NIRF

Communication pubs.acs.org/bc

Avidin/Biotin Bioinspired Platform for Dual In Vivo Molecular Imaging

18

F‑PET/NIRF

Annelaure Damont, Raphael Boisgard, Frédéric Dollé, Morgane Hollocou, and Bertrand Kuhnast* IMIV, Service Hospitalier Frédéric Joliot, CEA, Inserm, Université Paris Sud, CNRS, Université Paris-Saclay, Orsay, France S Supporting Information *

ABSTRACT: The complementary nature of positron emission tomography (PET) and near-infrared fluorescence (NIRF) imaging makes the development of innovative multimodal PET/NIRF probes a very exciting prospect. Herein, the bioinspired design of novel platform exploiting the strength and specificity of interactions between radioactive and fluorescent biotin derivatives and an avidin core is reported. The combination of an original [18F]fluoropyridinylated-biotin derivative and commercially available fluorescent biotin derivatives (Atto-425 and Atto-680) is investigated. The in vivo distribution of such a customized platform is also reported, for the first time, in healthy rodent using PET and ex vivo fluorescence imaging.

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information in terms of spatial resolution at the histologic and superficial levels. Major advantages of this unique combination are as follows: (i) both radiolabeled and fluorescent probes can be detected in the same range of concentrations (nanomolar and even below);1 (ii) matched dynamic whole body (PET) and intraoperative or histologic (OI) images can be produced; and (iii) quantitative PET imaging allows for the quantification of fluorescent images. Conceivably, a patient would undergo a noninvasive whole body nuclear scan (PET) to assess the distribution of the radioactive imaging agent to the targeted (diseased) tissue and observe its (dys)function, and subsequently, the NIRF signal would allow for a refined observation with in vivo endomicroscopy systems which generate optical biopsies or with ex vivo fluorescence microscopy.2−4 These recent evolutions and even revolutions in medical molecular imaging have lead to reinvention of some of the imaging agents. The challenge of implementation of such combined imaging is to design a probe that combines, in a single monomolecular structure, a targeting moiety, a positronemitting radionuclide, and a NIRF dye. Such a platform avoids the weaknesses of approaches based on a sequential or simultaneous “cocktail” injection of the radioactive and fluorescent probes separately, both suffering from the possibly

olecular medical imaging is able today to provide multiple and complementary information (anatomic, physiologic, metabolic) for accurate diagnosis of disease, treatment response prediction, and patient followup. In other words, molecular medical imaging is the cornerstone of contemporary stratified and personalized medicine. Imaging is indeed a precious component of medical diagnosis and patient care either in observing tissue anatomy and morphological changes using, for example, Magnetic Resonance Imaging (MRI), Ultra Sound (US), or Computerized Tomography (CT), or by tracking functional and molecular events with Positron Emission Tomography (PET), Single Photon Emission Computerized Tomography (SPECT), or Optical Imaging (OI). In spite of their wide use in preclinical research and clinical diagnosis, these imaging modalities considered individually present weaknesses. However, when cleverly combined, these techniques can not only concomitantly provide complementary information, but also synergistically conjugate their advantages in terms of spatial and temporal resolution, depth penetration, sensitivity, and cost. Besides the classical combination of PET and CT used today, or more recently PET and MRI, dual PET/near-infrared fluorescence (NIRF) imaging holds great promise. PET is currently considered the gold standard in functional molecular nuclear imaging without depth limitation (thanks to the high 511 keV energy photons emitted and tomographic reconstruction) and fluorescence contrast imaging nicely provides complementary © XXXX American Chemical Society

Received: September 4, 2017 Published: September 20, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00536 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry Scheme 1. Synthesis/Radiosynthesis of the Fluorinated Biotin Derivatives (3/[18F]3)a

a Reagents and conditions: (a) EtOH, H2SO4, r.t. 4 h; (b) LiAlH4, CH2Cl2, −78 °C to r.t., 1 h; (c) TsCl, pyridine, r.t., 3 h; (d) K2CO3, 3-hydroxy-2fluoropyridine, DMF, 70 °C, 2 h; (e) K2CO3, 3-hydroxy-2-nitropyridine, DMF, 70 °C, 2 h; (f) K[18F]F-K222, DMSO, 160 °C, 5 min.

