Persistent luminescence nanoparticles excitable in situ for in vivo optical imaging, in vivo multimodal optical—MRI imaging, and theranostics

Abstract

Multimodal optical and magnetic resonance imaging methods based on the use of persistent luminescence nanoparticles. Use of mesoporous persistent luminescence <<core-shell>> complexes for theranostic applications.

Claims

1. An in vivo optical imaging method of a human or animal body, comprising the following steps: a) exciting the persistent luminescence of nanoparticles by in vivo irradiation of all or part of the human or animal body at a wavelength between 550 and 1000 nm, said nanoparticles being previously administered to the human or animal body, and said nanoparticles emitting photons at wavelengths between 550 and 1000 nm for at least 0.01 second, after light excitation at wavelengths between 550 and 1000 nm, and said nanoparticles comprising a nanomaterial formed of a matrix selected from among gallates, aluminates, indates, and their mixed compounds gallo-germanates, gallo-aluminates, gallo-indates, gallium oxides, indium oxides, magnesium oxides, zinc and gallium oxysulfides, zinc and gallium oxyselenides, zinc and gallium oxytellurides, said matrix being doped with a transition metal or lanthanide selected from among chromium, europium, cerium, nickel, iron, copper and cobalt; and b) detecting the nanoparticles in vivo in all or part of the human or animal body by measuring the persistent luminescence of the nanoparticles via optical imaging.

2. The in vivo optical imaging method of a human or animal body according to claim 1, wherein said nanoparticles comprise the nanomaterial ZnGa.sub.2(1-x)Cr.sub.2xO.sub.4 with x between 0.001 and 0.0075.

3. The in vivo optical imaging method of a human or animal body according to claim 1, wherein said nanoparticles comprise the nanomaterial ZnGa.sub.1.995Cr.sub.0.005O.sub.4.

4. The in vivo optical imaging method of a human or animal body according to claim 1, wherein the administering of the nanoparticles is previously performed via intravenous, intra-arterial, intramuscular, intraperitoneal or retro-orbital route.

5. The in vivo optical imaging method of a human or animal body according to claim 1, wherein the size of the nanoparticles is between 1 and 10.sup.3 nm.

6. The in vivo optical imaging method of a human or animal body according to claim 1, wherein the nanoparticles are surface grafted or coated.

7. The in vivo optical imaging method of a human or animal body according to claim 1, wherein the nanoparticles are surface grafted with a ligand.

8. The in vivo optical imaging method of a human or animal body according to claim 1, wherein the nanoparticles are encapsulated in mesoporous silica allowing the loading and release of molecules of interest.

9. A bimodal in vivo imaging method of a human or animal body comprising the following steps: a) exciting the persistent luminescence of the nanoparticles by in vivo irradiation of all or part of the human or animal body at a wavelength between 550 and 1000 nm, said nanoparticles being previously administered to the human or animal body, and said nanoparticles emitting photons at wavelengths between 550-1000 nm for at least 0.01 second under excitation light at wavelengths between 550 and 1000 nm, said nanoparticles having paramagnetic properties and said nanoparticles comprising a nanomaterial formed of a matrix from among gallates, aluminates, indates, gallium oxides, indium oxides, magnesium oxides, gallo-germanates, alumina-gallates, zinc and gallium oxysulfides, zinc an gallium oxyselenides, zinc and gallium oxytellurides, said matrix being doped with a transition metal or lanthanide selected from among chromium, europium, cerium, nickel, iron, copper and cobalt and with at least one paramagnetic element selected from among Cr.sup.3+; Mn.sup.2+; Gd.sup.3+; Fe.sup.3+ and Ni.sup.3+; b) detecting the nanoparticles in vivo in all or part of the human or animal body by measuring the persistent luminescence of the nanoparticles using optical imaging; and c) detecting the nanoparticles in vivo in all or part of the human or animal body by magnetic resonance imaging.

10. The in vivo bimodal imaging method of a human or animal body according to claim 9 wherein said nanoparticles comprise the nanomaterial ZnGa.sub.2(1-x-y)Cr.sub.2xGd.sub.2yO.sub.4 with x between 0.001 and 0.0075 and y between 0.01 and 0.08.

11. The in vivo bimodal imaging method of a human or animal body according to claim 9, wherein said nanoparticles comprise the nanomaterial ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4.

12. The in vivo bimodal imaging method of a human or animal body according to claim 9, wherein the administering of the nanoparticles is previously performed via intravenous, intra-arterial, intramuscular, intraperitoneal or retro-orbital route.

