Exploring Light-Sensitive Nanocarriers for Simultaneous Triggered Antibiotic Release and Disruption of Biofilms Upon Generation of Laser-Induced Vapor Nanobubbles.

Impaired penetration of antibiotics through bacterial biofilms is one of the reasons for failure of antimicrobial therapy. Hindered drug diffusion is caused on the one hand by interactions with the sticky biofilm matrix and on the other hand by the fact that bacterial cells are organized in densely packed clusters of cells. Binding interactions with the biofilm matrix can be avoided by encapsulating the antibiotics into nanocarriers, while interfering with the integrity of the dense cell clusters can enhance drug transport deep into the biofilm. Vapor nanobubbles (VNB), generated from laser irradiated nanoparticles, are a recently reported effective way to loosen up the biofilm structure in order to enhance drug transport and efficacy. In the present study, we explored if the disruptive force of VNB can be used simultaneously to interfere with the biofilm structure and trigger antibiotic release from light-responsive nanocarriers. The antibiotic tobramycin was incorporated in two types of light-responsive nanocarriers-liposomes functionalized with gold nanoparticles (Lip-AuNP) and graphene quantum dots (GQD)-and their efficacy was evaluated on Pseudomonas aeruginosa biofilms. Even though the anti-biofilm efficacy of tobramycin was improved by liposomal encapsulation, electrostatic functionalization with 70 nm AuNP unfortunately resulted in premature leakage of tobramycin in a matter of hours. Laser-irradiation consequently did not further improve P. aeruginosa biofilm eradication. Adsorption of tobramycin to GQD, on the other hand, did result in a stable formulation with high encapsulation efficiency, without burst release of tobramycin from the nanocarriers. However, even though laser-induced VNB formation from GQD resulted in biofilm disruption, an enhanced anti-biofilm effect was not achieved due to tobramycin not being efficiently released from GQD. Even though this study was unsuccessful in designing suitable nanocarriers for simultaneous biofilm disruption and light-triggered release of tobramycin, it provides insights into the difficulties and challenges that need to be considered for future developments in this regard.

Laser-induced vapour nanobubbles improve drug diffusion and efficiency in bacterial biofilms.

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.

Development and validation of an LC tandem MS assay for the quantification of ß-lactam antibiotics in the sputum of cystic fibrosis patients.

OBJECTIVES: Antibiotic therapy is of vital importance for the control of infectious exacerbations in cystic fibrosis (CF) patients. However, very little is known regarding the fraction of systemically administered antibiotics reaching the lower respiratory tract secretions. We developed and validated a method to measure the concentrations of piperacillin, ceftazidime, meropenem and aztreonam in CF sputum, and present the validation data. METHODS: Ultra-performance LC coupled to tandem MS was used. A single sample can be measured in 2.5 min with multiple reaction monitoring in positive electrospray ionization mode. Deuterated internal standards were used and a concentration range of 0.7-160 mg/L was covered. The method was validated according to the EMA guideline on analytical method validation. RESULTS: The boundaries within which a reliable measurement in CF sputum can be performed were determined. A few constraints are linked to the instability of the antibiotics in sputum. Piperacillin showed limited stability at room temperature and during freeze-thaw cycles. Autosampler instability was observed after 15 h for aztreonam at low concentrations. CONCLUSIONS: The method allows a reliable measurement of the selected antibiotics, if precautions are taken regarding the limited stability of piperacillin at room temperature. Due to freeze-thaw instability, piperacillin should always be analysed on the day of sampling. Quick review of the analytical data and reanalysis are needed as low concentrations of aztreonam are not stable in the autosampler.

Characterization data on the topical carrier DDC642.

This article contains original data, figures and methods used in the characterization of the liposomal carrier 'DDC642' for topical applications, described in "An elastic liposomal formulation for RNAi-based topical treatment of skin disorders: proof-of-concept in the treatment of psoriasis" (Desmet et al., 2016) [1]. Several elastic liposomal formulations have been evaluated for their ability to encapsulate and deliver RNA interference (RNAi) molecules to cultured primary skin cells. The efficiency and effectiveness of these liposomes were compared to that of our previously characterized liposomes, the 'SECosomes' (SEC) (Geusens et al., 2010) [2]. After selection of a potential superior carrier, based on encapsulation and transfection efficiency data (Desmet et al., 2016) [1], the selected DDC642 liposomes were characterized more in-depth. Herein, a detailed characterization of the DDC642 liposome and RNAi-loaded lipoplexes is given, including the matching protocols.

An elastic liposomal formulation for RNAi-based topical treatment of skin disorders: Proof-of-concept in the treatment of psoriasis.

RNA interference (RNAi) is a rapidly emerging approach for targeted gene silencing to alleviate disease pathology. However, lack of efficient carriers for targeted delivery delays the clinical translation of RNAi. An interesting target for local RNAi therapeutics is the skin as it allows direct access to target cells. Still, applications are limited due to the effective skin barrier which hinders penetration. Herein, a description is given of a liposomal carrier, called 'DDC642', capable of delivering RNAi molecules to the epidermis of impaired and intact human skin, without targeting the dermis or circulatory system. In a psoriasis tissue model, down-regulation of the psoriasis marker human beta-defensin 2 by DDC642-delivered siRNA was confirmed, providing proof-of-concept. These liposomes thus hold great potential as topical delivery system for RNAi therapeutics in the treatment of numerous skin diseases.

