、丙醇、丁醇等;乙二醇醚类(溶纤剂)如乙二醇单乙醚、乙二醇单甲基醚、乙二醇单丁醚、二乙二醇单乙醚、二乙二醇单甲醚、二乙二醇单丁醚、丙二醇单甲醚、丙二醇单乙醚、二丙二醇二乙醚、三乙二醇单乙醚等;其它化合物包括,例如,甲基乙酰胺(DMAc)、N,N-二乙基乙酰胺、二甲基甲酰胺(DMF)、二乙基甲酰胺(DEF)、N,N-二甲基乙酰胺(DMAc)、N-甲基吡咯烷酮(NMP)、N-乙基吡咯烷酮(NEP)、1,3-二甲基-2-咪唑啉酮、N,N-二甲基甲氧基乙酰胺、二甲基亚砜、吡啶、二甲基砜、六甲基磷酰胺、四甲基脲、N-甲基己内酰胺、四氢呋喃、间二噁烷、对二噁烷、或1,2-二甲氧基乙烷等。可优选使用醇,更优选乙醇,但不限于此。
可在例如6,000至10,000rpm的离心下进行清洗。清洗可进行3至60分钟,在上述范围内的3至30分钟、4至30分钟、或5至30分钟等,但不限于此。
可选地,可以在通过过滤器无离心地过滤颗粒的同时进行清洗。本文使用的过滤器可设置有具有等于或小于多孔二氧化硅颗粒直径的孔径的孔。通过过滤反应溶液,仅颗粒保留在过滤器中,并且可通过将水和/或有机溶剂注入过滤器中来清洗。
清洗期间,水和有机溶剂可轮流使用一次或多次。另外,可单独使用水或有机溶剂清洗一次或多次。多次可以是2次以上且10次以下,优选地,3次以上且10次以下、4次以上且8次以下、或4次以上且6次以下等。
干燥可在例如20℃至100℃下进行,但不限于此。可选地,干燥可在真空条件下进行。
其后,可对得到的多孔二氧化硅颗粒进行孔扩张。
可使用孔扩张剂来进行孔扩张。
本文使用的孔扩张剂可包括,例如,三甲基苯、三乙基苯、三丙基苯、三丁基苯、三戊基苯、三己基苯、甲苯、或苯等。优选地,可使用三甲基苯,但不限于此。
此外,本文使用的孔扩张剂可包括,例如,N,N-二甲基十六烷基胺(DMHA),但不限于此。
可通过,例如,将溶剂中的多孔二氧化硅颗粒与孔扩张剂混合并且加热以进行反应来进行孔扩张。
本文使用的溶剂可包括水和/或有机溶剂。有机溶剂可包括,例如:醚类(具体地,环醚类)如1,4-二噁烷等;卤代烃类如氯仿、氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷等;酮类如丙酮、甲基异丁酮、环己酮等;芳香烃类如苯、甲苯、二甲苯等;烷基酰胺类如N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等;醇类如甲醇、乙醇、丙醇、丁醇等。优选使用醇,更优选乙醇,但不限于此。
多孔二氧化硅颗粒可以以10至200g对1升溶剂的量添加,例如,在上述范围内的10至150g、10至100g、30至100g、40至100g、50至100g、50至80g、或60至80g等,但不限于此。
多孔二氧化硅颗粒可均匀分散在溶剂中。例如,可以将多孔二氧化硅颗粒添加至溶剂中,接着超声分散。当使用混合溶剂时,多孔二氧化硅颗粒可分散在第一溶剂中,接着将其添加至第二溶剂中。
孔扩张剂可以以10至200体积份(‘vol.parts’)对100体积份的溶剂的量来添加,例如,在上述范围内的10至150体积份、10至100体积份、10至80体积份、30至80体积份、或30至70体积份等,但不限于此。
反应可在120至180℃下进行,例如,在上述范围内的120至170℃、120至160℃、120至150℃、130至180℃、130至170℃、130至160℃、或130至150℃等,但不限于此。
反应可进行24至96小时,例如,在上述范围内的30至96小时、30至96小时、30至90小时、30至80小时、30至72小时、24至80小时、24至72小时、36至96小时、36至80小时、36至72小时、36至66小时、36至60小时、48至96小时、48至88小时、48至80小时、或48至72小时等,但不限于此。
通过在上述示例范围内调节时间和温度,可充分但不过度地进行反应。例如,随着反应温度降低,反应可随反应时间的增加而进行。另一方面,当反应温度降低时,反应时间可降低。如果反应进行不充分,孔扩张也会是不充分的。另一方面,如果过度进行反应,颗粒会由于孔的过度扩张而崩解。
可在例如逐步提高反应温度的同时进行反应。更具体地,可通过从室温以0.5℃/分钟至15℃/分钟的速率逐步提高温度至上述温度来进行反应,例如,在上述范围内的1℃/分钟至15℃/分钟、3℃/分钟至15℃/分钟、3℃/分钟至12℃/分钟、或3℃/分钟至10℃/分钟等,但不限于此。
反应后,可逐渐冷却反应溶液,例如,可逐步降低温度来冷却反应溶液。具体地,可通过将上述温度以0.5℃/分钟至20℃/分钟的速率逐步降低至室温来进行冷却,例如,在上述范围内的1℃/分钟至20℃/分钟、3℃/分钟至20℃/分钟、3℃/分钟至12℃/分钟、或3℃/分钟至10℃/分钟等,但不限于此。
冷却后,可对反应产物进行清洗和干燥来制备具有扩张的孔的多孔二氧化硅颗粒。
必要时,可在清洗前去除未反应的材料。
例如,可通过离心分离上清液来进行这样的除去。
离心可以在6,000至10,000rpm下进行。此外,离心可进行3至60分钟,例如,在上述范围内的3至30分钟、4至30分钟、或5至30分钟等,但不限于此。
清洗可用水和/或有机溶剂进行。更具体地,由于不同类型的材料溶于不同溶剂中,水和有机溶剂可以轮流使用一次或多次。可选地,可单独使用水或有机溶剂清洗一次或多次。多次可以是2至10次,例如,3次、4次、5次、6次、7次、或8次等。
有机溶剂可包括,例如,醚类(具体地,环醚类)如1,4-二噁烷等;卤代烃类如氯仿、氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷等;酮类如丙酮、甲基异丁酮、环己酮等;芳香烃类如苯、甲苯、二甲苯等;烷基酰胺类如N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等;醇类如甲醇、乙醇、丙醇、丁醇等。优选使用醇,更优选乙醇,但不限于此。
可在6,000至10,000rpm的离心下进行清洗。此外,清洗可以进行3至60分钟,例如,在上述范围内的3至30分钟、4至30分钟、或5至30分钟等,但不限于此。
还可通过无离心地过滤颗粒来进行清洗。过滤器可具有等于或小于多孔二氧化硅颗粒直径的孔径。通过过滤反应溶液,仅颗粒保留在过滤器中,并且通过将水和/或有机溶剂注入过滤器中来清洗颗粒。
水和有机溶剂在清洗期间可轮流使用一次或多次。另外,可单独使用水或有机溶剂清洗一次或多次。多次可以是2次以上且10次以下,优选地,3次以上且10次以下、4次以上且8次以下、或4次以上且6次以下等。
可在20至100℃下进行干燥,但不限于此。此外,还可在真空条件下进行干燥。
其后,可对得到的颗粒进行煅烧。
煅烧是加热颗粒来赋予颗粒的表面和内部更致密的结构同时去除填充在颗粒的孔的内部的任何有机物质的工艺。煅烧例如在400℃至700℃下进行3小时至8小时,具体地,在500℃至600℃下进行4小时至5小时,但不限于此。
其后,可对得到的颗粒进行表面修饰。
可在表面上和/或孔的内部进行表面修饰。颗粒的表面和颗粒的孔的内部可以以彼此相同的方式或不同的方式进行修饰。
修饰可使得颗粒带电或具有亲水性和/或疏水性。可以通过将具有待引入的亲水性、疏水性、阳离子性或阴离子性取代基的化合物与颗粒反应来进行修饰,但不限于此。上述化合物可以是,例如,具有C1至C10烷氧基的烷氧基硅烷,但不限于此。烷氧基硅烷可具有至少一个烷氧基,例如,1至3个烷氧基并且在烷氧基未键合的位点具有待引入的取代基或由上述取代基取代的其它取代基。
烷氧基硅烷与多孔二氧化硅颗粒反应可形成硅原子和氧原子之间的共价键,因此使得烷氧基硅烷连接至多孔二氧化硅颗粒的表面或颗粒的孔的内部。此外,由于烷氧基硅烷具有待引入的取代基,所述取代基可引入多孔二氧化硅颗粒的表面或颗粒的孔的内部。
上述反应可通过将分散在溶剂中的多孔二氧化硅颗粒与烷氧基硅烷反应来进行。
使用的溶剂可以是水和/或有机溶剂。该有机溶剂可包括,例如:醚类(具体地,环醚类)如1,4-二噁烷等;卤代烃类如氯仿、氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷等;酮类如丙酮、甲基异丁酮、γ-丁内酯、1,3-二甲基-咪唑啉酮、甲基乙基酮、环己酮、环戊酮、4-羟基-4-甲基-2-戊酮等;芳香烃类如苯、甲苯、二甲苯、四甲基苯等;烷基酰胺类如N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等;醇类如甲醇、乙醇、丙醇、丁醇等;乙二醇醚类(溶纤剂)如乙二醇单乙醚、乙二醇单甲基醚、乙二醇单丁醚、二乙二醇单乙醚、二乙二醇单甲醚、二乙二醇单丁醚、丙二醇单甲醚、丙二醇单乙醚、二丙二醇二乙醚、三乙二醇单乙醚等;其它化合物包括,例如,甲基乙酰胺(DMAc)、N,N-二乙基乙酰胺、二甲基甲酰胺(DMF)、二乙基甲酰胺(DEF)、N,N-二甲基乙酰胺(DMAc)、N-甲基吡咯烷酮(NMP)、N-乙基吡咯烷酮(NEP)、1,3-二甲基-2-咪唑啉酮、N,N-二甲基甲氧基乙酰胺、二甲基亚砜、吡啶、二甲基砜、六甲基磷酰胺、四甲基脲、N-甲基己内酰胺、四氢呋喃、间二噁烷、对二噁烷、或1,2-二甲氧基乙烷等,具体地,可使用甲苯,但不限于此。
例如,对阳离子取代基的修饰可通过将颗粒与具有碱性基团、即含氮基团如氨基或氨基烷基的烷氧基硅烷反应来进行。更具体地,可使用N-[3-(三甲氧基甲硅烷基)丙基]乙二胺、N1-(3-三甲氧基甲硅烷基丙基)二乙基乙二胺、(3-氨基丙基)三甲氧基硅烷、N-[3-(三甲氧基甲硅烷基)丙基]苯胺、三甲氧基[3-(甲基氨基)丙基]硅烷、和3-(2-氨基乙基氨基)丙基二甲氧基甲基硅烷等,但不限于此。
例如,对阴离子取代基的修饰可通过将颗粒与具有酸性基团如羧基、磺酸基、或硫醇基的烷氧基硅烷反应来进行。更具体地,可使用(3-巯基丙基)三甲氧基硅烷等,但不限于此。
亲水性可通过将颗粒与具有亲水性官能团的烷氧基硅烷反应来引入,所述亲水性官能团例如,羟基、羧基、氨基、羰基、巯基、磷酸基、硫醇基、铵基、酯基、酰亚胺基、硫代酰胺基、酮基、醚基、茚基、磺酰基、或聚乙二醇基等。更具体地,可使用N-[3-(三甲氧基甲硅烷基)丙基]乙二胺、N1-(3-三甲氧基甲硅烷基丙基)二亚乙基三胺、(3-氨基丙基)三甲氧基硅烷、(3-巯基丙基)三甲氧基硅烷、三甲氧基[3-(甲基氨基)丙基]硅烷、和3-(2-氨基乙基氨基)丙基二甲氧基甲基硅烷等,但不限于此。
疏水性可通过将颗粒与具有疏水性官能团的烷氧基硅烷反应来引入,疏水性官能团例如取代或未取代的C1至C30烷基、取代或未取代的C3至C30环烷基、取代或未取代的C6至C30芳基、取代或未取代的C2至C30杂芳基、卤素、C1至C30酯、或含卤素的基团。更具体地,可使用三甲氧基(十八烷基)硅烷、三甲氧基正辛基硅烷、三甲氧基(丙基)硅烷、异丁基(三甲氧基)硅烷、三甲氧基(7-辛烯-1-基)硅烷、三甲氧基(3,3,3-三氟丙基)硅烷、三甲氧基(2-苯乙基)硅烷、乙烯基三甲氧基硅烷、氰基甲基、3-(三甲氧基甲硅烷基)丙基]三硫代碳酸酯、和(3-溴丙基)三甲氧基硅烷等,但不限于此。
