Complex Structures Made Simple - Continuous Flow Production of Core Cross-Linked Polymeric Micelles for Paclitaxel Pro-Drug-Delivery.

Translating innovative nanomaterials to medical products requires efficient manufacturing techniques that enable large-scale high-throughput synthesis with high reproducibility. Drug carriers in medicine embrace a complex subset of tasks calling for multifunctionality. Here, the synthesisof pro-drug-loaded core cross-linked polymeric micelles (CCPMs) in a continuous flow processis reported, which combines the commonly separated steps of micelle formation, core cross-linking, functionalization, and purification into a single process. Redox-responsive CCPMs are formed from thiol-reactive polypept(o)ides of polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine) and functional cross-linkers based on dihydrolipoic acid hydrazide for pH-dependent release of paclitaxel. The precisely controlled microfluidic process allows the production of spherical micelles (Dh  = 35 nm) with low polydispersity values (PDI < 0.1) while avoiding toxic organic solvents and additives with unfavorable safety profiles. Self-assembly and cross-linking via slit interdigital micromixers produces 350-700 mg of CCPMs/h per single system, while purification by online tangential flow filtration successfully removes impurities (unimer ≤ 0.5%). The formed paclitaxel-loaded CCPMs possess the desired pH-responsive release profile, display stable drug encapsulation, an improved toxicity profile compared to Abraxane (a trademark of Bristol-Myers Squibb), and therapeutic efficiency in the B16F1-xenotransplanted zebrafish model. The combination of reactive polymers, functional cross-linkers, and microfluidics enables the continuous-flow synthesis of therapeutically active CCPMs in a single process.

Modular Synthesis of Semiconducting Graft Copolymers to Achieve "Clickable" Fluorescent Nanoparticles with Long Circulation and Specific Cancer Targeting.

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

Π-Π interactions stabilize PeptoMicelle-based formulations of Pretomanid derivatives leading to promising therapy against tuberculosis in zebrafish and mouse models.

Tuberculosis is the deadliest bacterial disease globally, threatening the lives of millions every year. New antibiotic therapies that can shorten the duration of treatment, improve cure rates, and impede the development of drug resistance are desperately needed. Here, we used polymeric micelles to encapsulate four second-generation derivatives of the antitubercular drug pretomanid that had previously displayed much better in vivo activity against Mycobacterium tuberculosis than pretomanid itself. Because these compounds were relatively hydrophobic and had limited bioavailability, we expected that their micellar formulations would overcome these limitations, reduce toxicities, and improve therapeutic outcomes. The polymeric micelles were based on polypept(o)ides (PeptoMicelles) and were stabilized in their hydrophobic core by π-π interactions, allowing the efficient encapsulation of aromatic pretomanid derivatives. The stability of these π-π-stabilized PeptoMicelles was demonstrated in water, blood plasma, and lung surfactant by fluorescence cross-correlation spectroscopy and was further supported by prolonged circulation times of several days in the vasculature of zebrafish larvae. The most efficacious PeptoMicelle formulation tested in the zebrafish larvae infection model almost completely eradicated the bacteria at non-toxic doses. This lead formulation was further assessed against Mycobacterium tuberculosis in the susceptible C3HeB/FeJ mouse model, which develops human-like necrotic granulomas. Following intravenous administration, the drug-loaded PeptoMicelles significantly reduced bacterial burden and inflammatory responses in the lungs and spleens of infected mice.

Antibiotic-Loaded Amphiphilic Chitosan Nanoparticles Target Macrophages and Kill an Intracellular Pathogen.

