Antibacterial Plasma Coating with Aggregation-Induced Emission Photosensitizers to Prevent Surgical Site Infections

Surgical site infections (SSI) are major post-operative complications following surgery. Reducing SSI is a global urgency as they account for huge pecuniary, physiological, and emotional burdens for patients. Antibiotic resistance is the main challenge for surgeons dealing with SSIs. Aggregation-induced emission photosensitisers (AIE PS) with their distinct optical characteristics, biocompatibility, low toxicity, and target speciﬁcity, hold the potential for the treatment of SSI. Herein, a synergetic strategy combining plasma polymerization and AIE PS is adopted to develop coatings that can eradicate SSI-causing bacteria. These coatings can preferentially kill Gram-negative and Gram-positive bacteria over mammalian cells after triggered by light irradiation. The cell viability and immunostaining studies conﬁrmed their biocompatibility on mammalian cells. The antibacterial mechanistic studies explored their ability to generate reactive oxygen species (ROS), which is crucial for overcoming antibiotic resistance. Thus, this study opens an avenue toward antibacterial coatings to decrease the prevalence of SSI.


Introduction
[3][4] They are mainly caused by bacteria that enter the surgical incisions via contaminated surgical instruments or infected skin and may lead to morbidity when left untreated. [5,6]According to the World Health Organization DOI: 10.1002/admi.202400053(WHO), the incidence of SSI accounts for 11% in low and middle-income countries. [7,8]Around 20% of women undergoing caesarean in Africa suffer from SSI which seriously impacts their health.SSI contributes to 3.5-10 billion in the United States that includes extra stays at hospitals. [9]Antibiotics are mostly used to treat patients with SSIs. [10,11]Even though antibiotics can mitigate SSIs for a short period, the altered metabolism and genetics of bacteria lead to loss of therapeutic efficiency of these drugs after a prolonged period. [12]Such treatment exacerbates the danger of the rise of multidrug resistance and endangers the physiology of gut microbes. [13,14]For instance, the methicillin-resistant S.aureus and Ceftriaxone-resistant E. coli bacteria are responsible for a variety of infectious illnesses. [15]Scientists have proposed several strategies in response to this serious predicament.Nanoparticle-based strategies have gained lots of attention due to their physiochemical properties, synthesis routes, mechanism of antibacterial action etc. [16,17] However, as different strains from the same species of bacteria have variable levels of antibiotic resistance, antibacterial activities and cytotoxicity, a better solution was needed to tackle this challenge. [18][21] Their rational synthesis route, photothermal ability and photosensitization characteristics are explored widely in the biomedical field. [22,23]The unique characteristic of these materials is that they are nonemissive in their molecular state but fluoresce when they aggregate. [24]Traditional photosensitizer (PS) can do selfquenching in their aggregated state by non-radioactive decay, which causes big problems when they are used in the solid state.Whereas AIE PS exhibits less nonradiative decay and higher photosensitization and emission at solid state, which provides a potential solution for SSIs. [25,26]CN-TPAQ-PF6 (cyano-2,2,6,6-tetramethylpiperidine −1-oxyl-hexafluorophosphate) is the aggregation-induced emission photosensitizer (AIE PS) employed in this study as these cationic derivatives can eradicate drug-resistant bacteria due to their ability to generate reactive oxygen species (ROS). [27]Triphenylamine is the electron donor in this molecule.Two electron-withdrawing groups namely, a cyano group and quinolinium hexafluorophosphate are conjugated to triphenylamine, thereby adopting an acceptor-donor-acceptor (A-D-A) configuration.This asymmetric configuration will enhance the light-harvesting and ROS generation ability of this molecule. [27]The main advantage of such ROS-generating materials over antibiotics is that they can overcome multi-drug resistance, as ROS can damage various cellular components including lipids, proteins and nucleic acids, whereas antibiotics target particular metabolic pathway. [28,29]Incorporation of these molecules in fibers or coatings can further enhance their antibacterial properties.32][33] The combination of nanoparticle-based strategies with antibacterial coatings is a promising approach to prevent biofilm formation on surgical devices and thereby prevent SSIs.[38] Other unique characteristics include an environmentally friendly nature, with no organic waste and minimum use of precursors.The substrates were plasma coated with polyoxazoline (POX) due to its stability, low fouling properties, biocompatibility and ability to bind antibodies, proteins, nanoparticles etc. [39,40] The plasmacoated surfaces were then immobilized with AIE PS to generate photodynamic antibacterial coatings.