另外,在易用性和制剂化方面,为了增加颗粒对不可溶(疏水性)生物活性物质的结合力,可将疏水性取代基引入孔的内部并且可将亲水性取代基引入颗粒的表面上,并且可以通过修饰将用于结合除生物活性物质以外的其它物质的取代基引入表面上。
此外,修饰可以以组合方式进行。例如,可在颗粒的外表面或颗粒的孔的内部进行两次以上的表面修饰。在具体实例中,带正电的颗粒可通过将含有羧基的化合物通过酰胺键与具有氨基的二氧化硅颗粒结合来带电以具有不同表面特性,但不限于此。
可以在例如加热下进行颗粒与烷氧基硅烷的反应。
在这种情况下,加热可以在例如80℃至180℃下进行。例如,反应可以在上述范围内的80℃至160℃、80℃至150℃、100℃至160℃、100℃至150℃、或110℃至150℃等下进行,但不限于此。
此外,颗粒与烷氧基硅烷的反应可以进行例如4至20小时。例如,反应可以进行在上述范围内中的4至18小时、4至16小时、6至18小时、6至16小时、8至18小时、8至16小时、8至14小时、或10至14小时等,但不限于此。
关于上述修饰,反应温度、反应时间、用于修饰的化合物的量等可根据修饰程度来适当选择。更具体地,在基于生物活性物质的亲水性、疏水性和/或电荷水平的不同反应条件下,可调节多孔二氧化硅颗粒的亲水性、疏水性和/或电荷水平,从而控制生物活性物质的释放速率。例如,一旦生物活性物质在中性pH下高度带负电,可增加反应温度或反应时间或可增加处理的化合物的量使得多孔二氧化硅颗粒高度带正电,但不限于此。
关于本发明的组合物,多孔二氧化硅颗粒可通过,例如,用于制备具有小孔的颗粒、孔扩张、表面修饰和孔的内部修饰的方法来制备。
用于制备具有小孔的颗粒和用于孔扩张的工艺可以通过上述工艺进行。其后,可进行清洗和干燥。
必要时,可在清洗前去除未反应的材料。
例如,可通过离心分离上清液来进行这样的除去。
离心可以在6,000至10,000rpm下进行3至60分钟,例如,在上述范围内的3至30分钟、4至30分钟、或5至30分钟等,但不限于此。
制备具有小孔的颗粒之后的清洗过程可通过所述方法/在上述示例范围内的条件下进行,但不限于此。
与上述示例方面相比,孔扩张后的清洗过程可在更缓和的条件下进行。例如,清洗可进行3次以下,但不限于此。
可通过上述方法进行颗粒的表面和/或颗粒的孔的内部的修饰。颗粒的表面修饰和然后颗粒的孔的内部修饰可以以该顺序顺次进行。可选地,可在上述两个过程之间进一步进行颗粒的清洗过程。
当在制备具有小孔的颗粒和孔扩张之后在更缓和的条件下清洗时,将反应溶液例如用于颗粒制备和/或孔扩张的表面活性剂填充在孔的内部。因此,在表面修饰期间孔的内部不修饰,反之,可仅修饰表面。其后,孔的内部的反应溶液可通过清洗颗粒来去除。
可使用水和/或有机溶剂进行表面修饰和孔的内部修饰之间的颗粒清洗。更具体地,由于不同种类的材料溶于不同溶剂中,水和有机溶剂可轮流使用一次或多次。可选地,可单独使用水或有机溶剂清洗一次或多次。多次可以是2次以上且10次以下,优选地,3次以上且10次以下、4次以上且8次以下、或4次以上且6次以下等。
清洗可在离心下进行。离心可以在6,000至10,000rpm下进行3至60分钟,例如,在上述范围内的3至30分钟、4至30分钟、或5至30分钟等,但不限于此。
可选地,可以在通过过滤器无离心地过滤颗粒的同时进行清洗。本文使用的过滤器可含有具有等于或小于多孔二氧化硅颗粒直径的孔径。通过过滤反应溶液,仅颗粒保留在过滤器中,并且可通过将水和/或有机溶剂注入过滤器中来清洗。
在清洗期间,水和有机溶剂可轮流使用一次或多次。可选地,可单独使用水或有机溶剂清洗一次或多次。多次可以是2次以上且10次以下,优选地,3次以上且10次以下、4次以上且8次以下、或4次以上且6次以下等。
干燥可以在,例如,20℃至100℃下进行,但不限于此。可选地,干燥可以在真空条件下进行。
生物活性物质的负载
可在颗粒的表面和/或孔的内部负载生物活性物质。
例如,可通过在溶剂中将多孔二氧化硅颗粒与生物活性物质混合来进行负载。
使用的溶剂可以是水和/或有机溶剂,并且有机溶剂可包括,例如:醚类(具体地,环醚类)如1,4-二噁烷等;卤代烃类如氯仿、氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷等;酮类如丙酮、甲基异丁酮、环己酮等;芳香烃类如苯、甲苯、二甲苯、四甲基苯等;烷基酰胺类如N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮等;醇类如甲醇、乙醇、丙醇、丁醇等。
本文使用的溶剂可进一步包括磷酸盐缓冲生理盐水溶液(PBS)、模拟体液(SBF)、硼酸盐缓冲生理盐水、或tris缓冲生理盐水等。
多孔二氧化硅颗粒和生物活性物质的相对比例没有特别限制,但可以为重量比1:0.05至0.8,例如,在上述范围内的1:0.05至0.7、1:0.05至0.6、1:0.1至0.8、1:0.1至0.6、1:0.2至0.8、或1:0.2至0.6等。
生物活性物质的释放
多孔二氧化硅颗粒可以长时间逐渐释放其中携带的生物活性物质。
颗粒中携带的生物活性物质可被生物降解并且释放。在这种情况下,可以缓慢降解颗粒来缓释携带的生物活性物质。例如,可以通过调整多孔二氧化硅颗粒的表面积、粒径和/或孔径、调节颗粒表面和/或颗粒的孔的内部的取代基、或表面致密性等来控制该释放,但不限于此。
此外,颗粒中携带的生物活性物质可从多孔二氧化硅颗粒中逃脱并且还在扩散的同时释放。多孔二氧化硅颗粒和生物活性物质、或生物活性物质的释放环境等之间的关系可影响该过程。因此,生物活性物质的释放可通过调节上述条件来控制。例如,生物活性物质的释放可通过表面修饰加强或减弱多孔二氧化硅颗粒和生物活性物质之间的结合力来控制。
根据更优选的实例,当携带的生物活性物质为难溶性(疏水性)时,颗粒的表面和/或颗粒的孔的内部可具有疏水性取代基,由此增加颗粒对生物活性物质的结合力,从而可以缓释生物活性物质。例如,可以用具有疏水性取代基的烷氧基硅烷表面修饰上述颗粒。
在本公开中,术语“难溶性”可包括“不溶”、“几乎不溶”或“仅微溶”于水等含义,其为"Pharmaceutical Science"第18版中定义的术语(由U.S.P.,Remington,MackPublishing Company出版)。
难溶性生物活性物质可具有,例如,在1atm和25℃下的小于10g/L的水溶性、优选小于5g/L、更优选小于1g/L,但不限于此。
当携带的生物活性物质为水溶性(亲水性),颗粒的表面或颗粒的孔的内部可具有亲水性取代基,由此增加颗粒对生物活性物质的结合力,从而可以缓释生物活性物质。例如,可以用具有亲水性取代基的烷氧基硅烷表面修饰多孔二氧化硅颗粒。
水溶性生物活性物质可具有,例如,在1atm和25℃下的10g/L以上的水溶性,但不限于此。
当携带的生物活性物质带电时,颗粒的表面或颗粒的孔的内部可以是抗衡带电的,由此增加多孔二氧化硅颗粒和生物活性物质之间的结合力,从而可以缓释生物活性物质。例如,可以用具有酸性基团或碱性基团的烷氧基硅烷表面修饰多孔二氧化硅颗粒。
更具体地,如果生物活性物质在中性pH下带正电,颗粒的表面和/或颗粒的孔的内部可在中性pH下带负电,由此增加多孔二氧化硅颗粒和生物活性物质之间的结合力,从而可以缓释生物活性物质。例如,可以用具有例如羧基(-COOH)、或磺酸基(-SO3H)等酸性基团的烷氧基硅烷表面修饰多孔二氧化硅颗粒。
此外,如果生物活性物质在中性pH下带负电,颗粒的表面和/或颗粒的孔的内部可在中性pH下带正电,由此增加多孔二氧化硅颗粒和生物活性物质之间的结合力,从而可以缓释生物活性物质。例如,可以用具有例如氨基、或其它含氮基团等碱性基团的烷氧基硅烷表面修饰多孔二氧化硅颗粒。
取决于释放环境、和用于携带生物活性物质的多孔二氧化硅颗粒等,携带的生物活性物质可以经,例如,7天至1年或更长的时间释放。
关于本发明的组合物,多孔二氧化硅颗粒可以是生物降解性并且可以完全降解至约100%,因此,本文携带的生物活性物质可100%释放。
生物活性物质载体的配制和施用
本发明的生物活性物质载体可配制成用于通过任何给药途径递送。“给药途径”可以包括气溶胶、鼻内、口服、转化粘液质、经皮肤、肠胃外或肠内途径,然而,还可以指本领域已知的任何给药,但不限于此。
本发明的多孔二氧化硅颗粒可以是生物降解性的并且降解高达100%,因此可具有优异的体内稳定性,从而肠胃外施用,以及制造成用于肠胃外施用的制剂。
“肠胃外(非口服)”途径是指通常与注射相关的给药途径,其包括眼眶内的、眼内的、输注、动脉内的、关节内的、心内的、皮内的、肌内的、腹膜内的、肺内的、脊柱内的、胸骨内的、鞘内的、子宫内的、静脉内的、蛛网膜下的、被膜下的、皮下的、转化粘液质的或跨器官的给药。通过肠胃外途径,载体可以是用于输注、注射或冻干的溶液或悬浮液形式。通过肠途径,生物活性物质载体可以是任何形式:片剂、凝胶胶囊、糖衣片剂、糖浆、悬浮液、溶液、粉末、颗粒、乳剂、微球或纳米微球、或可控制释放的脂质小囊泡或聚合物小囊泡。通常,通过静脉内或腹膜内途径之一注射给药。用于通过这些途径给药的方法是本领域技术人员已知的。
根据本发明所述的生物活性物质载体可进一步包括任何药学上可接受的载体。本公开中使用的“药学上可接受的载体”可以指药学上可接受的材料、组合物或运载体,其与从组织、器官或身体的部分携带或运输目标化合物至其它组织、器官或身体的部分相关。例如,载体可以是液体或固体填充物、稀释剂、赋形剂、溶剂或包封材料、或其组合。载体中的各个成分应该是“药学上可接受的”,即,可与制剂的其它成分相容。此外,载体应适用于当与可与载体接触的任何组织或器官接触时。事实上,载体应不涉及毒性、辐射、过敏反应、免疫原性或任何其它并发症的风险,其比载体实现的治疗优势更重要。
根据本发明所述的生物活性物质载体还可胶囊化、成形为片剂或以乳剂或糖浆形式制备而用于口服给药。可以加入药学上可接受的固体或液体载体来增强或稳定组合物、或促进组合物的产生。液体载体可包括糖浆、花生油、橄榄油、甘油、生理盐水、醇和水。固体载体可包括淀粉、乳糖、硫酸钙、二水合物、石膏粉、硬脂酸镁或硬脂酸、滑石、果胶、阿拉伯树胶、琼脂或明胶。载体还可包括缓释材料如单独的单硬脂酸甘油酯或二硬脂酸甘油酯或与蜡一起。
生物活性物质载体可通过常规制药技术制造,包括:必要时,在片剂形式的情况中,粉碎、混合、造粒和压缩;或在硬明胶胶囊形式的情况中,粉碎、混合和填充。当使用液体载体时,制剂可以是糖浆剂、酏剂、乳液剂或水性或非水性悬浮液形式。这类液体型制剂可以直接或者口服施用,或可填充在软明胶胶囊中。
根据本发明的生物活性物质载体可以以治疗有效量递送。确切的治疗有效量是得到对给定受试者的治疗疗效的最有效结果的组合物的量。该量可根据以下变化:治疗化合物的特性(包括活性、药代动力学、药效学和生物活性)、受试者的生理状况(包括年龄、性别、疾病类型和阶段、一般身体健康状况、给定剂量的药物的反应和类型)、药学上可接受的载体在制剂、给药途径中的特征,并且可取决于许多不限于此的其它因素而变化。在临床药理学领域,本领域技术人员将监测受试者对化合物给药的反应并且基于监测结果调节剂量,从而通过常规实验确定治疗有效量。对于其它指导,在此引入文献(参见[Remington:The Science and Practice of Pharmacy(Gennaro编辑,第20版,Williams&Wilkins PA,USA)(2000)])
