Polyoxometalate-polypeptide nanoassemblies as peroxidase surrogates with antibiofilm properties

Nanoscale rsc.li/nanoscale ISSN 2040-3372 PAPER Scott G. Mitchell, Rafael Martín-Rapún et al. Polyoxometalate–polypeptide nanoassemblies as peroxidase surrogates with antibiofi lm properties Volume 14 Number 16 28 April 2022 Pages 5931-6270 Nanoscale PAPER Cite this: Nanoscale, 2022, 14, 5999 Received 14th December 2021, Accepted 23rd March 2022 DOI: 10.1039/d1nr08223j rsc.li/nanoscale Polyoxometalate–polypeptide nanoassemblies as peroxidase surrogates with antibiofilm properties† Héctor Soria-Carrera, a,b,c Elena Atrián-Blasco, a,b Jesús M. de la Fuente, a,b Scott G. Mitchell * a,b and Rafael Martín-Rapún * a,b,c Developing artificial metalloenzymes that possess a superior performance to their natural counterparts is an attractive concept. Polyoxometalates (POMs) are a class of anionic molecular metal–oxides with excellent redox properties and bioactivity. We have recently introduced “POMlymers”–covalently conjugated POM–peptide hybrid materials –where the polypeptide chain is obtained through a ring-opening polymerisation (ROP) of α-amino acid N-carboxyanhydrides (NCA) on an inorganic POM scaffold. Attracted by the idea of preparing artificial metalloenzymes, here we report the supramolecular self-assembly of POMlymer hybrids into nanoparticles where an optimal environment for catalysis is created. Our results demonstrate that the self-assembly of covalent POMlymers, enhances the peroxidase-like activity of the parent POM anion whereas, in contrast, the catalytic activity for nanoparticles obtained by ionic selfassembly of the same peptide and POM components practically disappears. Furthermore, POMlymer nanoparticles also present antimicrobial and antibiofilm activity against the skin bacterium Staphylococcus epidermidis; whereas, ionic POM–peptide hybrids significantly increase biofilm production and endogenous production of reactive oxygen species. In summary, we present the self-assembly of POMlymer hybrids into nanoparticles and a combination of peroxidase activity and microbiology assays that show that the POM–peptide covalent bond is essential for the stability of the self-assembled nanoparticles and therefore for their catalytic and biological activity. Introduction Self-assembly is the ultimate degree of complexity designed by Nature to foster activity development and protein evolution underpins such an idea. It is accepted that enzymes emerged from simpler peptides that eventually started to self-arrange into catalytically active assemblies. 1 Peptide folding can further evolve by sequestering metal ions, that once in the structure could synergistically favour catalysis. For instance, Gazit 2 and co-workers prepared a single amino acid enzyme (phenylalanine) that self-assembles into cross-β-sheet nanotubes in the presence of Zn 2+ and displays hydrolase-like activity. Likewise, Korendovych 3 reported that Zn 2+ favours β-sheet formation in heptapeptides containing a metal binder residue (histidine) displaying esterase activity. Peptides capable of binding Zn 2+ should have been important in primitive metabolism. For instance, Moran et al. 4 discovered that Zn 2+ could carry out hydrolytic reactions. Short peptides prone to fold into β-sheets are likely to form amyloids which display catalytic pockets. In consequence, these structures have been proposed to be primitive enzymes on the early Earth. Polyoxometalates (POMs) –redox-active molecular metal– oxide anions –are extraordinary building blocks that have been widely employed to promote the self-assembly of small molecules and polymers. 5,6 One unique and applicable property of POMs is their excellent redox properties which means that they can potentially display enzymatic behaviour. For example, the group of Parac-Vogt has developed a set of artificial proteases based on POMs which reach a remarkable degree of selectivity. 7 Meanwhile, Wang and co-workers systematically evaluated POMs as peroxidase surrogates, finding comparable efficiencies as horseradish peroxidase (HRP). 