Two-photon fluorescence and magnetic resonance specific imaging of

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Two-photon fluorescence and magnetic resonance specific imaging of A# amyloid using hybrid nano-GdF3 contrast media Francis Mpambani, Andreas Aslund, Frederic Lerouge, Sofie Nystrom, Nina Reitan, Else Marie Huuse, Marius Wideroe, Frederic Chaput, Cyrille Monnereau, Chantal Andraud, Marc Lecouvey, Susann Handrick, Stefan Prokop, Frank L Heppner, K. Peter R. Nilsson, Per Hammarström, Mikael Lindgren, and Stephane Parola ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.8b00191 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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ACS Applied Bio Materials

Two-photon fluorescence and magnetic resonance specific imaging of Aβ amyloid using hybrid nanoGdF3 contrast media Francis Mpambania‡, Andreas K.O. Åslundb,c‡, Frederic Lerougea*, Sofie Nyströmc, Nina Reitanb, Else Marie Huused, Marius Widerøed, Frederic Chaputa, Cyrille Monnereaua, Chantal Andrauda, Marc Lecouveye, Susann Handrickf, Stefan Prokopf, Frank L. Heppnerf, Peter Nilssonc, Per Hammarströmc, Mikael Lindgrenb, Stephane Parolaa* a

Laboratoire de Chimie ENS Lyon, Université de Lyon, Ecole Normale Supérieure de Lyon,

Université Claude Bernard Lyon 1, CNRS UMR 5182, 46 allée d’Italie, 69364 Lyon, France. b

Department of Physics, Norwegian University of Science and Technology (NTNU), 7491

Trondheim, Norway c

IFM-kemi, Linköpings Universitet, 581 83 Linköping, Sweden

d

Department of Circulation and Medical Imaging, NTNU, 7491 Trondheim, Norway

e

Laboratoire CSPBAT, UMR 7244, CNRS, Université Paris 13, 74 Rue Marcel Cachin, 93017

Bobigny, France

ACS Paragon Plus Environment

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f

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Department of Neuropathology, Charité - Universitätsmedizin Berlin, Charitéplatz 1,

Virchowweg 21, 10117 Berlin, Germany KEYWORDS. Gadolinium fluoride, MRI, two-photon imaging, oligothiophene, Amyloid β.

ABSTRACT. Real time in vivo detection of Amyloid β (Aβ) deposits at an early stage may lead to faster and more conclusive diagnosis of Alzheimer’s disease (AD) and can facilitate the follow up of the effect of therapeutic interventions. In this work, the capability of new hybrid nanomaterials to target and detect Aβ aggregates using magnetic resonance (MRI) and fluorescence imaging is demonstrated. These smart contrast agents contain paramagnetic nanoparticles surrounded by luminescent conjugated oligothiophenes (LCOs) known to selectively bind to Aβ aggregates, with emission spectra strongly dependent on their conformations, opening the possibilities for several fluorescence imaging modes for AD diagnostics. Relaxivity is evaluated in vitro and ex vivo. The capability of these contrast media to link to Aβ fibrils in stained sections is revealed using transmission electron microscopy and fluorescence microscopy. Preliminary in vivo experiments show the ability of the contrast agent to diffuse through the blood brain barrier of model animals and specifically stain amyloid deposits.

1. INTRODUCTION Alzheimer’s disease (AD) is the most important illness at the origin of cognitive decline among the aged people. This pathology is correlated with two specific histological


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characteristics, amyloid β plaques (Aβ pathology)1 and neurofibrillary tangles (tau pathology).2 Important developments are currently ongoing to propose early diagnostic markers and ultimately a disease modifying treatment, before the severe onset of neurodegeneration. In the case of Aβ, pathology arises from aggregates of the Aβ-peptide. Due to β- and γ-secretase sequential cleavage of the Amyloid-β Precursor Protein (APP) and proteolytic processing, a mixture of different Aβ peptides (31–46 amino acids long) is obtained in humans. The most prevalent alloforms being 1-40 and 1-42, where 1-42 is experimentally associated with the most potently neurotoxic aggregated states.3 In AD, these long peptides assemble into insoluble amyloid fibrils that are resistant to degradation. Senile plaques and cerebrovascular deposits are mainly constituted by these fibrils.4 Nowadays, the accurate diagnosis of AD can only be performed post-mortem using brain histology. In addition to the cognition tests, promising diagnostic tools are appearing nowadays such as the NINCDS-ADRDA criteria from 19845 together with the recently revised version proposal6, positron emission tomography (PET)7-10 and magnetic resonance imaging (MRI).11-15 More recently, several approaches were proposed to develop specifically designed compounds allowing amyloid imaging. Such systems should meet several criteria: (i) nondestructive crossing of the blood brain barrier (BBB); (ii) in vivo stability; (iii) moderate lipophilicity; (iv) limited uptake of metabolites into the brain; (v) specific binding to the plaques; (vi) increased sensitivity (imaging of low amount of Aβ).16 Among possible imaging technologies, fluorescence appears as a tool of choice for its high sensitivity and resolution. Several fluorescent probes have been used in vivo via multiphoton microscopy in animal models.17 For AD such fluorescent probes are useful as research tools but with strong potential for future applications.10,18,19 Luminescent conjugated oligomers, LCOs, are interesting due to their specific spectroscopic response which is typical of their conformation and they


