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A promising family of fluorescent water-soluble aza-BODIPY dyes for in vivo molecular imaging Jacques Pliquett, Adrien Dubois, Cindy Racoeur, Nesrine Mabrouk, Souheila Amor, Robin Lescure, Ali Bettaieb, Bertrand Collin, Claire Bernhard, Franck Denat, Pierre-Simon Bellaye, Catherine Paul, Ewen Bodio, and Christine Goze Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00795 • Publication Date (Web): 07 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019
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Bioconjugate Chemistry
A promising family of fluorescent water-soluble aza-BODIPY dyes for in vivo molecular imaging Jacques Pliquetta,b, Adrien Duboisa, Cindy Racoeurb, Nesrine Mabroukb, Souheila Amora, Robin Lescurea, Ali Bettaïebb, Bertrand Collinc, Claire Bernharda, Franck Denata, Pierre Simon Bellayec, Catherine Paulb*, Ewen Bodioa*, Christine Gozea*
AUTHOR ADDRESS [a] Department of Chemistry, ICMUB, 9 avenue Alain Savary, 21000 Dijon, France. [b] Laboratoire d’Immunologie et Immunothérapie des Cancers, EPHE, PSL Research University, 75000 Paris ; LIIC, EA7269, Université de Bourgogne Franche Comté, 21000 Dijon, France. [c] Centre Georges François Leclerc, Service de médecine nucléaire, 1 rue Professeur Marion, BP77980, 21079 Dijon Cedex KEYWORDS: molecular Imaging • Fluorescent probes • Water-soluble fluorophores • aza-BODIPY • boron functionalization ABSTRACT: A new family of water-soluble and bioconjugatable aza-BODIPY fluorophores was designed and synthesized using a boron- functionalization strategy. These dissymmetric bis-ammonium aza-BODIPY dyes present optimal properties for a fluorescent probe, i.e. they are highly water-soluble, very stable in physiological medium, they do not aggregate in PBS, possess high quantum yield and finally they can be easily bioconjugated to antibodies. Preliminary in vitro and in vivo studies were performed for one of these fluorophores to image PD-L1 (Programmed Death-Ligand 1), highlighting the high potential of these new probes for future in vivo optical imaging studies.
These last few years have witnessed the surge of interest for medical imaging. The different imaging modalities help physicians to establish a diagnostic, to monitor the evolution of a disease, and may even assist them during surgery. Molecular imaging is also a powerful tool for a better understanding of biological phenomena and mechanism of action of therapeutic agents. Among the different techniques, optical imaging is very popular due to its high resolution, high sensitivity, its easy and relatively cheap implementation, and its non-ionizing character compared to nuclear imaging (PET – Positron Emission Tomography – or SPECT – Single-Photon Emission Computed Tomography –). Therefore, optical molecular imaging represents the technique of choice for in vitro and ex vivo investigations. It is also experiencing a growing interest for preclinical studies and fluorescence-guided surgery in oncology.[1] However, it has certain limitations for clinical applications due to its limited penetrability.[2] The highest penetration depth can be achieved when the fluorophore absorbs and emits in a region of the light spectrum where biological tissues exhibit less absorption and less autofluorescence, the main investigated field is the NIR I (Near InfraRed I: 650-900 nm). The fluorophore usually needs to be highly conjugated to reach these emission wavelengths, thus involving tedious and low yielding syntheses, and, more importantly, resulting in increased hydrophobicity and lowered chemical and photochemical stability of the dye. This explains why only very few optical probes have been approved for clinical trials. There is currently only one FDA approved compound being used in clinic: IndoCyanine Green (ICG). However, despite numerous advantages, ICG is not the ideal fluorophore for clinical applications because of its low chemical and photochemical stability, toxicity, high plasma protein binding, and nonspecific accumulation.[3] New optimized
cyanines, such as IRDye800 CW, have been recently highlighted for their clinical potential. [4] Among the different fluorophores, we focused our work on boron-dipyrromethene dyes (BODIPY), more precisely on azaBODIPY – derivatives which contain a nitrogen atom at the C8 position of the BODIPY core –. Recently, this family of dyes has gained considerable attention and have been widely studied due to their unique properties: outstanding photostability, high molar absorption coefficients, good quantum yield, absorption/emission bands in red/NIR region, and ability to be structurally modified.[5] Therefore, aza-BODIPY dyes fulfill all the requirements for being the ideal class of fluorophores, except water solubility. Indeed, the presence of numerous aromatic rings and the highly planar structure of the azaBODIPY core cause aggregation phenomena and are responsible of poor water solubility. This drawback is probably the reason why aza-BODIPY dyes have yet been very rarely in in vivo imaging studies. Recently, very elegant strategies were reported, which improve significantly the water-solubility of aza-BODIPYs. Unfortunately, the reported compounds still suffer from aggregation in water, and the use of surfactants was mandatory to prevent their aggregation in water or physiological media (see ESI section for a list of all the additive, which were used).[1,6-16]. In order to take advantage from the potential of these fluorophores, we tried to develop a new methodology to solve this aggregation problem.
