EMIM 2018 ControlCenter

Online Program Overview Session: PS-11

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Imaging Infection

Session chair: Catherine Chapon - Fontenay-aux-Roses, France; Jan R. Zeevaart - Pretoria, South Africa
 
Shortcut: PS-11
Date: Wednesday, 21 March, 2018, 6:15 PM
Room: Lecture Room 03 | level -1
Session type: Parallel Session

Abstract

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6:15 PM PS-11-1

Introductory Talk by Greetje Vande Velde - Leuven, Belgium

This talk provides an overview of state-of-the-art research and refers to the following presentations selected from abstract submissions.

6:35 PM PS-11-2

Elucidation of oropharyngeal and tracheal human papillomavirus infection by in vivo bioluminescence imaging (#276)

S. Schelhaas1, 3, M. Becker2, 3, S. Eligehausen1, M. Schelhaas2, 3

1 Westphalian Wilhelms-University Münster, European Institute for Molecular Imaging, Münster, Germany
2 Westphalian Wilhelms-University Münster, Institute of Cellular Virology, Münster, Germany
3 Westphalian Wilhelms-University Münster, Cluster of Excellence 'Cells in Motion', Münster, Germany

Introduction

Human papillomaviruses (HPV) have not only been recognized as the etiological cause of cervical cancer, but they are also associated with oropharyngeal cancer and recurrent juvenile papillomatosis of the upper respiratory tract. Incidents of oropharyngeal cancers caused by HPV are notably increasing, whereas the rare juvenile papillomatosis is complicated to treat and may result in airway obstruction and death due to recurrence. To date, no animal model exists to evaluate HPV infections in these tissues in vivo

Methods

We employed HPV type 16 (HPV16) pseudovirions, which are capable of infecting cells and tissues, but are abrogated in completion of the viral life cycle and cannot cause pathogenicity or carcinogenesis. Instead, the particles harbor pseudogenomes that allow reporter gene expression, such as the firefly luciferase (lucf). We infected isoflurane-narcotized BalbC mice with HPV-lucf virions via the nose or through a tracheal tubus. Bioluminescence imaging (BLI) was performed using an IVIS Spectrum. Tissue was collected to verify the presence of mCherry-expressing pseudovirions by immunohistochemistry.

Results/Discussion

HPV16-lucf infected cells expressed firefly luciferase, as has been verified by BLI in cell culture experiments. After nasal virus application, infection of the snout was detected by in vivo BLI, whereas tracheal infection was observed after tracheal virus application only. Interestingly, with both infection methods, bioluminescence signal in the lung was observed. The peak light emission occurred two days after infection. At this time point, mCherry expressing HPV pseudovirions were detected in the lung and in the lacrimal glands after nasal infection. 

Conclusions

We demonstrate that HPV pseudovirions are capable of infecting respiratory tissues of mice. In the future, this will allow in depth preclinical work on HPV infections in the oropharynx and in the trachea. HPV infection of the lung was surprising, as epidemiologically no pathogenic HPV infections of this organ have been reported. This indicates that HPV infections of the lung may be abortive in nature. Interestingly, as infectivity of HPV pseudovirions in the lung appears high, it may be worthwhile to explore HPV16 as vehicle for novel gene therapies in the lung.

Acknowledgement

This work was supported by the Deutsche Forschungsgemeinschaft (DFG), Cells-in-Motion Cluster of Excellence (EXC1003 – CiM), University of Münster, Germany and within the Infect-ERA initiative by funding from the Federal Ministry for Education and Research (BMBF, 031L0095A).

Keywords: human papillomavirus, mouse model, bioluminescence imaging
6:45 PM PS-11-3

In vivo imaging of B. pertussis infection and interactions in the airways of non-human primates in a model of whooping cough (#41)

T. Naninck1, C. Mayet1, V. Contreras1, S. Langlois1, L. Bossevot1, L. Coutte2, C. Locht2, N. Klimova1, 3, P. Sebo3, R. Le Grand1, C. Chapon1

1 CEA, IDMIT, Fontenay aux roses, France
2 Institut Pasteur de Lille, INSERM U1019, Lille, France
3 Institute of Microbiology, CASL, Prague, Czech Republic

Introduction

Whooping cough, due to Bordetella pertussis infection, is today a public health problem. Recent studies on non-human primates (NHP) indicate that acellular vaccines protect from symptoms but not from infection, confirming clinical data1,2. To develop more effective vaccines, a better understanding of the bacterial interactions with the host is needed. We thus developed fluorescence imaging techniques including in vivo Fibered Confocal Fluorescence Microscopy (FCFM)3 to explore the respiratory tract and assess interactions between B. pertussis and antigen presenting cells (APCs) in baboons.

