Enzyme-Responsive Nanoparticle Systems

Enzyme-Responsive Nanoparticle Systems

(Parte 1 de 2)

DOI: 10.1002/adma.200703158

Enzyme-Responsive Nanoparticle Systems**

By James E. Ghadiali and Molly M. Stevens*

1. Introduction

Whilst inorganic nanoparticles can be utilized in electronic and optical devices and chemical catalysis, their integration into biological systems provides the possibility of creating new high-sensitivity biological labels and drug-delivery systems.[1] Another key application of biofunctional metallic nanoparticles is the development of biosensor devices for the rapid detection of disease-related proteins, with a view to creating new diagnostic and high-throughput screening techniques. Such approaches often rely on utilizing the size-dependent phenomena associated with metallic nanoparticles, namely localized surface plasmon resonance, superparamagnetism, and quantum confinement, to devise high-sensitivity readout platforms. Furthermore, prior to integration into any biosensor device, inorganic nanoparticles need to be rendered watersoluble and appended with various biofunctional surface ligands.[2,3] For an up-to-date discussion of the numerous available bioconjugation strategies involved in interfacing inorganic nanoparticles with biological systems, Rotello et al. provide a comprehensive and detailed Review in this issue.[4]

High-sensitivity detection of enzymes and determination of their activity and kinetic parameters is of great importance in the development of novel pharmaceuticals and medical diagnostic devices. Protein detection methods have largely been dominated by enzyme-linked immunological assays (i.e., ELISA), where specific antibody binding to a target analyte is amplified by a secondary colorimetric enzyme reaction. Although this is a well-established procedure found in many clinical settings, the technique, in its traditional sense, is limited by the requirement of producing high-quality antibodies, multiple washing steps, and low capacity for multiplexed analyte detection. Many of these limitations are now being addressed by the development of novel detection strategies that incorporate the magnetic, optical, and electronic properties associated with metallic nanoparticles into high-sensitivity sensing devices for a broad range of target analytes. For instance, the amenability of antibody-conjugated magnetic particles to physical separation, coupled with the enormous amplification potential of enzymatic DNA replication (polymerase chain reaction) can be used to detect ultralow concentrations of disease-related proteins in serum.[5,6] Furthermore, protein detection using optical methods can be carried out by homogeneous nanoparticle assays, including whole blood, without the need for additional separation and purification.[7] For example, antibody-functionalized gold nanoshells have been tailored to specifically detect target analytes in a protein-rich background using the principles of a sandwich-immunoassay, where analyte-driven aggregation of the nanoshells results in a red-shift in their absorption spectra.[6]

[*] Dr. M. M. Stevens, J. E. Ghadiali

Department of Materials, Imperial College London London, SW7 2AZ (UK) E-mail: m.stevens@imperial.ac.uk

Dr. M. M. Stevens, J. E. Ghadiali Institute of Biomedical Engineering, Imperial College London London, SW7 2AZ (UK)

[**] We gratefully acknowledge the support of the EPSRC (EP/E007627/1) and The Leverhulme Trust.

Inorganic nanoparticles and their accompanying diverse physical properties are now virtually in routine use as imaging tools in cell-biology. In addition to serving as excellent contrast agents, their size- and environment-dependent optical and magnetic properties can beharnessed tocreate enzyme biosensor devices of extremely high sensitivity, whilst circumventing the numerous technical limitations associated with traditional enzyme assays. In this Research News article we discuss recent advances in field of enzyme-responsive nanoparticle systems, where the activity of an enzyme elicits a specific response in the nanoparticle assembly to produce a signal relating to enzyme activity, focusing on three important systems: DNA-structured nanoparticles, protein kinases and proteases.