13. The in vivo bimodal imaging method of a human or animal body according to claim 9, wherein the size of the nanoparticles is between 1 and 10.sup.3 nm.

14. The in vivo bimodal imaging method of a human or animal body according to claim 9, wherein the nanoparticles are surface grafted or coated.

15. The in vivo bimodal imaging method of a human or animal body according to claim 9, wherein the nanoparticles are grafted with a ligand.

16. The in vivo bimodal imaging method of a human or animal body according to claim 9, wherein the nanoparticles are encapsulated in mesoporous silica allowing the loading and release of molecules of interest.

17. A nanoparticle comprising the nanomaterial ZnGa.sub.2(1-x-y)Cr.sub.2xGd.sub.2yO.sub.4 with x between 0.001 and 0.0075 and y between 0.01 and 0.08.

18. The nanoparticle according to claim 17 characterized in that the nanomaterial is ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4.

19. The nanoparticle according to claim 17, characterized in that its size is between 1 and 10.sup.3 nm.

Description

FIGURES

(1) FIG. 1: Diffractogram of ZnGa.sub.1.995Cr.sub.0.005O.sub.4 (left) and ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4 (right) compounds. The vertical lines indicate the reference diffraction peaks of ZnGa.sub.2O.sub.4 (reference code: 00-038-1240).

(2) FIG. 2: Emission spectrum of LED lamp used for visible range excitation (orange-red) of the nanoparticles (A). Excitation spectrum of photoluminescence at 705 nm of the compound ZnGa.sub.1.995Cr.sub.0.005O.sub.4 (B). Emission spectrum of persistent luminescence after UV and visible excitation (C). Compared decay of persistent luminescence after UV excitation or using the LED system (D).

(3) FIG. 3: Compared decay of persistent luminescence according to material composition and type of excitation.

(4) FIG. 4: Electron microscope image of nanoparticles of ZnGa.sub.1.995Cr.sub.0.005O.sub.4 (the bold line represents 80 nm).

(5) FIG. 5: Functionalization scheme of ZnGa.sub.2(1-x-y)Cr.sub.2xGd.sub.2yO.sub.4 nanoparticles.

(6) FIG. 6: Top picture: On the left, mouse injected with hydroxylated particles (negative charge). On the right, mouse injected with PEGylated particles (neutral). Bottom pictures: On the left, optical image of a mouse carrying three sub-cutaneous CT26 tumours and injected (intravenous) with nanoparticles of ZnGa.sub.1.995Cr.sub.0.005O.sub.4. On the right, persistent luminescence signal obtained after illumination of the animal under an orange-red light source (see FIG. 2.A), 4 hours after injection of the probe.

(7) FIG. 7: In vivo images of the biodistribution of stealth nanoparticles at different times. The mouse was injected with 2 mg of PEGylated nanoparticles having a core diameter of 80 nm. Long-length times are accessible via re-excitation of the persistent luminescence of the particles for 2 minutes under an orange-red light source (see FIG. 2.A) through animal tissue.

(8) FIG. 8: Acquisition under persistent luminescence of the signal from a suspension of RAW macrophages tagged with persistent luminescence nanoparticles.

(9) FIG. 9: Compared in vivo biodistribution 30 minutes after systemic injection of amino persistent luminescence nanoparticles (left) and RAW cells (murine macrophages) tagged with the same persistent luminescence particles (right). These images were recorded after excitation of persistent luminescence in the visible range (between 550 and 700 nm).

(10) FIG. 10: Ex vivo biodistribution 15 hours after systemic injection of amino nanoparticles (top row) and RAW cells (murine macrophages) tagged with persistent luminescence nanoparticles (bottom row). These images were recorded after excitation of dissected organs in the visible range for 2 minutes. The majority locating of cells in the lungs can be seen confirming the in vivo biodistribution in FIG. 9.

(11) FIG. 11: MRI image obtained under T1 contrast for different concentrations (mg/mL) of nanoparticles of ZnGa.sub.1.995Cr.sub.0.005O.sub.4 (left) and ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4 (right).

(12) FIG. 12: MRI image obtained under T2 contrast for different concentrations (mg/mL) of nanoparticles of ZnGa.sub.1.995Cr.sub.0.005O.sub.4 (left) and ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4 (right).

(13) FIG. 13: Relaxivities obtained from MRI images under T1 and T2 contrast for nanoparticles of ZnGa.sub.2(1-x-y)Cr.sub.2xGd.sub.2yO.sub.4.

