Synthetic Cargo Internalization Receptor System for Nanoparticle

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Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging ACS Nano 2018.12:11178-11192. Downloaded from pubs.acs.org by YORK UNIV on 12/02/18. For personal use only.

Sebastian Temme,†,# Paul Baran,‡,# Pascal Bouvain,†,# Christoph Grapentin,§ Wolfgang Krämer,§ Birgit Knebel,∥ Hadi Al-Hasani,∥ Jens Mark Moll,‡ Doreen Floss,‡ Jürgen Schrader,† Rolf Schubert,§ Ulrich Flögel,*,†,# and Jürgen Scheller*,‡,# †

Experimental Cardiovascular Imaging, Molecular Cardiology, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany Institute for Biochemistry and Molecular Biology II, Medical Faculty, Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany § Department of Pharmaceutical Technology and Biopharmacy, Albert Ludwig University Freiburg, 79104 Freiburg im Breisgau, Germany ∥ Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Leibniz Center for Diabetes Research at the Heinrich Heine University Düsseldorf, 40225 Düsseldorf, Germany ‡

S Supporting Information *

ABSTRACT: Specific detection of target structures or cells lacking particular surface epitopes still poses a serious problem for all imaging modalities. Here, we demonstrate the capability of synthetic “cargo internalization receptors” (CIRs) for tracking of individual cell populations by 1H/19F magnetic resonance imaging (MRI). To this end, a nanobody for green fluorescent protein (GFP) was used to engineer cell-surface-expressed CIRs which undergo rapid internalization after GFP binding. For 19F MR visibility, the GFP carrier was equipped with “contrast cargo”, in that GFP was coupled to perfluorocarbon nanoemulsions (PFCs). To explore the suitability of different uptake mechanisms for this approach, CIRs were constructed by combination of the GFP nanobody and three different cytoplasmic tails that contained individual internalization motifs for endocytosis of the contrast cargo (CIR1−3). Exposure of CIR+ cells to GFP-PFCs resulted in highly specific binding and internalization as confirmed by fluorescence microscopy as well as flow cytometry and enabled visualization by 1H/19F MRI. In particular, expression of CIR2/3 resulted in substantial incorporation of 19F cargo and readily enabled in vivo visualization of GFP-PFC recruitment to transplanted CIR+ cells by 1 H/19F MRI in mice. Competition experiments with blood immune cells revealed that CIR+ cells are predominantly loaded with GFP-PFCs even in the presence of cells with strong phagocytotic capacity. Importantly, binding and internalization of GFP-PFCs did not result in the activation of signaling cascades and therefore does not alter cell physiology. Overall, this approach represents a versatile in vivo imaging platform for tracking of individual cell populations by making use of cell-type-specific CIR+ mice. KEYWORDS: cell tracking, 19F magnetic resonance imaging, green fluorescent protein, perfluorocarbons, active targeting, endocytosis

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create contrast to surrounding tissue. Therefore, several contrast agents (CAs) have been developed to load cells and to enable their tracking by MRI,1 whereby most CAs make use

agnetic resonance imaging (MRI) is one of the most important key modalities for clinical diagnosis, and it is also widely used in preclinical research. The technique is free of radiation and generates excellent contrast between different soft tissues. However, individual cells or cell types cannot directly be detected because most cells do not possess any physical properties which can be exploited to © 2018 American Chemical Society

Received: July 27, 2018 Accepted: October 29, 2018 Published: October 29, 2018 11178

DOI: 10.1021/acsnano.8b05698 ACS Nano 2018, 12, 11178−11192

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Cite This: ACS Nano 2018, 12, 11178−11192

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ACS Nano of a modulation of the hydrogen (1H) MR signal.2−4 However, all of these approaches have the disadvantage that they create hypo-/hyperintense regions with the entire anatomy of the investigated object as background signal, which makes an unambiguous identification in vivo difficult or even impossible.5−7 As an alternative, noninvasive cell tracking by fluorine (19F) MRI has garnered significant interest over the past decade. Due to almost complete absence of fluorine from biological tissue acquired 19F signals are essentially background-free. Thus, the 19F signal generates an unequivocal “positive contrast” which does not interfere with the anatomical 1H image, and merging of morphological 1H with corresponding 19 F images enables the precise localization of the labeled target. As CAs with high 19F payload, emulsions of biochemically inert perfluorocarbons (PFCs) are widely used and have recently been exploited for cell tracking in a variety of disease models.9,10 For this, labeling of cells can be performed in vitro (which requires isolation, cultivation, and reimplantation) or in situ by intravenous application of PFCs. The latter is more straightforward but also more challenging because the CA has to get access to the target, and different cell types compete for the label. Furthermore, the time window for uptake of PFCs is limited by clearing mechanisms of the spleen, liver, or kidney.11−13 To confer specificity, distinct ligands are required to direct the CA to the target cell type. Ligands can be antibodies (Abs), Ab fragments, peptides, small molecules, aptamers, or even sugars.14 However, the targeting of distinct surface epitopes remains difficult as many of the endogenous receptors are not perfectly cell-specific and/or ligand binding induces undesired signaling cascades. Moreover, some surface receptors are not internalized upon binding, and therefore the ligand-coupled PFC is prone to detach over time from the target. Recently, also so-called nanobodies (Nbs) emerged as ligands for molecular imaging.15 Nbs are the variable domain of camelid or shark heavy-chain-only Abs that have a molecular weight of 12−15 kDa.15 Nbs have several advantages over conventional Abs as they are smaller in size, weakly immunogenic, stable, and exhibit rapid as well as specific binding. In the present study, we utilized a Nb against green fluorescent protein16 (GFP) to develop a highly specific targeting system which rapidly internalizes PFCs attached to GFP. To this end, we designed synthetic cargo internalization receptors (CIRs) comprising the GFP-Nb and selected extracellular, transmembrane, and cytoplasmic domains that internalize the coupled PFCs but do not result in any induction of signaling pathways. The functionality of this system was confirmed in adherent (CHO, COS) and nonadherent cells (Ba/F3), making this approach suitable for targeting of cells with CIR expression at the vessel surface as well as in the circulating blood. Generation of cell-type-specific CIR+ mice will thus provide a general and versatile platform for cell tracking, strongly extending the frontiers of molecular MRI.

Figure 1. CIR expression and GFP binding. (A) CIRs are composed of an extracellular N-terminal myc-tag (white), the GFP nanobody (green), and a short linker (black). The receptors differ in their transmembrane domain (TM) and the cytoplasmic sequences (CTD). CIR1 contains the CTD of the IL-6 receptor, CIR2 of Endo180, and CIR3 has the CTD of the phagocytic receptor FcγRII receptor. Yellow dots represent internalization motifs; red dots are tyrosine activation motifs (TAMs). The CTDs differ according to the number and properties of the endocytic motifs: Whereas CIR1 does not contain any known endocytic motifs, CIR2 possesses a tyrosine-based (FEGARY) as well as a dihydrophobic motif with an upstream acidic residue (EKNILV, yellow circle). The cytoplasmic domain of CIR3 contains two tyrosine activation domains (red dots) which is responsible for the phagocytic properties of the FcγRII receptor. (B) Flow cytometric analysis of GFP binding to stably transfected CHO-CIR1−3+ cells. Gray histograms represent controls, and green histograms show CIR+ expressing cells treated with GFP.