High oxygen tension increases global methylation in bovine 4-cell embryos and blastocysts but does not affect general retrotransposon expression.

Retrotransposons are transposable elements that insert extra copies of themselves throughout the genome via an RNA intermediate using a 'copy and paste' mechanism. They account for more than 44% of the bovine genome and have been reported to be functional, especially during preimplantation embryo development. In the present study, we tested whether high oxygen tension (20% O2) influences global DNA methylation analysed by immunofluorescence staining of developing bovine embryos and whether this has an effect on the expression of some selected retrotransposon families. High oxygen tension significantly increased global DNA methylation in 4-cell embryos and blastocysts. A significant expression difference was observed for ERV1-1-I_BT in female blastocysts, but no significant changes were observed for the other retrotransposon families tested. Therefore, the study indicates that global DNA methylation is not necessarily correlated with retrotransposon expression in bovine preimplantation embryos.

Effect of Native Gastric Mucus on in vivo Hybridization Therapies Directed at Helicobacter pylori.

Helicobacter pylori infects more than 50% of the worldwide population. It is mostly found deep in the gastric mucus lining of the stomach, being a major cause of peptic ulcers and gastric adenocarcinoma. To face the increasing resistance of H. pylori to antibiotics, antimicrobial nucleic acid mimics are a promising alternative. In particular, locked nucleic acids (LNA)/2'-OMethyl RNA (2'OMe) have shown to specifically target H. pylori, as evidenced by in situ hybridization. The success of in vivo hybridization depends on the ability of these nucleic acids to penetrate the major physical barriers-the highly viscoelastic gastric mucus and the bacterial cell envelope. We found that LNA/2'OMe is capable of diffusing rapidly through native, undiluted, gastric mucus isolated from porcine stomachs, without degradation. Moreover, although LNA/2'OMe hybridization was still successful without permeabilization and fixation of the bacteria, which is normally part of in vitro studies, the ability of LNA/2'OMe to efficiently hybridize with H. pylori was hampered by the presence of mucus. Future research should focus on developing nanocarriers that shield LNA/2'OMe from components in the gastric mucus, while remaining capable of diffusing through the mucus and delivering these nucleic acid mimics directly into the bacteria.

A beneficiary role for neuraminidase in influenza virus penetration through the respiratory mucus.

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.

Probing the size limit for nanomedicine penetration into Burkholderia multivorans and Pseudomonas aeruginosa biofilms.

Encapsulation of antibiotics into nanoparticles is a potential strategy to eradicate biofilms. To allow further optimization of nanomedicines for biofilm eradication, the influence of the nanoparticle size on the penetration into dense biofilm clusters needs to be investigated. In the present study, the penetration of nanoparticles with diameters ranging from 40 to 550 nm into two biofilms, Burkholderia multivorans LMG 18825 and Pseudomonas aeruginosa LMG 27622, was evaluated using confocal microscopy. Through image analysis, the percentage of particles able to penetrate into dense biofilm clusters was calculated. The size cut off for optimal penetration into biofilm clusters was located around 100-130 nm for both biofilms. The mesh size of the biofilm matrix and the size of the channels in between the bacteria of the clusters are two factors which likely play a role in the exclusion of the larger particles. For B. multivorans, a sharp drop in the penetration into the clusters is seen for particles larger than 130 nm while for P. aeruginosa, a more gradual decrease in penetration could be observed. The overall penetration of the nanoparticles was slightly lower for P. aeruginosa than for B. multivorans. Based on these results, it could be concluded that nanocarriers of about 100 nm and smaller are good candidates to improve the treatment of chronic pulmonary biofilms in CF patients. Furthermore, the confocal microscopy method demonstrated here is a useful tool to assess the penetration of nanomedicines in biofilm clusters. Such information is important to optimize nanomedicine formulations for the treatment of biofilm infections.

Single-particle tracking for studying nanomaterial dynamics: applications and fundamentals in drug delivery.

Many macromolecular therapeutics could potentially treat genetic disorders and cancer. They have, however, not yet reached the clinical stage owing to a lack of suitable carriers that can bring the therapeutics from the administration site to the subcellular site in target cells. One of the reasons that is hindering the development of such carriers is the limited knowledge of their transport dynamics and intracellular processing. Single-particle tracking (SPT) microscopy, thanks to its single molecule sensitivity and its broad applicability, has found its entry in the field of drug delivery to get an answer to these questions. This review aims to introduce the fundamentals of SPT to the drug delivery community and highlight the most recent discoveries obtained with SPT in the field of drug delivery.

Lipid and polymer nanoparticles for drug delivery to bacterial biofilms.

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.

DNA counterstaining for methylation and hydroxymethylation immunostaining in bovine zygotes.