Methods

FCFM in airways was first evaluated ex vivo on NHP explants. A solution of anti-HLA-DR mouse antibody labelled with AlexaFluor647 and a solution of acriflavine were dropped topically in the bronchus of a lung lobe and in a tracheal ring to label specifically the APCs and the other cellular structures, respectively. For in vivo studies, GFP-expressing B. pertussis was inoculated by intranasal and intra-tracheal routes in young baboons. Besides, monoclonal anti-HLA-DR antibody, labelled with AF647, was administered by topical application in the trachea to specifically target and label APCs. FCFM, coupled with bronchoscopy, was performed in the lower respiratory tract at day -2, 2, 7, 14 and 21 post-infection. Validation by immunohistofluorescence was then performed on tissues post-mortem. 

Results/Discussion

Animals infected with a GFP-expressing B. pertussis B1917 strain developed the classical clinical symptoms for whooping cough as previously described in baboons infected with wild-type strains4,2. We were also able to specifically label and detect cells of interest like APCs in the airways of NHP ex vivo using FCFM (Figure 1). Furthermore, in vivo FCFM coupled with bronchoscopy allowed us to detect bacterial and APC localizations and interactions in the lower respiratory tract of young baboons after B. pertussis-GFP infection in baboons (Figure 2 A-B). Ex vivo analyses also confirmed the interactions between B. pertussis and APCs in lungs (Figure 2 C) and allowed the detection of the bacteria 27 days post-infection in bronchi (Figure 2 D).

 

Conclusions

These findings confirm previous published in vitro data about strong interactions between Bordetella pertussis and APCs5. This approach using fluorescence imaging will then be a useful tool to describe the mechanisms of action of the bacteria during infection to develop more effective vaccines against pertussis. Moreover, this imaging protocol may also be implemented to study many diverse respiratory infectious diseases like tuberculosis or influenza for instance.

References

1.         Klein, N. P., Bartlett, J., Rowhani-Rahbar, A., Fireman, B. & Baxter, R. Waning Protection after Fifth Dose of Acellular Pertussis Vaccine in Children. N. Engl. J. Med. 367, 1012–1019 (2012).

2.         Warfel, J. M., Zimmerman, L. I. & Merkel, T. J. Acellular pertussis vaccines protect against disease but fail to prevent infection and transmission in a nonhuman primate model. Proc. Natl. Acad. Sci. U. S. A. 111, 787–792 (2014).

3.         Thiberville, L. et al. In vivo imaging of the bronchial wall microstructure using fibered confocal fluorescence microscopy. Am. J. Respir. Crit. Care Med. 175, 22–31 (2007).

4.         Warfel, J. M., Beren, J., Kelly, V. K., Lee, G. & Merkel, T. J. Nonhuman Primate Model of Pertussis. Infect. Immun. 80, 1530–1536 (2012).

5.         Lamberti, Y., Gorgojo, J., Massillo, C. & Rodriguez, M. E. Bordetella pertussis entry into respiratory epithelial cells and intracellular survival. Pathog. Dis. 69, 194–204 (2013).

Ex vivo imaging of airways by Fibered Confocal Fluorescence Microscopy

Fig. 1: Imaging of airways by Fibered Confocal Fluorescence Microscopy (FCFM). Ex vivo FCFM pictures on tracheal (A,B) and lung (C,D) explants. Prior to ex vivo imaging, tissues were stained with acriflavine (green) and with either anti-human HLA-DR AF647 (A,C) or isotypic control -IgG2a AF647 antibodies- (B,D) (red).