Endo180, a member of the mannose receptor family. Finally, CIR3 was constructed with TM/CTD of the phagocytic FcγRIIA receptor.17 The detailed amino acid sequences of CIR1−3 are provided in supplemental Figure S1. After transfection, we verified cellular expression of the CIRs by Western blot analysis against the myc-tag (supplemental Figure S2A). Flow cytometry confirmed that CIRs were expressed at the cell surface of both stably and transiently transfected cells (supplemental Figure S2B). Interestingly, CIR2/3 exhibited cell surface signals stronger than those of CIR1 in each case. Next, we validated the binding of GFP to the cell surface of CIR+/− cells by flow cytometry. As expected, after incubation with GFP, no fluorescence was observed for CIR− control cells, whereas CIR1−3+ cells showed substantial GFP binding (Figure 1B). Again, the strongest signal was detected in CIR2/3+ cells. Similar results were found in the timedependent binding of GFP (supplemental Figure S2C). In separate experiments, we could demonstrate that the CIR system, in principle, is also applicable to primary cells (supplemental Figure S3). Internalization of GFP by CIRs. To explore whether GFP is actively internalized by the CIRs, transfected cells were first exposed to GFP at 4 °C (allows binding to the surface) and subsequently switched to 37 °C to initiate energy-dependent

RESULTS Generation and Characterization of Cargo Internalization Receptors. In a first step, we engineered three different CIRs (Figure 1A) all composed of an N-terminal myc-tag, the GFP-Nb, a short peptide spacer, and different transmembrane (TM) and cytoplasmic domains (CTD). Whereas CIR1 consists of TM and CTD of the human IL-6 receptor (IL-6R), CIR2 contains TM of IL-6R and CTD of 11179


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Figure 2. Internalization of GFP. (A) Immunofluorescence of transiently transfected COS-CIR+ cells incubated with GFP at 4 °C (top panel) and subsequently shifted to 37 °C for 30 min. Note the homogeneous GFP staining on the cell surface at 4 °C, whereas the GFP signals transitioned to a vesicular pattern at 37 °C, indicating accumulation in endosomes. Nuclei are counterstained with DAPI. Scale bar represents 10 μm. (B) CIR+ cells were prelabeled with GFP at 4 °C followed by varying incubation periods at 37 °C to enable the internalization of cell-surface-located GFP/CIR complexes. Thereafter, cells were incubated with antimyc mAb to stain residual cell surface CIRs and subsequently analyzed by flow cytometry. Data are mean values ± SD of n = 3 independent experiments.

internalization processes. At 4 °C, CIR1+ or CIR3+ cells displayed a homogeneous GFP staining of the cell surface, whereas CIR2+ cells showed a more vesicular pattern (Figure 2A, top panel). Increasing the temperature to 37 °C diminished surface signals in all cells and strongly enhanced staining of vesicular structures inside the cell, indicating that GFP is internalzed to the endosomal system (Figure 2A, bottom panel). In order to quantify the internalization rates of the different CIRs, we repeated the temperature-controlled experiments described above but varied the incubation period at 37 °C and subsequently stained cell surface CIRs with anti-Myc tag mAb for analysis by flow cytometry (Figure 2B). Upon GFP exposition at 37 °C, CIR2 and CIR3 were strongly internalized over time, whereas only a moderate decrease of CIR1 at the cell surface was observed (Figure 2B). Interestingly, CIR2 showed very rapid endocytosis, which was then maintained on a constant level, and CIR3 showed a slower internalization rate, but the magnitude was higher as compared to that of CIR2. Visualization of CIR-Mediated Cargo Uptake by 19F MRI. In a next step, we generated GFP-targeted PFC

nanoemulsions (GFP-PFCs) for 19F MRI studies. For this, lysine residues at the surface of GFP β-barrels (supplemental Figure S4A) were functionalized with free −SH groups and conjugated to preformed PFCs containing free maleimide groups at the lipid shell (supplemental Figure S4B). Binding of GFP-SH to PFCs resulted in a slight increase in size as compared to neat PFCs; however, size distribution was unchanged, whereas the observed decrease of ζ-potential is in line with the added charge of GFP (supplemental Figure S5). Cryo-TEM confirmed particle integrity and presence of only very small amounts of liposomes. For selected batches, rhodamine was additionally attached directly to PFCs to prove internalization of the entire cargo and not only GFP. After incubation of CIR+ cells with GFP-PFCs, cells were separated from remaining free GFP-PFCs by density gradient centrifugation and analyzed by 1H/19F MRI. In T2-weighted 1H MR images, the cell layer in the upper part of the tube (Figure 3A, first column) can easily be identified as a small dark film within the “light” PBS band overlaying the dark and “dense” Percoll layer (Figure 3A, second column). Merging of 1H and corresponding 19F images revealed that GFP-PFC incubation resulted in strong 19F signals that exactly colocalized with the 11180


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Figure 3. 19F MRI of CIR+ cells. (A) CHO-CIR1−3+ cells were incubated with GFP-PFCs, washed and separated by Percoll gradient. The left row displays photos of the centrifugation tubes, where the cells appear as narrow white line. T2-weighted 1H MRI enabled the precise visualization of the cellular layer as a darker structure superimposed to the Percoll. Corresponding 19F MRI (middle) revealed a weak 19F signal for control cells, a slightly stronger signal for CIR1+, and strong 19F signals for CIR2+ and CIR3+ cells. (B) Quantification of the 19F signal in stably transfected CHO-CIR+ cells (left), transiently transfected CHO-CIR+ cells (middle) and stably transfected Ba/F3-CIR3+ cells (right). Data are mean values of n = 6 individual experiments. Asterisks indicate statistical significance with * = P < 0.05; ** = P < 0.01, *** = P < 0.001.

narrow layers of CIR+ cells (Figure 3A, third and fourth column, rows 2−4), whereas control cells showed only marginal 19F signal (Figure 3A top). Quantification of the 19 F signals confirmed a substantial cellular uptake of GFPPFCs under CIR2/3 expression (0.6/0.4 fmol 19F nuclei per cell), whereas CIR1 led only to a moderate internalization of the 19F cargo (Figure 3B left). Separate experiments indicated that our approach could also be applied to transiently transfected cells, where only a fraction of the cells expressed the CIRs. Even with varying transfection rates (between 10 and 30%), we found significantly increased 19 F incorporation under CIR2/3 expression as compared to controls (Figure 3B, middle). Finally, we investigated whether GFP-PFC uptake could also be enforced in nonadherent cells cultured in suspension. To this end, murine Ba/F3 cells were transduced with CIR3, which after incubation with the targeted

PFCs again resulted in a strongly enhanced 19F signal as compared to CIR− cells (Figure 3B, right). Competition of CIR+ with Immune Cells for Uptake of Targeted PFCs. In order to evaluate whether CIR+ cells can compete with the native phagocytic capabilities of blood immune cells for uptake of the targeted 19F cargo, we incubated a coculture of CIR+ and murine immune cells with rhodamine-labeled GFP-PFCs at 37 °C. Subsequently, cells were stained with CD45/CD11b to discriminate between lymphocytes (T cells, B cells), myeloid immune cells (monocytes, neutrophil granulocytes), and CIR+ cells. Flow cytometry demonstrated that CIR2/3 expression led to a massive uptake of GFP-PFCs, which clearly exceeded the uptake by myeloid cells (Figure 4). Similar results were obtained when primary macrophages were used for comparison of cargo uptake (supplemental Figure S6). 11181


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Figure 4. Coculture of CHO-CIR+ cells with murine blood immune cells. Top panel: CHO-CIR+ cells could be clearly distinguished from the immune cells by the lack of CD45 expression and a high SSC/FSC value. Left: Immune cells without CHO. Middle: CHO/CHO-CIR+ cells. Right: Coculture of immune and CHO/CHO-CIR+ cells. Bottom panel: Quantification of GFP-PFC uptake by CHO, CHO-CIR+, and murine blood immune cells (CD11b−, CD11b+). Data are mean values ± SD of n = 3 individual experiments.

