Enhanced Permeability and Retention-Like Extravasation of

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Enhanced Permeability and Retention-Like Extravasation of Nanoparticles from the Vasculature into Tuberculosis Granulomas in Zebrafish and Mouse Models. Federico Fenaroli, Urska Repnik, Yitian Xu, Kerstin Johann, Simon Van Herck, Pradip Dey, Frode Miltzow Skjeldal, Dominik M Frei, Shahla Bagherifam, Agnese Kocere, Rainer Haag, Bruno G. De Geest, Matthias Barz, David G. Russell, and Gareth Griffiths ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b04433 • Publication Date (Web): 06 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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Enhanced Permeability and Retention-Like Extravasation of Nanoparticles from the Vasculature into Tuberculosis Granulomas in Zebrafish and Mouse Models. Federico Fenaroli1, Urska Repnik1, Yitian Xu2, Kerstin Johann3, Simon Van Herck4, Pradip Dey5, Frode Miltzov Skjeldal1, Dominik M. Frei1, Shahla Bagherifam6, Agnese Kocere1, Rainer Haag5, Bruno G. De Geest4, Matthias Barz3 , David G. Russell2 and Gareth Griffiths1 1 Department of Biosciences, University of Oslo, Blindernveien 31, 0371 Oslo, Norway 2 Department of Microbiology and Immunology, Cornell University College of Veterinary Medicine, C5 109 VMC, Ithaca NY 14853, USA. 3 Institute for Organic chemistry, Johannes Gutenberg-University Mainz, Duesbergweg 10-14 55099 Mainz, Germany 4 Faculty of Pharmaceutical Sciences, Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium 5 Institute of Chemistry and Biochemistry – Organic Chemistry, Free University of Berlin, Takustrasse 3, 14195 Berlin, Germany 6 Department of Radiation Biology, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway

Keywords: nanoparticles, tuberculosis, zebrafish, mouse, epr, mycobacteria Abstract The enhanced permeability and retention (EPR) effect is the only described mechanism enabling nanoparticles (NP) flowing in blood to reach tumours 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 NP 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 nm and 190 nm NP concentrated most in granulomas even ≈700 nm liposomes reached these infection sites in significant numbers. We show by confocal microscopy that NP 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 NP on the ablumenal side of these blood vessels. EM analysis suggests that NP cross the endothelium via the paracellular route. PEGylated NP 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 behaviour in mammalian hosts. In earlier studies we and others showed that uptake of NP by macrophages that are attracted to infection foci is one pathway for NP to reach TB granulomas. This study reveals that when NP are designed to avoid macrophage uptake they can also efficiently target granulomas via an alternative mechanism that resembles EPR.


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The use of nanoparticles (NP) for drug delivery has enormous potential to improve treatment of different diseases. 1, 2 Among these, cancer attracts the most intensive research effort. 3 A crucial factor for NP-mediated chemotherapy is that NP circulating in the vasculature must reach tumors in sufficient concentrations for the released drug to have an enhanced therapeutic effect. Extensive observations from mouse and human systems have converged upon one dominant hypothesis for rationalizing how NP cross the endothelium lining the blood vessels and reach tumors, namely the enhanced permeability and retention (EPR) effect.4 This relatively simple concept relies on the fact that endothelia adjacent to tumors are not tight barriers but have gaps between cells sufficiently large to allow NP up to a critical size to pass through and reach the tumors.5 The upper size limit varies between different solid tumors but estimates have ranged from below 100 nm to over 1µm,6-9 although most publications emphasize a preference for sub-100 nm particles10 for the most effective tumor penetration. Accumulation of NP in tumors is also facilitated by poor lymphatic drainage, which is known to occur in solid tumors.11-13 While the EPR theory is supported by many lines of evidence the concept is arguably of limited efficiency;14 recently, Chan and colleagues surveyed the published literature on the EPR effect and concluded that on average below one percent of drugs administered via NP reaches tumors in mice.15 It should be noted that this review was itself criticized16 and other reviews in the literature report on studies in humans and mouse in which the accumulation can be much higher 17 as for example in (Harrington et al. 2001)18 whose study quantified the uptake of radioactive liposomes in different human tumors. When they expressed the liposomes uptake relative to kg of tumor mass, the values obtained varied from 5.3% for breast tumors and up to 33% for head and neck tumors. Importantly, the actual mechanism and kinetics by which NP are supposed to cross extracellular spaces between endothelial cells in the EPR process are illdefined, according to some authors.19, 20 It has recently been suggested, without supporting evidence that the EPR effect might operate to deliver NP to bacterial infected tissues.21, 22 Our focus here is the granuloma, the specialized assembly of different cells, especially infected macrophages, that is a prominent feature of tuberculosis (TB), the disease caused by the bacterium Mycobacterium tuberculosis (Mtb).23 At present this is the bacterial infection of humans that causes the highest mortality (1.7 million deaths in 2016; WHO: Global tuberculosis Report 2017). Jain’s group, having pioneered our understanding of how newly formed blood vessels access and feed tumors, recently provided compelling data concerning the similarities between TB granulomas and solid tumors.24 For example, both structures are often hypoxic and, consequently, both have the potential to stimulate angiogenesis, the outgrowth of blood vessels towards and into the tumor or granuloma. These new blood vessels are the conduits for drugs, either in free form or NP-linked to reach the


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diseased site. The work of Jain and Tobin24-26 provided evidence that this TB-induced angiogenesis can be manipulated with drugs to increase the efficacy of delivery of small molecules and free antibiotics to the granulomas. Datta and colleagues used a rabbit TB model that, like mouse, limits the possibilities for live imaging of the granulomas and angiogenesis at high resolution. In contrast, Oehlers and colleagues took advantage of the transparency of the zebrafish embryo model of TB. This system, pioneered by the Ramakrishnan group,27 consists in infection via injection of the fish pathogen Mycobacterium marinum (Mm) into zebrafish embryos. They injected mycobacteria intravenously (referred to here as the ‘standard’ infection method) resulting in rapid phagocytosis by blood resident macrophages - which form small, dynamic, granuloma-like structures associated with the endothelium. Our previous study using this system showed that intravenously injected poly lactide co-glycolide (PLGA) NP are avidly taken up by blood resident macrophages which can subsequently migrate to the granulomas. The natural ability of the macrophages to rapidly and efficiently remove all the NP from the circulation and deliver them to the small, endothelium-associated granulomas was striking.28 This study is one of many focused on mycobacteria, including Mtb, that shows promising therapeutic data using NP containing antibiotics.29 An important innovation introduced by Oehlers and colleagues was to inject the bacteria into the dorsally positioned ‘trunk’ of the zebrafish embryo at 2 days post-fertilization (pf) (identified here as the ‘neural tube’ model). This gave rise to relatively large and static granulomas that facilitated the development of newly formed vessels - that were seen in great detail as they grew towards the granuloma, highly reminiscent of angiogenesis growth towards mammalian tumors.25 In the present study we focused specifically on this neural tube model of infection. We first made a detailed histological and ultrastructural analysis of these granulomas which revealed that they have many similarities to the most common form of the human disease, pulmonary TB. After our first experiments revealed that (non PEGylated) PLGA NP were surprisingly inefficient in accessing the neural tube granulomas, we made a detailed analysis of liposomes with surface polyethylene glycol (PEG), or other stealth factors; PEG has been widely used to avoid macrophage uptake of NP, that consequently stay longer in the circulation.30 Our results argue compellingly that various NP having a range of sizes are able to extravasate from blood vessels adjacent to granulomas and accumulate in significant amounts next to these infection foci. We discuss the similarities between this process and the EPR mechanism. Subsequent experiments in a mouse model of TB revealed that these different types of NP were also able to accumulate significantly in lung granulomas containing Mtb. This supports our contention that the zebrafish embryo model is well-suited to serve as a rapid screening tool to select the most effective NP for subsequent testing in mouse. ACS Paragon Plus Environment

