• Edith Dalgaard posted an update 1 year, 2 months ago

    Despite vast efforts to detect and treat pancreatic cancer, the incidence and mortality rates remain virtually the same. Early diagnosis and efficient delivery of therapeutic agents to malignant cells remain the two major challenges in cancer management strategies. Monoclonal antibodies against growth factor receptors have been shown to be viable treatments for inhibiting cancer growth. Utilizing these monoclonal antibodies as targeting agents for tumor specific delivery is evolving as a promising approach to selectively deliver chemotherapeutics. Inorganic nanomaterials are being studied as the delivery vehicle for targeted drug delivery. Gold nanomaterials are of particular interest due to the unique physico-chemical and optoelectronic properties, ease of synthesis and surface modification. Gold nanoparticles have recently been used to kill tumor cells by hyperthermia using non-invasive radiofrequency. Their utility as a contrast agent has also been demonstrated by clear delineation of blood capillaries in a preclinical model by CT in comparison to the conventional iodine based contrast agents. Both studies are hopeful and their utility is further encouraged by the safety profile. Epidermal growth factor receptor is an important target in cancer research. It is overexpressed in a number of human malignancies including pancreatic cancer. Human EGFR is a transmembrane glycoprotein. It consists of an extracellular ligand binding domain, a hydrophobic transmembrane domain and an intracellular tyrosine kinase domain. Ligand binding to EGFR induces receptor homo/heterodimerization leading to the phosphorylation of tyrosine residues. Phosphorylation of EGFR activates complex down stream signaling events leading to proliferation, migration, invasion, and inhibition of apoptosis of cancer cells. The monoclonal anti-EGFR antibody, Cetuximab, is a unique targeting agent to target WZ4002 1213269-23-8 EGFR-positive cancer cells. Cetuximab was approved by the FDA for the treatment of patients with EGFR positive colorectal cancer. It has also been either approved or is in different phases of clinical trials in many other malignancies such as NSCLC, SCCHN and pancreatic cancer. Cetuximab is a chimeric human:murine immunoglobulin G1 monoclonal antibody. The binding of C225 to EGFR leads to receptor internalization and degradation without receptor phosphorylation, thus inhibiting EGFR-associated pathways. Despite the emerging utility of GNPs in targeted delivery fundamental questions remain unanswered. What are the design criteria for fabricating nanoconjugates that will ensure maximum uptake in cancer cells? Herein, we utilize cetuximab as a targeting agent and GNP as a model system. We demonstrate that the number of C225 antibodies on a GNP, the hydrodynamic size, the available reactive surface area and the ability of the nanoconjugate to sequester EGFR, all play critical roles in effectively targeting tumor cells in vitro and in vivo in an orthotopic model of pancreatic cancer. Uptake studies with the isotype control, GNP-IgG, indicate that the specificity of tumor cell targeting is dependent on the nanoparticle surface coverage by C225. Non-specific uptake decreases when the C225 to GNP ratio increases. These studies are critical to develop an efficient targeted delivery system for future clinical use. The work presented herein shows the systematic characterization of GNP-C225 nanoconjugates. The specific targeting potential was investigated, both in vitro and in vivo in an orthotopic model of pancreatic cancer. Our results demonstrate that the hydrodynamic radius, accessible reactive surface area and loading capacity of C225 on GNPs play critical roles for efficient targeting of tumor cells. These findings highlight key parameters to be considered for a promising nanoparticle based drug delivery system for future clinical application. In order to understand the parameters required for specific targeting of nanoconjugates to tumor cells, we selected cetuximab as a targeting agent. C225 is a chimeric human-murine monoclonal antibody that binds to the extracellular domain of EGFR. EGFR is overexpressed in a number of human malignancies including pancreatic cancer, rendering it an attractive target. To determine the optimum valency of C225 on GNPs for intracellular uptake, we synthesized various nanoconjugates with multiple C225:GNP ratios. Characterization of these nanoconjugates was performed using several physicochemical techniques: UV-Visible spectroscopy, transmission electron microscopy, dynamic light scattering and radioiodination of C225 with 125I. The GNPs used in this study were synthesized by sodium borohydride reduction of tetrachloroaurate. As previously reported, the presence of a surface plasmon resonance band at ca. 510 nm confirms the formation of spherical gold nanoparticles. The formation of spherical GNPs and their 5 nm size diameter was further confirmed by TEM. The GNP-C225 conjugates were then synthesized using this naked GNP solution and purified as described in the materials and methods section. The antibody spontaneously binds to the GNPs through Au-S and Au-N bonding. The production of GNPC225 was monitored by UV-Vis spectroscopy. It is evident that with the addition of C225 there is a gradual red shift in the SPR band of the naked gold, from 510 nm to 519 nm. Such a red shift in the SPR band of the GNPs suggests the perturbation of the electrical double layer by the antibody surrounding the GNPs and thus indicates binding of the antibody to the nanoparticles. To further confirm the GNP-C225 conjugation we challenged the nanoconjugates against salt induced aggregation. Addition of 140 mM NaCl has been reported to result in aggregation of naked or partially covered particles, such aggregation leads to a dramatic red shift in the SPR band.