Peptide-lipid Nanoprobes For The Theranostics Of Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is an epidemic malignant cancer in Southern China and Southeast Asia with hidden location and early distant metastasis, which results to its treatment failure as one of the main reasons. Cisplatin-based combination chemotherapy remains the main treatment for locoregional advanced NPC, but over30%of patients fail with local recurrent or distant metastases in the treatment and their5-year survival rate is just25%-30%. Anti-epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab combined with chemotherapy improves the overall survival of metastatic NPC patients to some extent but still with side effects induced by human anti-mouse antibody (HAMA) response. Compared with small-molecule chemothera-peutic agents, nanocarrier-based drugs can extend the half-time of drugs in the blood, improve drugs utilization and increase intracellular concentration of drugs but with low side effects. Thus, it is valuable in clinical applications for developing nanodrugs targeted NPC with high efficiency to treat metastatic NPC. The innovation results in this thesis can be summarized as follows.1) We develop an octavalent peptide fluorescent nanoprobe (Octa-FNP) which can be used to evaluate peptides in vivo and utilize it to identify a NPC-targeting peptide TTP. This was accomplished by combining a tetrameric far-red fluorescent protein (tfRFP) as a scaffold with a tumor-targeting peptide to engineer an Octa-FNP. Whole-body imaging in vivo demonstrated Octa-FNP could specifically target5-8F subcutaneous tumor with enhanced tumor accumulation and high-contrast tumor imaging which was valuable for assessing the targeting capability of peptides. We used the methods to identify a NPC-targeting peptide LTVSPWY (TTP), which could high efficiently accumulate in the tumor tissue when radioimaging with125I-labeled Octa-FNP.2) We creat a novel NPC-targeting peptide R4F-TTP with synergistic enhancement and selective killing. It was designed by conjugating a tandem NPC-targeted peptide (LTVSPWY) with an amphipathic a-helical peptide R4F. Confocal imaging and FACS analysis demonstrated the uptake of R4F-TTP by5-8F cells after incubated24h is3.1-fold of TTP,10.4-fold of R4F, indicating R4F-TTP exhibit a synergistic effect on enhancing the targeting to5-8F cells. Propidium iodide (PI) staining further demonstrated the selective killing tumor cells of R4F-TTP.3) We develop a ultra-small nanocarrier for5-8F tumors with high-specific targeting and significant therapeutic effects. Both transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed NP-TTP with an average size of11nm and spherical structure. FACS analysis demonstrated the uptake of NP-TTP by5-8F cells after incubated3h is20.5-fold of TTP,3.0-fold of R4F-scrTTP (scrTTP is the scramble peptide of TTP). The enhanced specific uptake of NP-TTP by5-8F cells suggested that targeting peptide TTP was presented on the surface of the NP-TTP. Whole-body imaging demonstrated NP-TTP loaded with near infrared fluorescent dyes DiR-BOA could be efficiently accumulated in the5-8F-mRFP tumor and diffused into entire tumor tissue, which resulted in extremely high contrast between the tumor tissue and normal tissues, such as liver and kidney. When administered twice every other day by post-injection, NP-TTP achieved88±7%inhibition rate in subcutaneous growth of5-8F tumors and significant higher survival rate when compared with PBS. The inhibition of tumor growth was demonstrated to be related with the induction of apoptosis and autophagy formation by NP-TTP. When synchronized with curcumin, NP-TTP showed a strong preventive effect on5-8F metastasis.In sumarry, we invented an octavalent peptide fluorescent nanoprobe based on tetrameric far-red fluorescent protein and used it to identify a target-specific peptide TTP for NPC. Conjugating the TTP peptide with an amphipathic a-helical peptide R4F created a novel NPC-targeting peptide R4F-TTP, which not only synergistically enhance the target-specific cellular upatke by5-8F cells and their selective killing, but only can control the formation of lipid nanoparticles and their function. The novel lipid nanoparticle could achieve high-contrast imaging in vivo and therapeutic effects for5-8F tumors. This study provides new methods for the evaluation in vivo of therapeutic and targeted peptides, tumor-targeted imaging for diagnosis and multi-targeted therapies for cancer treatment.