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RESULTS AND DISCUSSION Mycobacterium marinum neural tube infection. We first established the infection by injecting red fluorescent Mycobacterium marinum (Mm, DsRed, 200 CFU) at 48 h post-fertilization (pf) into a structure referred to as the ‘trunk’- that our studies confirmed to be the neural tube of zebrafish embryos. This is situated in the dorsal side of the embryo, above the notochord and below the dorsal longitudinal anastomotic vessel (DLAV, Fig 1 A and 1 B). The zebrafish embryos used were genetically modified to express green fluorescent protein (GFP) throughout the blood vasculature.31 The work of Oehlers and colleagues has already shown that Mm-infected macrophages are the first cells to be recruited to this area, where the infection proceeds with the formation of a slowly growing granuloma, which in turn stimulates the assembly of new vasculature.25 In Supporting Fig 1 we show that the newly formed vessels and the primary, parental ones appear dilated and, as a consequence, have a stronger blood supply (Supporting Fig 1 A). Here, we reveal other striking features unique to the neural tube granulomas. During the later stages of infection, between day three and day five after Mm injection (Fig 1 C-H), the granuloma progresses, causing loss of organization of the infected tissues. This is accompanied by necrosis of blood vessels (Fig 1 D-E; Supporting Fig 1 B), which is evident from the loss of green fluorescence of the GFP-labeled endothelial cells next to the granuloma. Around day 5 post-infection extensive bacterial growth led to release of the fluorescent Mm into the medium, leaving a cavity (Fig 1 E, H). This process, which does not occur in the ‘standard’ blood infection model, is highly reminiscent of adult pulmonary TB, the main form affecting humans, whereby bacteria are released from necrotic granulomas of the lungs.32 Another important feature we observed at late stages of infection was the significant higher numbers of neutrophils compared to macrophages in the TB granulomas at day 5 post-infection (Fig 1 I, J). This is also a prominent feature of TB granulomas in mice. Moreover, high numbers of neutrophils have been detected in lung exudates of patients with active TB.33-35


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Figure 1. Overview of neural tube granuloma. A. Schematic drawing of a zebrafish embryo to show the position of the neural tube (green). B shows an enlargement of the boxed area in A in order to indicate the relationship between the dorsal lateral anastomotic vessel (DLAV), neural tube (NT), notochord (N), dorsal artery (DA) and posterior caudal vein (PCV). The arrow shows the Mm injection site. C-E reveal the fluorescence images of the progression of the neural tube infection in the zebrafish embryo (Tg: fli1a:EGFP). C reveals a compact intact granuloma (red) at day 3 post-infection. Green indicates the endothelium of the blood vessels. D. At day 4 postinfection the granuloma becomes highly necrotic and protrudes away on the dorsal region of the ACS Paragon Plus Environment

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fish. E. At day 5 post-infection, the disrupted granuloma is evident after release of some of the bacteria into the medium. F-H show the corresponding transmission images relative to C-E. I shows a granuloma from a fish with non-fluorescent Mm at day 3 post-infection. The dense granuloma can still be seen in transmission light (inset). Both macrophages (red) and neutrophils (green) are present in the granuloma. A quantification (J) confirms the significantly higher number of neutrophils than macrophages in the granulomas. N = 15. Scale bars: 200µm. We then prepared semi-thin sections of five-day infected embryos (overview, Fig 2 A) that were stained with toluidine blue for light microscopy, or prepared for ultrathin sections for EM analysis. This confirmed that the granulomas were within the neural tube, directly adjacent to the transparent notochord, and muscle tissue, which appeared normal (Fig 2 B). Within the neural tube one could distinguish between the uninfected spinal cord precursor cells (Fig 2 C, E, F), which had spherical nuclei and the bacterial-rich infected cells, presumably macrophages, with more irregular nuclear profiles (Fig 2 C-F). A vast area of the infected macrophages appeared to be necrotic (Fig 2 C, D). EM sections of the Mm granulomas confirmed the presence of huge aggregates of necrotic cells containing Mm , areas of extracellular bacteria and cytoplasmic bacteria (not within phagosomes) in the necrotic cells (Fig 2 F, 2 G and Supporting Fig 2 A and B). Many of these bacteria grew as long filaments that were aligned into tight stringlike bundles (Fig 2 G). These structures, mostly seen with virulent mycobacteria in culture have been referred to as ‘cords’ whose formation is stimulated by a ‘cord factor’ made from trehalose dimycolate36 and other components.37 Taken together, all these features make the zebrafish ‘neural tube’ model a very powerful system that more closely resembles pulmonary TB, the most common form of the disease in humans, than does the zebrafish ‘standard model’. This set the stage for evaluating whether intravenously injected NP could access these prominent neural tube granulomas.


