Colloidal Gold Modified with a Genetically Engineered Nitroreductase

Oct 20, 2011 - Directed enzyme prodrug therapy is an extensive area of research in cancer chemotherapy. Although very promising, the current directed ...
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Colloidal Gold Modified with a Genetically Engineered Nitroreductase: Toward a Novel Enzyme Delivery System for Cancer Prodrug Therapy Vanessa V. Gwenin, Chris D. Gwenin,* and Maher Kalaji School of Chemistry, Bangor University, Bangor, Gwynedd, LL57 2DG, Wales, United Kingdom

bS Supporting Information ABSTRACT: Directed enzyme prodrug therapy is an extensive area of research in cancer chemotherapy. Although very promising, the current directed approaches are still hampered by inefficient enzyme expression and tumor targeting. This work investigates the viability of using metal nanoparticles as a novel delivery vehicle for prodrug-activating enzymes. Using genetically incorporated amino acid sequences, a nitroreductase from E. coli was directly immobilized onto a 50 nm gold colloid, as confirmed by gel electrophoresis, DLS, and UV vis spectroscopy. The resulting conjugates showed excellent stability in changing proton and sodium chloride environments, including PBS at 37 °C. Remarkably, the immobilized nitroreductase retained more than 99% activity to the CB1954 prodrug without the need for stabilizers. This work provides the foundation for attaching prodrug-activating enzymes to metal nanoparticles for future use in directed enzyme prodrug therapy.

1. INTRODUCTION Cancer is globally responsible for 30% of all deaths each year and is second only to cardiovascular disease as a world killer, which is more than HIV/AIDS, TB, and malaria combined.1 Chemotherapy for cancer has evolved significantly since its first intended use in 1946,2 but most drugs in use today still target any rapidly dividing cells, causing systemic toxicity. In order to overcome this limitation, either the current strategies need to be improved or new ways devised to selectively target solid tumors. To increase tumor selectivity, current targeting strategies rely on prodrugs,3 which are relatively harmless at the point of administration but become activated by specific enzymes within the tumor or the organ containing the tumor. One intensely investigated approach uses specific antigens to locate the drug/ prodrug directly at the solid tumor.4 A second strategy comprises the delivery of bacterial prodrug-activating enzymes or their encoding genes to the tumor before administering the prodrug. The latter is generally referred to as directed enzyme prodrug therapy (DEPT).5 Carriers for bacterial enzymes or their encoding DNA include antibodies6 (ADEPT), viruses7 9 (VDEPT), cationic lipids,10 peptides,11 or the naked DNA.12 The limitations of the current DEPT are the scarcity of tumor-specific antigens, the immunogenicity of the drug carrier combination, and the inefficient expression of enzymes from the targeted DNA.4 A novel carrier investigated for DEPT is gold-coated magnetic nanoparticles (MNDEPT).13 Use of a magnetic core could allow prodrug-activating enzymes to be directed to a solid tumor using an external magnetic field, as illustrated for other chemotherapy agents,14 and of which treatment localization and progress could be monitored using real-time magnetic resonance (MR) imaging.15 In 2006, an esterase was attached to gold-coated magnetic nanoparticles (MNPs),16 presumably for use in industrial processes such r 2011 American Chemical Society

degradation of plastics and other toxic compounds.17 The hexaarginine-tagged esterase was tethered to the gold-coated surface through electrostatic interaction with a long carboxyaliphatic thiol linker. Although the enzyme showed good reusability, only 60% of the activity was retained. Direct immobilization has the possibility to overcome the denaturation and distortion seen when trying to attach proteins to linker molecules on the colloid surface. To investigate the feasibility of directly immobilizing a prodrug-activating enzyme onto a metal colloid, a genetically modified nitroreductase (NTR) from E. coli (NfnB)18 was immobilized onto a 50 nm gold colloid with the future aim of applying this methodology to gold-coated MNPs.19 Direct immobilization of enzymes onto gold colloids has previously been studied for fabrication of enzyme electrodes.20 The enzymes glucose oxidase, horseradish peroxidase, xanthine oxidase, and carbonic anhydrase were directly adsorbed onto a 50 nm gold colloid surface and managed to retain nearly all activity, except for carbonic anhydrase. Enzyme adsorption was however very poor and nonspecific. Direct immobilization of enzymes using inserted amino sequences with high affinity for the metal colloid is a very new area of research, and to the best of our knowledge, only one example exists.21 This group used an N-terminal peptide repeat (RRTVKHHVN) for direct immobilization of their enzyme onto iron oxide nanoparticles. The enzyme retained very little activity after immobilization and required addition of stabilizers to restore enzyme activity to 99%.