(14) FIG. 14: Top picture: Monitoring by optical imaging of the biodistribution of non-functionalised nanoparticles doped with gadolinium, 2 mg of ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4, 24 h after injection. Signal acquisition was obtained during 5-minute period after mouse exposure time of 2 minutes to a halogen source emitting at between 550 and 700 nm. Bottom picture: MRI slice (7T) of the liver of a control mouse without particles (red arrow, left picture) and of a mouse injected with the compound doped with gadolinium (red arrow, right picture). The MRI images were obtained by T2* weighting. Homogeneous negative contrast can be seen at the bulk of the liver (dark region in the right-hand picture compared with the particle-free control liver on the left).

(15) FIG. 15: Principle of the use of a mesoporous persistent luminescence structure. PLNP: <<persistent luminescence nanoparticles>>; MPLNP: <<mesoporous persistent luminescence nanoparticles>>.

(16) FIG. 16: Electron microscope image of ZnGa.sub.1.995Cr.sub.0.005O.sub.4 (dark region) after formation of the mesoporous layer (light region). The bold line represents 100 nm.

(17) FIG. 17: Porosity parameters of mesoporous persistent luminescence nanoparticles (mean diameter about 100 nm).

(18) FIG. 18: Release kinetics of doxorubicin after loading DOX in mesoporous persistent luminescence nanoparticles.

(19) FIG. 19: MTT test after 24 h contact between mesoporous particles without doxorubicin (MPLNP), or loaded with doxorubicin (MPLNP-Dox), and two cancer cell lines. U87MG: human glioblastoma line; CT26: murine colon carcinoma line.

(20) FIG. 20: Optical imaging monitoring of the biodistribution of non-functionalised mesoporous nanoparticles, 24 h after injection of 2 mg of MPLNP. Signal acquisition was obtained for 5 minutes after mouse exposure time of 2 minutes to a LED source (see FIG. 2.A for the emission spectrum).

EXAMPLES

1Synthesis of ZnGa2(1-x-y)Cr2xGd2yO4 with x[0.001; 0.0075]; y[0.01; 0.08]

(21) The nanoparticles were prepared by hydrothermal synthesis. A mixture of gallium, chromium, zinc and gadolinium nitrates in desired proportions, adapted for the desired composition were dissolved in water under agitation and at ambient temperature. The addition of concentrated ammonia to this solution of cations, up to a value of pH=7.5 allowed the precipitation of a precursor of zinc gallate. The suspension was left under agitation at ambient temperature for 3 hours then transferred to a Teflon reactor to undergo treatment under pressure at 120 C. for 24 hours. The compound obtained after treatment was washed several times in water and ethanol and dried in vacuo. Finally the dried compound was ground to a fine powder and calcined at 750 C. for 5 hours.

(22) The structure of the crystal was confirmed by X-ray diffraction. It can be seen in FIG. 1 that the compound without gadolinium, ZnGa.sub.1.995Cr.sub.0.005O.sub.4, and the compound with gadolinium, ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4, have the structure of zinc gallate: ZnGa.sub.2O.sub.4. It is also observed that the addition of these two cations (Cr.sup.3+ and Gd.sup.3+) does not cause the formation of any parasitic phase.

(23) The optical properties of the nanoparticles obtained are shown in FIG. 2. It can be seen that excitation focused on 605 nm (FIG. 2.A) allows activation of the persistent luminescence signal, that is weaker but characterized by an emission spectrum centred around 700 nm (FIG. 2.C) and decay kinetics (FIG. 2.D) similar to those obtained after UV excitation. The excitation spectrum of photoluminescence at 705 nm of the compound ZnGa.sub.1.995Cr.sub.0.005O.sub.4 can be seen (FIG. 2.B).

(24) The addition of gadolinium leads to a drop in persistent luminescence but allows the maintaining of the same type of decay and comparable kinetics (FIG. 3).

2Extraction of a Monodisperse Suspension of Nanoparticles of ZnGa2(1-x-y)Cr2xGd2yO4

(25) The nanoparticles obtained were crushed for about ten minutes then taken up in a sodium hydroxide solution (5 mM) to allow surface hydroxylation. The suspension was passed through an ultrasound bath then left under agitation overnight. Finally the nanoparticles were extracted by selective centrifugations. Precise adjustment of the centrifugation parameters allows selection of the diameter of interest. An example of nanoparticles having a diameter of 80 nm is given in FIG. 4.

