Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing, Zhejiang, PR China; CICS-UBI, Health Sciences Research Center, University of Beira Interior, Covilhã, Portugal; Department of Medical Biology, Cancer Center Amsterdam, Amsterdam UMC Location Academic Medical Center, Amsterdam, the Netherlands; Laboratory of Experimental Oncology, Department of Pathology, Erasmus MC, Rotterdam, the Netherlands.
Jiaxing Key Laboratory for Photonanomedicine and Experimental Therapeutics, Department of Pharmaceutics, College of Medicine, Jiaxing University, Jiaxing, Zhejiang, PR China; Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Utrecht, the Netherlands; Membrane Biochemistry and Biophysics, Department of Chemistry, Faculty of Science, Utrecht University, Utrecht, the Netherlands.
J Photochem Photobiol B. 2022 Sep;234:112500. doi: 10.1016/j.jphotobiol.2022.112500. Epub 2022 Jun 17.
A photosensitizer (PS) delivery and comprehensive tumor targeting platform was developed that is centered on the photosensitization of key pharmacological targets in solid tumors (cancer cells, tumor vascular endothelium, and cellular and non-cellular components of the tumor microenvironment) before photodynamic therapy (PDT). Interstitially targeted liposomes (ITLs) encapsulating zinc phthalocyanine (ZnPC) and aluminum phthalocyanine (AlPC) were formulated for passive targeting of the tumor microenvironment. In previous work it was established that the PEGylated ITLs were taken up by cultured cholangiocarcinoma cells. The aim of this study was to verify previous results in cancer cells and to determine whether the ITLs can also be used to photosensitize cells in the tumor microenvironment and vasculature. Following positive results, rudimentary in vitro and in vivo experiments were performed with ZnPC-ITLs and AlPC-ITLs as well as their water-soluble tetrasulfonated derivatives (ZnPCS4 and AlPCS4) to assemble a research dossier and bring this platform closer to clinical transition.
Flow cytometry and confocal microscopy were employed to determine ITL uptake and PS distribution in cholangiocarcinoma (SK-ChA-1) cells, endothelial cells (HUVECs), fibroblasts (NIH-3T3), and macrophages (RAW 264.7). Uptake of ITLs by endothelial cells was verified under flow conditions in a flow chamber. Dark toxicity and PDT efficacy were determined by cell viability assays, while the mode of cell death and cell cycle arrest were assayed by flow cytometry. In vivo systemic toxicity was assessed in zebrafish and chicken embryos, whereas skin phototoxicity was determined in BALB/c nude mice. A PDT efficacy pilot was conducted in BALB/c nude mice bearing human triple-negative breast cancer (MDA-MB-231) xenografts.
The key findings were that (1) photodynamically active PSs (i.e., all except ZnPCS4) were able to effectively photosensitize cancer cells and non-cancerous cells; (2) following PDT, photodynamically active PSs were highly toxic-to-potent as per anti-cancer compound classification; (3) the photodynamically active PSs did not elicit notable systemic toxicity in zebrafish and chicken embryos; (4) ITL-delivered ZnPC and ZnPCS4 were associated with skin phototoxicity, while the aluminum-containing PSs did not exert detectable skin phototoxicity; and (5) ITL-delivered ZnPC and AlPC were equally effective in their tumor-killing capacity in human tumor breast cancer xenografts and superior to other non-phthalocyanine PSs when appraised on a per mole administered dose basis.
AlPC(S4) are the safest and most effective PSs to integrate into the comprehensive tumor targeting and PS delivery platform. Pending further in vivo validation, these third-generation PSs may be used for multi-compartmental tumor photosensitization.
本研究开发了一种基于光敏化实体瘤（癌细胞、肿瘤血管内皮细胞及肿瘤微环境中的细胞和非细胞成分）中关键药理靶点的光敏剂（PS）递药和综合肿瘤靶向平台，用于光动力疗法（PDT）之前。我们制备了包载锌酞菁（ZnPC）和铝酞菁（AlPC）的间质靶向脂质体（ITL），用于被动靶向肿瘤微环境。在之前的工作中，我们已经证实 PEG 化的 ITL 被胆管癌细胞摄取。本研究的目的是在癌细胞中验证之前的结果，并确定 ITL 是否也可用于光敏化肿瘤微环境和脉管系统中的细胞。在获得阳性结果后，我们用 ZnPC-ITL 和 AlPC-ITL 及其水溶性四磺化衍生物（ZnPCS4 和 AlPCS4）进行了初步的体外和体内实验，以构建研究档案并使该平台更接近临床转化。
采用流式细胞术和共聚焦显微镜检测胆管癌细胞（SK-ChA-1）、内皮细胞（HUVEC）、成纤维细胞（NIH-3T3）和巨噬细胞（RAW 264.7）摄取 ITL 和 PS 分布。在流动腔中通过流动条件下的内皮细胞摄取来验证 ITL 的摄取。通过细胞活力测定法确定暗毒性和 PDT 疗效，通过流式细胞术测定细胞死亡方式和细胞周期停滞。在斑马鱼和鸡胚中评估体内全身性毒性，在 BALB/c 裸鼠中评估皮肤光毒性。在荷人三阴性乳腺癌（MDA-MB-231）异种移植瘤的 BALB/c 裸鼠中进行了 PDT 疗效初步研究。
主要发现如下：（1）光动力学活性 PS（即除 ZnPCS4 以外的所有 PS）能够有效光敏化癌细胞和非癌细胞；（2）PDT 后，光动力学活性 PS 具有高度的细胞毒性-效力，符合抗癌化合物分类；（3）在斑马鱼和鸡胚中，光动力学活性 PS 未引起明显的全身毒性；（4）ITL 递药的 ZnPC 和 ZnPCS4 与皮肤光毒性有关，而含铝的 PS 则未显示出可检测的皮肤光毒性；（5）在评估每摩尔给药剂量时，ITL 递药的 ZnPC 和 AlPC 在杀伤人肿瘤乳腺癌异种移植瘤方面同样有效，优于其他非酞菁 PS。
AlPC（S4）是整合到综合肿瘤靶向和 PS 递药平台中最安全和最有效的 PS。在进一步的体内验证之前，这些第三代 PS 可用于多隔室肿瘤光敏化。
