Cyrille Richard

Thomas Maldiney

Quentin Le Masne De Chermont

Johanne Seguin

Nicolas Wattier

Gabriel Courties

Florence Apparailly

Michel Bessodes

Daniel Scherman
email: daniel.scherman@univ-paris5.fr

Nicolas Wat- Tier

Persistent Luminescence Nanoparticles for Bioimaging

Keywords: Nanoparticle, Persistent luminescence, Silicate, Surface coating, Biodistribution, Tumor targeting, Inflammation, Biomedical imaging

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Introduction

From fluorescent organic dyes to luminescent nanocrystals, optical imaging raises growing interest for the understanding of physiological mechanisms or the development of new diagnosis and therapeutic applications [START_REF] Weissleder | Imaging in the era of molecular oncology[END_REF]]. Photonic probes offer not only practicable living sensors, but also rely on lowcost or affordable imaging devices and techniques. The semiconductor Quantum Dots (QDs) [START_REF] Medintz | Quantum dot bioconjugates for imaging, labelling and sensing[END_REF]][Smith et al. 2008] exhibit high quantum yield, good photostability and tunable emission wavelength. Luminescent porous silicon nanoparticles can act as both diagnostic [START_REF] Li | Water-soluble poly(acrylic acid) grafted luminescent silicon nanoparticles and their use as fluorescent biological staining labels[END_REF]] and therapeutic tools [START_REF] Salonen | Mesoporous silicon in drug delivery applications[END_REF]. At last, near-infrared fluorescent molecules [START_REF] Chen | Arthritis Imaging Using a Near-Infrared Fluorescence Folate-Targeted Probe[END_REF] offer the possiblity to work with very low autofluorescence from tissues under constant illumination.

Each of the above-mentioned probes displays characteristics that limit preclinical use or future therapeutic application. First, the emission wavelength of semiconductor QDs must be tuned by changing the particle diameter (ranging from 2 nm to 10 nm) or its composition. On the one hand, this opens alternatives for renal clearance [Choi 2007], but on the other hand it also shortens circulation time of the probe and impairs effective targeting, levying strict regulations on its design [START_REF] Longmire | Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats[END_REF]. Luminescent porous silicon nanoparticles show great promise as non-toxic, self-destructive, and traceable cargo for anti-cancer drugs, but they suffer from limited quantum yield [START_REF] Park | Biodegradable luminescent porous silicon nanoparticles for in vivo applications[END_REF]] (∼ 10%) compared to QDs (> 80% in organic solvent). As for near-infrared fluorescent probes, it is now well established that organic dyes are susceptible to photobleaching, and are most often unstable under physiological conditions, which make them hardly the best candidate for a long-term biological or biomedical purpose [START_REF] Resch-Genger | Quantum dots versus organic dyes as fluorescent labels[END_REF]].

To overcome these difficulties, we have recently developed persistent luminescence silicate nanoparticles [le Masne de Chermont et al. 2007], referred to as PLNP, suitable for in vivo imaging, and specially designed to avoid most inherent problems encountered in classical optical systems. The key element of this technology is based on a new generation of long-luminescence nanoparticles, emitting in the nearinfrared region, that can be optically excited before in vivo local or systemic injection. This long-lasting afterglow prevents residual background noise originating from in situ excitation and can last several hours. Thus, the significant signal-tonoise ratio improvement allows detection in rather deep organs and real-time biodistribution monitoring of active elements hours after injection.