here exploiting that LPS-induced inflammation opens the endothelial barrier to enable access of GFP-PFCs to matrigelresident CIR+ cells. Twenty-four hours after implantation of the plug doped with LPS and CIR2/3+ cells, GFP-PFCs were applied i.v., and mice were subjected to 1H/19F MRI. Using T2weighted 1H MRI, the matrigel plug could easily be identified in proximity of the injection site as a bright, oval structure in the dorsal region of the 1H MR image (Figure 5B, left). Anatomically matching 19F data sets concomitantly demonstrated the presence of a robust 19F signal in the border zone of plugs doped with CIR2/3+ cells (Figure 5B, middle and bottom panels; 19F integral CIR2/3 = 3.7 ± 1.8/11.1 ± 1.8 au, n = 3). The detected 19F patterns are consistent with previous observations in this model and clearly indicate the uptake of GFP-PFCs by CIR+ cells (Figure 5B, right). In parallel, we analyzed the biodistribution of GFP-PFCs by 1 H/19F MRI up to 72 h after i.v. injection (supplemental Figure S7). GFP-PFCs were rapidly cleared from the blood pool within 24 h and, as expected, concurrently accumulated in

Next, we made use of a murine model with a defined inflammatory hot spot20 to confirm that CIR+ cells can also compete with activated immune cells for cargo internalization under in vivo-like conditions. To this end, a matrigel plug containing 50 μg of LPS as well as CIR2/3+ cells was subcutaneously implanted into the neck of mice. Constituting a depot for LPS, the plug triggered an acute inflammatory response, resulting in the local accumulation of activated neutrophil granulocytes.20 After 24 h, both neutrophil granulocytes and CIR2/3+ cells were isolated from the plug and exposed to GFP followed by flow cytometric analysis. Whereas almost no GFP was found in neutrophil granulocytes, CIR2/3+ cells were clearly labeled with GFP (Figure 5A). Thus, CIR+ cells can preferentially be loaded with targeted PFCs in the presence of phagocytic immune cells and outcompete even activated neutrophils for cargo uptake. In Vivo Targeting of CIR+ Cells at Inflammatory Hot Spots. Subsequently, proof of concept in vivo 19F MRI was performed using the matrigel/LPS model described above, 11182


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Figure 5. In vivo competition of CIR+ cells with activated neutrophils for recruitment of GFP-PFCs. (A) Top: Local inflammation was induced by subcutaneous implantation of a matrigel plug doped with 50 μg of LPS and containing additionally Ba/F3-CIR2+ or −CIR3+ cells into the neck of C57BL/6 mice. Ba/F3-CIR2/3+ cells and neutrophil granulocytes were isolated from the plug after 24 h, incubated with GFP, and analyzed by flow cytometry. The inset displays mean values ± SD (GFP+ cells (%), n = 5). (B) Twenty-four hours after implantation of matrigel doped with 50 μg of LPS and Ba/F3-CIR2+ or -CIR3+ cells, GFP-PFCs were i.v. injected followed by 1H/19F MRI the next day. Left: Anatomical sagittal slice of the head and neck region, where the matrigel injection site (arrow) can be detected as a bright structure. The dashed lines indicate the area of the axial sections displayed on the right. Right: Axial 1H (top), 19F (middle), and merged (bottom) images of the matrigel area demonstrating a robust 19F signal at the border of the implanted plug.

all cells were isolated from the plug and analyzed by flow cytometry. Here, we identified in addition to the implanted cells only very low numbers of CD45+/CD11b+ immune cells. This amount was similar for unmodified and CIR2/3+ cells, whereas LPS as a positive control induced a massive infiltration of immune cells into the plug (supplemental Figure S8, bottom). Taken together, these experiments clearly demonstrate the physiological reconcilability of the CIR system. Evaluation of CIR-Mediated Uptake Mechanisms. In a last step, we used rhodamine-labeled GFP-PFCs to analyze the uptake characteristics of the distinct CIRs in more detail. Flow cytometry showed uptake kinetics for GFP-PFCs by CIR1−3 (supplemental Figure S9A,B) as could be expected from their internalization rates (Figure 2A). Confocal microscopy revealed a strong accumulation of GFP-PFCs in perinuclear regions, confirming that the majority of GFP-PFCs were internalized and localized in cellular endosomes/lysosomes (supplemental Figure S9C). In another approach, we preincubated CIR1−3+ cells with GFP, which strongly inhibited the incorporation of GFP-PFCs (Figure 7). Interestingly, there was no further increase in the GFP-PFC signal over time, indicating that a blockade of CIRs did not lead to significant receptor recycling over the observation period. Finally, we coupled GFP to aldehyde-activated latex beads (GFP-LBs) of different size (30, 100, or 4000 nm,

liver and spleen, but also in bone marrow, lymph nodes, and to a very low degree in kidneys (not shown). However, at no time adverse effects of GFP-PFCs were observed in these animals. CIR-Mediated Uptake of GFP-PFCs Does Not Induce Signaling or Immunogenic Responses. The biocompatibility of the CIR system was further corroborated by gene expression analysis after exposure of CIR1−3+ cells to GFPPFCs (Figure 6). Whereas hyper-IL-6a fusion molecule of IL-6R and IL-6 known to activate gp13021resulted in an increase of 151 transcripts and stimulated the TNF and Jak/ STAT pathways (see supplemental Tables 1 and 7), GFPPFCs only led to a negligible number of altered genes and did not activate any specific signaling cascades in CIR+ cells (supplemental Tables 2−6 and 8−11). Of note, CIR1 was not presumed to induce signaling, but surprisingly, CIR2/3 did not lead to activation of signaling pathways, although they contain the cytoplasmic tails of Endo180 or FcγRIIA. To exclude that the artificial receptors may provoke adverse immune reactions in vivo, we implanted matrigel plugs without LPS but with CIR+ cells into the neck of mice. Following injection of neat PFCs for passive 19F loading of circulating immune cells, mice were subjected to 1H/19F MR inflammation imaging,9,10 but we could detect not any 19F signal in the area of the matrigel plug containing the CIR+ cells (supplemental Figure S8, top). To verify these in vivo findings, 11183


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Figure 6. Holistic gene expression analyses. Top panel: Schematic overview of the conditions used for the gene expression analysis (left). Volcano plot analysis of gene expression data from Ba/F3 cells stimulated with hyper-IL-6 (HIL-6; middle) or GFP-PFCs (right). Bottom panel: Volcano plot analysis of gene expression data from Ba/F3-CIR1−3+ cells incubated with GFP-PFCs. For the sake of clarity, only data points are shown that exceeded a threshold of ±1.50-fold change (P < 0.05). The number of differently expressed genes after stimulation with GFP-PFCs and hyper-IL-6 (HIL-6) is indicated as a number. Statistical analysis of gene expression from each group was performed by one-way between-subject ANOVA analysis.

