catalysts

Article Increased Stability of Oligopeptidases Immobilized on Gold Nanoparticles

Marcelo Yudi Icimoto 1,*, Adrianne Marlise Mendes Brito 1,2, Marcos Paulo Cyrillo Ramos 1, Vitor Oliveira 1 and Iseli Lourenço Nantes-Cardoso 2,*

1 Departamento de Biofísica, Universidade Federal de São Paulo, São Paulo CEP 04024-002, Brazil; [email protected] (A.M.M.B.); [email protected] (M.P.C.R.); [email protected] (V.O.) 2 Centro de Ciências Naturais e Humanas, Universidade Federal do ABC, Santo André CEP 09210-580, Brazil * Correspondence: [email protected] (M.Y.I.); [email protected] (I.L.N.-C.)

 Received: 21 October 2019; Accepted: 16 December 2019; Published: 4 January 2020 

Abstract: The metallopeptidases (THOP, EC 3.4.24.25) and (NEL, EC 3.4.24.26) are that belong to the M13 family. Numerous studies suggest that these peptidases participate in the processing of bioactive such as and . Efforts have been conducted to develop biotechnological tools to make possible the use of both to regulate blood pressure in mice, mainly limited by the low plasmatic stability of the enzymes. In the present study, it was investigated the use of nanotechnology as an efficient strategy for to circumvent the low stability of the proteases. Recombinant THOP and NEL were immobilized in gold nanoparticles (GNPs) synthesized in situ using HEPES and the enzymes as reducing and stabilizing agents. The formation of rTHOP-GNP and rNEL-GNP was characterized by the surface plasmon resonance band, zeta potential and atomic force microscopy. The gain of structural stability and activity of rTHOP and rNEL immobilized on GNPs was demonstrated by assays using fluorogenic substrates. The enzymes were also efficiently immobilized on GNPs fabricated with sodium borohydride. The efficient immobilization of the oligopeptidases in gold nanoparticles with gain of stability may facilitate the use of the enzymes in therapies related to pressure regulation and stroke, and as a tool for studying the physiological and pathological roles of both proteases.

Keywords: THOP; NEL; gold nanoparticles; oligopeptidases

1. Introduction Gold nanoparticles (GNPs) are attractive for biological applications because of the stability, biocompatibility, facile functionalization and compatibility with organic molecules containing groups [1]. GNPs encompass both the intrinsic properties of nanoscale, surface effects and quantum confinement and the capacity to undergo surface functionalization that can increase stability and provide specific targeting [2]. Surface functionalization of gold nanoparticles has opened new frontiers for the delivery of drugs such as anticancer and antimicrobial drugs as well as functional studies of biomolecules such as enzymes and antibodies [3–6]. GNPs can also be synthesized by one-pot, rapid processes, including the in-situ synthesis using the organic functionalizing agent as the reducing compound to convert gold ions (Au3+) to the metallic form [7–9]. A diversity of biomolecules and their mimetics has been used for in-situ synthesis of GNPs, but the use of proteins is particularly attractive because these biomolecules act as a template and reducing agents for GNP fabrication [10–13]. Several enzymes have the potential for therapeutic applications that can be limited by their stability, and this is the case of the enzymes thimet oligopeptidase (THOP; EC 3.4.24.15) and neurolysin (NEL; EC 3.4.24.16). THOP and NEL promise as hypotensive therapeutics despite their low structural

Catalysts 2020, 10, 78; doi:10.3390/catal10010078 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 78 2 of 17 and functional stability. THOP and NEL are members of the -like mammalian zinc endopeptidase family [14], which is maximally active at neutral pH and is among the most studied groups of peptidases responsible for hydrolytic processing of bioactive peptides in both intra and extracellular environment [15]. These enzymes are found in animals and plants, showing distinctive functions [16]. Biochemical and pharmacological features have been well characterized in vivo and in vitro [14,15,17,18] with well-known endogenous substrates such as hemopressin, bradykinin, angiotensins I and II, , substance P, dynorphin A (1–8), somatostatin and Aβ (amyloid peptide). The processing of these substrates is related to the regulation of a myriad of physiological processes [19]. Despite the capacity to process a variety of endogenous substrates, the functional significance of THOP and NEL in pathophysiological conditions is poorly understood, linking their function to neurotensin-dependent nociception, bradykinin-mediated hypotension, microvascular permeability and hyperalgesia [20,21]. Neurotensin (NT) is a tridecapeptide that exerts a broad range of cardiovascular and endocrine effects when administered intravenously, including hypotension, analgesia and hypothermia [22–24]. The roles of THOP and NEL in the inactivation of neurotensin in vitro were determined by several studies. NEL was the only peptidase ubiquitously contributing to its inactivation, being present in all tissue and established cell lineages where NT receptors are located [23,25]. THOP, on the other hand, cleaves NT at the Arg–Arg bond and reduces NT in rat brain synaptosomes [26]. The use of specific inhibitors also increases the evidence for the involvement of both enzymes in the physiological degradation of NT, as the dipeptide Pro-Ile [27] and phosphinic peptides [28]. In addition to possible functions within the brain, THOP may also play a role in the periphery. The potent vasodilatory peptide bradykinin (BK) is a powerful influence in stimulating extravascular smooth muscle contraction, inducing hypotension, dilating blood vessels and increasing vascular permeability [29,30]. THOP has been shown to efficiently degrade BK in vitro at the Phe–Ser bond [31,32]. The use of the THOP specific inhibitor JA2 was shown to potentiate BK-induced hypotension, without affecting the hypertensive effects of angiotensins I and II, strongly suggesting its role in blood pressure control [22]. THOP also significantly affects BK in the kidney, having a role in blood flow, glomerular filtration rate, urinary sodium and potassium excretion and urine volume [33–36]. Furthermore, THOP also converts I in vitro to the biologically active peptide angiotensin 1–7 [37] synergizing with the effects of bradykinin in vascular smooth muscle cells culture [38,39] and in rat hindlimb perfusate ex vivo [40]. Thus, THOP may participate in cardiovascular homeostasis via the activation (angiotensin 1–7) and inactivation (bradykinin) of vasodilatory peptides. Therefore it is necessary, both in vivo and in cell culture approaches, to perform functional studies of these enzymes when administered as exogenous agents [41,42]. The lack of cytotoxicity, low blood stability, systemic bioavailability and pharmacokinetics of intraperitoneally administered recombinant neurolysin, and its effects on critical physiological parameters in mice have already been demonstrated [43]. These studies suggest that NEL can serve as a research tool and could contribute to a better comprehension of the functional significance of this peptidase in diseases [44,45]. The potential for the therapeutic application of THOP and NEL allied to the necessity of prolonged structural stability motivated the present study to develop THOP, and NEL capped GNPs. The results demonstrate a significant gain of stability necessary for therapeutic applications. In fact, the redox balance is closed related to the mitochondrial permeability transition. Furthermore, it was demonstrated that THOP activity can be regulated by peroxide concentrations in cells that in turn are modulated by the cellular thiol oxidation status [46–48].