Persistent luminescence nanoparticles from silicate host

Great progress have been made in long-lasting phosphorescence since the early 20th century. Long-lasting phosphors were widely used in emergency lighting, safety in-dications, road signs and so on. Most of the long-lasting phosphors are based on sulfides and aluminates. Recently silicate phosphors have been paid considerable attention because of their bright phosphorescence, notably in blue and green regions [START_REF] Jiang | Luminescent properties of CaMgSi 2 O 6 and Ca 2 MgSi 2 O 7 phosphors activated by Eu 2+ , Dy 3+ and Nd 3+[END_REF]]. In 2003, Wang et al. reported the synthesis of MgSiO 3 enstatite, doped with Eu 2+ , Dy 3+ and Mn 2+ luminescent ions, showing a long red persistent luminescence [START_REF] Wang | Mn 2+ activated green, yellow, and red long persistent phosphors[END_REF]]. This article initiated in our laboratory the preparation of several luminescent silicates intended for bioimaging applications. For example, for the synthesis of Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 (where 10% of Zn and Mg atoms were substituted for Ca) doped with 0.5% Eu 2+ , 1% Dy 3+ , and 2.5 % Mn 2+ (the percentages are based on metallic cations content), we used: 125 mg of calcium chloride, 990 mg of magnesium nitrate, 525 mg of zinc chloride, 16 mg of europium chloride, 39 mg of dysprosium nitrate, 44 mg of manganese chloride, 4 mL of deionized water at pH 2 and 2 mL of TEOS. The mixture is vigourously stirred at room temperature for 1 hour and then for 2 hours at 70 • C until the sol-gel transition occured. The wet gel was then dried in an oven at 110 • C for 20 h to remove water and ethanol (Figure 1).

Synthesis of enstatite-like silicates: MgSiO

Heating to get crystals

The resulting opaque dry gel is then calcined in a zircone crucible under a weak reductive atmosphere using 10% H 2 , 90% Ar (Noxal 4, Air Liquide, Düsseldorf, Germany). Depending on the material we want, the final sintering temperature must be adapted. To prepare Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 (Eu 2+ , Dy 3+ , Mn 2+ ), the gel is fired at 1050 • C for 10 h and slowly cooled down to room temperature to give white crystals (Figure 2).

Characterisation of synthesized nanomaterials

Fluorescence spectra

Optical spectra, shown in Figure 3, were recorded with a Varian (Palo Alto, CA) Cary-Eclipse Fluorescence spectrophotometer by using the phosphorescence mode with a delay and a gate time of 300 ms (for excitation spectrum: em = 690 nm; excitation slit = 5 nm; emission slit = 20 nm) (for excitation spectrum: ex = 340 nm; excitation slit = 20 nm; emission slit = 5 nm). About 50 mg of each powder were deposited on a glass plate, stuck with a few drops of ethanol and inserted into the spectrophotometer.

Luminescence

To record persistent luminescence decline curves, 10 mg of each material were deposited on a 96-wells plate, excited for 2 min with a UV lamp, and placed under the photon-counting system consisting of a cooled GaAs intensified charge-coupled device (ICCD) camera (Photon Imager; Biospace Lab, Paris, France) without any external illumination system. Luminescence intensity was detectable for more than 24 h when kept in the dark. The decay kinetics (Figure 4) were found to be close to a power law I ≈ I 0 • t -n (n = 0.96, R 2 = 0.996) after the first 100 s.

Fig. 3 Emission spectra of the three synthesized materials doped with Eu 2+ , Dy 3+ , Mn 2+ . MgSiO 3 (λ em = 645 nm), ZnMgSi 2 O 6 (λ em = 660 nm), Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 (λ em = 690 nm).

Fig. 4 Afterglow decays after UV excitation of the materials doped with Eu 2+ , Dy 3+ , Mn 2+ . MgSiO 3 , ZnMgSi 2 O 6 , Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 .

For bioimaging application, we selected the Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 composition since it had the best characteristics in terms of emission wavelength (690 nm, which is located within the transparency window) and of luminescence intensity (the highest among all three synthesized silicates).

3 Chemical surface functionalization of silicates

Obtention of nanoparticles of different sizes

Nanometer-sized particles were obtained by basic wet grinding of the solid (500 mg) for 15 minutes with a mortar and pestle in a minimum volume of 5 mM NaOH solution. Hydroxylation was then performed overnight by dispersing the ground powder (Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 doped with Eu 2+ , Dy 3+ , and Mn 2+ ) in 50 mL of the same NaOH solution to get hydroxyl-PLNP. Nanoparticles with a diameter of 180 nm were selected from the whole polydisperse colloidal suspension by centrifugation on a SANYO MSE Mistral 1000 at 4500 rpm for 5 minutes (centrifugation time was lengthened to 30 min in order to obtain 120 nm PLNP). They were located in the supernatant (assessed by Dynamic Light Scattering). The supernatants were gathered and concentrated to a final 5 mg/mL suspension. PLNP with a diameter of 80 nm were selected from the 120 nm concentrated suspension by centrifugation on an Eppendorf MiniSpin Plus at 8000 rpm for 5 minutes. Following a similar approach, centrifugation steps were repeated 4 times and the resulting suspension concentrated to a final amount of 5 mg/mL [START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF]].