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Figure 2. Neural tube granuloma ultrastructure. A shows an overview of a fish with green vasculature at day 4 post-infection showing the red fluorescent Mm granuloma protruding upwards on the dorsal side of the embryo. The inset shows a bright field image of the same embryo. The granuloma is indicated by a red arrow. B is a semi-thin Epon vertical section of the same fish as shown in A (rectangle) that has been stained with toluidine blue. NT indicates the neural tube, N notochord and M muscle. The arrowheads denote the upper boundaries of the neural tube. The three boxed regions are enlarged in C, D and E. In C the arrowhead indicates the infected/necrotic tissue that protrudes on the dorsal side of the fish; the arrows in C-E reveal aggregates of bacteria, and the asterisks in C and E indicate uninfected neural tube precursors cells. M, muscle. F shows a low magnification overview of a part of the infected neural tube. Bacteria are indicated by an arrow. NT shows the typical spherical nucleus of uninfected NT cells, which have very little cytoplasm. Note the different shaped profile of the nucleus of the macrophage (M). Fig G shows a high magnification of a typical necrotic region of the granuloma full of bacteria, arrow. The inset shows a high magnification view of one Mm, with contains multiple lipid bodies (arrow). In F and G the arrowheads indicates the cording phenomenon. Scale bars: A 1mm, A-inset 1mm, B 100µm, C-E 20µm, D 10µm, E 5µm, E-inset 500nm. NP accumulate in the vicinity of granulomas during neural tube infection An overview of NP details is provided in Table 1 while a schematic representation of NP chemistry used in this study can be found in Supporting Figure 3. In order to monitor NP interactions with granulomas we first injected 200 colony-forming units (CFU) of wild type (WT) (non-fluorescent) Mm into the neural tube of zebrafish having green fluorescent vasculature (fli1a:EGFP) at 48h post-fertilization. Granulomas formed after a further 4 days and could be easily seen in transmission light by their dark and swollen appearance (Fig 3A, B) and by the extensive neovascularization (Fig 3 C, D). We then first tested red fluorescent, non-PEGylated PLGA NP (hydrodynamic diameter (Dh) = 396 nm) by injecting them into the caudal vein. These were the same type of NP used in our previous study28 that accumulated readily in macrophages and granulomas in the standard blood model of infection. To our surprise only a low fluorescence signal could be detected in the area of the neural tube granuloma after 4 hours in circulation (Results not shown).


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Nanoparticle type

Polymers and components, MW(Da)

Hydrodynamic diameter (Dh) / nm

PDI

Zeta potential / mV

1) Liposomes NO PEG

PC, 779 Chol, 386

101.5 ± 7

0.05 ± 0.02

-4.88 ± 0.08

2) Liposomes PEG

PC, 779 DSPE-PEG, 2800 Chol, 386

101 ± 4

0.05 ± 0.01

-3.35 ± 0.2

PC, 779 DSPE-PEG, 2800 Chol, 386 PC, 779 DSPE-PEG, 2800 Chol, 386 PLGA, 7000-17000 PVA, 57000-66000

190 ± 1

0.09 ± 0.01

-7.83 ± 0.7

0.35 ± 0.035

-10.7 ± 0.42

0.37 ±0.043

23.95±0.35

6) Quantum dots PEG

NG

20 nm

ND

ND

7) Polyglycerol nanogels

dPG, 6800

8) Colloidal PLApSar

216 ± 5

9) Benzyl Ketal nanoparticles

PLA, 18000-28000 pGlu(OBn)-b-PSar, 18900 pDMA-pBzKEA, 12200

10) Polymer Micelles

pGlu(OBn)-b-PSar, 18900

102 ± 1

3) Liposomes PEG

4) Liposomes PEG

5) PLGA NO PEG

703 ± 31

387 ±10

Animals tested

Zebrafish Zebrafish and Mouse Zebrafish

Zebrafish

Zebrafish Zebrafish

108.9 ± 0.7

65.8 ± 0.6

0.074 ± 0.013 0.28 ± 0.02

-7 ± 0.5

0.174 ± 0.006 0.18 ± 0.03

-28.9 ± 16

Zebrafish

-7.02 ± 0.38 Zebrafish

Zebrafish

-8.33 ± 0.33 Zebrafish and Mouse

Table 1 Overview of NP used in this study and their properties. NG-not given. ND-not determined. Hydrodynamic diameter measures the peak intensity obtained by DLS; +/- represent Standard Deviation.

Having seen how rapidly 70K dextran-Texas red that was injected into the blood localized in neural tube granulomas in the study by Oehlers and colleagues, we asked whether different types of NP, including long-circulating ones could also accumulate there. For this, we used PEGylated liposomes of about 100 nm in hydrodynamic diameter. As controls we used red fluorescent non PEGylated PLGA and far-red labeled PEGylated quantum dots (20 nm); an analysis of the circulation times of the used NP revealed that PEGylated 100 nm liposomes ACS Paragon Plus Environment

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circulated longer than quantum dots and PLGA, with the latter essentially not circulating at all already at 4 hours after the injection (Supporting Figure 4 A, B). When injected in zebrafish larvae infected with M.marinum, the fluorescence signal from PEGylated liposomes in the area of the granuloma (Fig 3 E-K) increased significantly with time while the low fluorescence signal in control uninfected areas remained constant. We determined that on average 6.5% of the injected liposomes localized in the granuloma area 4 h after injection. This value is almost 10 times higher than the uninfected control area next to granulomas which had a value of 0.73 %. In comparison, non-PEGylated PLGA NP accumulated significantly less, with 1.6 % at the infected area 4 hours after the injection. To our surprise, the smallest NP we tested, PEGylated quantum dots localized significantly less in the granulomas than the PEGylated liposomes, with a value of 2.6 % (Fig 3 K) after 4 h. A more detailed analysis of the kinetics of NP accumulation in granulomas showed that the concentration peaked at 4 h for the liposomes with only a slight increase at twelve hours while the quantum dots accumulate rapidly (10 minutes) but then do not further increase in the following hours. Despite their poor circulation, non PEGylated PLGA steadily accumulated with time, even though the values remained relatively low even at 12 h after injection (Supporting Figure 5A).


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Figure 3. Time-dependent accumulation of NP in one neural tube granuloma. A shows the transmission image of a neural tube infected embryo at day 5 post-infection. The boxed area shows the granuloma enlarged in Fig B. C-J show the fluorescence images of the same embryo


