Selective Labeling of Individual Neurons in Dense Cultured Networks With Nanoparticle-Enhanced Photoporation.

Neurodevelopmental and neurodegenerative disorders are characterized by subtle alterations in synaptic connections and perturbed neuronal network functionality. A hallmark of neuronal connectivity is the presence of dendritic spines, micron-sized protrusions of the dendritic shaft that compartmentalize single synapses to fine-tune synaptic strength. However, accurate quantification of spine density and morphology in mature neuronal networks is hampered by the lack of targeted labeling strategies. To resolve this, we have optimized a method to deliver cell-impermeable compounds into selected cells based on Spatially resolved NAnoparticle-enhanced Photoporation (SNAP). We show that SNAP enables efficient labeling of selected individual neurons and their spines in dense cultured networks without affecting short-term viability. We compare SNAP with widely used spine labeling techniques such as the application of lipophilic dyes and genetically encoded fluorescent markers. Using SNAP, we demonstrate a time-dependent increase in spine density in healthy cultures as well as a reduction in spine density after chemical mimicry of hypoxia. Since the sparse labeling procedure can be automated using an intelligent acquisition scheme, SNAP holds promise for high-content screening campaigns of neuronal connectivity in the context of neurodevelopmental and neurodegenerative disorders.

Endosomal Size and Membrane Leakiness Influence Proton Sponge-Based Rupture of Endosomal Vesicles.

In gene therapy, endosomal escape represents a major bottleneck since nanoparticles often remain entrapped inside endosomes and are trafficked toward the lysosomes for degradation. A detailed understanding of the endosomal barrier would be beneficial for developing rational strategies to improve transfection and endosomal escape. By visualizing individual endosomal escape events in live cells, we obtain insight into mechanistic factors that influence proton sponge-based endosomal escape. In a comparative study, we found that HeLa cells treated with JetPEI/pDNA polyplexes have a 3.5-fold increased endosomal escape frequency compared to ARPE-19 cells. We found that endosomal size has a major impact on the escape capacity. The smaller HeLa endosomes are more easily ruptured by the proton sponge effect than the larger ARPE-19 endosomes, a finding supported by a mathematical model based on the underlying physical principles. Still, it remains intriguing that even in the small HeLa endosomes, <10% of the polyplex-containing endosomes show endosomal escape. Further experiments revealed that the membrane of polyplex-containing endosomes becomes leaky to small compounds, preventing effective buildup of osmotic pressure, which in turn prevents endosomal rupture. Analysis of H1299 and A549 cells revealed that endosomal size determines endosomal escape efficiency when cells have comparable membrane leakiness. However, at high levels of membrane leakiness, buildup of osmotic pressure is no longer possible, regardless of endosomal size. Based on our findings that both endosomal size and membrane leakiness have a high impact on proton sponge-based endosomal rupture, we provide important clues toward further improvement of this escape strategy.

Quantifying the Average Number of Nucleic Acid Therapeutics per Nanocarrier by Single Particle Tracking Microscopy.

Nucleic acid biopharmaceuticals are being investigated as potential therapeutics. They need to be incorporated into a biocompatible carrier so as to overcome several biological barriers. Rational development of suitable nanocarriers requires high-quality characterization techniques. While size, concentration, and stability can be very well measured these days, even in complex biological fluids, a method to accurately quantify the number of nucleic acid therapeutics encapsulated in nanocarriers is still missing. Here we present a method, based on concentration measurements with single particle tracking microscopy, with which it is possible to directly measure the number of plasmid DNA molecules per nanoparticle, referred to as the plasmid/NP ratio. Using DOTAP/DOPE liposomes as a model carrier, we demonstrate the usefulness of the method by investigating the influence of various experimental factors on the plasmid/NP ratio. We find that the plasmid/NP ratio is inversely proportional with the size of the pDNA and that the plasmid/NP decreases when lipoplexes are prepared at lower concentrations of pDNA and nanocarrier, with values ranging from 6.5 to 3 plasmid/NP. Furthermore, the effect of pre- and post-PEGylation of lipoplexes was examined, finding that pre-PEGylation results in a decreased plasmid/NP ratio, while post-PEGylation did not alter the plasmid/NP ratio. These proof-of-concept experiments show that single particle tracking offers an extension of the nanoparticle characterization toolbox and is expected to aid in the efficient development of nanoformulations for nucleic acid-based therapies.

