Final Report Summary - MIPC (Microsphere Based Cellular Manipulation – From Protein Delivery to Intracellular Palladium Catalysis)
Summary description of the project objectives: The objectives of the project were the application, exploitation and development of a generic microsphere cellular delivery system for the efficient and benign delivery of a broad range of cargos into cells.
Description of the work performed since the beginning of the project:
(1). Microspheres were covalently functionalised with nitrilotriacetic acid (NTA) and imido diaceticacid (IDA) to allow His-tag protein capture and subsequent cellular delivery. Model peptides based on hexa-His attached to a nuclear localisation sequence were successfully delivered to HeLa and HEK 293T cells. However cellular delivery of His-tag-enhanced green fluorescent protein (GFP) met with limited success with loss of the GFP from the microspheres (in the media) and non-specific binding of the GFP observed to the cell membrane. Effort to optimise the properties of microspheres led to the synthesis of two unique nanaoparticles: Fluorescent nanoparticles with enhanced fluorescence properties1 and Dual fiunctionalised nanaoparticles.
(2). Loading and intra-cellular release of cargos using palladium mediated coupling reactions were explored. The boronic acid groups on the dual functionalised particles allowed the conjugation of complex peptide-reporter cargos by Pd-mediated Suzuki–Miyaura cross coupling and allowed the delivery of a range of cargos into specific organelle targets.
(3). Various polymer supported Pd(0) catalyst system were prepared (see Figure 3) for “in-situ” controlled synthesis of drugs from inert intermediates or prodrugs. The catalytic activities of the Pd0 catalysts, under physiological conditions, were investigated using caged fluorophore (non-fluoroscent). As a result of Pd(0) catalytic activity of biocompatible catalysts, the decaging of fluorophore was quantified via fluorescence measurements. Further, the application of biocompatible Pd(0) catalysts was explored by a cell-based experiments for “in-situ” synthesis of various inhibitors that resulted in inhibition of the proliferation of cervical carcinoma (HeLa) and prostate cancer (PC3) cells and induces apoptosis in vitro.
Description of the work performed since the beginning of the project:
(1). Microspheres were covalently functionalised with nitrilotriacetic acid (NTA) and imido diaceticacid (IDA) to allow His-tag protein capture and subsequent cellular delivery. Model peptides based on hexa-His attached to a nuclear localisation sequence were successfully delivered to HeLa and HEK 293T cells. However cellular delivery of His-tag-enhanced green fluorescent protein (GFP) met with limited success with loss of the GFP from the microspheres (in the media) and non-specific binding of the GFP observed to the cell membrane. Effort to optimise the properties of microspheres led to the synthesis of two unique nanaoparticles: Fluorescent nanoparticles with enhanced fluorescence properties1 and Dual fiunctionalised nanaoparticles.
(2). Loading and intra-cellular release of cargos using palladium mediated coupling reactions were explored. The boronic acid groups on the dual functionalised particles allowed the conjugation of complex peptide-reporter cargos by Pd-mediated Suzuki–Miyaura cross coupling and allowed the delivery of a range of cargos into specific organelle targets.
(3). Various polymer supported Pd(0) catalyst system were prepared (see Figure 3) for “in-situ” controlled synthesis of drugs from inert intermediates or prodrugs. The catalytic activities of the Pd0 catalysts, under physiological conditions, were investigated using caged fluorophore (non-fluoroscent). As a result of Pd(0) catalytic activity of biocompatible catalysts, the decaging of fluorophore was quantified via fluorescence measurements. Further, the application of biocompatible Pd(0) catalysts was explored by a cell-based experiments for “in-situ” synthesis of various inhibitors that resulted in inhibition of the proliferation of cervical carcinoma (HeLa) and prostate cancer (PC3) cells and induces apoptosis in vitro.