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Synthesis of water soluble aza-BODIPYS We first tried to improve the hydrophilicity of aza-BODIPYs following a strategy reported by the group of O-Shea, by adding polyethylene glycol (PEG) chain(s) or sulfonate groups on the aza-BODIPY core (e.g. aza-BODIPY A, Figure 1). [6,7] At first glance, this strategy is attractive but it is timeconsuming, yields a very limited number of desired compounds, and, more problematically, aggregation phenomena are not totally prevented. Therefore, the probe A required, as for the previous reported systems, the use of formulation agents, such as SDS (Sodium DodecylSulfate), Triton X-100, or Tween 10, to prevent aggregation in physiological media.[1,6-16]
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EtMgBr, THF
N N
reflux, 1h30 90%
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O
N
Cl
3Cl H N
A
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H N
NHBoc
4
Figure 1: Aza BODIPY developed by O’shea (left) and our azaBODIPY A (right).
Consequently, we decided to investigate the functionalization of the boron atom of the aza-BODIPY, a strategy which has been widely investigated on BODIPY dyes, but almost not reported on aza-BODIPY (Figure 2).[16] This approach offers many advantages: (1) increased water solubility by addition of two hydrophilic arms; (2) prevention of aggregation phenomena thanks to steric hindrance of aza-BODIPY faces; (3) increased chemical stability especially towards acidic and basic conditions; (4) improved photophysical properties; (5) introduction of different functionalities on the aza-BODIPY, without changing absorption and emission maximal
O
1.
O S O O CH3CN, NaHCO3, r.t.
OEt
EtOH, 5eq DIPEA, 50°C, 5h
N B
O
EtO
84%
2. HCl 1M
O
NH3 3
Wazaby2
38%
N
O
O
3
N N
N
N
O
N
O
B
N
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2Cl
2Cl O 3S
H N
O
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3
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O
DMSO/Carbonate, 37°C
OEt
EtOH, 5eq DIPEA
NH2
Overnight
30°C, 5h
N
N N
O
N
B
N
N
O
O
N
N
B
N
O
N
2Cl
Cl O 3S O
NH
O O
H N
Wazaby3
3 EtO
NH O
O O
Wazaby4-anti-PD-L1 (43% yield)
=
H N
O
HN
O
3
anti PD-L1 antibody
wavelengths. [17] Figure 2: General structure of the targeted aza-BODIPY
Scheme 1: Synthetic pathway for the different Wazabys Being fully convinced by the potential of this approach, we challenged ourselves to construct new optical imaging probes by doing all the chemistry on the boron atom of the azaBODIPY precursor. Thus, we designed a new kind of aza-
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0,2
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600
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800
Wavelength [nm]
Photophysical studies The photophysical properties of the Wazaby derivatives were investigated in DMSO (dimethyl sulfoxide) and in physiological medium (PBS) and compared to the aza-BODIPY A (Table 1). Table 1. Photophysical studies of the new aza-BODIPYs. Compoun d