Received: December 3, 2010 Revised: October 20, 2011 Published: October 20, 2011 14300

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Langmuir Here, we disclose the first report of direct immobilization of an enzyme onto a gold colloid using genetically inserted amino acid sequences utilizing two modified forms of the NfnB. The first contained a His-tag purification sequence (MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS), which contains a large percentage of amino acids with high affinity for gold.22 The second modified form of NfnB contained an additional Cystag23 in a position between the His-tag and the N-terminal of the NTR. Direct immobilization was confirmed using dynamic light scattering (DLS), agarose gel electrophoresis, and UV vis spectroscopy, and the stability of the gold colloid conjugates was assessed in changing ionic and pH environments, including the solutions emulating physiological conditions. Enzyme activity to the CB1954 prodrug was determined before and after immobilizing the enzymes to the gold colloid.

2. MATERIALS AND METHODS All chemicals were obtained from VWR (Lutterworth, U.K.) unless otherwise stated.

2.1. Cloning, Expression, and Purification of Recombinant NTRs. The cloning and expression of the recombinant NfnB proteins was carried out as previously described.23 Briefly, the wild-type nfnB gene (gene ID 945778) from Escherichia coli K12 (Genbank accession number NC_00913) was amplified from genomic DNA using designed primers during a PCR protocol. To generate the Cys-nfnB gene, a Cystag was introduced during PCR by using a primer coding for six cysteine residues downstream from the nfnB start codon. Both the wild-type (nfnB) and modified (Cys-nfnB) genes were then cloned into the pET28a(+) expression vector (Novagen, Merck, U.K.), which adds an N-terminal histidine purification tag (His-tag). An E. coli Rosetta strain (Novagen, U.K.) was then transformed with the two recombinant plasmids and both proteins expressed after isopropyl-β-D-thio-galactoside (IPTG) induction. The recombinant NfnB containing only the histidine purification tag (His-NfnB) and the NfnB with the additional Cys-tag (Cys-NfnB) were purified from the cell debris by centrifugation and metal ion affinity chromatography (IMAC)24 before finally removing small molecule contaminants with ultrapure water during size exclusion chromatography (SEC). The purity and molecular weight of the protein fractions were assessed after separation on a 12% SDS-PAGE gel during electrophoresis (Mini-PROTEAN Electrophoresis System, Bio-Rad) and visualized with Coomassie blue before use in further experiments.25 Protein concentration was determined from a BSA standard curve using the ProPure Biuret protein assay (Amresco, NBS Biologicals, U.K.) according to the manufacturer’s instructions. 2.2. Formation of Enzyme Gold Colloid Conjugates. A solution of 50 nm gold colloid (1.5 nM, Naked Gold, BioAssay Works, USA) was incubated with varying molar equivalents (90:1, 180:1, 270:1, 360:1, and 450:1) of purified recombinant Cys-NfnB or His-NfnB in ultrapure water (pH ≈ 7.00, ELGA purification system) for 1 h at 25 °C26 or overnight at 4 °C.23 The ratios were achieved by changing the enzyme concentration and keeping the gold colloid concentration constant. The interaction of enzyme with the gold colloid surface was subsequently investigated by UV vis spectroscopy (750 200 nm) using a Jasco V-550, and spectra were analyzed with the Spectra Manager Software. 2.3. Agarose Gel Electrophoresis. Enzyme gold colloid conjugates of molar ratios 90, 180, 270, 360, and 450 enzymes to gold were prepared as stated above and stored at 4 °C until required. The enzyme gold colloid conjugates or gold colloid alone (50 μL) were first mixed with 20 μL of loading buffer (50% glycerol in 1.5 M tris-HCL, pH 8.8) before loading onto a 2% agarose gel. The gel was run in 0.5% TBE running buffer (10 dilution of TBE stock consisting of 450 mM Tris/450 mM Borate/10 mM EDTA) at 220 V for 30 min with the gel