Fast spatial-selective delivery into live cells.

Intracellular delivery of functional compounds into living cells is of great importance for cell biology as well as therapeutic applications. Often it is sufficient that the compound of interest (being a molecule or nanoparticle) is delivered to the cell population as a whole. However, there are applications that would benefit considerably from the possibility of delivering a compound to a certain subpopulation of cells, or even in selected single cells. Here we report on an integrated platform for high-throughput spatially resolved nanoparticle-enhanced photoporation (SNAP) of adherent cells. SNAP enables safe, intracellular delivery of exogenously administered nanomaterials in selected subpopulations of cells, even down to the single cell level. We demonstrate the power of SNAP by selectively delivering a safe contrast agent into a subpopulation of polynucleated keratinocytes, enabling their downstream purification for unraveling their role in neoplasm formation. The flexibility and speed with which individual cells can be labeled make SNAP an ideal tool for high-throughput applications, not only for selective labeling but also for targeted drug delivery.

The role of intracellular trafficking of CdSe/ZnS QDs on their consequent toxicity profile.

BACKGROUND: Nanoparticle interactions with cellular membranes and the kinetics of their transport and localization are important determinants of their functionality and their biological consequences. Understanding these phenomena is fundamental for the translation of such NPs from in vitro to in vivo systems for bioimaging and medical applications. Two CdSe/ZnS quantum dots (QD) with differing surface functionality (NH2 or COOH moieties) were used here for investigating the intracellular uptake and transport kinetics of these QDs. RESULTS: In water, the COOH- and NH2-QDs were negatively and positively charged, respectively, while in serum-containing medium the NH2-QDs were agglomerated, whereas the COOH-QDs remained dispersed. Though intracellular levels of NH2- and COOH-QDs were very similar after 24 h exposure, COOH-QDs appeared to be continuously internalised and transported by endosomes and lysosomes, while NH2-QDs mainly remained in the lysosomes. The results of (intra)cellular QD trafficking were correlated to their toxicity profiles investigating levels of reactive oxygen species (ROS), mitochondrial ROS, autophagy, changes to cellular morphology and alterations in genes involved in cellular stress, toxicity and cytoskeletal integrity. The continuous flux of COOH-QDs perhaps explains their higher toxicity compared to the NH2-QDs, mainly resulting in mitochondrial ROS and cytoskeletal remodelling which are phenomena that occur early during cellular exposure. CONCLUSIONS: Together, these data reveal that although cellular QD levels were similar after 24 h, differences in the nature and extent of their cellular trafficking resulted in differences in consequent gene alterations and toxicological effects.

Effect of hyaluronic acid-binding to lipoplexes on intravitreal drug delivery for retinal gene therapy.

Intravitreal administration of nanomedicines could be valuable for retinal gene therapy, if their mobility in the vitreous and therapeutic efficacy in the target cells can be guaranteed. Hyaluronic acid (HA) as an electrostatic coating of polymeric gene nanomedicines has proven to be beneficial on both accounts. While electrostatic coating provides an easy way of coating cationic nanoparticles, the stability of electrostatic complexes in vivo is uncertain. In this study, therefore, we compare electrostatic with covalent coating of gene nanocarriers with HA for retinal gene therapy via intravitreal administration. Specifically, DOTAP:DOPE/plasmid DNA lipoplexes coated with HA are evaluated in terms of intravitreal mobility using a previously optimized ex vivo model. We find that both electrostatic and covalent HA coating considerably improve the mobility of the lipoplexes in the vitreous humor of excised bovine eyes. In addition we evaluate in vitro uptake and transfection efficiency in ARPE-19 cells. Contrary to PEGylated lipoplexes it is found that HA coated lipoplexes are efficiently internalized into ARPE-19 cells. Covalent HA-coated lipoplexes had an 8-fold increase of transgene expression compared to the uncoated lipoplexes. We conclude that covalent HA-coating of gene nanomedicines is a promising approach for retinal gene therapy by intravitreal administration.