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reliably report on amyloid fibrillation, providing unprecedented contrast and new insight into the time-changes of plaque inhomogeneity.20-23 This optical feature of the LCO depends on the conformational variations in protein aggregates16,22,24,25 and is not observed with the usual amyloidotropic dyes.23 Furthermore, previous studies have shown that functionalized LCOs have a Kd of 10 nM towards Aβ fibrils.26 MRI is a broadly used medical imaging technology based on the relaxation time of protons excited under an external magnetic field. It allows the detection of the modification of rotational orientation of protons from water constituting biological tissues.27 Several studies have reported the use of various strategies to image amyloid plaques using MRI without contrast agents.28-34 Iron rich plaques in brain areas were observed28 but the correlation between iron concentrations and AD plaque density remained inconclusive.29 Moreover, the technique could not provide an earlier diagnosis as compared to other existing diagnosis protocols. PET shows promising premises with three β-amyloid probes approved by FDA (F-18 flutemetamol (Vizamyl), F-18 florbetapir (Amyvid), and F-18 florbetaben (Neuraceq))35,36 but it is generally limited by the local availability of short-lived and expensive isotopes. MRI is a good candidate for a clinically available diagnostic tool of AD. However, MRI has quite low sensitivity and the related image contrast is dependent on a significant signal change caused by altered magnetic properties (relaxation) in pathological tissues. The use of contrast media will increase the particularity of this signal variations in the tissues. The most commonly used magnetic nanoparticles are iron oxides,37 manganese oxide38-40 and systems based on high spin gadolinium, for instance gadolinium-hydroxide,41 -oxides,42 -phosphates43,44 and -fluorides.45-47 Rare earth fluoride nanoparticles have excellent electronic, chemical, magnetic and optical characteristics, together


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with high stability due to the fluoride shield, making them potentially useful as MRI contrast agents.45 In this study, a novel hybrid contrast media is proposed, based on MRI active magnetic nanoparticles (GdF3),47 combined with Aβ-amyloid highly specific fluorescent probes based on LCOs.22,23 By doing so, we aim to reach selective accumulation of the nanoparticles onto amyloid deposits and consequently increase the MRI-contrast in those regions, in order to achieve multimodal detection of those AD markers both ex vivo and, ultimately, in vivo.

2. RESULTS AND DISCUSSION The strategy for making fluorescence and MR imaging multifunctional systems is based on the combination of two merging technologies: paramagnetic nanoparticles and luminescent oligomers. We first describe the preparation of water-soluble magnetic nanoparticles (MNP – (3) in figure 1) followed by their functionalization with LCO based linkers. These hybrid nanomaterials consist of GdF3 nanoparticles co-functionalized with both poly(ethylene glycol) (PEG) and LCO moieties. Finally, the resulting LCO labeled particles (4) (Figure 1) are evaluated for imaging, in vitro and in vivo.


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GdF3

GdF3

(1) 3

O

O

NaO

ONa O S

S

O

S S

O

N H

O

O

N H

O P OH OH

(2)

GdF3 GdF3

+ (1)

95 % (1) + 5 % (2)

4

Figure 1. The prepared nanoparticles: GdF3@PEG as reference (3) and GdF3@(PEG+LCO) (4).

Synthesis of GdF3 nanoparticles, surface modification and characterization. The GdF3 NPs were prepared following a previously reported procedure, easy to handle and applicable to essentially all rare earth salts.47 Transmission electron microscopy (TEM) and Dynamic Light Scattering (DLS) showed that the nanocrystals exhibited an average size of about 15-20 nm with a low polydispersity index (PDI = 0.10) and a hydrodynamic diameter centered at 35 nm (Figure S1 in SI). High magnification TEM pictures (Figure S1) showed that particles are not aggregated and well crystallized (see figure S2 for X-ray diffraction (XRD) patterns). The diffraction patterns of GdF3 are in good agreement with the presence of mainly orthorhombic phase (spheroidal particles), and minor hexagonal phase (elongated particles, also visible in the right frame of figure S1). Surface modification of the nanoparticles. Although the nanoparticles exhibit high stability in water, they are not suitable for biological applications as metal ions can potentially leak from their surfaces, hence the necessity to stabilize those with ligands.48-50 Phosphonates and bisphosphonates based ligands are indeed more suitable than classically used carboxylic acid derivatives (carboxylate salts, acid chlorides, esters, amides) due to their stronger affinity to the


