School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Quai Ernest Ansermet 30, CH-1211 Geneva 4, Switzerland.
Eur J Pharm Sci. 2009 Oct 8;38(3):230-7. doi: 10.1016/j.ejps.2009.07.006. Epub 2009 Jul 24.
Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).
紫杉醇（Tx）负载的抗 HER2 免疫纳米粒子（NPs-Tx-HER）通过将人源化单克隆抗 HER2 抗体（曲妥珠单抗，赫赛汀）共价偶联到 Tx 负载的聚（dl-乳酸）纳米粒子（NPs-Tx）来制备，用于主动靶向过表达 HER2 受体的肿瘤细胞。将 NPs-Tx-HER 的理化性质与未负载的免疫纳米粒子（NPs-HER）进行比较，以评估药物对抗体与 NP 表面偶联的影响。用磺基-MBS 激活的抗 HER2 mAbs 测试了 Sulfo-MBS 激活的抗 HER2 mAbs 的免疫反应性和 NPs-Tx-HER 的体外功效，并在过表达 HER2 抗原的 SKOV-3 卵巢癌细胞上进行了测试。通过盐析法获得的负载 Tx 的纳米粒子（NPs-Tx）的粒径为 171+/-22nm（PI=0.1），包封效率约为 78+/-10%，对应于 7.8+/-0.8%（w/w）的载药量。然后将 NPs-Tx 巯基化并与激活的抗 HER2 mAbs 缀合，得到 237+/-43nm 的免疫纳米粒子（PI=0.2）。使用 125I 放射性标记的 mAbs 在 SKOV-3 细胞上测试了活化步骤对 mAbs 免疫反应性的影响，并且磺基-MBS 功能化后抗 HER2 mAbs 的活性几乎没有受到影响。每个纳米粒子大约结合了 270 个抗 HER2 mAbs。NPs-Tx-HER 的 zeta 电位为 0.2+/-0.1mV。负载 Tx 的免疫纳米粒子的理化性质与未负载的免疫纳米粒子非常相似，这表明药物的包封并不影响 mAbs 与 NPs 的偶联。在制备过程中未观察到药物损失。DSC 分析表明，包封的 Tx 处于无定形或无序结晶相。这些结果表明，Tx 被包埋在聚合物基质中，而不是吸附在 NPs 的表面。在 SKOV-3 卵巢癌细胞上的体外研究表明，与其他 Tx 制剂相比，NPs-Tx-HER 具有更强的细胞毒性作用。结果表明，在 1ngTx/ml 时，与孵育在含有 NP-Tx-HER 药物的细胞相比，孵育在 NP-Tx（97.4+/-12%）、涂有 Mabthera 的免疫纳米粒子（NPs-Tx-RIT）（93.8+/-12%）或游离药物（92.3+/-9.3%）的细胞的存活率较低（77.32+/-5.48%）。
