Subscriber access provided by UNIV OF WESTERN ONTARIO
Article
A Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging Sebastian Temme, Paul Baran, Pascal Bouvain, Christoph Grapentin, Wolfgang Krämer, Birgit Knebel, Hadi Al-Hasani, Jens Mark Moll, Doreen Floss, Jurgen Schrader, Rolf Schubert, Ulrich Flögel, and Jürgen Scheller ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b05698 • Publication Date (Web): 29 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
A Synthetic Cargo Internalization Receptor System for Nanoparticle Tracking of Individual Cell Populations by Fluorine Magnetic Resonance Imaging
Sebastian Temme#1, Paul Baran#2, Pascal Bouvain#1, Christoph Grapentin3, Wolfgang Krämer3, Birgit Knebel4, Hadi Al-Hasani4, Jens Mark Moll2, Doreen Floss2, Jürgen Schrader2, Rolf Schubert3, Ulrich Flögelǂ1* and Jürgen Schellerǂ2* #,ǂ = Equal contribution
AUTHOR ADDRESS: 1Experimental Cardiovascular Imaging, Molecular Cardiology, University of Düsseldorf, 40225 Düsseldorf, Germany; 2Institute for Biochemistry and Molecular Biology II, Medical Faculty, Heinrich-Heine University of Düsseldorf, 40225 Düsseldorf, Germany;
3
Department of
Pharmaceutical Technology and Biopharmacy, Albert Ludwig University Freiburg, 79104 Freiburg im Breisgau, Germany. 4Institute for Clinical Biochemistry and Pathobiochemistry, German Diabetes Center, Leibniz Center for Diabetes Research at the Heinrich-Heine-University Düsseldorf, 40225 Düsseldorf, Germany.
* Corresponding author Name:
Ulrich Flögel Ph.D. or Jürgen Scheller Ph.D.
Mailing address:
Department of Molecular Cardiology or Biochemistry and Molecular Biology II, Heinrich-Heine-University Düsseldorf, Universitätsstraße 1, 40225 Düsseldorf, Germany.
Phone:
+49 211 81 10187 or 11553.
Fax:
+49 211 81 01610187 or 12726.
E-mail:
[email protected] or
[email protected].
KEYWORDS: cell tracking,
19
F magnetic resonance imaging, green fluorescent protein, perfluoro-
carbons, active targeting, endocytosis.
ACS Paragon Plus Environment
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 2 of 38
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
F cargo and readily enabled in vivo
19
visualization of GFP-PFC recruitment to transplanted CIR+ cells by 1H/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.
ACS Paragon Plus Environment
2
Page 3 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
Magnetic 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 create contrast to surrounding tissue. Therefore, several contrast agents (CA) have been developed to load cells and to enable their tracking by MRI,1 whereby most CA make use 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 alternative, non-invasive cell tracking by fluorine (19F) MRI has garnered significant interest over the last decade. Due to almost complete absence of fluorine from biological tissue acquired 19F signals are essentially background-free. Thus, 19F signal generates an unequivocal ‘positive contrast’ which does not interfere with the anatomical 1H image and merging of morphological 1H with corresponding 19F images enables the precise localization of the labelled target. As CA with high
19
F 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, labelling of cells can be performed in vitro (which requires isolation, cultivation and re-implantation) or in situ by intravenous application of PFCs. The latter is more straightforward, but also more challenging since 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 the 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, since 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, since they are smaller in size, weakly immunogenic,
ACS Paragon Plus Environment
3
ACS Nano 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 4 of 38
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 non-adherent 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.
RESULTS Generation and characterization of cargo internalization receptors (CIRs) In a first step, we engineered three different CIRs (Fig. 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 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 Fig. S1. After transfection we verified cellular expression of the CIRs by Western blot analysis against the myc-tag (Supplemental Fig. S2A). Flow cytometry confirmed that CIRs were expressed at the cell surface of both stably and transiently transfected cells (Supplemental Fig. S2B). Interestingly, CIR2/3 exhibited stronger cell surface signals than 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 (Fig. 1B). Again, the strongest signal was detected in CIR2/3+ cells. Similar results were found in the time-dependent binding of GFP (Supplemental Fig. S2C). In separate experiments, we could demonstrate that the CIR system in principle is also applicable to primary cells (Supplemental Fig. S3).
