Design, Synthesis And Biological Activities Of Antibody-Targeted Multifunctionalized Gold Nanopariticles

Chapter1IntroductionFor decades, nanotechnology has grown by leaps and bounds. Many great breakthroughs of nanotechnology in medicine have been achieved. As a result, lots of excellent nanoplatforms such as liposome, polymer, protein-bound particles, gold nanoparticles (GNPs), superparamagnetic iron oxides, and quantum dots have been developed for tumor's diagnosis and therapy. Of these, gold nanoparitcles show the unique physical and chemical properties including biocompatibility, size-and shape-dependent optical and electronic features, and readily modifiable surfaces. Consequently, GNPs present realistic potential for clinical translation. This chapter reviewed the latest studies on the synthesis, modification and application of multifuncitonalized gold nanoparticles in medicine.Chapter2Sensitizing neuroblastoma cells to X-ray radiation by using antibody-targeted cell cycle-modulated gold nanoparticlesNeuroblastoma is the most common extracranial solid tumor in childhood. More than50%of neuroblastoma cases occur in children younger than2years old. Current treatments, including surgery, chemotherapy, radiation, or stem cell transplantation, induce significant side effects including growth delays, hearing loss, and learning disabilities. Despite multimodality therapy, many patients with advanced-stage disease will ultimately succumb to relapse. In St Jude Children's Research Hospital, the five-year survival rate is still not more than55%. Therefore, more effective and cancer-specific therapy is urgently needed, especially in this fragile patient population. GD2disialoganglioside is an antigen expressed on tumors of neuroectodermal origin, including neuroblastoma, whereas on normal tissues its presence is restricted to neurons, skin melanocytes, and peripheral pain fibers. Many anti-GD2antibodies (e.g.3F8,14G2a, ch14.18, hu14.18, and hu14.18K322A) have been developed and used in clinical trials. Among these antibodies, hu14.18K322A is a humanized anti-GD2antibody developed by Merck KGaA incorporation, currently being investigated in a Phase â…  immunotherapy study in neuroblastoma patients at St Jude Children's Research Hospital. Compared to hu14.18, hu14.18K322A has a single amino acid mutation at K322which results in substantially less complement activation and less side effects. In our study, the hu14.18K322A antibody is used as targeting molecule for GNPs to recognize neuroblastoma cells. Its discovery, chemical structure and clinical trial results are not included in this thesis.Lots of studies have shown that gold nanoparticles (GNPs), like other high atomic (Z) number elements, enhance radiation-induced cell death. The mechanism is that GNPs can enhance the DNA damage to tumor cells. However, there are still no studies on the interactions of GNPs with tumor cells in different phase of cell cycle (e.g G1, S, G2/M), which remains great chance for chemist and biologist. In addition, all cells are not equally sensitive to radiation during all stages of the cell cycle. For example, cells are radiation-resistant in the S-phase, whereas they become very sensitive to radiation in the G2/M phase. Consequently, design and synthesis of antibody targeted and cell cycle modulated gold nanoparticle play important roles in the study of interactions of GNPs with tumor cells in a given cell cycle and the development of tumor specific radiosensitizers. Based on the above knowledge, we designed an antibody targeted and cell cycle modulated gold nanoparticle to effectively kill neuroblastoma cells.Objective:Design and synthesis of antibody targeted and cell cycle modulated gold nanoparticles, HPGNP.Significance:(1) Coming up with materials for the study of interactions of GNPs with tumor cells in G2/M phase;(2) Developing novel and more powerful radiosensitizer that is neuroblastoma cell specific to minimize the side effect in radiotherapy.Results:(1) Neuroblastoma cells were specifically targeted by HPGNP. The specificity depended on the hu14.18K322A-GD2interaction;(2) HPGNP was internalized into neuroblastoma cells but not GD2negative cells, and the tumor cells were artificially arrested in the G2/M phase of the cell cycle (HPGNP could not arrest GD2negative