Tracking of Defined Cell Populations by 19F MRI. Intravenous injection of PFCs has been extensively utilized for in vivo imaging of inflammation in a variety of different settings.22−30 After injection, PFCs are taken up by the monocyte/macrophage system and transported to areas of inflammation, resulting in highly specific signals for infiltrating immunocompetent cells loaded with PFCs. However, although the majority of the 19F signal is related to monocytes and macrophages, the particles can also be internalized by other phagocytic cells. For example, we have recently shown that PFCs are predominantly taken up by neutrophil granulocytes in a model of LPS-induced inflammation and also in arthritis.20,27 Furthermore, administration of PFCs after myocardial infarction can also lead to a pronounced labeling of epicardium-derived cells.31 In order to enable a more specific in vivo labeling of distinct cell types, an active targeting of the PFCs is required, which should be highly specific and result in a rapid internalization of the 19F cargo. The most widely used targeting ligands are Abs or derivatives of them,14 but many of the recognized surface molecules are not specific to a certain cell type, and crosslinking antigens on the cell surface may result in unwanted activation of cells. In addition, targeting particular receptors may also fail to internalize the contrast agent, or the receptors are not efficiently expressed on the cell surface and therefore only low amounts of cargo bind to the cells. To overcome this limitation, we have designed synthetic CIRs based on a GFP-

supplemental Figure S10) to explore whether distinct cytoplasmic tails of CIR1−3 result in different endocytic properties. Whereas all CIR+ cells internalized 30 and 100 nm GFP-LBs, only CIR3 led to uptake of 4 μm GFP-LBs (Figure 8). 3D reconstruction clearly confirmed that the big GFP-LBs are indeed located within CIR3+ cells (Figure 8, bottom). Of note, incubation of CIR1/2+ cells with 4 μm GFP-LBs induced binding to the receptor but was not followed by incorporation into the cell (Figure 8C, right panel).

DISCUSSION In the present study, we developed a targeting system for specific tracking of individual cell types by 19F MRI. To this end, we constructed three types of CIRs expressed on the cell surface for rapid binding, internalization, and trapping of GFPPFCs within the cell (Figure 9). In particular, expression of CIR2/3 resulted in substantial incorporation of 19F cargo and readily enabled in vivo visualization of GFP-PFC recruitment to CIR+ cells by 1H/19F MRI. Importantly, engineered CIRs do not induce any acute immune responses, and binding/ internalization of GFP-PFCs did not result in the induction of signaling pathways. Furthermore, competition experiments revealed that CIR+ cells are preferentially loaded with GFPPFCs even in the presence of activated, phagocytic immune cells, indicating that this approach is highly suitable for in vivo tracking of individual cell populations using cell-type-specific CIR+ mice. 11184


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Figure 7. Blocking GFP-PFC uptake: Recombinant GFP (5 μg) was added to CHO-CIR+ cells for 30 min at 4 °C prior to GFP-PFC incubation. Thereafter, rhodamine-labeled GFP-PFCs were added, and cells were analyzed by flow cytometry after 0, 5, 10, 20, 40, and 80 min. The graphs display the mean fluorescence of rhodamine (left) and GFP (right). CIR-PFC = CIR+ cells incubated with GFP-PFC; CIRPFC block = CIR+ cells preincubated with recombinant GFP. Data are mean values ± SD of n = 3 individual experiments.

nonadherent cells. Transplantation of CIR+ cells into an inflammatory environment did not impact their binding and internalization properties; upon i.v. injection of GFP-PFCs, we could monitor in vivo the recruitment of 19F signals to the inflammatory hot spot containing the CIR+ cells. Importantly, under these conditions, the CIR system successfully competed with phagocytic immune cells for incorporation of the targeted cargo. This is caused not only by the highly specific binding of GFP to the Nb but also by PEGylation of the GFP-PFCs, which strongly impairs opsonization by serum proteins and therefore reduces nonspecific phagocytic uptake.8,32,33 After i.v. application of GFP-PFCs, we found a rapid clearance from the blood and a concurrent accumulation in the reticuloendothelial system without any adverse side effects, which is in line with previous PFC studies.18,19,33,34 Thus, the

Nb, which confer excellent specificity, high cell surface expression, and efficient internalization. Using this system, we were able to efficiently load, in particular, CIR2/3+ cells with GFP-PFCs, which enabled their visualization using 19F MRI anddue to the presence of GFPalso via its fluorescence signal. Of note, we were able to detect incorporation of the cargo even after transient transfection where only 10−30% of the cells express the receptor to a varying degree. This indicates that our approach is also suitable to detect CIR+ cells in a mixed population of CIR± cells, such as in inflammation when a variety of immune cells are invading, and shows that background-free 19F MRI can visualize even low amounts of cells. The feasibility of CIRmediated cargo uptake was demonstrated in different cell lines but also primary cells and can be applied to both adherent and 11185


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Figure 8. Immunofluorescence of CIR-mediated uptake of GFP latex beads. Top: GFP was coupled to aldehyde-activated latex microspheres of different size (30 nm, 100 nm, 4 μm) and exposed to COS-7 cells transiently transfected with CIR1−3. Bottom: 3D reconstruction of a zstack of a CIR3+ cell which internalized 4 μm GFP beads. Note that the vesicles are found within the cells. Gray = cell surface; green = GFP beads; blue = nucleus.

short blood half-life of GFP-PFCs and the rapid binding/ internalization render the CIR system particularly suitable for labeling of circulating immune cell subsets which cannot be specifically targeted by conventional approaches. CIR-Mediated Uptake Mechanisms of Cargo. All CIRs generated in this study are identical in the N-terminal myc tag linked to the GFP-Nb sequence, which is fused to parts of the IL-6R. However, they differ in their TM and CTD, which strongly impacts their internalization properties and the capability to endocytose GFP cargo of different size. Obviously, CIR2/3 with strong cell surface expression and good internalization properties are better suited for targeting strategies than CIR1 with weak expression and internalization rates. Despite CIR1 expression led to efficient binding of GFP-

PFC, there was inefficient cargo internalization most likely due to lacking endocytic motifs in TM/CTD of the IL-6Rα receptor subunit gp80.35 Interestingly, CIR1 displayed a weaker cell surface expression than CIR2/3, although total expression levels were quite similar. Therefore, the cytoplasmic tail not only determines internalization but also impacts the distribution of the CIRs inside and on the surface of the cell a crucial issue for in vivo targeting strategies. This is of particular importance for polarized cells (e.g., endothelial cells) with a basolateral and apical side. Because only the latter is interfaced to the flowing blood, it requires a CTD to direct the CIR construct to the apical side, for example, from the IL-11R, which exhibits endocytic capacity and is sorted to the apical side.36 11186


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monocyte subsets but also even nonphagocytic T-cells, helping to unravel the role of distinct immune cell subtypes in disease progression/resolution without activation of any major signaling pathways. Furthermore, the CIR system is not restricted to 19 F MRI and can be used to track cells by other imaging modalities such as optical, CT, PET, or ultrasound because GFP can easily be conjugated to other compounds, providing contrast for the respective technique. Finally, the CIR system could also be adopted to improve theranostic approaches for simultaneous, site-specific delivery of drugs and imaging agents.

