Silver and Gold Nanoparticles Alter Cathepsin Activity In vitro
© Speshock et al. 2010
Received: 21 May 2010
Accepted: 6 August 2010
Published: 29 August 2010
Nanomaterials are being incorporated into many biological applications for use as therapeutics, sensors, or labels. Silver nanomaterials are being utilized for biological implants and wound dressings as an antiviral material, whereas gold nanomaterials are being used as biological labels or sensors due to their surface properties and biocompatibility. Cytotoxicity data of these materials are becoming more prevalent; however, little research has been performed to understand how the introduction of these materials into cells affects cellular processes. Here, we demonstrate the impact that silver and gold nanoparticles have on cathepsin activity in vitro. Cathepsins are important cellular proteases that are imperative for proper immune system function. We have selected to examine gold and silver nanoparticles due to the increased use of these materials in biological applications. This manuscript depicts how both of these types of nanomaterials affect cathepsin activity, which could impact the host's immune system and its ability to respond to pathogens. Cathepsin B activity decreases in a dose-dependent manner with all nanoparticles tested. Alternatively, the impact of nanoparticles on cathepsin L activity depends greatly on the type and size of the material.
Cathepsins are lysosomal proteases that are present in many different types of mammalian cells. Cathepsins B and L are cysteine proteases localized in the lysosomes and endosomes and are mainly involved in protein degradation and antigen presentation . Cathepsin B is mainly expressed in antigen-presenting cells (APCs) and is involved in protein processing for presentation and is responsible for the degradation of the invariant chain (Ii), a chaperone molecule essential for major histocompatibility complex (MHC) class II molecules assembly and transport [2, 3]. Cathepsin B also functions to process MHC-II binding peptides in the endosome . In addition to antigen presentation, cathepsin B is also involved in many other physiological processes such as inflammation, wound repair, apoptosis, and activation of thyroxine and renin . Cathepsin L is present in all mammalian tissues and degrades both exogenous and endogenous proteins . Cathepsin L also regulates MHC class II antigen presentation by cleaving Ii, especially in the thymic endothelium where it generates epitopes required for T-cell selection [3, 4]. Due to the many immune system processes that these enzymes regulate, alteration in their function could be detrimental to the host.
Nanotechnology is a growing field of study focused on the development of materials sized <100 nm. These nano-sized particles exhibit unique physical and chemical properties that are often different than the bulk material from which they are derived . Nanomaterials are being adapted for many uses, but until recently not much effort has been put toward examining the biological effects of these unique materials. Recent studies have demonstrated that silver nanoparticles (Ag-NPs), which are receiving considerable attention due to their antimicrobial properties, are actually quiet toxic to mammalian cells at low doses [6–8]. Bulk and nanosilver have been shown to interact readily with mammalian proteins and inactivate cellular enzymes, which could contribute to Ag toxicity in vitro [9–11]. In this current study, we examined the interaction of Ag-NPs with cathepsins B and L and determined the extent of enzymatic inhibition caused by the nano-Ag using both purified cathepsins and a cell culture model. The effects of gold nanoparticles (Au-NPs) on cathepsin activity were also assessed due to recent concerns involving the effects of cathepsin L on functionalized Au-NPs . Au-NPs are generally found to be non-toxic to mammalian cells in culture , and there is little evidence indicating how they interact with enzymes and whether they alter enzymatic function.
Materials and Methods
Plasma gas-synthesized 25-nm Ag-NPs were a kind gift from Dr. Karl Martin (Novacentrix, Austin, TX). Colloidal Ag-NPs at 10 nm and Au-NPs at 10 or 30 nm were synthesized by Dr. Steven Oldenburg (NanoComposix, San Diego, CA). Polysaccharide-coated Ag-NPs (PS-Ag) 10 and 25 nm were a generous gift from Dr. Dan Goia (Clarkson University, Center for Advanced Materials Processing, Potsdam, NY). These PS-Ag NPs were synthesized by the reduction of Ag ions in solution by a polysaccharide (acacia gum), which leads to a polysaccharide surface coating . All NPs were well characterized in our laboratory and were diluted in deionized water to 1 mg/ml, sonicated with a probe sonicator, and then further diluted in PBS or cell culture media.