Characterization of the nanoparticles by DLS and TEM

Dynamic light scattering measurements

Photon correlation spectroscopy is a technique used to determine the diffusion coefficient of small particles in a liquid. The coefficient is determined by accurately measuring the light scattering intensity of the particles as a function of time. Dynamic light scattering (DLS) was performed on a Nano ZS (Malvern Instruments, Southborough, MA) equipped with a 632.8 nm helium neon laser and 5 mW power, with a detection angle at 173 • (noninvasive back scattering).

Transmission electron microscopy

Electronic microscopy analysis was used to show both the shape and size of Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 nanoparticles doped with luminescent ions (Figure 6).

Surface functionalization

In vivo biodistribution of nanoparticles highly depends on their charge and surface properties. Chemical modification of the surface of the probe is critical to better control its circulation and hope for a future targeting to a specific region of interest. For this reason, the PLNP were first reacted with 3-aminopropyl-triethoxysilane (APTES), in order to provide positively charged PLNP (referred to amino-PLNP) resulting from the presence of free amino groups at the surface. The APTES in excess was removed by several sedimentation washing procedures. The global surface charge of the amino-PLNP was reversed by condensation with diglycolic anhydride, which reacted with amines to give free carboxyl groups (carboxy-PLNP). As the use of polyethylene glycol (PEG) capping has already been shown to increase the circulation time of several colloidal systems [START_REF] Moghimi | Long-circulating and targetspecific nanoparticles: theory to practice[END_REF], we also conducted coupling of different PEG moieties (either 5, 10 or 20 kDa) with the amino-PLNP (Figure 7).

Characterization of the functionalized PLNP

Zeta potential

The success of the grafting procedure was assessed by zeta potential measurements (Figure 8). The zeta potential of amino-PLNP was positive (+35.8 mV at pH 7), the zeta potential of carboxyl-PLNP was negative (-50.3 mV at pH 7). Finally as expected, PEGylation of PLNP led to neutral particles (+1 mV).

Thermogravimetric analysis

It exists several chemical methods to determine the amount of a given molecule grafted on nanoparticles. They usually rely on the reactivity of known chemical motifs on the ligand to dose, generally amines, carboxyl or carbonyl groups. In the case of PEG coverage, these methods could not be considered since the polymer lacked reactive functions. However, the relative high molecular weight of the chain opened alternative to a sensitive dosing of PEG moiety through thermogravimetric analysis (TGA). Briefly, TGA analysis was performed by gradually heating the sample from 20 • C to 780 • C under Argon atmosphere. Precision scales allowed to monitor the weight loss as a function of temperature. Weight loss curves for 120 nm PLNP were recorded after each functionalization step. From amino-PLNP to PEGylated PLNP we clearly distinguish two stages in surface decomposition. First, water trapped in the organic layer on the surface of PLNP evaporates before 325 • C. The second stage begins after 325 • C and corresponds to the decomposition of organic APTES and PEG coverage on the particles. When comparing TG curves for different PEG, we notice that the longer the PEG chain length, the fewer PEGylated chains remain grafted on the surface. Calculated values from weight loss percentages indicate that in the case of 5 kDa PEG, polymer concentration reaches about 15 nmol/mg of PLNP. This value drops significantly when increasing the size of the PEG to 9 nmol/mg for 10 kDa PEG and 3 nmol/mg for 20 kDa PEG. Despite the 20 fold molar excess of PEG (compared to the amount of free amino groups previously estimated on the surface: about 60 nmol/mg), it seems that steric hindrance hampers the approach of larger PEG near the particle, thus preventing quantitative reaction as well as equimolar loadings of the different PEG.