Despite the sensitivity and versatility of immunological detection methods for enzymes, it is often useful to directly measure their activity. Nanoparticle-based materials are attractive alternatives to the fluorescence- or radiolabeled enzyme substrates of traditional enzymatic assays, owing to their photostability, ease of synthesis, and ability to conjugate to biological molecules (Table 1). In the vast majority of circumstances, excess of a thiolated derivative of a biological molecule of interest (e.g. DNA/peptide)coordinates directly to the nanoparticle’s surface, namely gold or silver, whilst any excess ofthe molecule is readily removed by centrifugation and washing steps. Nanoparticle enzyme sensors, in the most basic sense, consist of a biological substrate molecule immobilized onto the nanoparticle surface. The activity of an enzyme results in some form of modification of the substrate and a subsequent change in the nanoparticles’ local environment, leading to the generation of a user-readable signal, for example, optical or electronic. Being able to probe the activity of the enzyme directly, as opposed to simply detecting its presence as in immunological assays, yields several key benefits, as the enzyme’s activity and continuous turnover serves as an inherent means of signal amplification and also provides a route to screening potential inhibitors.

Here, we provide an overview of recent examples of enzymes that have been examined using a range of nanoparticle-based strategies, focusing on enzymes involved in DNA metabolism, protein kinases, and proteases owing to the extent of their characterization and important implications in numerous disease states.

2. Enzyme-Responsive Nanoparticle Systems 2.1. Enzymatic Control of DNA Nanostructures

The site-specific addressability of DNA base-pairing is now well-known as a tool to drive the assembly of many diverse higher-order structures and controlled aggregation of inorganic nanoparticles.[8,9] There are now several examples demonstrating that such DNA–nanoparticle conjugates can serve as substrates for a range of enzymes involved in DNA processing, with the resultant changes in particle dispersion used to drive precise structural rearrangements as a signaloutput for enzyme activity.[10]

Disassembly of gold nanoparticles crosslinked by complementary duplex strands of DNA can be achieved by incubation with an endonuclease that cleaves double-stranded DNA in a site-specific manner.[1] The resultant dispersions can be subsequently characterized by absorption spectroscopy, transmission electron microscopy (TEM), and polyacrylamide gel electrophoresis. Furthermore, employing DNA ligases, in conjunction with similar specific endonucleases to generate cohesive DNA ends, provides a means to selectively link DNA-functionalized gold nanoparticles and form higher-order assemblies (Fig. 1).[12]

These principles of enzyme-driven assembly/disassembly have also been extended to real-time colorimetric screening of endonuclease inhibitors and to discriminate chemically modified DNA from an unmodified form.[13,14] In the first example, a DNA-cleaving enzyme was added to a DNA– gold-nanoparticle crosslinked network, in the presence or absence of various DNA binding agents known to inhibit the enzyme. Monitoring the intensity of the plasmon absorbance band as a function of time provided rapid, semiquantitative information on relative inhibitor potency and kinetics. For a more in-depth profiling of reaction kinetics and mechanistic details of the cleavage reaction, surface-immobilization and single-molecule imaging of DNA–nanoparticle conjugates is

J. E. Ghadiali and M. M. Stevens / Enzyme-Responsive Nanoparticle Systems

Table 1. Key points for consideration during the development of ‘‘traditional’’ and nanoparticle-based enzyme assays.

Radioisotope and fluorometric assays Nanoparticle-based assays

Labelled substrate analogues can be costly. Nanoparticles are synthetically accessible using routine methods. Substrates are often commercially available. Many nanoparticle derivatives are commercially available at reasonable cost. Substrate can be added to enzyme reaction medium without any additional preparative steps. Nanoparticle-substrate conjugates must be prepared.

Decay of substrate and photochemical stability of substrates can limit shelf-life and necessitates careful handling.

Nanoparticles are often chemically robust. Colloidal stability is important factor with respect to shelf-life and compatibility with enzyme reaction media.

Requires scintillation counting, autoradiography, or fluorometric detection equipment. Fluorometric, SERS, relaxivity, absorbance, or visual detection. Multistep procedures are often required. Possibility for homogeneous, one-step reactions.

Figure 1. Enzyme-driven modification of DNA nanostructures by a) endonuclease/ligation mechanisms, and b) DNA methyltransferase enzymes.

normally necessary.[13] By using gold nanoparticles tethered to a surface via a double-stranded DNA molecule, Glass and co-workers have investigated the effect of enzymatically methylating the DNA prior to endonuclease cleavage, using dark-field imaging to monitor release of the particles as the cleavage reaction proceeds (Fig. 1).[14] It has also been shown that it is possible to interrogate the structural details of the restriction enzyme reaction using the distance-dependent plasmon coupling observed in individual pairs of gold nanoparticles.[15] The robust photophysical properties of gold nanoparticles, compared to organic fluorophores, allow long reaction-time trajectories to be monitored in addition to observing larger distance changes in biological molecules that have been previously inaccessible to single-molecule fluorescence techniques, namely FRET – the distance-dependent nonradiative energy transfer between two fluorophores with overlapping excitation/emission spectra.