Systems). Average production yields were 44% ± 9% (n > 10, decay-corrected) in a 50 to 55 min synthesis time. Chemical and radiochemical purity of [18F]3 exceeded 95%, with an average decay-corrected MA of 153 ± 45 GBq/μmol (see details in SI section 3). Other groups have described the synthesis of fluorine-18labeled biotin derivatives, where the radionuclide was borne by either a sp3-carbon or a boron atom. In the first case, Shoup et al. synthesized two [18F]fluoroalkyl-biotin derivativesusing a mesyloxy precursor for labelingbut the yields were lower (12% to 20% non-decay-corrected) than those we obtained for longer synthesis times (90 min).5 Blom et al. proposed three [18F]fluoroalkyl- and [18F]fluoroalkoxy-biotin derivatives prepared in moderate to good yields (13% to 35% decaycorrected) and with high MA (320 GBq/μmol).6 Compared to these two examples, the strategy that we have developed allows the production of an original [18F]pyridinyl-biotin derivative in 30% to 80% higher radiochemical yields. Claesener et al. also synthesized another [18F]fluoroalkyl-biotin derivative, using a tosyloxy precursor too. Whereas comparable yields (45%) and reaction times (45 min) were reported, the MA they observed was drastically lower (16 GBq/μmol) than the one we obtained.7 None of these studies proposed a whole body PET image of the [18F]biotin derivative/avidin complex, in spite of the fact that the affinity of all these radioactive biotinderivatives for the avidin was assessed. The biodistribution of only two [18F]biotin detivatives was evaluated.5,7 When a boron center was considered, two examples were reported in the literature.8,9 In both cases, the radiolabeling was performed by a 19F/18F isotopic exchange leading to the radiolabeled biotin derivatives with expectedly low MA, even if these values are not precisely reported. As a consequence, the low MA may limit the binding efficiency to avidin. Moreover, it has recently been observed that defluorination of 18F-boronbased radiotracers may occur in vivo,10 restraining therefore the interest for such labeling processes. Strategically, we have chosen to introduce fluorine-18 on a sp2-carbon center and at the alpha-position of a pyridine ring to benefit from (i) the high carbon−fluorine bond stability observed for this motive11 and (ii) the high yielding radiofluorination of the pyridine moiety at the ortho position.12,13 Binding Potency to Avidin. We have then evaluated in vitro (i) the binding kinetics of [18F]3 to avidin, (ii) the binding specificity of [18F]3 regarding other proteins, (iii) the stability

distinct pharmacokinetics and imaging time windows of each agent leading to discrepancies in final (combined) images. In this communication, we describe the preparation of the first fully modular avidin/biotin-based bioinspired platform, for dual 18F-PET/NIRF molecular imaging. To that purpose, we have synthesized and radiolabeled at high molar activity (MA) with the short-lived positron-emitter fluorine-18 (T1/2: 109.8 min) an original fluorinated biotin-derivative (3, Scheme 1) of which we have confirmed the binding potency (affinity, selectivity) to avidin. Then, using two commercially available fluorescent dye-containing biotin derivatives (Atto-425, a bluefluorescent dye, and Atto-680, a near-IR fluorescent dye) and our newly developed radioactive derivative, we have demonstrated the possibility to decorate in a fully modular fashion avidin, leading to customized 18F-PET/NIRF platforms. Finally, for the first time, we have evaluated the biodistribution in vivo ̈ of one of these “naive” dual probes with PET imaging in healthy rats, including a set of ex vivo fluorescence images recorded after a single injection of the 18F-PET/NIRF platform.

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RESULTS AND DISCUSSION Chemistry/Radiochemistry. Starting from natural biotin (1), the carboxylic acid function was first esterified with ethanol in acidic conditions (81%) and the resulting ethyl ester reduced with lithium aluminum hydride to the corresponding alcohol (93%) (Scheme 1). The latter was then converted to the tosyloxy key-intermediate 2 (78%) from which both the nonradioactive fluorinated biotin derivative 3 and its nitro analogue 4 as precursor for labeling with fluorine-18 could be analogously obtained, from 2-fluoro- and 2-nitro-3-hydroxypyridine, respectively. Using this straightforward pathway, intermediate 2 was obtained in an overall 59% yield (3 steps), and compounds 3 and 4 in 70% and 54% yields (from compound 2), respectively (see details in SI). Radiolabeling with fluorine-18 was performed in standard reaction conditions, involving a nitro-for-fluorine aromatic nucleophilic substitution. Precursor 4 (4 to 5 mg) was reacted for 5 min at 160 °C in DMSO with K[18F]F−K222 complex, as a source of no-carrieradded and activated [18F]fluoride. The crude was prepurified using an Alumina N cartridge before semipreparative RP-HPLC and final formulation in an in vivo compatible excipient (0.9% aq. NaCl/EtOH: 80/20 (v/v)). The whole process (radiosynthesis, purification, and formulation) has been fully automated on a TRACERLab Fx N Pro synthesizer (GE Medical B