ACS Paragon Plus Environment
4
Page 5 of 38
CIR1
A
CIR2
CIR3 Extracellular space
GFP nanobody
Cytosol IL-6R Myc-tag GFP nanobody
Endo180 IL-6 receptor FcγRIIA
FcγRIIA Endocytic motif Tyrosine activation motif (TAM)
CHO-CIR1-3+
B
Control
CIR1
CIR2
CIR3
Cell count
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
GFP fluorescence
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 FcRII 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: While CIR1 does not contain any known endocytic motifs, CIR2 possesses a tyrosine-based (FEGARY) as well as a di-hydrophobic motif with an upstream acidic residue (EKNILV, yellow circle). The cytoplasmic domain of CIR3 contains nonclassical internalization motifs and two tyrosine activation domains (red dots) which are responsible for the phagocytic properties of the FcRII receptor. (B) Flow cytometric analysis of GFP binding to stably transfected CHO-CIR1-3+ cells. Grey histograms represent controls and green histograms show CIR+ expressing cells treated with GFP.
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 internalization processes. At 4 °C, CIR1+ or CIR3+ cells displayed a homogenous GFP staining of the cell
ACS Paragon Plus Environment
5
ACS Nano
surface, whereas CIR2+ cells showed a more vesicular pattern (Fig. 2A upper panel). Raising 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 interiorized to the endosomal system (Fig. 2A lower panel).
A
CIR1
CIR2
CIR3
4°C
37°C
B
125
CIR cell surface level [%]
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 6 of 38
CIR1 CIR2 CIR3
100
75
50
25
0 0
50
100
150
200
Time [min]
Figure 2: Internalization of GFP. (A) Immunofluorescence of transiently transfected COS-CIR+ cells incubated with GFP at 4 °C (upper panel) and subsequently shifted to 37 °C for 30 min. Note the homogenous GFP staining on the cell surface at 4 °C whereas the GFP signals turned 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 pre-labelled 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 anti-myc mAb to stain residual cell surface CIRs and subsequently analyzed by flow cytometry. Data are mean valuesSD of n=3 independent experiments.
ACS Paragon Plus Environment
6
Page 7 of 38 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Nano
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 mAb for analysis by flow cytometry (Fig. 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 (Fig. 2B). Interestingly, CIR2 showed very rapid endocytosis which then maintained on a constant level, while CIR3 showed a slower internalization rate but the magnitude was higher as compared to 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 Fig. S4A) were functionalized with free SH groups and conjugated to preformed PFCs containing free maleimide groups at the lipid shell (Supplemental Fig. S4B). Binding of GFP-SH to PFCs resulted in a slight increase in size as compared to neat PFCs, however, size distribution was unchanged, while the observed decrease of potential is in line with the added charge of GFP (Supplemental Fig. 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 proof 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 (Fig. 3A, 1st column) can easily be identified as small dark film within the ‘light’ PBS band overlying the dark and ‘dense’ percoll layer (Fig. 3A, 2nd column). Merging of 1H and corresponding 19
F images revealed that GFP-PFC incubation resulted in strong 19F signals that exactly co-localized with
the narrow layers of CIR+ cells (Fig. 3A, 3rd and 4th column, rows 2-4), while control cells showed only marginal 19F signal (Fig. 3A top). Quantification of the 19F signals confirmed a substantial cellular uptake of GFP-PFCs under CIR2/3 expression (0.6/0.4 fmol
19
F nuclei per cell), whereas CIR1 led only to a
moderate internalization of the 19F cargo (Fig. 3B left).
ACS Paragon Plus Environment
7
ACS Nano
A
1H
19F
1H+19F
CHO
CIR1
CIR2
CIR3
B
Stable CHO
Transient CHO
**
40
Stable Ba/F3 15
25
*** * F signal [a.u.]
15
10
19
F signal [a.u.] 19
F Signal [a.u.]
20
***
*
20 30
19
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Page 8 of 38
10
5
10 5
0
0
0
CHO
CIR1
CIR2
CIR3
CHO
CIR1
CIR2
CIR3
Ba/F3
CIR3
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 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 CHOCIR+ 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