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at different times. C and D show the green fluorescence of blood vessel endothelial cells; note the increased density of disorganized vessels in and around the granuloma (box) - enlarged in D. This embryo was injected in the caudal vein with PEG 101 nm liposomes (red) and imaged at 10 minutes, 30 min and 4 h after injection (E, G, I respectively). The boxes in E-I are enlarged in FK; note the striking accumulation of liposomes. A quantitation of this experiment is shown in K. We quantified the fluorescence relative to the liposomes at different times in the granuloma region or from an equivalent uninfected area (background). Also quantified from a separate group of larvae were Non-PEGylated PLGA and PEGylated quantum dots, both after 4 h. For each NP used, N = 9. Scale bars: A, C, E, G, I- 500µm B, D, H, J: 1 µm Effect of NP size and surface properties on granuloma accumulation There is general consensus from cancer studies that an NP coated with stealth-like polymers, e.g. PEG or polysarcosine30, 38 results in a reduced uptake by macrophages, relative to non-coated NP. This results in a longer circulation time, which increases the probability for any passive accumulation process to take place.39 Further, smaller NP (below 100 nm) are often believed to be the most effective at concentrating in tumors.40 We therefore decided to test whether these criteria also applied to our granuloma system. For this, we compared liposomes of 101 nm, with and without PEG, as well as PEGylated liposomes of about 190 nm and 703 nm. A quantitative fluorescence analysis in the zebrafish embryo confirmed that liposomes 101 nm circulated much better when provided with a PEG group and that these could also flow in the blood for longer times than the 190 and 703 PEGylated liposomes (Supporting Figure 4 C, D). At 4 h after injection the fluorescence signal in the neural tube granulomas (7.6%), relative to the total signal from the 101nm PEGylated liposomes, was modestly, albeit significantly higher than that of non-PEGylated 101nm Liposomes (6.5%) (Fig 4 A). Consequently, the presence of PEG on the NP seems to increase their localization in TB granulomas. However, to our surprise, 190 nm PEGylated liposomes accumulated significantly more (9.7%) than 101 nm PEGylated ones while 703 nm PEGylated liposomes had a lower level of accumulation (4.1%). All these NPs accumulated in the granuloma areas significantly more than in uninfected areas of the zebrafish (Fig 4 A). In addition, we conducted an analysis of accumulation of these different NP at different time points. This revealed that liposomes 101 PEG and Liposomes 190 PEG accumulated similarly at different times while liposomes 101 NO PEG and Liposomes 703 PEG concentrated markedly less (Supporting figure 5 B, C). In order to establish whether different types of NP shared the propensity of liposomes to accumulate in granulomas we analyzed a wide range of NP possessing different chemistry. We therefore tested polyglycerol nanogels (NG) (108 nm), polylactic acid NP coated with polysarcosine-block-poly(O-benzylglutamic acid) (PLA-pSar) NP (216 nm), benzyl ketal NP (65 nm), and Polymer micelles (102 nm), all known for their long circulating properties.41, 42 All these NP accumulated adjacent to neural tube granulomas with values comparable to those ACS Paragon Plus Environment

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obtained for liposomes at 4h after injection (4.5, 6.3 and 4.8, 6.5 %, respectively). Again, there was essentially no signal in adjacent uninfected areas of the embryos (Fig 4 B).

Fig 4. Role of NP size and chemistry in accumulation in granulomas. A shows the comparison of 101nm liposomes with and without PEG and, in addition, 190 and 703 nm PEGylated liposomes. As in Fig 3 the background signal for each type of Liposome represents the signal for an equivalent size of an uninfected area (N ≥ 40). In B a comparison is shown of four different NP with different compositions for their ability to accumulate in granulomas after 4 hours (N ≥ 12).

Details of NP extravasation from vessels neighboring granulomas


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Our next step was to use light microscopy of live larvae to analyze the pathway that allows the NP from the vasculature to localize in the vicinity of the neural tube granuloma so efficiently. Mm was first injected into the neural tube and, after four days we injected red fluorescent 101 nm liposomes into the caudal vein, and followed their fate over time. Confocal stacks reconstructed in 3D using the image analysis software package IMARIS allowed us to observe in detail the process of extravasation of the liposomes after 10, 20 and 30 min. This process is shown as maximal intensity projections (Fig 5 A-C) and as a 3D reconstruction for each time point (Supporting video 1, 2 and 3); Figures 5 D-I show snapshots of these videos. The red liposomes appeared to adhere in small aggregates to the luminal surface of the green endothelial cells and to traverse these green cells (Fig 5 A, D, G; Fig 5 B, E, H; Supporting videos 1 and 2) in zones (presumably gaps) that appeared to connect the luminal to the ablumenal (outside) aspect of the endothelium. The aggregates that accumulated on the latter surface were much bigger than those found on the inner surface, suggesting that they were somehow trapped on the ablumenal side. Our analysis revealed that NP were localized outside of blood vessels already after 10 minutes and that this extravasation increased at later time points (5 C, F, I and Supporting video 3). After 4 h post injection more liposomes concentrated outside the vessels; at this time point some NP aggregates could be found completely detached from the vessels (Supporting Fig 6 and Supporting video 4). Overall, these fluorescence data argue that a significant fraction of injected NP extravasated and accumulated in close vicinity of the granuloma after 4h. The Supporting video 4 revealed that even at this relatively early time point a fraction of the NP appeared to be in contact with the foci enriched in mycobacteria.


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Fig 5. Confocal microscopy imaging of dynamic NP extravasation from blood vessels. A-C are maximum projections of stacks of confocal sections relative to 101 nm Liposomes (red) with


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PEG extravasating from a vessel (green) at 10, 20 and 30 min after injection. White arrowheads indicate NP accumulations outside the endothelium. Lower fluorescence intensity in green and red channel at 30 min is due to photo bleaching. D-I are snapshots of 3D videos made with the program IMARIS (see Supporting video 1, 2 and 3), where NP accumulation and the endothelium are seen as Iso-surfaces. D, G and E, H are enlargements of the boxes in A and B. White arrowheads indicate liposomes accumulating outside the endothelium while white arrows indicate NP aggregations in the lumen of the vessel. F, I are enlargement of the boxes in C. Arrowheads indicate liposomes concentrations outside the vessel. N= 20. Scale bars: A, B, C 4µm; D, E, F 1µm, G, H, I - 2µm. Electron microscopy of liposome localization adjacent to granulomas. In order to better follow the fate of 101 PEGylated Liposomes in vivo, we analyzed blood vessels adjacent to granulomas in ultrathin sections by transmission EM. This type of analysis was performed at 4h, a time point in which the volume of NP that extravasated was high enough to be easily detected in ultrathin sections. A low magnification overview (Supporting Fig 7 A and B) shows that the blood vessels can be close to, or even in contact with the neural tube, that is heavily infected with Mm. By our approach individual liposomes, as well as aggregates could be easily identified due to their uniform size and their strong reactivity with osmium, which cross-links lipids and made the NP appear highly electron dense. However the specimen preparation procedure seemed to affect their shape, with many appearing oval rather than spherical (Fig 6 B-E, Supporting Fig 7 C-E). With this approach, using samples in which only a tiny volume of the tissue of interest was analyzed, we mostly visualized the regions where the majority of NP accumulated, namely outside the vessels. There, we noted three different types of liposome aggregations. First, NP clusters were found in between endothelial cells and contained by the basal lamina (Fig 6 A and C and E). While NP in the blood lumen were surrounded by an electron dense background (Supporting Fig 7 C), those trapped between endothelial cells and basal lamina had an electron translucent one (Fig 6 C), suggesting that the two sites are functionally distinct. Second, accumulation of NP was seen in paracellular regions between two cells distal to the tight junctions, that appeared to be structurally intact (Fig 6 B, Supporting Fig 7 D, E). Such images suggest that the NP had crossed open tight junctions that then closed, leaving the extravasated NP to accumulate between opposing plasma membranes separating two cells. The third type of observation was of NP that had crossed the basal lamina and had accumulated in electron translucent areas of extracellular space (Fig 6 A, B, D, Supporting figure 7 A, B). These zones were clearly distinct from the blood vessels and the endothelial cells, as well as the basement membrane, which were all more electron dense than the extracellular sites where the NP accumulated. We saw no evidence of NP crossing the cells by transcytosis. We occasionally saw


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some extracellular spaces (gaps) between endothelial cells but little evidence that NP were associated with these sites (Results not shown).


