Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

Department of Biomedical Engineering, Vanderbilt University, Nashville, TN 37235, USA.
International Journal of Nanomedicine (Impact Factor: 4.2). 02/2008; 3(1):95-103.
Source: PubMed

ABSTRACT The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure.

Download full-text


Available from: Todd D Giorgio, Jun 17, 2015
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The use of activable cell-penetrating peptides (ACPPs) as molecular imaging probes is a promising new approach for the visualization of enzymes. The cell-penetrating function of a polycationic cell-penetrating peptide (CPP) is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a proteinase-sensitive substrate present between the CPP and polyanionic peptide affords dissociation of both domains and enables the activated CPP to enter cells. This ACPP strategy could also be used to modify antitumor agents for tumor-targeting therapy. Here, we aimed to develop a conjugate of ACPP with antitumor drug doxorubicin (DOX) sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for tumor-targeting therapy purposes. The ACPP-DOX conjugate was successfully synthesized. Enzymatic cleavage of ACPP-DOX conjugate by matrix metalloproteinase (MMP)-2/9 indicated that the activation of ACPP-DOX occurred in an enzyme concentration-dependent manner. Flow cytometry and laser confocal microscope studies revealed that the cellular uptake of ACPP-DOX was enhanced after enzymatic-triggered activation and was higher in HT-1080 cells (overexpressed MMPs) than in MCF-7 cells (under-expressed MMPs). The antiproliferative assay showed that ACPP had little toxicity and that ACPP-DOX effectively inhibited HT-1080 cell proliferation. These experiments revealed that the ACPP-DOX conjugate could be triggered by MMP-2/9, which enabled the activated CPP-DOX to enter cells. ACPP-DOX conjugate may be a potential prodrug delivery system used to carry antitumor drugs for MMP-related tumor therapy.
    International Journal of Nanomedicine 03/2012; 7:1613-21. DOI:10.2147/IJN.S30104 · 4.20 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Small interfering RNA (siRNA) has significant potential to evolve into a new class of pharmaceutical inhibitors, but technologies that enable robust, tissue-specific intracellular delivery must be developed before effective clinical translation can be achieved. A pH-responsive, smart polymeric nanoparticle (SPN) with matrix metalloproteinase (MMP)-7-dependent proximity-activated targeting (PAT) is described here. The PAT-SPN is designed to trigger cellular uptake and cytosolic delivery of siRNA once activated by MMP-7, an enzyme whose overexpression is a hallmark of cancer initiation and progression. The PAT-SPN is composed of a corona-forming polyethylene glycol (PEG) block, an MMP-7-cleavable peptide, a cationic siRNA-condensing block, and a pH-responsive, endosomolytic terpolymer block that drives self-assembly and forms the PAT-SPN core. With this novel design, the PEG corona shields cellular interactions until it is cleaved in MMP-7-rich environments, shifting the SPN ζ-potential from +5.8 to +14.4 mV and triggering a 2.5 fold increase in carrier internalization. The PAT-SPN exhibits pH-dependent membrane disruptive behavior that enables siRNA escape from endo-lysosomal pathways. Intracellular siRNA delivery and knockdown of the model enzyme luciferase in R221A-Luc mammary tumor cells is significantly increased by MMP-7 pre-activation (p < 0.05). These combined data indicate that the PAT-SPN provides a promising new platform for tissue-specific, proximity-activated siRNA delivery to MMP-rich pathological environments.
    Advanced Functional Materials 06/2013; 23(24). DOI:10.1002/adfm.201202215 · 10.44 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: An estimated 16 million people in the United States have coronary artery disease (CAD), and approximately 325,000 people die annually from cardiac arrest. About two-thirds of unexpected cardiac deaths occur without prior recognition of cardiac disease. A vast majority of these deaths are attributable to the rupture of 'vulnerable atherosclerotic plaques'. Clinically, plaque vulnerability is typically assessed through imaging techniques, and ruptured plaques leading to acute myocardial infarction are treated through angioplasty or stenting. Despite significant advances, it is clear that current imaging methods are insufficiently capable for elucidating plaque composition-which is a key determinant of vulnerability. Further, the exciting improvement in the treatment of CAD afforded by stenting procedures has been buffered by significant undesirable host-implant effects, including restenosis and late thrombosis. Nanotechnology has led to some potential solutions to these problems by yielding constructs that interface with plaque cellular components at an unprecedented size scale. By leveraging the innate ability of macrophages to phagocytose nanoparticles, contrast agents can now be targeted to plaque inflammatory activity. Improvements in nano-patterning procedures have now led to increased ability to regenerate tissue isotropy directly on stents, enabling gradual regeneration of normal, physiologic vascular structures. Advancements in immunoassay technologies promise lower costs for biomarker measurements, and in the near future, may enable the addition of routine blood testing to the clinician's toolbox-decreasing the costs of atherosclerosis-related medical care. These are merely three examples among many stories of how nanotechnology continues to promise advances in the diagnosis and treatment of vulnerable atherosclerotic plaques. WIREs Nanomed Nanobiotechnol 2011 DOI: 10.1002/wnan.158 For further resources related to this article, please visit the WIREs website.
    Wiley Interdisciplinary Reviews Nanomedicine and Nanobiotechnology 11/2011; 3(6). DOI:10.1002/wnan.158 · 4.24 Impact Factor