METHODS Cell Lines, Primary Cells, and Animals. Ba/F3-gp130 cells (Ba/ F3) were obtained from Immunex (Seattle, WA, USA), and COS-7 (COS; ACC-60) and CHO-K1 (CHO; ACC-110) were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). All cells were cultivated in DMEM high-glucose medium (GIBCO, Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (GIBCO, Thermo Fisher Scientific), 60 mg/L penicillin, and 100 mg/ L streptomycin (Genaxxon Bioscience, Ulm, Germany) at 37 °C with 5% CO2 in a water-saturated atmosphere. Primary rat epicardium-derived cells (EPDCs) were prepared as described previously,31 whereas primary rat fibroblasts were obtained by conventional explant culture. To this end, subcutaneous rat skin tissue was cut into small pieces (∼1 mm3), transferred to a culture dish, covered by medium, and placed at 37 °C in an incubator. After approximately 1 week, the tissue samples were removed, and the fibroblasts were released by trypsinization and transferred to a new culture dish. One day later, the cells were seeded into 6-well dishes (∼3 × 105 per well) and transfected as described below. Animal experiments were performed in accordance with the European Union guidelines described in the directive 2010/63/EU and were approved by the local authorities. Male C57BL/6 mice used in this study were obtained from Janvier (Le Genest-Saint-Isle, France) and were fed with a standard chow diet receiving tap water ad libitum. Murine blood was extracted from the inferior vena cava from male C57BL/6J mice (10−14 weeks of age and 20−25 g). The blood was collected in heparinized tubes, and erythrocytes were lysed using ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) for 10 min at room temperature. Afterward, the remaining leukocytes were centrifuged for 10 min at 350g and resuspended in culture medium. Cloning of CIR Receptors and Generation of CIR-Expressing Cells. Cloning of CIRs. The nucleotide sequence of CIR1 was purchased from GeneArt (Regensburg, Germany). CIR1 was cloned with a 5′-EcoRV site and 3′-NotI site into the pcDNA3.1 plasmid. The nucleotide sequence of the CTD of CIR2 was amplified by PCR and cloned via a 5′-BglII site and a 3′-NotI site into a pcDNA3.1CIR1 plasmid (the CIR1 CTD was removed by BglII/NotI restriction). The nucleotide sequence of the CIR3 TM/CTD was amplified from Jukat cells (T-cell leukemia) (DMSZ, Braunschweig, Germany) cloned using 5′-EcoRI and 3′-NotI restriction sites into a pcDNA3.1 CIR1 plasmid. Cloning all CIR receptors into the lentiviral vector pMOWS was done by digestion of the pcDNA3.1 plasmids with PmeI.42 After cloning into the pcDNA or the pMOWS vectors, we verified all CIR-sequences by Sanger sequencing. All CIRs are composed of an N-terminal hIL-11R signal peptide (AA: 1−24), a myc-tag (AA: 25−34), followed by a GFP nanobody (GFP-NB, AA: 35−149), a 5 amino acid long linker (AS 150−154), the stalk region (AS 155−161), the transmembrane region (TMD; CIR1/2, AA: 162−179; CIR3 AA: 162−187), and the cytoplasmic domain (CTD; CIR1 AA: 180−262 from hIL-6R; CIR2 AA: 180− 224 from hEndo180 receptor; CIR3 AA: 187−264 from the human FcγRIIA receptor). An alignment of the amino acid sequences with detailed description of the individual parts of the CIRs is provided in the supplemental Figure S1.

Figure 9. Binding, uptake, and internalization of GFP-PFCs. CIRs are expressed on the cell surface where they bind GFP-PFC cargo (1). Upon binding, CIRs become internalized (2) and accumulate within the endosomal system of CIR+ cells, which ensures trapping of the cargo inside the cells (3), giving rise to a 19F signal that is specifically associated with the desired target cell.

Upon binding of GFP-PFCs, CIR2/3 led to efficient internalization of the cargo and its subsequent deposition in late endosomes/lysosomes. CIR2 was designed to include the CTD of Endo180, a member of the mannose receptor family which binds collagen and contains tyrosine-based and dihydrophobic motifs for internalization.37 The first motif is connected to endocytic adapter proteins, whereas the second is important for targeting to endocytic vesicles. In the absence of collagen, the receptor recycles between early endosomal compartments and the plasma membrane,38 whereas binding of collagen reroutes the receptor to lysosomal compartments.39 As GFP-PFCs internalized by CIR2 strongly accumulate within the endosomal system, it can be presumed that GFP-PFC binding also targets CIR2 from the early endosomal system to late endosomes/lysosomes. The underlying mechanism is unclear, but most likely, binding of the ligand results in receptor clustering that segregates recycling from the lysosomal pathway. The notion that the intracellular sorting and trafficking patterns of CIR2 are mainly derived from the CTD of Endo180 is further supported by the vesicular pattern of CIR2 on the cell surface, which has similarly been found for Endo180 localized in clathrin-coated pits.40,41 CIR3 was constructed with the CTD of the phagocytic FcγRIIA receptor that contains two ITAM-like motifs (immunoreceptor tyrosine-based activation motifs), which determine the phagocytic properties of the receptor enabling the uptake of large IgG-decorated sheep erythrocytes.37 However, these two ITAM motifs are also YxxL motifs which can bind to the clathrin adapter AP2, inducing clathrin-mediated endocytosis. Therefore, cargo uptake by CIR3 can be mediated via different signaling pathways to initiate phagocytosis or alternatively conventional receptor-mediated endocytosis.

CONCLUSIONS Here, we have shown that the CIR systemin particular CIR2/3is suitable for targeting and tracking of individual cell types by GFP-PFCs and 19F MRI. In the next step, CIR constructs are cloned into the Rosa locus of mice to be crossed with strains expressing Cre-recombinase under tissue-specific promoters (e.g., LysM-Cre, CD19-Cre, CD3-Cre). This will not only enable the specific targeting of neutrophil or 11187


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ACS Nano Transient Transfection of CIRs. For transient transfection, 2 × 105 cells were seeded in 6-well plates. Twenty-four hours later, the medium was replaced with fresh culture medium. One microgram of plasmid DNA and 4 μL of PEIMax (Polysciences) were suspended in 50 μL of saline (150 mM NaCl). Subsequently, the DNA and the PEIMax solutions were mixed and incubated for 15 min at room temperature. One hundred microliters of the transfection reagent was added per well of a 6-well plate. The medium was replaced after 24 h, and the cells were cultivated for further 24 or 48 h and then subjected to further experiments. Generation of Stably Transfected CHO-CIR+ Cells. To generate stably transfected CHO-CIR+ cells, cells were transfected as described above and 48 h after transfection, and G418 (Carl Roth; 0.5 mg/mL) was added to the cells. The medium/G418 was replaced each 72 h. After the death of untransfected cells, CIR-expressing cell clones were grown until they could be picked by a pipet tip and then transferred into 96-well plates for further culture. Cell clones were tested for CIR expression by incubation with GFP (1 μg/mL) and analysis by flow cytometry. The process of generating stable CHO-CIR CIR+ lines took about 4 months. We propagated at least three different clones for each CIR. Generation of Stably Transduced Ba/F3-CIR+ Cells. Retroviral transduction of murine pre-B cell line Ba/F3 was described previously.42 The plasmid DNAs of CIR1−3 (pMOWS) were transiently transfected to phoenix-Eco cells. Twenty-four hours after transfection, the supernatant containing the retrovirus was collected; 250 μL was mixed with 8 μg/mL Polybrene (Sigma-Aldrich) and 1 × 105 Ba/F3 cells and centrifuged (2 h, 1800 rpm, 4 °C). In the following, cells were suspended in 10 mL of DMEM with 10 ng/mL hyper-IL6 (HIL6). Forty-eight hours after transfection, 1.5 μg/mL puromycin (Carl Roth) was used to select the transduced cells, at least for 2 weeks. SDS-PAGE and Western Blotting. CIR+ and CIR− control cells were harvested by PBS/2.5 mM EDTA detachment, collected by centrifugation, and suspended in 250 μL of lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, complete protease inhibitor). After 60 min incubation at 4 °C, the lysed cells were centrifuged at 20000g at 4 °C to remove cellular debris, and the clear supernatant (lysate) was transferred into a new tube. Equal amounts of protein lysates were used (25 μg), and the protein lysates were separated by SDS-PAGE under reducing conditions. The separated proteins were transferred to a PVDF membrane (Carl Roth, Karlsruhe, Germany), and the membranes were blocked with 5% skim milk powder in TBS-T (10 mM Tris-HCl, pH 7.6, 150 mM NaCl and 0.05% Tween 20) for 2 h at room temperature. Then the membrane was probed with an antimyc mAb (Cell Signaling, #2278, Danvers, MA, USA; 1:1000 dilution) overnight in 5% skim milk powder in TBS-T at 4 °C. After several washing steps with TBS-T, the membranes were incubated with a secondary anti-rabbit antibody conjugated to horseradish peroxidase (Cell Signaling, #7074S; 1:5000 dilution) in 5% skim milk powder in TBS-T for 1 h at room temperature. Finally, the proteins were detected with the Immobilin Western Chemiluminescent HRP substrate (Millipore, Billerica, MA, USA) according to the manufacturer’s instructions and the ChemoCam Imager (INTAS Science Imaging Instruments, Göttingen, Germany) for signal detection. Preparation and Characterization of GFP-PFCs and GFPLBs. Expression and Purification of Recombinant GFP. The cDNA encoding the sequence of GFP was cloned in a pQE plasmid. The resulting protein sequence was flanked by a C-terminal hexahistidine sequence for purification. Proteins were expressed in Escherichia coli BL21-plys. Bacterial cells were incubated in 2 L of LB medium containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol at 37 °C with 120 rpm, until an optical density of 0.6−0.9 was reached. Protein production was induced by adding 1 mM isopropyl 1-thio-βgalactopyranoside (Carl Roth), and the bacteria were harvested 5 h after induction by centrifugation (5000g, 30 min at 4 °C). Bacteria were resuspended in PBS (pH 7.4) and mechanically lysed using a microfluidizer (Model M110S, Microfluidics Corp, Newton, MA, USA). Lysates were centrifuged (20000g, 20 min, 4 °C) to collect