8 Eventually, the combination with biomolecules will lead to hybrid materials with synergistic properties. For instance, Wu reported the co-assembly of K 5 PV 2 Mo 10 O 40 with folic acid in nanospheres that promote 3,3′,5,5′-tetramethylbenzidine (TMB) oxidation. 9 Ma et al., reported the preparation of dipeptide–POM–graphene oxide hybrid materials that display peroxi- †Electronic supplementary information (ESI) available: Comprehensive characterisation and antimicrobial activity data. See DOI: https://doi.org/10.1039/ d1nr08223j a Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, c/Pedro Cerbuna 12, 50009 Zaragoza, Spain. E-mail: [email protected], [email protected] b CIBER de Bioingeniería, Biomateriales y Nanomedicina, Instituto de Salud Carlos III, 28029 Madrid, Spain c Departamento de Química Orgánica, Facultad de Ciencias, Universidad de Zaragoza, c/Pedro Cerbuna 12, 50009 Zaragoza, Spain This journal is © The Royal Society of Chemistry 2022 Nanoscale, 2022, 14,5999–6006 | 5999 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online View Journal | View Issue dase activity superior to the parent POM (Keggin anion PW 12 ). 10 Co-assembling POMs with peptides has also been recently explored for a wide range of applications in catalysis 11 or biomedicine. 12a,b For example, in facially amphipathic peptides the addition of POMs triggers fibre formation through β-sheet folding, enhancing the antimicrobial properties of both individual components. 12b Covalent linking is an alternative strategy to tune hybrid self-assembly. Cronin, 13,14 Coutsolelos, 15 Lacôte 16,17 and Carraro 18,19 have investigated these structures and how both components influence the folding. Ionic interactions between connected POMs and peptides play a key role in the assembly of such hybrids. Recently, Carraro et al. 18 reported the use of an anionic spacer, between the POM and the peptide, to preserve the pristine peptide folding. Likewise, Nature has incorporated metallic clusters in proteins which display crucial roles in metabolic pathways like photosynthesis 20 (Mn 4 CaO 5 ) or oxidative phosphorylation (Fe– S). 21 POMs are present in some natural proteins as well, for example, the molybdenum storage protein (MoSto), involved in the Mo metabolism in N 2 fixating bacteria, displays two sets of polyoxomolybdate POMs: one ionically linked and the other covalently bonded. Remarkably, the protein scaffold not only allocates the POMs inside the peptide chain but also prevents their hydrolysis. 22 We are interested in developing new synthetic approaches to combining inorganic POM cores with peptides and have recently reported an innovative “on-POM polymerisation” route to obtain covalent POM–polypeptide hybrids, POMlymers. The well-known bis-amino functionalised MnAnderson POM derivative [MnMo 6 O 18 ((OCH 2 ) 3 CNH 2 ) 2 ] 3− acts as an initiator for the ring-opening polymerisation (ROP) of amino acid N-carboxyanhydrides (NCAs). The first examples, such as POMK 10 (Fig. 1), display polypeptides comprising cationic and hydrophobic lateral groups along with the central anionic POM. We envisioned that the folding of the polypeptides present in POMlymers 23 could be used to direct the solution-based self-assembly of nanoscale materials to appropriate environments for catalysis. Furthermore, such studies would mean we could expand on the role of the different components involved in generating the antibacterial activity of the POMlymers as well as in their self-assembly. In recent years, numerous studies have demonstrated that the self-assembly of antimicrobial peptides (AMPs) can boost their antimicrobial activity and also improve their stability. 24–26 This could be one of the many strategies proposed for the development of new antimicrobial agents, necessary to combat antimicrobial resistance (AMR), which is currently deemed to be one of the most important global health threats and is estimated to cause more than 33 000 deaths annually just in Europe. 