Communication

Bioconjugate Chemistry of the [18F]3/avidin complex, (iv) the affinity of [18F]3 for avidin in competition binding assays, and (v) the multiplexing of avidin with [18F]3 and two (commercially available) fluorescent biotinylated dyes. For all these in vitro experiments, the formation of the biotin/avidin complexes was systematically assessed in a double-analysis approach, using radio-iTLC and size exclusion chromatography (SEC) (see details in SI sections 1.2.2; 1.2.3; 4.1). Binding kinetics was established by analyses of a mixture of [18F]3 and avidin (in excess), every minute over a total incubation period of 5 min at room temperature. Both iTLCs and SEC analyses showed that more than 98% of the radioactivity was bound to avidin as early as 1 min of incubation (iTLC: >98% of radioactivity detected at Rf = 0.0, corresponding to [18F]3/avidin complex; SEC: >98% of the radioactivity eluted in the high-MW fraction, corresponding to [18F]3/avidin complex; see SI section 4.2). These results not only confirmed that the introduction of a fluoropyridine moiety at the alkyl-terminal position of biotin does not alter the potency of this derivative to bind to avidin, but also demonstrated that the formation of such avidin complexes is compatible with the short half-life of fluorine-18. The stability of the [18F]3/avidin complex has been evaluated at different time points after complex formation and purification. More than 97% of the radioactivity was associated with the high-MW-fraction over a period of time of 90 min in a SEC analysis (see details in SI section 4.3; Table S1). These results showed that there is neither release of [18F]3 (as expected) nor generation of radioactive low-MW byproducts that would be retained in the gel column. The nonspecific binding of [18F]3e.g., binding of 3 to a protein lacking the binding site of the characteristic bicyclic motive (oxo-tetrahydro-1H-thieno[3,4-d]imidazo) borne by all biotin derivativeswas evaluated by incubating [18F]3 with a Bovin Serum Albumin (BSA) buffered solution for 5 min at room temperature. iTLCs and SEC showed that no radioactivity was associated with the protein (iTLC: radioactivity was only detected at Rf = 0.7, corresponding to free [18F]3; SEC: no radioactivity was eluted with the high-MW fraction and more than 98% of the radioactivity was retained on the column, which is compatible with free [18F]3; see SI section 4.4). As natural biotin, the fluoropyridinilated analogue 3 is devoid of nonspecific binding properties to proteins. Affinity of [18F]3 for avidin was finally evaluated in a binding competition assay. [18F]3 was incubated with a constant amount of avidin (7.5 nmol, thus corresponding to 30 nmol in terms of binding sites for biotin) and increasing amounts of (nonradioactive) 3 or natural biotin (Figure 1) (see SI section 4.5 and Table S2 and Table S3). Competition assays were analyzed according to different methods: two of them were analyzed by iTLC and the third one by SEC. Graphical determination allowed establishing the IC50 to 30 μM and 45 μM for biotin and 3, respectively. This result definitively confirmed that the chemical structure of our biotin derivative, and thus the modifications introduced when compared to natural biotin, do not affect its binding affinity for avidin. Multiplexing and Customized Constructions. Finally, we have evaluated in vitro the multiplexing ability of this bioinspired platform by simultaneous complexation of various biotin derivatives with avidin (Figure 2): [18F]-3 as well as two commercially available fluorescent biotins (Atto-680 and Atto425).

Figure 1. Binding competition assay.

