Thermo-resistance of ESKAPE-panel Pathogens, Eradication and Growth Prevention of an Infectious Biofilm by Photothermal, Polydopamine-nanoparticles In Vitro

Nanotechnology offers many novel infection-control strategies that may help prevent and treat antimicrobial-resistant bacterial infections. Here, we synthesized polydopamine, photothermal-nanoparticles (PDA-NPs) without further surface-functionalization to evaluate their potential with respect to biofilm-control. Most ESKAPE-panel pathogens in suspension with photothermal-nanoparticles showed three- to four-log-unit reductions upon Near-Infra-Red (NIR)-irradiation, but for enterococci only less than two-log unit reduction was observed. Exposure of existing Staphylococcus aureus biofilms to photothermal-nanoparticles followed by NIR-irradiation did not significantly kill biofilm-inhabitants. This indicates that the biofilm mode of growth poses a barrier to penetration of photothermal-nanoparticles, yielding dissipation of heat to the biofilm-surrounding rather than in its interior. Staphylococcal biofilm-growth in the presence of photothermal-nanoparticles could be significantly prevented after NIR-irradiation because PDA-NPs were incorporated in the biofilm and heat dissipated inside it. Thus, unmodified photothermal nanoparticles have potential for prophylactic infection-control, but data also constitute a warning for possible development of thermo-resistance in infectious pathogens.

additional antimicrobial surface modifications to PDA-NPs can provide benefits on top of photothermal killing, they often come at the expense of increased cytotoxicity [39]. Moreover, surface modification can present a hurdle for clinical translation, making regulatory approval more difficult and endangering return of investment for interested market parties due to higher costs.
Considering that the development of photothermal nanoparticles for bacterial infection control has largely "skipped" in-depth evaluation of the merits of unmodified PDA-NPs, the aim of this chapter is to determine the potential of photothermal PDA-NPs without any surface modification with respect to bacterial infection-control. Two modes of clinical infection treatment will be studied using in vitro models: eradication of an existing infectious biofilm (the "therapeutic"-mode") or prevention of the development of an infectious biofilm (the "prophylactic"-mode"). First, we will describe the synthesis of photothermal PDA-NPs and measure their photothermal conversion efficiency, after which their killing efficacy towards planktonic ESKAPE-panel pathogens (in suspension) will be evaluated. ESKAPE is an acronym for the names of six pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter spp.) requiring focus in the development of new infection-control strategies because of their ability to escape killing of commonly used antibiotics [39][40][41][42]. Next, an existing S. aureus biofilm will be exposed to suspensions of PDA-NPs (therapeutic-mode) and upon NIR-irradiation biofilm viability will be assessed. Note that S. aureus is a prominent ESKAPE-panel member, causing a wide variety of human infections [43]. Finally, staphylococcal biofilm growth in the absence and presence of PDA-NPs and NIR-irradiation will be evaluated (prophylactic-mode), followed by assessment of biofilm viability.

Synthesis and characterization of polydopamine-NPs
The synthesis and characterization of polydopamine nanoparticles (PDA-NPs) can be found in the Supplementary Materials.