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Fig 6: Ultrastructure of 101nm PEG liposomes crossing the blood endothelial barrier. (A) A low magnification overview image of an inter-segmental blood vessel in the peri-granuloma region outside the neural tube. Red: blood lumen, yellow: vessel wall (endothelial cells) and possibly a pericyte; blue: basal lamina (BL), RBC: red blood cell, Lip: liposomes (coloured in green) Scale bar, 10 µm. (B-E) The boxed regions in A are magnified in B-E to show different localizations of liposomes: between endothelial cells (B, D), between endothelial cells and the basal lamina (C, E), or across the basal lamina outside the vessel (B, D, E). The red arrowhead in B indicates the intact (closed) junctional region between two endothelial cells. The accumulation of the liposomes is distal from the junction. N=22. Scale bars B-E, 500 nm. NP accumulation in mice Finally, we wanted to establish whether our findings in zebrafish larvae correlated with those in established mouse models. For this, two different NP from the above studies were tested in a mammalian infection system using the human pathogen Mycobacterium tuberculosis (Mtb) and the mouse, an important preclinical model in the TB field. For this, mice infected via aerosol with Mtb for four weeks were injected intravenously with either PEGylated 101 nm liposomes or 136 nm polymer micelles via the tail vein. The animals were sacrificed 24 h after NP injection and their lungs sectioned for confocal microscopy analysis. Both types of NP accumulated strongly in granulomas, enriched in Mtb while few NP were found in areas of the lung that were devoid of Mtb (Fig 7 A-D). Quantification of the fluorescence signal in different areas confirmed that the amount of liposomes and polymer micelles in granulomas was about 20, and 5 times higher, respectively, than that found in healthy tissues of the lung (Fig 7 E). These data indicated that, also in the mouse model, NP can effectively extravasate out of blood vessels and accumulate in the tissues where the pathogen resides, but not in the normal surrounding tissue devoid of bacteria.


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Fig 7: Accumulation of 101 nm PEGylated liposomes and polymer micelles in lung granulomas of mouse infected with Mtb. Accumulation of liposomes (A) or polymer micelles (C) in sections of infected lungs enriched in Mtb. B and D show the significantly lower concentration of liposomes (B) or polymer micelles (D) found in non-infected parts of the lungs. E indicates quantitation of this experiment for liposomes (left) or polymer micelles (right) expressed either as total fluorescence (intensity) or voxels. In both cases, significantly more NP accumulate in infected areas than uninfected ones. N ≥ 6 for analysis of both liposomes and polymer micelles. Sale bars: 20 µm.

Using the superior imaging capacity of the zebrafish embryos system we provide evidence here that a process reminiscent of EPR operates to allow long-circulating NP to extravasate from the blood circulation and reach TB granulomas in zebrafish embryos. In our earlier studies we showed the potential of the zebrafish larvae to be a useful tool for monitoring the fate of NP, for models of both TB28 and cancer.43 In those studies Mm bacteria (TB) (or human cancer cells) were injected into the vasculature of 2-3 day old zebrafish embryos. One to three days later NP were injected into the blood. In the TB model, the small granulomas that formed were intimately associated with the blood vessel endothelium. Within minutes of being injected, the (nonPEGylated) NP were taken up by blood macrophages and by 24 h they co-localized extensively with all the granulomas. Here, we used a different model of Mm infection where the bacteria are injected into a dorsal site that had previously been referred to as the ‘trunk’ of the embryo.26, 44, 45 Our detailed immunofluorescence, histological and ultrastructural analysis identified this injection site as the neural tube, the developing spinal cord of the zebrafish embryo.46, 47 In contrast to the previous blood vessel infection the neural tube is a better model for pulmonary TB in humans, the most common clinical form.48 Whereas the blood vessel granulomas in the standard model are small and dynamic,49 the neural tube model produces granulomas that are much larger, and relatively stable, with infected macrophages being responsible for the bulk of the volume of the early granuloma stages.26 Within a few days of infection, the infected macrophages become highly necrotic, leading to formation of cavities and release of bacteria into the exterior of the fish, a process highly reminiscent of human pulmonary TB. Another striking feature of this fish model is the massive accumulation of neutrophils and large areas full of extracellular bacteria that appear to be dividing in the granulomas; again, both are phenomena that have been described in human TB granulomas.50, 51 At this late stage when massive necrosis and extracellular bacteria predominate, the innate immune system of the zebrafish larvae has clearly lost the ability to control the infection. These neural tube granulomas are also very potent in inducing angiogenesis, a process occurring in a hypoxic environment, that is dependent on VEGF secretion from infected


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macrophages.26 Angiogenesis related to granulomas is a topic that has been little discussed in the TB field; see, for example52 but is evident in striking radiographic images of bronchial arteries of TB patients.53 A better understanding of the permeability of the vessels surrounding granulomas is paramount for the development of new drugs both in free form or encapsulated into NP.54 Nanomedicine has attracted increasing interest in the TB field in the last two decades and several groups have shown improvement in NP-encapsulated drugs over the free drugs for therapy, as well as for a decrease in side-effects.29 In addition, the severe toxicity of the (free) anti TB drugs not only affects the patients’ well-being during treatment but is also a major driving force for acquired drug-resistance, when doses are not strictly adhered to. Given this background, the ability to see even small blood vessel adjacent to granulomas in great detail in live animals is a crucial advantage of the zebrafish model for following the fate of NP that are aimed at TB granulomas. Whereas in the standard blood vessel injection model of zebrafish embryos, nonPEGylated PLGA NP access macrophages and the entire population of granulomas rapidly and efficiently, in the neural tube model they access the granulomas very inefficiently. We attribute this to the fact that standard model granulomas are in direct contact with the vasculature and that blood macrophages efficiently take up these PLGA NP flowing in the lumen of the vessels.28 In contrast, in the neural tube model, while vessels grow around the granuloma these are not in direct contact with the sites of the bacterial aggregates; the NP need to exit from the vessels in order to access the granuloma. The PLGA NP, at 4 hours post-injection, were probably too rapidly removed by blood-borne macrophages to have a chance to accumulate in the neural tube granulomas. We nevertheless saw that accumulation of PLGA NP increased at 12 h, suggesting that also in the neural tube granulomas uninfected macrophages taking up NP could be migrating into the infected tissue. In the present study, we systematically tested NP, mostly liposomes, that were PEGylated in order to reduce or avoid uptake by macrophages. These are precisely the conditions aimed for in cancer therapy with clinical anti-cancer NP such as Doxil, doxorubicinenclosing liposome particles which are PEGylated in order to increase their circulation time, and thereby the chance that the EPR process can operate in well-vascularized tumors.55 In our system, the PEGylated liposomes accumulated rapidly, as observed in some cancer studies,56 and efficiently near the neural tube granulomas. In fact, these NP crossed the endothelial barrier adjacent to the granulomas within minutes. In the following hours as much as 20 percent of the injected liposomes accumulated in sites contiguous with the granulomas. Although many reviews on EPR have a tendency to cite an upper size range in the 100200 nm range for NP to take advantage of the EPR process the actual limit by which particles can extravasate to reach tumors is reported to vary significantly with the tumor type.57 In fact, the measured pore sizes are surprisingly broad and the upper size limit is more than 1µm in