与药物载体即本发明的载体一起施用的对象可包含包括人类在内的哺乳动物,具体地,为人类。
在向对象施用之前,可添加制剂至制剂。优选液体型制剂。制剂可包括,例如,油、聚合物、维生素、碳水化合物、氨基酸、盐、缓冲液、白蛋白、表面活性剂、扩链剂、或其组合。
碳水化合物制剂可包括糖或糖醇,例如单糖、二糖或多糖、或水溶性葡聚糖。糖类或葡聚糖可包括果糖、右旋糖、乳糖、葡萄糖、甘露糖、山梨糖、木糖、麦芽糖、蔗糖、葡聚糖、普鲁兰多糖、糊精、α和β环糊精、可溶性淀粉、羟乙基淀粉、和羧甲基纤维素、或其混合物。将“糖醇”定义为具有–OH基团的C4至C8烃,可包括半乳糖醇、肌醇、甘露醇、木糖醇、山梨醇、甘油和阿拉伯糖醇。上述糖或糖醇可单独使用或使用其组合。如果糖或糖醇可溶于水性制剂,则待使用量没有固定限制。在一个实施方案中,糖或糖醇的浓度范围可以是1.0w/v%至7.0w/v%,并且更优选为2.0至6.0w/v%。
氨基酸制剂可包括左旋(L)形式的肉毒碱、精氨酸和甜菜碱,然后,也可添加其它氨基酸。
在一些实施方案中,制剂可以是含有平均分子量为2,000至3,000的聚乙烯吡咯烷酮(PVP)的聚合物、或平均分子量为3,000至5,000的聚乙二醇(PEG)。
此外,为了最小化冻干前或重构后的溶液中的pH变化,优选在组合物中使用缓冲液。缓冲液可包括柠檬酸盐、磷酸盐、琥珀酸盐和谷氨酸盐缓冲液或其混合物,然而,还可使用大多数生理缓冲液而没有限制。在一些实施方案中,其浓度范围可为0.01至0.3mol。欧洲专利Nos.270,799和268,110中显示了可以添加至制剂中的表面活性剂。
此外,载体可以是,例如,通过共价缀合聚合物的化学修饰,来增加循环半衰期。在美国专利Nos.4,766,106;4,179,337;4,495,285;和4,609,546中公开了将载体连接至肽的优选的聚合物和方法,并且其内容通过引用完整并入本文。优选的聚合物可包括聚氧乙烯化多元醇和聚乙二醇(PEG)。PEG在室温下溶于水,并且在一些实施方案中,具有500至40,000、2000至20,000或3,000至12,000的平均分子量。在一些实施方案中,PEG具有至少一个羟基,例如末端羟基。可以激活羟基使得其与抑制剂上的游离氨基反应。然而,可改变反应器的类型和量来实现根据本发明共价缀合的PEG/抗体。
另外,水溶性聚氧乙烯化多元醇可用于本发明。这些可包括,例如,聚氧乙烯化山梨醇、聚氧乙烯化葡萄糖、和聚氧乙烯化甘油(POG)等。优选使用POG。一个原因是,聚氧乙烯化甘油的甘油骨架是单-、二-或三酸甘油酯,其是天然衍生的,即,与动物或人中的骨架相同。因此,不一定将该支链视为体内的外来试剂。POG具有与PEG相同范围的分子量。在文献[Knauf等人,1988,J.Bio.Chem.263:15064-15070]中公开了POG的结构,并且在美国专利No.4,766,106中发现了关于POG/IL C2缀合物的讨论,其内容通过引用完整并入本文。
在制造液体生物活性载体后,可冻干载体来防止降解并且保持无菌。本领域技术人员已知用于冻干液体组合物的方法。在使用前即刻,可在可含有另外成分的无菌稀释剂(例如,林格氏溶液、蒸馏水、或无菌生理盐水)中重构载体。重构后,使用本领域技术人员已知的任何方法向受试者施用载体。
生物活性物质载体的用途
上述生物活性物质载体可包括药物和多孔二氧化硅颗粒,并且本发明提供了上述多孔二氧化硅颗粒在制造生物活性物质载体的方法中的用途。
如上所述,根据本发明所述的多孔二氧化硅颗粒可以是生物降解性的并且在体内缓慢降解,因此以持续方式释放携带的生物活性物质,从而用于制造缓释型生物活性物质载体。
多孔二氧化硅颗粒的物理性质、规格、和表面修饰的细节可在上述范围内,并且多孔二氧化硅颗粒可以是通过在上述范围内的条件下的方法制造的那些。
实施例
1.多孔二氧化硅颗粒的制备
(1)多孔二氧化硅颗粒的制备
1)小孔颗粒的制备
将960mL蒸馏水和810mL MeOH置于2L圆底烧瓶中。在将7.88g CTAB置于烧瓶中后,快速添加4.52mL的1M NaOH同时搅拌。在形成均质混合物同时搅拌10分钟后,添加2.6mLTMOS。在搅拌6小时并且均匀混合后,老化混合物24小时。
其后,在25℃和8000rpm下离心反应溶液10分钟来去除上清液,接着在25℃和8000rpm下进一步离心10分钟并且用乙醇和蒸馏水清洗,可选地5次。
然后,在70℃下在烘箱中干燥产物来得到1.5g的粉末型小孔二氧化硅颗粒(具有2nm平均孔径和200nm粒径)。
2)孔扩张
将1.5g的小孔二氧化硅颗粒添加至10ml乙醇中,并且进行超声分散。然后,将10ml水和10ml三甲基苯(TMB)添加至反应溶液中,接着超声分散。
然后,将分散液置于高压釜中并在160℃下反应48小时。
反应在25℃下开始,继续反应同时以10℃/min的速率升高温度,然后,在高压釜中以1至10℃/min的速率逐步冷却反应的分散液。
在25℃和8000rpm下对冷却的反应溶液进行10分钟离心来去除上清液,接着在25℃和8000rpm下进一步离心10分钟并且用乙醇和蒸馏水清洗,可选地5次。
然后,在70℃下在烘箱中干燥产物来得到粉末型小孔二氧化硅颗粒(具有10至15nm平均孔径和200nm粒径)。
3)煅烧
将在上述第2)节中制备的多孔二氧化硅颗粒置于玻璃小瓶中,在550℃下加热5小时,并且在反应完成后,缓慢冷却至室温,从而得到颗粒。
(2)多孔二氧化硅颗粒的制备
除了将反应条件改变为在140℃和72小时以外,以如上述实施例1-(1)中所述的相同方法制备多孔二氧化硅颗粒。
(3)多孔二氧化硅颗粒的制备(10L规模)
除了使用5倍大的容器并且使用5倍体积的各个材料以外,以如上述实施例1-(1)中所述的相同方法制备多孔二氧化硅颗粒。
(4)多孔二氧化硅颗粒的制备(粒径300nm)
除了在小孔颗粒的制备中使用920ml蒸馏水和850ml甲醇以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(5)多孔二氧化硅颗粒的制备(粒径500nm)
除了在小孔颗粒的制备中使用800ml蒸馏水、1010ml甲醇和10.6g CTAB以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(6)多孔二氧化硅颗粒的制备(1000nm粒径)
除了在小孔颗粒的制备中使用620ml蒸馏水、1380ml甲醇和7.88g CTAB以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(7)多孔二氧化硅颗粒的制备(4nm孔径)
除了在孔扩张中使用2.5mL TMB以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(8)多孔二氧化硅颗粒的制备(7nm孔径)
除了在孔扩张中使用4.5mL TMB以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(9)多孔二氧化硅颗粒制备(17nm孔径)
除了在孔扩张中使用11mL TMB以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(10)多孔二氧化硅颗粒的制备(23nm孔径)
除了在孔扩张中使用12.5mL TMB以外,以如上述第(1)节中所述的相同方法制备多孔二氧化硅颗粒。
(11)多孔二氧化硅颗粒的制备(双重修饰)
1)小孔颗粒的制备
以如实施例(1)-1)中所述的相同方法制备小孔颗粒。
2)孔扩张
在如实施例(1)-2)中所述的相同方法中,将小孔颗粒与TMB反应,然后冷却并离心来去除上清液。然后,在如实施例(1)-2)中的相同条件下,进行离心,接着用乙醇和蒸馏水清洗,可选地3次。其后,在如实施例(1)-2)中的相同条件下干燥反应产物,从而得到粉末型多孔二氧化硅颗粒(具有10至15nm孔径、和200nm粒径)。
3)表面修饰
在将0.8g至1g的具有扩张的孔的多孔二氧化硅颗粒在50mL甲苯中分散后,将5mL的(3-氨基丙基)三乙氧基硅烷置于分散液中并且在120℃下加热12小时同时回流。该过程通过以下来进行:在上述清洗和干燥过程后,将1mL的三甘醇(PEG3,2-[2-(2-甲氧基乙氧基)乙氧基]乙酸)、100mg EDC(1-乙基-3-(3-二甲基氨基丙基)碳二亚胺)和200mg N-羟基丁二酰亚胺(NHS)在30mL PBS中分散,进行反应12小时同时在室温下搅拌。然后,对反应产物进行清洗和干燥。
由于先前的反应溶液保留在孔的内部,因此孔的内部未被修饰。
4)孔的内部的清洗
将800mg的表面修饰的颗粒粉末在40ml 2M HCl/乙醇中溶解,接着回流同时剧烈搅拌12小时。
然后,以8000rpm离心冷却的反应溶液10分钟来去除上清液,在25℃和8000rpm下进一步离心10分钟,接着用乙醇和蒸馏水清洗,可选地5次。
其后,在70℃下在烘箱中干燥反应产物,从而得到粉末型多孔二氧化硅颗粒。
5)孔的内部的修饰
(i)以后述实施例2-(2)-1)中所述的相同方法将丙基引入孔的内部。
(ii)以后述实施例2-(2)-2)中所述的相同方法将辛基引入孔的内部。
2.表面修饰
(1)带电至正电荷
1)具有300nm粒径的颗粒
将实施例1-(4)中的多孔二氧化硅颗粒与(3-氨基丙基)三乙氧基硅烷(APTES)反应使其带正电。
具体地,通过水浴超声波仪(bath sonicator)将100mg多孔二氧化硅颗粒在100mL圆底烧瓶中的10ml甲苯中分散。然后,添加1mL APTES,将反应混合物在130℃下反应同时以400rpm搅拌12小时。
反应后,将反应产物缓慢冷却至室温,以8000rpm离心10分钟来去除上清液,在25℃和8000rpm下进一步离心10分钟,接着用乙醇和蒸馏水清洗,可选地5次。
其后,在70℃下在烘箱中干燥反应产物,从而得到在颗粒表面和孔的内部具有氨基的粉末状多孔二氧化硅颗粒。
2)200nm粒径的颗粒
(i)除了将实施例1-(1)中的多孔二氧化硅颗粒与(3-氨基丙基)三乙氧基硅烷(APTES)反应使其带正电,添加0.4ml APTES并且进行反应3小时以外,以如上述实施例2-(1)-1)中所述的相同方法进行修饰。
(ii)除了将实施例1-(9)中的多孔二氧化硅颗粒与(3-氨基丙基)三乙氧基硅烷(APTES)反应使其带正电以外,以如上述实施例2-(1)-1)中所述的相同方法进行修饰。
(iii)除了将实施例1-(10)中的多孔二氧化硅颗粒与(3-氨基丙基)三乙氧基硅烷(APTES)反应使其带正电以外,以如上述实施例2-(1)-1)中所述的相同方法进行修饰。
(2)疏水性基团的引入
1)丙基