Photothermal properties of PDA-NPs
Different suspensions (250 μL) of PDA-NPs (0.05 to 1 mg/mL) in phosphate buffered saline (PBS, 5 mM K2HPO4, 5 mM KH2PO4, 150 mM NaCl, pH 7.0) were added in a 96 wells-plate and irradiated at 808 nm using a NIR-laser (Thorlabs, USA). After irradiation for different durations up to 20 min under gentle shaking, suspension temperatures were measured with a digital thermometer (MOSEKO, Gauteng, South Africa). Temperatures were measured at different laser power densities of 260, 520, 780, and 1300 mW/cm 2 , as established by optically defocusing the laser beam into a parallel beam with controllable diameter. Temperature measurements were done at pH 7.0 and 5.0 (pH adjusted with HCl) to mimic physiological pH conditions and pH conditions in a biofilm, respectively.
In addition, in order to measure heat losses in the system required for the calculation of the photothermal conversion efficiency, PDA-NPs suspensions were photo-activated at a laser power density of 1300 mW/cm 2 for 5 min after which photo-activation was arrested and temperature decreases due to heat loss to the environment monitored as a function of time. The photothermal conversion efficiency of PDA-NPs was calculated as described in previous studies using measured temperatures [32,44]. Neglecting heat uptake by the PDA-NPs, the temperature change of the system upon NIR-irradiation equals where m H 2 O and C P,H 2 O are the mass and specific heat of water, respectively. T is the suspension temperature. Q s is the heat uptake per unit time associated with the light absorbed by the suspension fluid, Q NPs is the photothermal heat generated by the PDA-NPs per unit time and Q loss represents the heat loss of the system per unit time. Q NPs can be derived from the NIR absorption spectrum according to where I is the laser power, A 808 nm is the absorbance of PDA-NPs at the wavelength of 808 nm and η is the photothermal conversion efficiency. The heat loss can be expressed as where h is the heat transfer coefficient, A is the surface area of the system exposed to its surrounding and ΔT is the temperature difference between the system and its surrounding, i.e. T -Tenv , in which T and Tenv are the suspension and surrounding temperatures, respectively. At equilibrium, in absence of photothermal PDA-NPs but upon NIR-irradiation, combination of Eqs.

Bacterial culturing and harvesting
ESKAPE-panel pathogens [41], including S. aureus ATCC 12600 were stored in 7% (v/v) DMSO at −80 °C. Of each panel strain, a single colony from a blood agar plate was inoculated in 10 mL of Tryptone Soya Broth (TSB) and incubated aerobically at 37 °C for 24 h. Bacterial suspension were then transferred into fresh 200 mL of TSB and incubated for 17 h. Bacteria were harvested by centrifugation at 5000 g for 5 min at 10 °C and washed twice with PBS. Bacteria were resuspended in 10 mL PBS and sonicated for 3 x 10 s at 30 W (Vibra Cell model 375, Sonics and Materials Inc., USA) while cooling in an ice/water bath to break possible aggregates. Final concentrations of bacterial suspensions were determined using a Bürker-Türk counting chamber.

Photothermal killing of planktonic ESKAPE-panel pathogens by PDA-NPs
To determine the killing efficiency of PDA-NPs on planktonic ESKAPE-panel pathogens, 2.5 μL of a bacterial suspension in PBS (3 × 10 8 bacteria/mL) was diluted with a PDA-NPs suspension (0.5 mg/mL PDA-NPs) in 96 well-plates (Greiner Bio-One, Austria) to make ratios of PDA-NPs to bacteria of 4.2 × 10 5 or 2.1× 10 5 nanoparticles per bacterium (for details, see Supplementary Materials). The total volume in the wells was 250 μL. Next, the mixed suspensions were irradiated for 10 min with a NIR-laser at a power density of 1300 mW/cm 2 . After irradiation, bacterial suspensions were serially diluted and plated on TSB agar plates. After overnight incubation at 37 °C, the number of colony-forming units (CFU) were counted. All experiments were carried out with bacteria grown from three separate bacterial cultures.