In vivo imaging with FCFM coupled with bronchoscopy in B. pertussis B1917-GFP infected baboons
Fig.2: In vivo FCFM (A) coupled with bronchoscopy (B) of the trachea of baboons 2 days post-infection with B.pertussis B1917-GFP.  (APCs in red; bacteria in green). Ex vivo confocal microscopy in lung tissue of (C) the interactions between APCs (CD163+, in red) and GFP-B.pertussis (green) after lung-bacteria co-culture and (D) the B.pertussis bacteria detected post-mortem with anti-LOS-A antibody.
Keywords: Whooping cough, pertussis, FCFM, bronchoscopy, bacterial infection, fluorescence imaging
6:55 PM PS-11-4

Multimodal imaging allows non-invasive assessment of the progression of lung and brain infection in mouse models of Cryptococcus neoformans infection (#231)

L. Vanherp1, A. Ristani1, J. Poelmans1, A. Hillen1, M. Brock2, K. Lagrou3, G. Janbon4, U. Himmelreich1, G. Vande Velde1

1 KU Leuven, Imaging and Pathology, Biomedical MRI, Leuven, Belgium
2 University of Nottingham, School of Life Sciences, Fungal Biology Group, Nottingham, United Kingdom
3 KU Leuven, Microbiology and Immunology, Clinical Bacteriology and Microbiology, Leuven, Belgium
4 Pasteur Institute, Mycology, RNA Biology of Fungal Pathogens, Paris, France

Introduction

Fungal infections caused by Cryptococcus species typically start in the lungs but can disseminate to the brain via the bloodstream. Preclinical studies assessing disease progression and dissemination are often limited to end-point analysis of the fungal burden or histology, which precludes longitudinal studies. By combining lung computed tomography (CT) and brain magnetic resonance imaging (MRI) with bioluminescence imaging (BLI), our aim was to gain insights in the progression of both lung and brain infection in different mouse models of C. neoformans infection.

Methods

As model of disseminated disease, Balb/C mice (n=3) were infected by intravenous injection of 50 000 cells of a newly engineered luciferase-expressing C. neoformans strain (NE1270). Mice were scanned with BLI and brain MRI (9.4T or 7T, Bruker Biospec) on day 3, 5 and 7. To study lung infection and a potential progression to brain infection, mice were infected with 500 or 50 000 bioluminescent C. neoformans cells via intranasal or oropharyngeal route (n=3-6 per group). These mice were scanned using BLI (1-2x/week), lung CT (1/week) and brain MRI (1/week). After sacrificing, ex vivo BLI was performed and the fungal burden in the organs was quantified (colony-forming unit counting). Additional animals were sacrificed at intermediate time points to correlate imaging readouts with fungal load.

Results/Discussion

In the model of disseminated disease, brain infection could be detected with BLI on day 3, while MRI only showed brain lesions on day 5. In addition, bioluminescent signals could be observed in other organs such as the spleen and kidneys. In the intranasal model, the progression of lung disease could be followed by BLI, while CT confirmed the presence of lung lesions (Fig. 1). Concurrently, we observed an increase in the BLI signal from the nose region. This complicated further assessment and quantification of brain involvement, as MRI could detect brain lesions in some animals. Mice infected via the oropharyngeal route did not present with this nose signal. Currently, we are investigating whether BLI can be used to further define the timeframe of lung to brain transition in this model. Quantification of the BLI signal and lesion volumes (CT/MRI) in the lungs (intranasal model) or the brain (intravenous model) showed a strong correlation with the fungal burden in the respective organs.

Conclusions

The combination of anatomical imaging techniques (CT/MRI) with BLI allowed monitoring of the progression of lung and brain infection. Imaging readouts were found to correlate strongly with the fungal burden in these organs, indicating that this approach is a non-invasive alternative that can lead to longitudinal insights in the evolution of the fungal load. Future studies will focus on the application of these techniques to further narrow down the timeframe of blood-brain barrier crossing in cryptococcosis models and to non-invasively assess therapeutic approaches.

Acknowledgement

This work was supported by funding provided by the European ERA-NET project CryptoVIEW.