2. Results and Discussion

2.1. Expression and Purification The optimum growth of Escherichia coli BL21-DE3 containing the pET28a (+) constructs for expression of both recombinant thimet oligopeptidase and neurolysin (rTHOP and rNEL) was achieved Catalysts 2020, 10, x FOR PEER REVIEW 3 of 17

The optimum growth of Escherichia coli BL21-DE3 containing the pET28a (+) constructs for Catalysts 2020, 10, 78 3 of 17 expression of both recombinant thimet oligopeptidase and neurolysin (rTHOP and rNEL) was achieved using LB broth medium at 20 °C, 180 rpm, induced by 1 mM IPTG for 24 h. Active proteasesusing LB brothwere isolated medium from at 20 lysed◦C, 180 bacteria rpm, induced culture byusing 1 mM affinity-based IPTG for 24 HisTrap h. Active FF proteases column (GE) were elutedisolated in froma 50 lysedmM Tris-buffer bacteria culture 100 mM using NaCl affinity-based pH 7.4 and HisTrap 250 mM FF imidazole column (GE) (rTHOP) eluted or in 200 a 50 mM mM imidazoleTris-buffer (rNEL) 100 mM resulting NaCl pH in 7.4 protein and 250 purity mM imidazole greater than (rTHOP) 90% or(Figure 200 mM 1a). imidazole Purified (rNEL)proteases resulting were observedin protein as purity single greater bands thanon SDS-PAGE 90% (Figure with1a). apparent Purified molecular proteases wereweights observed as 78 kDa as single for THOP bands and on NEL.SDS-PAGE The structural with apparent profile molecular was confirmed weights using as 78 the kDa circular for THOP dichroism and NEL. (CD; The Figure structural 1b), profileconsistent was withconfirmed previous using literature the circular data dichroism [49]. The (CD; far-UV Figure CD1b), spectra consistent of rTHOP with previous and rNEL literature show datathat [the49]. recombinantThe far-UV CD enzymes spectra ofare rTHOP folded and with rNEL high show α that-helix the recombinantcontent and enzymesconsequently are folded suitable with highfor activity-basedα-helix content assays and consequently that are described suitable herein. for activity-based assays that are described herein.

FigureFigure 1. 1. PurificationPurification of of recombinant recombinant oligopeptidases oligopeptidases THOP THOP (EC (EC 3.4.24.15) 3.4.24.15) and and NEL NEL (EC (EC 3.4.24.16). 3.4.24.16). ((aa)) Twelve Twelve percent percent SDS-Page SDS-Page of of purified purified recombinant recombinant THOP THOP and and NEL NEL followed followed by by Coomassie Coomassie Blue Blue staining,staining, indicating thethe purificationpurification homogeneity. homogeneity. (b )(b Circular) Circular dichroism dichroism profile profile of rTHOP of rTHOP and rNEL, and rNEL,demonstrating demonstrating a proper a proper fold after fold purification. after purifica Alltion. samples All samples were prepared, were prepared, and the spectraand the obtained, spectra obtained,as described as described in Materials in Materials and Methods. and Methods.

2.2. Enzyme Thermostability 2.2. Enzyme Thermostability TheThe thermostability thermostability of of THOP THOP and and NEL NEL were were assessed assessed by by previous previous incubation incubation in in defined defined temperaturetemperature in in buffered buffered aqueous aqueous solutions solutions (not (not immobilized) immobilized) and and immobilized immobilized in in a a 1% 1% agarose agarose matrix.matrix. Activity Activity assays assays were were pe performedrformed by by measurement measurement at at 37 37 °C◦C on on the the fluorogenic fluorogenic substrate substrate Abz-GFSPFRQ-EDDnpAbz-GFSPFRQ-EDDnp after after each each step step of of enzyme enzyme challenging challenging to toa temperature a temperature ramp ramp of 5 of °C 5 per◦C per20 min20 min (Figure (Figure 2).2 ).Figure Figure 2a2 ashows shows that that in in solution, solution, rTHOP rTHOP and rNELrNEL havehave thethe turningturning pointpoint (half-activity)(half-activity) of temperature-induced denaturationdenaturation and and loss loss of of activity activity at at 60 60◦ C.°C. When When immobilized immobilized in ina 1%a 1% agarose agarose matrix, matrix, the half-activitiesthe half-activities of THOP of THOP and NEL andincreased NEL increased to 75 ◦ Cto (Figure 75 °C2 b).(Figure A plausible 2b). A plausibleexplanation explanation for the gain for ofthe thermal gain of stability thermal in stability an agarose in an matrix, agarose at matrix, least in at the least case in of the THOP, case isof a THOP,probable is a impairment probable impairment of inactivating of inactivating enzyme oligomerization enzyme oligomerization that is observed that is observed after a few after hours a few of hoursincubation of incubation in solution. in Consideringsolution. Considering that oligomerization that oligomerization involves the formationinvolves the of intermolecularformation of intermoleculardisulfide [50,51 disulfide], the reducing [50,51], agent the reducing DTT was agent also usedDTT aswas control. also used It also as usedcontrol. the It THOP also used cysteine the THOPmutant cysteine 6 M, where mutant all 6 sixM, surface-exposedwhere all six surface-ex cysteineposed residues cysteine were residues mutated were to serinemutated (Figure to serine2c). (FigureDTT preincubation 2c). DTT preincubation and THOP and mutations THOP mutation promoteds promoted a small increase a small ofincrease rTHOP of andrTHOP rNEL and thermal rNEL thermalresistance. resistance. This result This can result be rationalized can be rationalized considering considering that the that oligomerization the oligomerization promoted promoted by heating by heatingis preceded is preceded by unfolding, by unfolding, which exposes which internal exposes cysteine internal residues cysteine and residues results in and an intermolecularresults in an intermolecularreaction. The better reaction. thermal The resistance better thermal obtained resistan for thece peptidases obtained for in the the agarose peptidases matrix in demonstratesthe agarose matrixthe importance demonstrates of immobilization the importance for of theimmobilization gain of stability. for the The gain aspects of stability. of THOP The and aspects NEL of stability THOP andregulation NEL stability by reducing regulation agents by are reducing already well agents discussed are already in the literature,well discussed and they in the were literature, the result and of a combined effect of mixed forms of inactive oligomerized enzymes and oxidation as post-translational modification [47,50].

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Catalyststhey were2020 , the10, 78 result of a combined effect of mixed forms of inactive oligomerized enzymes4 ofand 17 oxidation as post-translational modification [47,50].