Photothermal killing of existing S. aureus biofilms by PDA-NPs
Therapeutic use of photothermal nanoparticles implies the eradication of an existing infectious biofilm. To this end, a staphylococcal biofilm was grown on a glass surface (0.4 cm × 0.4 cm × 0.1 cm), cut from a microscope slide (ThermoFisher, Germany). Before biofilm growth, glass surfaces were cleaned with a piranha solution (3:10:3, v:v:v, NH4OH: ultrapure water: H2O2) rinsed with copious amounts of water, followed by rinsing twice with absolute ethanol. Cleaned glass samples were stored in ethanol and dried with filtered nitrogen immediately before use. Cleaned and dried glass samples were placed in a 96 well-plate and 100 μL of a S. aureus ATCC 12600 suspension (1 × 10 9 bacteria/mL) in PBS was added to the wells and left to sediment for 1 h at 37 °C to allow bacteria to adhere. Next, the suspensions were removed, and the wells were washed once with 100 μL of PBS. Subsequently, 200 μL of TSB was added and staphylococci were grown at 37 °C for 48 h and after 24 h the TSB was refreshed. After 48 h, the biofilms were washed once with 100 μL PBS, and 200 μL PDA-NPs (0.5 mg/mL) in PBS were added at 37 °C for 20 min and irradiated for 10 min (808 nm, 1300 mW/cm 2 ). Importantly, TSB is a protein-rich nutrient source for bacteria to grow in, possibly affecting the stability of our PDA-NPs. The observation of heat generation under these conditions negates this assumption.
For confocal laser scanning microscopy (CLSM), the biofilm was stained with LIVE/DEAD BacLight (3 μL STYO9 and 3 μL propidium iodine in 1 mL demineralized water) at room temperature for 30 min in the dark. After staining, the samples were transferred from 96 well-to 12 well-plates and PBS was added for observation by CLSM. CLSM images were taken using a Leica microscope (LEICA TCS SP2 Leica, Wetzlar, Germany) and 3D reconstructions were created using IMAGE J software (version 1.50b).
A series of similar experiments were carried out, but instead of evaluating the percentage of live/dead staphylococci using staining, biofilm was removed from the glass surfaces by pipetting and sonication for 30 s on ice (30 W) and bacteria suspended in 2.5 mL of PBS. Staphylococcal suspensions were serially diluted and plated on TSB agar plates. After overnight incubation at 37 °C, the numbers of CFU were counted. All experiments were carried out with biofilms grown from three separate bacterial cultures.

Photothermal prevention of S. aureus biofilm formation by PDA-NPs
Prophylactic use of photothermal nanoparticles implies the prevention of infectious biofilm formation, as currently achieved e.g. by post-operative administration of antibiotics to prevent the growth of per-operatively introduced bacteria into an infectious biofilm. In analogy with this prophylactic use of antibiotics, staphylococcal biofilms were grown as described above, but in the presence of PDA-NPs. PDA-NPs (0.5 mg/mL) were suspended in the growth medium for the first 24 h or entire growth period of 48 h. After 24 h, the growth medium was refreshed (with or without PDA-NPs), and the biofilm was grown for another 24 h. Control 48 h staphylococcal biofilms were grown in absence of PDA-NPs. All biofilms were irradiated by a NIR-laser (10 min, 808 nm, 1300 mW/cm 2 ). All experiments were carried out with biofilms grown from three separate bacterial cultures.

Tissue cell compatibility
Tissue cell compatibility was evaluated towards L929 fibroblasts using the Cell Counting Kit-8 (CCK-8) assay. Fibroblasts were obtained from the American Type Culture Collection (ATCC-CRL-2014, Manassas, USA) and grown in 75 cm 2 tissue culture polystyrene flasks in RPMI-1640 medium (ThermoFisher Scientific, Inc., Carlsbad, CA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco), 100 U/mL penicillin (Genview) and 100 μg/mL streptomycin (Solarbio) at 37 °C in 5% CO2. Culture media were changed every two days. Cells were grown to 70-80% confluence and detached from the cell-culture flask by trypsinization, collected by centrifugation at 1200 rpm for 5 min and re-suspended in fresh medium to a concentration of 4×10 4 cells/mL, as determined using a Bürker-Türk counting chamber. Then 200 μL cell suspension was placed in 96 well-plates and left to incubate in a humidified 5% CO2, atmosphere at 37 °C for 12 h, after which growth medium was replaced by 200 μL fresh RPMI-1640 medium, supplemented with different concentrations of PDA-NPs, yielding nanoparticle concentrations up to 1 mg/m. Importantly, suspension of PDA-NPs in this nutrient-rich source supplemented with serum proteins did not affect heat generation or cause visual disassembly of the nanoparticles. After another 24 h of incubation, the medium was removed, and cells washed with PBS for 3 times and CCK-8 solution (20 μL) diluted 1:10 with FBS-free RPMI-1640 (200 μL) was added to each well at 37 °C for 1.5 h. Absorbance at 450 nm was subsequently measured using a microplate reader (Thermo, Varioskan Flash) and tissue cell compatibility was expressed as where A experiment and A control are the absorbances of a cell suspension with and without being exposed to PDA-NPs, respectively and A blank is the absorbance of a solution containing FBSfree RPMI-1640 medium and CCK-8 solution, is the mean absorbance of control which contain cells. Each concentration of nanoparticles was evaluated in six-fold using cells from one culture.