Microfabricated devices for single objective single plane illumination microscopy (SoSPIM).

Light sheet microscopy is a relatively new form of fluorescence microscopy that has been receiving a lot of attention recently. The strong points of the technique, such as high signal to noise ratio and its reduced photodamage of fluorescently labelled samples, come from its unique feature to illuminate only a thin plane in the sample that coincides with the focal plane of the detection lens. Typically this requires two closely positioned perpendicular objective lenses, one for detection and one for illumination. Apart from the fact that this special configuration of objective lenses is incompatible with standard microscope bodies, it is particularly problematic for high-resolution lenses which typically have a short working distance. To address these issues we developed sample holders with an integrated micromirror to perform single lens light sheet microscopy, also known as single objective single plane illumination microscopy (SoSPIM). The first design is based on a wet-etched silicon substrate, the second on a microfabricated polished polymer plug. We achieved an on-chip light sheet thickness of 2.3 µm (FWHM) at 638 nm with the polymer micromirror and of 1.7 µm (FWHM) at 638 nm with the silicon micromirror, comparable to reported light sheet thicknesses obtained on dedicated light sheet microscopes. A marked contrast improvement was obtained with both sample holders as compared to classic epi-fluorescence microscopy. In order to evaluate whether this technology could be made available on a larger scale, in a next step we evaluated the optical quality of inexpensive replicas from both types of master molds. We found that replicas from the polished polymer based mold have an optical quality close to that of the master component, while replicas from the silicon based mold were of slightly lower but still acceptable quality. The suitability of the replicated polymer based sample holder for single-lens light sheet microscopy was finally demonstrated by imaging breast cancer spheroids.

Cytosolic Delivery of Nanolabels Prevents Their Asymmetric Inheritance and Enables Extended Quantitative in Vivo Cell Imaging.

Long-term in vivo imaging of cells is crucial for the understanding of cellular fate in biological processes in cancer research, immunology, or in cell-based therapies such as beta cell transplantation in type I diabetes or stem cell therapy. Traditionally, cell labeling with the desired contrast agent occurs ex vivo via spontaneous endocytosis, which is a variable and slow process that requires optimization for each particular label-cell type combination. Following endocytic uptake, the contrast agents mostly remain entrapped in the endolysosomal compartment, which leads to signal instability, cytotoxicity, and asymmetric inheritance of the labels upon cell division. Here, we demonstrate that these disadvantages can be circumvented by delivering contrast agents directly into the cytoplasm via vapor nanobubble photoporation. Compared to classic endocytic uptake, photoporation resulted in 50 and 3 times higher loading of fluorescent dextrans and quantum dots, respectively, with improved signal stability and reduced cytotoxicity. Most interestingly, cytosolic delivery by photoporation prevented asymmetric inheritance of labels by daughter cells over subsequent cell generations. Instead, unequal inheritance of endocytosed labels resulted in a dramatic increase in polydispersity of the amount of labels per cell with each cell division, hindering accurate quantification of cell numbers in vivo over time. The combined benefits of cell labeling by photoporation resulted in a marked improvement in long-term cell visibility in vivo where an insulin producing cell line (INS-1E cell line) labeled with fluorescent dextrans could be tracked for up to two months in Swiss nude mice compared to 2 weeks for cells labeled by endocytosis.

Sizing nanomaterials in bio-fluids by cFRAP enables protein aggregation measurements and diagnosis of bio-barrier permeability.