Multimodal imaging of pulmonary Cryptococcus infection
BLI and CT allowed non-invasive assessment of the progression of lung disease in the intranasal model after infection with 50 000 or 500 bioluminescent Cryptococcus cells. A) BLI showed a progressing lung disease and signal from the nasal region. B) CT showed the presence and increase of hyperintense lesions in the infected lungs.
Keywords: Fungal infection, Multimodal imaging, Cryptococcus, Murine Models
7:05 PM PS-11-5

Dynamic assessment of immune cell recruitment in pulmonary aspergillosis models using perfluorocarbon particles and 19F MRI (#142)

S. Saini1, J. Poelmans1, S. Liang1, 2, R. Verbeke3, B. Attili4, G. Vande Velde1, H. Korf5, I. Lentacker3, S. de Smedt3, K. Lagrou6, U. Himmelreich1

1 KU Leuven, Biomedical MRI unit, Leuven, Belgium
2 Philips Research China, Shanghai, China
3 University of Gent, General Biochemistry and Physical Pharmacy, Gent, Belgium
4 KU Leuven, Radiopharmaceutical Research laboratory, Leuven, Belgium
5 KU Leuven, Hepatology laboratory, Leuven, Belgium
6 KU Leuven, Clinical Bacteriology and Mycology, Department of Microbiology and Immunology, Leuven, Belgium

Introduction

Depending on the type of immunosuppression, progression of pulmonary fungal infection can lead to invasive pulmonary aspergillosis (IPA) in immunocompromised subjects, recruiting certain types of innate immune cells to lungs. It is crucial to understand particularly the interplay between the host’s immune system & pathogen. We aimed to understand the dynamics of disease progression & migration of inflammatory immune cells to the lungs in A. fumigatus infection mouse models using two clinically applied immunosuppressive (IS) drugs with the help of fluorine magnetic resonance imaging (19F MRI).

Methods

For immunosuppression in Balb/c mice, hydrocortisone acetate (9mg/mouse) was injected s.c. in the HCA group (n=10) on d-3/d -1. Alternatively, cyclophosphamide (200mg/mouse) was i.p. injected on d-4/d-1 (CY group, n=10) prior to infection. On d0, A. fumigatus (fluc+) infection was induced by intranasal administration of 1x106 (HCA group) or 5x105 spores (CY group), retrospectively [1]. Infected immunocompetent (I-IC group, n=4) received 1x106 spores. Non-infected IC mice (n=3) served as control. 19F MRI was performed with a purpose-built double-tuned coil 1H/19F coil commencing 1h post IV injection (d0/d1) of PFCE particles [2] containing fluorosurfactant Zonyl® FSP (ZPFCE) using Bruker 9.4T scanner. In vivo CT was performed on d1/d3. Ex vivo BLI was performed on d3 on the excised lungs.

Results/Discussion

The HCA group showed higher pulmonary 19F MRI signal intensity compared to the CY group, corresponding to higher in vivo ZPFCE labeled immune cell infiltration post fungal infection. We also observed transient 19F signal in the lungs of I-IC group, which was dissolved after d2 (fig 1). No detectable 19F MRI signal was observed in the lungs of N-IC group. 19F hot spots were also identified in the ‘lymph node’ region of HCA group and were not found in I-IC, CY or control groups. We noticed differences in ex vivo BLI in two different immunocompromised groups where a significantly larger increase in BLI signal intensity from lungs was observed in CY group when compared to the HCA group. No BLI signal was found in I-IC and N-IC mice groups indicating clearance of fungal burden from the lungs by the immune system. Quantitative CT imaging data confirmed lesion development in both HCA & CY groups but not in control group indicating differences in terms of lesion volume from whole lungs(fig 2).

Conclusions

We showed distinctive interactions between host & pathogen in IPA models by tracking pulmonary recruitment of immune cells (19F MRI), which correlated with fungal load of A. fumigatus (CT, BLI & CFUs). Our findings indicate that the HCA group developed excessive inflammation & less fungal infection in contrast to the CY group where infection predominates [3], based on peculiar immune environment created by different clinical IS drugs. This indicates the potential of 19F MRI for providing sensitive and quantitative data on immune cells infiltration in infectious diseases models in vivo.

References

[1] Poelmans J, Hillen A, Vanherp L, Govaerts K, Maertens J, Dresselaers T, Himmelreich U, Lagrou K, Vande Velde G. Longitudinal, in vivo assessment of invasive pulmonary aspergillosis in mice by computed tomography and magnetic resonance imaging. Laboratory Investigation (2016) 96, 692–704.