Thus, this study aims to design and fabricate smart photodynamic coatings that generate ROS when exposed to light and exhibit antibacterial properties.To achieve this, we prepared substrates plasma coated with polyoxazoline (POX).AIE PS, for the first time, was then immobilized onto these plasma-coated surfaces.The physicochemical characterization of these functionalized surfaces was studied in detail.The antibacterial properties together with a mechanistic understanding of the mode of killing are investigated in depth.The cytocompatibility and immunofluorescence images confirmed the low toxicity of these surfaces.To the best of our knowledge, there are no reports on plasma-functionalized surfaces immobilized with AIE PS for treating SSIs.

Results and Discussion
Plasma-functionalized substrates immobilized with AIE PS were designed and developed using a combination of plasma polymerization and phototheranostics (Scheme 1).To accomplish the proposed strategy, the substrates were plasma coated with a nano-thin layer of polyoxazoline (POX).The thickness of the coating was measured using an ellipsometer, yielding a result of 23.7 ± 1.5 nm.POX coatings retain many oxazoline rings on their surface that aid in the covalent binding of nanoparticles and biomolecules. [34]The coated samples were then immersed in AIE PS solution at two different concentrations (0.46 and 0.33 mg mL −1 ) for 24 h.After AIE PS immobilization, the surfaces acquired a yellowish color.We named AIEa for 0.46 mg mL −1 and AIEb for 0.33 mg mL −1 .The binding of AIE PS on plasma-coated surfaces was first confirmed using UVvis absorption spectroscopy.AIE PS exhibited a characteristic absorption peak ≈457 nm (Figure 1B).Interestingly, plasma-coated quartz slides functionalized with AIE PS showed an absorption peak ≈459 nm, confirming their successful immobilization.The presence of AIE PS on plasma-functionalized surfaces was further established by measuring fluorescence intensity.Figure 1D showed that untreated and POX-coated surfaces showed no fluorescence, whereas both POX-AIEa and POX-AIEb showed good binding of AIE PS on POX-coated surfaces.As reported previously, the emission peak of AIE PS (CN-TPAQ-PF6) is at 592 nm and has been attributed to this fluorescence.The fluorescence intensity of POX-AIEa was higher than that of POX-AIEb as confirmed in Figure 1D.
Fourier transform infrared spectroscopy (FTIR) was used to detect the chemical functionalities present in AIE PS, POX and POX-AIE.A typical infrared (IR) spectrum of POX plasma coating shows many functional groups including C═N (1627 cm −1 ) and C─O (1008 cm −1 ), attributed to the oxazoline ring (Figure 1C).The band at 2354 cm −1 indicates the presence of isocyanate nitrile or alkyne group as reported previously. [41]The unique chemical functionalities of AIE PS include nitrile groups at 2351 cm −1 , N─H bending at 1584 cm −1 and aromatic rings at 1485 cm −1 .The peak at 840 cm −1 in the fingerprint region is attributed to PF 6 groups.The presence of all major vibrational features associated with AIE PS can be seen in the spectrum of POX-AIE that confirms the successful immobilization of AIE PS on plasma coated surfaces.The shift of the FTIR peak of POX from 1600 to 1584 cm −1 in the case of POX-AIE suggests a possible interaction of intact oxazoline functionalities in POX with nucleophilic sites of AIE PS.