Statistical analysis
One-way ANOVA statistical analyses were performed using the Bonferroni multiple comparison correction (GraphPad Prism v. 8.1.1) with statistical significance accepted at p < 0.05 for comparing different groups with respect to planktonic bacterial killing and biofilm thickness. Bacterial killing data were log-transformed before performing the ANOVA analyses. Biofilm killing in different groups was compared using a one-way ANOVA with Bonferroni multiple comparison correction, selected for all comparisons changing a single variable (15 comparisons).

PDA-NP characterization and photo-thermal conversion efficiency
The PDA-NPs synthesized had an average hydrodynamic diameter of 85 nm ( Figure 1A) and were fully tissue cell compatible ( Figure S1). UV-vis absorption spectroscopy indicated strong absorption of NIR wavelengths by PDA-NPs as compared with dopamine ( Figure 1B), confirming their photothermal potential. The A808 nm required in Eq. 6 to calculate the photothermal conversion efficiency was taken from Figure1B to equal 0.66.  presents the necessary temperature data to calculate the photothermal conversion efficacy of the PDA-NPs synthesized. Higher concentrations of PDA-NPs yielded higher system temperatures, reaching equilibrium within 10 min (Figure 2A). For a PDA-NP concentration of 0.5 mg/mL and an NIR-irradiation of 0.5 W, it can be read that ΔT max, suspension as occurring in Eq. 6. amounts 28.5 °C. System temperatures also increased with increasing irradiation power (Figure 2B), while the suspension pH (7.0 under physiological conditions [40] and around 5.0 in a biofilm [45]) had no influence upon the photothermal efficiency of the nanoparticles ( Figure  2C). Note that also NIR-irradiation of the system in absence on PDA-NPs yielded a minor increase in temperature (see also Figure 2A), reaching a maximum equilibrium temperature ΔT max, H 2 O as occurring in Eq. 6. after NIR-irradiation at 0.5 W, that amounted 4.9 °C. Heat losses of the system were evaluated by switching the NIR-laser on and off, while monitoring temperature increases and decreases, respectively ( Figure 2D). Presentation of the logarithm of temperature decrease as a function of time yielded a linear relation (Figure S2), according to Eq. 8. Using the mass (0.25 × 10 -3 kg) and the specific heat (4.2 × 10 3 J kg -1 . °C -1 ) of water, hA follows from the slope and Eq. 8 (0.0034 J/(s cm 2 )) and the photothermal conversion efficiency η of our PDA-NPs synthesized can be calculated to be 21 %. This is lower than the conversion efficiency of core-shell nano-plates of Pd and Au (29%) [46], but similar to gold nano-rods (22%) and higher than of gold nano-shells (13%) [47].

Killing of planktonic ESKAPE-panel pathogens by photo-activated polydopamine nanoparticles
Bacterial killing efficacy of PDA-NPs was evaluated against ESKAPE-panel pathogens in suspension (3 x 10 6 CFU/mL), including E. faecium W54, S. aureus ATCC 12600, K. pneumonia-1, A. baumanni-1, P. aeruginosa PA01, and Enterobacter cloacae BS 1037 and additionally Enterococcus faecalis 1396 (NIR-irradiation at 808 nm and 1300 mW/cm 2 for 10 min). NIRirradiation at a PDA-NP concentration of 0.5 mg/mL in the volume employed (250 μL) yielded a temperature increase to 50.1 °C (Figure 2A). Neither the presence of PDA-NPs in absence of NIR-irradiation nor NIR-irradiation in absence of PDA-NPs yielded killing of ESKAPE member pathogens in relevant numbers (< 0.2 log-unit reductions). However, NIR-irradiation of suspensions with PDA-NPs and ESKAPE-panel members caused three to five log-unit reductions, with the exception of enterococcal spp. (Figure 3A). For E. faecium and E. faecalis species, photothermal killing was limited to maximally two-log unit reductions. Photothermal killing was significantly less at a lower nanoparticle to bacteria ratio, as illustrated in Figure 3B for S. aureus ATCC 12600.