Solvent
abs (nm)
(M-1.cm-1)
em (nm)
[a]
Br [b] (/1000)
Wazaby1
DMSO
703
79 700
731
0.41
32.4
Wazaby1
PBS
676
51 600
712
0.09
4.7
Wazaby2
DMSO
696
58 800
726
0.32
18.9
Wazaby2
PBS
673
50 500
714
0.07
3.2
A
DMSO
672
50 000
725
0.04
2.2
A
PBS
700
35 900
-
[a]: reference: dimethoxy BODIPY (Q = 36% in CHCl3, exc = 670nm).[19] [b]: Brightness = ε.Φf
The two aza-BODIPY dyes absorb and emit in the window of transparency (650-900 nm), with emissions between 712 and 731 nm (Figure 3). It is noteworthy that besides the blue shifts of around 20 nm of the absorption and emission spectra due to different solvent polarities, the absorption, emission and excitation profiles of Wazaby1 and 2 in DMSO and in PBS are strictly identical, meaning that there is no aggregation of the aza-BODIPY dyes
500
600
700
normalized Absorption
0,6
0,4
0,2
0,0 300
800
400
0,2 0,2
0,2 0,2
0,0 0,0
0,0 0,0 400 400
500 500
600 600
Wavelength [nm] Wavelength [nm]
700 700
0,8
Wazaby1, DMSO PBS Wazaby2, Wazaby1, PBS
1,0 1,0
0,6
0,6 0,6 0,6
0,6 0,6
0,4 0,4 0,4
0,4 0,4 0,4
0,2 0,2 0,2
0,2 0,2 0,2
0,0 0,0 0,0
0,0 0,0 0,0 400 400 400
500 600 700 500 600 500 600 [nm] 700 700 Wavelength
800 800 800
1,0 1,0
0,8 0,8 0,8
0,8 0,8 0,8
0,8 0,8
0,8
0,6 0,6 0,6
0,6 0,6 0,6
0,6 0,6
0,6
0,4 0,4 0,4
0,4 0,4 0,4
0,4 0,4
0,4
0,2 0,2 0,2
0,2 0,2 0,2
0,2 0,2
0,2
0,0 0,0 0,0
0,0 0,0 0,0
0,0 0,0
0,0
1,0
500 600 500 600 500 600[nm] Wavelength
Wavelength [nm] Wavelength [nm]
700 700 700
800 800 800
Wazaby2, PBS
300 300
400 400
500 500
1,0 1,0
600 600
Wavelength [nm] Wavelength [nm]
700 700
0,8
0,6
0,4
0,2
0,0
800 800
1,0
0,8
0,8
0,6
0,6
0,4
0,4
0,2
0,2
The quantum yields of the fluorescence emission and the resulting brightness of the two Wazabys are lower in PBS, due to non-emissive interactions between the dyes and different species present in the PBS media, but still sufficient for in vitro and in vivo use. When the TOTA group was introduced onto the periphery of the aza-BODIPY (compound A, Figure 1) using the “classical” approach, the resulting fluorophores exhibited very little fluorescence in DMSO and no fluorescence at all in PBS solution (Table 1), thus demonstrating the added value of our strategy. 0,0
0,0
300
400
500
600
700
800
Wavelength [nm]
Bioconjugation and preliminary in vitro and in vivo investigations The next step of our study was to validate the ability of Wazabys to be easily conjugated to biomolecules such as antibodies. Several strategies were investigated and the most efficient one implies the use of diethyl squarate, which enables the synthesis of bioconjugatable Wazaby3 and Wazaby4 in good yields (Scheme 1). Moreover, this activating group is less prone to hydrolysis compared to the classical ones such as NHS-ester or isothiocyanates.