Colloidal stability

Before any use of these nanoparticles for bioimaging, colloidal stability in biological media had to be checked. As shown in Figure 9, the addition of polyethylene glycol significantly changes the average value of hydrodynamic diameter, leading to particles from 40 to 70 nm larger than the initial hydroxyl-PLNP, and providing high colloidal stability in normal saline solution. 4 Bioimaging with persistent luminescence nanoparticles

Principle

As explained in the introduction, the major difference between our luminescent silicate and other classical optical probes (used for bioimaging) comes from the ability of PLNP to store excitation energy and to emit at higher wavelength from dozen of minutes to hours. This property allows to excite the probe before its systemic injection (Figure 10).

Influence of coating on the biodistribution of 180 nm PLNP

Biodistribution of 180 nm PLNP with different surface coatings (either amino, carboxy, or PEG) was monitored 30 minutes after systemic injection in mice. Acquired images are shown in Figure 11. For positive amino-PLNP, an important lung retention was observed (Figure 11a). During the first hour, there was little change in this distribution, except a progressive PLNP release from lungs to liver and spleen. Two reasons can explain this biodistribution pattern [START_REF] Song | Enhanced gene expression in mouse lung by prolonging the retention time of intravenously injected plasmid DNA[END_REF]]. The first one is a nonspecific electrostatic interaction of amino-PLNP with the negative charges displayed by plasmatic membranes of capillary endothelial cells, such as sulfated proteoglycans and glycosylaminoglycans. Another explanation could be provided by aggregation of amino-PLNP with negatively charged blood components, leading to trapping agglomerates of increased size in the narrow lung capillaries. Negative carboxy-PLNP were rapidly cleared from blood flow by liver uptake (Figure 11b). This biodistribution pattern presumably resulted from a rapid opsonisation and an uptake by endothelial and Kupffer cells of the reticuloendothelial system (RES), as is generally observed for nanoparticles or liposomes [START_REF] Kamps | Massive targeting of liposomes, surface-modified with anionized albumins, to hepatic endothelial cells[END_REF]][Krieger et al. 1994]. Neutral PEG-PLNP were able to circulate longer, as assessed by a diffuse signal throughout the mouse body that lasted for the rest of the acquisition time (Figure 11c). However, biodistribution images clearly show liver accumulation after 60 minutes. When Kupffer cells were saturated by prior administration of anionic liposomes, the neutral PEG-PLNP circulation time increased further (images not shown).

Influence of nanoparticle diameter and PEG chain length on the biodistribution in healthy mice

The biodistribution of PLNP was studied in function of nanoparticle diameter (80, 120 and 180 nm) and PEG chain length (5, 10 and 20 kDa). Relative long-term biodistribution in healthy mice was achieved six hours after systemic injection to validate a potential use of this probe for in vivo applications. Delayed fluorescence from europium ions trapped in the core of the nanoparticles allowed an ex vivo quantitative analysis through tissue homogenates after animal sacrifice. As shown in Figure 12, similarly to several classes of nanoparticles, and regardless of surface coverage, long term PLNP biodistribution occurs predominantly within liver and spleen [START_REF] Minchin | Nanoparticles for molecular imaging -an overview[END_REF]. Combined uptake in the kidneys and lungs remains below 5 % of the injected dose. The PEG chain length seems to have little influence on the global RES accumulation (combined uptake in liver and spleen), and is only responsible for a slight change in the liver/spleen ratio (only noticeable for 120 nm PLNP). On the contrary, the nanoparticle core diameter appears to be critical. As shown in Figure 12, decreasing the particle core diameter from 180 nm to 80 nm causes a much lower combined RES uptake of PEG-PLNP from 100 % down to around 35 % of the injected dose [START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF]].