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bed placed on ice (Midi horizontal electrophoresis unit, Sigma-Aldrich, U.K.). Migration of the pink-colored samples was visible by eye. 2.4. Dynamic Light Scattering and Zeta Potential. Enzyme gold colloid conjugates were prepared at 4 °C, as described above. Particle size and zeta-potential measurements were performed using a HORIBA SZ-100Z system (Kyoto, Japan). Particle size analysis measurements were made at a 90° scattering angle and standard Dynals analysis algorithm at 25 °C. Results are reported as the z average from the intensity distribution. None of the samples required dilution or other sample preparation steps. The system was first verified using both a 100 nm PSL standard (Duke Scientific cat no. 3100A) and a 10 nm colloidal gold reference material from NIST (NIST reference material 8011). Zeta-potential measurements were made on the same system using disposable measurement cells with carbon-coated electrodes in order to minimize sample interaction at the electrode surface. The samples were also analyzed without dilution for zeta-potential analysis. 2.5. Enzyme Gold Colloid Conjugate Stability. His-NfnB and Cys-NfnB gold colloid conjugates were prepared as stated above at both 25 and 4 °C. To assess the ability of the conjugates to resist a change in ionic environment, the UV vis absorbance spectra (750 200 nm) of the enzyme gold colloid conjugates or gold colloid alone were measured every 15 min after sequential addition of sodium chloride (NaCl, 6 M stock). To assess the effect of the pH on stability, enzyme gold colloid conjugates or gold colloid alone were added to cuvettes containing phosphate-buffered solutions of pH 2 11 and incubated for 15 min before the UV vis absorbance spectra (750 200 nm) were measured. Ultrapure water served as a reference against which all spectra were measured. For long-term stability measurements, all conjugate samples were prepared in ultrapure water as stated above and stored either at room temperature or at 4 °C, and spectra were analyzed after 1 month and 1 year.

2.6. Enzyme Gold Colloid Conjugates Stability under Physiological Conditions. To determine whether the enzyme gold colloid conjugates could remain stable in an environment emulating physiological conditions, the His-NfnB and Cys-NfnB 270:1 gold colloid conjugates were prepared as stated above and incubated at 4 °C overnight. The next day the samples were spun down at 13 000 rpm for 5 min and resuspended in 1 mL of cold phosphate-buffered saline (PBS, 0.1 M) pH = 7.4. Samples were equilibrated to room temperature over a period of 30 min. Absorbance spectra were recorded before and after centrifugation and measured every 30 min during incubation in PBS at 37 °C for 2 h.

2.7. Enzyme Reactivity to CB1954 before and after Immobilization. Purified recombinant Cys-NfnB and His-NfnB, at a molar ratio of 90:1, were prepared as described above and incubated at 4 °C overnight. The next day each solution was divided in half (to form the test and reference solutions) and made up to a total volume of 970 μL using ultrapure water. To each solution, 20 μL of NADH (10 mM stock) was added and incubated at 37 °C for 5 min, after which either 10 μL of CB1954 (10 mM stock) or 10 μL of DMSO was added and absorbance spectra (750 nm- 200 nm) recorded every 1 1/2 min for 20 min. The same procedure was followed for the enzyme gold colloid samples and free enzyme purified by size exclusion chromatography. For the free enzyme controls, the gold colloid was substituted for ultrapure water. The specific activity was calculated for both the enzyme gold colloid conjugates and enzymes free in solution using the molar absorptivity of the CB1954 hydroxylamine products at 420 nm (ε = 1200 M 1 cm 1).27

2.8. Size Exclusion Chromatography of Enzyme Gold Colloid Conjugates. For this purpose, Sephadex G-75 (SigmaAldrich, U.K.) was used with a molecular weight cut off of 70 KDa. The Sephadex (3 g) was mixed with filtered, distilled water (50 mL) and allowed to swell for 24 h. The next day the solution was degassed for 30 min using N2 (g) and the slurry poured into a glass column (1.5 cm in 14301

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Figure 2. Agarose gel electrophoresis of gold colloid incubated with different ratios of Cys-NfnB. The gold colloid alone sample (0:1) aggregated within the well as a dark black band. The Cys-NfnB gold colloid conjugates migrated within the agarose gel to different degrees based on the amount of protein present on the colloid surface (90:1 450:1).