In this work, levofloxacin (LVX), a third-generation fluoroquinolone antibiotic, is encapsulated within amphiphilic polymeric nanoparticles of a chitosan-g-poly(methyl methacrylate) produced by self-assembly and physically stabilized by ionotropic crosslinking with sodium tripolyphosphate. Non-crosslinked nanoparticles display a size of 29 nm and a zeta-potential of +36 mV, while the crosslinked counterparts display 45 nm and +24 mV, respectively. The cell compatibility, uptake, and intracellular trafficking are characterized in the murine alveolar macrophage cell line MH-S and the human bronchial epithelial cell line BEAS-2B in vitro. Internalization events are detected after 10 min and the uptake is inhibited by several endocytosis inhibitors, indicating the involvement of complex endocytic pathways. In addition, the nanoparticles are detected in the lysosomal compartment. Then, the antibacterial efficacy of LVX-loaded nanoformulations (50% w/w drug content) is assessed in MH-S and BEAS-2B cells infected with Staphylococcus aureus and the bacterial burden is decreased by 49% and 46%, respectively. In contrast, free LVX leads to a decrease of 8% and 5%, respectively, in the same infected cell lines. Finally, intravenous injection to a zebrafish larval model shows that the nanoparticles accumulate in macrophages and endothelium and demonstrate the promise of these amphiphilic nanoparticles to target intracellular infections.

Block Length-Dependent Protein Fouling on Poly(2-oxazoline)-Based Polymersomes: Influence on Macrophage Association and Circulation Behavior.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Targeting Cancer Chemotherapy Resistance by Precision Medicine-Driven Nanoparticle-Formulated Cisplatin.

Therapy resistance is the major cause of cancer death. As patients respond heterogeneously, precision/personalized medicine needs to be considered, including the application of nanoparticles (NPs). The success of therapeutic NPs requires to first identify clinically relevant resistance mechanisms and to define key players, followed by a rational design of biocompatible NPs capable to target resistance. Consequently, we employed a tiered experimental pipeline from in silico to analytical and in vitro to overcome cisplatin resistance. First, we generated cisplatin-resistant cancer cells and used next-generation sequencing together with CRISPR/Cas9 knockout technology to identify the ion channel LRRC8A as a critical component for cisplatin resistance. LRRC8A's cisplatin-specificity was verified by testing free as well as nanoformulated paclitaxel or doxorubicin. The clinical relevance of LRRC8A was demonstrated by its differential expression in a cohort of 500 head and neck cancer patients, correlating with patient survival under cisplatin therapy. To overcome LRRC8A-mediated cisplatin resistance, we constructed cisplatin-loaded, polysarcosine-based core cross-linked polymeric NPs (NPCis, Ø âˆ¼ 28 nm) with good colloidal stability, biocompatibility (low immunogenicity, low toxicity, prolonged in vivo circulation, no complement activation, no plasma protein aggregation), and low corona formation properties. 2D/3D-spheroid cell models were employed to demonstrate that, in contrast to standard of care cisplatin, NPCis significantly (p < 0.001) eradicated all cisplatin-resistant cells by circumventing the LRRC8A-transport pathway via the endocytic delivery route. We here identified LRRC8A as critical for cisplatin resistance and suggest LRRC8A-guided patient stratification for ongoing or prospective clinical studies assessing therapy resistance to nanoscale platinum drug nanoformulations versus current standard of care formulations.

Core Cross-Linked Polymeric Micelles for Specific Iron Delivery: Inducing Sterile Inflammation in Macrophages.

Iron is an essential co-factor for cellular processes. In the immune system, it can activate macrophages and represents a potential therapeutic for various diseases. To specifically deliver iron to macrophages, iron oxide nanoparticles are embedded in polymeric micelles of reactive polysarcosine-block-poly(S-ethylsulfonyl-l-cysteine). Upon surface functionalization via dihydrolipoic acid, iron oxide cores act as crosslinker themselves and undergo chemoselective disulfide bond formation with the surrounding poly(S-ethylsulfonyl-l-cysteine) block, yielding glutathione-responsive core cross-linked polymeric micelles (CCPMs). When applied to primary murine and human macrophages, these nanoparticles display preferential uptake, sustained intracellular iron release, and induce a strong inflammatory response. This response is also demonstrated in vivo when nanoparticles are intratracheally administered to wild-type C57Bl/6N mice. Most importantly, the controlled release concept to deliver iron oxide in redox-responsive CCPMs induces significantly stronger macrophage activation than any other iron source at identical iron levels (e.g., Feraheme), directing to a new class of immune therapeutics.