[2] Dewitte H, Geers B, Liang S, Himmelreich U, Demeester J, De Smedt SC, Lentacker I. Design and evaluation of theranostic perfluorocarbon particles for simultaneous antigen-loading and 19F-MRI tracking of dendritic cells. Journal of Controlled Release 169 (2013) 141–149.

[3] Helioswilton SC, Tonani L, Ribeiro Barros Cardoso C and Regina Von Zeska Kress M. The Immune Interplay between the Host and the Pathogen in Aspergillus fumigatus Lung Infection. BioMed Research International (2013) 693023, 14.

Acknowledgement

The research leading to these results was supported by Marie Curie’s ITN BetaTrain project and was partly funded by FWO.

Fig. 1: In vivo longitudinal 19F MRI follow-up of immune cell in A. fumigatus model
19F MRI signal is shown as hot spots and was overlaid on anatomical MRI (1H) images from hydrocortisone acetate (HCA), cyclophosphamide (CY) and infected immunocompetent (I-IC) groups. The non-infected control group did not show detectable 19F MR signal in the lungs, (L=lungs, H= heart, R= 30mM reference).
Fig.2: In vivo quantification of A. fumigatus infected mice models using computed tomography & CFUs
Quantitative CT data showing the differences in lesion volume on day 1 and day 3 post infection in HCA (hydrocortisone acetate) model and CY (Cyclophosphamide) model. Colony forming units (CFUs) were counted from the homogenised lungs from all the experimental models  and cross validated with the in vivo 19F and CT data.
Keywords: Immune cells, 19F MRI, immunosuppression, imaging, Infectious diseases
7:15 PM PS-11-6

Neutrophil dynamics in the pulmonary vasculature (#281)

J. Secklehner1, 2, J. B. G. Mackey1, 2, K. De Filippo2, J. Vuononvirta2, M. B. Headley3, M. F. Krummel3, N. Guerra4, L. M. Carlin1, 2

1 Cancer Research UK Beatson Institute, Glasgow, United Kingdom
2 Imperial College London, Inflammation, Repair and Development, London, United Kingdom
3 University of California, San Fransisco, Department of Pathology, San Fransisco, California, United States of America
4 Imperial College London, Life Sciences, London, United Kingdom

Introduction

Lung immune cells must be regulated to limit pathology while mounting a robust defence. Paradoxically, the immune system can both antagonise and benefit tumours. In cancer, neutrophils are associated with poor prognosis and linked to lung metastasis in breast cancer. During severe and/or chronic inflammation and cancer, blood neutrophils increase, where a subset are developmentally immature. Neutrophil maturation is essential to the development of effector mechanisms, however, methods for identifying murine immature neutrophils and analysing their activity in vivo are required.

Methods

Neutrophils are easily activated ex vivo; hence, we used lung intravital microscopy (IVM) to visualize interactions in vivo. We analysed localization, speed and duration of neutrophil: NK cell interactions in live mice in homeostasis or acute endotoxin-induced lung inflammation.  We used confocal laser-scanning microscopy of the live mouse lung in vivo and ex vivo, agarose inflated precision cut lung slices with multicolour labelling by fluorescent antibodies and dyes, transgenic reporter mice and adoptive transfer of cells in combination with multicolour flow cytometry to investigate the regulation and behaviour of lung leukocytes.

Results/Discussion

Immature and mature neutrophils have been identified degree of Ly6G. We confirmed their phenotypes based on nuclear morphology and recent mitotic activity within bone marrow. We developed methods that distinguish neutrophil maturity for their direct study in vivo by IVM of the lung and other experiments to test behaviour. Interactions with other immune cells can regulate neutrophils. Natural Killer (NK) cells are enriched in the lung where they may form a ‘resident’ intravascular population. We hypothesised NK cells regulate neutrophils in the lung vasculature. We found that lung NK:neutrophil interactions frequently occur for 5-10 minutes and remarkably, at times, material transfers from the neutrophils to NK cells. As predicted, endotoxin led to a rapid increase in neutrophil numbers in the lung, but, importantly, in the NK cell-depleted group this effect was substantially intensified. Cell-tracking of neutrophils revealed that NK cells also affect steady-state neutrophil motility.