27,28 While POMs, 29 POM-hybrids 30–32 and POM– peptide assemblies have been described as tailorable new hybrid materials with antimicrobial activity, 12,23,33,34 their mechanism of action has not been explored in detail and has been related, among others, to induced oxidative stress resulting from the production of reactive oxygen species (ROS). ROS can result in hormesis in bacteria, which is characterised by low-dose stimulation and high-dose inhibition. Consequently, high levels of ROS, either internal or external to the cell, can lead to cell death; whereas low levels can trigger an adaptive response from the bacteria, such as increased proliferation or secretion of enzymes and polysaccharides to facilitate the production of biofilm and biofilm matrix. 35 Therefore, understanding how to control the level of ROS produced by antimicrobial materials will aid the development of new antimicrobial agents. Herein we report our systematic study of the self-assembly of POMlymer POMK 10 –where K 10 refers to the number of positively charged lysine residues covalently linked to the Anderson–Evans POM –into hybrid nanoparticles and their peroxidase-like properties in the oxidation of TMB. We demonstrate that the covalent hybrid POMK 10 enhances the catalytic properties of the parent POM Na 3 [Mn III Mo 6 O 18 ((OCH 2 ) 3 CNH 2 ) 2 ], while the ionic assembly of the parent POM and the polypeptide, K 10 /POM, has a detrimental effect on the catalysis (Fig. 1). Our data suggest that the POM moieties are buried deep inside the self-assembled POMlymer nanoparticle creating a favorable catalytic pocket. Finally, we evaluated the antimicrobial activity of the POMK 10 nanoparticles against the Gram-positive skin bacterium Staphylococcus epidermidis and measured the dose-dependent bacterial response to this redox-active hybrid material by characterising its biofilm production and generation of endogenous ROS. Fig. 1 Structures of POMlymer POMK 10 , polypeptide K 10 and parent POM, [Mn III Mo 6 O 18 ((OCH 2 ) 3 CNH 2 ) 2 ] 3− ; and representations of the selfassembled materials under study: POMlymer nanoparticles (red), polypeptide K 10 assemblies (blue) and ionic K 10 /POM assemblies (yellow). Z = benzyloxycarbonyl. Paper Nanoscale 6000 |Nanoscale, 2022, 14, 5999–6006 This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Results and discussion Characterisation of POMlymer nanoassemblies Self-assembly of amphiphilic polymers is a combination of various interactions that results in burying the non-soluble parts of the polymer. In our system, POMK 10 , we have positively charged lysine and hydrophobic lysine –protected as benzyloxycarbonyl (Z) –randomly distributed along the polymer chain (Fig. 1). This combination results in an amphiphilic polymer that can self-assemble in water into nanoparticles of diameter 70.3 ± 18.4 nm, as observed and characterised by dynamic light scattering (DLS) and scanning electron microscopy (SEM) (Fig. 2A & D and Fig. S1 & S2†). The POMK 10 nanoparticles display the typical behaviour of polymer-based nanoobjects whose assembly is concentrationdependent (Fig. S3†). The critical aggregation concentration (CAC), which determines the concentration at which the components start to aggregate, could be measured using the hydrophobic solvatochromic probe Nile Red (Fig. S4†)–which also proved the presence of hydrophobic pockets. The corresponding CAC was determined to be 0.34 mg mL −1 . Systematic DLS and ζ-potential studies showed that increasing temperature had a detrimental effect on the stability of the POMK 10 nanoparticles and led to precipitation of the hybrids selfassemblies above 37 °C (Fig. S5†). Based on previous research 13 we explored how different milieu influence the assembly. We found that increasing the ionic strength (NaCl concentration) led to screening of the polymer charges, which provoked the aggregation of POMK 10 and low ζ-potentials (Fig. S6†) associated with nanoparticle disassembly. To evaluate the importance of hydrogen bonding in the assembly, we used two well-known chaotropic agents, hexafluoroisopropanol (HFIP) and thiourea (TU), to disrupt the hydrogen-bonding within the assemblies. In general, we observed a variation in size, tending towards larger aggregates and an increase in ζ-potential towards more positive values for both HFIP and TU (Fig. S7 and S8†). We ascribe this effect to the exposure of more lysine groups to the outside since both agents compete for hydrogen bonding. Altogether these studies indicated that ionic interactions must play a crucial role