新型肝靶向去甲斑蝥素修饰物纳米粒的研制
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摘要
目的:本课题旨在利用体内肝细胞表面的去唾液酸糖蛋白受体(ASGP-R)能特异性识别药物分子的半乳糖残基,将治疗原发性肝癌的去甲斑蝥素(NCTD)进行乳糖化修饰得到具有主动肝靶向性的修饰物乳糖化-去甲斑蝥素(Lac-NCTD),并初步评价了该新化合物的安全性,同时以其为模型药物制备壳聚糖纳米粒(Lac-NCTD-NPs),并对纳米粒的体内外抗肿瘤活性进行了研究,以期得到一种安全有效的主被动肝靶向相结合的去甲斑蝥素修饰物纳米粒。
方法:(1)利用乙二胺为连接臂合成Lac-NCTD,并测定了药物的溶解度、油水分配系数和稳定性等基本理化性质。(2)通过急性毒性实验,计算Lac-NCTD腹腔给药的最大耐受量(MTD)。(3)采用离子诱导法制备Lac-NCTD-NPs,以粒径分布、包封率、载药量为综合指标,采用L9(34)正交实验优化制备工艺。(4)采用红外光谱(FT-IR)、差示扫描量热法(DSC)、粉末X-ray衍射和透射电镜等方法和技术,对Lac-NCTD-NPs的表面特征、外观形态进行验证,并以透析袋法考察纳米粒胶体的体外释放特性。(5)通过MTT法考察Lac-NCTD及Lac-NCTD-NPs对肿瘤细胞株HepG2、SMMC-7721和SGC-7901的细胞毒作用和半乳糖化小牛血清(Gal-FBS)的竞争性抑制作用。(6)采用HPLC法评价SMMC-7721细胞对Lac-NCTD的摄取过程。(7)通过建立H22肝癌细胞小鼠荷瘤模型考察药物的体内抗肿瘤活性。
结果:(1)Lac-NCTD是一种具有良好水溶性的多羟基化合物,其合成产率为68.35%。(2)急性毒性实验结果得出Lac-NCTD单次给药时MTD>20 g?kg-1,多次给药时MTD>30 g?kg-1,远大于人临床去甲斑蝥素的拟用量。(3)经正交优化后,Lac-NCTD-NPs制备工艺条件为:壳聚糖溶液浓度2.0 mg?mL-1、药物浓度2.0 mg?mL-1、壳聚糖和TPP质量比3:1,优化工艺制备的纳米粒的平均粒径(149.46±1.79) nm,包封率(80.29±0.56)%,载药量(9.58±0.09)%。(4)纳米粒的FT-IR、DSC、X衍射图谱中,LCS与TPP的特征吸收峰均消失或转移;透射电镜(TEM)照片显示纳米粒近似球形、边缘清晰;纳米粒体外释放符合Higuch方程。(5)体外细胞毒作用结果显示:对HepG2和SMMC-7721,当细胞培养48 h时Lac-NCTD-NPs的细胞毒作用最强,其次是Lac-NCTD,且都能显著的被Gal-FBS抑制;对SGC-7901,Lac-NCTD-NPs和Lac-NCTD的细胞毒作用并不比NCTD强,且不受Gal-FBS的影响。(6)培养12 h后,SMMC-7721对Lac-NCTD的摄取量为3.89μg?106cell-1。(7)体内抗肿瘤实验表明Lac-NCTD-NPs能有效地抑制H22肿瘤的生长。
结论:(1)Lac-NCTD合成简单,产率高,吸湿性强,保存时需防潮。(2)急性毒性实验初步证实Lac-NCTD是安全的。(3)该新化合物上大量的-OH能与壳聚糖上正电荷以分子间氢键结合,从而以吸附和包裹的方式形成纳米粒以致Lac-NCTD-NPs的包封率明显地优于相同条件下去甲斑蝥素壳聚糖纳米粒(NCTD-NPs)。(4)FT-IR、DSC、X-衍射图谱和TEM照片显示,纳米粒已经形成,形成机理为三聚磷酸根与壳聚糖氨基发生交联,药物被包裹其中,Lac-NCTD-NPs体外释放具有良好的缓释特性。(5)HepG2和SMMC-7721表面的ASGP-R能识别Lac-NCTD上的半乳糖残基,Lac-NCTD及其纳米粒对HepG2和SMMC-7721的毒性比NCTD强,且能被Gal-FBS抑制,NCTD的作用与ASGP-R的识别无关,它对三种细胞的毒杀作用不受Gal-FBS的影响。(6)SMMC-7721对Lac-NCTD的摄取实验结果进一步证实了部分药物是通过ASGP-R的识别吞噬作用而发挥抗癌效果的。(7)Lac-NCTD-NPs结合了药物的主动靶向性和纳米粒自身的被动靶向性,在体内被肝细胞ASGP-R特异性识别,达到药物在体内的双重肝靶向作用,在小鼠体内表现出强的抑瘤活性。
Objective: Norcantharidin (NCTD) against primary carcinoma of liver was lactosylated to obtain lactosyl-norcantharidin(Lac-NCTD)as initiative derivative, based on the asialoglycoprotein receptor (ASGP-R), situated on the hepatocyte membrane, can recognize the galactose residue of drug, then the safety of Lac-NCTD was preliminarily evaluated. Lac-NCTD-NPs were prepared and their anti-tumor activity in vivo and in vitro were evaluated. As a result, the safe and effective nanoparticles loaded norcantharidin derivative, which combine both active and passive liver-targeting were achieved.