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Gd metal.50-53 Moreover, it has recently been shown that such bisphosphonate groups lead to much stronger anchoring on nanoparticles than phosphonates in buffer solutions.54 Thus, to ensure both biocompatibility51,52 and stability of the contrast agents, nanoparticles were functionalized with bisphosphonates linkers consisting of PEG molecules with 10 ethylene glycol units long. Two systems were prepared for this study: one control, consisting in GdF3 covered with PEG chains GdF3@PEG (3) and one active nanoparticle, functionalized with a mixture of 5 % LCO ligand and 95% PEG chains GdF3@PEG+LCO (4), as depicted in figure 1. Synthesis of both ligands is reported in the SI. Fourier transformed Infrared (FT-IR) spectra of the surface modified NPs 3 and 4 clearly verified the successful grafting of the different linkers on the GdF3 surface (Figure 2). Table 1 summarizes the phosphonate groups mains bands observed, with the characteristic shifts arising from complexation indicating their strong affinity for the nanoparticles surface.

Figure 2. FT-IR spectra of the nanoparticles (1) (black line) and (2) (red line) with zoom on the 1600-800 cm-1 region.


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Table 1. Representative vibration bands in FT-IR for the phosphonates BP-PEG (1)

GdF3@PEG (3)

GdF3@PEG+LCO (4)

P=O

1246 cm-1

1246 cm-1

1225 cm-1

P-O

1064 cm-1

1105 cm-1

1058 cm-1

P-OH

923 cm-1

945 cm-1

971 cm-1

The presence of LCO on the particles was evidenced by a signal at 1549 cm-1 related to the anti-symmetric stretching mode of the aromatic C=C bond and a band at 1721 cm-1 attributed to the C=O bond. In the case of free BP-PEG-CH3 spectra the P=O vibration is observed at 1246 cm-1 and two signals at 1064 cm-1 and 923 cm-1 are respectively assigned to the P-O and the POH bonds.53,55 After coupling with the GdF3, band shifts are observed for P-O vibration from 1064 to 1105 cm-1, and for P-OH vibration from 923 to 945 cm-1 with an important decrease of the intensity. The P=O vibration peak at 1246 cm-1 becomes negligible. The FTIR results highlight the role of P-OH functions in the binding at the particles surface. In the case of 4 results are similar and phosphonate groups are evidenced by the bands at 1225 (P=O), 1058 (PO) and 971 cm-1 (P-OH). According to elemental analysis, the coverage of PEG unit per particle of ca 1300 could be evaluated, which corresponds to an average of 1.8 PEG unit per nm2. DLS results showed an increase of the hydrodynamic diameter of the nanoparticles from 35 nm for non-functionalized GdF3 to 60 nm for the final systems 3 (PDI = 0.07) and 4 (PDI = 0.11) attesting the presence of the PEG shell (Figure S3). Grafting was also confirmed with zeta potential measurements, which also provided important information on the stability of these


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systems. For a fixed value of pH=7, GdF3 particles (4) show a potential at +40 mV, which, upon surface functionalization, change to -15 mV (Figure S4). Both the zeta potential and size of the particles are stable in a broad range of pH, from pH 12 to 5 for 3 and 12 to 6.5 for 4. Dispersions in PBS solution or water of particles 4 give highly stable colloidal suspensions for months without aggregation. The presence of the LCO on the particle does not affect their stability. Furthermore, the ionic LCO moiety contributes to steric and electrostatic stabilization. Measurements of relaxivities at 7T. Relaxivity is the main parameter that defines the suitability for MRI applications. It was studied at 7T for compounds 3 and 4 along with two pegylated gadolinium oxide variants and two commercially available MRI contrast agents (Table 2). For both systems 3 and 4 the longitudinal relaxivity (r1) measured as per Gd atom is relatively low, whereas they both exhibit a very high per particle transversal (r2) relaxivity. This is due to the larger particle size, where the ratio of surface-to-core Gd atoms is low. Inner sphere relaxation mechanisms are most important for efficient r1 relaxation and require the interaction of diffusing water with Gd.56 This effect is limited to the surface Gd atoms. On the other hand, transverse relaxation (r2) is less dependent on the proximity of diffusing water, and a larger and slower particle complex usually gives high r2 relaxivity, as can be seen with both system 3 and 4. These effects increase with the field strength giving these particles interesting potential for imaging. A 3-fold enhancement in r2 relaxivity for 4 compared to 3 is also noted, which is likely related to reduced tumbling rates of the system due to increased size after the addition of both the linker and LCO. The relaxivity values are thus on the same level as commercially available superparamagnetic iron particles. Moreover, r2/r1 ratios are very high, suggesting that particles are particularly suitable as negative contrast agents. The ratio is better compared to gadolinium oxide57 or fluoride58 and similar to already reported USPIO.44


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Table 2. Relaxivity rates at 7T, and comparison with reported and commercial particles. Relaxivity per Gd atom (mM-1s-1)

