Methods for attaching targeting ligands to drug delivery vehicles have been under investigation for decades. Antibodies are considered to be the strongest of ligands available in terms of targeting strength and selectivity. The goal of this work is to create an actively targeting and site-specific vehicle for the delivery of anticancer drugs. An ideal method for attachment would be one covalent in nature and capable of being run in mildly reactive conditions that do not harm the biological activity of the antibody selected. In this work a PLGA-PEG nanoparticle was attached covalently to an anti-HER2 monoclonal antibody via click chemistry. The targeting ability of the conjugated system was analyzed via flow cytometry and fluorescent microscopy to determine its effectiveness and activity.
Perhaps the most important pieces of information that will allow for a controlled system are being able to determine the number of antibodies bound to each particle and to understand the effectiveness of different quantities of bound antibody. Activity levels are determined by way of evaluating samples of varied nanoparticle:antibody ratios, analyzed through the use of flow cytometry. A filtration step is introduced to remove unbound antibody from the antibody-nanoparticle conjugates and the recovered protein is measured via Bradford assay, a colorimetric protein quantification assay method. Particle concentration is determined by a modified coulter counter made by Izon® called the qNano. This collection of data allows for determination of the number of molecules of antibody bound to each individual nanoparticle which can be then compared to their activity levels.
Finally, the formation of nanoparticles must be explored so that a method can be developed which yields particles in the desired size ranges with a narrow size distribution. Different methods and parameters that are known to affect the resultant particles are examined and trends determined. The method which is the most promising for future work is nanoprecipitation by means of injection. The most important parameters which affect the outcome of this method are shear forces and surface tension. Trends between different needle diameters used for injection, the flow rates, and corresponding Reynolds numbers indicate that there is an ideal Reynolds number for optimum particle formation.
To demonstrate the specific targeting abilities of the conjugates combined and individual cultures of MCF7, a breast cancer cell which expresses the HER2 antigen, and fibroblast cells, which do not express the HER2 antigen, are examined with the confocal microscope and the flow cytometer. The results show that the antibody-particle conjugates specifically target only cells that exhibit the HER2 antigen. A series of images taken by a confocal microscope display the internalization of particles into the cells. Experiments with increasing quantities of antibody yield an attachment rate of antibody on to particles of approximately 30% and an average of 1.92e5 molecules of antibody per particle. This work shows that we have successfully utilized click chemistry to bind a monoclonal antibody to a polymeric nanoparticle, creating a novel drug delivery vehicle which could be utilized to deliver cytotoxic drugs specifically to the offending cells.