Preparation Of Hybrid Nanogel Based On Genetically Engineered Polypeptide And Its Applications In Biomedicine

Theragnostics is a treatment strategy that combines therapeutics with diagnostics. In recent years, nanogel as a nanocarrier for loading therapeutic drug and diagnostic reagent has attracted much attention due to the unique properties of high drug loading efficiency and controlled release, especially nanogel with high biocompatibility and tunable properties was prepared with genetically engineered polypeptides. In addition, inorganic nanometer materials are wildely used in bioimaging, drug delivery and cancer therapy because of their advantages of easy prepareation, stably and uniquely electrical, optical and mechanical properties. In this thesis, we focus on the hybrid nanogels based on genetically engineered polypeptides and inorganic nanometer materials and its applications in medicine. The main contents and results are summarized as follows:(1) A new type of multifunctional quantum dots (QDs)-polypeptide hybrid nanogel that combines the capability of targeted tumor imaging with drug delivery were designed and prepared. Coiled-coil polypeptide (PC10A or PC10ARGD), which consists of an associative domain P, a zipper domain A, and a random coil midblock (C10), was chosen to modify the surface of QDs by specific metal-affinity interaction between polypeptides appended with N-terminal polyhistidine sequences and hydrophilic CdSe-ZnS core-shell QDs. Rigid PC10A or PC10ARGD on the surface of QDs forms a sandwich hydrogel which consists of two hydrophobic layers (formed by P domain and A domain) and one hydrophilic layer (formed by C10 domain) between them by self-assembly. We demonstrated that surface ligands consisting self-assembly of coiled-coil domains on the QDs-PC10A and QDs-PC10ARGD nanogels can form the sandwich microstructure by CE and DLS measurement. We are able to tune the physical properties of QD-polypeptide including size, shape, binding domain, and surface potential by constructing the different structures of polypeptide with genetic engineering method. The successfully simultaneous loading of hydrophobic and hydrophilic drugs into the hydrophobic and hydrophilic layer of QDs-PC10A or QDs-PC10ARGD nanogel demonstrated their potential for drug delivery applications. QDs-polypeptide will provide a general avenue for the fabrication of dual drug vehicular nanocarriers. Temperature- and pH-sensitive polypeptide monolayer on the QDs can serve as a trigger to control the release of the loaded hydrophobic cargo. Uptake of drug-loaded QDs-PC10ARGD nanogel in ?w?3 overexpressing cells was remarkably greater than in control cells, and the QDs-PC10A revealed lower non-specific binding than GSH-capped QDs. Compared with the original QDs, the QDs-polypeptide nanogels showed lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Furthermore, cytotoxicity of targeted QDs-polypeptide nanogel is lower for normal NIH 3T3 cells than that for HeLa cancer cells. The results reported here open up new perspectives for targeting imaging and drug delivery.(2) A new NIR-light-triggered ligand release and photothermal therapy system was fabricated using gold nanorods (GNRs) functionalized with engineered polypeptides. Bioactive ligands of interest are fused to C terminus of the leucine zipper A and immobilized on the surface of GNRs through a thiol-gold bond. The multifunctional GNRs allow coimmobilization of a B-PEG through a pair of complementary leucine zipper domains, which shields the bioactive ligands on the A on the molecular level. After functionalized GNRs accumulating in the targeted cancer cells or tumors, coimmobilized B-PEG could be removed to release the shielded bioactive ligands for cancer therapy through a photothermal mechanism of GNRs. In addition, functionalized GNRs presented simultaneously photothermal therapy effects. Results demonstrated that coimmobilized B-PEG could be removed to release the bioactive ligand RGD with 810nm NIR light irradiation, and the polypeptides-functionalized GNRs showed an enhanced photothermal therapy for HeLa cancer cells. Thus, such GNRs-based controlled-release systems have great potential in the fields of targeted biomolecule delivery and photothermal therapy.(3) A new cancer therapeutic system with the combination of targeted chemotherapy and photothermal therapy using multifunctional GNRs-polypeptide hybrid nanogel was fabricated with a triblock engineered polypeptide PC10ARGD and GNRs by the electrostatic adsorption. The immobilized PC10ARGD forms hydrogel through self-assembly. Hydrogel formed by PC10ARGD can not only increase the biocompatibility, but also increase the stability of hybrid nanogel. The chemotherapic drug doxorubicin (DOX) was loaded into the hydrogel of PC10ARGD. GNRs@PC10ARGD@DOX presented good stability in water, PBS, serum-free DMEM and DMEM with serum. Hybrid nanogel still remains high photothermal efficiency. A fast release rate and high release amounts of the DOX from the hybrid nanogel were observed at low pH and high temperature. Compared with CTAB-GNRs, the GNRs-polypeptide hybrid nanogels showed lower in vitro cytotoxicity for HeLa cells. The results of chemotherapy and photothermal therapy showed that the GNRs@PC10ARGD@DOX hybrid nanogel had the combined effect of the cytotoxicity of DOX and the photothermal effect of GNRs. The effects of chemotherapy and photothermal therapy are related with the dose of DOX and GNRs. In addition, the combined treatment of chemo-photothermal therapy showed a synergistic effect for cancer therapy. This type of hybrid nanogel is expected to provide some new ideas and approaches for cancer therapy.(4) Triblock engineered polypeptides PC10A and PC10ARGD can form nanogel at low concentration. The sizes of PC10A and PC10ARGD nanogel can be tuned by the simple changing the concentration of polypeptides. One-step in situ photochemical synthesis of gold nanoparticles in PC10A nanogel was performed using gold ion and photoinitiator 1-2959 under 365 nm UV light irradiation. The effect of gold ion concentration on gold nanoparticles in the hybrid nanogel was studied. The results showed that the size of gold nanoparticles in hybrid nanogel increased gradually and its concentration increased gradually with the increase of the gold ion concentration. The concentration of polypeptide has little effect on the gold nanoparticles in hybrid nanogel and only effect on the size of the hybrid nanogel. The concentration of gold nanoparticles in hybrid nanogel increased with increasing of irradiation time. pH also has little effect on the gold nanoparticles in hybrid nanogel. Results of cytotoxicity showed PC10A, PC10ARGD, and hybrid nanogel had lower in vitro cytotoxicity for both HeLa cells and NIH 3T3 cells. Preliminary CT imaging experiments showed that the gold nanoparticles-PC10ARGD hybrid nanogel presented good in vitro CT imaging result.