UPAR-Targeted Upconversion Nanoparticles For In Vivo Imaging Of Pancreatic Cancer

Part I:Construction of uPAR Targeted Upconversion Nanopar-ticlesBackground:Complete removal of tumors by surgery is the most important prognostic factor for pancreatic cancer patients with the early stage cancers. The ability to identify invasive tumor edges of the primary tumor, locally invaded small tumor lesions during surgery should significantly reduce the incidence of local tumor recurrence and improve survival of cancer patients. Molecular imaging probes based on upconversion nanoparticles have provided great hope for pancreatic cancer margins detection. The unique anti-Stokes mechanism of the upconversion luminescence probes offers greater penetration of tissue by the NIR excitation and eliminates the interference of endogenous fluorescence, giving rise to an enhanced signal-to-noise ratio (SNR). Moreover, with the incorporation of gadolinium in the nanoparticle matrix, dual-modality imaging probes can be constructed with integrated magnetic and upconversion properties. Urokinase plasminogen activator receptor (uPAR), a cellular receptor that is highly expressed in pancreatic cancer and tumor stromal cells, is an excellent surface molecule for receptor-targeted imaging. It is a serine protease that interacts with its activator to regulate multiple pathways involved in matrix degradation, cell motility, and metastasis. uPAR-targeted upconversion nanoparticles integrated nanotechnology, molecular targeting technology and multi-modality molecular imaging technology would hold a promising for identification of tumor margins and change the traditional single downconversion luminescent imaging mode, as near-infrared excitation can better penetrate tissue than visible light while giving rise to greatly reduced background noise.Purpose:The major objective of the investigation was to designed and prepared uPAR-targeted probe by conjugating amino-terminal fragment (ATF) of the receptor binding domain of human urokinase plasminogen activator (uPA) to the surface of upconversion nanoparticles to achieve uPAR-targeting specificity.Methods:The NaGdF4:Yb,Er nanoparticles were synthesized through a high temperature approach. To further increase the photo luminescence intensity, core/shell structured NaGdF4:Yb,Er@NaGdF4 nanoparticles were synthesized by using the NaGdF4:Yb,Er nanoparticles as growth seeds. A ligand exchange process by using asymmetric PEG simultaneously bearing with phosphate and maleimide groups as ligands was applied for rendering the as prepared core/shell nanoparticles with water solubility and biocompatibility. The properties of core and core@shell nanoparticles were examined by transmission electron microscopy (TEM), spectral scanner and 3.0T MRI. A cDNA fragment encoding amino acids 1-135 of human uPA was isolated by PCR amplification using a PCR primer pair. Recombinant ATF peptides were expressed in E. coli DH5α and purified from bacterial extracts under native conditions using a Ni2+ NTA-agarose column. The uPAR-targeted probe was contructed between the maleimide residue on the surface of the nanoparticle and peptide ATF. Hydrodynamic size of the probes and conjugates dispersed in PBS were characterized by dynamic light scattering(DLS). By using the same approach, the NaGdF4:Yb,Er@NaGdF4-mIgG conjugate was also constructed and used as a negative control. After incubation with the targeted and non-targeted probes, the cells were subjected to MarkⅡ and MRI. The typical image-streams were captured. The MRI contrast enhancement effect of the cells after incubation was also characterized.Results:Photoluminescence (PL) intensity of the core@shell nanoparticles, normalized according to Er3+ concentration, is increased by a factor of 70 compared with that for the core nanoparticles. The uniformity of size and shape remain almost unchanged after shell growth. Optical studies revealed that the PL intensity of the nanoparticles dropped significantly after being transferred into water, likely due to the high-energy vibration effect of water, however, it remained 57 times higher than that of the PEGylated core nanoparticles. The slight decrease of the r1 value for the core@shell particles can be attributed to the reduced relative surface coverage of Gd3+. A reasonable increment in the hydrodynamic size of the conjugates indicates the successful construction of the NaGdF4:Yb,Er@NaGdF4-ATF (UCNP-ATF) probe. The typical ImageStream MarkⅡ images indicate that the UCNP-ATF probes possess outstanding specific binding specificity for the target cells. MRI scan of the cells after incubating with uPAR-targeted nanoparticles and non-targeted nanoparticles further demonstrated target specificity, because MRI signal enhancement was detected in uPAR expressing cells.Conclusions:In this study, we prepared the nanoparticle-based dual-modal molecular probes for detection of tumor margin. The probes taking biocompatibility upconversion nanoparticles with unique magnetic properties as a carrier, conjugated with ATF peptide specifically targeting the uPAR.Part II:uPAR-targeted Upconversion Nanoparticles for In Vivo Imaging of Pancreatic CancerBackground:Apart from imaging modalities, tumor models that can better represent vasculature, pathophysiological processes, and tumor microenvironment are essentially required. Owing to the advances in the biocompatible and biofunctionalizable modifications of rare-earth particles, NaGdF4:Yb,Er nanoparticles were successfully used for optical detection of tiny subcutaneous tumors less than 2 mm in diameter via active targeting. In comparison with subcutaneous tumor models, orthotopic models are highly preferable by showing higher metastatic rates and a more human-analogous micro-environment. Nevertheless, due to the complexity of the model construction and the poor performance of the available nanoprobes, very limited success has been achieved. In a recent study, uPAR-specific fluorescence-labeled peptide probe was reported for imaging an orthotopically transplanted pancreatic cancer. However, the signal was heavily interfered with by the autofluorescence of the surrounding tissue. Therefore, developing advanced optical probes suitable for orthotopic tumor detection remains challenging.Purpose:The major objective of the investigation was to further validate the effect of detection of tumor margins of uPAR-targeted upconversion nanoparticles in an orthotopic human pancreatic cancer xenograft model by pathology and dual-modal imaging.Methods:An orthotopic pancreatic tumor model was established by a surgical procedure. To monitor the tumor growth, a stably GFP-Luciferase-expressing SW1990 cell line (GFP-Luc-SW1990) was used to enable constant monitoring of tumor development. We transduced Lentiviral vector (PRRL-EF1a-GFPluc-WPRE) into the SW1990 cells, which is a typical pancreatic cancer cell line, and analyzed cells for GFPluc expression using flow cytometer. The nude mice were screened by bioluminescence imaging (BLI) to verify successful inoculation. Tumor-bearing mice were subjected to MRI measurements at specific time points of 2,4,6h after intravenous delivery of the targeted probe through tail vein injection. The upconversion luminescence images were acquired at specific time points of 2,4,6h as well, which were carefully chosen based on the aforementioned MRI results. The mice were sacrificed after the imaging study was completed. The tumor tissue sections were examined by HE. Other normal tissues were collected, and ex vivo ICP test was performed at the same time.Results:Vetor transduced SW1990 effeciency is 99.9%. Systemic delivery of the uPAR-targeted imaging probes in mice bearing orthotopic human pancreatic tumor xenografts tumors led to the accumulation of the probes in the tumor, resulting in strong signals for optical imaging of tumors and identification of tumor margins. Histological analysis showed that a high level of uPAR-targeted nanoparticles was present in the tumor. Moreover, it has been demonstrated that primary sites in an orthotopic mouse model of human pancreatic cancer can be MRI detected by using uPAR-specific upconversion luminescence nanoprobes.Conclusions:Our results revealed that the probe could be useful for small tumor lesion diagnosis, indicating potential clinical applications in the early pancreatic cancer diagnosis and image-guide surgery.