In a first set of experiments, [18F]3 (4.06 nmol) and Atto-680 (8.9 nmol) or [18F]3 (4.12 nmol) and Atto-425 (14.06 nmol) were incubated with an excess of avidin (7.57 nmol avidin corresponding to 30 nmol binding sites). In these conditions the binding of all biotin-derivatives exceeds 84%, indicating an almost quantitative complexation (Table 1, entries 1/2, and SI section 4.6). In a second set of experiments, avidin (7.57 nmol or 30.3 nmol) was incubated with equimolar amounts of the three biotin derivatives. When an excess of avidin−biotin’s binding sites (121.2 nmol, e.g., 30.3 nmol avidin) was incubated with a total amount of 36 nmol of biotin derivatives ([18F]3 = 13.3 nmol; Atto-680 = 8.9 nmol; Atto-425 = 14.1 nmol), the binding of each biotin derivative exceeds 92%, demonstrating again a quantitative complexation of the biotin derivatives (Table 1, entry 3, and SI section 4.6). In a third and final set of experiments, a default of avidin−biotin’s binding sites (30 nmol, e.g., 7.57 nmol avidin) was incubated with a 20% excess of biotin derivatives (condition 1: total amount = 36 nmol distributed according to [18F]3 = 13.1 nmol; Atto-680 = 8.9 nmol; Atto-425 = 14.1 nmol; condition 2: total amount = 38 nmol distributed according to [18F]3 = 13.1 nmol; Atto-680 = 10.7 nmol; Atto-425 = 14.1 nmol), a partial binding was observed but the values were equally distributed between the different biotin derivatives (61%, 70%, and 65% (condition 1) and 53%, 53%, and 52% (condition 2) for [18F]3, Atto-680, and Atto-425, respectively (Table 1, entries 4/5 and SI section 4.6). These results show that it is possible to perform a fine-tuning of the design of the platform with different biotin derivatives and to reach the desired balance between imaging dyes and targeting entities in the final construction, in particular for dual 18 F-PET/NIRF imaging applications. Fluorine-18, as radioactive isotope in dual PET/OI platforms, appeared in the literature concomitantly with 18F/boron chemistry9,14 and was notably used to radiolabel the dye BODIPY, which contains a BF2 moiety.15−17 Other dual imaging dyes containing B[ 18 F]F 3 entities were also reported.14,18 However in all cases, the final imaging dyes were obtained with low MA either due to the need for nonradioactive fluoride carrier to boost the radiolabeling process or simply due to 18F/19F exchange. The maximum MA reported was 2.7 GBq/μmol,16 a very low value compared to the 100−500 GBq/μmol usually reported for non-carrieradded 18F-labeled radiotracers. Such low MAs drastically limit the potential applications of these probes in receptor-based imaging strategies due to the minute amounts of targets to visualize. Moreover, classical BODIPY dyes are not emitting fluorescence light in the NIR spectrum, which is strongly recommended for in vivo imaging. It was also recently reported C


Communication

Bioconjugate Chemistry

Figure 2. Schematic representation of the bioinspired platforms.

Table 1. Evaluation of Multiplexed [18F]3/Fluorescent-Biotin Avidin Platforms biotin-derivatives 1 2 3 4 5

% of biotin-derivatives bound with avidin 18

18

Atto-680 (nmol)

Atto-425 (nmol)

[ F]3 (nmol)

Avidin (nmol)

[ F]3

8.9 0 8.90 10.7 8.90

0 14.06 14.06 14.06 14.06

4.06 4.12 13.3 13.2 13.1

7.57 7.57 30.3 7.57 7.57

93a 94a 92a 53a 61a

Atto-680-biotin 0.240b 0.292b 0.176b 0.19b

86c 100c 53c 70c

Atto-425-biotin 0.1625b 0.1906b 0.1013b 0.1253b

84c 99c 52c 65c

a % calculated as the ratio between the amount of radioactivity associated with the high-MW fraction (γ counting) and the initial amount of [18F]3 introduced in each assay. bOptical measurement using a UV detector at λ = 680 nm for Atto-680-biotin and λ = 425 nm for Atto-425-biotin. c% calculated as the ratio between the optical measurement associated with the high-MW fraction and the standard OD values for each fluorescent biotin-derivative.

Figure 3. In vivo PET biodistribution in rats of a [18F]3/Atto-680 bioinspired platform (n = 3; injected dose = 41 ± 3 MBq, equivalent to 1.11 nmol (MA = 20 GBq/μmol at injection time)).