Surface wettability is an important parameter that controls cell adhesion and affects cell behavior.Wettability is affected by several factors including surface chemistry and topographical features.The static water contact of POX is ≈51.00 ± 0.04 (Figure 1A).After AIE PS immobilization, both POX-AIEa and POX-AIEb showed increased water contact angle due to the hydrophobic groups present in AIE PS.Literature has shown that cells prefer moderately hydrophilic surfaces for cell attachment between 40˚and 70˚. [42]The WCA of POX-AIEa and POX-AIEb are in this range and may be suitable for cell adhesion.The ability of functionalized surfaces to preferentially kill bacterial cells over mammalian cells was investigated under dark conditions and after exposure to light for 30 min and 1 h.The representative Gram-positive and Gram-negative bacteria used in the study were Staphylococcus aureus and Pseudomonas aeruginosa respectively, as they were the principal causative agents of SSI.In vitro antibacterial assays showed that both S. aureus and P. aeruginosa showed good viability on untreated surfaces.The percentage of dead bacteria following light irradiation in the case of POX-AIEa and POX-AIEb is greater than in dark conditions, demonstrated by the discernible rise in dead cells.Again, the bactericidal effect of POX-AIEa and POX-AIEb after 1 h of light irradiation is >30 min of exposure.The percentage of dead S aureus treated with POX-AIEa and POX-AIEb after 1 h of light irradiation was 93 ± 2.48% and 73 ± 11%, respectively (Figure 2).After 1 h of light irradiation, the percentage killing of P. aeruginosa on POX-AIEa and POX-AIEb was 99 ± 0.39% and 99 ± 0.78%, respectively (Figure 3).Previous literature proves that cationic AIE PS had a good binding affinity to Gram-negative bacteria with thinner peptidoglycan compared to Gram-positive bacteria with thicker peptidoglycan. [27]Among the various samples, POX-AIEa and POX-AIEb showed higher bactericidal activity compared to POX alone.The results of the colony enumeration assay further confirmed that POX-AIEa exhibited higher antibacterial capacity (Figure S1, Supporting Information).
Next, we examined the cytotoxicity of these materials on mammalian cells via 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay, where viable cells convert tetrazolium dye to insoluble purple-colored formazan (Figure 4B).Immotalized human keratinocytes (Hacat) cells were chosen as the model cell line for this assay, as they play a major role in wound healing and re-epithelialization. [43] Both POX and POX-AIEb showed negligible cytotoxicities under dark and light conditions.However, POX-AIEa showed a significant reduction in cell viability due to higher concentrations of surface-bound AIE PS that might have generated an enormous amount of ROS.The cell viability of POX-AIEb was 73 ± 12% and 96 ± 15% under light and dark conditions.We further investigated the morphology of Hacat cells treated with the different plasma functionalized surfaces using an immunostaining technique (Figure 4A).In this assay, Phalloidin is the fluorescent dye that stains the actin filaments of live cells and DAPI is a nuclear staining dye.Hacat cells showed their normal flat and healthy morphology for untreated, POX and POX-AIEb.However, very few cell densities were observed in the case of POX-AIEa, which confirms the toxicity of this surface.Abiding by ISO 10993-5 standards, we chose untreated, POX-AIEb and POX for the remaining studies.
The antibacterial mechanisms of untreated, POX and POX-AIEb were investigated using ROS assay and representative scanning electron microscopy (SEM) images.One of the key drivers of antibacterial activity is the production of ROS, which supports one of our theories about the AIE PS's ability to combat Grampositive S. aureus and Gram-negative P. aeruginosa.Alpha-oxygen (-O), singlet oxygen ( 1 O 2 ), hydroxyl radicals (˙OH), superoxide anions (O 2 −), hydrogen peroxide (H 2 O 2 ), and other highly reactive molecules and free radicals produced from molecular oxygen are together referred to as ROS. [44]From previous studies, CN-TPAQ-PF6 was able to generate more ROS than its neutral counterparts in a short period. [27]This suggests that cationization is a progressive strategy to increase the efficiency of ROS generation.The insertion of the cyano group in CN-TPAQ-PF6 to enable this kind of A-D-A configuration is also advantageous for enhancing ROS production. [27]Oxidative stress in bacterial cells was identified and quantified using a fluorescent dichlorofluorescein (DCF) dye.Greater amounts of intracellular ROS are indicated by green fluorescence signaling intensity.In this investigation, bacterial strains (S. aureus and P. aeruginosa) treated with POX-AIEb showed higher levels of intracellular ROS than POX (Figure 5A,B).The fluorescence intensity profile showed that POX-AIEb generated more ROS in P. aeruginosa compared to S. aureus.This was in agreement with the antibacterial results discussed in Figure 3.
