Proteolytic Surface Functionalization Enhances in Vitro Magnetic Nanoparticle Mobility through Extracellular Matrix Sam J. Kuhn, † Stephanie K. Finch, † Dennis E. Hallahan, †,‡,§ and Todd D. Giorgio* ,†,§ Department of Biomedical Engineering, Vanderbilt UniVersity, NashVille, Tennessee, 37235, Department of Radiation Oncology and Department of Cancer Biology, Vanderbilt UniVersity School of Medicine, NashVille, Tennessee 37232-5671, and Vanderbilt-Ingram Cancer Center, Vanderbilt UniVersity, NashVille, Tennessee 37235 Received November 13, 2005; Revised Manuscript Received December 22, 2005 ABSTRACT Steric barriers such as collagen I sharply limit interstitial delivery of macromolecular and nanoparticle (NP) based therapeutic agents. Collagenase- linked superparamagnetic NPs overcame these barriers and moved through in vitro extracellular matrix (ECM) at 90 μmh -1 , a rate similar to invasive cells, under the influence of a magnetic field. NP migration in ECM diminished linearly over 5 days. The collagenase-NP construct overcame two of the most significant barriers to nano- and microscale therapeutics deployment: proteolytic enzyme stability was maintained during a clinically useful time frame by immobilization on the NP surface and degradation of interstitial barriers to tissue biodistribution was enabled by the conjugated microbial protease. Nanoscale and microscale therapeutic structures have poor mobility when administrated to tissue interstitium. Clinical application of gene therapy, bioactive proteins, and other large molecular therapeutics are often limited by geometric effects such as steric barriers, negligible diffusion, and poor interstitial convective mobility. 1,2 Viscous nonspecific mo- lecular and physical interactions with tissue components also restrict transport and further limit the efficacy of these novel therapeutic structures. 3,4 We propose to overcome these limits to nanoscale and microscale therapeutic tissue distribution by development of a protease-functionalized superparamagnetic nanoparticle (SPM NP) vehicle. The magnetic character of the SPM NP provides spatial and temporal control of NP localization in the interstitial space using an external magnetic field to facilitate intratissue mobility. 5,6 A surface-linked protease enables degradation of adhesive and steric barriers in the interstitial space. 7,8 Protease degradation of biological barriers is a common strategy employed by motile and invasive cells. Involvement of a broad spectrum of metalloproteases in metastasis has been documented, including various collagenases. 9,10 Migrat- ing neural crest cells in embryos utilize matrix metallopro- tease-type 2, tissue plasminogen activator, and urokinase plasminogen activator to migrate to the developing neural crest. 11,12 Embryo implantation following fertilization is marked by dramatic increase in protease secretion. 13 Patho- genic microorganisms and viruses, including S. pyogenes, human papillomavirus, P. insidiosum, and P. aeruginosa secrete proteases or induce the release of endogenous proteases as an invasive mechanism in host tissues. 14-17 Reported literature values for in vitro cell migration rates through purified extracellular matrix substrates average 104 ( 44 µmh -1 (4-434 µmh -1 ), 18-20 a rate that is consistent with a significant increase in tissue biodistribution of large molecules and nanoparticles. While collagenase is an approved salve by the United States Food And Drug Administration for tissue debridement, exploration of proteases for enhanced interstitial mobility is still an emerging area of study. Netti et al., for example, documented a 2-fold increase in diffusion rates of antibodies following in situ collagenase treatment of rigid human glioblastoma (U87) and human soft tissue sarcoma (HSTS 26T) tumors. 7 Nanoparticle platforms enable development of multifunc- tional therapeutic vehicles. Intrinsic material properties such as superparamagnetism or X-ray absorption can be combined with surface modifications that minimize nonspecific interac- tions with biological structures. 21,22 The NP surface can be * To whom correspondence may be addressed: todd.d.giorgio@ vanderbilt.edu. † Department of Biomedical Engineering, Vanderbilt University. ‡ Department of Radiation Oncology and Department of Cancer Biology, Vanderbilt University School of Medicine. § Vanderbilt-Ingram Cancer Center, Vanderbilt University. NANO LETTERS 2006 Vol. 6, No. 2 306-312 10.1021/nl052241g CCC: $33.50 © 2006 American Chemical Society Published on Web 01/25/2006