in the POMK 10 self-assembly process. POMK 10 nanoparticles showed a wide range of stability between pH 2 and pH 8.8, as can be seen by the inflection point in the ζ-potential values and the stable hydrodynamic diameter obtained by DLS (Fig. S9†). In addition, circular dichroism (CD) measurements showed a modification in the folding –from an α-helix to a less organised structure –at pH 6 with a subsequent loss of conformation upon further increasing pH (Fig. S10C†). DLS measurements showed that polypeptide K 10 and the ionic combination of K 10 and POM, K 10 /POM, self-assembled to form 62.6 nm and 60.5 nm nanoparticles respectively, both smaller than those of covalent POMK 10 (92.3 nm) (Fig. 2A). While POMK 10 and K 10 nanoparticles displayed similar ζ-potential (Fig. 2A and S1†), K 10 /POM displayed a larger ζ-potential. In addition, when we varied the POM-to-K 10 ratio we observed that ζ-potential linearly increased with POM concentration (Fig. S11B†) despite the anionic POM conferring a large negative charge. Bearing in mind that POMs are also chaotropic agents, we can assume that thiourea and POM behave similarly. In consequence, the POM molecules act to effectively disrupt the peptide folding entering inside the structure which leads to the surface display of previously buried lysine residues. Accordingly, the helical structure of the peptide K 10 was disrupted upon incorporation of POM molecules into the ionic assemblies K 10 /POM, as proven with CD spectroscopy measurements (Fig. 2C). Helicity decreased because POM interfered with the hydrogen bonding interactions needed for the α-helix. This unfolding leads to less structured nanoparticles, in which more cationic lysine side chains can emerge to the surface, therefore increasing the ζ-potential value. Peroxidase-like activity POMK 10 assemblies displayed peroxidase-like activity with the Mn(III) as the redox centre. 36 Steady-state kinetic analysis showed a non-Michaelis–Menten behaviour obtaining a sigmoidal curve in the oxidation of TMB (Fig. 3A and S12–S14†), with partial inhibition at high concentrations. A sigmoidal curve was also observed in the parent POM (Fig. 3A). This behaviour typically appears in allosteric enzymes and can be associated either with multiple catalytic centres or molecular interactions in the catalytic centre. TMB is positively charged in the reaction buffer and so it is likely that it reaches close to the POM, which facilitates the subsequent oxidation. Fitting to the Hill equation we quantified that POMK 10 had more affinity Fig. 2 (A) DLS comparison of the assemblies formed by POMK 10 , polypeptide K 10 , and the mixture K 10 /POM (D H is in log scale, measurements are in water). (B) CD comparison of POMK 10 ,K 10 ,K 10 /POM in water. (C) Variation in the CD of K 10 upon increasing POM concentration with K 10 concentration fixed at 1 mg mL −1 (ca. 192 µM). (D) SEM micrograph of POMK 10 particles (coated with Pd) possessed a corresponding particle size of 70.3 ± 18.4 nm (N> 200 particles). All experiments were performed at least in duplicate and represented as mean ± SD. Nanoscale Paper This journal is © The Royal Society of Chemistry 2022 Nanoscale, 2022, 14, 5999–6006 | 6001 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online for TMB (K half ) than the parent POM, although not statistically significant (KPOMK10 half ¼69:05 +0:15 μM and KPOM half = 79.2 ± 0.3 µM). The POMK 10 nanoparticles significantly reduced in size after the reaction, from 92.3 to 67.1 nm, (Fig. S15†) but retained their secondary structure (Fig. 3D). The reduction in size likely arises to a larger hydrophobic content due to the presence of TMB. The catalytic activity of POMK 10 is enhanced when selfassembled. We observed a significant increase in catalytic activity above 0.2 mg mL −1 POMK 10 concentration (Fig. S20†). This concentration is close to the CAC of POMK 10 in the presence of TMB, which is smaller than for POMK 10 alone due to the increase of the hydrophobic content (Fig. S21†). These experiments support our hypothesis that the presence of hydrophobic pockets within the assemblies could enhance catalysis. We further verified this concept by disassembling the particles prior