Particle

r1

r2

r2/r1

DOTA-Gd (Dotarem)

4.1

4.9

1.2

Gd2O3@PEG56

4.4

28.9

6.6

Gd2O3@PEG57

10.9

15.9

1.5

Fe2O3-Dextran (Endorem)

2.2

182

83

3

0.55

55

100

4

0.40

149

372

Absorbance and fluorescence in solution. Basic photo-physical characterizations were carried out to investigate the impact of the surface modification of the particles, on the optical properties of the LCO (absorption, fluorescence, quantum efficiency). Spectroscopic study of LCO with spacer and LCO grafted to MNP is shown in Figure 3. The spectra have been normalized to allow a simple comparison of the peak positions. Compared to the pure LCO 2, nanoparticle 4 shows a different overall absorption spectrum due to contribution from scattering induced by the nanoparticles, and the small apparent blue-shift is partially due to this phenomenon. The fluorescence emission spectra are similar for both 2 and 4, only a slight blue shift of 20 nm is seen upon attachment to the MNP, along with a small signal ca 460 nm that corresponds to a


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Raman diffusion peak of the water. The blue shift suggests a different environment around the MNP as compared to the free LCO-spacer in solution. This was further supported from the measured quantum efficiency (QE) that differed substantially: with a value of 0.11 for 2 and a decrease down to 0.06 upon grafting onto the NP to form 4. Those two effects could be due either to interactions of the probe with PEG chains or to some degree of π-stacking between neighboring LCO units at the particle surface, but QY remains high enough to enable fluorescence microscopy experiments.

Figure 3. Normalized absorbance (solid lines) and fluorescence emission (dashed lines) of LCO (2) (black) and LCO coated nanoparticles (4) (red).

Staining of Aβ with fluorescent-MNP. To assess whether the LCO could still selectively bind to Aβ aggregates after its grafting on the particles, 3 and 4 were associated with amyloid fibrils obtained from recombinant Aβ1-42 peptide. In vitro preparations of recombinant Aβ1-42 fibrils stained with aqueous solutions of 3 and 4 respectively were analyzed by TEM (Figure 4). The resulting images demonstrate that 4 binds as clusters to Aβ1-42 fibrils but not to other parts of


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the grid whereas 3 is found both on the Aβ fibrils and on other parts of the grid as singular particles. The results indicate that the attached LCO efficiently targets the nanoparticle to the Aβ1-42 fibril.

Figure 4. TEM images of negative stained Aβ 1-42 fibrils binding to 3 (upper panel) and 4 (lower panel). Scale bar = 2 µm. Green arrows indicate NPs associated to Aβ fibrils. Red arrows indicate NPs adsorb randomly on the copper mesh grid.


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To validate probe binding to pathologic Aβ in tissue, cryosections of APPPS1 transgenic mice59 were stained with 2, 3 and 4 (Figure 5). Consistently with the results from TEM, the fluorescence and binding properties of the LCO to amyloid in brain tissue are retained upon conjugation to the PEG spacer as well as to the nanoparticle (Figure 5a, 5b). Fluorescence can not be detected from amyloid tissue using 3, since it lacks the fluorescent LCO-moiety (Figure 5c). However, using 4, the fluorescent signal gives contrast and expected abundance (Figure 5b, 5d). Application of 4 to a negative control, lacking amyloid pathology, yield no fluorescence (Figure 5e), demonstrating that the LCO binding is amyloid specific and attaches the NP to the amyloid in ex vivo samples.

Figure 5. Ex vivo staining of murine cerebral cryosections from a 540 days old APPPS1 mouse by 2 (a) and 4 (b). APPPS1 brain stained with 3 displayed no fluorescence (c). APPPS1 brain


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stained with 4 displayed LCO fluorescence (green) from AD plaques (d). Non-transgenic brain displayed no LCO fluorescence when stained with 4 (e).

Next, the binding efficiency of 4 in vivo was studied using fluorescence microscopy (Figure 6). The intensity of fluorescence associated to the plaques in the brain of APP/PS1 mice is higher when the animals are treated in vivo with the LCO p-FTAA23 or 4 than in the case of PBS. pFTAA is chosen as the reference substance since it binds to a wider variety of plaque morphologies, thereby revealing the total plaque load. Comparably, q-FTAA is more discriminatory in its plaque affinity and binds to a subset of plaques.60 In the case of PBS control, the signal related to amyloid plaque is observed (auto-fluorescence) under excitation at 489 nm, but remains less intense than the fluorescence in the presence of probes . Despite this auto-fluorescence (AF), the images in Figure 6 show that the emission due to the nanoparticles bonded to Aß deposits compares to the more concentrated pure LCOs (reference). Two-photon imaging was tested with excitation at 780, 800 and 830 nm for p-FTAA, in order to prevent AF. Biomolecules, such as Aβ, usually have very small two-photon absorption (TPA) cross-section compared to fluorescent probes such as the oligothiophenes used here, or other variants.61 Thus, AF originating from the plaque is therefore lower by comparison with LCO (Figure 7) for compound 4 (see also Figure S5). The signal to noise ratio (SNR) performance of two-photon excitation vs. single photon excitation (458 nm) was calculated from the average intensity of multiple ROIs (Figure S6). At single photon excitation, 458 nm, the image interpretation is complicated due to a high AF and a SNR at only 3.0. From multiphoton excitation, 780 nm and higher excitation wavelengths, the SNR regularly increase to reach a maximum value of 6.9 at 830 nm (Figures S5 and S6). In the latter case, AF is negligible and the plaques are easily