Photothermal effects on existing staphylococcal biofilms exposed to PDA-NPs in suspension
Eradication of an existing 48 h S. aureus ATCC 12600 biofilm on glass surfaces was evaluated by exposing biofilms to a suspension of PDA-NPs (0.5 mg/mL in a volume of 200 μL) with and without 10 min NIR-irradiation at 808 nm (1300 mW/cm 2 ). Growth of biofilms on a glass sample added a second heat-absorbing component, i.e. the glass sample, to the system, but its heat capacity (0.04 J/°C) is negligible compared to the heat capacity of the water (1.05 J/°C) and it can be assumed that the same maximal temperature can be reached as in absence of the glass sample (50.1 °C; see Figure 2A). LIVE/DEAD staining of the biofilms grown followed by CLSM imaging (Figure 4), showed that the biofilms had an average thickness of 36 ± 7 μm, corresponding well with clinical thicknesses of biofilm infections [24]. S. aureus biofilm thickness was neither affected by NIR-irradiation in the absence of PDA-NPs nor in presence of PDA-NPs in suspension above the biofilm. Regardless of the absence or presence of PDA-NPs or their NIR-irradiation, staphylococcal biofilms were predominantly green-fluorescent, indicative of live bacteria, with very little dead, red-fluorescent bacteria (see also Figure 4). Note that although live/dead staining is generally applied to demonstrate bacterial cell death, it technically only implies cell wall damage [48]. Therefore, conclusions derived from live/dead staining were verified by CFU enumeration. Removal of biofilms and subsequent CFU enumeration, only indicated slightly lower numbers of CFUs in presence of PDA-NPs before and after NIR-irradiation (Figure 5). This supports our conclusions drawn from live/dead staining. Figure 4) and during growth in the presence of PDA-NPs during the initial 24 h of growth or during the entire 48 h period of growth (Figures 6 and S3). NIR-irradiation was always done after 48 h of growth. Error bars represent standard deviations over triplicate experiments with different bacterial cultures. Significance was tested using a one-way ANOVA test with Bonferroni correction, * p < 0.05, ** p < 0.01.

Photothermal effects on staphylococcal biofilm formation grown in the presence of PDA-NPs
Prophylactic use of antimicrobials implies preventing growth of a bacterial biofilm while being exposed to antimicrobials. Therefore, in order to mimic prophylactic conditions, staphylococcal biofilms were grown in the presence of PDA-NPs in the growth medium during the initial 24 h of growth (Figure 6) or during the entire 48 h period of growth ( Figure S3), with or without NIRirradiation. NIR-irradiation in absence of PDA-NPs during growth did not affect the number of green-fluorescently stained staphylococci (Figures 6C and D and Figures S3C and D). Growth in the presence of PDA-NPs yielded red-fluorescent staphylococci also in absence of NIRirradiation (Figures 6B and S3B). Verification of staphylococcal killing by PDA-NPs in absence of NIR-irradiation using agar-plating did not yield any reduction in CFUs (Figure 5), supporting again the conclusions from live/dead staining and indicative of bacterial survival despite the cell wall damage done by PDA-NPs. NIR-irradiation and subsequent heat dissipation by PDA-NPs incorporated during growth in the biofilm yielded red-fluorescence (i.e. membrane damage) in nearly all staphylococci (Figures 6A and S3A), accompanied by a reduced number of CFUs ( Figure 5). CFU reduction was larger when biofilms were grown in the presence of PDA-NPs during the entire 48 h of growth than when solely present during the initial 24 h of growth.