Fig. 12 PLNP tissue distribution 6 hours after systemic injection to healthy mice (n=6). Error bars correspond to standard deviation. [START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF] 4.4 Biodistribution of PEG-PLNP in tumor bearing mice

Malignant tumours display both increased angiogenesis and chaotic microenvironment growth, mainly responsible for hypervascularization, leaky vasculature, and poor lymphatic drainage [START_REF] Maeda | Tumor vascular permeability and the EPR effect in macromolecular therapeutics: a review[END_REF]]. The pore size in the vasculature ranges from 200 to 600 nm in diameter, leading to an enhanced permeability and retention (EPR) effect [START_REF] Brannon-Peppas | Nanoparticle and targeted systems for cancer therapy[END_REF]. It was reported that long-circulating nanoparticles with PEG modifications on their surface were able to conduct passive tumour targeting [START_REF] Moghimi | Long-circulating and targetspecific nanoparticles: theory to practice[END_REF]][Greenwald 2001]. The injection of our 180 nm PEG-PLNP to C57BL/6 mouse bearing an s.c. implanted Lewis lung carcinoma (3LL) tumour in the inguinal region, allowed us to detect the tumour vasculature the first 2 minutes after injection (Figure 13a). However no passive accumulation of our PLNP-PEG could be observed at longer time (Figure 13b). In order to favour a passive accumulation of PEG-PLNP in the tumour area we have recently reported the influence of both the diameter of the PLNP (80, 120 and 180 nm) and the length of the PEG chain (5,10 and 20 kDa). This study revealed that after intravenous injection of 80 nm PLNP-PEG 10kDa in 3LL tumour bearing mice, 5.9 ± 2.8 % of the injected dose were located within the tumour micro-environment 6 hours after the injection [START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF]]. Another way to target tumour vasculature is to use nanoparticles coated with targeting ligands [START_REF] Richard | Nanoparticles for imaging and tumor gene delivery[END_REF]][Byrne et al. 2008][Loomis et al. 2011]. Efforts in our laboratory are actually directed towards surface functionalization of PLNP with ligands able to target receptors of interest.

PLNP for imaging inflammation in a mouse model of arthritis

Rheumatoid synovium is characterized by hyperplasia of the synovial lining layer and marked infiltration by lymphocytes, macrophages and plasma cells. Locally produced inflammatory mediators and subsequent up-regulation of adhesion molecules within inflammation sites are both pivotal to the development of rheumatoid arthritis [START_REF] Choy | Cytokine pathways and joint inflammation in rheumatoid arthritis[END_REF]][Tak et al. 1996].

Neoangiogenesis is also a vital component of pannus formation. The early diagnosis of rheumatoid arthritis is made problematic by heterogeneity in its clinical presentation and uncertainty about which patients will respond to treatment. Sensitive and specific imaging methods are required for the detection of early inflammatory changes to the synovium in patients with arthritis and for monitoring response to treatment.

We have evaluated the capacity of carboxy and PEG-PLNP to reveal inflammatory sites in an experimental mouse model of arthritis. The pathology was induced in the DBA/1 mice by immunization with 100 µg of bovine collagen type II at the base of the tail on day 0 and day 21 [START_REF] Courties | RNA interference-based gene therapy for successful treatment of rheumatoid arthritis[END_REF]]. Thirty days after immunization, 100 µg of PLNP (carboxy or PEG) previously excited with a UV lamp for 5 min were injected in the tail vein. Their biodistribution was then monitored under the photon-counting system (Figure 14). As shown in Figure 14-A, carboxy-PLNP were quickly trapped in liver, even in the presence of GdCl 3 (generally used to saturate liver macrophages). In contrast, PEGylated PLNP evade liver (Figure 14-B) to accumulate in the hind legs, at sites of inflammation, and in the oral cavity which is a highly vascularized area. Surface and diameter of the PLNP are key elements that we are actually modifying to improve these results.

Conclusion

We described the synthesis of several persistent luminescence nanoparticles from silicate host. Among them, Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 doped with Eu 2+ , Dy 3+ , and Mn 2+ appeared as the most interesting material for bioimaging applications in terms of emission wavelength and intensity of luminescence. We extracted different monodisperse nano-sized distributions (from 80 to 180 nm) and showed that surface functionalization could not only drastically improve their biodistribution in healthy and tumor-bearing mice, but could also allow to monitor inflammation in a mouse model of arthritis. These results are encouraging, and additional works are actually in progress to heighten image analysis of the acquired data by the means of more reliable softwares and computational tools, but also to improve PLNP optical performances, biodegradability and targeting capabilities.