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distinguishable from the background, as expected because of the low TPA efficiency of endogeneous molecules.

Figure 6. Fluorescence images (exc. 489 nm, em. 509 nm) of Aβ amyloid plaques in brain tissue sections acquired with identical exposure times of in vivo injected 15-month-old APPPS1 mice with: 4 (top) at 35 µM, PBS control (middle), reference LCO (pFTAA) at 20 µM (bottom).23 Mice were injected intravenously with 100 µL of respective compound or control twice within 48h and sacrificed 24h after the final injection.

TPA fluorescence imaging of APPPS1 mouse was carried out at 780 nm on brain tissue sections after in vivo injection of 4 (Figure 7). The signal is clearly observed despite a high AF. By collecting data from the plaques and the background (as in Figures S5 and S6) it was possible to un-mix the spectral contributions, as shown in Figure 7, right panel. Acquisition of the


15


fluorescence spectra of a control sample (PBS injection) and the sample stained with 4 was performed at the center of the amyloid plaques. Thereafter, average spectra from several ROIs (plaque and background AF) were fitted to a Gaussian (dashed curves) to accentuate the differences of the spectra. An unmixed spectral analysis was achieved to improve the observation of the nanoparticles. The very clear difference between labeled and unlabeled region, as obtained in the unmixed image in figure 7, can even be improved with more sophisticated signal treatment procedures. Those are however beyond the scope of this article. It has been shown that by using advanced spectral correlation algorithms that are better for noisy background, the staining of the chromophore

Raw image:

can

be

better

ascertained60

in

Unmixed image:

more

systematic

studies.

1,0

Relative Intensity

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

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0,8 0,6 0,4 0,2 0,0 400

450

500

550

600

650

700

Wavelength (nm)

Figure 7. Two-photon fluorescence (exc. 780 nm) (left), unmixed image (middle), spectral separation of 4’s signal (green) from the auto-fluorescence (red) on brain tissue section after in vivo injection of 4. Right spectrum is a fit of the spectral signal of 4 (black squares) together with the AF (red dots). Gaussian fitting functions (dashed) are used to accentuate the spectral difference. An apparent peak difference of 10 nm is deduced from this fitting analysis. The scale bars are 40 µm.


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MRI experiments. Ex vivo MRI experiments were performed in mice brain tissues, to evaluate the capacity of 3 to generate contrast. The particles were tested by injection ex vivo in wild type (WT) mice brains (Figure 8A). When using a T2-weighted spin echo sequence, the contrast from the nanoparticle is clearly visible (Figure 8, red arrow). In the case of T1-weighted gradient echo sequence, the dark contrast is observed as a consequence of T2* effects, mostly due to a higher value of r2 versus r1 (Figure 8C). When T1-weighted spin echo sequence is performed (Figure 8B), almost no contrast is observed which is in agreement with the low r1 relaxivity measured in vitro. Such result confirms that the studied particles can be used as efficient MRI negative contrast media.

A

B

D

C

Figure 8. Wild type mice brains ex vivo MRI image after direct injections of 3 into the brains ex vivo (A, B, C). Red arrows indicate injection sites. A: T2-weighted spin echo MRI shows reduced signal at injection site. T1-weighted spin echo MRI (B) shows no contrast, while T1weighted gradient echo MRI (C) signal reduction due to nanoparticles at the injection site. T2 measurements of APPPS1 and WT after intravenous injection of 4 and p-FTAA as control (D).