除了将实施例1-(1)中的多孔二氧化硅与三甲氧基(丙基)硅烷反应来引入丙基至颗粒的表面和孔的内部,添加0.35ml的三甲氧基(丙基)硅烷来代替APTES,并且反应进行12小时以外,以如上述实施例2-(1)中所述的相同方法进行修饰。
2)辛基
除了将实施例1-(1)中的多孔二氧化硅与三甲氧基正辛基硅烷反应来引入丙基至颗粒的表面和孔的内部,添加0.5ml的三甲氧基正辛基硅烷来代替APTES,并且反应进行12小时以外,以如上述实施例2-(1)中所述的相同方法进行修饰。
(3)带电至负电荷
1)羧基
除了将实施例1-(1)中的多孔二氧化硅与丁二酸酐反应使其带负电荷以外,以如上述实施例2-(1)-1)中所述的相同方法进行修饰。
使用二甲基亚砜(DMSO)代替甲苯,添加80mg的丁二酸酐代替APTES并且在室温下反应同时搅拌24小时,使用DMSO代替蒸馏水来清洗。
2)硫醇基
除了使用1.1mL的MPTES代替APTES以外,以如上述实施例2-(1)-1)中所述的相同方法进行修饰。
3)磺酸基
除了将100mg的第(3)-2)节中的多孔二氧化硅颗粒在1mL的1M硫酸溶液和20mL的30%过氧化氢中分散并且在室温下搅拌来诱导氧化反应,其反过来将硫醇基氧化至磺酸基。其后,以如实施例2-(1)-1)中所述的相同方法清洗并干燥反应产物以外,以如上述实施例2-(1)-1)中所述的相同方法进行修饰。
3.生物活性物质负载
(1)阿霉素
使实施例1-(1)中的多孔二氧化硅颗粒负载有阿霉素。
具体地,在将5mg多孔二氧化硅颗粒粉末和2mg阿霉素在蒸馏水中混合后,将混合物在室温下静置1小时。
(2)伊立替康
在将5mg的实施例2-(3)-1)中的带负电的多孔二氧化硅颗粒粉末在1mL的1xPBS中分散后,添加2mg的伊立替康至分散液中并且分散15分钟,将混合物在室温下静置1小时。
(3)索拉非尼
使实施例1-(11)-5)(i)中的多孔二氧化硅颗粒负载有索拉非尼。
具体地,在将5mg多孔二氧化硅颗粒粉末和2mg索拉非尼在1ml的具有5:5混合比(体积比)的去离子水/乙醇中混合后,将混合物在室温下培养1小时。其后,用1ml去离子水清洗产物3次。
(4)维A酸
将1ml的维A酸溶液(50mM乙醇)添加至100μg的实施例2-(1)-2)(i)中的多孔二氧化硅颗粒粉末中,接着在室温下静置4小时。其后,用1ml乙醇清洗产物3次。
(5)p53肽
此处使用的多孔二氧化硅颗粒是实施例1-(11)-5)(i)中的颗粒。
此处使用的p53肽模拟与细胞死亡(凋亡)机制相关的p53蛋白序列的一部分。模拟序列涉及其中p53蛋白与hMDM2蛋白组合的疏水二级螺旋结构的序列。因此,p53肽用作hMDM2蛋白的拮抗剂。
下式1表示p53肽的氨基酸序列(N末端->C末端)(计算分子量(Cal.m.w.)2596.78,MALDI-TOF实测值2597.92)。
[式1]
Z-Gly-Gly-Qln-Ser-Qln-Qln-Thr-Phe-Y-Asn-Leu-Trp-Arg-Leu-Leu-X-Qln-Asn-NH2
(其中X是引入有叠氮官能团的非天然氨基酸并且表示2-氨基-5-叠氮基-戊酸;并且Y使引入有炔烃官能团的非天然氨基酸,其中将4-戊炔酸引入D-Lys的侧链;
X和Y通过与反应环的叠氮-炔烃环加成反应(或点击反应)连接来形成三唑官能团;以及
Z表示5(6)-羧基荧光素(FAM))。
将1.3mg(500nmole)的p53肽在15mL的锥形管中100μl的DMSO中溶解并且与5mL的含有5mg多孔二氧化硅颗粒粉末的溶液混合,接着在室温下培养12小时。
对负载有p53肽的多孔二氧化硅颗粒进行离心(9289rcf,8500rpm,20分钟,和15mL锥形管)并且用水清洗3次,从而纯化。
(6)siRNA
根据要求合成并从Bioneer Co.Ltd.购买靶向绿色荧光蛋白(GFP)的21碱基对双链siRNA(序列:正义;5'-GGCUACGUCCAGGAGCGCACC-3’(SEQ ID NO:1),反义;5'-UGCGCUCCUGGACGUAGCCUU-3’(SEQ ID NO:2))。
将10μg的实施例2-(1)-2)(ii)中的多孔二氧化硅颗粒和50pmol siRNA在1xPBS条件下混合,接着在室温下静置30分钟来完成负载。
(7)质粒DNA
从细菌中产生设计用于在pcDNA3.3骨架中表达GFP的6.7k碱基对的质粒DNA(SEQID NO:5)并且在纯化后使用。
将10μg的实施例2-(1)-2)(iii)中的多孔二氧化硅颗粒和0.25μg的质粒DNA在1xPBS条件下混合,接着在室温下静置30分钟来完成负载。
(8)线性DNA
使用了以正向引物-CMW启动子-eGFP cDNA-反向引物顺序设计的并且通过PCR扩增得到的1.9k碱基对的线性DNA(SEQ ID NO:6)。
将12.5μg的实施例2-(1)-2)(iii)中的多孔二氧化硅颗粒和0.25μg的线性DNA在1xPBS条件下混合,接着在室温下静置30分钟来完成负载。
(9)蛋白质
1)BSA
将100μg的实施例2-(1)-2)(ii)中的多孔二氧化硅颗粒粉末和10μg BSA(SigmaAldrich,A6003)在200μl的1xPBS中混合,接着在室温下培养1小时。
2)IgG
将100μg的实施例2-(1)-2)(ii)中的多孔二氧化硅颗粒粉末和10μg的抗-twistIgG(Santacruz,sc-81417)在200μl的1xPBS中混合,接着在室温下培养1小时。
3)RNase A
将100μg的实施例1-(9)中的多孔二氧化硅颗粒粉末和10μg的RNase A(SigmaAldrich,R6513)在200μl的1xPBS中混合,接着在室温下培养1小时。
4)Cas9
将40μg的实施例2-(1)-2)(i)中的多孔二氧化硅颗粒粉末、4μg的Cas9蛋白(SEQID NO:3)和2.25μg的指导BSA(SEQ ID NO:4)在10μl的1xPBS中混合,接着在室温下培养1小时。
实验实施例
1.颗粒形成和孔扩张的识别
根据通过显微镜对实施例1-(1)至(3)中制备的小孔颗粒和多孔二氧化硅的观察,确定小孔颗粒是否均匀产生、孔是否充分扩张、以及多孔二氧化硅颗粒是否均匀形成(图1至4)。
图1是显示实施例1-(1)中多孔二氧化硅颗粒的照片,并且图2是显示实施例1-(2)中多孔二氧化硅颗粒的照片,表明均匀形成了孔充分扩张的球状多孔二氧化硅颗粒。
图3是显示实施例1-(1)中小孔颗粒的照片,并且图4是显示实施例1-(1)和1-(3)中的小孔颗粒的比较的照片,表明均匀形成了球状小孔颗粒。
2.BET表面积和孔体积的计算
对于实施例1-(1)中的小孔颗粒和实施例1-(1)、(7)、(8)和(10)中的多孔二氧化硅颗粒,计算表面积和孔体积。通过Brunauer-Emmett-Teller(BET)法计算表面积,并且通过Barrett-Joyner-Halenda(BJH)法计算孔体积的分布。
图5中显示各个颗粒的显微照相,并且在下表1中显示计算结果。
[表1]
3.多孔二氧化硅颗粒的生物降解性的识别
为了识别实施例1-(1)中的多孔二氧化硅颗粒的生物降解性,通过显微镜在第0小时、120小时和360小时观察在37℃下在SBF(pH 7.4)中的生物降解程度,图6中显示其结果。
参见图6,可以看到多孔二氧化硅颗粒被生物降解并且在360小时后几乎降解。
4.多孔二氧化硅颗粒的吸光度比的测定
根据下式1测定吸光度随时间变化的比。
[式1]
At/A0
(其中A0表示在将5ml的含有1mg/ml多孔二氧化硅颗粒的悬浮液置于具有50kDa直径的孔的圆筒状渗透膜中后测定的多孔二氧化硅颗粒的吸光度,
其中,15ml的与渗透膜接触并且与悬浮液实质上相同的溶剂存在于渗透膜的外部,在37℃和60rpm下水平搅拌渗透膜的内部和外部,悬浮液的pH为7.4,并且
其中At表示在测定A0后‘t’小时测定的多孔二氧化硅颗粒的吸光度。
具体地,将5mg的多孔二氧化硅颗粒粉末在5ml SBF(pH 7.4)中溶解。然后,将制备的5ml多孔二氧化硅颗粒溶液置于图7中所示的具有50kDa直径的孔的渗透膜中。将15mlSBF添加至外膜并且每12小时更换外膜中的SBF。在37℃和60rpm的水平搅拌下进行多孔二氧化硅颗粒的降解。
其后,通过紫外-可见光吸收光谱(UV-vis spectroscopy)测定吸光度并且在λ=640nm下分析。
(1)吸光度比的测定
根据上述方法测定实施例1-(1)中的多孔二氧化硅颗粒的吸光度比,并且在图8中显示其结果。
参考图8,其中吸光度比达到1/2的‘t’为约58小时,并且可看到多孔二氧化硅非常缓慢的降解。
(2)根据粒径的吸光度
根据上式1分别测定实施例1-(1)、(5)和(6)中多孔二氧化硅的吸光度,并且在图9中显示其结果(SBF用作悬浮液和溶剂)。
参考图9,可看到‘t’随颗粒直径的增加而减少。
(2)根据平均孔径的吸光度
根据上式1分别测定实施例1-(1)和(9)中多孔二氧化硅颗粒的吸光度,以及作为对照组的实施例1-(1)中小孔二氧化硅颗粒的吸光度,并且在图10中显示其结果(SBF用作悬浮液和溶剂)。
参考图10,可看到实施例中的多孔二氧化硅颗粒的‘t’显著大于对照组。
(3)根据pH的吸光度
测定实施例1-(4)中多孔二氧化硅颗粒的根据pH的吸光度。在pH 2、5和7.4下在SBF和Tris中测定吸光度,并且在图11中显示其结果。
参考图11,可看到,尽管根据pH的‘t’存在差异,但其中全部吸光度比达到1/2的‘t’为24以上。
(4)带电
测定实施例2-(1)-1)中多孔二氧化硅颗粒的吸光度,并且在图12中显示其结果(Tris(pH 7.4)用作悬浮液和溶剂)。
参考图12,可看到当吸光度比达到1/2时带正电的颗粒也具有24以上的‘t’。
5.生物活性物质的释放
(1)阿霉素
将0.5mg的负载有阿霉素(0.1mg)的多孔二氧化硅颗粒在PBS中分散。在用于在37℃和200rpm下水平搅拌的动态条件下维持该溶液。在各个时间点通过离心分离器沉降阿霉素负载的多孔二氧化硅溶液,测定上清液的吸光度(λab=480nm)来确定释放的阿霉素的量,在图13中显示其结果。
参考图13,可看到,阿霉素以相对弱的结合力负载于颗粒的表面并且由于在PBS中的高溶解度而相对快地释放,并且生物活性物质经70小时以上连续释放。
(2)伊立替康
将1mg的负载有伊立替康(0.2mg)的多孔二氧化硅颗粒在1mL的人血浆中分散。在用于在37℃和200rpm下水平搅拌的动态条件下维持该溶液。在各个时间点通过离心分离器沉降伊立替康负载的多孔二氧化硅溶液,测定上清液的吸光度(λab=255或278nm)来确定释放的伊立替康的量,在图14中显示其结果。
参考图14,可看到约50%的伊立替康在5.5小时后释放,并且生物活性物质经120小时以上连续释放。
(3)索拉非尼
将1mg的负载有索拉非尼(0.1mg)的多孔二氧化硅颗粒在10mL的1xPBS中分散。在用于在37℃和200rpm下水平搅拌的动态条件下维持该溶液。在各个时间点通过离心分离器沉降索拉非尼负载的多孔二氧化硅溶液,测定上清液的吸光度(λab=270nm)来确定释放的索拉非尼的量,在图15中显示其结果。
