\u003ctitle\u003eSilicates doped with luminescent ions: useful tools for optical imaging applications\u003c/title\u003e

$Page 1: \u003ctitle\u003eSilicates doped with luminescent ions: useful tools for optical imaging applications\u003c/title\u003e$
Silicates doped with luminescent ions: useful tools for optical imaging

applications

Quentin le Masne de Chermonta, Cyrille Richardb, Johanne Seguinb, Corinne Chanéacc, Michel Bessodesb, Daniel Scherman*b

a Biospace Lab, 10 rue Mercoeur, Paris, F-75011 France; b Unité de Pharmacologie Chimique et

Génétique; CNRS, UMR 8151, Paris, F-75270 cedex France; Inserm, U 640, Paris, F-75270 cedex France; Université Paris Descartes, Faculté des Sciences Pharmaceutiques et Biologiques, Paris, F-

75270 cedex France; ENSCP, Paris, F-75231 cedex; c Laboratoire de Chimie de la Matière Condensée de Paris, CNRS UMR 7574, Université Pierre et Marie Curie, Paris, F-75005 France.

*[email protected]

ABSTRACT

Fluorescence is increasingly used for in vivo imaging and has provided remarkable results. Howerver this technique presents several limitations, especially due to tissue autofluorescence under external illumination and weak tissue penetration of low wavelength excitation light. We have developed an alternative optical imaging technique using persistent luminescent nanoparticles suitable for small animal imaging. These nanoparticles can be excited before the injection, and their in vivo distribution can be followed in real-time for several hours. Chemical modifications of their surface is possible leading to lung or liver targeting, or to long-lasting blood circulation.

Keywords: Optical imaging, silicates, persistent luminescence, nanoparticles, in vivo imaging , small animal, targeting

INTRODUCTION

Because of a growing demand of imaging tools for biomedical research and medicine, existing imaging systems have been rapidly improved and new techniques have been developed during the past decades. Nowadays, MRI, microcomputed tomography (microCT), ultrasound, positron emission tomography (PET), optical coherence tomography (OCT), and other major imaging systems are available to scientists and clinicians. Each technique has advantages and limitations, thus making them complementary (1). These techniques are increasingly used for biodistribution studies, because they can significantly reduce the number of required animals and increase the experimental data harvested for each animal in longitudinal studies. However, the high cost of several of these techniques and a number of technological barriers impair their widespread use. Optical imaging, in which photons are the information source, represents a rapidly expanding field, with direct applications in pharmacology, molecular and cellular biology, and diagnostics. With the development of more sensitive optical sensors and new powerful probes such as semiconductor nanocrystals (2-4), fluorescent proteins (5), or near-infrared fluorescent molecules (6), optical imaging is widely used for in vivo studies. In vivo optical imaging using fluorescent probes is commonly used (7) but still presents numerous disadvantages. The first one is the autofluorescence (8) from tissue organic components due to constant probe illumination during signal acquisition. This autofluorescence often results in poor signal-to-noise ratio. In addition, deep tissue imaging is difficult because of intrinsic tissue signal attenuation. The probe's emission has thus to be tuned in the tissue transparency window (9) (wavelength from 650 nm to the infrared), in which light attenuation is largely due to scattering rather than to absorption.

To overcome these difficulties, we have recently developed persistent luminescent silicate nanoparticles (PLN), which are suitable for in vivo imaging and can avoid most of inherent problems encountered in classical optical systems

Invited Paper

Colloidal Quantum Dots for Biomedical Applications IV, edited by Marek Osinski, Thomas M. Jovin, Kenji Yamamoto,Proc. of SPIE Vol. 7189, 71890B · © 2009 SPIE · CCC code: 1605-7422/09/$18 · doi: 10.1117/12.819316

Proc. of SPIE Vol. 7189 71890B-1

(10). The key element of this technology is based on a new generation of long luminescent nanoparticles emitting in the red to near-infrared range, which can be optically excited before in vivo local or systemic injection. The long-lasting afterglow (also called persistent luminescence) can reach several hours and permits the removal of the background noise originating from in situ excitation. Thus, the significant signal-to-noise ratio improvement allows detection in rather deep organs and real-time biodistribution monitoring of active elements hours after injection.