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Next, to investigate if plaque formation could be detected by the particles, 4 was injected intravenously in APPPS1 and WT mice with the same dose, and APPPS1 mice were treated with 2 as control. MRI experiments were then performed on the brain hemispheres ex vivo. To be able to quantify the changes in tissue relaxation caused by the presence of LCO labeled particles bound in tissue, a series of images with different T2 weighting were acquired during MRI in order to create voxel-wise maps. Figure 8D shows the dependence of T2 on the dose of Gd(III) injected with the different probes. There are no differences between WT mice when 4 was injected and APPPS1 mice when 2 was injected, indicating that the particle alone does not accumulate in the brain tissue in sufficient amounts to provide a noticeable contrast. Conversely, T2 is approximately 4 ms (11.5%) lower in APPPS1 mice (32ms) compared to WT mice (36ms) when both were treated with 4 (containing 5.8 µmol Gd(III)). These results show that 4 accumulates in the brain tissue of APPPS1 mice giving a noticeable reduction in T2. When a lower dose of 4, is injected (eq.1.33 µmol Gd (III)), T2 is in the same order as WT and APPPS1 (with the LCO p-FTAA23). However, to conclude that a reduction in T2 could be due to the binding of 4 to amyloid plaques, the assumptions that the particles have passed the blood-brainbarrier and been retained in the brain tissue of APPPS1 mice while not in the WT mice have to be fulfilled. Also, the resulting reduction in T2 after injection of high dose of 4 is not as strong as expected. In fact, considering individual variation of T2 in tissue between animals, water content of the sample and other measurement conditions, the measured values are in the order of the error margin. Although fluorescent imaging of the tissue has shown the presence of LCO, the small reduction in T2 indicates that the particles are not present in high enough concentrations


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for unambiguous detection. In order to verify if the T2 reduction is related to the presence of GdF3 particles, ICP-MS was performed on the brain hemispheres. The concentration of Gd(III) in the mice brain was measured using ICP-MS (Figure S7), after in vivo injection of 4. In the case of APPPS1 mice treated with pure LCO, Gadolinium is not detected. On the other hand, when 4 was injected in both APPPS1 and WT mice, concentrations up to 22 µg/Kg are measured. Thus, there is a tendency towards increased Gd(III) concentration in the brain with increased Gd(III) dose given (Figure S7), with practically the same concentrations in WT and APPPS1 mice. The measured difference in T2 between APPPS1 and WT mice, when 4 was injected, is not possible to explain by a difference in Gd(III) in the brain. We speculate that binding of 4 to amyloid deposits in the APPPS1 mice may have increased the relaxivity of the GdF3 leading to the enhancement of a T2 reduction. Finally, the low amount of Gd(III) in the brain may have different causes: fast clearance from blood circulation in the liver making few particles available to enter the brain; too low diffusion through the blood-brainbarrier; or clearance from the brain after entrance.

Biodistribution. To further delineate the low signals from in vivo labeled mouse brains by fluorescence and MRI as compared to the ex vivo stained tissue, biodistribution of the nanoparticles was studied. Livers were collected from the animals used in the various experiments and sections were examined with the fluorescence microscope. Judging from LCO fluorescence it becomes clear that large amounts of 4 accumulate in the liver (Figure 9a and 9b).


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Figure 9. In vivo, 4 accumulates in the liver of injected mice. APPPS1 mice ~150 days old were injected in vivo with PBS and 4 respectively. The liver was collected post-mortem and examined for LCO fluorescence. The fluorescence signal collected from the PBS injected control mouse is low and displays the spectral properties of tissue autofluorescence (a). The LCO signal collected from the liver of a mouse injected with 4 is slightly shifted compared to free LCO,23 indicating that it is conjugated to the nanoparticle (b). Fluorescence intensity (c) and spectra (d) are shown in bottom figures. (PBS control in black and 4 in green).

Fluorescence emission from the particles at 520 nm can be compared with the intensity from autofluorescence and a 6-fold higher signal is observed for mice treated with 4 compared to PBS


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mock-control (Figure 9c). Fluorescence spectra of the accumulated fluorescence particles correlate well with spectra from 4 compared to autofluorescence (Figure 9d). It is therefore evident that a major fraction of 4 accumulates in the liver and is hence inaccessible for brain imaging. This is a common observation when using such hybrid nanoparticles in vivo, where hepatic uptake occurs frequently due to the reticulo-endotelial system. One way to overcome that issue is to lower the size of the nanoparticles, this is currently under investigation.

3. CONCLUSION The development of original targeted hybrid nano-agents for in vivo multimodal (fluorescence and MRI) imaging of Aβ amyloid fibrils is reported. Besides the experimental evidence that gadolinium fluorides are interesting T2 contrast agents with high chemical stability, optimized surface modification allowed the preparation of colloidal and physiologically stable hybrid architectures that retained the amyloid specific targeting of the native fluorescent probe. The hybrid contrast media is used to efficiently target and visualize Aβ-amyloid, in vitro, ex vivo and in vivo. The crucial issues with current design for in vivo imaging is that a major fraction of the probe is trapped in liver, hence hampering accessibility for BBB penetration and cerebral amyloid targeting. Nonetheless, efficient labeling and multimodal imaging of Aβ, using the prepared nanoprobe, is demonstrated. This opens up for future theranostic approaches towards neurodegenerative diseases.