Figure 2. Thermostability of in solution and agarose-immobilized rTHOP and rNEL. Recombinant Figure 2. Thermostability of in solution and agarose-immobilized rTHOP and rNEL. Recombinant enzymes were preincubated for 20 min at each temperature in activity buffer (a) in solution or (b) enzymes were preincubated for 20 min at each temperature in activity buffer (a) in solution or (b) immobilized in 1% agarose, indicating the positive effect of agarose in enzyme stability. The effect immobilized in 1% agarose, indicating the positive effect of agarose in enzyme stability. The effect of of the reducing agent DTT and THOP cysteine mutant (6 M) was also observed in solution (without the reducing agent DTT and THOP cysteine mutant (6 M) was also observed in solution (without immobilization) at the fixed temperature of 65 ◦C(c), suggesting that the increase in stability observable immobilization) at the fixed temperature of 65 °C (c), suggesting that the increase in stability in (b) is mainly due to immobilization and not to enzyme oligomerization or other protein–protein observable in (b) is mainly due to immobilization and not to enzyme oligomerization or other interactions. Activities of both THOP and NEL were accessed by using the fluorogenic substrate protein–protein interactions. Activities of both THOP and NEL were accessed by using the Abz-GFSPFRQ-EDDnp (5 µM) in Tris-NaCl buffer at 37 ◦C in a spectrofluorimeter Hitachi F7000. fluorogenic substrate Abz-GFSPFRQ-EDDnp (5 µM) in Tris-NaCl buffer at 37 °C in a 2.3. In-Situspectrofluorimeter Synthesis of Hitachi rTHOP-GNPs F7000. and rNEL-GNPs The temperature for the synthesis of GNPs in situ using rTHOP and rNEL as reducing agents was 2.3. In-Situ Synthesis of rTHOP-GNPs and rNEL-GNPs established at 40 ◦C in which the enzymes had 100% of activity. The preservation of enzyme activity is associatedThe temperature with the nativefor the folded synthesis structure of GNPs of thein si enzymestu using thatrTHOP assures and thatrNEL the as proteins reducing can agents also actwas as established a template at for 40 GNP °C in growing. which the rTHOP- enzymes and ha rNEL-assistedd 100% of activity. synthesis The of preservation GNPs was performedof enzyme atactivity different is associated enzyme concentrations with the native (Figures folded3 structureand4, respectively). of the enzyme Considerings that assures that boththat THOPthe proteins and NELcan also have act di ffaserent a template exposed for cysteine GNP growing. content, di rTHOP-fferent proteinand rNEL-assisted concentration synthesis implicates of inGNPs variable was reductiveperformed power at different of the system,enzyme whichconcentrations may affect (Figur the physicochemicales 3 and 4, respectively). properties Considering of the nanoparticles that both generated.THOP and WeNEL considered have different the reductive exposed capacity cysteine of cont theent, system different as the protein molar concentrationconcentration ofimplicates exposed cysteinein variable residues reductive and power protein of concentration the system, which during may protein-GNP affect the physicochemical synthesis. The spectra properties of rTHOP- of the andnanoparticles rNEL-GNPs, generated. synthesized We at considered 40 ◦C, using the protein redu concentrationsctive capacity at of 2, 8,the 40 andsystem 200 nMas arethe shownmolar inconcentration Figure3 (rTHOP-GNP) of exposed cysteine and Figure residues4 (rNEL-GNP), and protein respectively concentration as well during as appropriatedprotein-GNP controls.synthesis. InThe order spectra to guarantee of rTHOP- any and unbounded rNEL-GNPs enzymes, synthesized in protein-GNP at 40 °C, using preparations, protein concentrations the synthesized at 2, GNPs 8, 40 wereand 200 three nM times are washedshown in by Figure ultracentrifugation 3 (rTHOP-GNP) (50.000 and g, Figure 5 min at4 4(rNEL-GNP),◦C) followed respectively by resuspension. as well GNP as synthesisappropriated was controls. successful In in order all tested to guarantee protein concentrations,any unbounded evidenced enzymes in by protein-GNP the appearance preparations, of surface plasmonthe synthesized resonance GNPs (SPR) were spectral three bands times peaking washed around by ultracentrifugation 535 nm in the UV-vis (50.000 spectra. g, 5 The min spectra at 4 °C) of THOP1followed (200 by nM) resuspension. and THOP2 GNP (40 nM) synthesis generate was broader succ SPRessful bands, in all possibly tested indicatingprotein concentrations, polydispersed particles.evidenced The by same the appearance observation wasof surface suggested plasmon for NEL1 resonance (200 nM). (SPR) However, spectral the bands relatively peaking low-intensity around 1 rNEL-GNP535 nm in the spectrum UV-vis spectra. obtained The from spectra the synthesisof THOP1 using (200 nM) 200 nmol.Land THOP2− of (40 protein nM) reflectsgenerate colloidal broader instability,SPR bands, as possibly observable indicating in the stability polydispersed assay (Figure particles.5). The same observation was suggested for NEL1 (200 nM). However, the relatively low-intensity rNEL-GNP spectrum obtained from the synthesis using 200 nmol.L−1 of protein reflects colloidal instability, as observable in the stability assay (Figure 5).

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1.4

1.2 0.5 1.4 THOP GNP 2 nM 1.4 sca 1.2 1.2 0.025 0.5 1.0 THOP GNP 2 nM 1.4 σ

sca 1.2 1.2 0.025 0.4 1.0 1.0 1.0 + σ 1.2 1.0 0.020 0.4 0.8 1.0 1.0 + 0.8 1.0 0.020 0.3 0.8 0.8

abs 0.015

0.6 0.8 0.3 0.6 0.8 abs 0.6 0.015 σ

0.6 0.5 0.2 0.4 0.6 0.010

= 0.6 σ 0.4 0.5 0.4 0.2 0.4 0.010 = 0.4 0.4 0.005 0.1 0.2 0.2 0.2 0.005 0.1 total 0.2 0.2 0.2

σ 0.0 0.0 0.0 0.0 0.000 0.0 total 300 400 500 600 700 300 350 400 450

σ 0.0 0.0 0.0 0.0 0.000 0.0 300 400 500Wavelength 600 700 (nm) 300 350 400 450 Wavelength (nm) Figure 3. UV-vis spectra of rTHOP-gold nanoparticles (GNPs) synthesized with different THOP Figure 3. UV-vis spectra of rTHOP-gold nanoparticles (GNPs) synthesized with different THOP concentrations.Figure 3. UV-vis Spectra spectra of THOP-GNPsof rTHOP-gold were nanoparticles obtained from (GNPs) synthesis synthesized using 200, with 40, different8 and 2 nMTHOP of concentrations. Spectra of THOP-GNPs were obtained from synthesis using 200, 40, 8 and 2 nM of recombinantconcentrations. THOP. Spectra These of THOP-GNPsvalues correspond were obtainto 1200,ed 240,from 48 synthesis and 12 nMusing of 200,exposed 40, 8 sulfhydryls.and 2 nM of recombinant THOP. These values correspond to 1200, 240, 48 and 12 nM of exposed sulfhydryls. Red: Red:recombinant THOP-GNP THOP. 2 nM. These Green: values THOP-GNP correspond 8 nM. to Blue: 1200, THOP-GNP 240, 48 and 40 12 nM. nM Orange: of exposed THOP-GNP, sulfhydryls. 200 THOP-GNP 2 nM. Green: THOP-GNP 8 nM. Blue: THOP-GNP 40 nM. Orange: THOP-GNP, 200 nM. nM.Red: Gray THOP-GNP dashed line:2 nM. GNP-HEPES Green: THOP-GNP (without 8 nM.protein). Blue: Blac THOP-GNPk: THOP 40protein. nM. Orange: Pink: reaction THOP-GNP, without 200 Gray dashed line: GNP-HEPES (without protein). Black: THOP protein. Pink: reaction without HEPES. HEPES.nM. Gray The dashed wavelength line: GNP-HEPES of 300 nm corresponds (without protein). mainly toBlac Au-proteink: THOP complexesprotein. Pink: absorption reaction and without 535 The wavelength of 300 nm corresponds mainly to Au-protein complexes absorption and 535 nm to nmHEPES. to the The surface wavelength plasmonic of 300resonance nm corresponds (SPR) band mainly of the to nanoparticle. Au-protein complexes The samples absorption were prepared, and 535 the surface plasmonic resonance (SPR) band of the nanoparticle. The samples were prepared, and the andnm theto thespectra surface obtained plasmonic as described resonance in Materials(SPR) band and of Methods.the nanoparticle. The samples were prepared, andspectra the obtainedspectra obtained as described as described in Materials in Materials and Methods. and Methods.