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many studies. The most dramatic example involved the accumulation of significant numbers of roughly 1µm length, rod shaped bacteria- Bifidobacterium bifidum into mouse tumors; the bacteria were injected into the tail vein of tumor bearing mice.58 More recently, similar results were shown with Lactobacillus casei.59 In our results where we quantified the accumulation of different NP into granulomas in zebrafish larvae, 190 nm liposomes actually accumulated slightly better than the 100 nm ones while the 700 nm liposomes had less access, but nevertheless still concentrated significantly at the granuloma sites. A range of different types of NP with different chemistry and sizes did not show significant differences in their ability to concentrate at the granuloma site. Collectively, these data argue that the zebrafish larval vascular endothelium adjacent to the granulomas is rather promiscuous in allowing different NP particles to access the infection foci. Our light microscopy live imaging of NP shed some light on the mechanism by which aggregates of NP are able to extravasate from the blood vessels adjacent to the TB granulomas. Small aggregates of liposomes collected in foci on the inner surface of the blood vessel, especially intersegmental vessels and new vessels sprouting from them. These aggregates were continuous with (mostly) larger aggregates on the outside, abluminal side of the endothelium. The thin section EM analysis gave more insights into the pathway by which liposomes extravasated from the blood vessels. The most striking accumulation of liposomes was evident in enlarged extracellular spaces in between the plasma membranes of two adjacent endothelial cells (Fig 6 and Supporting Figure 7). Intriguingly, these NP aggregates were distal to the (closed) tight junctions leading us to suggest that from the luminal aggregates these NP had crossed the (open) junctions that then closed, leaving the NP in pockets between two plasma membrane domains of adjacent endothelial cells. For such a model to work, the tight junctions would need to open and close. Consistent with this idea, it has been shown that VEGF, that is secreted by infected macrophages,26 can bind to endothelial cells and dynamically open and close their tight junctions, thereby transiently opening the paracellular pathway between adjacent cells.60 If this interpretation is correct it implies that a barrier, most likely the basal lamina serves to delay the exit of the NP on the abluminal side of the endothelium, leading to a concentration in the extracellular space relative to the blood vessels lumen. The extracellular space where NP concentrated was easy to distinguish from the (partially electron dense) cytoplasm of the endothelial cells and from the vessel lumen since it was electron transparent. Moreover, by EM, we saw no evidence of NP crossing the cells by transcytosis, nor of large spaces (gaps) between endothelial cells, which were rare. It should also be stated that the classical EPR mechanism represents a combination of accumulation of NP in tumors with a block in lymphatic drainage, which in normal tissues has the capacity to remove the NP.4 In the zebrafish larvae whereas the blood flow develops within the first 2 days, the lymphatic system is likely not yet fully functional at day 6 post-fertilization,


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the time in which we injected our NP.61 This is thus a fortuitous feature of our TB model in that the lymph system is non-functional, as it is in solid tumors. Our observations are reminiscent of recent findings using confocal microscopy revealing a dynamic release of puncta of (sub-100 nm) fluorescent NP from blood vessels adjacent to tumors in a mouse skin models of cancer; this appears to be a highly dynamic process referred to as ‘vascular bursts’.62, 63 These authors described fluorescent NP explosively extravasating from the blood vessels in stochastic pulses lasting around 10 minutes at apparently random sites. Whether this process involves the same mechanism that is responsible for the extravasation of NP that we observe remains to be determined. Taken together, our results in the neural tube zebrafish model of TB argue that the use of long-circulating NP for treating this disease appeared promising and further testing in a mammalian model of TB was the logical next step. We therefore proceeded by injecting our fluorescent NP into C57/BL6 mice infected with Mtb, a widely used model for investigating the host response to infection, as well at the bacterial response to the immune environment.64, 65 Our findings in mice confirmed our zebrafish observations and we quantified a significant accumulation of two types of NP in Mtb granulomas relative to non-infected areas of the mouse TB-infected lungs. CONCLUSION We consider the results of this study to be highly relevant for two reasons. First, enrichment of NP in granulomas via an EPR-like process in zebrafish embryos could be confirmed in a standard preclinical TB infection model, the mouse. This extends the promising data in the literature showing the potential of NP for tuberculosis therapy. Second, the validation in mouse of the data obtained first in the zebrafish model show that this easy to visualize vertebrate system is a powerful tool that should be considered as a general intermediate model between in vitro cell cultures and mammalian systems for rapidly screening and characterizing drug-encapsulated NP. MATERIALS AND METHODS Nanoparticles preparation: Preparation of Liposomes: Phosphatidyl choline (PC) was bought from Lipoid (product E PC3), Cholesterol (Chol) from Sigma and Phosphoethanolamine PEG 2000 (PE-PEG) from Avanti lipids (product 880120P). Briefly, for PEGylated liposomes, 3 ml of chloroform was placed in a Florence flask containing 2.4 mg of PC, 0.64 mg of Chol and 0.69 mg of PE-PEG and 5 µl of ATTO 550 or ATTO 633 labelled DOPE (ATTO-TEC). Non-PEGylated liposomes had 3.09 mg of PC, 0.64mg of Chol and 5 µl of ATTO 550 labelled DOPE. The prepared mixtures were then dried at room temperature in the dark using a rotatory evaporator. The drying step conditions