Figure 1. SDS-PAGE gel indicating purification of the recombinant Cys-NfnB using metal ion affinity chromatography: Lane 1, protein marker I, Applichem; lane 2, blank; lane 3, supernatant before separating column; lane 4, flow through after applying supernatant to separating column; lane 5, 100 mM imidazole eluent; lane 6, 200 mM imidazole eluent; lane 7, 300 mM imidazole eluent; lane 8, 500 mM imidazole eluent; lane 9, blank; lane 10, protein marker. diameter containing frit with 16 μm cut off) to a height of 15 cm. The slurry was allowed to settle in the column under gravity, before passing a column volume (26.5 dm3) of water through the column using slight pressure. The 360:1 Cys-NfnB and His-NfnB gold colloid conjugates were individually applied to the top of the Sephadex bed and allowed to enter the column before filling the reservoir with water. During elution of the samples, slight pressure was applied and fractions (1 mL) were collected which were then analyzed by UV vis spectroscopy. The fractions containing the enzyme gold colloid conjugates were pooled, and the concentration of the conjugates was determined using the molar absorptivity for colloidal gold at 520 nm (ε = 1  109 M 1 cm 1). Samples of free Cys-NfnB and His-NfnB enzyme were also passed through the SEC column. Absorbance at 280 nm was measured, and the fractions containing the free enzyme were combined and protein concentration determined as mentioned above.

3. RESULTS AND DISCUSSION Purification of Recombinant Enzymes. The modified nfnB genes obtained during PCR were cloned into an expression vector which adds a His-tag to the N-terminal of the proteins for the purpose of protein purification. The His-tag of the proteins complexes with the Ni2+ ions adsorbed onto a Sephadex matrix during IMAC, separating them from other cellular proteins. The recombinant proteins were then eluted with a competitive binder, imidazole, resulting in a normal distribution of eluting protein, as seen in the SDS-PAGE gel (Figure 1, lanes 6 8). Recombinant proteins were finally purified from the imidazole and other small ion contaminants using SEC and ultrapure water. As seen from the supernatant fraction in Figure 1 (lane 3), the recombinant Cys-NfnB monomer (∼27.3 kDa) was successfully overexpressed and purified from the supernatant during IMAC. Although there was no visible difference in molecular weight between the Cys-NfnB and the His-NfnB recombinant proteins using SDS-PAGE, the Cys-NfnB protein required greater amounts of imidazole to elute from the IMAC column due to the affinity of cysteine amino acids for metal ions.28 The CysNfnB eluted with imidazole concentrations of 200, 300, and 500 mM (Figure 1, lanes 6 8), whereas the His-NfnB eluted between 100 and 200 mM imidazole (data not shown). The