FigureFigure 4. 4. UV-visUV-vis spectra spectra of of rNEL-GNP rNEL-GNP synthesized synthesized with with different different NEL NEL concentrations. concentrations. Spectra Spectra of of NEL-GNPsFigureNEL-GNPs 4. UV-vis were were obtained spectra of from rNEL-GNP thethe synthesissynthesis synthesized using using NEL NEL with at at 200,different 200, 40, 40, 8 and NEL8 and 2 nM,concentrations. 2 nM, corresponding corresponding Spectra to 1000, to of 1000,NEL-GNPs200, 40200, and 40 were 10and nM 10obtained of nM exposed of exposedfrom sulfhydryls. the sulfhydryls. synthesis Red: usin Re NEL-GNPgd: NEL NEL-GNP at (2200, nM). (2 40, nM). Green:8 and Green: 2 NEL-GNP nM, NEL-GNP corresponding (8 nM). (8 nM). Blue: to Blue:1000,NEL-GNP NEL-GNP200, 40 (40 and nM). (40 10 Orange:nM).nM ofOrange: exposed NEL-GNP NEL-GNP sulfhydryls. (200 nM).(200 Re GraynM).d: NEL-GNP dashedGray dashed line: (2 nM). GNP-HEPES line: Green: GNP-HEPES NEL-GNP without without Protein.(8 nM). Protein.Blue:Black: NEL-GNP NELBlack: protein. NEL (40 protein. Pink:nM). reaction Orange:Pink: reaction without NEL-GNP without HEPES. (200 HEPES. The nM). wavelength Gray The wadashedvelength of 300 line: nm of correspondsGNP-HEPES300 nm corresponds mainly without to mainlyAu-proteinProtein. to Black: Au-protein complex NEL protein. absorptioncomplex Pink absorption and: reaction 535 nm and without to the535 plasmonic nmHEPES. to the The bandplasmonic wa ofvelength the nanoparticle.band of of300 the nm nanoparticle. Spectracorresponds were obtained in a spectrophotometer Evolution 200 (Thermo Scientific) at room temperature. Spectramainly towere Au-protein obtained complex in a spectrophotometeabsorption and 535r Evolutionnm to the plasmonic200 (Thermo band Scientific) of the nanoparticle. at room temperature.Spectra were obtained in a spectrophotometer Evolution 200 (Thermo Scientific) at room temperature.The position of SPR bands of GNPs between 525 and 535 nm that was obtained for all THOP- and NEL-GNPs synthesized it was possible an estimative of the GNP diameter between 20 and The position of SPR bands of GNPs between 525 and 535 nm that was obtained for all THOP- 60 nm based in literature data [52]. The stoichiometry protein/nanoparticles can also be calculated. and NEL-GNPsThe position synthesized of SPR bands it was of GNPspossible between an estimative 525 and of 535 the nm GNP that diameter was obtained between for 20all andTHOP- 60 Considering the mean diameter of 40 nm, in atomic spherical metal clusters [53,54], the concentration nmand based NEL-GNPs in literature synthesized data [52].it was The possible stoichiometry an estimative protein/nanoparticles of the GNP diameter can also between be calculated. 20 and 60 of protein-GNPs obtained in our synthesis is 1 nM, resulting in a THOP/NEL:GNP ratio of 2:1 rTHOP Consideringnm based in theliterature mean datadiameter [52]. Theof 40stoichiometry nm, in at omicprotein/nanoparticles spherical metal canclusters also be[53,54], calculated. the and rNEL GNPs synthesized in the presence of 2 nmol.L 1 of the proteins. The spherical shape of concentrationConsidering ofthe protein-GNPs mean diameter obtained of 40 in ournm, synthesisin atomic −is 1spherical nM, resulting metal in clusters a THOP/NEL:GNP [53,54], the rTHOP and rNEL GNPs with a mean diameter of 40 nm was corroborated by atomic force microscopy ratioconcentration of 2:1 rTHOP of protein-GNPs and rNEL GNPs obtained synthesized in our synthesisin the presence is 1 nM, of 2resulting nmol.L− 1in of a theTHOP/NEL:GNP proteins. The (AFM) measurements (Figure5a,b). Figure5a depicts a 2D representation of− rTHOP-GNP,1 while sphericalratio of 2:1 shape rTHOP of rTHOP and rNEL and GNPsrNEL synthesizedGNPs with ain mean the presence diameter of of 2 nmol.L40 nm was of thecorroborated proteins. Theby Figure5a, inset, depicts a 3D representation of the same bio-functionalized nanoparticle, indicating atomicspherical force shape microscopy of rTHOP (AFM) and measurementsrNEL GNPs with (Figure a mean 5a,b). diameter Figure 5aof depicts40 nm wasa 2D corroborated representation by monodispersed GNPs and suggesting spherical shapes of the particles. Similarly, Figure5b, inset, ofatomic rTHOP-GNP, force microscopy while Figure (AFM) 5a, measurementsinset, depicts a(Figure 3D representation 5a,b). Figure of 5a the depicts same abio-functionalized 2D representation represent rNEL-GNP in 2D and 3D perspectives, respectively. nanoparticle,of rTHOP-GNP, indicating while Figuremonodisp 5a, inset,ersed depictsGNPs anda 3D suggesting representation spheri of calthe shapes same bio-functionalized of the particles. Similarly,nanoparticle, Figure indicating 5b, inset, monodisprepresent ersedrNEL-GN GNPsP in and 2D andsuggesting 3D perspectives, spherical respectively.shapes of the particles. Similarly, Figure 5b, inset, represent rNEL-GNP in 2D and 3D perspectives, respectively.

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FigureFigure 5. 5. AtomicAtomic force force microscopy microscopy of of rTHOP-GNP rTHOP-GNP and and rNEL-GNP. rNEL-GNP. Atomic Atomic force force microscopy microscopy (AFM) (AFM) measurementmeasurement of of ( a)) rTHOP-GNP andand ((bb)) rNEL-GNP rNEL-GNP 2D 2D images images are are observable observable (red (red scale scale bar bar= 200 = 200 nm) nm)as the as centralthe central figure figure and 3D and rendering 3D rendering of as insets of as of insets (a,b) for of rTHOP-GNP(a,b) for rTHOP-GNP and rNEL-GNP, and rNEL-GNP, respectively. respectively. Despite the significant differences observed in the UV region, the SPR bands of GNP synthesized in the absenceDespite andthe presencesignificant of thedifferences enzymes areobserved very similar in the as wellUV asregion, the zeta the potential SPR bands values of that GNP were 1 synthesizeddetermined in for the GNPs absence synthesized and presence with HEPESof the enzyme and HEPESs are assistedvery similar by 2 as nmol.L well −as ofthe THOP zeta potential and NEL ( 25.4 +/ 5.48, 25.9 +/ 4.97 and 22.2 +/ 5.18 V, respectively). Therefore, some experiments were−1 values− that− were −determined− for GNPs− synthesi− zed with HEPES and HEPES assisted by 2 nmol.L ofcarried THOP out and to NEL check (− the25.4 binding +/− 5.48, capacity −25.9 +/ of− 4.97 THOP and and −22.2 NEL +/ on− 5.18 the V, surface respectively). of GNPs. Therefore, Bare GNPs some were experimentssynthesized were using carried sodium out borohydride, to check the and bindin the changesg capacity in theof UV-visTHOP and spectrum NEL on and the zeta surface potential of 1 GNPs.values Bare were GNPs determined were synthesized after the addition using ofsodium 2 nmol.L borohydride,− of the enzymes and the at changes pH 3.5, whichin the isUV-vis below spectrumtheir pI (isoelectric and zeta potential point) values. values Bare were gold determined nanoparticles after presentedthe addition spectral of 2 nmol.L changes−1 of consistent the enzymes with atnanoparticle pH 3.5, which aggregation is below by their changing pI (isoelectric the pH from point) 6.0 to values. 3.5 that Bare is accompanied gold nanoparticles by change presented of the zeta spectralpotential changes from 46.9 consistent+/ 2.45 with to 23.5 nanoparticle+/ 0.72 mV aggregation (Table S1 inby Supplemental changing the Material).pH from 6.0 The to addition3.5 that is of − − 1 accompaniedTHOP for a final by concentrationchange of the of zeta 2 nmol.L potential− promoted from 46.9 significant +/− 2.45 spectralto 23.5 changes+/− 0.72 thatmVare (Table compatible S1 in Supplementalwith nanoparticle Material). disaggregation The addition (Figure of S1TH inOP Supplemental for a final concentration Material). The of enzyme 2 nmol.L immobilization−1 promoted significanton bare GNPs spectral was alsochanges corroborated that are by compatible zeta potential with measurements nanoparticle ofdisaggregation bare gold nanoparticles (Figure S1 in thein Supplementalsame conditions Material). used for The the UV-visibleenzyme immobilization spectral measurements on bare GNPs at pH was 3.5. Thealso additioncorroborated of THOP by zeta for a final concentration of 2 nmol.L 1 changed the zeta potential of bare GNPs at pH 3.5 from 23.5 +/ potential measurements of bare− gold nanoparticles in the same conditions used for the UV-visible− − 0.72 mV to 34.2 +/ 1.85 mV (Table S1 in Supplemental Material). However, bare gold nanoparticles−1 spectral measurements− at pH 3.5. The addition of THOP for a final concentration of 2 nmol.L synthesized with sodium borohydride had a diameter of 5.7 +/ 1.7 nm (Figure S2 in Supplemental changed the zeta potential of bare GNPs at pH 3.5 from −23.5 +/−− 0.72 mV to 34.2 +/− 1.85 mV (Table S1Material) in Supplemental while the gold Material). nanoparticles However, fabricated bare usinggold HEPESnanoparticles were ca.synthesized six-fold larger. with Therefore,sodium borohydrideconsidering had the lowa diameter protein of concentration 5.7 +/− 1.7 nm that (Figure was compatibleS2 in Supplem withental a mean Material) of two while proteins the gold/gold nanoparticlesnanoparticles fabricated (Figure6), itusing was expectedHEPES were that the ca. protein six-fold adsorption larger. Therefore, on gold nanoparticles considering producedthe low proteinusing HEPESconcentration is unable that to was promote compatible significant with changes a mean inof zetatwo valuesproteins/gold because nanoparticles of the low superficial (Figure 6),area it ofwas GNPs expected covered that by the the protein enzymes. adsorption on gold nanoparticles produced using HEPES is unable to promote significant changes in zeta values because of the low superficial area of GNPs covered by the enzymes.