to the oxidation reaction of TMB, which led to poor catalytic activity (Fig. S22†). When we raised the pH, we observed an abrupt decrease in the catalysis above pH 7, which could be attributed to a less charged nanoparticle which could disrupt the catalytic pockets of the assembly (Fig. S16†). We can conclude that the activity of the parent POM was retained in the assembly of the covalent hybrid POMK 10 while the ionic hybrid K 10 /POM was considerably less active. We reasoned that the catalytic environment in POMK 10 nanoparticles is more favourable than in K 10 /POM. We explored cooperativity changing the chromogenic probe to 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), which is negatively charged and has a higher reduction potential. In these conditions, ABTS was only oxidised by POMK 10 (Fig. 3B). However, we observed precipitation of POMK 10 nanoparticles at high ABTS concentrations, which we ascribe to anionic ABTS acting as crosslinker among positively charged nanoparticles. This was also the case for K 10 /POM which led to strong scattering and an unreliable apparent increase of initial velocity at higher ABTS concentrations (Fig. 3B and S17–19, [ABTS] = 1.5 mM). Consequently, only POMK 10 nanoparticles were able to oxidise anionic ABTS, which is an improvement compared to parent POM and K 10 / POM. Antibacterial and antibiofilm activity In our original report on the synthesis of POMlymers, 23 we observed that POMK 10 could be coated on surfaces to prevent Bacilus subtilis biofilm formation. In this study we have researched the antibacterial activity of POMK 10 , as well as the parent POM and the ionic hybrid K 10 /POM, against the Grampositive skin bacterium Staphylococcus epidermidis. This generally non-pathogenic bacterium forms an important part of human skin microbiota, however it has an important role in nosocomial infections and sepsis in imunocompromised individuals, mainly due to its ability to form biofilms and attach to medical devices. 38 First, we determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the different assemblies and compounds, which determine respectively the bacteriostatic and bactericidal activity in solution. Briefly, optical density measurements were used to determine the corresponding MIC values, while a colorimetric resazurin cell viability assay in combination with colony plate-counting were used to verify the MBC values. Selfassembled POMK 10 possessed a MIC corresponding to 125 µg mL −1 (24 μM). The MIC of both polypeptide K 10 and ionic K 10 / POM was 62.5 µg mL −1 , lower than POMK 10 , while the parent POM had the poorest antibacterial activity with a MIC corresponding to 500 µg mL −1 (Table 1). The MBCs of K 10 and its hybrids were found to be double their corresponding MIC values, rendering all components and assemblies as effective bactericidal agents against the tested model bacterium. Importantly, the antimicrobial activity of the polypeptide and hybrids against Gram-positive bacteria was in the same range as other polypeptidic materials 39–42 and POM–peptide hybrids described in literature. 12,23,33,43 To better understand the different MIC and MBC values of each material we further studied the early-stage intracellular production of reactive oxygen species (ROS), closely linked to the oxidative stress suffered by the bacteria, in the first hours Fig. 3 Steady-state kinetic curves for POMK 10 , parent POM and K 10 / POM fitted using the Hill equation for (A) TMB and (B) ABTS as substrates. Concentration of POM moiety is 84 µM in all. (C) Initial reaction rates in the oxidation of TMB for different POMK 10 concentrations (log scale). The solid line represents a Boltzmann fitting whose inset point was calculated 37 as x 0 −2dx = 0.15 mg mL −1 . (D) CD before and after the reaction in a buffered solution (acetate buffer pH 4.6, 10 mM). All experiments were performed at least in duplicate and represented as mean ± SD. Table 1 Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC), in µg mL −1 , of the different materials against Gram-positive S. epidermidis MIC (µg mL −1 ) MBC (µg mL −1 ) K 10 62.5 125 POMK 10 125 250 K 10 /POM 62.5 125 POM 500 >500 Paper Nanoscale 6002 |Nanoscale, 2022, 