Methods: (1) Lac-NCTD was synthesized using ethanediamine as a truss arm and its physicochemical properties such as solubility, oil/water partition coefficient, stability, etc. were determined. (2) MTD for Lac-NCTD was calculated by acu-toxicity test by intraperitoneal injection. (3) Lac-NCTD-NPs were achieved by ionic cross-linkage process and orthogonal experiment design was applied to optimize the formulation and preparation procedure with particle distribution, entrapment rate and drug-loading rate as integrated indexes. (4) Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction, transmission electron microscopy (TEM), etc. were applied to evaluate nanoparticle apatial representable structure; the release in vitro was investigated by dialysis membrane. (5) MTT was used to study the cytotoxic effects of Lac-NCTD and Lac-NCTD-NPs on HepG2, SMMC-7721 and SGC-7901 and the inhibition effects of Gal-FBS. (6) Lac-NCTD accumulated in SMMC-7721 cells was established by high performance liquid chromatogram (HPLC). (7) The H22 hepatoblastoma cell line bearing mice was establishmented to evaluate the in vivo anti-tumor activity of Lac-NCTD and Lac-NCTD-NPs.
Results: (1) Lac-NCTD was a kind of polyhydroxy compound and the yield of it was 68.35%. (2) Single and multiple dosing of MTD for compound Lac-NCTD exceeded 20 g?kg-1 and 30 g?kg-1 respectively,which exceed the amount of human clinical usage of NCTD fairly far. (3) The optimal preparation condition was as follows: CS concentrations was 2.0 mg?mL-1, drug concentrations was 2.0 mg?mL-1 and CS:TPP was 3:1(m/m). The average particle size of the prepared nanoparticles were (149.46±1.79) nm, entrapment rate was (80.29±0.56)%, drug-loading rate was (9.58±0.09)%. (4) In FT-IR、DSC and X-ray diffraction graph, the diagnostic absorption peak of LCS and TPP vanished or shifted. TEM graph showed that nanoparticles were sphere and distinct. The curve of the release in vitro was suitable for Higuch equation. (5) The in vitro studies showed that the cytotoxic effects of Lac-NCTD-NPs against HepG2, SMMC-7721 cells were the most powerful, then Lac-NCTD and they were inhibited remarkably by Gal-FBS; as for SGC-7901, the cytotoxic effects of Lac-NCTD-NPs and Lac-NCTD were not stronger than NCTD and Gal-FBS had no influence on them at all. (6) The amount of Lac-NCTD accumulated in SMMC-7721 cells was 3.89μg?106cell-1 after treatment for 12 h. (7) The results of the anti-tumor activity in vivo suggested that the tumor-growth of H22 were inhibited effectively by Lac-NCTD-NPs.
Conclusions: (1) The synthesis procedure of Lac-NCTD was simple and the yield was high. It should be protected against the tide when stored because of its strong moisture absorption. (2) Acute toxicity test demonstrated the compound Lac-NCTD was safe. (3) The large scale hydroxy group on the new chemical, Lac-NCTD, could combine with the positive charge of chitosan, shaped in the diluted acid, through intermolecular hydrogen bond to form nanoparticles by the means of adsorbing and encapsulation. As a result, the entrapment rate of Lac-NCTD-NPs was significantly higher than the norcantharidin nanoparticles(NCTD-NPs)prepared in the same condition. (4) The results of FT-IR、DSC、X-ray diffraction and TEM graph showed that the nanoparticles were shaped by the linkage of tripolyphosphate acid radical and the -NH2+ on the surface of chitosan. Effect of sustained drug release of Lac-NCTD-NPs was evident. (5) The ASGP-R on the surface of HepG2 and SMMC-7721 can recognize the galactose residue of Lac-NCTD, therefore, the cytotoxic effects of Lac-NCTD and Lac-NCTD-NPs on these two cell lines were stronger than NCTD and they were inhibited significantly by Gal-FBS. (5) The results of Lac-NCTD accumulated in SMMC-7721 cells further indicated that Lac-NCTD can partly permeate the cell membrance with the prime type through the ASGP-R pathway. (6) Lac-NCTD-NPs combined both the active targeting of Lac-NCTD and passive targeting of nanoparticles itself and were recognized specially by ASGP-R on the hepatocyte surface to achive liver-targeting and show powerful anti-tumor activity in vivo.