4. EXPERIMENTAL SECTION


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The synthesis of the LCO (2) is detailed in SI. Synthesis of GdF3 nanoparticles.62 A solution of 1.49 g GdCl3,6H2O (4 mmol) in 3 ml of methanol is added to a mixture of 0.45 g hydrofluoric acid (9 mmol, HF) and 16 mL of Nmethyl-2-pyrrolidone (NMP) under stirring. The obtained clear solution is refluxed 16 hours at 170 °C leading to a stable suspension of GdF3 nanoparticles. The particles are washed 3 times with 60 mL acetone (centrifugation at 8000 rpm for 10 minutes and redispersion), before redispersion in 5 mL distilled water.

Synthesis of particles (3).62 A 10 ml solution of water and bisphosphonate-PEG-CH3 (2 mmol, BP-PEG-CH3 (1)) is filtrated (0.45 µm nylon filter) and 5 ml of the previous GdF3 particles suspension is added. The mixture is sonicated and kept under stirring for 5H at 70 °C. The transparent solution is dialyzed (10 days, 12-14 KDa cutoff) followed by freeze-drying.

Synthesis of particles (4).62 A suspension of GdF3 particles (1 mmol in 2 mL H2O) is added to an aqueous mixture of BP-PEG-CH3 (1) (0.475 mmol in 5 mL H2O) and BP-LCOs (2) (0.025 mmol in 0.5 mL H2O). In order to avoid aggregation, the solution is sonicated and kept 5H at 70 °C. The final suspension is dialyzed for purification (Typically after 10 days, dialysis is stopped when LCO ligands fluorescent signal totally disappear from the washing solution), followed by freeze-drying.

Transmission electron microscopy (TEM). Aβ1-42 (10 µM) (rPeptide) is fibrillated during agitation for 22 h at 37 °C in phosphate saline buffer (pH 7.4). Fibril formation is verified by thioflavin T fluorescence (Figure S8).63 Fibril suspension (100 µL) is mixed with water


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solutions of 1 and 2 in separate tubes. Samples for transmission electron microscopy (TEM) are deposited on copper mesh grids coated with carbon (Ted Pella, inc.). Here 5 µl nanoparticle suspension stained Aβ1-42 fibrils are deposited on the grid and left 2 min, dried, washed with 5 µl of deionized water. The sample is counterstained for 30 s with 5 µl of a solution of 2% (w/v) uranyl acetate in deionized water. A Jeol 1230 TEM (100 kV), CCD camera is used. Animals. Groups of 5 APPPS1 mice are individually housed in ventilated cages (Tecniplast) at controlled temperature and humidity (respectively 19-22 °C and 50–60%). Mice have free access to sterile water and food. Housing conditions are exempted of specific pathogens following the recommendations from the Federation of European Laboratory Animal Science Associations. All animals are submitted to experimental procedures which are in agreement with the approved protocols from the Norwegian National Animal Research Authorities. APPPS1 mice are provided through an MTA with the LUPAS project and with NTNU Trondheim. Mice injection. Nanoparticles are dispersed in aqueous PBS (10 mg/ml) prior to injection (intravenous in tail vein) in aged (14-15 months of age) APPPS1 mice. Two injections of contrast media suspension (100 µl) are performed consecutively within 48h. 24 h following the second injection, the animals are sacrificed without perfusion. PBS and pure LCOs (PBS, 10 mg/ml) are utilized for control. A brain hemisphere is kept at the temperature of -80°C for MRI imaging and further experiments. Brain hemispheres are used for fluorescence microscopy after preparing cryosections (unfixed). A Carl Zeiss Observer, Z1 system with Axio Vision® software (GFP (S65T) filter (excitation 489 nm, emission 509 nm) is used.

Rehydration and staining of mouse tissue. 10 µm thick sections of desired tissues are positioned on superfrost ultra plus slides (Thermo scientific) and stored at -20 °C. Rehydration of


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the sections is performed by consecutive 10 minutes dips of 96% ethanol, 70% ethanol, dH2O, PBS pH 7.4 (Medicago).

In vivo stained tissue is then mounted with Dako fluorescence

mounting medium and cover slip and kept at room temperature. For ex vivo staining, the respective probes are diluted to 6 µM in PBS, applied to the rehydrated tissue and left in the dark at room temperature for 30 minutes prior to washing 3x5 min in PBS. The sections are dried in air before mounting with Dako medium and cover slip. Fluorescence microscopy. Single-photon epifluorescence images of in vivo and ex vivo stained tissue are collected with a microscope Leica DM6000 B combined with a SpectraCube module (Applied spectral imaging). Images are collected in spectral mode to determine spectral properties and fluorescence intensity. Sections are also visualized through a Zeiss 510-LSM confocal microscope with a META spectral detector. Excitation is achieved using the 458 nm line of a He-Ne laser and a MIRA 900F fs laser tuned to 780, 800 or 830 nm used for two-photon excitation. For further details, see ref. 25.