14, 5999–6006 This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online of their treatment with the materials. We assessed ROS production using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) (Fig. 4A). DCFH-DA is a non-fluorescent molecule that is internalised by bacteria and then hydrolysed to produce DCFH. In the event of oxidative stress, DCFH becomes oxidised by H 2 O 2 producing fluorescent DCF, thus enabling direct correlation with intracellular production of ROS (Scheme S1†). Therefore, once bacteria had been incubated with DCFH-DA and properly washed to remove any excess of extracellular dye, we added K 10 , POMK 10 ,K 10 /POM and parent POM at a concentration of 125 µg mL −1 , which corresponds to the MIC of POMK 10 and MBCs of K 10 and K 10 /POM (Fig. 4A). The increase of fluorescence intensity, corresponding to the production of DCF and proportional to the ROS formed inside the bacteria, was recorded for at least 90 minutes, and values presented in Fig. 4A were measured 90 minutes after addition of the different treatments, in at least three independent experiments. At first sight, while K 10 does not promote production of intracellular ROS, the parent POM induces a significant amount of ROS in the first 90 minutes of incubation. What is more relevant is that production of intracellular ROS is increased in an independent manner to the amount of POM added (Fig. S23†). Treatment with the ionic hybrid K 10 / POM at 125 µg mL −1 leads to a 12-fold increase of ROS, just slightly lower than the parent POM. We reasoned that in the bacterial milieu the ionic assembly loses the POM due to the interactions with the proteins. As a result, the induction of ROS production in K 10 /POM behaves similarly to the parent POM, and the antibacterial activity the same as K 10 . In the presence of the covalent hybrid, POMK 10 , the increase in ROS is equal to half that of K 10 /POM. The origin of the induced intracellular ROS production could be behind an increase in oxidative stress as a defence of the bacteria or as the redox activity of internalized POM. Further studies, which are beyond the scope of this work, will be needed to determine the origin of the increase in intracellular ROS production. Nevertheless, the observed level of ROS production during the first hours of treatment is not lethal to the bacteria, as shown by the corresponding MIC & MBC values obtained for the parent POM. Thus, the antibacterial activity of POMK 10 is induced by other mechanisms, most likely via interaction with the cell membrane, as previously described for cationic AMPs and lysine-based polypeptides. 42,44 To further assess the effect of ROS and oxidative stress induced by the different materials under study, we conducted the MIC & MBC assays in presence of ascorbic acid (2.5 mM), an important antioxidant and ROS scavenger. ROS quenching by ascorbic acid led to a loss of antibacterial activity of the parent POM and the ionic hybrid, K 10 /POM, as reflected by the increase of their MBC (Fig. 4C): the MBC of K 10 /POM doubles from 62.5 µg mL −1 to over 125 µg mL −1 , and in the case of the parent POM, the MBC value is greater than 500 µg mL −1 . This effect has been previously observed in hybrid systems with an important involvement of oxidative stress in the antimicrobial mechanism of action. 35 On the contrary, addition of ascorbic acid seems to have an additive effect on the activity of both K 10 and POMK 10 because ascorbic acid greatly reduces S. epidermidis growth at concentrations ≥5 mM (Fig. S24†). As such we observed that the MBC value of K 10 decreased by half and there was a visible decrease of bacterial growth for POMK 10 at 125 µg mL −1 (Fig. S25†). These observations from the DCFH-DA and ROS quenching assays seem to indicate that the parent POM has oxidative stress-related activity, although the levels are not enough to produce cell death, whereas the polypeptide and POM–peptide hybrids exert their antibacterial mode of action through an alternative mechanism, or the combination of both. The morphology of the bacteria inoculated with POM– peptide hybrids and components were studied using SEM to obtain more information about the mechanism of action of the