Conclusions

We propose that NK:neutrophil interactions in the pulmonary vasculature mediate alterations to neutrophil behaviour and may signify a check-point for restricting neutrophilic infiltration during lung inflammation. This might enable us to develop new strategies to locally modify neutrophil behaviour in the lung without affecting systemic function. Additionally, we are now able to identify immature and mature neutrophils efficiently in the vasculature and we are using these methods to study their behaviour and putative roles in pathology.

Acknowledgement

Medical Research Council, Imperial College London, National Heart & Lung Institute Foundation and Cancer Research UK

Keywords: Immunology, Cancer, Tumor immunology, Inflammation, Intravital microscopy
7:25 PM PS-11-7

Red-light-switchable antibiotics: towards theranostics of bacterial infections (#37)

W. Szymanski1, 2, M. Wegener2, M. J. Hansen2, A. J. M. Driessen3, B. L. Feringa2

1 University Medical Centre Groningen, Department of Radiology, Groningen, Netherlands
2 University of Groningen, Centre for Systems Chemistry, Groningen, Netherlands
3 University of Groningen, Groningen Biomolecular Sciences and Biotechnology Institute, Groningen, Netherlands

Introduction

Medical treatments employ using bioactive compounds that evoke a pharmacological response by interacting with molecular targets in the human body. The selectivity of this interaction is crucial and the lack of it leads to the emergence of severe side-effects in the body and toxicity in the environment.[1]

Theranostics[2] aims at solving this issue using a combination of: advanced molecular diagnostics, which allows the doctors to precisely locate the disease; and locally-activatable treatment which can be locally activated, avoiding the emergence of side effects, toxicity and drug resistance.

Methods

Here, new photopharmacological[3] antibiotics will be introduced[4] that can be reversibly activated using deep-tissue-penetrating red light. The activating light could be delivered externally to an infection site discovered by imaging (yellow arrows, Fig. 1). Alternatively, optical imaging agents for infection can be used, and the light emitted from them can be used for unbiased local activation of the antibiotic (red arrow, Fig. 1).

Trimethoprim was chosen as the bioactive component. It is active against a broad spectrum of Gram-positive and Gram-negative bacteria and widely used in the clinic. It was modified with photoswitchable units (azobenzenes). Subsequent modification of the photoswitch moiety lead to structures that allowed us to control their activity with visible light.

Results/Discussion

A library of photoresponsive antibiotics was created and screened for the bactericidal activity against a model E. coli CS1562 strain. For the best compound, irradiation with red light at 652 nm effected photoisomerization to a photostationary state (PSS) of cis:trans = 87:13 (Fig. 2A).

In antibiotic activity assays, one half of a divided stock solution in DMSO was irradiated for 2.5 h with red light at 652 nm, before treating bacteria with the two separate samples in two-fold dilution series. To our delight, this experiment revealed a dramatic photoactivation effect: Whereas non-irradiated compound remained largely inactive with a MIC50 > 80 μM (Fig. 2B), red light-irradiated compound induced bacteriostasis down to 20 μM, with an observed MIC50 of 10 μM (Fig. 2B). It is worth noting at this point that photoisomerization with red light at close proximity also works effectively in aqueous medium.

Conclusions

We successfully developed diaminopyrimidines bearing azobenzene photoswitches, whose activity can be controlled by light. Remarkably, these compounds allowed for the full in situ photocontrol of antibacterial activity with green and violet light, making it possible to trigger both the activation and deactivation in the presence of bacteria. Apart from showcasing the activation of a biological agent otherwise inactive within the investigated concentration range, we were able to do so while also shifting the wavelength of activation from the UV range into the near-infrared therapeutic window.