NTR dimer (54.6 kDa) was also visible in fractions containing a high concentration of protein (Figure 1, lane 7). Binding of Recombinant Proteins to 50 nm Gold Colloid. Migration of a gold colloid within an agarose gel conjugated to various amounts of DNA,29 peptide,30 or protein,31 has previously been used to confirm the successful immobilization of the ligand on a colloid surface. Here, gel electrophoresis was also used to confirm the direct binding of the recombinant NTRs to the gold colloid by mixing the gold colloid alone and enzyme gold colloid conjugates with a Tris-HCL buffer of pH 8.8 prior to loading on a 2% agarose gel for separation during electrophoresis. As can be seen from Figure 2, the gold colloid alone (0:1) did not migrate toward the anode but instead aggregated within the well of the agarose gel. Aggregation was due to loss of charge imparted by the neutral tris molecule, which replaces surfaceassociated citrate at high pH.32 Both the Cys-NfnB (Figure 2) and the His-NfnB gold colloid conjugates (data not shown) migrated toward the anode in a manner that appeared to be dependent on the number of immobilized enzymes, except for the 360:1 NfnB gold and 450:1 Cys-NfnB gold colloid conjugate fractions. The minimum number of enzymes which caused the greatest retardation within the gels was 270, which was taken to be the minimum number of enzymes required to cover the gold colloid surface. The apparently contradictory increase in migration of the 450:1 Cys-NfnB gold colloid conjugate (Figure 2) and 360:1 NfnB gold colloid conjugates (data not shown) was thought to be due to excess enzymes forming a double layer on the colloid, causing a greater increase in overall negative charge with respect to the increase in hydrodynamic diameter. In order to investigate the hypothesis of an enzyme double layer in some of the gold colloid conjugates, dynamic light scattering was performed for all the conjugate samples, and the summarized results have been shown in Table S1 and accompanying figures within the Supporting Information. Dynamic light scattering measures the hydrodynamic diameter of particles in solution, which includes the diffuse electric Debye layer surrounding the conjugate particles. Thus, the diameter of the gold colloid is not only increased by adsorbed enzyme but also by the counterion and solvent molecules associated with the enzyme layer. The DLS analysis showed the gold colloid alone to have a hydrodynamic diameter of 51 nm, indicating that the associated citrate and counterion cloud contributed 1 nm to the overall diameter. For a gold colloid completely covered by protein, the diameter would be expected to increase by at least two times the diameter of the protein.33 The diameter of the wild-type NfnB is 5.7 nm,34 and hence, the hydrodynamic diameter of the gold 14302

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Figure 3. Change in the absorbance profile of gold colloid upon interaction with NTR. (A) Absorbance of gold colloid before (solid black line) and after interacting with a molar ratio of 90:1 recombinantNfnB vs gold colloid (dotted black line). (B) Change in gold colloid λmax after incubation with different molar ratios of either of the recombinant NTR enzymes (n = 3, standard error of the mean = 0).

colloid was expected to increase by an estimated 11.4 nm. The diameter of the Cys-NfnB gold colloid conjugates increased nonlinearly, and at the 270:1 molar ratio of enzyme vs gold, the particle diameter had increased by 11.1 nm, indicating full monolayer coverage. In the presence of excess Cys-NfnB enzyme, a double layer had formed, increasing the diameter of the particles by a further 8 nm. The hydrodynamic diameter of the NfnB gold colloid conjugates also increased in diameter with an increase in enzyme concentration, but at and above the 270:1 molar ratio of enzyme vs gold colloid, double and triple layers were formed (17 30 nm increase in diameter), suggesting that monolayer coverage occurred somewhere between the 180:1 and the 270:1 molar ratios. From the DLS results it appeared that the His-NfnB protein bound in a less ordered manner compared to the Cys-NfnB protein. These results suggested that the CysNfnB preferentially bound to the gold colloid through the thiols from the Cys-tag, forming a more ordered and tightly packed arrangement of enzymes on the gold colloid surface, as would be expected for monolayers formed with thiols.35 The zeta potentials were also determined for all of the conjugate samples and compared to the gold colloid alone (Table S1 and Figures 4 and 5, Supporting Information). The zeta potential of the colloid particles is the electric potential between the tightly associated counterion cloud which migrates with the conjugates in an electric field and the more diffuse layer which migrates in the opposite direction.36 From the results it was noted that it only required a small number of Cys-NfnB