Vero cells were obtained from the ATCC (CCL-81; Manassas, VA). The Vero cells were maintained in Dulbecco's modified Eagle's Medium (DMEM; Biowhitaker, Basel, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Carlsbad, CA) and 1% penicillin-streptomycin (PS; Invitrogen, Carlsbad, CA).
Vero cells were seeded into 96-well tissue culture-treated plates at a concentration of 50,000 cells/well. Twenty-four hours post-seeding, the Vero cells were exposed to Ag- or Au-NPs. The NPs were diluted to the various doses in DMEM + PS and sonicated for 30 s prior to exposure. At 24-h post-exposure, a standard MTS assay (Promega, Madison, Wisconsin) measuring mitochondrial function was performed to determine cell viability.
Cellular Cathepsin Activity Assay
Vero cells were treated with the above NPs at various doses and were incubated for 24 h at 37°C in 5% CO2. The NPs were removed and excess NPs were washed off with PBS. CV-(RR)2 or CV-(FR)2 reagents were added to the treated cells (diluted 1:250 in PBS) to detect changes in cathepsin B or cathepsin L activity, respectively (Biomol International, Plymouth Meeting, PA). Biomol's cathepsin detection kit utilizes the fluorophore, cresyl violet (CV), linked to two peptides moieties (arginine-arginine for cathepsin B and phenylalanine-arginine for cathepsin L), which are cleaved when the enzyme is active to release the fluorophore. The cathepsin substrate was incubated with the cells for 45 min at 37°C in 5% CO2 and was then read using a fluorometer (BioTeck Synergy HT with Gen5 software) at excitation/emission wavelengths of 594/625 nm. Alternatively for the imaging, Hoechst (Molecular Probes – Invitrogen, Carlsbad, CA) was added directly to the CV-(RR)2 or CV-(FR)2 reagents (1:1,000), and the cells were imaged following the 45-min incubation with the Becton Dickinson Pathway 435 spinning disc Confocal microscope (BD, Franklin Lakes, NJ). The CV-(RR)2 or CV-(FR)2 reagents were detected with a rhodamine filter with settings normalized to the control (untreated) sample.
Purified Cathepsin Activity Assay
Purified cathepsins B and L were purchased from Biomol International (SE198 and SE201, respectively). Three micrograms of each protein was incubated with the 4 types of Ag-NPs at either 10 or 50 μg/mL in 300 μL sterile H2O for 1 h at room temperature with rotation. Following this incubation, the mixture was added in triplicate to a 96-well black-sided plate (100 μL/well). Hundred microliters of the respective Biomol CV reagent (diluted 1:125 in water) was then added to each well of the enzyme-NP mixture. The reaction was incubated for 45 min at 37°C in 5% CO2 and was then read in a fluorometer. The process was repeated to obtain a total of six replicates.
Data were expressed as the mean ± standard error of the mean (SEM). The one-way ANOVA (Prism 5 statistical and graphing software; GraphPad Software, Inc, La Jolla, CA) was used for the data analysis. The one-way ANOVA was used to determine the effect of test compound concentration on the mean number of treatments/well. P ≤ 0.05 was used as the level for significance.