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Figure 6.6. CartoonCartoon representative representative of aof putative a putative effect effect of nanoparticle of nanoparticle size on size the zetaon potentialthe zeta promotedpotential promotedby the adsorption by the adsorption of the proteases. of the proteases.

The immobilization of THOP and NEL on the GNPs fabricated with HEPES and assisted by the enzymes was also corroborated by differences differences in the ki kineticsnetics of synthesis, colloidal stability, and the presence of proteolytic activity inin thethe GNPsGNPs producedproduced withwith HEPESHEPES andand assistedassisted byby thethe enzymes.enzymes.

2.4. Kinetics Kinetics of rTHOP-GNP and rNEL-GNP Formation Considering the interest in the use of THOP- and NEL-GNPs as catalysts, it is essential to have effectiveeffective enzymeenzyme immobilizationimmobilization and and activity activity in thein the same same conditions conditions in which in which free enzymesfree enzymes are active. are Therefore,active. Therefore, the kinetics the kinetics of HEPES-GNP of HEPES-GNP synthesis sy assistednthesis assisted by the enzymes by the enzymes were compared were compared with the withkinetics the of kinetics GNPs synthesizedof GNPs synthesized with HEPES with alone. HEPES Figure alone.7a shows Figure the 7a kinetic shows of the GNP kinetic synthesis of GNP by synthesisusing 30 mMby using HEPES 30 asmM a reducingHEPES as and a reducing stabilizing and agent stabilizing in the agent absence in ofthe THOP absence and of NELTHOP that and is 1 NELcompared that is with compared the respective with the respective synthesis kineticssynthesi ofs kinetics GNP formation of GNP formation assisted byassisted 2 nmol.L by 2− nmol.LTHOP−1 THOPand NEL and (Figure NEL 7(Figureb,c respectively). 7b,c respectively). In all the In conditions all the conditions used, it was used, observed it was that observed the kinetics that the of kineticsGNP synthesis of GNP insynthesis the presence in the ofpresence HEPES of had HEPE threeS had essential three observableessential observable steps: progressive steps: progressive spectral spectralchanges inchanges the UV in region the UV that region corresponded that corresponded to the occurrence to the occurrence of gold ion reductionof gold ion and reduction formation and of formationnanoclusters of (stepnanoclusters 1), SPRband (step formation 1), SPR band (step form 2) andation the (step progressive 2) and increasethe progressive of a broad increase band at of the a broadspectral band range at of the 700–800 spectral nm. range Considering of 700–800 the three nm. steps Considering of synthesis the that three involves steps changesof synthesis in the that UV involvesregion, followed changes successively in the UV region, by increasing followed of SPR successively bands and by aband increasing around of 750 SPR nm, bands Figure and7a–c a showband aroundthe kinetics 750 ofnm, synthesis Figure 7a–c monitored show the at 310kinetics nm, 532–535of synthesis nm andmonitored around at 750 310 nm nm, and 532–535 the insets nm withand aroundthe corresponding 750 nm and spectra the insets obtained with the in corresponding the course of the spectra synthesis. obtained The in kinetics the course of GNP of the formation synthesis. in Thethe processeskinetics of assisted GNP formation by the enzymes in the processes was significantly assisted by di thefferent enzymes of that was promoted significantly by HEPES different alone, of thatespecially promoted at the by steps HEPES of involving alone, especially changes in at the the visible steps region. of involving Considering changes the in high the concentration visible region. of ConsideringHEPES relative the to high the enzymes, concentration it was of expected HEPES that relative the bu toff eringthe enzymes, agent is the it principalwas expected agent that for gold the bufferingion reduction agent and is nanoclustersthe principal nucleation agent for ingold step ion 1. Afterreduction the formation and nanoclusters of nanoclusters, nucleation the association in step 1. withAfter thethe enzymes formation at someof nanoclusters, nanoclusters the facets associatio drivesn thewith nanoparticles the enzymes growing at some and nanoclusters consequently facets the driveskinetics the of SPRnanoparticles and the 700–800 growing nm and band consequently increasing. It the is importantkinetics of to SPR note and that the for GNPs700–800 synthesized nm band increasing.in the absence It is of important enzymes to the note band that in thefor GNPs red region synthesized of the spectrum in the absence that appeared of enzymes in the the third band step in peaksthe red at region 715 nm of whereas the spectrum in the synthesisthat appeared assisted in the by thethird enzymes step peaks the peakat 715 was nm found whereas at 765 in nm.the Thesynthesis nature assisted of this by band the remains enzymes to the be peak determined was foun butd at it is765 suggestive nm. The nature of the formationof this band of remains elongated to benanostructures determined but or aggregates. it is suggestive of the formation of elongated nanostructures or aggregates.

Catalysts 2020, 10, 78 8 of 17 Catalysts 2020, 10, x FOR PEER REVIEW 8 of 17

FigureFigure 7. Kinetics 7. Kinetics of GNP of GNP synthesis synthesis with with HEPES HEPES in in the the absence absence and and presencepresence of enzymes. (a (),a ),(b ()b and) and (c) represent(c) represent the kinetics the kinetics of the three of the steps three of steps the GNPof the synthesis GNP synthesis using using HEPES HEPES alone, alone, and HEPESand HEPES assisted by THOPassisted and by NEL, THOP respectively. and NEL, respectively. In each spectra In ea thech bluespectra lines the represent blue lines kinetic represent and kinetic corresponding and