Sizing nanomaterials in complex biological fluids, such as blood, remains a great challenge in spite of its importance for a wide range of biomedical applications. In drug delivery, for instance, it is essential that aggregation of protein-based drugs is avoided as it may alter their efficacy or elicit immune responses. Similarly it is of interest to determine which size of molecules can pass through biological barriers in vivo to diagnose pathologies, such as sepsis. Here, we report on continuous fluorescence recovery after photobleaching (cFRAP) as a analytical method enabling size distribution measurements of nanomaterials (1-100 nm) in undiluted biological fluids. We demonstrate that cFRAP allows to measure protein aggregation in human serum and to determine the permeability of intestinal and vascular barriers in vivo. cFRAP is a new analytical technique that paves the way towards exciting new applications that benefit from nanomaterial sizing in bio-fluids.

Hitchhiking nanoparticles: Reversible coupling of lipid-based nanoparticles to cytotoxic T lymphocytes.

Following intravenous injection of anti-cancer nanomedicines, many barriers need to be overcome en route to the tumor. Cell-mediated delivery of nanoparticles (NPs) is promising in terms of overcoming several of these barriers based on the tumoritropic migratory properties of particular cell types. This guided transport aims to enhance the NP accumulation in the tumor and moreover enhance the infiltration of regions that are typically inaccessible for free NPs. Within this study, cytotoxic CD8(+) T cells were selected as carriers based on both their ability to migrate to the tumor and their intrinsic cytolytic activity against tumor cells. Many anti-cancer nanomedicines require tumor cell internalization to mediate cytosolic drug delivery and enhance the anti-cancer effect. This proof-of-concept therefore reports on the reversible attachment of liposomes to the surface of cytotoxic T lymphocytes via a reduction sensitive coupling. The activation status of the T cells and the liposome composition are shown to strongly influence the loading efficiency. Loading the cells with liposomes does not compromise T cell functionalities like proliferation and cytolytic function. Additionally, the triggered liposome release is demonstrated upon the addition of glutathione. Based on this optimization using liposomes as model NPs, a small interfering RNA (siRNA)-loaded NP was developed that can be coupled to the surface of CD8(+) T cells.

Hybrid pulmonary surfactant-coated nanogels mediate efficient in vivo delivery of siRNA to murine alveolar macrophages.

The local delivery of small interfering RNA (siRNA) to the lungs may provide a therapeutic solution to a range of pulmonary disorders. Resident alveolar macrophages (rAM) in the bronchoalveolar lumen play a critical role in lung inflammatory responses and therefore constitute a particularly attractive target for siRNA therapeutics. However, achieving efficient gene silencing in the lung while avoiding pulmonary toxicity requires appropriate formulation of siRNA in functional nanocarriers. In this study, we evaluated pulmonary surfactant-coated dextran nanogels for the delivery of siRNA to rAM upon pharyngeal aspiration in BALB/c mice. Both the surfactant-coated and uncoated nanogels achieved high levels of siRNA uptake in rAM, yet only the surfactant-coated formulation could significantly reduce gene expression on the protein level. Surfactant-coated nanogels induced a profound downregulation of target mRNA levels, reaching 70% knockdown with ~1mgkg(-1) siRNA dose. In addition, only mild acute pro-inflammatory cytokine and chemokine responses were detected one day after nanoparticle aspiration, accompanied by a moderate neutrophil infiltration in the bronchoalveolar lumen. The latter could be substantially reduced by removal of excess surfactant from the formulation. Overall, our hybrid core-shell nanoparticles have demonstrated safe and effective siRNA delivery to rAM, providing a new therapeutic approach for treatment of inflammatory pathologies in the lung.

Bio-inspired pulmonary surfactant-modified nanogels: A promising siRNA delivery system.