Ag-NP Biocompatibility in Vero cells
Cathepsin B Activity in Ag-NP-treated Cells
Cathepsin L Activity in Ag-NP-treated Cells
Effects of Ag-NPs on Purified Cathepsins
Au-NP Biocompatibility in Vero Cells
Au-NP effects on Cathepsin Activity
The research illustrated here demonstrates the effects of Ag- and Au-NPs on cathepsin enzymatic function. We have shown that Ag-NPs decrease the efficiency of cathepsin cleavage in a dose-dependent manner with significant effects observed at doses as low as 1–5 μg/ml, which is non-toxic to the cell itself. The inhibitory effects of the NPs on cathepsin activity were observed within as little as 1 h of incubation (data not shown); following 24 h of incubation with the NPs, the cathepsin activity was significantly affected in Vero cells. The Ag-NPs had a greater effect against the activity of purified cathepsins than that of Vero cell cathepsins. This may suggest a possible direct interaction of the Ag-NPs with the proteins; however, the internalized Ag-NPs are likely to interact with many proteins in the cell and not just the cathepsins, therefore diluting the dose actually exposed to the cathepsins themselves, which could also account for this difference. Bulk silver and Ag-NPs have previously been shown to inhibit the sodium-potassium ATPase [9, 10] and FYN kinase , respectively, indicating that Ag inhibits different types of enzymatic function, which should be considered prior to use in living systems. Au-NPs also inhibited cathepsin B activity both in a cellular model and with the purified enzyme. However, even more interesting was the impact of Au-NPs on cathepsin L activity. When the Vero cells were exposed to Au-NPs, a stimulatory effect of cathepsin L activity was observed. Conversely, a decline in cathepsin L activity was observed when the Au-NPs were incubated with the purified enzyme. This may indicate that the Au-NPs are interacting with the cathepsins indirectly, possibly through a signaling cascade, inside of the cells.
The biological applications of both Au- and Ag-NPs are very promising, but little effort has been put into determining how they may affect the natural function of the cells in which they are incorporated. Both Ag- and Au-NPs are capable of recognizing and readily binding thiol groups of proteins and other ligands, suggesting that they would likely have similar effects on protein activity [14, 15]. However, the two types of nanomaterials have substantially different effects on cytotoxicity. Au-NPs are considered to be relatively benign to most cells, which make them an attractive tool for biomedical applications . Conversely, Ag-NPs have been shown to be toxic to cell lines at low concentrations  and appear to have a more significant affect on altering enzymatic activity.
Biological molecules are being added to Au- and Ag-NPs to improve their biocompatibility or functionality for biological applications. However, recently, it was determined that cathepsin L can cleave biological moieties off functionalized nanoparticles . See et al utilized functionalized gold nanoparticles to assess the ability of cathepsin L to cleave substrates off the NPs and confirmed that upon internalization into the cell, the substrate is cleaved off the Au-NPs by cathepsin L . We utilized similar sized Au-NPs and Ag-NPs to determine the effects of these NPs on cathepsin activity. We concluded that the Au-NPs, but not the Ag-NPs, stimulated cathepsin L activity in cells, confirming that functional groups added to the surface of Au-NPs may be lost upon cell internalization. Alternatively, cathepsin B activity was decreased by both types of NPs testing, indicating that cathepsin B directed cleavage of biomolecules will be inhibited in the presence of NPs. Our findings suggest that the use of cathepsin L inhibitors or alternate core metals may be necessary for use of intracellular nano-based therapeutics and sensors.
Cathepsin activity is important for many cellular processes, most notably for priming of the adaptive immune response through antigen processing . Therefore, using materials that are capable of inhibiting cathepsin activity for biological applications may perhaps cause harmful side-effects. Studies have suggested the potential for Ag-NPs as an antimicrobial: however, if they cause a dramatic reduction in cellular cathepsin activity, which can alter the adaptive immune response, they may actually interfere with the host's ability to clear the infection. There are a lot of beneficial biomedical applications for using nanomaterials, but careful consideration to avoid potential undesired effects must be determined before they are used in vivo.
We would like to thank the Defense Threat Reduction Agency (DTRA) for funding the project, the JSTO-NRC postdoctoral fellowship program for supporting Dr. Janice Speshock, the STEP summer student program for supporting Mr. Szymanski, and the Henry Jackson Foundation for funding Dr. Braydich-Stolle.
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