References

[1]    Lancet (London, England) 2000, 356, 1255–9.
[2]    Bioconjug. Chem. 2011, 22, 1879–903.
[3]    a) J. Am. Chem. Soc. 2014, 136, 2178–91; b) Nat. Chem. 2013, 5, 924–8; c) Angew. Chem. Int. Ed. 2016, 55, 10978-10999.
[4]    J. Am. Chem. Soc. 2017, DOI: 10.1021/jacs.7b09281

Acknowledgement

M. W. gratefully acknowledges a postdoctoral fellowship of the German Research Foundation (WE 5922/1). We gratefully acknowledge generous support from NanoNed, The Netherlands Organization for Scientific Research (NWO-CW, Top grant to B. L. F. and NWO VIDI grant no. 723.014.001 for W. S.), the Royal Netherlands Academy of Arts and Sciences (KNAW), the Ministry of Education, Culture and Science (Gravitation programme 024.001.035) and the European Research Council (Advanced Investigator Grant no. 694345 to B. L. F.).

Figure 1
Activation principles for light-controlled antibiotics in theranostic approach.
Figure 2
Structure of the optimised design, together with photostationary states at different wavelengths (A) and difference in antibacterial activity (growth curves) for the irradiated and non-irradiated samples.
Keywords: theranostics; optical imaging; antibiotics; photopharmacology; visible light
7:35 PM PS-11-8

In Vivo Tracking and Quantification of Inhaled Aerosol using Magnetic Particle Imaging towards Inhaled Drug Delivery Monitoring (#449)

Z. W. Tay1, P. Chandrasekharan1, X. Y. Zhou1, D. Hensley2, B. Zheng1, S. M. Conolly1

1 University of California, Berkeley, Bioengineering, Berkeley, California, United States of America
2 Magnetic Insight, Inc, Alameda, California, United States of America

Introduction

Pulmonary delivery of therapeutics is attractive but it is challenging to monitor and quantify the delivered aerosol / powder [1]. Currently, SPECT and PET are used but require inhalation of radioactive tracers [2]. Magnetic particle imaging (MPI) is an emerging medical imaging technique [3] that produces a sensitive tracer image [4] of superparamagnetic iron oxide nanoparticles (SPION) with zero ionizing radiation and robust imaging in the lung [5-7]. By mixing SPIONs into the aerosol, we can image & quantify aerosol mass deposited, delivery efficiency and evaluate lung clearance in vivo. 

Methods

Aerosol with droplet size smaller than 4.0 microns was generated by the Kent Scientific AeronebTM Nebulizer. Micromod PerimagTM SPIONs were added to the mix (final concentration of 5 mg / ml Fe) before aerosolization to provide an MPI-visible label of the aerosol. 40-week-old female Fischer 344 rats (180 - 200 g) were used. Delivery of the aerosol (net 0.06 mg Fe) was conducted by either forced ventilation (endotracheal intubation) or pulsed flow directed into the mouth cavity. Ventilation rate was varied and delivery efficiency evaluated. MPI imaging (respiratory gating) used a custom-built 3D 6.3 T/m field-free-line MPI scanner [8].  X-ray imaging was performed on a Kubtec Xpert 40. Timecourse, quantitative imaging of the lung and droppings was done to evaluate lung clearance. 

Results/Discussion

The lung phantom experiment in Fig 1b used aerosolized Doxorubicin HCl as a model drug and SPIONs mixed into the aerosol. The SPIONs have minimal effect on the droplet density / aerodynamic performance at the 5 mg/ml. MPI and Fluorescence Imaging shows that MPI image intensity is linearly quantitative of the Doxorubicin HCl deposited in the lung phantoms. Because MPI can image at depth without any tissue attenuation effects [9], MPI can track and quantitate aerosolized drug deposition. In vivo MPI scans in Fig 1c demonstrate that MPI can track and quantify the SPION biodistribution after delivery by aerosol. MPI images of rats with two forced ventilation rates shows that slower rates result in better delivery of aerosol throughout the lung while fast ventilation results in focal deposition. Timecourse imaging over 2 weeks (Fig 2) shows that MPI enables sensitive and quantitative measurement of SPION clearance from lung through the GI tract and into the droppings. 

Conclusions

MPI imaging is robust and quantitative in the lung and has zero ionizing radiation compared to current nuclear medicine methods for aerosol tracking. MPI monitoring of the mucociliary clearance is useful for long-term controlled release applications. SPIONs can also produce heat via RF excitation [10] to actuate drug release or perform hyperthermia therapy. With high sensitivity and high contrast images, MPI ventilation imaging could offer quantitative monitoring of the distribution and efficacy of drug aerosol delivery methods to inform and improve inhalable drug-delivery treatments.