spectracorresponding (inset) of HAuCl spectra4 immediately(inset) of HAuCl after4 immediately addition to after solution addition (time to solution zero) and (time the zero) formation and the of the complexformation HEPES of/gold the complex ions and HEPES/gold protein with ions subsequent and protein reduction with subsequent of Au3+ to reduction the atomic of formAu3+ to before the the appearanceatomic ofform Surface before Plasmon the appearance Resonance of (SPR,Surface first-step), Plasmon redResonance lines represents (SPR, first-step), the represent red lines kinetic and correspondingrepresents the spectrarepresent (inset) kinetic obtained and correspondi during theng GNPspectra formation (inset) obtained evidenced during by SPR the peakingGNP at formation evidenced by SPR peaking at 535 nm (second-step) and the black lines represent kinetic 535 nm (second-step) and the black lines represent kinetic and corresponding spectra (inset) GNP and corresponding spectra (inset) GNP aggregation and modification evidenced by a progressive aggregation and modification evidenced by a progressive increase of an additional band at 750 nm increase of an additional band at 750 nm (third-step). All samples were prepared, and the spectra (third-step). All samples were prepared, and the spectra carried out as described in Materials and Methods. The appearance of SRP bands during GNP synthesis assisted by rTHOP and rNEL was earlier than in the synthesis by HEPES in the absence of enzymes but the absorbance increasing is slower. The kinetics difference observed in the presence of the enzymes is consistent with these proteins participating in the GNP growing step as reducing agent and template. Catalysts 2020, 10, x FOR PEER REVIEW 9 of 17

carried out as described in Materials and Methods. The appearance of SRP bands during GNP synthesis assisted by rTHOP and rNEL was earlier than in the synthesis by HEPES in the absence of enzymes but the absorbance increasing is slower. The kinetics difference observed in the presence of the enzymes is consistent with these proteins participating in the GNP growing step as reducing Catalystsagent 2020and, template.10, 78 9 of 17

The association of GNPs with the enzymes THOP and NEL was also crucial for the colloidal stabilityThe of the association nanoparticles of GNPs as described with the herein. enzymes THOP and NEL was also crucial for the colloidal stability of the nanoparticles as described herein. 2.5. Colloidal Stability of rTHOP-GNP and rNEL-GNP 2.5. Colloidal Stability of rTHOP-GNP and rNEL-GNP The colloidal stability of GNPs associated with enzymes is essential for catalytic and therapeuticThe colloidalapplications. stability Therefore, of GNPs rTHOP associated- and withrNEL enzymes-GNP suspensions is essential forwere catalytic analyzed and by therapeutic naked eyesapplications. for 10 days Therefore,of resting at rTHOP- room temperature and rNEL-GNP (Figure suspensions 8, left panel) were. analyzedAll the purple by naked-red eyessuspensions for 10 days remainedof resting stable at room on the temperature day of synthesis. (Figure8 ,Gradually, left panel). colloidal All the purple-red suspensions suspensions produced remained with 40 stableand 8 on 1 nmol.Lthe day−1 of of rTHOP synthesis. and Gradually, 200 and colloidal 8 nmol.L suspensions−1 of rNEL produced lost colloidal with stability40 and 8 nmol.L and resulted− of rTHOP in the and 1 formation200 and of 8 nmol.Lvisually− detectableof rNEL lost precipitates colloidalstability after 10 anddays resulted of resting. in the In formationcontrast, GNPs of visually suspensions detectable 1 producedprecipitates with after 2 nmol.L 10 days−1 of of rTHOP resting., as In well contrast, as with GNPs 40 suspensionsand 8 nmol.L produced−1 of rNEL, with showed 2 nmol.L exceptional− of rTHOP, 1 colloidalas well stability. as with 40 The and protein 8 nmol.L-GNPs− of suspension rNEL, showed with exceptional colloidal stability colloidal after stability. 10 days The protein-GNPs (produced 1 1 withsuspension THOP 2 withnmol.L colloidal−1 and stabilityNEL 2 nmol.L after 10−1 days) are (producedalso evidenced with THOPby UV 2- nmol.Lvis spectra− and (Figure NEL 28, nmol.L right − ) panel)are alsoand evidencedwere elected by for UV-vis the determination spectra (Figure of8, rightthe synthesis panel) and kinetics were electedthat are for depicted the determination in the next of sectionthe synthesis. kinetics that are depicted in the next section.

FigureFigure 8. 8.ColloidalColloidal stability stability of of THOP THOP-GNP-GNP and NEL-GNP. NEL-GNP. The The stability stability of GNPsof GNPs produced produced by HEPES by HEPESassisted assisted by proteases by proteases at the at four the four tested tested concentrations concentrations was was followed followed for for10 days. 10 days. The The left left panel paneldepicts depicts the the visual visual aspects aspects of fresh of fresh synthesized synthesized (day (day 0) and 0) and protein-GNP protein-GNP after after 10 days. 10 days The. UV-visThe UV-vis spectra of rTHOP (2 nmol. L1−1) and rNEL (2 nmol.L−1)1 nanoparticles after 10 days (black lines) spectra of rTHOP (2 nmol. L− ) and rNEL (2 nmol.L− ) nanoparticles after 10 days (black lines) indicateindicate the thecolloidal colloidal stability stability of both of both rTHOP rTHOP-GNP-GNP and and rNEL rNEL-GNP-GNP preparations preparations compared compared to freshly to freshly synthesizedsynthesized protein protein-GNPs-GNPs (day (day 0, 0,blue blue line). line). The The lower lower stability stability of of rTHOP rTHOP-GNP-GNP produced produced with with 200, 200, 40 40 and 8 n nmol.L−1,1 and rNEL-GNP produced with rNEL at 200 nmol.L−1 and1 8 nmol.L−1 leading1 to and 8 n nmol.L− , and rNEL-GNP produced with rNEL at 200 nmol.L− and 8 nmol.L− leading to nanoparticlenanoparticle precipitation precipitation is noteworthy. is noteworthy.

It isIt interesting is interesting to relate to relate the the differential differential colloidal colloidal stability stability of THOP of THOP-GNP-GNP and and NEL NEL-GNP-GNP when when wewe consider consider their their structures. structures. Figure Figure 9 shows9 shows the the Protein Protein Data Data Bank Bank structure structure of ofTHOP THOP (1S4B) (1S4B) and and NEL (1I1I) in which is evident a highly similar structure of these enzymes. However, it is well-known

that free THOP and NEL have different stability. Contrary to NEL, THOP loses activity due to interchain disulfide bonds oligomerization or post-translational modifications of oxidative nature in its surface-exposed cysteine residues. These changes have been associated with the reactivity of three acidic cysteine residues (low pKa of lateral chain thiol group) that are in proximity to the basic amino Catalysts 2020, 10, x FOR PEER REVIEW 10 of 17

NEL (1I1I) in which is evident a highly similar structure of these enzymes. However, it is well-known that free THOP and NEL have different stability. Contrary to NEL, THOP loses activity Catalystsdue to 2020interchain, 10, 78 disulfide bonds oligomerization or post-translational modifications of oxidative10 of 17 nature in its surface-exposed cysteine residues. These changes have been associated with the reactivity of three acidic cysteine residues (low pKa of lateral chain thiol group) that are in proximity acid residues, arginine, and lysine. In NEL structure, however, there is only a solvent-exposed acidic to the basic residues, arginine, and lysine. In NEL structure, however, there is only a cysteine residue Cys247 that, similarly to Cys246 of THOP, is sided by arginine and lysine residues. solvent-exposed acidic cysteine residue Cys247 that, similarly to Cys246 of THOP, is sided by The difference of colloidal stability between THOP- and NEL-capped gold nanoparticles is one more arginine and lysine residues. The difference of colloidal stability between THOP- and NEL-capped evidence of protein immobilization. gold nanoparticles is one more evidence of protein immobilization.

FigureFigure 9.9. ComparativeComparative cartooncartoon ofof ProteinProtein DatabankDatabank (PDB)(PDB) structurestructure ofof THOPTHOP andand NELNEL highlightinghighlighting cysteinecysteine andand basicbasic aminoamino acidacid residuesresidues ofof eacheach enzyme.enzyme. InIn THOPTHOP structure,structure, therethere areare threethree exposedexposed acidicacidic cysteinecysteine residues:residues: C246, C248 and C253, whereaswhereas NELNEL hashas onlyonly oneone exposedexposed acidicacidic cysteinecysteine residueresidue thatthat is is C247. C247. In In the the cartoon cartoon structures, structures, cysteines cysteines are are highlighted highlighted in red in whilered while the neighbor the neighbor basic aminobasic amino acids Racids and R K and are highlightedK are highlighted in blue. in blue.