Inhalation therapy with small interfering RNA (siRNA) is a promising approach in the treatment of pulmonary disorders. However, clinical translation is severely limited by the lack of suitable delivery platforms. In this study, we aim to address this limitation by designing a novel bioinspired hybrid nanoparticle with a core-shell nanoarchitecture, consisting of a siRNA-loaded dextran nanogel (siNG) core and a pulmonary surfactant (Curosurf®) outer shell. The decoration of siNGs with a surfactant shell enhances the colloidal stability and prevents siRNA release in the presence of competing polyanions, which are abundantly present in biofluids. Additionally, the impact of the surfactant shell on the biological efficacy of the siNGs is determined in lung cancer cells. The presence of the surfactants substantially reduces the cellular uptake of siNGs. Remarkably, the lowered intracellular dose does not impede the gene silencing effect, suggesting a crucial role of the pulmonary surfactant in the intracellular processing of the nanoparticles. In order to surmount the observed reduction in cellular dose, folate is incorporated as a targeting ligand in the pulmonary surfactant shell to incite receptor-mediated endocytosis. The latter substantially enhances both cellular uptake and gene silencing potential, achieving efficient knockdown at siRNA concentrations in the low nanomolar range.

Coating nanocarriers with hyaluronic acid facilitates intravitreal drug delivery for retinal gene therapy.

Retinal gene therapy could potentially affect the lives of millions of people suffering from blinding disorders. Yet, one of the major hurdles remains the delivery of therapeutic nucleic acids to the retinal target cells. Due to the different barriers that need to be overcome in case of topical or systemic administration, intravitreal injection is an attractive alternative administration route for large macromolecular therapeutics. Here it is essential that the therapeutics do not aggregate and remain mobile in the vitreous humor in order to reach the retina. In this study, we have evaluated the use of hyaluronic acid (HA) as an electrostatic coating for nonviral polymeric gene nanomedicines, p(CBA-ABOL)/pDNA complexes, to provide them with an anionic hydrophilic surface for improved intravitreal mobility. Uncoated polyplexes had a Z-averaged diameter of 108nm and a zeta potential of +29mV. We evaluated polyplexes coated with HA of different molecular weights (22kDa, 137kDa and 2700kDa) in terms of size, surface charge and complexation efficiency and noticed their zeta potentials became anionic at 4-fold molar excess of HA-monomers compared to cationic monomers, resulting in submicron ternary polyplexes. Next, we used a previously optimized ex vivo model based on excised bovine eyes and fluorescence single particle tracking (fSPT) microscopy to evaluate mobility in intact vitreous humor. It was confirmed that HA-coated polyplexes had good mobility in bovine vitreous humor, similar to polyplexes functionalized with polyethylene glycol (PEG), except for those coated with high molecular weight HA (2700kDa). However, contrary to PEGylated polyplexes, HA-coated polyplexes were efficiently taken up in vitro in ARPE-19 cells, despite their negative charge, indicating uptake via CD44-receptor mediated endocytosis. Furthermore, the HA-polyplexes were able to induce GFP expression in this in vitro cell line without apparent cytotoxicity, where coating with low molecular weight HA (22kDa) was shown to induce the highest expression. Taken together our experiments show that HA-coating of nonviral gene complexes is an interesting approach towards retinal gene therapy by intravitreal administration. To our knowledge, this is the first time electrostatic HA-coating of polyplexes with different molecular weights has been evaluated in terms of their suitability for intravitreal delivery of therapeutic nucleic acids towards the retina.

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.

The performance of gradient alloy quantum dots in cell labeling.

The interest in using quantum dots (QDots) as highly fluorescent and photostable nanoparticles in biomedicine is vastly increasing. One major hurdle that slows down the (pre)clinical translation of QDots is their potential toxicity. Several strategies have been employed to optimize common core-shell QDots, such as the use of gradient alloy (GA)-QDots. These particles no longer have a size-dependent emission wavelength, but the emission rather depends on the chemical composition of the gradient layer. Therefore, particles of identical sizes but with emission maxima spanning the entire visible spectrum can be generated. In the present study, two types of GA-QDots are studied with respect to their cytotoxicity and cellular uptake. A multiparametric cytotoxicity approach reveals concentration-dependent effects on cell viability, oxidative stress, cell morphology and cell functionality (stem cell differentiation and neurite outgrowth), where the particles are very robust against environmentally-induced breakdown. Non-toxic concentrations are defined and compared to common core-shell QDots analyzed under identical conditions. Additionally, this value is translated into a functional value by analyzing the potential of the particles for cell visualization. Interestingly, these particles result in clear endosomal localization, where different particles result in identical intracellular distributions. This is in contrast with CdTe QDots with the same surface coating, which resulted in clearly distinct intracellular distributions as a result of differences in nanoparticle diameter. The GA-QDots are therefore ideal platforms for cell labeling studies given their high brightness, low cytotoxicity and identical sizes, resulting in highly similar intracellular particle distributions which offer a lot of potential for optimizing drug delivery strategies.