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enzymes to dramatically change the zeta potential from 52 (gold colloid alone) to 23 mV (90:1 molar ratio of enzyme vs gold colloid). Thereafter, the zeta potentials of the CysNfnB gold colloid conjugate samples were similar up to a 450:1 molar ratio of enzyme vs gold, at which a significant change in zeta potential was again observed. The large change in zeta potential between the 360:1 and the 450:1 Cys-NfnB gold colloid conjugate samples, although the hydrodynamic diameter was the same, suggested that the double-layer packing was significantly different. Similarly, the 360:1 NfnB gold colloid conjugate had a double-layer packing with a very similar zeta potential to the 450:1 Cys-NfnB gold colloid conjugate. These results confirmed the hypothesis that the increased migration seen in the agarose gel of some conjugate samples in the presence of excess enzyme was due to the packing of the enzyme double layer resulting in a greater increase in overall negative charge compared to the increase in hydrodynamic diameter. To summarize, migration of conjugates within the agarose gel demonstrated not only that the enzymes had bound directly to the gold colloid but also that the conjugates had an overall negative charge (also confirmed with the zeta potentials). The results presented here support a previous observation in which migration within an agarose gel depended on the number of immobilized proteins and hydrodynamic diameter of the protein colloid particles36 but only until a full monolayer had formed on the colloid surface. A difference in the migration of particles containing multiple layers of protein is only seen if the packing is significantly altered to yield a more negative zeta potential. UV Vis Spectroscopy of Conjugates. The absorbance spectra of gold colloids have previously been used to indicate immobilization of the proteins onto the colloid surface.37,38 The delocalized electrons (conduction band electrons) of the gold colloid resonate with incoming light, which leads to the phenomenon known as the localized surface plasmon resonance (LSPR). The localized surface plasmon resonance (LSPR, λmax) is routinely used as an indication of the molecular interactions occurring at the surface of the nanoparticles and depends on particle shape and size,39 interparticle distance,40 immobilized ligand,41 and constitution of the surrounding solvent.42 Here, using the change in the LSPR at around 523 nm (λmax),43,44 the interaction between various molar equivalents of purified Cys-NfnB and His-NfnB with a spherical 50 nm gold colloid was measured by spectroscopy (Figure 3). As could be seen from Figure 3A, there was a red shift and an increase in the peak intensity of the gold colloid in the presence of an NTR, which indicated a change in the dielectric constant of the particle surface.41 The λmax of the gold colloid shifted toward the red as the amount of enzyme added increased and reached a maximum at the 270:1 enzyme gold colloid molar ratio, after which no more change was observed (Figure 3B). The degree of red shift was the same for the Cys-NfnB and NfnB gold colloid conjugates and supported the agarose and DLS data, indicating that at the 270:1 molar ratio of enzyme vs gold a full monolayer had formed on the colloid surface. After full coverage of the gold colloid surface, the LSPR was no longer influenced by excess protein, as supported by observations for other protein gold colloid conjugates.37,38 Enzyme Gold Colloid Conjugate Stability. The ability of enzyme gold colloid conjugates to withstand a change in ionic environment is important if such particles are to be used within a clinical setting. The change in the λmax of gold colloid conjugates 14303

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Figure 4. (A) UV vis spectrum of gold colloid alone upon addition of 0.7 M NaCl. (B) UV vis spectra of an enzyme gold colloid conjugate exposed to increasing amounts of NaCl.

is routinely used to indicate colloid stability either after addition of sodium chloride (NaCl) or during changes in pH.38 Similarly, the absorbance spectra of a range of recombinantNfnB gold colloid conjugates and gold colloid alone were monitored following addition of increasing amounts of electrolyte up to a concentration of 5 M NaCl. As expected the gold colloid alone aggregated upon first addition of NaCl (0.7 M)45 as indicated by the large shift in λmax from 523 nm to a peak at 600 700 nm (Figure 4A). All molar ratios of both the HisNfnB and the Cys-NfnB gold colloid conjugates were stable up to a concentration of 5 M NaCl, with no signs of aggregation between 600 and 750 nm (Figure 4B). The only change seen was a red shift of 4 nm, due to a change in the dielectric constant of the solution.46 The decrease in absorbance intensity seen in Figure 4B was due to the sample becoming more dilute upon every addition of NaCl. The stability of the enzyme nanoparticle conjugates seen here, up to a concentration of 5 M NaCl, is to our knowledge the highest reported thus far for proteins on nanoparticles. The stability of the enzyme gold colloid conjugates in changing pH environments was also investigated. The gold colloid alone and each of the Cys-NfnB and His-NfnB gold colloid conjugates were mixed with potassium phosphatebuffered solutions of different pH’s and the absorbance spectra measured after 15 min. Any changes in λmax of more than 5 nm with an increased absorbance between 600 and 800 nm were used as indicators of instability. The gold colloid alone was stable at all pH values above pH 3 (data not shown), as were all conjugates formed from

Figure 5. Change in λmax at different pH’s of Cys-NfnB gold colloid conjugates of molar ratios from 90:1 to 450:1 (n = 3 and error bars indicate the standard deviation).