方法:(1)利用乙二胺为连接臂合成Lac-NCTD,并测定了药物的溶解度、油水分配系数和稳定性等基本理化性质。(2)通过急性毒性实验,计算Lac-NCTD腹腔给药的最大耐受量(MTD)。(3)采用离子诱导法制备Lac-NCTD-NPs,以粒径分布、包封率、载药量为综合指标,采用L9(34)正交实验优化制备工艺。(4)采用红外光谱(FT-IR)、差示扫描量热法(DSC)、粉末X-ray衍射和透射电镜等方法和技术,对Lac-NCTD-NPs的表面特征、外观形态进行验证,并以透析袋法考察纳米粒胶体的体外释放特性。(5)通过MTT法考察Lac-NCTD及Lac-NCTD-NPs对肿瘤细胞株HepG2、SMMC-7721和SGC-7901的细胞毒作用和半乳糖化小牛血清(Gal-FBS)的竞争性抑制作用。(6)采用HPLC法评价SMMC-7721细胞对Lac-NCTD的摄取过程。(7)通过建立H22肝癌细胞小鼠荷瘤模型考察药物的体内抗肿瘤活性。
结果:(1)Lac-NCTD是一种具有良好水溶性的多羟基化合物,其合成产率为68.35%。(2)急性毒性实验结果得出Lac-NCTD单次给药时MTD>20 g?kg-1,多次给药时MTD>30 g?kg-1,远大于人临床去甲斑蝥素的拟用量。(3)经正交优化后,Lac-NCTD-NPs制备工艺条件为:壳聚糖溶液浓度2.0 mg?mL-1、药物浓度2.0 mg?mL-1、壳聚糖和TPP质量比3:1,优化工艺制备的纳米粒的平均粒径(149.46±1.79) nm,包封率(80.29±0.56)%,载药量(9.58±0.09)%。(4)纳米粒的FT-IR、DSC、X衍射图谱中,LCS与TPP的特征吸收峰均消失或转移;透射电镜(TEM)照片显示纳米粒近似球形、边缘清晰;纳米粒体外释放符合Higuch方程。(5)体外细胞毒作用结果显示:对HepG2和SMMC-7721,当细胞培养48 h时Lac-NCTD-NPs的细胞毒作用最强,其次是Lac-NCTD,且都能显著的被Gal-FBS抑制;对SGC-7901,Lac-NCTD-NPs和Lac-NCTD的细胞毒作用并不比NCTD强,且不受Gal-FBS的影响。(6)培养12 h后,SMMC-7721对Lac-NCTD的摄取量为3.89μg?106cell-1。(7)体内抗肿瘤实验表明Lac-NCTD-NPs能有效地抑制H22肿瘤的生长。
结论:(1)Lac-NCTD合成简单,产率高,吸湿性强,保存时需防潮。(2)急性毒性实验初步证实Lac-NCTD是安全的。(3)该新化合物上大量的-OH能与壳聚糖上正电荷以分子间氢键结合,从而以吸附和包裹的方式形成纳米粒以致Lac-NCTD-NPs的包封率明显地优于相同条件下去甲斑蝥素壳聚糖纳米粒(NCTD-NPs)。(4)FT-IR、DSC、X-衍射图谱和TEM照片显示,纳米粒已经形成,形成机理为三聚磷酸根与壳聚糖氨基发生交联,药物被包裹其中,Lac-NCTD-NPs体外释放具有良好的缓释特性。(5)HepG2和SMMC-7721表面的ASGP-R能识别Lac-NCTD上的半乳糖残基,Lac-NCTD及其纳米粒对HepG2和SMMC-7721的毒性比NCTD强,且能被Gal-FBS抑制,NCTD的作用与ASGP-R的识别无关,它对三种细胞的毒杀作用不受Gal-FBS的影响。(6)SMMC-7721对Lac-NCTD的摄取实验结果进一步证实了部分药物是通过ASGP-R的识别吞噬作用而发挥抗癌效果的。(7)Lac-NCTD-NPs结合了药物的主动靶向性和纳米粒自身的被动靶向性,在体内被肝细胞ASGP-R特异性识别,达到药物在体内的双重肝靶向作用,在小鼠体内表现出强的抑瘤活性。
Objective: Norcantharidin (NCTD) against primary carcinoma of liver was lactosylated to obtain lactosyl-norcantharidin(Lac-NCTD)as initiative derivative, based on the asialoglycoprotein receptor (ASGP-R), situated on the hepatocyte membrane, can recognize the galactose residue of drug, then the safety of Lac-NCTD was preliminarily evaluated. Lac-NCTD-NPs were prepared and their anti-tumor activity in vivo and in vitro were evaluated. As a result, the safe and effective nanoparticles loaded norcantharidin derivative, which combine both active and passive liver-targeting were achieved.