1 SILICATES AS PERSISTENT LUMINESCENCE NANOPARTICLES

Great progress has been made in long-lasting phosphorescence since the early 20th century. Long-lasting

phosphors were widely used in emergency lighting, safety indications, road signs and so on. Most of long-lasting phosphors are based on sulfides and aluminates. Recently silicate phosphors have been paid considerable attention because of their multi-color phosphorescence, notably in blue, green (11). In 2003, Wang et al. reported the synthesis of MgSiO3 doped with Eu2+, Dy3+and Mn2+ luminescent ions, showing a long red persistent luminescence (12). This article initiated the preparation of a series of luminescent silicates for small animal optical imaging applications in our lab.

1.1 Preparation of three luminescent silicates: MgSiO3, ZnMgSi2O6 and Ca0.2Zn0.9Mg0.9Si2O6 doped with Eu2+, Dy3+and Mn2+

1.1.1 The sol-gel synthesis

Persistent luminescent materials are generally synthesized by a solid-state reaction, giving micrometer-sized particles. Such a process cannot be used for our applications since in our case as the nanomaterials should be injected into the tail vein of small animals. For this reason, we used a Sol-Gel approach synthesis in order to decrease the particles size (13). All the reactants we used are analytical-grade and are used without any purification. The raw materials used in our synthesis are: magnesium nitrate (Mg(NO3)2, 6H2O), zinc chloride (ZnCl2), calcium chloride (CaCl2, 2H2O), europium chloride (EuCl3, 6H2O), dysprosium nitrate (Dy(NO3)3, 5H2O), manganese chloride (MnCl2, 4H2O), and tetraethoxysilane (TEOS).

Depending on the desired nanomaterial, different amount of reagents were used. For example, for the preparation of Ca0.2Zn0.9Mg0.9Si2O6 (Eu2+, Dy3+, Mn2+) we need: 126 mg of calcium chloride, 993 mg of magnesium nitrate, 527 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).


Figure 1. The sol-gel transition. Sol on the left and the gel on the right.

1.2.2 Heating to get nanocristals

The resulting opaque dry gel was then fired in a zircone crucible in a weak reductive atmosphere using 10% H2, 90% Ar (Noxal 4, Air Liquide, Düsseldorf, Germany). Depending on the material we want, the final temperature varies. For preparing Ca0.2Zn0.9Mg0.9Si2O6 (Eu2+, Dy3+, Mn2+) the gel is warmed to 1050°C for 10 h and then cool done to room temperature to give a white powder.

1.2.3 Obtention of small nanoparticles

The powder was then ground by using an agate mortar and a pestle, and the smallest nanoparticles were isolated by selective sedimentation. The powder was first dispersed by sonication in 5 mM NaOH solution at a concentration of 10 mg of PLN per ml. The suspension was then diluted with distilled water at a concentration of 2.5 mg/ml and gently centrifuged with a SANYO MSE Mistral 1000 (670 x g for 15 min) to eliminate the largest particles. Acetone corresponding to 25% of the supernatant volume was added to promote sedimentation of the PLN. The suspension was then centrifuged at 3400 x g for 30 min. After removal of the supernatant, in which no NPs were detected by using Dynamic Light Scattering, the PLN were dried in a vacuum oven. Electronic microscopy analysis showed that the PLN exhibited a quite narrow size distribution, with particles diameter ranging from 50 to 100 nm (Figure 2).

