Biofilms are matrix-enclosed communities of bacteria that show increased antibiotic resistance and the capability to evade the immune system. They can cause recalcitrant infections which cannot be cured with classical antibiotic therapy. Drug delivery by lipid or polymer nanoparticles is considered a promising strategy for overcoming biofilm resistance. These particles are able to improve the delivery of antibiotics to the bacterial cells, thereby increasing the efficacy of the treatment. In this review we give an overview of the types of polymer and lipid nanoparticles that have been developed for this purpose. The antimicrobial activity of nanoparticle encapsulated antibiotics compared to the activity of the free antibiotic is discussed in detail. In addition, targeting and triggered drug release strategies to further improve the antimicrobial activity are reviewed. Finally, ample attention is given to advanced microscopy methods that shed light on the behavior of nanoparticles inside biofilms, allowing further optimization of the nanoformulations. Lipid and polymer nanoparticles were found to increase the antimicrobial efficacy in many cases. Strategies such as the use of fusogenic liposomes, targeting of the nanoparticles and triggered release of the antimicrobial agent ensured the delivery of the antimicrobial agent in close proximity of the bacterial cells, maximizing the exposure of the biofilm to the antimicrobial agent. The majority of the discussed papers still present data on the in vitro anti-biofilm activity of nanoformulations, indicating that there is an urgent need for more in vivo studies in this field.

Swine influenza virus (SIV) has a strong tropism for pig respiratory mucosa, which consists of a mucus layer, epithelium, basement membrane and lamina propria. Sialic acids present on the epithelial surface have long been considered to be determinants of influenza virus tropism. However, mucus which is also rich in sialic acids may serve as the first barrier of selection. It was investigated how influenza virus interacts with the mucus to infect epithelial cells. Two techniques were applied to track SIV H1N1 in porcine mucus. The microscopic diffusion of SIV particles in the mucus was analyzed by single particle tracking (SPT), and the macroscopic penetration of SIV through mucus was studied by a virus in-capsule-mucus penetration system, followed by visualizing the translocation of the virions with time by immunofluorescence staining. Furthermore, the effects of neuraminidase on SIV getting through or binding to the mucus were studied by using zanamivir, a neuraminidase inhibitor (NAI), and Arthrobacter ureafaciens neuraminidase. The distribution of the diffusion coefficient shows that 70% of SIV particles were entrapped, while the rest diffused freely in the mucus. Additionally, SIV penetrated the porcine mucus with time, reaching a depth of 65 µm at 30 min post virus addition, 2 fold of that at 2 min. Both the microscopic diffusion and macroscopic penetration were largely diminished by NAI, while were clearly increased by the effect of exogenous neuraminidase. Moreover, the exogenous neuraminidase sufficiently prevented the binding of SIV to mucus which was reversely enhanced by effect of NAI. These findings clearly show that the neuraminidase helps SIV move through the mucus, which is important for the virus to reach and infect epithelial cells and eventually become shed into the lumen of the respiratory tract.

Hindered penetration of antibiotics through biofilms is one of the reasons for the alarming increase in bacterial tolerance to antibiotics. Here, we investigate the potential of laser-induced vapour nanobubbles (VNBs) formed around plasmonic nanoparticles to locally disturb biofilm integrity and improve antibiotics diffusion. Our results show that biofilms of both Gram-negative (Burkholderia multivorans, Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) bacteria can be loaded with cationic 70-nm gold nanoparticles and that subsequent laser illumination results in VNB formation inside the biofilms. In all types of biofilms tested, VNB formation leads to substantial local biofilm disruption, increasing tobramycin efficacy up to 1-3 orders of magnitude depending on the organism and treatment conditions. Altogether, our results support the potential of laser-induced VNBs as a new approach to disrupt biofilms of a broad range of organisms, resulting in improved antibiotic diffusion and more effective biofilm eradication.

An ex vivo eye model is presented for studying nanoparticle mobility in intact vitreous humor by single-particle tracking microscopy. These results give important guidelines for developing gene- and drug-delivery nanomedicines that are compatible with intravitreal administration.

This study's data suggest that for future nanocarrier design it will be essential to combine PEGylation with a targeting moiety to ensure sufficient mobility in mucus and a better accumulation of the nanoparticles in the biofilm.

No abstract

Due to the intrinsic resistance of Burkholderia cepacia complex (Bcc) to many antibiotics and the production of a broad range of virulence factors, lung infections by these bacteria, primarily occurring in cystic fibrosis (CF) patients, are very difficult to treat. In addition, the ability of Bcc organisms to form biofilms contributes to their persistence in the CF lung. As Bcc infections are associated with poor clinical outcome, there is an urgent need for new effective therapies to treat these infections. In the present study, we investigated whether liposomal tobramycin displayed an increased anti-biofilm effect against Bcc bacteria compared to free tobramycin. Single particle tracking (SPT) was used to study the transport of positively and negatively charged nanospheres in Bcc biofilms as a model for the transport of liposomes. Negatively charged nanospheres became immobilized in close proximity of biofilm cell clusters, while positively charged nanospheres interacted with fiber-like structures, probably eDNA. Based on these data, encapsulation of tobramycin in negatively charged liposomes appeared promising for targeted drug delivery. However, the anti-biofilm effect of tobramycin encapsulated into neutral or anionic liposomes did not increase compared to that of free tobramycin. Probably, the fusion of the anionic liposomes with the negatively charged bacterial surface of Bcc bacteria was limited by electrostatic repulsive forces. The lack of a substantial anti-biofilm effect of tobramycin encapsulated in neutral liposomes could be further investigated by increasing the liposomal tobramycin concentration. However, this was hampered by the low encapsulation efficiency of tobramycin in these liposomes.

Pseudorabies virus (PRV) initially replicates in the porcine upper respiratory tract. It easily invades the mucosae and submucosae for subsequent spread throughout the body via blood vessels and nervous system. In this context, PRV developed ingenious processes to overcome different barriers such as epithelial cells and the basement membrane. Another important but often overlooked barrier is the substantial mucus layer which coats the mucosae. However, little is known about how PRV particles interact with porcine respiratory mucus. We therefore measured the barrier properties of porcine tracheal respiratory mucus, and investigated the mobility of nanoparticles including PRV in this mucus. We developed an in vitro model utilizing single particle tracking microscopy. Firstly, the mucus pore size was evaluated with polyethylene glycol coupled (PEGylated) nanoparticles and atomic force microscope. Secondly, the mobility of PRV in porcine tracheal respiratory mucus was examined and compared with that of negative, positive and PEGylated nanoparticles. The pore size of porcine tracheal respiratory mucus ranged from 80 to 1500 nm, with an average diameter of 455±240 nm. PRV (zeta potential: −31.8±1.5 mV) experienced a severe obstruction in porcine tracheal respiratory mucus, diffusing 59-fold more slowly than in water. Similarly, the highly negatively (−49.8±0.6 mV) and positively (36.7±1.1 mV) charged nanoparticles were significantly trapped. In contrast, the nearly neutral, hydrophilic PEGylated nanoparticles (−9.6±0.8 mV) diffused rapidly, with the majority of particles moving 50-fold faster than PRV. The mobility of the particles measured was found to be related but not correlated to their surface charge. Furthermore, PEGylated PRV (-13.8±0.9 mV) was observed to diffuse 13-fold faster than native PRV. These findings clearly show that the mobility of PRV was significantly hindered in porcine tracheal respiratory mucus, and that the obstruction of PRV was due to complex mucoadhesive interactions including charge interactions rather than size exclusion.