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多形性胶质母细胞瘤(GBM)是恶性程度最高的脑肿瘤之一,呈强侵袭生长特点,患者中位生存期一般不超过15~17个月,5年生存率低于5%[1-3]。肿瘤最新机制研究聚焦高能量代谢特征,加州大学的研究人员揭示了癌症基因扩增和表观遗传重塑依赖于烟酰胺腺嘌呤二核苷酸(NAD+)途径,干预NAD+代谢成为癌症精准治疗的重要靶点[4]。肿瘤细胞通过生物合成上调来增加NAD+水平,对NAD+生物合成的限速酶——烟酰胺磷酸核糖转移酶(NAMPT)非常敏感[5]。Kim等[6]发现NAD+代谢在胶质瘤中过度活跃,GBM患者肿瘤细胞中NAMPT基因表达水平异常升高。PROTAC技术是一种化学诱导靶蛋白(POI)多聚泛素化,通过蛋白酶体通路降解POI的药物分子靶向新技术[7]。PROTAC分子由靶蛋白结合配体、连接链和E3泛素连接酶配体三部分组成,能特异性结合靶蛋白,同时招募E3泛素连接酶使POI多聚泛素化,最终通过蛋白酶体系统降解。并且标记POI促使蛋白降解后,可与POI解离在细胞内循环利用,起到亚化学计量催化的效果,减少药物剂量从而减少体内药物暴露量[8]。合作课题组合成了一种新型NAMPT酶抑制PROTAC分子NPT-B2,小鼠卵巢癌药效学研究表明NPT-B2具有比FK866更显著的抑制肿瘤效果。但是NPT-B2水溶性差,影响其适用性及吸收。纳米药物利用纳米尺度效应,对药物进行包载形成水分散的纳米粒子系统,具有较好的稳定性,降低药物毒性,并由载体特性具有缓控释能力。白蛋白(BSA)是一种生物安全性良好、可降解的纳米载体材料,已经被美国FDA收录可以用于人体[9]。此外,肿瘤组织对白蛋白具有摄取特性,白蛋白与肿瘤血管内皮细胞表面的Gp60受体结合,激活细胞内小窝蛋白-1,引起细胞膜内陷形成胞转小泡,将白蛋白载体从内皮细胞转运进入肿瘤组织间。同时肿瘤在生长过程中分泌的富含半胱氨酸的酸性蛋白功能类似于白蛋白受体,可吸引粘附白蛋白,促进其从组织间隙转运到肿瘤细胞内,聚集于肿瘤细胞内释放药物[10-11]。
本研究以牛血清白蛋白包载PROTAC药物NPT-B2,通过热驱动法[12]制备白蛋白纳米粒,提高其载药量和包封率,改善了PROTAC药物的水溶性不佳的问题,提高其适用性;并考察载药纳米粒对胶质瘤细胞增殖活性及NAMPT酶的抑制。
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如图1A所示,NPT-B2对U251细胞的IC50值为61.16 nmol/L,表明NPT-B2对其有一定毒性作用。Western Blot结果如图1B所示,当NPT-B2给药浓度为100 nmol/L时,NPT-B2对U251细胞中NAMPT具有显著降解效果。
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如图2所示,方法专属性良好,NPT-B2保留时间为8.5 min,载体对NPT-B2测定无干扰。6.25~100 ng/ml的浓度范围内,NPT-B2浓度(C, ng/ml)与曲线下面积(Area)呈良好线性关系,线性回归方程为A=24 998 C+30 006,R2=0.9997。
由表1可知,B2-BSA-NPs在低、中、高三个浓度都具有较好的精密度,日间精密度和日内精密度RSD值均小于5%。由表2可知,B2-BSA-NPs在低、中、高三个浓度的回收率在95%~115%范围内,且RSD值均小于5%,表明该方法稳定可靠,可用于样品中NPT-B2含量的测定。
表 1 NPT-B2的HPLC方法精密度(Mean±SD,n=3)
加样量
(ng/ml)日内精密度 日间精密度 测得量
(ng/ml)RSD
(%)测得量
(ng/ml)RSD
(%)12.5 11.78±0.04 0.34 11.78±0.02 0.20 50 50.47±0.14 0.28 51.29±0.70 1.36 80 82.18±0.11 0.14 82.91±1.00 1.22 表 2 NPT-B2的HPLC方法提取回收率(Mean±SD,n=3)
加样量
(ng/ml)测得量
(ng/ml)平均回收率
(%)RSD
(%)25 25.97±0.45 103.89±1.81 1.74 50 56.77±0.08 113.54±0.17 0.15 80 89.06±0.24 111.33±0.30 0.27 -
结果如表3所示,随着载体比例的降低,B2-BSA-NPs粒径逐渐增大,包封率逐渐减小,载药量先增大后减小。故选择药载比=1∶10作为最佳制备条件。
表 3 不同药载比对B2-BSA-NPs粒径、包封率、载药量的影响(Mean±SD,n=3)
药载比
(w/w)粒径
(nm)包封率
(%)载药量