Methods: (1) Lac-NCTD was synthesized using ethanediamine as a truss arm and its physicochemical properties such as solubility, oil/water partition coefficient, stability, etc. were determined. (2) MTD for Lac-NCTD was calculated by acu-toxicity test by intraperitoneal injection. (3) Lac-NCTD-NPs were achieved by ionic cross-linkage process and orthogonal experiment design was applied to optimize the formulation and preparation procedure with particle distribution, entrapment rate and drug-loading rate as integrated indexes. (4) Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), X-ray diffraction, transmission electron microscopy (TEM), etc. were applied to evaluate nanoparticle apatial representable structure; the release in vitro was investigated by dialysis membrane. (5) MTT was used to study the cytotoxic effects of Lac-NCTD and Lac-NCTD-NPs on HepG2, SMMC-7721 and SGC-7901 and the inhibition effects of Gal-FBS. (6) Lac-NCTD accumulated in SMMC-7721 cells was established by high performance liquid chromatogram (HPLC). (7) The H22 hepatoblastoma cell line bearing mice was establishmented to evaluate the in vivo anti-tumor activity of Lac-NCTD and Lac-NCTD-NPs.
Results: (1) Lac-NCTD was a kind of polyhydroxy compound and the yield of it was 68.35%. (2) Single and multiple dosing of MTD for compound Lac-NCTD exceeded 20 g?kg-1 and 30 g?kg-1 respectively,which exceed the amount of human clinical usage of NCTD fairly far. (3) The optimal preparation condition was as follows: CS concentrations was 2.0 mg?mL-1, drug concentrations was 2.0 mg?mL-1 and CS:TPP was 3:1(m/m). The average particle size of the prepared nanoparticles were (149.46±1.79) nm, entrapment rate was (80.29±0.56)%, drug-loading rate was (9.58±0.09)%. (4) In FT-IR、DSC and X-ray diffraction graph, the diagnostic absorption peak of LCS and TPP vanished or shifted. TEM graph showed that nanoparticles were sphere and distinct. The curve of the release in vitro was suitable for Higuch equation. (5) The in vitro studies showed that the cytotoxic effects of Lac-NCTD-NPs against HepG2, SMMC-7721 cells were the most powerful, then Lac-NCTD and they were inhibited remarkably by Gal-FBS; as for SGC-7901, the cytotoxic effects of Lac-NCTD-NPs and Lac-NCTD were not stronger than NCTD and Gal-FBS had no influence on them at all. (6) The amount of Lac-NCTD accumulated in SMMC-7721 cells was 3.89μg?106cell-1 after treatment for 12 h. (7) The results of the anti-tumor activity in vivo suggested that the tumor-growth of H22 were inhibited effectively by Lac-NCTD-NPs.
Conclusions: (1) The synthesis procedure of Lac-NCTD was simple and the yield was high. It should be protected against the tide when stored because of its strong moisture absorption. (2) Acute toxicity test demonstrated the compound Lac-NCTD was safe. (3) The large scale hydroxy group on the new chemical, Lac-NCTD, could combine with the positive charge of chitosan, shaped in the diluted acid, through intermolecular hydrogen bond to form nanoparticles by the means of adsorbing and encapsulation. As a result, the entrapment rate of Lac-NCTD-NPs was significantly higher than the norcantharidin nanoparticles(NCTD-NPs)prepared in the same condition. (4) The results of FT-IR、DSC、X-ray diffraction and TEM graph showed that the nanoparticles were shaped by the linkage of tripolyphosphate acid radical and the -NH2+ on the surface of chitosan. Effect of sustained drug release of Lac-NCTD-NPs was evident. (5) The ASGP-R on the surface of HepG2 and SMMC-7721 can recognize the galactose residue of Lac-NCTD, therefore, the cytotoxic effects of Lac-NCTD and Lac-NCTD-NPs on these two cell lines were stronger than NCTD and they were inhibited significantly by Gal-FBS. (5) The results of Lac-NCTD accumulated in SMMC-7721 cells further indicated that Lac-NCTD can partly permeate the cell membrance with the prime type through the ASGP-R pathway. (6) Lac-NCTD-NPs combined both the active targeting of Lac-NCTD and passive targeting of nanoparticles itself and were recognized specially by ASGP-R on the hepatocyte surface to achive liver-targeting and show powerful anti-tumor activity in vivo.
引文
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