Interaction between Engineered Pluronic Silica Nanoparticles and Bacterial Biofilms: Elucidating the Role of Nanoparticle Surface Chemistry and EPS Matrix.

Nanoparticles (NPs) are considered a promising tool in the context of biofilm control. Many studies have shown that different types of NPs can interfere with the bacterial metabolism and cellular membranes, thus making them potential antibacterial agents; however, fundamental understanding is still lacking on the exact mechanisms involved in these actions. The development of NP-based approaches for effective biofilm control also requires a thorough understanding of how the chosen nanoparticles will interact with the biofilm itself, and in particular with the biofilm self-produced extracellular polymeric matrix (EPS). This work aims to provide advances in the understanding of the interaction between engineered fluorescent pluronic silica (PluS) nanoparticles and bacterial biofilms, with a main focus on the role of the EPS matrix in the accumulation and diffusion of the particles in the biofilm. It is demonstrated that particle surface chemistry has a key role in the different lateral distribution and specific affinity to the biofilm matrix components. The results presented in this study contribute to our understanding of biofilm-NP interactions and promote the principle of the rational design of smart nanoparticles as an important tool for antibiofilm technology.

Enzyme-Functionalized Mesoporous Silica Nanoparticles to Target Staphylococcus aureus and Disperse Biofilms.

BACKGROUND: Staphylococcus aureus biofilms pose a unique challenge in healthcare due to their tolerance to a wide range of antimicrobial agents. The high cost and lengthy timeline to develop novel therapeutic agents have pushed researchers to investigate the use of nanomaterials to deliver antibiofilm agents and target biofilm infections more efficiently. Previous studies have concentrated on improving the efficacy of antibiotics by deploying nanoparticles as nanocarriers. However, the dispersal of the extracellular polymeric substance (EPS) matrix in biofilm-associated infections is also critical to the development of novel nanoparticle-based therapies. METHODS: This study evaluated the efficacy of enzyme-functionalized mesoporous silica nanoparticles (MSNs) against methicillin-resistant S. aureus (MRSA) and methicillin-sensitive S. aureus (MSSA) biofilms. MSNs were functionalized with the enzyme lysostaphin, which causes cell lysis of S. aureus bacteria. This was combined with two other enzyme functionalized MSNs, serrapeptase and DNase I which will degrade protein and eDNA in the EPS matrix, to enhance eradication of the biofilm. Cell viability after treatment with enzyme-functionalized MSNs was assessed using a MTT assay and CLSM, while crystal violet staining was used to assess EPS removal. RESULTS: The efficacy of all three enzymes against S. aureus cells and biofilms was significantly improved when they were immobilized onto MSNs. Treatment efficacy was further enhanced when the three enzymes were used in combination against both MRSA and MSSA. Regardless of biofilm maturity (24 or 48 h), near-complete dispersal and killing of MRSA biofilms were observed after treatment with the enzyme-functionalized MSNs. Disruption of mature MSSA biofilms with a polysaccharide EPS was less efficient, but cell viability was significantly reduced. CONCLUSION: The combination of these three enzymes and their functionalization onto nanoparticles might extend the therapeutic options for the treatment of S. aureus infections, particularly those with a biofilm component.

Tailoring Nanoparticle-Biofilm Interactions to Increase the Efficacy of Antimicrobial Agents Against Staphylococcus aureus.

BACKGROUND: Considering the timeline required for the development of novel antimicrobial drugs, increased attention should be given to repurposing old drugs and improving antimicrobial efficacy, particularly for chronic infections associated with biofilms. Methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) are common causes of biofilm-associated infections but produce different biofilm matrices. MSSA biofilm cells are typically embedded in an extracellular polysaccharide matrix, whereas MRSA biofilms comprise predominantly of surface proteins and extracellular DNA (eDNA). Nanoparticles (NPs) have the potential to enhance the delivery of antimicrobial agents into biofilms. However, the mechanisms which influence the interactions between NPs and the biofilm matrix are not yet fully understood. METHODS: To investigate the influence of NPs surface chemistry on vancomycin (VAN) encapsulation and NP entrapment in MRSA and MSSA biofilms, mesoporous silica nanoparticles (MSNs) with different surface functionalization (bare-B, amine-D, carboxyl-C, aromatic-A) were synthesised using an adapted Stöber method. The antibacterial efficacy of VAN-loaded MSNs was assessed against MRSA and MSSA biofilms. RESULTS: The two negatively charged MSNs (MSN-B and MSN-C) showed a higher VAN loading in comparison to the positively charged MSNs (MSN-D and MSN-A). Cellular binding with MSN suspensions (0.25 mg mL-1) correlated with the reduced viability of both MSSA and MRSA biofilm cells. This allowed the administration of low MSNs concentrations while maintaining a high local concentration of the antibiotic surrounding the bacterial cells. CONCLUSION: Our data suggest that by tailoring the surface functionalization of MSNs, enhanced bacterial cell targeting can be achieved, leading to a novel treatment strategy for biofilm infections.