Scanning electron microscopy (SEM) was utilized to see the morphological alterations of the S. aureus and P. aeruginosa bacteria after exposure to untreated, POX and POX-AIEb.Under SEM, the bacteria had their normal, healthy cell morphologies; rod-shaped for P. aeruginosa and spherical for S. aureus for untreated samples.The bacterial cells, however, were distorted and wrinkled when treated with POX-AIEb (Figure 5C).The integrity of the cell was compromised by damage to the cell membrane.POX-AIEb had extremely strong antibacterial effects on both Gram-positive and Gram-negative bacteria with ruptured and swollen cell membranes, according to SEM.Furthermore, P. aeruginosa cells were more disrupted than S. aureus, in agreement with previous observations.The cell permeability experiment was performed to demonstrate that POX-AIE also caused cellular architecture destruction and the extrusion of all intramolecular components into the culture fluid compared to POX (Figure S2, Supporting Information).
These coatings can have numerous practical applications in clinical settings.Apart from preventing SSI, these coatings can be applied to surgical instruments to improve their longevity and performance.Reusable surgical instruments with antibacterial coatings can prevent cross-contamination.Such coatings can prevent biofilm evasion on stents and catheters, reducing bacterial infections.Antibacterial coatings on wound dressings can reduce the requirement for recurrent dressing changes.These coatings can improve optical clarity and reduce fogging in surgical cameras and endoscopes.

Conclusion
Bacterial infections are major threats to human health because of the number of antibiotic-resistant bacteria causing significant mortality and morbidity in the general population.SSI is one of the major bacterial infections that appear at the site of surgery.All the current treatment stratagems for SSI, however, are seriously threatened by antibiotic-resistant microorganisms.In this work, we could successfully develop photodynamic coatings immobilized with AIE PS that can eradicate SSI-causing bacteria.Initially, nano-thin films of polyoxazoline-based plasma coatings were deposited on suitable substrates.CN-TPAQ-PF6 (or AIE PS) was immobilized on the surfaces of plasma-coated samples at two different concentrations.The intact oxazoline functionalities in POX are anticipated to interact with nucleophilic sites of AIE PS.The presence of AIE PS on the surface of plasma-coated samples was confirmed using characteristic UV absorption peak and fluorescence intensity data.The plasma functionalized surfaces demonstrated bactericidal activity against S. aureus and P. aeruginosa, after light irradiation.POX-AIEb has also shown outstanding biocompatibility in both light and dark conditions.The mechanistic studies exposed the ROS generation potential of surface-bound AIE PS when irradiated by light, which imparted its bactericidal effect.This photodynamic coating with antibacterial properties and desirable cytocompatibility is promising for wide applications in a range of medical devices to combat SSI.

Experimental Section
Materials: The AIE PS used for this study was a kind gift from the Aggregation Induced Emission Institute, China. [27]Ultrahigh-purity water was obtained from a Milli Q system (Millipore Milli-Q Academic, USA).2-Methyl-2-oxazoline, phosphate buffer saline (PBS), 2′,7′dichlorodihydrofluorescein diacetate (DCF dye) and Dulbecco's Modified Eagle's Medium (DMEM) were purchased from Sigma-Aldrich, Australia and used without further purification.Ethanol and acetone were bought from ChemSupply, Australia.Streptomycin and penicillin were procured from Life Technologies.Fetal bovine serum (FBS) and Tryptone Soy Broth (TSB) were purchased from Thermo Scientific and Oxoid.Live/dead Ba-cLight viability kit (L7007) was purchased from Thermo Fischer Scientific.Silicon wafers, quartz microscope slides, sodium cacodylate buffer and glass coverslips (G400-13) were procured from ProSciTech, Australia.For anti-bacterial experiments, Pseudomonas aeruginosa ATCC 15692 and Staphylococcus aureus ATCC 25923 were used as they were causative agents for SSIs.These strains were obtained from American Type Culture Collec-tion.Human epidermal keratinocyte cells (HaCaT, CLS 300493) were used for cell viability and immunostaining studies.
Preparation of Plasma Polymerized Surfaces: The substrates (1.0 cm 2 silicon wafer, 1.0 cm diameter coverslips, quartz glass slides), which were thoroughly cleaned with acetone and ethanol and dried using nitrogen flow, were coated with a thin layer of plasma polymer using a specially constructed bell chamber reactor.The substrates were treated with air plasma set at 50 W and pressure of 1 × 10 −1 mbar for 5 min.A thin layer of polyoxazoline (POX) was deposited on these substrates at 1.3 × 10 −1 mbar pressure and 50 W for 2 min.The coated substrates were stored in vacuumsealed bags.