different materials and their possible regulation of biofilm production. K 10 and POMK 10 both induced obvious signs of damage to the cell membrane, which collapses in on itself to produce hollow semi-spheres (Fig. 4E). Importantly, S. epidermidis treated with K 10 and POMK 10 also displayed a greater variability in sizes of bacteria, where significant shrinkage and enlargement of bacteria were both observed (Fig. 4D and E). Furthermore, S. epidermidis treated with the parent POM and the ionic hybrid showed an increased production of biofilm matrix, whose components can be seen overlaying and attaching to cell surfaces (yellow arrows in Fig. 4E). Following this observation, we opted to use a crystal violet staining method to quantify the formation of biofilm upon different treatments. Using the crystal violet method, the amount of biofilm produced by S. epidermidis was assessed after 72 hours of treatment with the different hybrid nanoparticles or parent POM at their 1 2× MIC values, a concentration which does not kill the bacteria or drastically affect their growth but can have an impact on the production of biofilm (Fig. S26†). The results showed that both K 10 and POMK 10 effectively halved the biofilm mass being produced compared with the non-treated bacterial biofilm (Fig. 4B). On the other hand, both the ionic hybrid and the parent POM increased biofilm production, with respect to the non-treated bacteria. First, this could corroborate, as well, the more probable loss of POM from the ionic hybrid in bacterial cell culture broth. Second, the increased biofilm production upon treatment with POM and K 10 /POM could be related to the increase of the oxidative stress in bacteria treated with these materials: it has been shown that exposure to mild oxidative stress, at least below toxic levels as those presented by POM, could enhance the release of extracellular DNA 45 –an important biofilm matrix component – and bacteria could increase biofilm production as a defence response to oxidative stress. 35 Production of biofilm matrix is also connected to mechanisms of antimicrobial resistance. 46 Therefore, it is important that new antimicrobial materials can inhibit or at least not stimulate the production of biofilm matrix, as well as present an effective antimicrobial mechanism. In this case, our data demonstrate that the POMK 10 hybrid presents the advantages of both POM and polypeptide in their antimicrobial activities as well as an enhanced inhibiNanoscale Paper This journal is © The Royal Society of Chemistry 2022 Nanoscale, 2022, 14, 5999–6006 | 6003 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online Fig. 4 (A) Intracellular ROS produced by S. epidermidis in the presence of 125 µg mL −1 of POMK 10 (red), K 10 (blue), ionic K 10 /POM (yellow) and parent POM (green), measured by the fluorescence intensity of produced DCF at λ ex = 488 nm, λ em = 530 nm. The intensity values have been normalized taking as a value of 1 that of the positive control of S. epidermidis treated with 1.5 µL of HFIP. (B) Biofilm mass produced by S. epidermidis in the presence of 1/2× MIC of POMK 10 (red), K 10 (blue), ionic K 10 /POM (yellow) and parent POM (green) as measured by the absorption of crystal violet. The % values have been normalized, where 0% is the negative control (−), only NB, and 100% is normal S. epidermidis growth without any treatment. For both A and B, significance respect to POMK 10 was evaluated using ANOVA (p< 0.002 ***, p< 0.0002 ***, p< 0.0001 ****). (C) Verification of MBCs of K 10 /POM and POM on agar plate, with and without addition of 2.5 mM (aq.) ascorbic acid. Values on the left correspond to the concentration of K 10 /POM and on the right, to the concentration of parent POM. (D) Diameter size distribution of S. epidermidis cells without treatment (control) and after treatment with the different compounds, obtained by measurement of at least 100 cells from SEM images. (E) Scanning electron microscopy (SEM) images of S. epidermidis without treatment (control) and after incubation with the different compounds at their 1/2× MIC (scale bar = 500 nm) and MIC (scale bar = 1 µm). Yellow arrows indicate biofilm matrix, blue outlined arrows show