cells in G2/M phase, and the final location of HPGNP in neuroblastoma cells were endosome, lysosome and cytoplasm which was very important to the study of antigen-mediated cellular uptake of gold nanoparticles);(3) Compared with HGNP or PTX, HPGNP significantly increased the cytotoxicity of X-ray to neuroblastoma cells, and decreased the cytotoxicity of X-ray to GD2negative cells. Cell viability under X-ray: IMR-32(GD2positive neuroblastoma cell line):82%(HGNP),77%(PTX),19%(HPGNP); CHLA-20(GD2positive neuroblastoma cell line):80%(HGNP),83%(PTX),18%(HPGNP); PC-3(GD2negative cell line):99%(HGNP),63%(PTX),97%(HPGNP). These results indicated that HPGNP could recognize and be internalized into neuroblastoma cells. The internalized HPGNP could release paclitaxel and push cells to vulnerable phase of the cell cycle, and greatly enhance radiation-induced cells death. On the other hand, HPGNP could not recognize and be internalized into GD2negative cells. Consequently, HPGNP decrease the cytotoxicity of X-ray to GD2negative cells. These results had great significance in developing novel cancer therapy approaches, getting better treatments, and minimizing the side effects of chemotherapy and radiotherapy. The success of this multipronged attack approach launched by cell-embedded nanoconstructs demonstrates the power and flexibility of nanomedicine in treating cancer.Chapter3Hu14.18K322A-targeted gold nanoparticles as molecular computed tomography (CT) imaging contrast agent for GD2positive tumor cellsCurrent CT contrast agents are typically iodine compounds. These compounds still have many disadvantages such as very short imaging time (5-10min), renal toxicity, idiosyncratic responds (e.g. nettle-rash, angio-edema, laryngeal-edema, bronchospasm, severe drops in blood pressure, and sudden death) and physical or chemical responds (e.g. nausea, vomiting, flushing, fever, and local pain). What's more, iodine-based contrast agents are not tumor cell specific and not molecular CT imaging contrast agents.Gold is an excellent absorber of X-rays (100keV:gold,5.16cm2g-1; iodine,1.94cm2g-1) and the X-ray attenuation of GNPs is found to be much higher than that of the iodine-based contrast agent at the same molar concentration. As a result, GNPs have been widely studied as CT imaging contrast agent, and targeted GNPs will be promising molecular CT contrast agent. Based on the above knowledge, we synthesized the hu14.18K322A targeted GNPs-HGNP. We also explored its application in molecular CT imaging in vitro.Objective:Design and synthesis of molecular CT imaging contrast agent, HGNP.Significance:(1) Developing GD2specific molecular CT contrast agent to improve the sensitivity of CT in imaging neuroblastoma and melanoma cells;(2) Coming up with one powerful labeling approach for the study of hu14.18K322A in clinical trials (gold nanoparticles as labeling agent).Results:HGNP was specifically recognized by and internalized into the GD2positive neuroblastoma and melanoma cells. HGNP did not show considerable cytotoxicity to either GD2positive tumor cells or GD2negative cells (cell viability>96%), which indicated that HGNP would show high biocompatibility in vivo. Antibody-dependent cell-mediated cytotoxicity (ADCC) result showed HGNP could kill neuroblastoma cells when NK cells were present. This result indicated that HGNP maintained the ADCC effect, and would find out and remove neuroblastoma or melanoma cells in vivo. This result also indirectly demonstrated that the conjugation of hu14.18K322A on the surface of GNP core was random, but not directional. HGNP could visualize the GD2antigen which was expressed on the surface of melanoma and neurblastoma cells by CT imaging (distinguish GD2antigen from the surroundings). This result suggested that HGNP would be one promising molecular CT imaging contrast agent for neuroblastoma or melanoma, and would enable CT to be the molecular modality. Molecular CT imaging would contribute to exploring expression and activity of tumor-related markers and tacking tumor-related molecular process, which indicated that molecular CT imaging would be one powerful modality for diagnosis of microtumors including early tumors, postoperative tumors and metastatic tumors. These preliminary in vitro data was very important for the further in vivo studies.