In summary, many DNA-modifying enzymes, once traditionally restricted to the molecular biologist’s toolkit, are seeing increased use as a method to precisely control DNA- based nanostructures, and the optical properties associated with some nanoparticles show growing potential in the fields of high-throughput drug screening and structural biophysics.

2.2. Protein Kinases

One class of enzymes that has attracted much recent attention as candidate targets for nanoparticle biosensors are protein kinases. Kinases catalyze the transfer of the a-phosphate group from adenosine triphosphate (ATP) to nucleophilic amino-acid side chains (serine, tyrosine, threonine) and are intimately involved with the regulation of many key cellular processes. One of the most important manifestations of the deregulation of kinase activity is loss of cell-cycle control and the development of cancer.[16] As such, kinases are considered to have excellent potential as therapeutic targets, and numerous techniques have been developed to determine their activity. The standard method of measuring kinase activity has long since been the use of radiolabelled g-32P-ATP; however, recent reports have demonstrated that by using a g-biotin-ATP analogue, the kinase reaction can be used to drive the crosslinking of gold nanoparticles functionalized with a peptide substrate and streptavidin, respectively (Fig. 2).[17] The enzyme-mediated nanoparticle aggregation could be monitored by following the red-shift and peak broadening of the gold plasmon absorbance band and further applied to multiplexed screening of kinase inhibitors.

This approach has also been extended to a microarray format in conjunction with silver enhancement and resonance light-scattering as the detection method.[19]

Further efforts have been made to determine kinase activity from whole-cell lysates. One recent example has utilized the changes in overall peptide charge on addition of the phosphate group and its effect on gold nanoparticle stability as a kinase sensor. In this case, nonphosphorylated cationic substrate peptide leads to rapid aggregation of the nanoparticles on chemisorption to the anionic nanoparticle surface, whereas addition of the phosphorylated peptide maintains a stable dispersion (Fig. 2).[18]

In addition to monitoring the activity of purified enzymes, this simple colorimetric approach could also be extended to observing the effects of stimulatory drugs on cellular signal-transduction pathways that enhance kinase activity. An analogous procedure has also been developed to detect the activity of phosphatases; enzymes that catalyze the removal of phosphate groups from proteins.[20]

With a view to imaging kinase activity within individual living cells, polymeric nanoparticle carriers have been developed, which are both biocompatible and cell-permeable. These carriers consist of a kinase peptide substrate and near-infrared (NIR) fluorophores chemically coupled to poly(ethyleneimine), and self-assemble in the presence of a second negatively charged polymer to form a solution of monodisperse particles.[21] In the nonphosphorylated state, these particles have low levels of fluorescence owing to the short interfluorophore distances and quenching, however, phosphorylation of the peptide leads to nanoparticle solubilization, dequenching, and NIR fluorescence recovery.

J. E. Ghadiali and M. M. Stevens / Enzyme-Responsive Nanoparticle Systems

Figure 2. Nanoparticle-based enzyme sensing strategies. a) Kinasemediated crosslinking of gold nanoparticles by means of a biotinylated substrate analogue [17]. b) Utilization of changes in peptide charge following phosphorylation to influence nanoparticle aggregation [18].

Adv. Mater. 2008, 20, 4359–4363 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim w.advmat.de 4361

Considering that kinase deregulation occurs in essentially every cancerous cell, it is likely that these new screening methods will attract increasing notice in the near future as tools in research and drug development.