Immunostaining is the preferred technique to assess differences in methylation and hydroxymethylation status of both pronuclei in single zygotes. DNA counterstaining is needed to delimitate the pronuclear area for quantification purposes. For a correct epitope retrieval of 5-methylcytosine and 5-hydroxymethylcytosine in bovine zygotes, 1h of denaturation with 4N HCl is needed. However, DNA stains are sensitive to denaturation. Therefore, four DNA stains were tested after 1h of denaturation with 4N HCl in this study. After this treatment, DAPI (4',6-diamidino-2-phenylindole) and Hoechst failed to bind DNA, but both propidium iodide and ethidium homodimer-2 successfully bound it and both pronuclei were stained.

Equine oviduct explant culture: a basic model to decipher embryo-maternal communication.

Equine embryos remain for 6 days in the oviduct and thus there is a need for an in vitro model to study embryo-oviductal interactions in the horse, since this subtle way of communication is very difficult to analyse in vivo. Until now, no equine oviduct explant culture model has been characterised both morphologically and functionally. Therefore, we established a culture system for equine oviduct explants that maintained epithelial morphology during 6 days of culture, as revealed by light microscopy and transmission electron microscopy. We demonstrated the presence of highly differentiated, tall columnar, pseudostratified epithelium with basal nuclei, numerous nucleoli, secretory granules and apical cilia, which is very similar to the in vivo situation. Both epithelium and stromal cells originating from the lamina propria are represented in the explants. Moreover, at least 98% of the cells remained membrane intact and fewer than 2% of the cells were apoptotic after 6 days of culture. Although dark-cell degeneration, which is a hypoxia-related type of cell death, was observed in the centre of the explants, quantitative real-time PCR failed to detect upregulation of the hypoxia-related marker genes HIF1A, VEGFA, uPA, GLUT1 and PAI1. Since the explants remained morphologically and functionally intact and since the system is easy to set up, it appears to be an excellent tool for proteome, transcriptome and miRNome analysis in order to unravel embryo-maternal interactions in the horse.

Transport of nanoparticles and tobramycin-loaded liposomes in Burkholderia cepacia complex biofilms.

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.

Measuring the intravitreal mobility of nanomedicines with single-particle tracking microscopy.

AIM: To develop a robust assay to evaluate and compare the intravitreal mobility of nanoparticles in the intact vitreous body. MATERIALS & METHODS: Excised bovine eyes were prepared to preserve the fragile structure of the vitreous humor, while permitting high-resolution fluorescence microscopy and single-particle tracking analysis of intravitreally injected nanoparticles. This assay was validated by analyzing polystyrene beads and further employed to evaluate gene nanomedicines composed of poly(amido amine)s and plasmid DNA. RESULTS: The assay was able to distinguish immobilized cationic nanoparticles from mobile PEGylated nanoparticles. PEGylation of the polyplexes resulted in a drastic improvement of their mobility. CONCLUSION: 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.

Transport of nanoparticles in cystic fibrosis sputum and bacterial biofilms by single-particle tracking microscopy.

AIM: The aim of this study was to evaluate the effect of the surface functionalization of model nanoparticles on their mobility in bacterial biofilms and cystic fibrosis sputum. MATERIALS & METHODS: With single-particle tracking microscopy, the mobility of 0.1- and 0.2-µm fluorescent polyethylene glycol (PEG) modified, carboxylate- and N,N-dimethylethylenediamine-modified polystyrene nanospheres were evaluated in fresh cystic fibrosis sputum, as well as Burkholderia multivorans and Pseudomonas aeruginosa biofilms. RESULTS: PEGylation increased the mobility of the particles in sputum and biofilms, while the charged nanospheres were strongly immobilized. However, the transport of the PEGylated nanoparticles was lower in sputum compared with biofilms. Furthermore, the particle transport showed heterogeneity in samples originating from different patients. CONCLUSION: 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.

Immobilization of pseudorabies virus in porcine tracheal respiratory mucus revealed by single particle tracking.

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.

Fluorescence single particle tracking for the characterization of submicron protein aggregates in biological fluids and complex formulations.

PURPOSE: To evaluate the potential of fluorescence single particle tracking (fSPT) for the characterization of submicron protein aggregates in human serum, plasma and formulations containing human serum albumin (HSA). METHODS: A monoclonal IgG was covalently labeled with a fluorescent dye and cross-linked with glutaraldehyde. IgG aggregates and fluorescent beads of 0.1 µm (control) were diluted in buffer, serum and plasma, and their size distributions were analyzed by fSPT and nanoparticle tracking analysis (NTA). In a separate experiment, IgG and HSA, fluorescently labeled with different dyes, were mixed and subjected to heat stress. The stressed sample was analyzed by fSPT using a dual color mode and by NTA. RESULTS: The accuracy and precision of fSPT proved to be comparable to NTA. fSPT was able to successfully measure all the samples in buffer, serum and plasma. The average size of the cross-linked protein aggregates showed a slight increase in biological fluids. Moreover, fSPT analysis showed that a significant proportion of the aggregates formed by subjecting an IgG/HSA mixture to heat stress were composed of both proteins. CONCLUSION: fSPT is a powerful technique for the characterization of submicron protein aggregates in biological fluids and complex formulations.