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HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents
Journal of Liposome Research ( IF 3.6 ) Pub Date : 2008-01-01 , DOI: 10.1080/08982100802457377
Anu Puri 1 , Gabriela Kramer-Marek , Ryan Campbell-Massa , Amichai Yavlovich , Shrikant C Tele , Sang-Bong Lee , Jeffrey D Clogston , Anil K Patri , Robert Blumenthal , Jacek Capala
Affiliation  

Thermosensitive liposomes are attractive vehicles for the delivery and release of drugs to tumors. To improvethe targeting efficacy for breast cancer treatment, an 8.3-kDa HER2-specific Affibody molecule (ZHER2:342-Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nanoparticles denoted as “Affisomes” were investigated. Thermosensitive small unilamellar vesicle (SUV) liposomes of (80–100 nm) a diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41°C) as the matrix lipid and a maleimide-conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered-release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (e.g., tributylphosphine) for 16–20 hours under an argon atmosphere. Lipid-conjugated affibody molecule was visible as an 11.3-kDa band on a 4–12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were ∼70% at a protein-lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermosensitivity of Affisomes was determined by monitoring the release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as nontargeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90–100%) at 41°C. In contrast, liposomes prepared from Egg phosphatidyl choline (Egg PC, Tm ∼0°C) under similar conditions released only 5–10% calcein at 41°C. Affisomes, when stored at room temperature, retained > 90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in phosphate-buffered saline, supplemented with 10% heat-inactivated serum (fetal bovine serum) did not result in a destabilization of liposomes. Therefore, Affisomes present promising, novel drug-delivery candidates for breast cancer targeting.

中文翻译:

HER2 特异性 Affibody 偶联热敏脂质体 (Affisomes),用于改善抗癌剂的递送

热敏脂质体是用于向肿瘤递送和释放药物的有吸引力的载体。为了提高乳腺癌治疗的靶向功效,将 8.3-kDa HER2 特异性 Affibody 分子 (ZHER2:342-Cys) 偶联到脂质体表面。研究了这种修饰对表示为“Affisomes”的所得纳米粒子的物理特性和稳定性的影响。由二棕榈酰磷脂酰胆碱(DPPC,Tm 41°C)作为基质脂质和马来酰亚胺偶联的聚乙二醇化磷脂(DSPE-MaL-PEG2000)组成的直径为(80-100 nm)的热敏小单层囊泡(SUV)脂质体是通过以下方法制备的探头超声。荧光探针被掺入脂质体中,用于生物物理和/或生化分析和/或触发释放测定。通过在还原剂(例如三丁基膦)存在下在氩气气氛下孵育 16-20 小时,Affibody 通过其 C 端半胱氨酸与这些脂质体结合。在还原条件下,在 4–12% Bis/Tris 凝胶上,脂质偶联的亲和体分子以 11.3 kDa 的条带可见。在 20 μg/mg 的蛋白质 - 脂质比下,Affibody 缀合产量约为 70%,每个 Affibody 平均有 200 个 Affibody 分子。Affibody 与热敏脂质体的结合对脂质体的流体动力学尺寸分布没有任何显着影响。Affisomes 的热敏性通过监测作为温度函数的包埋的钙黄绿素(一种水溶性荧光探针,λex/em 490/515 nm)的释放来确定。钙黄绿素以温度依赖性方式从 Affisomes(具有 Affibody 靶向 SUV 的热敏脂质体)和非靶向 SUV(无 Affibody 的热敏脂质体)中释放,在 41°C 下具有最佳泄漏(90-100%)。相比之下,在类似条件下由卵磷脂​​酰胆碱(Egg PC,Tm ∼0°C)制备的脂质体在 41°C 下仅释放 5-10% 的钙黄绿素。Affisomes 在室温下储存时,可保留 > 90% 的钙黄绿素长达 7 天。此外,在补充有 10% 热灭活血清(胎牛血清)的磷酸盐缓冲盐水中孵育脂质体不会导致脂质体不稳定。因此,Affisomes 为乳腺癌靶向提供了有前景的新型药物递送候选药物。在 41°C 下具有最佳泄漏 (90–100%)。相比之下,在类似条件下由卵磷脂​​酰胆碱(Egg PC,Tm ∼0°C)制备的脂质体在 41°C 下仅释放 5-10% 的钙黄绿素。Affisomes 在室温下储存时,可保留 > 90% 的钙黄绿素长达 7 天。此外,在补充有 10% 热灭活血清(胎牛血清)的磷酸盐缓冲盐水中孵育脂质体不会导致脂质体不稳定。因此,Affisomes 为乳腺癌靶向提供了有前景的新型药物递送候选药物。在 41°C 下具有最佳泄漏 (90–100%)。相比之下,在类似条件下由卵磷脂​​酰胆碱(Egg PC,Tm ∼0°C)制备的脂质体在 41°C 下仅释放 5-10% 的钙黄绿素。Affisomes 在室温下储存时,可保留 > 90% 的钙黄绿素长达 7 天。此外,在补充有 10% 热灭活血清(胎牛血清)的磷酸盐缓冲盐水中孵育脂质体不会导致脂质体不稳定。因此,Affisomes 为乳腺癌靶向提供了有前景的新型药物递送候选药物。在磷酸盐缓冲盐水中孵育脂质体,并补充 10% 热灭活血清(胎牛血清)不会导致脂质体不稳定。因此,Affisomes 为乳腺癌靶向提供了有前景的新型药物递送候选药物。在磷酸盐缓冲盐水中孵育脂质体,并补充 10% 热灭活血清(胎牛血清)不会导致脂质体不稳定。因此,Affisomes 为乳腺癌靶向提供了有前景的新型药物递送候选药物。
更新日期:2008-01-01
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