A high throughput method to investigate nanoparticle entrapment efficiencies in biofilms.

The commercial use of nanoparticles has increased in recent years due to their unique characteristics, including high surface area, modifiable shape and surface charge and size-dependent properties. Consequently, a greater number of nanomaterials are now being released into the environment and inevitably interact with the natural ecosystem. Bacterial biofilms have the potential to capture and retain nanoparticles, however the factors determining the specific nanoparticle entrapment efficiencies of biofilms are not yet fully understood. Based on fluorescent intensity measurements we developed a simple and straightforward method that allowed the entrapment of different silica nanoparticles by two Pseudomonas strains to be quantified. It was determined that, regardless of nanoparticle size or surface functionalisation, Pseudomonas putida biofilms showed enhanced entrapment efficiencies compared to Pseudomonas fluorescens biofilms. It was also noted that both biofilms showed a higher entrapment capacity towards positively charged NPs. The method developed has the potential to be utilized for high throughput biofilm screening studies in order to develop a new understating of the relationship between nanoparticle characteristics and its uptake by bacterial biofilms.

Enhancing curcumin's solubility and antibiofilm activity via silica surface modification.

Bacterial biofilms are microbial communities in which bacterial cells in sessile state are mechanically and chemically protected against foreign agents, thus enhancing antibiotic resistance. The delivery of active compounds to the inside of biofilms is often hindered due to the existence of the biofilm extracellular polymeric substances (EPS) and to the poor solubility of drugs and antibiotics. A possible strategy to overcome the EPS barrier is the incorporation of antimicrobial agents into a nanocarrier, able to penetrate the matrix and deliver the active substance to the cells. Here, we report the synthesis of antimicrobial curcumin-conjugated silica nanoparticles (curc-NPs) as a possibility for dealing with these issues. Curcumin is a known antimicrobial agent and to overcome its low solubility in water it was grafted onto the surface of silica nanoparticles, the latter functioning as nanocarrier for curcumin into the biofilm. Curc-NPs were able to impede the formation of model P. putida biofilms up to 50% and disrupt mature biofilms up to 54% at 2.5 mg mL-1. Cell viability of sessile cells in both cases was also considerably affected, which is not observed for curcumin delivered as a free compound at the same concentration. Furthermore, proteomics of extracted EPS matrix of biofilms grown in the presence of free curcumin and curc-NPs revealed differences in the expression of key proteins related to cell detoxification and energy production. Therefore, curc-NPs are presented here as an alternative for curcumin delivery that can be exploited not only to other bacterial strains but also to further biological applications.

Ratiometric Imaging of the in Situ pH Distribution of Biofilms by Use of Fluorescent Mesoporous Silica Nanosensors.

Biofilms are communities of microorganisms enclosed in a self-generated matrix of extracellular polymeric substances. While biofilm recalcitrance and persistence are caused by several factors, a reduction in antimicrobial susceptibility has been closely associated with the generation of pH gradients within the biofilm structure. Cells embedded within the biofilm create a localized acidic microenvironment, which is unaffected by the external pH. Therefore, pH monitoring is a promising approach for understanding the complexities of a three-dimensional heterogeneous biofilm. A fluorescent pH nanosensor was designed through the synthesis of mesoporous silica nanoparticles (47 ± 5 nm diameter) conjugated to a pH-sensitive dye (fluorescein) and a pH-insensitive dye (rhodamine B) as an internal standard (dye-MSNs). The fluorescence intensity of fluorescein (IF) reduced significantly as the pH was decreased from 8.5 to 3.5. In contrast, the fluorescence intensity of rhodamine B (IR) remained constant at any pH. The ratio of IF/IR produced a sigmoidal curve with respect to the pH, in a working pH range between 4.5 and 7.5. Dye-MSNs enabled the measurement of pH gradients within Pseudomonas fluorescens WCS 365 biofilm microcolonies. The biofilms showed spatially distinct low-pH regions that were enclosed into large clusters corresponding to high-cell-density areas. Also present were small low-pH areas that spread indistinctly throughout the microcolony caused by the mass transfer effect. The lowest detected pH within the inner core of the microcolonies was 5.1, gradually increasing to a neutral pH toward the exterior of the microcolonies. The dye-MSNs were able to fully penetrate the biofilm matrix and allowed a quantitative ratiometric analysis of pH gradients and distribution throughout the biofilm, which was independent of the nanoparticle concentration.