ForFor thethe applicationapplication ofof rTHOP-rTHOP- andand rNEL-GNPsrNEL-GNPs forfor catalysiscatalysis andand therapeuticstherapeutics itit isis fundamentalfundamental thatthat thethe enzymesenzymes adsorbedadsorbed onon thethe GNPsGNPs retainsretains andand preferentiallypreferentially improvedimproved thethe catalyticcatalytic propertiesproperties andand activity.activity. TheThe activityactivity ofof GNP-immobilizedGNP-immobilized rTHOPrTHOP andand rNELrNEL waswas comparedcompared withwith thethe respectiverespective solublesoluble formsforms ofof thethe enzymes.enzymes.

2.6.2.6. Increased Stability-Activity ofof ImmobilizedImmobilized rTHOPrTHOP andand rNELrNEL inin GNPsGNPs TheThe temporaltemporal activityactivity preservationpreservation ofof bothboth freefree andand immobilizedimmobilized proteasesproteases waswas compared.compared. FreeFree enzymesenzymes showedshowed higherhigher activityactivity thanthan thethe immobilizedimmobilized ones,ones, butbut thethe stabilitystability ofof inin solutionsolution rTHOPrTHOP andand rNELrNEL atat roomroom temperaturetemperature decreased until completecomplete loss of activity in a few hours (Figure 10).10). However,However, goldgold nanoparticles-enzymes nanoparticles-enzymes preparations preparations of rTHOP-GNPof rTHOP-GNP and and rNEL-GNP rNEL-GNP maintained maintained their proteolytictheir proteolytic activity activity even aftereven 24after h (retaining24 h (retaining 8.1% 8.1% and 7.3%and 7.3% of their of their activity, activity, respectively). respectively). We also We testedalso tested the possibility the possibility that HEPES-based that HEPES-based GNP (without GNP (without protein) couldprotein) also could be capable also be of stabilizingcapable of THOPstabilizing activity. THOP As activity. observed, As it observed, resulted in it a resulted retention in of a 22%retention of its activityof 22% of at its the activity time of at 6 h,the indicating time of 6 theh, indicating presence of the in solutionpresence free of GNPsin solution are capable free GNPs of stabilizing are capable THOP of activity. stabilizing The extensionTHOP activity. of stability, The however,extension wasof stability, not comparable however, to was GNP-immobilized not comparable THOP, to GNP-immobilized still active after THOP, 24 h. still active after 24 h.

Catalysts 2020, 10, x FOR PEER REVIEW 11 of 17

Catalysts 2020, 10, 78 11 of 17

Figure 10. Increased activity based on stability of rTHOP-GNP and rNEL-GNP. Both THOP and NEL Figure 10. Increased activity based on stability of rTHOP-GNP and rNEL-GNP. Both THOP and NEL enzyme ultimately lose their activity after 6 h in solution at room temperature. When immobilized in enzyme ultimately lose their activity after 6 h in solution at room temperature. When immobilized in the gold nanoparticles, the maintenance of activity is notable. GNP synthesized without any the gold nanoparticles, the maintenance of activity is notable. GNP synthesized without any protease (HEPES-GNP) and incubated with in solution THOP was also capable of retaining THOP proteolytic (HEPES-GNP) and incubated with in solution THOP was also capable of retaining THOP proteolytic activity. Samples were prepared and data obtained as described in the Material and Methods section. activity. Samples were prepared and data obtained as described in the Material and Methods section. Recently, there has been a growing interest in protein-driven nanoparticles synthesis for a variety of applicationsRecently, there due to has their been unique a growing properties interest of controllable in protein-driven morphology nanoparticles and size dispersion synthesis [55 for,56 a]. Proteinsvariety of and applications amino acids due are to considered their unique safe, non-toxicproperties compounds of controllable and themorphology production and of goldsize nanoparticlesdispersion [55,56]. GNPs Proteins using these and materials amino acids are categorized are considered as green safe, and non-toxic biocompatible compounds materials and andthe techniques.production Theseof gold systems nanoparticles can be used GNPs both forusing newly these nanoparticle materials synthesis are categorized as well as immobilizationas green and techniquesbiocompatible in order materials to increase and techniques. protein stability These and syst conductems can functional be used studies.both for Enzymes newly nanoparticle are relevant andsynthesis useful as targets well as for immobilization selective cancer techniques drugs and in imagingorder to increase probe delivery proteindue stability to their and substrate conduct specificityfunctional thestudies. as well Enzymes as the ability are relevant to perform and usef biologicalul targets catalysis. for selective A wide cancer variety drugs of enzyme and imaging classes areprobe overexpressed delivery due in tumorsto their and substrate other pathological specificity th microenvironmentse as well as the ability acting asto potentialperform targetsbiological for therapycatalysis. andA imagingwide variety studies of [ 57enzyme–60]. Responsive classes are drugs overex involvingpressed proteases in tumors can an bed designed other pathological for a target tomicroenvironments increase selectivity acting and e ffiascacy potential in a specific targets physiological for therapy process. and Inimaging the case studies of the mammalian [57,58–60]. oligopeptidasesResponsive drugs THOP involving and NEL, proteases their ability can be to designed regulate bioactivefor a target peptides, to increase as the selectivity processing and of efficacy the pro- andin a anti-angiogenicspecific physiological derived process. peptides In angiotensin the case of and thebradykinin, mammalian may oligop be usedeptidases in relevant THOP approaches and NEL, intheir order ability to better to regulate comprehend bioactive these peptides, systems as and the their processing biology. of A the significant pro- and limitation anti-angiogenic to study derived THOP andpeptides NEL functionsangiotensin resides and inbradykinin, their low stabilitymay be inused biological in relevant systems, approaches which makes in order it challenging to better tocomprehend study their these physiology systems and and biotechnological their biology. uses.A significant One possibility limitation to surpass to study these THOP problems and NEL is to immobilizefunctions resides these enzymesin their low in orderstability to conductin biological these systems, studies. which Here wemakes defined it challenging conditions to capable study oftheir generating physiology stable and nanometric biotechnological gold nanoparticle uses. One immobilized possibility toto bothsurpass THOP these and problems NEL proteases; is to and,immobilize importantly, these retainingenzymes in their order proteolytic to conduct activity. these Thestudies. observed Here positivewe defined effects conditions of immobilization capable of ingenerating the proteolytic stable nanometric activity of THOPgold nanoparticle and NEL may immobilized be in part to dueboth to THOP the impossibility and NEL proteases; to generate and, highimportantly, molecular retaining weight inactivetheir proteolytic forms, whose activity. reversibility The observed depends positive on specific effects cysteineof immobilization residues that in composethe proteolytic the disulfide. activity In of addition, THOP and and NEL noteworthy, may be thein part retention due to of the activity impossibility indicates ato gain generate of stability high inmolecular the structure-function weight inactive of bothforms, THOP whose and reversibility NEL, showing depends that immobilization on specific cysteine on gold nanoparticlesresidues that cancompose be used the as disulfide. a valuable In approach addition, to and different noteworthy applications, the ofretention both enzymes, of activity from indicates functional a gain studies of tostability therapeutic in the uses. structure-function of both THOP and NEL, showing that immobilization on gold nanoparticles can be used as a valuable approach to different applications of both enzymes, from 3.functional Material studies and Methods to therapeutic uses.