References

  1. Ibrahim M, Verma R, Garcia-Contreras L. Inhalation drug delivery devices: technology update. Med Devices. 2015 Feb 12;8:131–139. 
  2. Dolovich M, Labiris R. Imaging drug delivery and drug responses in the lung. Proc Am Thorac Soc. 2004;1(4):329–337. 
  3. Gleich B, Weizenecker J. Tomographic imaging using the nonlinear response of magnetic particles. Nature. 2005 Jun 30;435(7046):1214–1217. 
  4. Graeser M, Knopp T, Szwargulski P, Friedrich T, von Gladiss A, Kaul M, Krishnan KM, Ittrich H, Adam G, Buzug TM. Towards Picogram Detection of Superparamagnetic Iron-Oxide Particles Using a Gradiometric Receive Coil. Sci Rep. 2017 Jul 31;7(1):6872. 
  5. Nishimoto K, Mimura A, Aoki M, Banura N, Murase K. Application of Magnetic Particle Imaging to Pulmonary Imaging Using Nebulized Magnetic Nanoparticles. Open Journal of Medical Imaging. Scientific Research Publishing; 2015;5(02):49.
  6. Zheng B, Yu E, Orendorff R, Lu K, Konkle JJ, Tay ZW, Hensley D, Zhou XY, Chandrasekharan P, Saritas EU, Goodwill PW, Hazle JD, Conolly SM. Seeing SPIOs Directly In Vivo with Magnetic Particle Imaging. Mol Imaging Biol. 2017 Jun;19(3):385–390
  7. Bulte JWM, Walczak P, Janowski M, Krishnan KM, Arami H, Halkola A, Gleich B, Rahmer J. Quantitative “Hot Spot” Imaging of Transplanted Stem Cells using Superparamagnetic Tracers and Magnetic Particle Imaging (MPI). Tomography. 2015 Dec;1(2):91–97.
  8. Yu EY, Chandrasekharan P, Berzon R, Tay ZW, Zhou XY, Khandhar AP, Ferguson RM, Kemp SJ, Zheng B, Goodwill PW, Wendland MF, Krishnan KM, Behr S, Carter J, Conolly SM. Magnetic Particle Imaging for Highly Sensitive, Quantitative, and Safe in Vivo Gut Bleed Detection in a Murine Model. ACS Nano. 2017 Nov 30
  9. Saritas EU, Goodwill PW, Croft LR, Konkle JJ, Lu K, Zheng B, Conolly SM. Magnetic particle imaging (MPI) for NMR and MRI researchers. J Magn Reson. 2013 Apr;229:116–126. 
  10. Hensley DW, Tay ZW, Dhavalikar R, Zheng B, Goodwill P, Rinaldi C, Conolly S. Combining magnetic particle imaging and magnetic fluid hyperthermia in a theranostic platform. Phys Med Biol [Internet]. 2016 Dec 29; 

Acknowledgement

We would like to acknowledge NIH funding and the A*STAR NSS-PhD and the Siebel Scholars fellowship (ZW Tay).

Magnetic Particle Imaging for Evaluation of Aerosol Drug Delivery Efficiency
a.  Photo of MPI scanner used.   b.  MPI image intensity is linear (R= 0.97) with the Doxorubicin HCl fluorescence (model drug) for DOX-SPION aerosol deposited on lung phantoms, indicating that the MPI image intensity can quantify the deposited drug.  c.   MPI can image and evaluate the aerosol delivery efficiency : inhaling the aerosol too fast results in focal deposition and poor C/P ratio. 
Magnetic Particle Imaging of the Timecourse Clearance of Deposited Aerosol From The Lung
a. Magnetic Particle Imaging (MPI) images show steady mucociliary clearance of deposited aerosol-SPIONs from the lung and into the GI tract to be excreted in droppings. MPI's high contrast and sensitivity enables tracking of the SPIONs at every stage of the clearance pathway, and is thus useful for long-term, controlled-release drugs. b. MPI's quantitative nature is useful for clearance kinetics. 
Keywords: Magnetic Particle Imaging, Drug Delivery, Lung Imaging, Magnetic Nanoparticles