2.3. Proteases

Protease-mediated cleavage of protein amide bonds is a central process to the regulation of many cellular processes, playing a key role in embryonic development and the progression of numerous disease states.[2] Whilst there are numerous high-sensitivity commercial assay kits for protease activity available on the market, they are typically fluorometric in nature or do not lend themselves to sensitive real-time monitoring. Some of the limitations of organic fluorophores can be addressed, to some extent, by using photochemically robust quantum dot (QD) probes or magnetic nanoparticles with relaxivity measurements to determine protease activity.[23,24] For example, protease substrate peptides labeled with aFRET-pair acceptordyecan beimmobilized onQDscaffolds that serve as the FRET donor. Peptide cleavage is accompanied by an increase in donor fluorescence intensity owing to a decrease in acceptor-mediated quenching.[25] A two-step gold-nanoparticle-based protease assay of nanomolar sensitivity has also been reported, in which a protease is preincubated with its corresponding peptide substrate. Upon addition to a dispersion of citrate-stabilized gold nanoparticles, in the absence of enzyme, the uncleaved peptide substrate induces nanoparticle aggregation via crosslinking, whereas addition of enzymatically cleaved peptide leads to the particles retaining dispersity.[26]

A rapid nonfluorometric protease sensing system that can be readily tailored for the detection of many different proteases has also been recently developed in our group.[27] In this system, protease substrate peptides are chemically anchored to gold nanoparticles via a thiolated amino-acid side chain. This is accompanied by the formation of a crosslinked peptide–nanoparticle network mediated by p–p interactions between aromatic moieties that normally serve as protecting groups during peptide synthesis. This self-assembly principle is also being developed to control enzyme-triggered gelation with many implications in controlled drug release and scaffolds for cell culture.[28,29] Protease activity results in cleavage of the cross-linking peptide groups and particle dispersion, as indicated by a color change of the solution from blue to red, owing to loss of hydrophobic interactions and electrostatic repulsion between exposed

NH3þ groups(Fig. 3). This versatile approach could be adapted by modifying the substrate-peptide amino acid sequence for different proteases, including prostate-specific antigen – a well known marker for prostate cancer.[30]

Whilst these methods can provide a direct colorimetric signal output, owing to changes in nanoparticle aggregation, Raman scattering (specifically, surface-enhanced Raman scattering, SERS) can also be utilized for in-solution determination of protease activity. For instance, gold nanocrescents generated on the surface of a polystyrene nanosphere can conjugate to protease peptide substrates carrying an additional Raman-active reporter group. Cleavage of the peptide leads to loss of the reporter groups from the vicinity of the nanocrescent surface and loss of their characteristic SERS peaks.[31]

3. Summary and Outlook

A plethora of enzyme-responsive nanoparticle systems are currently being explored. Nanoparticle-based enzyme sensing approaches show distinct advantages over traditional methods and immunological detection strategies in terms of their potential rapidity and sensitivity. Nonetheless, numerous technical challenges still remain in successfully interfacing biological molecules with inorganic nanoparticles to generate biosensing platforms.[32] For instance, development of a suitable repertoire of biocompatible bioconjugation strategies, greater control over ligand: nanoparticle stoichiometry, avoidance of nonspecific binding and uncontrolled aggrega-

J. E. Ghadiali and M. M. Stevens / Enzyme-Responsive Nanoparticle Systems

Figure 3. a) Schematic representation of Fmoc-peptide/gold nanoparticle protease sensing system. b) Rapid and sensitive detection of 10 ag mL–1 of prostate cancer-associated marker. c) Integrated spectral regions utilized for the aggregated/dispersed (A/D) ratiometric parameter [27].

tion, and long-term storage stability in physiological buffers. Numerous strategies are being investigated in order to control biomolecule:ligand stoichiometry. For instance, histidinetagged peptides and proteins can self-assemble on the surface of dihydrolipoic acid modified QDs, which permits some degree of control over the peptide: QD ratio whilst retaining the secondary structure of the peptide.[3] Issues of particle stability can also be addressed to some extent by the incorporation of hydrophilic polymers and biological molecules that confer resistance to nonspecific protein adsorption in addition to extreme tolerance towards high concentrations of electrolytes.[34,35] Despite the numerous current challenges in this field, there is enormous potential to be found in enzyme-responsive nanoparticle systems and it is likely that they will serve to greatly complement current enzymatic assay techniques employed in the research laboratory, clinical diagnostics, and drug discovery.

Published online: June 2, 2008

[3] E. Katz, I. Willner, Angew. Chem. Int. Ed. 2004,4 3, 6042.

[4] V. M. Rotello, Adv. Mater. 2008, in press.

(Parte 1 de 2)