soluble fractions, and proteins were isolated from the supernatant via IMAC purification. Isolated GFP was analyzed by SDS-PAGE. Preparation of Maleimide-PFC (Mal-PFC). Composition of the PFC nanoemulsions is 20% (w/w) PFCE (perfluoro-15-crown-5 ether; ABCR, Karlsruhe), 2.5% (w/w) Lipoid S75, 0.45% (w/w) DSPE-mPEG2000 (Lipoid), 0.05% (w/w) maleimide-PEG2000-DSPE (Avanti Polar Lipids) and buffer to 100%. For selected batches, about 0.025 mol % lissamine-rhodamine-DHPE (Molecular Probes) was incorporated into the lipid layer of the PFCs to prove internalization of the entire cargo and not only GFP. To ensure an equal distribution of the lipids, we used a film method. Lipids were dissolved in chloroform and added to a round-bottom flask. The chloroform was removed in a rotary evaporator at 200 mbar and 40 °C. After complete evaporation of the organic solvent, the evenly distributed lipids were resuspended in 10 mM phosphate buffer (pH 7.4). PFCE was added dropwise. A crude emulsion was formed by high shear mixing using a Micra D9 mixer (tool DS-8/P) at 15000 rpm for 3 min. The resulting crude emulsion was further processed on an LV1 microfludizer (Microfluidics) homogenizer for 10 cycles at 1000 bar process pressure. The final nanoemulsions were aliquoted and stored at −80 °C to prevent hydrolysis of the reactive maleimide group. Preparation of GFP-PFCs. For the generation of GFP-PFCs recombinant GFP was coupled to Mal-PFCs (see above). The GFP was used in a 10-fold lower concentration to the maleimide to ensure complete coupling of the GFP to the nanoemulsion droplets. For conjugating the GFP to the maleimide, GFP was first thiolated using a 50-fold molar excess of Trauts reagent (2-iminothiolane) in PBS/5 mM EDTA buffer adjusted to pH 8.0. After 1 h of incubation at room temperature, free Trauts reagent was removed by size-exclusion chromatography using a Zeba Spin column (Thermo Fischer). Subsequently, the thiolated GFP was incubated with Mal-PFCs at 20 °C overnight under constant motion. The GFP-PFC emulsions were purified by repeated centrifugation at 20000g for 20 min to remove unbound GFP and washing with PBS/EDTA buffer. Finally, the emulsion was resuspended in phosphate−glycerol buffer (10 mM, 2.5% glycerol) and stored at 4 °C under light protection. Generation of GFP-Labeled Latex Microspheres (GFP-LBs). Aldehyde-activated latex microspheres (Thermo Fischer Scientific) of different size (30 nm, 100 nm, 4 μm) were labeled with GFP to obtain GFP-LBs. To this end, 100 μL of latex beads was incubated with 40 μg of GFP in 500 μL of PBS overnight at 4 °C. Next, 500 μL of 5% BSA was added and incubated for 30 min at room temperature to block all free aldehyde reaction sites. The beads were washed three times with 2 mL of 1% BSA and two times with 2 mL of PBS. Finally, the beads were resuspended in 600 μL of PBS and subjected to uptake experiments or IVIS/electron microscopy. Centrifugation parameters differed for the individual latex beads (30 nm = 16000g for 30 min; 100 nm = 16000g for 20 min; 4 μm = 2000g for 5 min). To verify that unbound GFP does not spoil the GFP-LB preparations, free GFP without beads was also subjected to the coupling and washing procedure. No GFP signal was found in these samples, demonstrating that the GFP signal is exclusively associated with the LBs. Characterization of GFP-PFCs and GFP-LBs. Fluorescence. To verify the coupling of thiolated GFP to the Mal-PFCs, we purified the GFP-PFCs as described above. Then we analyzed the GFP fluorescence by using an IVIS Lumina-II system (PerkinElmer). To this end, 10 μL of the GFP-PFCs and 10 μL of the parent PFCs were spotted on a glass plate and analyzed with the IVIS spectrometer (FOV = A, GFP excitation and emission filters, 0.5 s excitation). Quantification of the fluorescence signals was done with the Caliper software. ROIs were drawn around the areas of the droplets, and the mean fluorescence signal was measured. The mean background signal was subtracted from the sample values. Dynamic Light Scattering. The mean intensity-weighted hydrodynamic diameter was determined by DLS on a Zetasizer Nano (Malvern Instruments, Malvern, UK). Prior to measurements, the nanoemulsions were diluted 1:100 (v/v) with 0.22 μm filtered sample buffer. The measurements were performed at 25 °C and at a scattering angle of 173°. Particle size is displayed as an averaged 11188