Figure 2. TEM image of Ca0.2Mg0.9Zn0.9Si2O6: Eu2+, Dy3+, Mn2+ (scale bar = 200 nm)


12

750 80"

2.2 CARACTERISATION OF THE THREE NANOMATERIALS

2.2.1 Fluorescence

Optical spectra 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: λem = 690 nm; excitation slit = 5 nm; emission slit = 20 nm) (for excitation spectrum: λex = 340 nm; excitation slit = 20 nm; emission slit = 5 nm). Around 50 mg of each powder was deposited on a glass plate, stick with few drops of ethanol and insert into the spectrophotometer. The different emision spectra are reported in figure 3.

Figure 3. Emission spectra of the three nanoparticles. Green: MgSiO3 (λem = 645 nm), blue: ZnMgSi2O6 (λem = 660 nm),

purple: Ca0.2 Zn0.9Mg0.9Si2O6 (λem = 690 nm).

2.2.2 Determination of the size of the nanoparticles

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 were 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). The sizes of the different PLN are reported in figure 4.


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1000

0 S00 1000 i;oo 2000 2500 000 3500 4000

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Figure 4. Size of the different nanoparticles determined by DLS. Green: MgSiO3 (size = 250 nm), blue: ZnMgSi2O6 (size =

100 nm), purple: Ca0.2 Zn0.9Mg0.9Si2O6 (size = 70 nm).

2.2.3 Luminescence

To determine the persistent luminescence, 10 mg of each PLN are deposited on a 96-wells plate, excited for 2 min with a UV lamp, and after removal of the excitation source, a photon-counting system based on a cooled GaAs intensified charge-coupled device (ICCD) camera (Photon Imager; Biospace Lab, Paris, France) without an external illumination system was used. The luminous intensity decay was typical of a persistent luminescence material (14) and was detectable for more than 24 h when kept in the dark. The decay kinetics (Figure 5) were found to be close to a power law I ≈ Io x t-n (n = 0.96 R2 = 0.996) after the first 100 s.

Figure 5. Afterglow decays after UV excitation. Green: MgSiO3, blue: ZnMgSi2O6, purple: Ca0.2 Zn0.9Mg0.9Si2O6

Size (nm)

Num

ber (

%)

Time (s)

Inte

nsity

(a.u

.)


(2) kT (3)Th CB

Velectron trap I

(1)

Hole trap

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2.2.4 Proposed mechanism of persistent luminescence

In these nanomaterials, trapped centers are created through the introduction of small amounts of Dy3+, and Mn2+ is the final emitting center receiving energy from electron-hole pair recombination. Rare-earth ions serve as primary acceptors of the energy, which is thermally released to the manganese for several hours. The symmetry and the crystal field strength at the Mn2+ site in the synthesized material are responsible for a Mn2+ emission in the red and the near-infrared part of the spectrum range, corresponding to the transition from the 4T1(4G) excited state to the 6A1(6S) fundamental state (16). The afterglow emission must be due to the persistence energy transfer from the rare earth ions. Long persistent phosphorescence in red (660 nm) was observed for several hours. The function of Eu2+ is to create electron-tarpped centers, Dy3+ is to create hole-trapped centers in the material (15), where the Mn2+ received energy from the excitation of the electron-hole pairs (Figure 6).

Figure 6. Proposed mechanism of the persistent luminescence

3. Use of Ca 0.2Zn 0.9Mg 0.9Si 2O6 for small animal optical imaging

3.1 SURFACE MODIFICATION OF THE PROBE

To use these nanoparticles for small animal optical imaging, modifications of the surface of the nanoparticles were realized. The PLN were first reacted with 3-aminopropyl-triethoxysilane (APTES), in order to provide positively charged PLN (referred to as amino-PLN) resulting from the presence of free amino groups at the surface. The success of the grafting procedure was assessed by zeta potential measurements (+35.8 mV at pH 7) and a positive test with trinitrobenzene sulfonate (TNBS). The APTES in excess was removed by several sedimentation washing procedures. The surface charge of the amino-PLN was reversed by reaction with diglycolic anhydride, which reacted with amines to give

free carboxyl groups. The zeta potential of these particles (carboxyl-PLN) at neutral pH was negative (–37.3 mV), as expected. We also conducted peptide coupling of amines with mPEG5,000-COOH (carboxyl-methoxy-polyethyleneglycol, molecular weight 5,000 g/mol). This reaction led to neutral particles (+5.1 mV), partly originating from the charge-shielding effect of PEG. The unreacted reagents were always removed by three or more centrifugation-washing steps (Figure 7).