参考图15,可看到由于索拉非尼与具有疏水性取代基的多孔二氧化硅颗粒的相互作用,作为难溶性生物活性物质的索拉非尼非常缓慢地释放。
(4)维A酸
将0.1mg的维A酸负载的颗粒置于含有5%乙醇的PBS(pH 7.4)溶液中并且在37℃的温度下保持同时水平搅拌。每24小时离心含有颗粒的溶液,并且在350nm波长下测定上清液的吸光度来确定释放的维A酸的量,在图16中显示其结果。
参考图16,可看到带负电的维A酸由于其与带正电的多孔二氧化硅颗粒相互作用而非常缓慢地释放,并且经10天几乎100%释放。
(5)p53肽
将5mg的负载有p53肽的颗粒置于5mL的含有10%FBS的1xPBS或5mL1xPBS中,并且在动态环境中保持同时在37℃和20rpm下旋转。在各个时间点在8500rpm下离心溶液,并且测定上清液中作为荧光标记与p53偶联的5(6)-羧基荧光素(FAM)的荧光强度(吸光度:480nm,发射:520nm)。
图17中显示其结果。
参考图17,可看到由于疏水作用,p53肽通过结合力负载于多孔二氧化硅颗粒中,并且不在PBS溶液中释放。然而,如果蛋白质例如胎牛血清(FBS)存在于溶液中,p53肽与FBS蛋白的疏水性部分结合并且可在溶液中溶解,因此,该肽可在颗粒外释放。另外,推测负载于颗粒内部的p53肽可释放至颗粒外并且因此将FBS蛋白引入颗粒中。
(6)siRNA
将10μl的负载有Cy5-siRNA的多孔二氧化硅颗粒在SBF(pH 7.4,37℃)中重悬,然后置于具有20kDa孔径的渗透膜中(图18中的管)。
其后,将可渗透管浸于1.5ml SBF中。
在37℃下进行siRNA的释放同时在60rpm下水平搅拌。
在24小时前,在经过0.5、1、2、4、8、12和24小时的时间恢复释放溶剂。其后,以24小时的间隔恢复0.5ml的释放溶剂用于荧光测定,接着以等量添加SBF。
在670nm波长(λex=647nm)下测定Cy5-siRNA的荧光强度来确定siRNA的释放程度,在图19中显示其结果。
参考图19,可看到在约48小时内释放了50%siRNA。
(7)质粒DNA
将负载有质粒DNA的多孔二氧化硅颗粒(质粒DNA1μg,多孔二氧化硅颗粒50μg)在PBS(pH 7.4,37℃)中重悬,然后置于具有20kDa孔径的渗透膜中(与图18中管相同的管)。
其后,将可渗透管浸于1.5ml PBS中。
在37℃下进行质粒DNA的释放同时在60rpm下水平搅拌。
在24小时前,在经过0.5、1、2、4、8、12和24小时的时间处回收释放溶剂。其后,以24小时的间隔回收0.5ml的释放溶剂用于Hoechst-结合分析,接着以等量添加PBS。
在460nm波长(λex=360nm)下测定Hoechst 33342的荧光强度来确定质粒DNA的释放程度,在图20和21中显示其结果。
参考图20和21,可看到在约24小时内释放了50%质粒DNA。
(8)线性DNA
将负载有线性DNA的多孔二氧化硅颗粒(线性DNA3μg,多孔二氧化硅颗粒100μg)在PBS(pH 7.4,37℃)中重悬,然后置于具有20kDa孔径的渗透膜中(与图18中管相同的管)。
其后,将可渗透管浸于1.5ml PBS中。
在37℃下进行线性DNA的释放同时在60rpm下水平搅拌。
在24小时前,在经过0.5、1、2、3、4、6、12和24小时的时间处回收释放溶剂。其后,以24小时的间隔回收0.5ml的释放溶剂用于Hoechst-结合分析,接着以等量添加PBS。
在460nm波长(λex=360nm)下测定Hoechst 33342的荧光强度来确定线性DNA的释放程度,在图22中显示其结果。
参考图22,可看到在约24小时内释放了50%线性DNA。
(9)蛋白质
1)BSA
将100μg的负载有具有荧光标记的BSA的多孔二氧化硅颗粒在200μl的SBF(pH7.4)或PBS(pH 7.4)中重悬。
在37℃下进行BSA的释放同时在60rpm下水平搅拌。
在6、12、24、48、96、144和240小时的时间点处,回收200μl的释放溶剂用于荧光测定,接着以等量添加SBF或PBS。
在517nm波长(λex=492nm)下测定标记有荧光的BSA的荧光强度来确定BSA的释放程度,在图23中显示其结果。
参考图23,可看到BSA可在SBF和PBS二者中以持续的方式释放,并且经250小时以上几乎100%释放。
2)IgG
将100μg的负载有具有荧光标记的IgG的多孔二氧化硅颗粒在200μl的SBF(pH7.4)或PBS(pH 7.4)中重悬。
在37℃下进行IgG的释放同时在60rpm下水平搅拌。
在6、12、24、48、96、144和240小时的时间点处,回收200μl的释放溶剂用于荧光测定,接着以等量添加SBF或PBS。
在517nm波长(λex=492nm)下测定标记有荧光的IgG的荧光强度来确定IgG的释放程度,在图24中显示其结果。
参考图24,可看到IgG可在SBF和PBS二者中以持续方式释放,并且在250小时以上中几乎100%释放。
3)RNase A
将100μg的标记有具有荧光标记的RNase A的多孔二氧化硅颗粒在200μl的SBF(pH7.4)或PBS(pH 7.4)中重悬。
在37℃下进行RNase A的释放同时在60rpm下水平搅拌。
在6、12、24、48、96、144和240小时的时间点处,回收200μl的释放溶剂用于荧光测定,接着以等量添加SBF或PBS。
在517nm波长(λex=492nm)下测定标记有荧光的RNase A的荧光强度来确定RNaseA的释放程度,在图25中显示其结果。
参考图25,可看到RNase A可在SBF和PBS二者中以持续的方式释放,并且在250小时以上中几乎100%释放。
4)Cas9
将40μg的负载有Cas9蛋白/指导RNA复合体的多孔二氧化硅颗粒在PBS(pH 7.4)中悬浮。
然后,在载玻片上的无血清培养基中用小鼠成纤维细胞处理多孔二氧化硅颗粒,即,将50,000NIH 3T3细胞压平,接着在5%CO2和37℃条件下培养。
在第1、3、6和24小时的时间点处,去除培养基,用1xPBS溶液清洗细胞,接着用4%多聚甲醛培养15分钟来固定细胞。
然后,在用PBS清洗后,在封闭缓冲液(1xPBS,5%正常山羊血清,0.3%triton X-100)中培养细胞1小时。
在用PBS清洗后,在His标签抗体(Santa Cruz,sc-8036)中培养细胞16小时。
再次,在用PBS清洗后,在与Alexa Fluor 488组合的抗小鼠二抗(Abcam,ab150113)中培养细胞2小时。
在用PBS清洗后,在载玻片上用DAPI(用于染色细胞核的染料)处理来染色细胞核。然后,在荧光显微镜下观察细胞来检测细胞中蛋白质的分布,在图26中显示其结果。
参考图26,用于染色细胞核的试剂DAPI在荧光显微图像中看到是蓝色,其表明细胞核的位置。在Cas9蛋白上标记的荧光染料Alexa Fluor 488在荧光显微图像中看到是绿色,其表明Cas9蛋白的位置。当用负载有Flexa Fluor 488标记的Cas9蛋白的二氧化硅颗粒处理细胞、接着用DAPI染色时,基于荧光显微图像,可以检测到Cas9蛋白是否通过二氧化硅颗粒引入细胞中、以及细胞核的位置。
参考上述结果,可以看到在引入3小时后,主要在细胞质部分观察到引入细胞中的Cas9蛋白,并且在24小时后存在于细胞核内。由于使用的二氧化硅颗粒实质上不可进入细胞核,可以理解为细胞中的Cas9蛋白是在24小时后从二氧化硅颗粒中释放然后引入已知为Cas9蛋白积累的细胞器的核中。
6.生物活性物质的递送
为了验证载体可以在动物水平的siRNA递送研究中充当合适的载体,测定通过释放生物活性物质在小鼠中的肿瘤抑制程度。
从Orient Bio Co.Ltd.购买Balb/c雄性裸鼠(5周龄),将3百万HeLa细胞(宫颈癌细胞)在无菌1xPBS中分散并且皮下注射至小鼠以在小鼠中生长异种移植肿瘤。当发现尺寸为70mm2的实体肿瘤时,通过注射分别将PBS、FITC-多孔二氧化硅颗粒(实施例2-(1)-2)(ii)中的多孔二氧化硅颗粒)和负载有Cy5-siRNA的FITC-多孔二氧化硅颗粒(实施例2-(1)-2)(ii)中的多孔二氧化硅颗粒)施用至小鼠的肿瘤中。然后,通过荧光体内成像系统(FOBI)(Neo science,Korea)在施用之前和之后即刻、以及施用48小时后观察其荧光强度和分布。
通过以下进行FITC标记:将50mg的二氧化硅颗粒在1mL二甲基亚砜(DMSO)中分散;添加25μg(10μl)的FITC-NHS(N-羟基丁二酰亚胺)溶液(2.5mg/mL)至分散液;在室温下反应混合物18小时同时用铝箔避光;通过离心(8500rpm,10分钟)纯化反应产物;弃去上清液同时收集沉降的颗粒并且将其在乙醇中均匀分散;并且用乙醇-蒸馏水重复纯化分散液,可选地,3或4次直至上清液中FITC颜色不可见。
图27中显示其结果。
参考图27,‘对照’表示仅施用PBS,同时‘cy5-siRNA’表示仅施用cy5-siRNA、‘FITC-DDV’表示仅施用以FITC标识的多孔二氧化硅颗粒,并且‘复合体’表示施用多孔二氧化硅颗粒,其负载有cy5-siRNA并且标记有FITC。参考这些结果,可以确证负载在颗粒上并且递送至体内的siRNA可以在更长时间内保持活性并且在注射点停留更长时间,因此即使在48小时后也表现强荧光。
<110> 雷莫内克斯生物制药有限公司(LEMONEX INC.)
<120> 生物活性物质递送载体
<130> 18P01046
<150> US 62/455,148
<151> 2017-02-06
<160> 6
<170> KoPatentIn 3.0
<210> 1
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> GFP siRNA正义
<400> 1
ggcuacgucc aggagcgcac c 21
<210> 2
<211> 21
<212> RNA
<213> 人工序列
<220>
<223> GFP siRNA反义
<400> 2
ugcgcuccug gacguagccu u 21
<210> 3
<211> 1386
<212> PRT
<213> 人工序列
<220>
<223> Cas9
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Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys
1010 1015 1020
Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser
1025 1030 1035 1040
Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu
1045 1050 1055
Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile
1060 1065 1070
Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser
1075 1080 1085
Met Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly
1090 1095 1100
Phe Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile
1105 1110 1115 1120
Ala Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser
1125 1130 1135
Pro Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly
1140 1145 1150
Lys Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile
1155 1160 1165
Met Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala
1170 1175 1180
Lys Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys
1185 1190 1195 1200
Tyr Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser
1205 1210 1215
Ala Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr
1220 1225 1230
Val Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His
1250 1255 1260
Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val
1265 1270 1275 1280
Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys
1285 1290 1295
His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu
1300 1305 1310
Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp
1315 1320 1325
Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp
1330 1335 1340
Ala Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile
1345 1350 1355 1360
Asp Leu Ser Gln Leu Gly Gly Asp Gly Gly Ser Gly Pro Pro Lys Lys
1365 1370 1375
Lys Arg Lys Val His His His His His His
1380 1385
<210> 4
<211> 130
<212> RNA
<213> 人工序列
<220>
<223> 指导RNA
<400> 4
gaaauuaaua cgacucacua uaggggccca guggaucuaa augagggguu uuagagcuag 60
aaauagcaag uuaaaauaag gcuaguccgu uaucaacuug aaaaaguggc accgagucgg 120
ugcuuuuuuu 130
<210> 5
<211> 6330
<212> DNA
<213> 人工序列
<220>
<223> 质粒DNA
<400> 5
gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 60
ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 120
ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 180
ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 240
cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 300
atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 360
cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 420
attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 480
cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 540
caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 600
cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg 660
agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg 720
actctagagg atcgaaccct tttggaccct cgtacagaag ctaatacgac tcactatagg 780
gaaataagag agaaaagaag agtaagaaga aatataagag ccaccatggt gagcaagggc 840
gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc 900
cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg 960
aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg 1020
acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc 1080
aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc 1140
aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag 1200
ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac 1260
tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac 1320
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 1380
aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag 1440
tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg 1500
accgccgccg ggatcactct cggcatggac gagctgtaca agtaagctgc cttctgcggg 1560
gcttgccttc tggccatgcc cttcttctct cccttgcacc tgtacctctt ggtctttgaa 1620
taaagcctga gtaggaagtg agggtctaga actagtgtcg acgcaagggt tcgatcccta 1680
ccggttagta atgagtttaa acgggggagg ctaactgaaa cacggaagga gacaataccg 1740
gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc 1800
gtttgttcat aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag 1860
accccattgg ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc 1920
gggtgaaggc ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct 1980
gcgcagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc 2040
gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc 2100
tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa 2160
tcggggcatc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact 2220
tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt 2280
gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa 2340
ccctatctcg gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt 2400
aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag 2460
ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc 2520
aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 2580
agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc 2640
ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat 2700
gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt 2760
ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat 2820
caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct 2880
ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc 2940
tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc 3000
gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc 3060
acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg 3120
ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag 3180
aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc 3240
ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt 3300
cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc 3360
gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc 3420
tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg 3480
ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag 3540
cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg 3600
cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg 3660
cgaaatgacc gaccaagcga cgcccaacct gccatcacga gatttcgatt ccaccgccgc 3720
cttctatgaa aggttgggct tcggaatcgt tttccgggac gccggctgga tgatcctcca 3780
gcgcggggat ctcatgctgg agttcttcgc ccaccccaac ttgtttattg cagcttataa 3840
tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca 3900
ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgta taccgtcgac 3960
ctctagctag agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc 4020
gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta 4080
atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa 4140
cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 4200
tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 4260
agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 4320
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 4380
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 4440
tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 4500
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 4560
ttcgggaagc gtggcgcttt ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt 4620
cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 4680
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 4740
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 4800
gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa 4860
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 4920
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 4980
agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 5040
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 5100
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt 5160
aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 5220
ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat 5280
gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg 5340
aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg 5400
ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat 5460
tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc 5520
ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt 5580
cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc 5640
agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga 5700
gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc 5760
gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa 5820
acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta 5880
acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg 5940
agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg 6000
aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat 6060
gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt 6120
tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtcg 6180
actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt 6240
gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg 6300
accgacaatt gcatgaagaa tctgcttagg 6330
<210> 6
<211> 1878
<212> DNA
<213> 人工序列
<220>
<223> 线性DNA
<400> 6
ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt 60
aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta 120
cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga 180
cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt 240
tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta 300
ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg 360
actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt 420
tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc 480
accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat 540
gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 600
atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt 660
ttgacctcca tagaagacac cgggaccgat ccagcctccg gactctagag gatcgaaccc 720
ttttggaccc tcgtacagaa gctaatacga ctcactatag ggaaataaga gagaaaagaa 780
gagtaagaag aaatataaga gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg 840
tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 900
gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 960
gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 1020
tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 1080
gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 1140
aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 1200
aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 1260
atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 1320
tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 1380
gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 1440
ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 1500
tcggcatgga cgagctgtac aagtaagctg ccttctgcgg ggcttgcctt ctggccatgc 1560
ccttcttctc tcccttgcac ctgtacctct tggtctttga ataaagcctg agtaggaagt 1620
gagggtctag aactagtgtc gacgcaaggg ttcgatccct accggttagt aatgagttta 1680
aacgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga 1740
cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg 1800
gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac 1860
gcccgcgttt cttccttt 1878
Claims (32)
1.一种生物活性物质载体,其包括:
生物活性物质;和
多孔二氧化硅颗粒,其负载所述生物活性物质,具有多个5nm至100nm直径的孔,并且具有在通过下式1测定的吸光度的比为1/2下的‘t’为24以上:
[式1]
At/A0
其中A0表示在将5ml的含有1mg/ml的所述多孔二氧化硅颗粒的悬浮液置于具有50kDa直径的孔的圆筒状渗透膜中后测定的所述多孔二氧化硅颗粒的吸光度,
其中15ml的与所述渗透膜接触并且与所述悬浮液实质上相同的溶剂存在于所述渗透膜的外部,在37℃和60rpm下水平搅拌所述渗透膜的内部和外部,所述悬浮液的pH为7.4,和
其中At表示在测定A0后‘t’小时测定的所述多孔二氧化硅颗粒的吸光度。
2.根据权利要求1所述的生物活性物质载体,其中所述悬浮液是选自由磷酸盐缓冲生理盐水(PBS)和模拟体液(SBF)组成的组的一种或多种。
3.根据权利要求1所述的生物活性物质载体,其中式1中的At在其中每隔预定时间更换所述渗透膜外部的溶剂的环境中测定。
4.根据权利要求1所述的生物活性物质载体,其中所述‘t’的范围为24至120。
5.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒是生物降解性的。
6.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒具有在通过式1测定的吸光度的比为1/5下的‘t’为70至120。
7.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒具有在通过式1测定的吸光度的比为1/20下的‘t’为130至220。
8.根据权利要求1所述的生物活性物质载体,其中通过式1测定的吸光度的比和‘t’之间的皮尔森相关系数为0.8以上。
9.根据权利要求1所述的生物活性物质载体,其中所述孔径的范围为7nm至30nm。
10.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒具有球状。
11.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒的平均直径的范围为150nm至1000nm。
12.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅的BET表面积的范围为200m2/g至700m2/g。
13.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅的BET表面积的范围为300m2/g至450m2/g。
14.根据权利要求1所述的生物活性物质载体,其中每克所述多孔二氧化硅颗粒的体积的范围为0.7ml至2.2ml。
15.根据权利要求1所述的生物活性物质载体,其中每克所述多孔二氧化硅颗粒的体积的范围为1.0ml至2.0ml。
16.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒在颗粒的外表面或孔的内部具有亲水性取代基或疏水性取代基。
17.根据权利要求1所述的生物活性物质载体,其中所述多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带正电或带负电。
18.根据权利要求1所述的生物活性物质载体,其中所述生物活性物质是难溶性的,并且所述多孔二氧化硅颗粒在颗粒的外表面或孔的内部具有疏水性取代基。
19.根据权利要求1所述的生物活性物质载体,其中所述生物活性物质是难溶性的,所述多孔二氧化硅颗粒在孔的内部具有疏水性取代基并且在颗粒的外表面具有亲水性取代基。
20.根据权利要求1所述的生物活性物质载体,其中所述生物活性物质在中性pH下带负电,并且所述多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带正电。
21.根据权利要求1所述的生物活性物质载体,其中所述生物活性物质在中性pH下带正电,并且所述多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带负电。
22.一种生物活性物质载体,其包括:
生物活性物质;和
球状多孔二氧化硅颗粒,其负载所述生物活性物质,粒径为150nm至500nm,并具有多个直径7nm至30nm的孔,并且具有在通过下式2测定的吸光度的比为1/2下的‘t’为24至120的范围:
[式2]
At/A0
其中A0表示在将5ml的含有1mg/ml的所述多孔二氧化硅颗粒的悬浮液置于具有50kDa直径的孔的圆筒状渗透膜中后测定的所述多孔二氧化硅颗粒的吸光度,
其中15ml的与所述渗透膜接触并且与所述悬浮液实质上相同的溶剂存在于所述渗透膜的外部,在37℃和60rpm下水平搅拌所述渗透膜的内部和外部,所述悬浮液为PBS或SBF并具有7.4的pH,和
其中At表示在测定A0后‘t’小时测定的所述多孔二氧化硅颗粒的吸光度。
23.根据权利要求22所述的生物活性物质载体,其中所述多孔二氧化硅颗粒的BET表面积的范围为300m2/g至450m2/g,并且每克的体积的范围为1.0ml至2.0ml。
24.根据权利要求22所述的生物活性物质载体,其中所述生物活性物质是难溶性的,并且所述多孔二氧化硅颗粒在颗粒的外表面或孔的内部具有疏水性取代基。
25.根据权利要求22所述的生物活性物质载体,其中所述生物活性物质是难溶性的,并且所述多孔二氧化硅颗粒在孔的内部具有疏水性取代基并且在颗粒的外表面具有亲水性取代基。
26.根据权利要求22所述的生物活性物质载体,其中所述生物活性物质在中性pH下带负电,并且所述多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带正电。
27.根据权利要求22所述的生物活性物质载体,其中所述生物活性物质在中性pH下带正电,并且所述多孔二氧化硅颗粒在中性pH下在颗粒的外表面或孔的内部带负电。
28.一种制造生物活性物质载体的方法,其包括在溶剂中将多孔二氧化硅颗粒与生物活性物质接触。
29.根据权利要求28所述的方法,其中所述溶剂为选自由以下组成的组的一种或多种:水、氯仿、二氯甲烷、四氯化碳、1,2-二氯乙烷、二氯乙烯、三氯乙烯、全氯乙烯、二氯丙烷、氯戊烷、1,2-二溴乙烷、丙酮、甲基异丁酮、环己酮、苯、甲苯、二甲苯、N,N-二甲基甲酰胺、N,N-二丁基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮、甲醇、乙醇、丙醇、丁醇、PBS、SBF、硼酸盐缓冲生理盐水和tris缓冲生理盐水。
30.根据权利要求28所述的方法,其中所述多孔二氧化硅颗粒与所述生物活性物质按重量计的比为1:0.05至0.8。
31.一种递送生物活性物质的方法,其包括向个体受试者肠胃外施用根据权利要求1至27任一项所述的生物活性物质载体。
32.根据权利要求31所述的方法,其中所述肠胃外施用是眼眶内、眼内、输注、动脉内、关节内、心内、皮内、肌内、腹膜内、肺内、脊柱内、胸骨内、鞘内、子宫内、静脉内、蛛网膜下、被膜下、皮下、转化粘液质或跨器官施用。
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US20200009054A1 (en) | 2020-01-09 |
US11129796B2 (en) | 2021-09-28 |
JP6883354B2 (ja) | 2021-06-09 |
EP3578171A1 (en) | 2019-12-11 |
CA3052561C (en) | 2023-02-14 |
AU2021232725B2 (en) | 2023-07-20 |
US20210369614A1 (en) | 2021-12-02 |
JP2020506972A (ja) | 2020-03-05 |
BR112019016281A2 (pt) | 2020-04-14 |
JP2021143177A (ja) | 2021-09-24 |
CA3052561A1 (en) | 2018-08-09 |
AU2018216591B2 (en) | 2021-07-22 |
AU2021232725A1 (en) | 2021-10-14 |
SG11201907260PA (en) | 2019-09-27 |
AU2018216591A1 (en) | 2019-08-29 |
EP3578171A4 (en) | 2020-12-30 |
US11793757B2 (en) | 2023-10-24 |
MX2019009271A (es) | 2019-10-30 |
JP7360727B2 (ja) | 2023-10-13 |
KR20180091768A (ko) | 2018-08-16 |
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