Biodistribution of surfactant-free poly(lactic-acid) nanoparticles and uptake by endothelial cells and phagocytes in zebrafish: Evidence for endothelium to macrophage transfer.

In the development of therapeutic nanoparticles (NP), there is a large gap between in vitro testing and in vivo experimentation. Despite its prominence as a model, the mouse shows severe limitations for imaging NP and the cells with which they interact. Recently, the transparent zebrafish larva, which is well suited for high-resolution live-imaging, has emerged as a powerful alternative model to investigate the in vivo behavior of NP. Poly(D,L lactic acid) (PLA) is widely accepted as a safe polymer to prepare therapeutic NP. However, to prevent aggregation, many NP require surfactants, which may have undesirable biological effects. Here, we evaluate 'safe-by-design', surfactant-free PLA-NP that were injected intravenously into zebrafish larvae. Interaction of fluorescent NPs with different cell types labelled in reporter animals could be followed in real-time at high resolution; furthermore, by encapsulating colloidal gold into the matrix of PLA-NP we could follow their fate in more detail by electron microscopy, from uptake to degradation. The rapid clearance of fluorescent PLA-NP from the circulation coincided with internalization by endothelial cells lining the whole vasculature and macrophages. After 30 min, when no NP remained in circulation, we observed that macrophages continued to internalize significant amounts of NP. More detailed video-imaging revealed a new mechanism of NP transfer where NP are transmitted along with parts of the cytoplasm from endothelial cells to macrophages.

Real-time imaging of polymersome nanoparticles in zebrafish embryos engrafted with melanoma cancer cells: Localization, toxicity and treatment analysis.

BACKGROUND: The developing zebrafish is an emerging tool in nanomedicine, allowing non-invasive live imaging of the whole animal at higher resolution than is possible in the more commonly used mouse models. In addition, several transgenic fish lines are available endowed with selected cell types expressing fluorescent proteins; this allows nanoparticles to be visualized together with host cells. METHODS: Here, we introduce the zebrafish neural tube as a robust injection site for cancer cells, excellently suited for high resolution imaging. We use light and electron microscopy to evaluate cancer growth and to follow the fate of intravenously injected nanoparticles. FINDINGS: Fluorescently labelled mouse melanoma B16 cells, when injected into this structure proliferated rapidly and stimulated angiogenesis of new vessels. In addition, macrophages, but not neutrophils, selectively accumulated in the tumour region. When injected intravenously, nanoparticles made of Cy5-labelled poly(ethylene glycol)-block-poly(2-(diisopropyl amino) ethyl methacrylate) (PEG-PDPA) selectively accumulated in the neural tube cancer region and were seen in individual cancer cells and tumour associated macrophages. Moreover, when doxorubicin was released from PEG-PDPA, in a pH dependant manner, these nanoparticles could strongly reduce toxicity and improve the treatment outcome compared to the free drug in zebrafish xenotransplanted with mouse melanoma B16 or human derived melanoma cells. INTERPRETATION: The zebrafish has the potential of becoming an important intermediate step, before the mouse model, for testing nanomedicines against patient-derived cancer cells. FUNDING: We received funding from the Norwegian research council and the Norwegian cancer society.

Polymersomes Eradicating Intracellular Bacteria.

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.

Zebrafish Embryos Allow Prediction of Nanoparticle Circulation Times in Mice and Facilitate Quantification of Nanoparticle-Cell Interactions.

The zebrafish embryo is a vertebrate well suited for visualizing nanoparticles at high resolution in live animals. Its optical transparency and genetic versatility allow noninvasive, real-time observations of vascular flow of nanoparticles and their interactions with cells throughout the body. As a consequence, this system enables the acquisition of quantitative data that are difficult to obtain in rodents. Until now, a few studies using the zebrafish model have only described semiquantitative results on key nanoparticle parameters. Here, a MACRO dedicated to automated quantitative methods is described for analyzing important parameters of nanoparticle behavior, such as circulation time and interactions with key target cells, macrophages, and endothelial cells. Direct comparison of four nanoparticle (NP) formulations in zebrafish embryos and mice reveals that data obtained in zebrafish can be used to predict NPs' behavior in the mouse model. NPs having long or short blood circulation in rodents behave similarly in the zebrafish embryo, with low circulation times being a consequence of NP uptake into macrophages or endothelial cells. It is proposed that the zebrafish embryo has the potential to become an important intermediate screening system for nanoparticle research to bridge the gap between cell culture studies and preclinical rodent models such as the mouse.