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4s

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oco

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Figure 7. Chemical modifications of the surface of the nanoparticles. (i) Treatment with APTES to get positive PLN, (ii)

treatment with diglycolic anhydride to get negative PLN, (iii) treatment with carboxy-PEG to get neutral PEG-PLN.

3.2 IN VIVO IMAGING

3.2.1 Principle Before being used for in vivo imaging, PLN are first excited by a UV light for two minutes outside the animal body, then injected to the animal, where they emit visible light for hours after the injection The signal is then acquired with an intensified CCD camera (Photon Imager, Biospace Lab) in complete darkness. Autofluorescence, resulting from external illumination during signal acquisition, is therefore avoided.

3.2.2 Results of in vivo biodistribution

The three type of PLN were dispersed in water (1 mg/ml) and 100 µl of each suspension were injected to anesthetized mice in the tail vein. The biodistribution of the different charged PLN are reported in Figure 8.


0 50 100 150 200 2.0 JOG

Concentration of NPs (g/rnL

25oco-.--NP-PEG--NP-B,otin

20000--a-NP-PE&BFotn

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15000

10000

5000

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Figure 8. Biodistribution of the differently charged nanoparticles. Left: (positive) amino-PLN, middle: (negative) carboxy-

PLN, right: (neutral) PEG-PLN.

As it can be seen on figure 8, the biodistribution is largely dependent on the the nanoparticle charge. Positive PLN (Figure 8, left) are rapidly capted by the lungs, negative PLN (Figure 8, middle) are rapidly capted by liver and neutral PEG-PLN can circulate in the blood as could be seen on figure 8 (right).

3.3 FIRST EXEMPLES OF TARGETING

Preliminary results based on the functionalization of the outer surface of these luminescent nanoparticles with biotin in order to target avidin (Ka = 1015 M-1) have been recently obtained in our lab. Three types of nanoparticles have been prepared: PLN-PEG3000, PLN-Biotin and PLN-PEG3000-Biotin, and dispersed in PBS. Increasing amount of these nanoparticles were deposited on a streptavidin plate, incubated for 30 min and washed. The quantity of PLN attached on the streptavidin plate was determined using time resolved fluorescence. As could be seen on Figure 10, the immobilization of the PLN on the plate is ligand specific. No immobilization occurred without biotin, and the fixation is PEG dependant since no immobilization of the PLN occurred when biotin was directly linked to the surface of the PLN. Moreover, we have shown that the immobilization of these PLN was specific to biotin since a dramatic decrease of PLN immobilized on the plate was observed when the plate was first incubated with a biotinylated antibody (Figure 9) (17).

Figure 9. Immobilization of targeting Ca0.2Zn0.9Mg.9Si2O6: Eu2+ Dy3+ Mn2+ on a streptavidin plate


3. Conclusion We have reported the synthesis of persistent luminescence silicate nanoparticles using the sol-gel process followed by an heating step. We are reported than the size and the luminescence properties are dependent on the composition of the nanomaterials. We have also shown that among them, Ca0.2Zn0.9Mg.9Si2O6: Eu2+ Dy3+ Mn2+ was the most interesting for us and have been used for small animals optical imaging. We have observed that the biodistribution of these PLN was charge dependent. Finally, we have shown than it is possible to functionalize the surface of these PLN with biotin to target streptavidin immobilize on a plate. These encouraging results led us to use these functionalized PLN to target other receptors in vitro and in vivo.

REFERENCES

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\u003ctitle\u003eSilicates doped with luminescent ions: useful tools for optical imaging applications\u003c/title\u003e

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