cell deformation or damage. Paper Nanoscale 6004 |Nanoscale,2022,14, 5999–6006 This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online tory effect in the production of biofilm. We propose that the covalent POM–peptide hybrids and their self-assembly into spherical nanoparticles represent a promising approach towards the development of antibiofilm materials. Conclusions The specific local interactions between components in selfassembled structures expand the possibilities for a material to foster different properties, particularly when compared to the non-assembled individual components. In this study we investigated the role of the covalent connectivity in the aqueous self-assembly of 70 nm diameter POM–polypeptide POMlymer nanoparticles. Our results show that the organisation of the covalent hybrid POMK 10 into nanoparticles is driven by electrostatic and hydrophobic interactions resulting in the POM being buried in the hydrophobic core. This encapsulation not only preserves the peroxidase-like activity of the parent POM towards the oxidation of TMB, but also permits the oxidation of ABTS –with higher reduction potential. The covalent POM– polypeptide linkage present in the POMlymers is crucial for this improvement, as demonstrated with the negligible catalytic activity of the ionic hybrid K 10 /POM. Consequently, the assembly of the POMlymer hybrids provides a more robust assembly pathway to preserve the parent POM behaviour and simultaneously expands the substrate scope. Since living organisms are sensitive to oxidative stress, we evaluated the self-assembled POMlymer against the Grampositive skin bacterium S. epidermidis. We found that the free polypeptide K 10 , the POMK 10 - POMlymer - and the ionic K 10 / POM assemblies all possess antibacterial activity against S. epidermidis, whereas the parent POM performs poorly. It is likely that the lower antibacterial activity of the POM can be explained by the production of a non-lethal amount of ROS that acts to stimulate bacterial cell division in order to overcome the external threat. Crucially, while the free polypeptide K 10 and ionic K 10 /POM hybrid possessed lower corresponding MIC & MBC values, covalent POMK 10 POMlymer hybrid significantly reduced biofilm formation, making them appropriate candidates for broad-spectrum antibacterial materials due to the combination of mechanisms of action, which could also help to prevent an antibacterial resistance response. Author contributions The idea for the work was conceived by HSC, RMR and SGM. Materials preparation, self-assembly and catalysis studies were performed by HSC and supervised by JMF, SGM, and RMR. Microbiology experiments were carried out by EAB and supervised by SGM. The main hypothesis development and data analysis were undertaken by HSC, EAB, SGM, and RMR. The first draft of the manuscript was written by HSC and EAB. All authors reviewed and edited the final version of the manuscript and approved its submission. Conflicts of interest There are no conflicts to declare. Acknowledgements This work was funded through the grant PID2019109333RB-I00 funded by MCIN/AEI/10.13039/501100011033 (Ministerio de Ciencia e Innovación/Agencia Estatal de Investigación, Spain), through project LINKA20270 i-Link+ 2019 funded by CSIC and through Fondo Social de la DGA (grupo DGA E15_20R). HSC is grateful for a predoctoral fellowship FPU2016/02456 funded by Ministerio de Universidades (Spain). E. A. B. and S. G. M. acknowledge funding from the European Union’s Horizon 2020 research and innovation program (Marie Skłodowska-Curie grant agreement no. 845427). Authors acknowledge Servicio General de Apoyo a la Investigación-SAI (Universidad de Zaragoza), and the use of instrumentation as well as the technical advice provided by the National Facility ELECMI ICTS, node “Laboratorio de Microscopías Avanzadas”at University of Zaragoza. 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Paper Nanoscale 6006 |Nanoscale, 2022, 14, 5999–6006 This journal is © The Royal Society of Chemistry 2022 Open Access Article. Published on 23 March 2022. Downloaded on 10/6/2022 12:08:48 PM. This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence. View Article Online