Colzani Barbara, Pandolfi Laura, Hoti Ada, Iovene Pietro Alessandro, Natalello Antonino, Avvakumova Svetlana, Colombo Miriam, Prosperi Davide
Department of Biotechnology and Biosciences, University of Milano Bicocca, Milano, Italy.
Nanomedicine Laboratory, ICS Maugeri S. p. A. SB, Pavia, Italy.
Int J Nanomedicine. 2018 Feb 14;13:957-973. doi: 10.2147/IJN.S152742. eCollection 2018.
We report the development of an efficient antibody delivery system for the incorporation of trastuzumab (TZ) into poly(lactic--glycolic) acid nanoparticles (PLGA NPs). The aim of the work was to overcome the current limitations in the clinical use of therapeutic antibodies, including immunogenicity, poor pharmacokinetics, low tumor penetration and safety issues.
Trastuzumab-loaded PLGA NPs (PLGA-TZ) were synthesized according to a double emulsion method. The same protocol was used to produce control batches of nonspecific IgG-loaded NPs and empty PLGA NPs. After release of TZ from PLGA NPs, the effects on the main biological activities of the antibody were evaluated on SKBR3 (human epidermal growth factor receptor 2 [HER2]-positive breast cancer cell line), including specific binding to HER2, phosphorylation of HER2 (Y1248), degradation of HER2 protein and antibody-dependent cell-mediated cytotoxicity (ADCC) mechanism. In addition, an MTT assay was performed for treating SKBR3 cells with PLGA NPs loaded with TZ and doxorubicin to evaluate the cytotoxic activity of the combined treatment.
TZ was gradually released in a prolonged way over 30 days. The physical characterization performed with circular dichroism, Fourier transform infrared and fluorescence spectroscopy of TZ after release demonstrated that no structural alterations occurred compared to the native antibody. In vitro experiments using SKBR3 cells showed that TZ released from PLGA NPs maintained the same biological activity of native TZ. PLGA NPs allowed a good co-encapsulation efficiency of TZ and doxorubicin resulting in improved therapy.
With the TZ case study, we demonstrate that the distinctive features of therapeutic monoclonal antibodies, including molecular targeting efficiency, capability to inhibit or properly affect the regulatory signaling pathways of cancer cells and stimulation of the ADCC, are fully preserved after loading into and release from PLGA NPs. In addition, PLGA NPs are shown to allow for the simultaneous incorporation of TZ and conventional chemotherapeutics, resulting in a potent antitumor nanodrug well suited for in situ combination and neoadjuvant therapy.
我们报道了一种高效抗体递送系统的研发，该系统用于将曲妥珠单抗（TZ）掺入聚乳酸-乙醇酸纳米颗粒（PLGA NPs）中。这项工作的目的是克服治疗性抗体临床应用中当前存在的局限性，包括免疫原性、药代动力学不佳、肿瘤穿透性低和安全性问题。
采用双乳液法合成负载曲妥珠单抗的PLGA NPs（PLGA-TZ）。使用相同方案制备负载非特异性IgG的NP和空PLGA NP的对照批次。在曲妥珠单抗从PLGA NPs释放后，在SKBR3（人表皮生长因子受体2 [HER2]阳性乳腺癌细胞系）上评估其对抗体主要生物学活性的影响，包括与HER2的特异性结合、HER2（Y1248）的磷酸化、HER2蛋白的降解以及抗体依赖性细胞介导的细胞毒性（ADCC）机制。此外，进行MTT试验，用负载曲妥珠单抗和阿霉素的PLGA NPs处理SKBR3细胞，以评估联合治疗的细胞毒性活性。
曲妥珠单抗在30天内逐渐持续释放。释放后对曲妥珠单抗进行圆二色性、傅里叶变换红外光谱和荧光光谱的物理表征表明，与天然抗体相比，未发生结构改变。使用SKBR3细胞的体外实验表明，从PLGA NPs释放的曲妥珠单抗保持了与天然曲妥珠单抗相同的生物学活性。PLGA NPs对曲妥珠单抗和阿霉素具有良好的共包封效率，从而改善了治疗效果。
通过曲妥珠单抗的案例研究，我们证明治疗性单克隆抗体的独特特性，包括分子靶向效率、抑制或适当影响癌细胞调节信号通路的能力以及刺激ADCC，在负载到PLGA NPs中并从其中释放后得到了充分保留。此外，PLGA NPs显示能够同时掺入曲妥珠单抗和传统化疗药物，从而形成一种适用于原位联合和新辅助治疗的强效抗肿瘤纳米药物。