[20] Wazaby3 and Wazaby4 were isolated as pure compounds and can be stored for several without any degradation. Their photophysical properties were studied in DMSO and PBS, revealing no change compared to their non-activated analogues (see ESI for more details on photophysical studies). After optimization of the bioconjugation conditions of Wazaby3 and Wazaby4 on small molecules and control antibodies (data not shown), we decided to tether Wazaby4 on a rat anti-PD-L1 monoclonal antibody (BioXCell, clone 10F.9G2). PD-L1 is a transmembrane protein overexpressed by numerous human and murine tumor cells (e.g. murine colon cancer CT26 cells)[21]; like an immune checkpoint inhibitor it is one of the key targets of immunotherapy. We chose this antibody for our pilot preclinical study because it allows the use of tumor-bearing syngeneic mice presenting an active immune system, which is ideal for a proof of concept (contrary to human
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Wavelength Wavelength[nm] [nm]
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W 1,0
Figure 3: Absorption (blue), Emission (red) and excitation (green) spectra of Wazaby 1 and 2 in DMSO, and in PBS, at 25°C. normalized Absorption
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0,6
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300
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0,4
0,8 0,8
0,8
1,0 1,0 1,0
1,0
1,0
0,6
300 300
0,2
600
0,8
1,0
Wazaby1, DMSO Wazaby1, DMSO
normalized Fluorescence [a.U.] normalized Fluorescence [a.U.] normalized Fluorescence [a.U.]
normalized Absorption normalized Absorption
1,0
0,6
500
0,2
1,0
normalized Fluorescence [a.U.] normalized Fluorescence [a.U.]
1,0
0,8
400
0,4
Wazaby1, P
0,8
Wavelength [nm]
0,6
300
0,6
0,4
300
0,8
0,4
0,6
normalized Fluorescence [a.U.]
normalized Absorption
1,0
0,8
in physiological media, and, very importantly, without adding any detergent.
normalized Fluorescence [a.U.]
The synthesis of Wazabys is depicted in Scheme 1. The starting aza-BODIPY 1 was obtained on a gram-scale by DMSO optimizing Vicente’s four-steps synthetic Wazaby1, pathway (see ESI [10] section). Then, the fluorine atoms on the boron center were substituted using the Grignard reagent of N,Ndimethylpropargylamine, and one of the dimethylamine moieties were quaternized with benzoic acid. The resulting dissymmetrical precursor 3 was then functionalized by peptide coupling reaction between the carboxylic acid function and the Wazaby2, DMSO pseudo – PEG-TOTA-Boc to give compound 4. Then the second dimethylamino group was alkylated with either propane sultone or iodomethane.[18] After removal of Boc protecting group in acidic conditions, two water-soluble compounds were obtained: Wazaby1 and Wazaby2.
1,0
1,0
Wazaby1, DMSO
0,8
normalized Fluorescence [a.U.]
normalized Absorption
BODIPY imaging probes, that we called Wazaby for Watersoluble aza-BODIPY dyes. Wazabys fulfill all the requirements for optical molecular imaging: they can be readily synthesized, are highly water soluble, do not show any aggregation in PBS (phosphate buffer saline), present very good photophysical properties in physiological media, and are chemically and photophysically robust.