Discussion
PDA-NPs were prepared with a photothermal conversion efficiency of 21% without further surface modification. Various types of surfaces modification have been applied to PDA-NPs (see also the Background section to this chapter), to allow blood circulation, targeting biofilm penetration and enhance bacterial killing [49,50]. Surface modifications can be applied to PDA-NPs through Michael addition or Schiff base reactions [35,51] but frequently yields loss of biocompatibility [30,52] therewith making clinical translation more difficult than with unmodified PDA-NPs possessing proven biocompatibility (Figure S1) [53]. Unfortunately, nanoparticle to bacteria ratios, but also suspension volumes and laser power densities vary across the literature, which makes comparison of our results with other studies difficult. Within the limitations of current literature description in which these essential features for adequate comparison with other studies are often missing, our unmodified PDA-NPs probably have a higher photothermal conversion efficacy and better cell tissue compatibility than surface modified PDA-NPs. In addition, unmodified PDA-NPs can be bio-degraded to pyrrole-2, 3-dicarboxylic acid, and pyrrole-2,3-dicarboxylic acid by hydrogen peroxide as widely distributed in phagocytes and various organs [32,36].
Most of our evaluation experiments were done in volumes between 200 -250 μL at a PDA-NP concentration of 0.5 mg/mL and 808 nm NIR-irradiation (1300 mW/cm 2 ) for 10 min, yielding a temperature increase to approximately 50 °C (Figure 2A). This is at the higher end of the therapeutic temperature range that does not produce collateral tissue damage [15]. For E. faecium and E. faecalis species, photothermal killing under these conditions was limited to maximally two-log unit reductions ( Figure 3A). Since two-log unit reductions are microbiologically and clinically meaningless, these strains may be classified as thermo-resistant human pathogens. This points to the potential danger of thermo-resistance in infectious pathogens if photothermal treatment of infections becomes large-scale used in the clinic. After all, thermo-resistant bacteria exist in natural environments [54] and industrial applications [55]. Horizontal gene-transfer [56] in infectious biofilms between more and less thermo-resistant inhabitants can easily convey thermo-resistance to an entire population, as common in the spreading of antibiotic resistance [57]. For other ESKAPE panel pathogens, three to five log-unit reductions were observed that may seem large, but photothermal killing of planktonic bacteria must always be judged in relation with the ratio at which photothermal nanoparticles and target bacteria are suspended, as illustrated for photothermal S. aureus killing ( Figure 3B). Overall however, other photothermal nanoparticles described in the literature showed less than two log-unit reduction in CFU upon NIR-irradiation [38]. This suggests, that PDA-NPs in absence of surface-modification are highly effective in photothermal killing of a wide variety of bacterial strains and species. This chapter shows that there is no therapeutic effect to be expected from photothermal treatment with PDA-NPs when applied to an existing biofilm, neither based on live-dead staining (Figure 4) nor on the basis of CFU enumeration (Figure 5). Conclusions on bacterial killing from live/dead were supported here by CFU enumeration which is important, because technically, livedead staining only implies cell wall damage [48] that can sometimes be self-repaired without impeding bacterial growth and colony formation on agar plates [58,59], which still is the gold standard for bacterial death in clinical microbiology [60,61]. Absence of therapeutic effects can be explained by lack of penetration of unmodified photothermal nanoparticles in the biofilms, causing heat dissipation in the aqueous surrounding of the biofilm rather than inside it.
Opposite to therapeutic benefits, prophylactic benefits of unmodified PDA-NP were demonstrated in our study (Figures 5, 6 and S3). Prophylactic benefits imply that photothermal treatment commences before or during the onset of biofilm growth, similar to the prophylactic use of antibiotics. PDA-NPS incorporated in a biofilm during its growth demonstrated minor bacterial killing ability even in absence of NIR-irradiation. This is in line with other studies, showing minor killing of bacteria adhering on polydopamine layers adsorbed to different substrata [62,63]. Antibacterial efficacy of PDA-NPs in absence of NIR-irradiation was not observed in planktonic evaluation (Figure 3) and existing biofilm eradication (Figure 4), probably because intimate contact between polydopamine and bacterial cell surfaces is needed that only occurs during growth of bacteria in presence of PDA-NPs. Bacterial growth in the presence of PDA-NPs was much more strongly reduced upon NIR-irradiation than in its absence. This suggests potential of unmodified PDA-NPs for infection prophylaxis, as after invasive surgery or trauma.
Exposure to PDA-NPs and subsequent NIR-irradiation of ESKAPE-panel pathogens demonstrated that particularly enterococci were more heat-resistant than other members of the ESKAPE-panel, most notably S. aureus. This constitutes a warning that development of thermo-resistance in human infectious pathogens may not a priori be excluded and warrants more research in the development of thermo-resistance by human pathogens if photothermal infectioncontrol is going to be large-scale clinically applied.
PDA-NPs in suspension above an existing biofilm did not cause significant killing of bacteria in the biofilm. This implies that clinically, photothermal nanoparticles without surface modification to enhance biofilm penetration have no therapeutic potential. This is different for their prophylactic potential: biofilm growth in the presence of photothermal nanoparticles and after NIR-irradiation, killed significant numbers of bacteria during biofilm formation. Currently, antibiotics are applied prophylactically to prevent infectious biofilm formation in the immediate period after invasive surgery or trauma. This type of prophylactic antibiotic administration is either orally or by local administration at a surgical-site, from which the antibiotics gradual diffuse away to become cleared from the body, enabling clinically-desired, short term antibiotic protection and infection prevention. Usually, broad spectrum antibiotics are given for these purposes which can cause collateral damage to the healthy microflora in the human body. Local administration at the surgical-site of highly biocompatible, unmodified photothermal nanoparticles and their temporary presence due to clearance from the body, would also be ideal to prevent surgical-site infection in the immediate period post-surgery. Photodynamic therapy avoids collateral damage to the healthy microflora as NIR-irradiation can be confined to the infection site.
Herewith, we have cleared a pathway for the clinical translation of unmodified photothermal PDA-NPs, identifying limitations and opportunities.