There remain several challenges associated with this innovative technique. First, as luminescence decreases with time, background signal becomes predominant. After approximately one hour, signal from PLNP is not detectable anymore. This first limitation requires to focus on persistent luminescence materials, and to find new compositions with improved optical properties. Then, two-dimensional optical detection raises the problem of photons diffusion, depth-dependent, that blurs biological structures contours and hampers organs distinction. Therefore, we sometimes observe distorted organs (such as liver or spleen) with variable shape and size. To circumvent this last issue, valuable efforts are being made to work on three-dimensional acquisition systems that would permit combined acquisition in several plans, as well as image reconstruction of the organ through highly developed softwares designed to return the corrected shape and size of the organ from acquired data.

Fig. 1

 1 Fig. 1 Hydrolysis of TEOS in the presence of the salts and sol-gel transition. Sol in the middle, gel on the right.

Fig. 2

 2 Fig. 2 Heating of the gel to get crystals.

Fig. 5

 5 Fig. 5 Size of the different nanoparticles determined by dynamic light scattering.

Fig. 6

 6 Fig.6TEM image of different size Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 : Eu 2+ , Dy 3+ , Mn 2+ . A: 80 nm. B: 120 nm. C: 180 nm. Scale bar = 100 nm.[START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF]

Fig. 7

 7 Fig. 7 PLNP surface modification with different molecules.

Fig. 8

 8 Fig.8Zeta potential of PLNP after each surface modification.[START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF]

Fig. 9

 9 Fig.9Colloidal stability of 5 kDa PEG-PLNP: evolution of the hydrodynamic diameter in 150 mM NaCl for 45 minutes.[START_REF] Maldiney | Effect of core diameter, surface coating and PEG chain length on the biodistribution of persistent luminescent nanoparticles in mice[END_REF]

Fig. 10

 10 Fig. 10 Bioimaging with PLNP: the principle.

Fig. 11

 11 Fig. 11 Biodistribution of PLNP 30 minutes after tail vein injection. a: amino-PLNP. b: carboxy-PLNP. c: PEG-PLNP. [le Masne de Chermont et al. 2007]

Fig. 13

 13 Fig. 13 Images of PLNP-PEG injected to a mouse bearing an s.c. implanted Lewis lung carcinoma (3LL) tumor. a: image obtained 2 minutes after injection. b: image obtained 20 minutes after injection of PLNP. [le Masne de Chermont et al. 2007]

Fig. 14

 14 Fig. 14 PLNP for imaging inflammation sites. A: image obtained with carboxy-PLNP 30 minutes after injection. B: image obtained with PEG-PLNP. In both cases GdCl 3 was injected before PLNP to increase the circulation time of the nanoparticles. The black arrow indicates the site of inflammation.

  3 , ZnMgSi 2 O 6 and Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 doped with Eu 2+ , Dy 3+ and Mn 2+ For this reason, we preferred a Sol-Gel approach to synthesize smaller particles[START_REF] Brinker | Sol-Gel Science: The physics and the chemistry of sol-gel processing[END_REF]]. Raw materials used in our routine synthesis are: magnesium nitrate (Mg(NO 3 ) 2 , 6H 2 O), zinc chloride (ZnCl 2 ), calcium chloride (CaCl 2 , 2H 2 O), europium chloride (EuCl 3 , 6H 2 O), dysprosium nitrate (Dy(NO 3 ) 3 , 5H 2 O), manganese chloride (MnCl 2 , 4H 2 O), and tetraethoxysilane (TEOS). Depending on the desired nanomaterial (MgSiO 3 , ZnMgSi 2 O 6 or Ca 0.2 Zn 0.9 Mg 0.9 Si 2 O 6 ) different amount of reagents were used [le Masne[START_REF] Le Masne De Chermont | Silicates doped with luminescent ions: useful tools for optical imaging applications[END_REF]].

2.1.1 The sol-gel process

Persistent luminescence materials are generally synthesized by a solid-state reaction, giving micrometer-sized particles. Such large particles would hardly circulate after systemic injection to small animals, thus limiting bioimaging applications.