Cytotoxicity of cadmium-free quantum dots and their use in cell bioimaging.

The use of quantum dots (QDots) as bright and photostable probes for long-term fluorescence imaging is gaining more interest. Thus far, (pre)clinical use of QDots remains limited, which is primarily caused by the potential toxicity of QDots. Most QDots consist of Cd2+ ions, which are known to cause high levels of toxicity. In order to overcome this problem, several strategies have been tested, such as the generation of cadmium-free QDots. In the present study, two types of cadmium-free QDots, composed of ZnSe/ZnS (QDotZnSe) and InP/ZnS (QDotInP), were studied with respect to their cytotoxicity and cellular uptake in a variety of cell types. A multiparametric cytotoxicity approach is used, where the QDots are studied with respect to cell viability, oxidative stress, cell morphology, stem cell differentiation, and neurite outgrowth. The data reveal slight differences in uptake levels for both types of QDots (maximal for QDotZnSe), but clear differences in cytotoxicity and cell functionality effects exist, with highest toxicity for QDotZnSe. Differences between cell types and between both types of QDots can be explained by the intrinsic sensitivity of certain cell types and chemical composition of the QDots. At concentrations at which no toxic effects can be observed, the functionality of the QDots for fluorescence cell visualization is evaluated, revealing that the higher brightness of QDotZnSe overcomes most of the toxicity issues compared to that of QDotInP. Comparing the results obtained with common Cd2+-containing QDots tested under identical conditions, the importance of particle functionality is demonstrated, revealing that cadmium-free QDots tested in this study are not significantly better than Cd2+-containing QDots for long-term cell imaging and that more work needs to be performed in optimizing the brightness and surface chemistry of cadmium-free QDots for them to replace currently used Cd2+-containing QDots.

Comparison of gold nanoparticle mediated photoporation: vapor nanobubbles outperform direct heating for delivering macromolecules in live cells.

There is a great interest in delivering macromolecular agents into living cells for therapeutic purposes, such as siRNA for gene silencing. Although substantial effort has gone into designing nonviral nanocarriers for delivering macromolecules into cells, translocation of the therapeutic molecules from the endosomes after endocytosis into the cytoplasm remains a major bottleneck. Laser-induced photoporation, especially in combination with gold nanoparticles, is an alternative physical method that is receiving increasing attention for delivering macromolecules in cells. By allowing gold nanoparticles to bind to the cell membrane, nanosized membrane pores can be created upon pulsed laser illumination. Depending on the laser energy, pores are created through either direct heating of the AuNPs or by vapor nanobubbles (VNBs) that can emerge around the AuNPs. Macromolecules in the surrounding cell medium can then diffuse through the pores directly into the cytoplasm. Here we present a systematic evaluation of both photoporation mechanisms in terms of cytotoxicity, cell loading, and siRNA transfection efficiency. We find that the delivery of macromolecules under conditions of VNBs is much more efficient than direct photothermal disturbance of the plasma membrane without any noticeable cytotoxic effect. Interestingly, by tuning the laser energy, the pore size could be changed, allowing control of the amount and size of molecules that are delivered in the cytoplasm. As only a single nanosecond laser pulse is required, we conclude that VNBs are an interesting photoporation mechanism that may prove very useful for efficient high-throughput macromolecular delivery in live cells.

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.