Development of Antibody-Conjugated Mesoporous Silica Nanoparticles for Biomedical Applications

Background:  There is significant interest in antibody-conjugated Mesoporous Silica Nanoparticles (MSNPs) due to their potential uses as drug/siRNA carriers, as well as imaging agents with enhanced detection limits. Current imaging techniques like immunofluorescence (IF) and flow cytometry methods require the use of a primary antibody and fluorescent secondary antibody and are known for having problems with photobleaching and imaging sensitivity. We believe that antibody-conjugated MSNPs are a promising alternative to both the primary and secondary antibody. For one, fluorescent MSNPs have been found to be more photostable than the fluorescent dye itself, and therefore less susceptible to photobleaching. These antibody-conjugated MSNPs may also aid in signal amplification, increasing their sensitivity for use in detecting low-abundance proteins in cells. Finally, antibody-conjugated MSNPs contain a variety of other valuable features including relative ease of synthesis, batch-to-batch reproducibility, modifiability, a large surface area relative to its size, and pores for drug/probe loading.

Methods/Characterization:  Our current synthesis procedure was divided into three distinct steps: 1) Micelle Template Formation, 2) Silica Condensation, and 3) Template Removal (by acidic reflux). After MSNP synthesis, DLS was used to measure the hydrodynamic size and charge of the MSNPs. Then, to functionalize the nanoparticles, the MSNPs were coated first with Polyethyleneimine (PEI) and then with Polyethylenegylcol (PEG) before conjugating an antibody targeting a given protein.

We found that it was possible to modify the surface groups of the nanoparticles by introducing different silanes 15 minutes after TEOS co-condensation. These modifications included: using (3-Mercaptopropyl) trimethoxysilane to for thiol surface groups and (3-Aminopropyl) trimethoxysilane for amine surface groups. We tested for the presence of these groups using Ellman’s Reagent and Fluorescamine assays. This surface modification was utilized for direct conjugation of antibody to nanoparticles, having PEG as a spacer. BCA/microBCA assays were also used to characterize the loading amount of antibody on the nanoparticles. We conjugated our developed MSNP with Trastuzumab (Herceptin) -- an antibody targeting the Human Epidermal Growth Factor 2 (HER-2) protein. This nanoconstruct was in turn evaluated for application in breast cancer targeting and treatment in our lab.

Results:  On average, we found that the size of nanoparticles synthesized this way were 70nm in diameter. The MSNPs were also found to have a negative charge of approximately -23mV in water. Additionally, due to our low Polydispersity Index (PDI) values (~0.002), and monodispersed size distribution, our nanoparticles hold great potential for development as antibody probes or drug carriers. The application of these developed mesoporous silica nanoparticles is currently under extensive investigation in our lab.