(%)1∶30 33.18±0.60 99.53±0.75 3.21±0.02 1∶20 55.48±5.29 98.06±1.69 4.67±0.08 1∶15 77.67±1.42 96.75±1.31 6.05±0.08 1∶10 86.86±1.50 94.41±1.28 8.58±0.12 1∶5 109.40±0.71 29.13±1.75 4.86±0.29 -
结果如表4所示,反应温度为37 ℃时,随着反应时间的延长,B2-BSA-NPs的粒径逐渐减小,包封率逐渐增大,载药量逐渐增大;反应温度为70 ℃时,随着反应时间的延长,B2-BSA-NPs的粒径逐渐增大,包封率逐渐增大,载药量逐渐增大。反应温度为37 ℃,反应时长2 h和3 h时,B2-BSA-NPs的粒径、包封率、载药量无显著性差异(P>0.05)。选择温度为37 ℃,反应时长为2 h作为最佳制备条件。
表 4 不同反应温度和时间对B2-BSA-NPs粒径、包封率、载药量的影响(Mean±SD,n=3)
反应温度和时间 粒径
(nm)包封率
(%)载药量
(%)37 ℃, 1 h 86.15±2.84 73.25±1.83 6.66±0.17 37 ℃, 2 h 54.92±1.61 94.74±1.25 8.61±0.11 37 ℃, 3 h 52.15±2.69 96.85±0.57 8.78±0.05 70 ℃, 3 min 54.29±0.68 47.33±2.31 4.30±0.21 70 ℃, 5 min 76.04±1.12 51.52±1.82 5.23±0.17 -
选择最优处方后制备B2-BSA-NPs并对其进行表征。如图3所示,B2-BSA-NPs的粒径、Zeta电位分别为55.48±5.30 nm,−12.9±1.21 mV,透射电镜照片显示B2-BSA-NPs为类圆形粒子,粒径约为50 nm。纳米粒包封率为94.74±1.25%,载药量为8.61±0.11%。
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如图4A所示,B2-BSA-NPs对U251细胞的IC50值为41.21 nmol/L,显著优于NPT-B2,表明B2-BSA-NPs对U251细胞有更强的毒性作用。如图4B所示,当B2-BSA-NPs给药浓度为100 nmol/L时,对U251中NAMPT具有非常显著降解效果。
Construction of albumin nanoparticles loading PROTAC and its inhibition effect on NAD+ in glioma cells
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摘要:
目的 制备载蛋白降解靶向嵌合体(PROTAC)分子的白蛋白纳米粒并优化处方,考察纳米粒对胶质瘤细胞增殖活性及烟酰胺腺嘌呤二核苷酸(NAD+)代谢的抑制。 方法 以热驱动法制备载NPT-B2的白蛋白纳米粒并表征,建立NPT-B2的高效液相色谱(HPLC)检测方法,以CCK8法和蛋白免疫印迹法分别考察NPT-B2和白蛋白纳米粒(B2-BSA-NPs)对胶质瘤细胞的增值活性抑制作用,及肿瘤细胞限速酶-烟酰胺磷酸核糖转移酶(NAMPT)的降解。 结果 HPLC方法线性良好,精密度、回收率符合测定要求。纳米粒粒径为55.48 nm、电位−12.9 mV,包封率94.74%,载药量8.61%。NPT-B2对胶质瘤细胞的IC50为61.16 nmol/L,同时具有NAMPT降解作用。B2-BSA-NPs对胶质瘤细胞的IC50为41.21 nmol/L,对NAMPT具有非常显著降解效果。 结论 构建了载PROTAC分子的白蛋白纳米粒,优化处方提高药物包封率,改善PROTAC分子水溶性差的问题,纳米粒对胶质瘤细胞增殖活性及NAD+能量代谢有显著抑制作用。 Abstract:Objective To prepare and optimize the formulation of Albumin nanoparticles loading PROTAC molecule and observe the inhibition effect of nanoparticles on the proliferation and NAD+ metabolism of glioma cells. Methods Albumin nanoparticles loading NPT-B2 were prepared and characterized with a thermal driving method, and the prescription was optimized. An HPLC method was established to determine the content of NPT-B2. The proliferation