Rab6 regulates cell migration and invasion by recruiting Cdc42 and modulating its activity.

Rab proteins are master regulators of intracellular membrane trafficking, but they also contribute to cell division, signaling, polarization, and migration. The majority of the works describing the mechanisms used by Rab proteins to regulate cell motility involve intracellular transport of key molecules important for migration. Interestingly, a few studies indicate that Rabs can modulate the activity of Rho GTPases, important regulators for the cytoskeleton rearrangements, but the mechanisms behind this crosstalk are still poorly understood. In this work, we identify Rab6 as a negative regulator of cell migration in vitro and in vivo. We show that the loss of Rab6 promotes formation of actin protrusions and influences actomyosin dynamics by upregulating Cdc42 activity and downregulating myosin II phosphorylation. We further provide the molecular mechanism behind this regulation demonstrating that Rab6 interacts with both Cdc42 and Trio, a GEF for Cdc42. In sum, our results uncover a mechanism used by Rab proteins to ensure spatial regulation of Rho GTPase activity for coordination of cytoskeleton rearrangements required in migrating cells.

Rifampicin Nanoformulation Enhances Treatment of Tuberculosis in Zebrafish.

Mycobacterium tuberculosis, the etiologic agent of tuberculosis, is an intracellular pathogen of alveolar macrophages. These cells avidly take up nanoparticles, even without the use of specific targeting ligands, making the use of nanotherapeutics ideal for the treatment of such infections. Methoxy poly(ethylene oxide)- block-poly(ε-caprolactone) nanoparticles of several different polymer blocks' molecular weights and sizes (20-110 nm) were developed and critically compared as carriers for rifampicin, a cornerstone in tuberculosis therapy. The polymeric nanoparticles' uptake, consequent organelle targeting and intracellular degradation were shown to be highly dependent on the nanoparticles' physicochemical properties (the cell uptake half-lives 2.4-21 min, the degradation half-lives 51.6 min-ca. 20 h after the internalization). We show that the nanoparticles are efficiently taken up by macrophages and are able to effectively neutralize the persisting bacilli. Finally, we demonstrate, using a zebrafish model of tuberculosis, that the nanoparticles are well tolerated, have a curative effect, and are significantly more efficient compared to a free form of rifampicin. Hence, these findings demonstrate that this system shows great promise, both in vitro and in vivo, for the treatment of tuberculosis.

Dual Transcriptomics of Host-Pathogen Interaction of Cystic Fibrosis Isolate Pseudomonas aeruginosa PASS1 With Zebrafish.

Pseudomonas aeruginosa is a significant cause of mortality in patients with cystic fibrosis (CF). To explore the interaction of the CF isolate P. aeruginosa PASS1 with the innate immune response, we have used Danio rerio (zebrafish) as an infection model. Confocal laser scanning microscopy (CLSM) enabled visualization of direct interactions between zebrafish macrophages and P. aeruginosa PASS1. Dual RNA-sequencing of host-pathogen was undertaken to profile RNA expression simultaneously in the pathogen and the host during P. aeruginosa infection. Following establishment of infection in zebrafish embryos with PASS1, 3 days post infection (dpi), there were 6739 genes found to be significantly differentially expressed in zebrafish and 176 genes in PASS1. A range of virulence genes were upregulated in PASS1, including genes encoding pyoverdine biosynthesis, flagellin, non-hemolytic phospholipase C, proteases, superoxide dismutase and fimbrial subunits. Additionally, iron and phosphate acquisition genes were upregulated in PASS1 cells in the zebrafish. Transcriptional changes in the host immune response genes highlighted phagocytosis as a key response mechanism to PASS1 infection. Transcriptional regulators of neutrophil and macrophage phagocytosis were upregulated alongside transcriptional regulators governing response to tissue injury, infection, and inflammation. The zebrafish host showed significant downregulation of the ribosomal RNAs and other genes involved in translation, suggesting that protein translation in the host is affected by PASS1 infection.