a molar ratio of at least 270:1 enzymes per gold colloid. Instability was seen for the bare gold colloid and enzyme gold colloid conjugates of 90:1 and 180:1 molar ratios at lower pH values. The same results were obtained for the His-NfnB gold colloid conjugates, but all conjugates formed at 4 °C overnight showed slightly greater pH stability than conjugates formed at 25 °C for 1 h (data not shown), indicating that the layer formed at 4 °C was more ordered. From the results presented in Figure 5, excellent stability was seen for all conjugates under pH conditions expected in vivo.47 These results together with the sodium chloride stability results 14304

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Figure 6. Activity of Cys-NfnB to CB1954 before (A) and after attachment to the gold colloid (B). Change in absorbance between 270 and 560 nm was measured over a period of 20 min for recombinant NTRs in the presence of NADH (0.3 mM) and CB1954 (0.1 mM).

indicate that the colloid surface need not be completely covered to confer good stability to the enzyme gold colloid conjugates. In order to determine the viability of using recombinant NfnB nanoparticle conjugates for in vivo use, enzyme gold colloid conjugates were subjected to conditions emulating the physiological environment with regard to temperature and ion composition. Both the His-NfnB and the Cys-NfnB gold colloid conjugates of the 270:1 molar ratio were spun down and resuspended in PBS pH = 7.4. Surprisingly, both His-NfnB and Cys-NfnB gold colloid conjugates were successfully spun down and easily resuspended in PBS without addition of any stabilizers and remained stable at 37 °C for the duration of the experiment. All molar ratio’s of enzyme gold colloid conjugates

were stable in water for 12 months when stored at 4 °C but became unstable at room temperature after 1 month (data not shown). Enzyme Reactivity to CB1954 before and after Immobilization onto the Gold Colloid. The viability of directing enzymes to solid tumors using nanoparticles depends on the enzyme retaining a sufficient amount of activity after immobilization, and hence, the ability of the recombinant proteins to retain activity to CB1954 after binding to the gold colloid was determined. The wild-type NfnB is active as a dimer in which each monomer is closely associated with a flavin mononucleotide (FMN) molecule and has two substrate binding pockets. During the enzymatic reaction of NfnB with CB1954, 2 mol of 14305

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Langmuir NAD(P)H are oxidized and the prodrug is reduced to either the 2- or the 4-hydroxylamine product.48,49 Both the Cys-NfnB and the His-NfnB enzymes were assessed for their activity to CB1954 before and after immobilization onto the 50 nm gold colloid by measuring formation of the hydroxylamine products at 420 nm using UV vis spectroscopy.50,51 As can be seen from Figure 6A, NADH (340 nm) was consumed and hydroxylamine products formed (420 nm) during reaction of the unbound Cys-NfnB with CB1954. The same was seen for the unbound His-NfnB (data not shown). Interestingly, both the Cys-NfnB (Figure 6) and the His-NfnB (data not shown) at the molar ratio of 90:1 enzymes vs gold colloid retained activity to the CB1954 prodrug after immobilization. In order to quantify the activity retained the specific activities of free and bound enzyme were compared. At the molar ratio of 90:1 enzyme to gold colloid the immobilized Cys-NfnB had a specific activity of 0.209 μmol/min/mg while that of the enzyme free in solution was 0.211 μmol/min/mg. The same was observed for the His-NfnB gold colloid conjugates. The enzymes had thus retained more than 99% activity to the CB1954 prodrug after direct immobilization. The ability of both the modified enzymes to retain nearly 100% of their activity after direct immobilization onto the gold colloid indicated that the three-dimensional structure of the proteins was preserved52 54 and that the enzyme active sites were easily solvent and substrate accessible. It was expected that the His-NfnB would retain less activity after immobilization compared with the Cys-NfnB because it could bind to the colloid through any of the hydroxyl,22 amino,55 or thioether56 amino acid functional groups within the His-NfnB protein. The Cys-tag was expected to orientate the Cys-NfnB with the N-terminal toward the gold and leave the active sites facing the solvent.13 The ability of the His-NfnB however to retain the same activity as the Cys-NfnB after immobilization suggested that the His-NfnB had in fact bound to the gold colloid with the inserted His-Tag, orientating the enzyme similar to that of the Cys-tagged enzyme. In an attempt to establish whether enzyme activity would be retained to the same degree when a full monolayer of enzyme was immobilized onto the gold surface, His-NfnB and CysNfnB gold colloid conjugates formed in a slight excess of enzyme (360:1 molar ratio) were purified using SEC. Solutions of free enzymes were also passed through the Sephadex column to determine what effect the process of purification had on the enzyme activity. Fractions containing either enzyme gold colloid conjugates or enzyme alone were analyzed for activity to the CB1954 prodrug as described above. Neither of the free His-nfnB nor Cys-NfnB enzymes retained any activity after passing through the Sephadex column. Similarly, no activity was detected for either of the purified HisNfnB nor Cys-NfnB gold colloid conjugates. There was however one difference between the two conjugate samples. The λmax of the His-NfnB gold colloid conjugates had decreased by 4 nm, indicating that most of the immobilized enzyme had desorbed from the colloid surface. To the contrary, the CysNfnB gold colloid conjugates had only a 1 nm loss in λmax, indicating that the majority of the Cys-NfnB protein was still immobilized on the colloid surface. Hence, the purification experiment highlighted the need for protein stabilization should purification be necessary. Thus, although enzymes could be immobilized on gold-coated MNPs using intrinsic or inserted metal binding amino acids,57