inhibition of NPT-B2 and B2-BSA-NPs on U251 cells were investigated by the CCK8 method, and the degradation effects of NPT-B2 and B2-BSA-NPs on NAMPT in glioma cells were investigated by western blotting. Results The HPLC method was stable, with good linearity, precision, and recovery rate. The nanoparticles had a particle size of about 55.48 nm, a potential of about −12.9 mV, an encapsulation rate of about 94.74%, and a drug loading amount of about 8.61%. The IC50 of NPT-B2 on glioma cells was 61.16 nmol/L, which had a degradation effect on NAMPT. The IC50 of B2-BSA-NPs on glioma was 41.21 nmol/L, which had a very significant degradation effect on NAMPT. Conclusion Albumin nanoparticles loading PROTAC molecules were constructed. The prescription was optimized to improve the drug encapsulation rate, and the low water solubility of PROTAC molecule was improved, which had a significant inhibitory effect on the proliferation and NAD+ energy metabolism of glioma cells. -
Key words:
- Glioma /
- Nanoparticle /
- Albumin /
- PROTAC /
- HPLC
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表 1 NPT-B2的HPLC方法精密度(Mean±SD,n=3)
加样量
(ng/ml)日内精密度 日间精密度 测得量
(ng/ml)RSD
(%)测得量
(ng/ml)RSD
(%)12.5 11.78±0.04 0.34 11.78±0.02 0.20 50 50.47±0.14 0.28 51.29±0.70 1.36 80 82.18±0.11 0.14 82.91±1.00 1.22 表 2 NPT-B2的HPLC方法提取回收率(Mean±SD,n=3)
加样量
(ng/ml)测得量
(ng/ml)平均回收率
(%)RSD
(%)25 25.97±0.45 103.89±1.81 1.74 50 56.77±0.08 113.54±0.17 0.15 80 89.06±0.24 111.33±0.30 0.27 表 3 不同药载比对B2-BSA-NPs粒径、包封率、载药量的影响(Mean±SD,n=3)
药载比
(w/w)粒径
(nm)包封率
(%)载药量
(%)1∶30 33.18±0.60 99.53±0.75 3.21±0.02 1∶20 55.48±5.29 98.06±1.69 4.67±0.08 1∶15 77.67±1.42 96.75±1.31 6.05±0.08 1∶10 86.86±1.50 94.41±1.28 8.58±0.12 1∶5 109.40±0.71 29.13±1.75 4.86±0.29 表 4 不同反应温度和时间对B2-BSA-NPs粒径、包封率、载药量的影响(Mean±SD,n=3)
反应温度和时间 粒径
(nm)包封率
(%)载药量
(%)37 ℃, 1 h 86.15±2.84 73.25±1.83 6.66±0.17 37 ℃, 2 h 54.92±1.61 94.74±1.25 8.61±0.11 37 ℃, 3 h 52.15±2.69 96.85±0.57 8.78±0.05 70 ℃, 3 min 54.29±0.68 47.33±2.31 4.30±0.21 70 ℃, 5 min 76.04±1.12 51.52±1.82 5.23±0.17 -
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