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  • Edith Dalgaard posted an update 4 days ago

    The absorption spectra of the nanoconjugates were recorded 15 minutes after incubation with 140 mM of sodium chloride. As expected, the naked GNPs showed a drastic red shift in the SPR band from 510 nm to around 600 nm confirming aggregation of uncovered nanoparticles. Salt induced aggregation was directly related to the increased loading of C225 on the GNP surface, hence the SPR band completely disappeared at a C225 to GNP ratio of 3 suggesting the absence of available reactive surface to salt induced aggregation. Targeted delivery of inorganic nanomaterials is an essential area of research for nanomedicine. Unique physicochemical properties of inorganic nanomaterials may be utilized for several biomedical applications such as detection/diagnosis, therapy and imaging. Thus, it is important to define the design parameters to specifically deliver nanoconjugates to the cells of interest. There are several key factors that may define the success of targeted delivery; selection of an appropriate model to study the delivery approach; selection of an effective targeting agent; optimization of the number of targeting agents per nanoparticle; availability of free reactive area on the particle surface that may initiate non-specific binding; ability of the targeting agent to sequester the target and hydrodynamic size of the nanoconjugates. Most of the delivery strategies are tested in non-orthotopic models where tumors are generated away from the original site. A major advantage to this model is tumors are easy to develop and growth can be monitored by manual measurement with slide calipers. A disadvantage of this model is that the tumor cells do not experience a true tumor microenvironment. In contrast, the orthotopic model implants tumor cells in the organ of tumor origin, rendering it a more realistic model. Thus, we selected the orthotopic model to study the design parameters necessary for nanoconjugates to successfully and specifically target a tumor. We selected cetuximab as a targeting agent and gold nanoparticles as the scaffold for targeting delivery. One of the major advantages to utilizing gold nanoparticles is their formation of stable bonds with biomolecules such as organothiols, organoamines, proteins, and antibodies. We demonstrated the significant binding of cetuximab to GNPs up to a ratio of 3. beyond which only a marginal increase in binding is observed. This marginal increase might be due to the lack of free reactive surface area remaining on the GNPs. The nature of bonding between gold and proteins has been the subject of intense investigation over the last several decades. It is now generally accepted that there are three main types of interactions that occur between a protein/WY 14643 Antibody and a gold nanoparticle; electrostatic interactions of negatively charged GNPs with positively charged proteins; covalent interactions between the thiol/amine groups present within amino acid residues in antibodies and the GNPs; and hydrophobic interactions between proteins and GNPs. Using X-ray photoelectron spectroscopy and thermogravimetric analysis we previously demonstrated that cetuximab utilizes thiol and amine functionalities to bind to the surface of gold nanoparticles. It is also imperative to validate the design parameters for different cell lines with variable expression of the target receptor. This is particularly important considering the heterogeneous nature of tumors. In these studies we employed three different pancreatic cancer cell lines with variable expression of EGFR. We also demonstrated that intracellular uptake of GNP-C225 is not only dependent on the number of cetuximab molecules, but also depends on their ability to bind EGFR, as well as the hydrodynamic size of the nanoconjugates. It is also evident that non-specific uptake can be minimized in vitro by optimizing the coverage of the targeting agent on the nanoparticle surface. Maximum specific uptake of GNP-C225 is observed when there are three C225 molecules on a GNP even though the overall uptake is reduced. This data further demonstrates the combined importance of the number of targeting agents per particle and the hydrodynamic size of the nanoconjugates. This is also reflected in the difference between the specific uptake of GNP-C225 and the non-specific uptake of GNP-IgG. Recently, several groups have been focusing on understanding the design parameters for efficiently targeting tumors with nanoconjugates in a preclinical model. It has also been reported that a nanoconjugate with a size of,100 nm is efficiently targeting tumor cells. Antibody targeted liposomes have been found to exert enhanced efficacy over their non-targeted counterparts. Furthermore, delivery of siRNA using transferring- targeted polymeric nanoparticles demonstrated better efficacy even though their kinetics, biodistribution and tumor uptake was similar. Similar observations were also made in a study of transferrin-coated gold nanoparticles; it was demonstrated that ligand content on the nanoparticles are important for targeting. Here, we explore an orthotopic model of pancreatic cancer and in turn, demonstrate the critical roles that the number of antibody molecules on the GNP, the ability of the nanoconjugates to bind the target receptor, the availability of the reactive surface for non-specific interaction and the hydrodynamic size of the nanoconjugates all play in efficient design. Taken together our data suggests that the specific targeting of tumor cells depends on a number of important factors; 1) targeting agent to nanoparticle ratio; 2) availability of the reactive surface area on the nanoparticle; 3) ability of the nanoconjugate to bind the target and 4) hydrodynamic diameter of the nanoconjugate.