Magnetic Resonance Imaging (MRI) and relaxivity measurements. MRI is achieved with a 7 Tesla magnet (Biospec 70/20 AS, Bruker Biospin MRI, Ettlingen, Germany) equipped with water-cooled (BGA-12, 400 mT/m) gradients. For in vitro relaxivity measurements a 72mm volume resonator is used for RF transmission and reception. For ex vivo imaging of the brain hemispheres, a 72mm volume resonator is used for RF transmission and an actively decoupled mouse head surface coil is used for RF reception (Bruker Biospin MRI).


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Relaxivity measurements are performed with probes diluted in 0.9% NaCl in 2ml Eppendorf tubes at 4 different concentrations. Acquisition of T2 weighted images is performed with a spin echo (MSME) sequence including 16 different echo times (TE): 11/22/33…/176ms and a repetition time (TR) of 15000ms. Acquisition of T1 weighted images is performed with a spin echo sequence including TE 8.3ms and varying TR 13ms-6400ms. R2 for the different concentrations of probes is estimated by fitting a mono-exponential model to the signal intensity of the images with different TE-values. R1 for the different concentrations of probes is calculated by fitting a mono-exponential model to the signal intensity of the images with different TR-values. The relaxivities, r1 and r2 of the different probes are calculated by linear fitting of the Gd/particle concentration to the measured R1 and R2 respectively. Frozen brain hemispheres from mice injected with different probes as described above are imaged. Brains are thawed for 2 hours in room temperature in 2 ml eppendorf tubes before placed in the MR scanner. After a gradient echo FLASH pilot scan (acquisition time 1min) a series of T2-weighted images are obtained using a spin echo (MSME) sequence with 43 different echo times (TE): 11/22/33…/473ms; repetition time: 5000ms. 10 consecutive slices of 0.4mm are sampled with an in-plane resolution of 125x125µm2, with an acquisition time of 1h20min. In-house developed software (MATLAB ver. R2010a, Math Works Inc, Natick MA, USA) is used for calculating T2-maps by fitting a mono-exponential model to the signal intensity of the images with different TE-values. A region of interest is placed in the parietal cortex and the mean T2 is calculated. After MRI the brains are refrozen and stored at -20°C before ICP-MS is performed.


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Tissue Gd measurements with ICP-MS. Brain hemispheres are thawed and transferred to 1.5ml eppendorf tubes. Then weighted in wet condition, dried overnight and the dry weight is measured. Samples are then lyophilized, weighted and transferred to PTFE digestion tubes. 2.00 ml 1:1 ultrapure water: 65% HNO3 (Merck – purified using a SubPUR system(Milestone)) are added to the lyophilized tissue and then digested using UltraCLAVE (Milestone, Italy). Ultrapure water is used to dilute samples. Evaluation of Gd amount is obtained with a HR-ICPMS (Element 2, Thermo Finnigan, Bremen, Germany) following the report from Gellein et al.64 The samples are introduced using a SC-2 autosampler with SC-FAST option and SYRIX syringpump (0.200 mL/min) (Elemental Scientific, Inc. Omaha, NE). A quartz cyclonic spray chamber, PC3 peltier chiller, aluminium sample and skimmer cones (Elemental Scientific) are used. The instrument is externally calibrated, using multi-element standards solutions (0.6 M HNO3). In order to minimize the interference generated by carbon, methane is added to the plasma.65 The quantification of Gadolinium present within the brains is calculated from concentration of Gd in tissue × 2 × tissue weight. The obtained value is divided by the injected dose of gadolinium and multiplied by 100 to obtain the gadolinium mass percentage in the brain versus the dose injected in the animal.

ASSOCIATED CONTENT Supporting Information. The synthesis of the LCOs, TEM images/DLS/FTIR/zeta potential//powder XRD of GdF3 nanoparticles, spectral imaging of plaques, spectral analysis of brain tissues of mouse, ICP-MS for Gd concentration in brain tissue, 1H NMR of LCOs are


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available in SI.

AUTHOR INFORMATION Corresponding Author E-mail addresses: [email protected], [email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. A. Å., K. P. R. N. and P. H. designed and synthesized the LCO. F. M., F. C., F. L. and S. Pa. designed, synthesized and characterized the magnetic nanoparticle and the final hybrid contrast agent. S. N., N. R. and M. Lind. achieved the optical characterization and the fluorescence microscopy. E. M. H. and M. W. performed the relaxivity measurements and the MRI imaging. S. H., S. Pr, F. L. H. participated to the in vivo experiments. M. Lec. helped with the BP functional ligands. C. A. and C. M. designed the LCO coupling. S. Pa., A. Å., M. L., M. W. and F. L. wrote the article.

ACKNOWLEDGMENT The LUPAS project, FP7 Health (Call Reference N°: FP7-HEALTH-2009-1.2-5, Project N°: 242098) is acknowledged for financial support. APPPS1 mice were provided by the group of M. Jucker, available through an MTA with the LUPAS project and with NTNU Trondheim.


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Two-photon fluorescence and magnetic resonance specific imaging of

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