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genetic incorporation of cysteines at predetermined positions within proteins for binding to colloids containing gold on their surface would most likely still be the preferred method of choice for use within an in vivo setting due to the unique character of the thiol gold bond.58

’ CONCLUSION Two recombinant forms of the E. coli nitroreductase, NfnB, were directly immobilized onto a 50 nm gold colloid while retaining nearly 100% activity to the CB1954 prodrug. The direct binding of the recombinant NTRs to a gold colloid without major loss in activity or the need for stabilizers is the first of its kind. The ability of the enzyme gold colloid conjugates to remain stable at and around physiological pH and withstand large changes in ionic concentration means that such conjugates should be suitable for in vivo application. It did however become apparent that enzymes immobilized onto the gold colloid using inherent or genetically inserted cysteine residues provides highaffinity binding and possibly greater robustness for universal use on colloids containing gold on the surface. The results given here illustrate the viability of attaching enzymes to gold-coated MNPs, which together is envisaged to become a novel enzyme-directed approach in cancer chemotherapy. ’ ASSOCIATED CONTENT

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Supporting Information. Table of the z-average diameter and zeta potentials of NfnB gold and Cys-NfnB gold colloid conjugates; figures of the size distribution (A) and zeta potential (B) of the gold colloid alone, size distributions of the His-NfnB gold and Cys-NfnB gold colloid conjugates as determined by DLS, and zeta potentials of His-NfnB gold and Cys-NfnB gold colloid conjugates. This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Phone: + 44 1248 383741. E-mail: [email protected].

’ ACKNOWLEDGMENT The authors thank the School of Chemistry at Bangor University for their support throughout this project, and our sincere thanks to Mark Bumiller from HORIBA Scientific for performing the DLS and zeta-potential measurements. ’ REFERENCES (1) The World Health Organization’s fight against cancer: strategies that prevent, cure and care; World Health Organisation: Geneva, Switzerland, 2007. (2) Goodman, L. S.; Wintrobe, M. M.; Dameshek, W.; Morton, M. J.; Gilman, A.; McLennan, M. T. J. Am. Med. Assoc. 1984, 251, 2255–2261. (3) Albert, A. Chemical aspects of selective toxicity. Nature 1958, 182, 421–423. (4) Kratz, F.; Muller, I. A.; Ryppa, C.; Warnecke, A. Chem. Med. Chem. 2008, 3, 20–53. (5) Berne, C.; Betancor, L.; Luckarift, H. R.; Spain, J. C. Biomacromolecules. 2006, 7, 2631–2636. (6) Bagshawe, K. D. In Prodrugs; Stella, V. J., Borchardt, R. T., Hageman, M. J., Oliyai, R., Maag, H.,Tilley, J. W., Eds.; Springer: New York, 2007; Vol. V, pp 527 528. 14306

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