Enhanced Permeability and Retention-like Extravasation of Nanoparticles from the Vasculature into Tuberculosis Granulomas in Zebrafish and Mouse Models.

The enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NPs) flowing in blood to reach tumors by a passive targeting mechanism. Here, using the transparent zebrafish model infected with Mycobacterium marinum we show that an EPR-like process also occurs allowing different types of NPs to extravasate from the vasculature to reach granulomas that assemble during tuberculosis (TB) infection. PEGylated liposomes and other NP types cross endothelial barriers near infection sites within minutes after injection and accumulate close to granulomas. Although ∼100 and 190 nm NPs concentrated most in granulomas, even ∼700 nm liposomes reached these infection sites in significant numbers. We show by confocal microscopy that NPs can concentrate in small aggregates in foci on the luminal side of the endothelium adjacent to the granulomas. These spots are connected to larger foci of NPs on the ablumenal side of these blood vessels. EM analysis suggests that NPs cross the endothelium via the paracellular route. PEGylated NPs also accumulated efficiently in granulomas in a mouse model of TB infection with Mycobacterium tuberculosis, arguing that the zebrafish embryo model can be used to predict NP behavior in mammalian hosts. In earlier studies we and others showed that uptake of NPs by macrophages that are attracted to infection foci is one pathway for NPs to reach TB granulomas. This study reveals that when NPs are designed to avoid macrophage uptake, they can also efficiently target granulomas via an alternative mechanism that resembles EPR.

Optical micromanipulation of nanoparticles and cells inside living zebrafish.

Regulation of biological processes is often based on physical interactions between cells and their microenvironment. To unravel how and where interactions occur, micromanipulation methods can be used that offer high-precision control over the duration, position and magnitude of interactions. However, lacking an in vivo system, micromanipulation has generally been done with cells in vitro, which may not reflect the complex in vivo situation inside multicellular organisms. Here using optical tweezers we demonstrate micromanipulation throughout the transparent zebrafish embryo. We show that different cells, as well as injected nanoparticles and bacteria can be trapped and that adhesion properties and membrane deformation of endothelium and macrophages can be analysed. This non-invasive micromanipulation inside a whole-organism gives direct insights into cell interactions that are not accessible using existing approaches. Potential applications include screening of nanoparticle-cell interactions for cancer therapy or tissue invasion studies in cancer and infection biology.

Thioridazine in PLGA nanoparticles reduces toxicity and improves rifampicin therapy against mycobacterial infection in zebrafish.

Encapsulating antibiotics such as rifampicin in biodegradable nanoparticles provides several advantages compared to free drug administration, including reduced dosing due to localized targeting and sustained release. Consequently, these characteristics reduce systemic drug toxicity. However, new nanoformulations need to be tested in complex biological systems to fully characterize their potential for improved drug therapy. Tuberculosis, caused by infection with the bacterium Mycobacterium tuberculosis, requires lengthy and expensive treatment, and incomplete therapy contributes to an increasing incidence of drug resistance. Recent evidence suggests that standard therapy may be improved by combining antibiotics with bacterial efflux pump inhibitors, such as thioridazine. However, this drug is difficult to use clinically due to its toxicity. Here, we encapsulated thioridazine in poly(lactic-co-glycolic) acid nanoparticles and tested them alone and in combination with rifampicin nanoparticles, or free rifampicin in macrophages and in a zebrafish model of tuberculosis. Whereas free thioridazine was highly toxic in both cells and zebrafish embryos, after encapsulation in nanoparticles no toxicity was detected. When combined with rifampicin nanoparticles, the nanoparticles loaded with thioridazine gave a modest increase in killing of both Mycobacterium bovis BCG and M. tuberculosis in macrophages. In the zebrafish, the thioridazine nanoparticles showed a significant therapeutic effect in combination with rifampicin by enhancing embryo survival and reducing mycobacterial infection. Our results show that the zebrafish embryo is a highly sensitive indicator of drug toxicity and that thioridazine nanoparticle therapy can improve the antibacterial effect of rifampicin in vivo.