Ratio of PDA-NPs to bacteria in suspensions with known mass concentrations of PDA-NPs
The ratio of PDA-NPs to bacteria is important as the heat generated upon NIR-irradiation of PDA-NPs dissipates over the bacteria present in their immediate surrounding. Bacterial concentrations in suspension in terms number of bacteria per mL (bacteria) are relatively easily measured, but owing to their small size, number concentrations cannot be directly measured for PDA-NPs. Nanoparticle concentrations in suspension only can be directly measured in terms of mass per mL. For a 1 mL suspension of nanoparticles, the number of the nanoparticles NPDA-NP equals: where mPDA-NP total is the measured, total mass of PDA-NPs per mL suspension volume and mPDA-NP is the mass of a single PDA nanoparticle. The mass of a single PDA nanoparticle mPDA-NP follows from the mass of a dopamine molecule mdopamine, i.e. its molar mass (153.178 g/mol) divided by Avogadro's number (6.02×10 23 ) and multiplied by Ndopamine molecules, the number of dopamine molecules in a nanoparticle The number of dopamine molecules in a nanoparticle Ndopamine molecules follows from the nanoparticle volume VPDA-NP (3.22 × 10 -16 cm 3 as calculated from its hydrodynamic diameter, i.e. 85 nm) and the volume of a dopamine molecule, i.e. its molar volume ( molar volume of dopamine hydrochloride, a substitute, 122.7 cm 3 /mol) divided by Avogrado's number. Subsequently, all unknowns in Eq. (1) are known and NPDA-NP can be directly calculated. Accordingly, for nanoparticle concentrations of 0.5 mg/mL and 0.25 mg/mL in suspension, this yields a number concentration of PDA-NPs equal to 1.25 × 10 12 and 6.25 × 10 11 nanoparticles/mL, respectively. Therewith upon addition of different volumes of nanoparticle suspensions and a bacterial suspension, these calculations have been employed to yield a nanoparticle to bacteria ratio of 4.2 × 10 5 and 2.1 × 10 5 nanoparticles per bacterium for nanoparticle in suspensions with nanoparticle concentrations of 0.5 mg/mL and 0.25 mg/mL, respectively. Figure S1. Viability of L929 cell exposed to different concentrations of PDA-NPs for 24 h as a measure for the tissue cell compatibility of PDA-NPs. Viability was measured using the CCK-8 assay kit and viability of a cell suspension not exposed to PDA-NPs was set at 100%. Error bars represent standard deviations over triplicate measurements.