3.1.3. Material Chemicals and Methods Culture medium supplies were purchased from Thermo Fisher DTT (dithiothreitol), HEPES, Tris, 4.1. Chemicals NaCl, IPTG (Isopropyl-β-d-thiogalactoside), sodium borohydride and metallic gold salt (tetrachloro auric acid trihydrate-HAuCl4) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Catalysts 2020, 10, 78 12 of 17

3.2. Enzyme Production THOP and NEL expression and purification were conducted as already described with few adaptations [47,61]. Briefly, the liquid culture of E. coli BL21-DE3 containing the pET28a (+) constructs for expression of wild type thimet oligopeptidase and neurolysin (rTHOP and rNEL) and THOP mutant 6 M were induced by IPTG. Sonicated soluble extracts of bacteria culture were filtered and purified using affinity-based HisTrap FF column (GE) in an AKta system (GE) and eluted in a 50 mM Tris-buffer 100 mM NaCl pH 7.4 using increasing concentrations of imidazole. Purity was followed by SDS-PAGE. The specific activity of both proteases was measured using the fluorogenic substrate Abz-GFSPFRQ-EDDnp, in a 50 mM Tris, 100 mM NaCl buffer (pH 7.4) at 37 ◦C using the specific inhibitor JA2 for THOP [22]. Fluorescent products were followed at the excitation wavelength of 420 nm and an emission wavelength of 320 nm in a spectrofluorimeter Hitachi F7000 (Tokyo, Japan). After 5 min pre-incubation, enzymes (0.1 nM) were added to the assay buffer with or without DTT (1 mM), and the fluorescence kinetic recorded for 10 min. The concentration of the substrate was 1 1 determined colorimetrically by absorbance of the 2, 4-dinitrophenyl group (ε365 = 17,300 M− .cm− ). Enzyme concentration for initial rate determination was chosen for hydrolyzing less than 5% of the substrate present. All the obtained data were fitted by nonlinear least-squares equations, using GRAFIT v.5.03 software [62].

3.3. Gold Nanoparticle Synthesis All syntheses were performed in a laminar flow cabinet using small lab glass vials clear with a black screw cap. The glassware for synthesis was previously cleaned to avoid undesired nucleation aggregation of colloidal gold solutions [56]. The addition of reagents was performed as follows: Milli-Q deionized water, 0.2 mM sodium phosphate buffer plus HEPES, pH 9.0, 100 µL of protein solution per mL and tetrachloroauric acid solution (400 µM). GNPs were synthesized at 40 ◦C for 2 h on a vat dry bath and let stabilize at room temperature. After 24 h of synthesis, the GNP colloidal suspensions were centrifuged and resuspended in 0.2 mM sodium phosphate buffer plus HEPES, pH 7.0 and stored at 4 ◦C. Several syntheses were performed and characterized by UV-vis to guarantee reproducibility. GNPs synthesized with sodium borohydride (GNP-BH) were prepared by a reaction between sodium borohydride (2 mM) in water and tetrachloroauric acid solution (0.25 mM) at 0 ◦C and maintained under agitation (200 rpm) for 15 min [63,64]. This solution was maintained for 24 h at 4 ◦C, and enzymes were added and incubated for 4 h before UV-vis, dynamic light scattering (DLS) and Zeta potential measurements [65].

3.4. Zeta Potential and DLS Measurements

The Zeta potential (ζ) of GNPs were all measured at 25 ◦C using a Zetasizer NanoZS (Malvern instruments Ltd., Worcestershire, UK). All GNPs solution was diluted in Milli-Q deionized water by 50% immediately before measurement. Three repeats for each sample were conducted to estimate the error in the measurements. Each measurement was taken by conducting 30 runs [66]. DLS (dynamic light scattering) measurements were acquired using the same instrument operating in the backward (a scattering angle of 173◦)[67].

3.5. UV-vis Spectra This spectroscopy was used to quantify proteins and characterize nanoparticles. UV-vis also characterized the optical properties of metallic nanoparticles, including absorbed radiation (σabs) and scattered radiation (σsca), where σtotal = σabs + σsca. Samples were analyzed in quartz cuvettes with optical path 1.0 or 0.5 cm. A UV-visible Photodiode Array Spectrophotometer MultiSpec-1501 spectrophotometer (Shimadzu Co, Kyoto, Japan) was used. The synthesis temperature of the GNPs was controlled by a thermal bath coupled to the spectrophotometer. Catalysts 2020, 10, 78 13 of 17

3.6. Circular Dichroism The CD measurements were carried out in a J-815 Circular Dichroism Spectrometer equipped with Peltier single cell holder (Jasco International Co Ltd., Tokyo, Japan) using quartz cuvettes with a 1.0 or 0.5 optical path; bandwidth, 1.0 nm; scanning speed, 50 nm/min and four accumulations.

3.7. Atomic Force Microscopy Samples were prepared by spreading the protein-GNP solution onto a freshly cleaved mica substrate and solvent dried using a nitrogen stream. AFM in the intermittent contact mode was performed using a scanning probe microscope equipped with a controller instruments and tip in an AFM XE7 Park System, Korea, using adjustable resonance frequency.

4. Conclusions In the present study, it was reported the synthesis of gold nanoparticles in situ using HEPES as reducing agent assisted by the presence of the recombinant proteolytic enzymes THOP and NEL that remained immobilized on the gold nanoparticles based on the following evidences: (i) The presence of significant spectral differences observed in the UV region that is consistent with different organic ligands on the GNP surfaces; (ii) proteolytic activity of GNPs produced using HEPES and assisted by THOP and NEL that is absent in GNPs produced exclusively by HEPES and (iii) significant gain of structural and catalytic stability of rTHOP and rNEL associated with GNP relative to the free enzymes. Free enzymes lost 70% and 95% after 2 and 6 h, respectively whereas the dropping of activity decreased to 30% and 60% after the same times of aging. Important to note that THOP gained stability even when it was added to gold nanoparticles previously fabricated with HEPES that was consistent with a binding capacity on gold nanoparticles; (iv) the significant gain of colloidal stability exhibited by gold nanoparticles produced with HEPES assisted by the enzymes and (v) in addition, the binding capacity of THOP on gold nanoparticles was also corroborated in bare GNPs synthesized with sodium borohydride that was evidenced by UV-visible spectroscopy and zeta potential.

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/10/1/78/s1, Figure S1: UV-vis spectra of bare GNPs synthesized using sodium borohydride, Figure S2: Characterization of GNPs synthesized using NaBH4, Table S1: Zeta potential and pH values. Author Contributions: Conceptualization, A.M.M.B., I.L.N.-C., M.Y.I. and V.O.; methodology, A.M.M.B., I.L.N.-C., M.Y.I. and V.O.; formal analysis, A.M.M.B., I.L.N.-C., M.Y.I., V.O.; investigation, A.M.M.B., M.P.C.R., M.Y.I.; writing—original draft preparation, M.Y.I. and I.L.N.-C.; writing—review and editing, M.Y.I., V.O. and I.L.N.-C.; supervision, M.Y.I. and I.L.N.-C.; project administration, M.Y.I. and I.L.N.-C.; funding acquisition, M.Y.I, V.O. and I.L.N.-C. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo, grants 2017/02317-3, 2018/09158-0 and 2019/01487-7 and by Conselho Nacional de Desenvolvimento Científico e Tecnológico, grant numbers 30924/2017-9 and 309247/2017-9. FAPESP 2015/017688-0, 2017/02317-2, SisNano (402289/2013-7), NBB/UFABC, CAPES grant 001, CEM/UFABC for access to facilities. Acknowledgments: We are grateful to the funding agencies FAPESP, CNPq, and CAPES. Paulo Costa and Nova Analítica are kindly acknowledged for lending the Park XE7 AFM instrument used in this work to the Structural Biophysics Laboratory (Emerson R. Silva). We also thank Emerson Silva and Lucas Mello for the valuable discussion concerning the AFM data and David da Mata Lopes for technical assistance. Conflicts of Interest: The authors declare no conflict of interest.

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