that the 18F-boron bond could be less stable than expected.10 To date, only one unique study proposes an alternative to the 18 F/boron-based dual PET/NIRF imaging dyes.19 It is based on a dual prosthetic reagent bearing fluorine-18 on a carbon center on one hand and a fluorescent dye on the other hand. Its conjugation to a vector and applicability in vitro or in vivo has not been demonstrated yet. Several examples using positronemitting radiometals have also been reported,20,21 and very recently, a 64Cu-PET/NIRF streptavidin/biotin-based platform was published using a dimeric RDG-containing peptide as targeting entity.22 Note that in this example, both imaging entities ([64Cu]Cu-DOTA and AlexaFluor-680) were covalently bound to streptavidin, and only the dimeric RGDcontaining peptide was derivatized with biotin. In this approach, like in most of other examples reported in the literature so far, the dual PET/fluorescence imaging moiety is a

covalent construction limiting the modular fashion of the bioinspired biotin−avidin platform. In our case, all imaging entities as well as the future targeting entities are biotinderivatives, allowing thus to tune on-demand and in a flexible manner the design and characteristics of the final platform. ̈ First In Vivo Experiments. Biodistribution of a “naive” platform, i.e., a platform free of a targeting entity, was evaluated in healthy rats (Wistar). For this, [18F]3 (407 MBq, 13 nmol) and Atto-680 (18 nmol) were incubated with avidin (1.34 mg, 20 nmol) for 5 min at room temperature, and the crude reaction mixture subjected to SEC purification. The resulting platform was then formulated as described above and injected, in part (10% based on radioactivity: 41 ± 3 MBq, 1.8 nmole Atto-680, 135 μg avidin; see SI, section 5.1), to animals (n = 3). In vivo distribution of the platform was followed by PET imaging (INVEON, Siemens) for 60 min. Images showed a D


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Bioconjugate Chemistry

Figure 4. A. Semiquantitative fluorescence images of collected organs at 60 min after injection of the [18F]3/Atto-680 bioinspired platform (exposure time 100 ms; equivalent Atto-680 injected dose = 1.8 nmol). B. Transverse PET images of the kidneys displaying a strong cortical distribution of the radioactivity.

combination of an original radiofluorinated biotin derivative and commercially available fluorescent biotin derivatives, all cross-connected thanks to the avidin-core, not only demonstrates for the first time the feasibility and versatility of such an approach, but also opens the route to almost unlimited combinations and connections of biotin derivatives with avidin, including the addition of a targeting vector.

rapid initial distribution through the vascular tree (Figure 3, 1 min) and elimination of the radioactivity through the liver and kidneys to a lesser extent (Figure 3, 2 min). At later time points (Figure 3, 30 and 60 min), the radioactivity was mainly accumulated in the liver and biliary tract suggesting a hepatobiliary elimination of the probe. Note the cortical distribution of the radioactivity in the kidneys that will largely persist until 60 min. Time activity curves (TACs) derived from dynamic PET images confirmed the previous observations (SI, section 5.2, Figure S1). Radioactivity could be mainly detected in elimination organs (liver and kidneys) from which a slow washout could be observed. Radioactivity did not accumulate in lungs and muscles (a rapid clearance was shown for these organs). The absence of any radioactivity in bones also suggests a good metabolic stability of the radiolabeling and absence of defluorination. Taken together, images and TACs both demonstrated the absence of nonspecific signal for the platform and strongly suggest that, once equipped with a relevant targeting entity (using another biotin link), such a construction will be amenable to detection in vivo, and, with the exception of the perimeter of the elimination organs, specific targets. Finally, 60 min after injection, animals were sacrificed and organs (liver, kidney, heart, muscle, and lungs) were withdrawn and exposed in darkness to a Fluobeam 700 camera (Fluoptics). In semiquantitative fluorescence images (Figure 4A), liver and kidneys could be detected but not lungs, muscle, and heart, confirming the biodistribution of the radioactivity observed in PET images. The fact that the fluorescence signal appeared stronger in the kidneys than in the liverin contrast to the PET imagescan be explained by the cortical (superficial) distribution of the platform in this organ, as it can be seen on the transversal PET images (Figure 3 30/60 min and Figure 4B). Combined in vivo PET and ex vivo fluorescence images demonstrate a good colocalization of the imaging platform in all examined organs. This set of imaging experiments demonstrates the possibility of dual PET/optical imaging based on a multiplexed avidin− biotin platform, where biotin is derivatized with both a radionuclide (fluorine-18) and a fluorescent dye (Atto-680).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00536. Additional details on syntheses, radiosyntheses, nonradioactive and radiolabeled compound characterizations, and chromatographies. Additional information on in vitro assays. Technical and ethical details on animal experiments (PET and optical), time-activity-curves derived from PET images. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: + 33 1 69 86 77 36. ORCID

Bertrand Kuhnast: 0000-0002-5035-4072 Notes

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSIONS The design of a fully modular and monomolecular dual PET/ OI platform, exclusively based on the strong and specific avidin−biotin recognition, was herein reported. Moreover, the E


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