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were: 150 millibar for 1h, 80 millibar for 30 min, 16 millibar for 10 min. After this process, a film can be seen on the flask that was then hydrated at 700 C with 1 ml of PBS and vortexed to allow complete solubilisation. After this passage, the re-suspended lipids are aspirated with a gas-tight syringe and the solution is passed through different sizes filters using a mini-extruder (Avanti lipids) mounted on a heating block at 70 0 C. Liposomes were analysed by dynamic light scattering (DLS) and Zetasizer in a solution 1 mg/ml in PBS pH 7.4. Preparation of PLGA-Nile red: See66 PEGylated Quantum Dots : QtrackerTM 655 vascular was purchased from ThermoFisher scientific. Preparation of polyglycerol nanogels (P-NGs). Dendritic polyglycerol azide dPG-N3 (3.0 mg, 3.52 µmol of N3) and dPG-cyclooctyne (2 mg, 2.3 µmol of cyclooctene) were dissolved separately in Milli-Q-water (0.5 mL). The solutions were cooled down to 4 °C, mixed and added quickly to a magnetically stirred acetone at 900 rpm (320 mL), (Supporting figure 8). The reaction was quenched by the addition of excess water after 30 min and the mixture was stored overnight at room temperature. Precipitated polyglycerol nanoparticles were obtained as shiny blue dispersions and. Acetone was evaporated to obtain blue shining nanogels dispersions in water. The nanogels were dialyzed in water for 2 days and concentrated by evaporation of water. Concentration was determined by lyophilizing the solution. Nanogels were analyzed by DLS and Zetasizer in a solution 1 mg/ml in PBS pH 7.4. Preparation of benzyl ketal nanoparticles: Block copolymers composed of a hydrophilic poly (N,N-dimethylacrylamide) block and a pH-sensitive poly(2-((2-(benzyloxy)propan-2yl)oxy)ethyl acrylate) block were synthesized by RAFT polymerization and subsequent trithiocarbonate end-group removal. For fluorescent labeling with Cyanine5, a small fraction of pentafluorophenylacrylate was incorporated in the hydrophobic block followed by reaction with Cyanine5-cadaverine (Supporting Figure 9). The Cyanine5 -labeled polymers were purified via dialysis and lyophilized. Block copolymer micelle NP were prepared by directly dissolving the polymer in aqueous medium. NP in a solution 2 mg/ml in PBS pH 7.4 were characterized by DLS and Zetasizer. Preparation of PLA-pSar: The preparation of poly-(D,L-lactide)-based (PDLLA-based) nanoparticles was adapted from literature and modified 41. A stock solution of nile red in chloroform with c = 1 mg/mL was prepared and 0.375 mL of the stock solution were added to 37.5 mg PDLLA (Mw = 18000-28000 g/mol) on a balance. Chloroform was added to give a total amount of 1.25 g chloroform. 10 mg pGlu (OBn)27-b-pSar182-Ac were dissolved in 3 mL Millipore water (MP-H2O). The aqueous phase was added to the organic phase and subjected to ultrasonication in a glass vial under ice cooling for 180 s at 70% amplitude in a pulse regime (10 s sonication, 10 s pause) for the preparation of colloidal nanoparticles. A Branson Sonifier II W-250 digital with a 1/4” tip was used for ultrasonication. The obtained milky-pink miniemulsion was slowly stirred overnight at room temperature until evaporation of the organic solvent was completed. To separate the nanoparticles from bigger aggregates, the colloidal


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solution was filtered through syringe filters with a pore size of 0.80 µm (Millex-AA Syringe Filter Unit, mixed cellulose esters) and afterwards stored at 4 °C. The PLA-pSar NP were analysed in a solution 0.3 mg/ml in a solution of PBS pH 7.4 by DLS and Zetasizer. Preparation of Polymer micelles: Polymeric micelles were prepared from the amphiphilic block copolymer pGlu(OBn)-block-pSar by dual asymmetric centrifugation. For the preparation of polymeric micelles labeled with the fluorescent dye Alexa Fluor 647 80% (20 mg) unlabeled and 20% (5 mg) AF647-labeled polymer were used. The synthesis of pGlu (OBn)-block-pSar-Ac and pGlu (OBn)-block-pSar-AF647 was adapted from literature and modified 41, 67 (Supporting Figure 10). For this, 200 µL (20 mg) of a 100 mg/mL stock solution of pGlu (OBn)26-b-pSar188Ac in chloroform and 5 mg of pGlu(OBn)27-b-pSar182-AF647 were added to a sterile 2 mL vial and the chloroform was evaporated overnight. The next day, 250 µL Millipore water (MP-H2O) and 350 mg ceramic beads (SiLibeads ZY-S, 0.3-0.4 mm) were added to the vial and the polymer was allowed to swell for four hours. Centrifugation was performed for 20 min at 3500 rpm with a dual asymmetric centrifuge (SpeedMixer DAC 150.1 CM, Hauschild & Co.KG). After centrifugation, the slightly turbid solution was separated from the beads with an Eppendorf Pipette and the vial was rinsed five times with 50 µL MP-H2O at a time. The polymer micelles were analysed in a 0.3 mg/ml PBS pH 7.4 solution by DLS and Zetasizer. Zebrafish experiments Zebrafish embryos husbandry: We used either zebrafish larvae with fluorescent vasculature, Tg (fli1a:EGFP)31 or wild type embryos. Zebrafish embryos were kept in Petri dishes containing embryo water68 supplemented with phenylthiourea (0.003%, Aldrich). The water was kept at a constant temperature of 28.50C. All experiments have been conducted according to the ethical standards and legislation for animal research in Norway. All activity was approved and overseen by the Norwegian food safety authority. Zebrafish injections: In order to perform microinjections, borosilicate needles (GC100T-10, Harvard instruments) were prepared using a pipette puller (P-97, Sutter instruments). The needles were then connected to an Eppendorf Femtojet Express pump and handled using a Narishige MN-153 micromanipulator. All the injection were performed on zebrafish larvae previously sedated with tricaine (Finquel; 0.02% in embryo water) and placed on a petri dish containing a hardened solution of 2% agarose in milliQ water. The injection of WT Mm or Mm – DsRed (pMSP12::dsRed2) were performed at 48 h post fertilization with an injection of 250 CFU in the neural tube. Nanoparticle injections were carried out in the posterior caudal vein at four days after the infection. Injection volumes were adjusted for each NP in order to inject 10 ng of fluorescent nanoparticles equivalent to a dose of 10 mg/kg. Zebrafish fluorescent imaging and nanoparticle accumulation analysis: Zebrafish embryos were imaged at different times after the injection using a Leica DFC365FX stereo microscope with a 1.0x planapo lens. For quantifying NP accumulation in granulomas images were taken at 30X, and the analysis was performed using the program Fiji (https://fiji.sc/). We first performed a ‘rolling ball’ background subtraction and measured the ACS Paragon Plus Environment

