Development and Characterization of Functionalized Superparamagnetic Nanoparticles for Interstitial Applications

Abstract

A persistent limitation to current and future anti-cancer therapies is an inability to deliver high levels of active agent throughout the entire target tumor tissue volume without adversely impacting normal tissues. Interstitial transport is compromised by the poor mobility of macromolecules and nanoscale structures. We developed an in vitro system to quantify the facilitated transport of superparamagnetic (SPM) nanoparticles (NPs) through model extracellular matrix (ECM) in vitro. SPM NP motion was induced by an external magnetic field. 135 nm radius NPs with a polyethylene glycol (PEG) surface moved through the ECM with an average velocity of 1.5 mm h-1, a velocity suitable for some clinical applications. Steric barriers such as collagen I sharply limit interstitial delivery of macromolecular and nanoparticle-based therapeutic agents. Collagenase-linked SPM NPs overcame these barriers and moved through ECM in vitro at 90 ìm hr-1, a rate similar to invasive cells, under the influence of a magnetic field. Temporal decay of collagenase activity shifted from an exponential behavior in solution to a linear relationship when NP-attached. NP platforms offer the opportunity to develop a unified synthesis method for formation of multifunctional agents. We have demonstrated a facile method of conjugating multiple enzyme species to a NP via sulfhydryl-maleimide reaction chemistry. Horseradish peroxidase, á-glucosidase, and collagenase were simultaneously conjugated to the 300 Da PEG surface of SPM NPs at 15:1, 127:1 and 103:1 functional enzyme:NP ratio, respectively. SATP addition of sulfhydryl groups to each enzyme was achieved without significant reduction in enzyme activity. Cross reactivity of enzymes between enzyme activity assay systems was negligible. Multifunctional NPs mimic complex invasive biological processes found in metastatic invasion and immune cell interactions. Isolated study of sets of enzymes in an invasion experimental system may make an ideal screening tool for blocking pathogenic invasive processes, including cancer metastasis and abnormal angiogenesis. Dispersion of otherwise immobile macromolecular or nanoscale therapeutic structures can be achieved with the proteolytic SPM NP carriers detailed in this work.