Dobiasch Sophie, Szanyi Szilard, Kjaev Aleko, Werner Jens, Strauss Albert, Weis Christian, Grenacher Lars, Kapilov-Buchman Katya, Israel Liron-Limor, Lellouche Jean-Paul, Locatelli Erica, Franchini Mauro Comes, Vandooren Jennifer, Opdenakker Ghislain, Felix Klaus
Department of Surgery, University of Heidelberg, Im Neuenheimer Feld 110, 69120, Heidelberg, Germany.
Department of Radiation Oncology, Technische Universität München, Munich, Germany.
J Nanobiotechnology. 2016 Dec 19;14(1):81. doi: 10.1186/s12951-016-0236-3.
Functionalized nanoparticles (NPs) are one promising tool for detecting specific molecular targets and combine molecular biology and nanotechnology aiming at modern imaging. We aimed at ligand-directed delivery with a suitable target-biomarker to detect early pancreatic ductal adenocarcinoma (PDAC). Promising targets are galectins (Gal), due to their strong expression in and on PDAC-cells and occurrence at early stages in cancer precursor lesions, but not in adjacent normal tissues.
Molecular probes (10-29 AA long peptides) derived from human tissue plasminogen activator (t-PA) were selected as binding partners to galectins. Affinity constants between the synthesized t-PA peptides and Gal were determined by microscale thermophoresis. The 29 AA-long t-PA-peptide-1 with a lactose-functionalized serine revealed the strongest binding properties to Gal-1 which was 25-fold higher in comparison with the native t-PA protein and showed additional strong binding to Gal-3 and Gal-4, both also over-expressed in PDAC. t-PA-peptide-1 was selected as vector moiety and linked covalently onto the surface of biodegradable iron oxide nanoparticles (NPs). In particular, CAN-doped maghemite NPs (CAN-Mag), promising as contrast agent for magnetic resonance imaging (MRI), were selected as magnetic core and coated with different biocompatible polymers, such as chitosan (CAN-Mag-Chitosan NPs) or polylactic co glycolic acid (PLGA) obtaining polymeric nanoparticles (CAN-Mag@PNPs), already approved for drug delivery applications. The binding efficacy of t-PA-vectorized NPs determined by exposure to different pancreatic cell lines was up to 90%, as assessed by flow cytometry. The in vivo targeting and imaging efficacy of the vectorized NPs were evaluated by applying murine pancreatic tumor models and assessed by 1.5 T magnetic resonance imaging (MRI). The t-PA-vectorized NPs as well as the protease-activated NPs with outer shell decoration (CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1) showed clearly detectable drop of subcutaneous and orthotopic tumor staining-intensity indicating a considerable uptake of the injected NPs. Post mortem NP deposition in tumors and organs was confirmed by Fe staining of histopathology tissue sections.
The targeted NPs indicate a fast and enhanced deposition of NPs in the murine tumor models. The CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1 interlocking steps strategy of NPs delivery and deposition in pancreatic tumor is promising.
功能化纳米颗粒（NPs）是检测特定分子靶点的一种有前景的工具，它结合了分子生物学和纳米技术以实现现代成像。我们旨在通过合适的靶向生物标志物进行配体导向递送，以检测早期胰腺导管腺癌（PDAC）。由于半乳糖凝集素（Gal）在PDAC细胞内和细胞表面强烈表达，且在癌前病变早期出现，而在相邻正常组织中不出现，因此是很有前景的靶点。
从人组织纤溶酶原激活物（t-PA）衍生的分子探针（10 - 29个氨基酸长的肽段）被选为与半乳糖凝集素的结合伙伴。通过微量热泳测定合成的t-PA肽段与Gal之间的亲和常数。带有乳糖功能化丝氨酸的29个氨基酸长的t-PA肽段-1显示出与Gal-1最强的结合特性，与天然t-PA蛋白相比高25倍，并且还显示出与Gal-3和Gal-4的强结合，这两种蛋白在PDAC中也过表达。t-PA肽段-1被选作载体部分，并共价连接到可生物降解的氧化铁纳米颗粒（NPs）表面。特别地，有前景作为磁共振成像（MRI）造影剂的掺铈磁赤铁矿NPs（CAN-Mag）被选作磁芯，并涂覆不同的生物相容性聚合物，如壳聚糖（CAN-Mag-壳聚糖NPs）或聚乳酸-羟基乙酸共聚物（PLGA），从而获得已被批准用于药物递送应用的聚合物纳米颗粒（CAN-Mag@PNPs）。通过流式细胞术评估，暴露于不同胰腺细胞系后测定的t-PA载体化NPs的结合效率高达90%。通过应用小鼠胰腺肿瘤模型评估载体化NPs的体内靶向和成像效果，并通过1.5 T磁共振成像（MRI）进行评估。t-PA载体化NPs以及具有外壳修饰的蛋白酶激活NPs（CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1）显示皮下和原位肿瘤染色强度明显可检测到下降，表明注射的NPs有相当程度的摄取。通过组织病理学组织切片的铁染色证实了NPs在肿瘤和器官中的死后沉积。
靶向NPs表明在小鼠肿瘤模型中NPs快速且增强的沉积。CAN-Mag@PNPs-PEG-REGAcp-PEG/tPA-pep1在胰腺肿瘤中NPs递送和沉积的联锁步骤策略很有前景。