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overall fluorescence of the zebrafish embryo. Using the rectangle tool, we then measured the fluorescence relative to the area of the neural tube granuloma and the fluorescence of an area that remained uninfected. These two values were divided by the overall fluorescence of the embryo and expressed as percentages representing the specific nanoparticle accumulation and the background, respectively. Zebrafish larvae without a visible blood flow were not analysed. For analysis of NP circulation times, images of the caudal region were taken at 120X and an image of the whole fish was taken at 30X. For obtaining absolute values, at each time point, the fluorescence was measured at three systematically placed sites along the caudal artery (imaged at 120X) using the line selection tool in Fiji. The average of these numbers was then divided by the average fluorescence of the whole fish when imaged at 30X. For conversion to percentages, the absolute value obtained at 5 minutes was considered 100% while 0% was the average value in the caudal artery of three zebrafish not injected with NP (background fluorescence). In depth imaging of the extravasation process was performed using a Zeiss LSM 880 with Fast AiryScan using a Leica LD LCI 63X/1.2 objective with glycerol immersion. Here zebrafish larvae after injection were placed on a glass bottom dish (MatTek) and covered with a solution of low melting point agarose (Sigma). After solidification of the solution, embryo water supplemented with Tricaine was added to the dish. Zebrafish electron microscopy: For Epon embedding, fish were fixed with 1% glutaraldehyde in 60 mM HEPES, pH 7.4 minimum overnight. Samples were post-fixed with 2% osmium tetroxide in 1.5% potassium ferricyanide for 2 h on ice, followed by incubation with 1% tannic acid for 30 min, inactivation of tannic acid with 1% sodium sulfate for 15 min, contrasting with 2% aqueous uranyl acetate for 2 h, and with 2% uranyl acetate in 70% ethanol for another 2 h, all at room temperature. Next, samples were dehydrated using a graded ethanol series (70/80/90/96/100%), progressively infiltrated with Epon, then flat embedded between two sheets of Aclar film, polymerized at 60 °C overnight. Fish were remounted in the appropriate orientation to cut longitudinal or sagittal sections using an ultramicrotome -Ultracut UCT (Leica Microsystems, Austria). For histological analysis, 1 µm thick sections were transferred to adhesion SuperFrost Plus glass slides (Menzel Gläser), stained with 0.1% toluidine blue diluted in 2.5% sodium carbonate, mounted using epon resin and after overnight heat polymerization imaged with an Axioplan 2 imaging light microscope with a Zeiss Plan-NEOFLUAR 40X/1,3 lens, equipped with an Axiocam camera and the AxioVision software (all Carl Zeiss Microscopy, Germany). For the ultrastructural analysis, 80 nm thin sections were transferred to formvar- and carbon-coated copper grids, contrasted with 0.2% lead citrate for 15 s and analyzed in a JEM1400 transmission electron microscope (JEOL, Japan); images were recorded with a TemCamF216 camera and the EM-MENU software (TVIPS, Germany). For fluorescence analysis on Tokuyasu thawed cryo-sections, fish were fixed with 4% paraformaldehyde and 0.1% glutaraldehyde in 60 mM HEPES, pH 7.4 minimum overnight. Samples were embedded in bovine gelatin blocks and infiltrated with 2.3 M sucrose minimum overnight. 400 nm thick sections were cut at - 80 °C and transferred to adhesion SuperFrost Plus glass slides. Sections were stained with 0.5 µg/ml DAPI, embedded in Mowiol 4-88 mounting ACS Paragon Plus Environment

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medium and examined with an Axioplan 2 imaging light microscope for endogenous GFP fluorescence to localize vessels, for endogenous red fluorescence to localize loaded liposomes and for DAPI fluorescence to visualize cell nuclei. Parallel sections were stained with toluidine blue and mounted in water to obtain a histological overview. Mice infection experiments: Mice were anesthetized with isoflurane and around 103 Erdman (smyc’::mCherry) bacteria in 25µl of PBS plus 0.5% Tween 80 were delivered intranasally as detailed in Tan et al.64 After 4-week infection, mice were injected 100ul of fluorescent liposome or polymer micelles (solutions were 4 mg/ml and 5 mg/ml in PBS, respectively) through retroorbital injection. 24h later, mice were euthanized in a 0.5% CO2 chamber until the animals stopped breathing. The thoracic cavity was opened and the lung lobes were removed from the cavity. The lung lobes were subsequently put in 4% paraformaldehyde for fixation overnight. Confocal analysis was performed as detailed previously.65 Briefly, lung tissue was cut into 1 mm thick slices using a razor blade. Then tissue was permeabilized in PBS plus 3% BSA plus 0.1% Triton X-100 buffer at room temperature for 1h in the dark. Samples were washed 3 times with PBS and mounted with mounting medium (Vectorshield). Images were taken using a Leica SP5 confocal microscope and quantified by Volocity software. Analysis of the data in mouse was performed without ‘Rolling ball’ fluorescence background subrtraction. Statistical analysis: The statistical analysis was carried out using the software GraphPad Prism 6. Comparisons of different NP (Figure 3 and 4) in infected and uninfected tissues had been carried out using a one way ANOVA with Tukey multiple comparison tests. For mice experiments (Figure 7) both liposomes and polymer NP for Total intensity and Voxel were analysed with an unpaired t test with Welch’s correction. Significance levels are indicated as, **p < 0.005, ***p < 0.001, ****p < 0.0001. Supporting information Supporting information files are available at the ACS Publications website. These include images showing additional features of the neural tube model (Supporting Fig 1 and 2), a schematic representation of the NP used (Supporting Fig 3), kinetics of NP flow in zebrafish embryos (Supporting Fig 4), kinetics of NP accumulation in granulomas (Supporting Fig 5), confocal 3D Stack of NP accumulating in a granuloma (Supporting Figure 6), additional TEM images of extravasating NP in proximity to granulomas (Supporting Figure 7) and additional characterization on the used NP. Three videos showing in detail the 3D arrangements of figure 5 are available (Supporting video 1, Supporting video 2, Supporting video 3) together with the 3D reconstruction relative to Supporting figure 6 (Supporting video 4). Acknowledgements Gareth Griffiths group acknowledges the generous funding by the Norwegian Cancer Society and Research council Norway. David G. Russell and Yitian Xu are supported by the National Institutes of Health USA, award AI 134183 and AI118582. Bruno De Geest and Simon Van


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Herck acknowledge Ghent University (BOF-GOA) for funding. The work of Pradip Dey in Haag’s group was supported by a DRS-Honors post-doctoral fellowship from the Free university of Berlin. Kerstin Johann and Matthias Barz received funding from the Max Plank Graduate Center. We are very grateful to David Tobin for his constant support and especially for his help in establishing the neural tube infection model. The generosity of cited members of the zebrafish community who have made their transgenic fluorescent fish lines available to the community is warmly applauded. Lars Herfindal was generous in his help in the preparation of liposomes. The generous support of the IBV EM facility (Head: Norbert Roos) and the Imaging platform (Head: Oddmund Bakke) is acknowledged.

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Enhanced Permeability and Retention-Like Extravasation of

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