normalized Absorption
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
1,0
Bioconjugate Chemistry
normalized Absorption normalized Absorption normalized Absorption
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Bioconjugate Chemistry 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
antibody such as trastuzumab, which implies the use of immunodeficient mice). 10 equivalents of Wazaby4 were reacted with the anti-PD-L1 antibody, yielding the desired bioconjugate Wazaby4-anti-PD-L1 after purification on FPLC (Fast protein liquid chromatography) with a DOL (Degree of Labeling) of 2.9 assessed by MALDI-TOF. As a reference for the in vitro and in vivo studies, we conjugated NHS-Cyanine 5 derivative (a fluorophore commonly used for optical imaging studies) to the same anti-PD-L1 antibody with a DOL of 2.5 (named Cy5-anti-PD-L1). Blue Coomassie staining of denaturing polyacrylamide gel electrophoresis (SDS-PAGE) showed that the Wazaby4-antiPD-L1 and Cy5-anti-PD-L1 were highly pure and resolved into bands of around 150 kDa in non-reducing conditions (fulllength antibody) or 50 and 25 kDa in reducing conditions (heavy and light chains respectively) (Figure 4). Fluorescence analysis of these gels demonstrated that both Cy5 and Wazaby4 were conjugated on the anti-PD-L1 antibody (non-reducing conditions). It is worth noting that Wazaby4 was preferentially conjugated on heavy chains while Cy5 was conjugated on both heavy and light chains (reducing conditions). The fact that Wazaby4 was not grafted on light chains is particularly valuable since it is more likely that this does not disturb the recognition of the epitope.
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nor the decrease of the fluorescence intensity on the antibody were observed. Fluorescence emission 1
2
3
4
5
Full-length Antibody
Figure 5: Stability of the Wazaby4-anti-PD-L1 in mice plasma were analyzed by electrophoresis on SDS-PAGE (7% acrylamide gel) in non-reducing conditions. The fluorescence analysis of the gel was performed on Odyssey CLx Infrared Imaging System (LICOR Biosciences) in pair filter mode (685/700 nm). 1: Wazaby4anti-PD-L1 without incubation in mice plasma, 2: mice plasma without Wazaby4-anti-PD-L1, 3 to 5: Wazaby4-anti-PD-L1 incubated in mice plasma during respectively 0, 24 and 48h.
After checking that Wazaby derivatives did not display significant cytotoxicity on cells (see antiproliferative assay in ESI section) and detecting them by confocal imaging on tumor cells CT26 (see Figure 64 in ESI section), the ability of this molecule to target PD-L1 expressing tumors was investigated in vivo in a model of colon cancer based on the subcutaneous xenograft of CT26 tumor cells in balb/c mice. Whole-body fluorescence images were recorded at different time points (1, 6, 24 and 48 h) post injection of Wazaby- or Cy5-anti-PD-L1 (Figure 6A and B), revealing, that the fluorescent aza-BODIPY probe emitted an intense signal in vivo.
Figure 4: The anti-PD-L1 antibodies uncoupled (UC) or coupled with cyanine 5 (Cy5) or Wazaby4 (W4) were analyzed by electrophoresis on a SDS polyacrylamide gel (SDS-PAGE analysis, 10% acrylamide gel) in non-reducing (without DTT (dithiothreitol), top panels) or reducing conditions (with heating and DTT, bottom panels). After Coomassie blue staining during 1h, the gels were visualized on a Chemidoc XRS+ analyzer (BioRad, left panels). PL: protein Ladders (180, 130, 95, 72, 55, 43, 34, 26, 17 kDa). The fluorescence analysis of the gels was performed on Odyssey CLx Infrared Imaging System (LI-COR Biosciences, right panels) in pair filter mode (685/700 nm).
Then, the stability of Wazaby4-anti-PD-L1 was demonstrated after incubation in mice plasma at 37°C and analysis on a SDS-PAGE in non-reducing conditions (Figure 5). Even after 48h of incubation at 37°C, neither the degradation
Figure 6: A and B: NIR fluorescence images of a subcutaneous CT26 tumor-bearing mouse at 1, 6, 24 and 48 h post injection of Wazaby4-anti-PD-L1 (A, Ex/Em filters: 660/710 nm, IVIS Lumina, Perkin Elmer) and Cy5-anti-PD-L1 (B, Ex/Em filters:
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Bioconjugate Chemistry 620/660 nm). Mice received a single injection of Wazaby4-antiPD-L1 (50 µg) or Cy5-anti-PD-L1 (50 µg). C and D: Quantification of fluorescence tumour uptake overtime. Results are presented as mean ± SEM, n=3, *p