Giselher Grabenweger

Sylvain Poggi

Nelson José

Oliveira Simoes

Leonardo Marianelli

Johannes Sauer

Nelson José De Oliveira Simões

How to fight biological invasions without compromising the aims of the European Green Deal?

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Detecting invasion

Invasive Alien Species pose a risk to plant health in the invaded area. Depending on their ability to establish and their specificity to certain host plants, some species may threaten the entire agricultural sector, as well as the biodiversity in the invaded area. After their detection, control of invasive alien species usually faces two constraints: (1) The possibilities to restrict movement of goods and people, a main driver of further expansion, are limited, and (2) successful eradication of once established populations is impossible. Against this background, it is paramount to develop measures, which confine the spread of the new pest, and prevent the build-up of high population densities that cause economic loss to agricultural crops. It is not a surprise that previous invasions have often triggered an excess use of pesticides for the sake of eradication or containment of an invasive alien species.

The Japanese beetle, Popillia japonica, is one of the worst invasive pests of North America.

Costs to control the pest in the US exceed $450 million per year. P. japonica was detected near Milano in 2014, and is now starting to spread in Europe. It is an enormous threat to Europe, since (1) it can feed on more than 300 host plants, including many important crops, (2) it is a good flyer and can be relocated via movement of goods and people, (3) climate suitability puts at risk an area ranging from the Atlantic to the Black Sea, and from the Mediterranean to Great Britain and Southern Scandinavia. EFSA and the JRC of the European Commission nominated P. japonica a high priority pest in the EU Plant health Law.

Independent from any concerns about the effects of excess pesticide use on human health or the environment, it is clear that a conventional control strategy against the P. japonica invasion into Europe, relying on the heavy use of insecticides, will never reach its containment goals anyway. Reasons are that, in contrast to other agricultural pests, P. japonica oviposition sites and larval populations are mainly found in grasslands. In these habitats, insecticide use is banned or has been uncommon for decades. Furthermore, Japanese beetles are abundant where pesticide use is technically not feasible or socially not acceptable, e.g. in nature conservation areas, forest margins, small-structured, fragmented landscape, or close to residential areas and private gardens. It is therefore clear that a sustainable reduction of P. japonica populations can be achieved only with a control strategy that is applicable in these crop and non-crop areas.

The H2020 project "IPM-Popillia" has the aim to counteract the invasion of the Japanese Beetle in Europe. IPM-Popillia is outstanding with the concept to counteract the invasion of a quarantine pest exclusively by means of sustainable and environmentally friendly control measures. IPM-Popillia will adduce evidence that it is possible to fulfill the obligations of quarantine regulations, without neglecting IPM principles of the EU, as outlined in the Directive on the sustainable use of pesticides.

The presentation will outline the main areas of research and development within the project "IPM-Popillia", which have been defined as the pillars of a successful IPM-Strategy against an invasive pest, and report on the progress made so far:

1.

Identifying the Japanese beetles' pathways of entry and spread This work package aims to make detection and monitoring of the new pest faster, more efficient and less labor intensive. It will provide plant health services with an optimal surveillance strategy, and raise public awareness of the new pest.

2.

Understanding the drivers of Japanese beetles' population development There is a range of biotic and abiotic factors, which play a role in success or failure of the establishment of an alien invader. It is a long-term goal of the project IPM-Popillia to contribute to the understanding of these factors from the gene to the ecosystem level.

3.

Providing an IPM Toolbox for Japanese beetle control A range of biocontrol options of larvae of the Japanese beetle in grassland soils and potting substrates, and of adult Japanese beetles in different crops, are developed and tested in this work package. In addition, measures must be suitable for application at sites with low and high pest abundance, respectively.

4.

Developing sustainable Japanese Beetle management in Europe The last work package goes one step further from development to implementation, and will examine economic and socioeconomic feasibility of the proposed IPM-strategy against P. japonica. Finally, it will focus on fast dissemination of the project's results as customized information and policy advice for a sustainable Japanese beetle management in Europe.



  Biological and biotecnical control  Low risk insecticides  IPM-toolbox for Popillia control TUM, Agric. Production and Resource Economics, 85354 Freising, Germany

This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 861852 This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 861852