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cells were washed to remove any unbound GFP and shifted to 37 °C. GFP internalization was stopped at distinct time points by adding icecold PBS, and cells were stained with antimyc mAb to detect cellsurface bond GFP. Cells were analyzed by flow cytometry using a FACS Canto II (BD Biosciences). GFP Competition Experiments. For the GFP inhibition experiments CHO-CIR1−3+ cells (∼5 × 105 cells) were used and each resuspended in 1 mL of DMEM. Afterward, the samples were incubated at 4 °C with or without 5 μg/mL GFP for 30 min. Finally, for the time kinetics, 10 μL of the GFP-PFCs was added to each sample, and at defined time points (0, 5, 10, 20, 40, 80 min), 100 μL of the cell suspension was transferred into 2 mL ice-cold PBS, centrifuged, and washed once with PBS. For FACS analysis, the cells were resuspended in MACS buffer and analyzed at a BD LSRFortessa. Competition Experiments of CIR+ Cells and Murine Immune Cells. Coculture with Blood Cells. Heparinized blood was withdrawn from the inferior vena cava from male C57BL/6 mice (10−14 weeks of age and 20−25 g) by venous puncture. Six hundred microliters of blood was subjected to hypotonic erythrocyte ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA) for 10 min at room temperature. Cells were washed and resuspended in 1 mL of DMEM medium supplemented with 10% FBS (Biochrom). CHO, CHOCIR2+, or CHO-CIR3+ cells, and GFP-PFCs were added to the immune cells (300 μL immune cells, 50 μL CHO/CIR; approximately 1−2 × 105, 10 μL GFP-PFCs) and incubated for 60 min at 37 °C in a MACS rotator. Afterward, cells were washed twice with PBS, resuspended in FACS buffer stained with CD45-PE.Cy7 (BD; 1:400), CD11b-APC (Miltenyi Biotec, 1:200), and incubated at 4 °C for 30 min. The cells were washed with PBS, stained with DAPI to exclude dead cells, and subjected to flow cytometry (LSR-Fortessa, BD-Biosciences). Coculture with Neutrophils from Matrigel/LPS. To isolate cells from the matrigel plug (see below), the matrigel was carefully isolated, digested with collagenase/DNase, and passed through a 40 μm mesh to obtain single cells. The cells were then collected by centrifugation and incubated with CD11b-PE.Cy7 (BD Biosciences), CD45-APC (BD Biosciences), and DAPI (1 μg/mL) to discriminate between viable neutrophil granulocytes immigrated into the plug (CD45+, CD11b+) and Ba/F3 cells (CD45+, CD11bneg). Cells were analyzed by flow cytometry using a FACS Canto II (BD Biosciences). Uptake of GFP-PFCs by Macrophages. For isolation of macrophages from matrigel plugs 10 days after implantation, the matrigel was processed as described above. In parallel peritoneal macrophages were isolated by injecting 10 mL of PBS into the peritoneum of the mouse followed by recollection of the fluid. The obtained cells (including matrigel macrophages and peritoneal macrophages) were collected by centrifugation and incubated with 10 μL/mL GFP/GFPPFCs or 1 μL/mL GFP for 30 min at 37 °C. Afterward, the cells were washed twice and stained against CD11b-APC (BD Biosciences), CD45-PECy.7 (BD Biosciences) and DAPI (1 μg/mL) to discriminate between viable and dead macrophages. Cells were analyzed by flow cytometry for their uptake of the GFP-PFCs by using a LSR Fortessa (BD Biosciences). Magnetic Resonance Imaging. Labeling of Cells with GFPPFCs. The stably transfected CHO-CIR+ cells (80% confluent in 10 cm cell culture plates) were incubated with 75 μL of GFP-PFCs overnight at 37 °C on a shaker. On the next day, the cells were washed five times with PBS to remove excess GFP-PFCs, trypsinized, resuspended in 1 mL of MACS buffer, carefully layered on 2 mL Percoll (Sigma-Aldrich), and centrifuged at 500g for 20 min. The falcon was immediately transferred into the MRI for analysis. For imaging of transiently transfected CIR+ cells, the cells were incubated with GFP-PFCs at 4 °C for 30 min to enable the binding of GFP-PFCs to CIR+ cells. After three washing steps, the temperature was increased to 37 °C and the cells were cultivated for another 2 h and released from the culture dish by trypsinization. After being fixed in PFA (0.5% PFA for 5 min) and centrifuged, cells were subjected to MRI. 1 19 H/ F MRI. Experiments were performed at a vertical 9.4 T Bruker AVANCEIII Wide Bore NMR spectrometer (Bruker, Rheinstetten,

hydrodynamic diameter, dz. The width of the particle size distribution is expressed by the polydispersity index (PI). ζ-Potential Measurements. The ζ-potential was measured as described previously43 at 25 °C by laser Doppler anemometry using a Zetasizer Nano (Malvern Instruments, Malvern, UK). Samples were diluted 1:200 (v/v) with 0.22 μm filtered sample buffer. The ζpotential was determined in three measurements each consisting of 10 subruns; data are given as the mean value. Cryo-Transmission Electron Microscopy. Sample preparation was done according to a formerly developed protocol.44 PFCs or GFP-LBs beads were diluted with sample buffer to minimize particle overlays and therefore enable size measurements. Approximately 5 μL diluted dispersion was applied on a 400 mesh Quantifoil S7/2 holey carbon film on copper grids (Quantifoil Micro Tools GmbH, Jena, Germany). Excess liquid was removed from the grid with filter paper. The sample was then immediately shock-frozen by injecting it into liquid ethane. These sample preparation steps were done in a climate-controlled room using a CryoBox 340719 (Carl Zeiss). The subsequent fixation of the grid on the sample rod (626-DH, Gatan) and transfer of the rod into the transmission electron microscope (Leo 912 Ω-mega, Carl Zeiss) were done under a nitrogen atmosphere at a temperature of 90 K (−183 °C). The instrument was operated at 120 kV, and pictures with a 6300−12500 magnification were taken from different positions of the grid to include all particles of the sample (Camera: Proscan HSC 2, Oxford Instruments). Nanoemulsion droplets can easily be distinguished from liposomes in cryo-TEM images. Due to different light refraction properties, buffer-filled liposomes appear as light vesicles, whereas perfluorocarbon-filled nanoemulsion droplets appear dark. Flow Cytometry and Immunofluorescence. Cell Surface Expression of CIRs by Flow Cytometry. COS and CHO cells as well as primary rat EPDCs and skin fibroblasts were transiently transfected and incubated for 48 h at 37 °C in an incubator. Alternatively, stably transfected CHO-CIR+ or Ba/F3-CIR+ cells were used. To assess the cell surface expression of the CIRs, cells were detached with PBS/2.5 mM EDTA, washed and resuspended in FACS buffer (PBS, 0.5% BSA). Approximately 5 × 105 cells were incubated in 100 μL of FACS buffer containing a 1:100 dilution of anti-Myc tag antibody (Cell Signaling, #2278) for 60 min at 4 °C, washed three times with FACS buffer, and stained with 0.5 μg/mL Alexa-488 coupled anti-rabbit secondary antibody (Invitrogen, #A11070) in 100 μL of FACS buffer for 60 min at 4 °C. Dead cells were counterstained with DAPI and excluded from the analysis. Finally, stained cells were washed three times and resuspended in 500 μL of FACS buffer and analyzed with a BD Canto II flow cytometer (BD Biosciences). All cytometry data were processed either with Flowing Software 2 (Turku Bioimaging) or FCS Express (Denovo Software). Binding and Internalization of GFP, GFP-PFCs, or GFP-LBs. To analyze the surface binding/internalization of GFP, cells were incubated with 1 μg/mL GFP for 30−60 min at 4 or 37 °C, detached with PBS/2.5 mM EDTA, washed three times with PBS, and subjected to flow cytometry. Dead cells were excluded from the analysis by staining with DAPI. To inspect the binding/uptake of GFP and GFP-PFCs/LBs by immunofluorescence, cells were incubated with GFP or GFP-PFCs/LBs, washed three times with PBS, fixed with 0.5% PFA for 15 min, permeabilized with 1% Triton X-100, and stained with DAPI to visualize nuclei. In some cases, an additional staining with anti-MHC class I antibody (W6/32, kindly provided by Prof. Dr. N. Koch, University of Bonn) and appropriate anti-mouse− Alexa594 secondary antibody was conducted. Finally, cells were embedded in MOWIOL and inspected using an Olympus BX61 fluorescence microscope (Olympus) or by confocal microscopy (Zeiss LSM710 meta, Zeiss). Images were analyzed and processed using Fiji.45 3D reconstructions from z-stack image packages were performed with the segmentation tool of Amira (FEI, Thermo Fischer Scientific). GFP Internalization. Ba/F3 and Ba/F3-CIR1−3+ cells were incubated with 5 μg/mL GFP at 4 °C for 30 min. Subsequently, 11189


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106 cells were stimulated with 50 μL of GFP-PFC emulsion and 100 ng/mL HIL6, and serum without any supplements was used as a control. Total RNA extraction was performed with RNeasy Mini Kit (Qiagen). RNA quality was evaluated using an Agilent 2100 Bioanalyzer, and only high-quality RNA (RIN > 8) was used for gene expression analysis. One hundred fifty nanograms of RNA was amplified using the Ambion WT expression kit and the WT terminal labeling kit (Affymetrix, Freiburg, Germany). The amplified cDNA was hybridized on Affymetrix Mouse Gene ST 1.0 arrays, containing a probe set of 28000 genes. Staining and scanning was done according to the Affymetrix expression protocol. For quality control and normalization of the RNA gene-level data, the expression console software (Affymetrix) was used. The statistical analysis of the data was performed with the Transcriptome Analysis Console 3.0 (Affymetrix). The full gene expression data set was uploaded to “gene expression omnibus (GEO)” (Project No. GSE106215; see supplemental data).