Zebrafish as a model system for characterization of nanoparticles against cancer.

Therapeutic nanoparticles (NPs) have great potential to deliver drugs against human diseases. Encapsulation of drugs in NPs protects them from being metabolized, while they are delivered specifically to a target site, thereby reducing toxicity and other side-effects. However, non-specific tissue accumulation of NPs, for example in macrophages, especially in the spleen and liver is a general problem with many NPs being developed for cancer therapy. To address the problem of non-specific tissue accumulation of NPs we describe the development of the zebrafish embryo as a transparent vertebrate system for characterization of NPs against cancer. We show that injection of human cancer cells results in tumor-like structures, and that subsequently injected fluorescent NPs, either made of polystyrene or liposomes can be imaged in real-time. NP biodistribution and general in vivo properties can be easily monitored in embryos having selective fluorescent labeling of specific tissues. We demonstrate in vitro, by using optical tweezer micromanipulation, microscopy and flow cytometry that polyethylene glycol (PEG) coating of NPs decreases the level of adhesion of NPs to macrophages, and also to cancer cells. In vivo in zebrafish embryos, PEG coating resulted in longer NP circulation times, decreased macrophage uptake, and reduced adhesion to the endothelium. Importantly, liposomes were observed to accumulate passively and selectively in tumor-like structures comprised of human cancer cells. These results show that zebrafish embryo is a powerful system for microscopy-based screening of NPs on the route to preclinical testing.

Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity.

Adipocyte fatty acid binding protein 4, aP2, contributes to the pathogenesis of several common diseases including type 2 diabetes, atherosclerosis, fatty liver disease, asthma, and cancer. Although the biological functions of aP2 have classically been attributed to its intracellular action, recent studies demonstrated that aP2 acts as an adipokine to regulate systemic metabolism. However, the mechanism and regulation of aP2 secretion remain unknown. Here, we demonstrate a specific role for lipase activity in aP2 secretion from adipocytes in vitro and ex vivo. Our results show that chemical inhibition of lipase activity, genetic deficiency of adipose triglyceride lipase and, to a lesser extent, hormone-sensitive lipase blocked aP2 secretion from adipocytes. Increased lipolysis and lipid availability also contributed to aP2 release as determined in perilipin1-deficient adipose tissue explants ex vivo and upon treatment with lipids in vivo and in vitro. In addition, we identify a nonclassical route for aP2 secretion in exosome-like vesicles and show that aP2 is recruited to this pathway upon stimulation of lipolysis. Given the effect of circulating aP2 on glucose metabolism, these data support that targeting aP2 or the lipolysis-dependent secretory pathway may present novel mechanistic and translational opportunities in metabolic disease.

Nanoparticles as drug delivery system against tuberculosis in zebrafish embryos: direct visualization and treatment.

Nanoparticles (NPs) enclosing antibiotics have provided promising therapy against Mycobacterium tuberculosis (Mtb) in different mammalian models. However, the NPs were not visualized in any of these animal studies. Here, we introduce the transparent zebrafish embryo as a system for noninvasive, simultaneous imaging of fluorescent NPs and the fish tuberculosis (TB) agent Mycobacterium marinum (Mm). The study was facilitated by the use of transgenic lines of macrophages, neutrophils, and endothelial cells expressing fluorescent markers readily visible in the live vertebrate. Intravenous injection of Mm led to phagocytosis by blood macrophages. These remained within the vasculature until 3 days postinfection where they started to extravasate and form aggregates of infected cells. Correlative light/electron microscopy revealed that these granuloma-like structures had significant access to the vasculature. Injection of NPs induced rapid uptake by both infected and uninfected macrophages, the latter being actively recruited to the site of infection, thereby providing an efficient targeting into granulomas. Rifampicin-loaded NPs significantly improved embryo survival and lowered bacterial load, as shown by quantitative fluorescence analysis. Our results argue that zebrafish embryos offer a powerful system for monitoring NPs in vivo and rationalize why NP therapy was so effective against Mtb in earlier studies; bacteria and NPs share the same cellular niche.