Immobilization of the Surfaces with AIE PS: Two different concentrations of AIE PS (CN-TPAQ-PF6) were prepared in an ethanol/water mixture, where AIEa was 0.46 mg mL −1 and AIEb was 0.33 mg mL −1 .The substrates coated with POX were immersed in the solution and incubated for 24 h.After the immobilization period, the samples were washed thrice with MilliQ water, dried using nitrogen gas and were named POX-AIEa and POX-AIEb respectively.Ellipsometry: The photographic ellipsometer from SENresearch, SEN-TECH, Germany was used to measure the thickness of the silicon wafers coated with POX.The contribution of a native 2 nm SiO 2 layer present on the silicon wafer surface was deducted during the analysis.The surfaces were positioned on the platform, and the ellipsometer's operating settings were configured.SpectraRay/4 software was used to evaluate the layer's thickness.
Water Contact Angle: The static water contact angle of untreated, POX, POX-AIEa, and POX-AIEb were investigated in this study.A water droplet of 2.0 μL was deposited on top of the substrate using the syringe provided in the RD-SDMO2 goniometer and the images were recorded.Additionally, the nature (hydrophobic/hydrophilic) of the functionalized surfaces was determined by measuring the contact angle using the drop snake analysis plugin of ImageJ software.
UV-vis Spectroscopy: The UV-vis spectra of AIE PS solution, quartz microscope slides functionalized with POX-AIEa and POX-AIEb, were obtained using a Perkin Lambda 350 spectrophotometer, over 200-900 nm.
Fourier Transform Infrared Microscopy (FTIR): The possible interactions among POX and AIE PS were investigated using Perkin Elmer FTIR over wavenumber ranging from 400 to 4000 cm −1 .
Fluorescent Microscope: The fluorescence intensity of untreated, POX, POX-AIEa, and POX-AIEb was evaluated from the images collected using Olympus IX83 Inverted Fluorescence.Zeiss Zen 3.8 software was used to analyse the data.
Scanning electron microscopy (SEM): The morphology of the samples was studied using FEI Inspect F50 Field Emission SEM.The biological samples were initially fixed using 4% glutaraldehyde for 45 min.Employing graded ethanol series (70, 80, 90, and 100%), the samples were dehydrated for 10 min each.The dehydrated samples were dried using nitrogen gas and platinum coated with Ion Sputter Coater, TB-SPUTTER from Quorum Technologies, UK.
Live/Dead Confocal Scanning Laser Microscopy: Pseudomonas aeruginosa and Staphylococcus aureus were inoculated in TSB and left for overnight incubation at 37 °C.After reaching the mid-log phase, the bacterial culture was diluted to 1 × 10 7 colony-forming units per ml (CFU/ml).Silicon wafers functionalized with POX, POX-AIEa and POX-AIEb were incubated with diluted bacterial suspension.They were exposed to light for 1 h (40 mW cm −2 ) and then incubated overnight.The silicon wafers were washed with PBS and stained with STYO9 and PI, for 10 min under dark conditions.They were then visualized under a ZEISS LSM 880 confocal microscope from Zeiss, Germany at excitation and emission wavelengths of 480 nm/500 nm for Syto 9 and 490 nm/635 nm for propidium iodide.Using Zen software, images were split into separate color channels (red and green).The stained cells were counted using ImageJ software v1.53 (NIH, Maryland, USA).The obtained data was converted to percentage viability using the following equation: Percentage of dead bacteria (%) = (Red cells) ∕ (Total cells) × 100 (1)   Colony Enumeration: The CN-TPAQ-PF6 immobilized plasma-coated samples were incubated with S. aureus and P. aeruginosa bacterial suspension (1 × 10 8 CFU mL −1 , 10 μL) for 1 h at 37 °C.The samples were then treated in the dark or with white light irradiation (40 mW cm −2 , for 1 h).After that, the suspension was shaken for 5 min and cleaned.The suspension was then serially diluted using the appropriate folds and plated on a nutrient broth agar plate.They were incubated for 20 h at 37 ˚C.The colonies were counted on the following day and tabulated.

Scheme 1 .
Scheme 1. Schematic illustration of the design of photodynamic antibacterial coatings using plasma polymerization and AIE PS.