Germany) operating at frequencies of 400.21 MHz for 1H and 376.54 MHz for 19F measurements using a Bruker microimaging unit Micro 2.5 with actively shielded gradient sets (1.5 T/m) as described previously.19 Data were acquired using a 25 mm quadrature 19F resonator with one channel tunable to both 1H and 19F resonator tunable to 1H and 19F. After acquisition of the morphological 1H images, the resonator was tuned to 19F and anatomically matching 19F images were recorded. For 19F MRI measurements always the same reference power and receiver gain were chosen to ensure a meaningful comparison of 19F signal intensities between data sets. Cellular Uptake. 1H MR reference images were acquired using a rapid acquisition and relaxation enhancement sequence (RARE; field of view (FOV) = 2.56 × 2.56 cm2, matrix 256 × 256, 0.1 × 0.1 mm2 in-plane resolution, 1 mm slice thickness; TR = 3000 ms; RARE factor = 128, 6 averages). Corresponding 19F images were recorded from the same FOV with a 19F RARE sequence (matrix 32 × 32, 0.8 × 0.8 mm2 in-plane resolution, 3 mm slice thickness; TR = 2500 ms, TE = 3.45 ms, RARE factor = 32, 256 averages). Biodistribution/Matrigel. One hundred microliters of GFP-PFCs was diluted in 200 μL of phosphate buffer and i.v. injected via the tail vein. Subsequently, 1H/19F MRI measurements were performed after 1, 3, 6, 24, and 72 h. We applied a 19F FLASH sequence (FOV = 2.56 × 2.56 cm2, matrix 32 × 32, 5 slices, 1 mm slice thickness, flip angle = 90°, TR 50 ms, TE = 1.62 ms, 188 averages) to visualize the 19F signal in the flowing blood.33 The accumulation of PFCs in liver and spleen was determined using a 19F RARE sequence (FOV = 2.56 × 2.56 cm2, matrix 64 × 64, 13 slices, 2 mm slice thickness, TR = 2500 ms, TE = 3.45 ms, RARE factor = 32, 64 averages). To image the area covering the matrigel/LPS area again, 1H and 19F RARE sequences were applied (19F RARE: FOV = 2.56 × 2.56 cm2, matrix 64 × 64, 9 slices, 1 mm slice thickness, TR = 2500 ms, TE = 3.45 ms, RARE factor = 32, 256 averages). The 19F MR data were analyzed using in-house developed software modules based on the LabVIEW package (National Instruments, Austin, TX). For quantification of the 19F signals ROIs was drawn around the area/tissue of interest, whereas background ROIs of similar geometry were placed outside the samples. The signal-to-noise ratio was calculated from the mean of the 19F signal divided by the standard deviation of the noise. Matrigel Model. LPS-Induced Inflammantion. Here, we adopted a recently developed model of localized subcutaneous inflammation.20 50 μL of ice-cold matrigel (BD Biosciences) were doped with 50 μg of LPS (Salmonella typhimurium, Sigma-Aldrich) and supplemented with 5 × 106 Ba/F3-CIR2+ or Ba/F3-CIR3+ cells were s.c. implanted into the neck of male C57BL/6 mice. Matrigel is liquid at 4 °C but transforms into a gel at 37 °C, which can be easily visualized by 1H MRI. After 24 h, GFP-PFCs were i.v. injected followed by 1H/19F MRI the next day. In order to confirm that CIR+ cells retain their GFP/GFP-PFC internalization properties after being embedded in the matrigel, in separate experiments, we isolated the cells from the plug (see above) and incubated the cells with GFP and subsequently analyzed the cells by flow cytometry. Immune Responses by CIR+ Cells. To evaluate the potential induction of CIR-mediated immune responses we implanted CIR+ Ba/F3 cells mixed with matrigel in the neck of mice. On the next day, neat (non-PEGylated) PFCs were intravenously injected for passive 19 F loading of circulating immune cells, and mice were analyzed 24 h later by 1H/19F MRI. As a negative control, Ba/F3 cells without CIR expression were used, whereas the standard LPS/matrigel mixture was utilized as a positive control. From all matrigel plugs, immune cells were isolated as described above and stained against CD11b-PE (BD Biosciences), CD45-PECy.7 (BD Biosciences), and DAPI (1 μg/mL) to discriminate between viable immune cells (CD45+, CD11b+, DAPI−) and viable Ba/F3 cells (CD45+, CD11b−, DAPI−). The amount of immune cells was determined by flow cytometry at a FACS Canto II. Gene Expression Analysis. Stably transduced Ba/F3-CIR+ cells were washed four times with 10 mL of PBS (centrifugation: 5 min, 1500 rpm, 4 °C). Cells were starved for 2 h in serum-free DMEM high glucose medium at 37 °C and 5% CO2. For every sample, 2 ×

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b05698. Figures S1−S10; alignment of the amino acid sequences of CIR1−3; cellular expression and GFP binding of CIR1−3; CIR transfection of primary cells; preparation of GFP-PFCs; characterization of GFP-PFCs; GFP and GFP-PFC uptake by CIR+ cells and macrophages; biodistribution of GFP-PFCs; absence of immunogenic effects by CIR+ cells; cellular uptake of GFP-PFCs; characterization of GFP-LBs; Tables S1−S11; gene expression analysis of Ba/F3 and Ba/F3-CIR1−3+ cells stimulated with hyper-IL-6 and GFP-PFCs, respectively, compared with unstimulated cells (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: fl[email protected]. *E-mail: [email protected]. ORCID

Sebastian Temme: 0000-0002-8807-2767 Ulrich Flögel: 0000-0001-7181-4392 Jürgen Scheller: 0000-0001-9932-1055 Author Contributions #

S. Temme, P. Baran, and P. Bouvain, as well as U. Flögel and J. Scheller contributed equally. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS We would like to thank Bodo Steckel and Sabine Barnert for excellent technical assistance, and the Center for Advanced Imaging (CAI, Düsseldorf) for providing technical assistance and access to the confocal microscope Zeiss LSM 710 Meta. This work was supported by the Deutsche Forschungsgemeinschaft DFG (German Research Foundation), CRC1116, project B02 to J.S. and U.F.; TE1209/1-1 to S.T.; FL303/61 to U.F., and SCHR 154/13-2 to J.S. REFERENCES (1) Srivastava, A. K.; Kadayakkara, D. K.; Bar-Shir, A.; Gilad, A. A.; McMahon, M. T.; Bulte, J. W. M. Advances in Using MRI Probes and Sensors for in Vivo Cell Tracking as Applied to Regenerative Medicine. Dis. Models & Mech. 2015, 8, 323−336. 11190


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