3Functionalization of Nanoparticles of ZnGa2(1-x-y)Cr2xGd2yO4

(26) After extraction of a monodisperse suspension, it is important to be able to modify the surface condition of the nanoparticles to promote their circulation within the body or to allow optional targeting by adding ligands of biological receptors. This functionalization is possible via successive chemical modifications on the surface of the persistent luminescence nanoparticles (FIG. 5) and allows controlling of the biodistribution of the probes after injection.

(27) Several characterization steps ensure that functionalization has effectively taken place. Measurement of potential allows evaluation of the surface charge of these persistent luminescence nanoparticles. In particular, the adding of polyethylene glycol (PEG) allows masking of surface charges and increases the circulation time of the nanoparticles after systemic injection into a small animal.

(28) Details and Protocols of the Functionalization Steps

(29) Functionalization of the Nanoparticles with 3-Aminopropyltriethoxysilane (APTES):

(30) The hydroxylated nanoparticles in suspension in dimethylformamide (DMF) at a concentration of 2.5 mg/mL were dispersed in an ultrasound bath for 5 minutes. The APTES was added thereto at a volume concentration of 5%. The suspension was again left in the ultrasound bath for 5 minutes, then left under strong agitation for 5 h. The nanoparticles were finally washed several times in ethanol to remove excess APTES.

(31) Functionalization of the Nanoparticles with Polyethylene Glycol (PEG):

(32) The amino nanoparticles were dissolved in DMF at a concentration of 2.5 mg/mL then dispersed in an ultrasound bath for 5 minutes. A solution of PEG 5 kDa (alpha-methoxy gamma-N-hydroxysuccinimide, 25 mg) was added to the suspension of nanoparticles. The mixture was again dispersed in an ultrasound bath 5 minutes, then left under strong agitation at 90 C. overnight. The nanoparticles were finally washed several times in DMF to remove excess APTES.

4Injection of the Nanoparticles of ZnGa1.995Cr0.005O4 Whether or not Functionalized, and Application of the Stealth Nanoparticles to Target CT26 Tumours

(33) It can be seen in FIG. 6 (top pictures) that there is a distinct difference in biodistribution between the negative nanoparticles and those whose charge is masked by PEG. This effect is characteristic of numerous types of nanoparticles. The negative nanoparticles, on account of their surface charge, undergo rapid opsonisation followed by capture by the liver macrophages (Kupffer cells), which can be seen in the picture on the left (FIG. 6, top picture). On the other hand, this recognition process is delayed when the surface charges are masked by adding PEG. This can be seen in the top right picture in FIG. 6. The particles are distributed more homogeneously within the body and are distributed in the general circulation.

(34) The intravenous injection of these PEGylated nanoparticles, called stealth nanoparticles, in a mouse carrying subcutaneous CT26 tumours (FIG. 6, bottom left) allows passive targeting of the tumour region by gradual accumulation of the probe at the diseased tissues. The persistent luminescence signal then allows clear viewing of the tumours implanted in the animal (FIG. 6, bottom right).

5Excitation of the Persistent Luminescence of the Nanoparticles Through an Animal Body

(35) One of the major weak points in the preceding generation of persistent luminescence nanoparticles lies in their incapability of being excited through animal tissue, thereby limiting observation to a time not exceeding one hour. The acquisitions of persistent luminescence signals presented below indicate that these compounds (ZnGa.sub.2(1-x-y)Cr.sub.2xGd.sub.2yO.sub.4), in the form of nanoparticles can be excited through animal tissue to obtain a persistent luminescence signal. With this innovation it is possible to avoid any time constraint and to observe the nanoparticles at any time.

(36) For this procedure the mice were anesthetised with a ketamine/xylazine mixture and injected with the nanoparticles of ZnGa.sub.1.995Cr.sub.0.005O.sub.4 in the tail vein. The mice were placed under the LED system (see FIG. 2.A for the emission spectrum) for 2 minutes to excite persistent luminescence, then placed under an ICCD camera (photon count system, Biospace Lab). It can be seen in FIG. 7 that the persistent luminescence signal allows the locating of the particles and monitoring of their biodistribution after several hours.

6Example of Application to Real-Time Cell Monitoring in Mice

(37) As an original example of the application of this technology, we also report on the possibility of marking the cells with these persistent luminescence nanoparticles, and of tracking their cell biodistribution after systemic injection into a small animal:

(38) Marking of RAW Cells (Murine Macrophages) and Injection:

(39) Marking of cells was obtained by incubating RAW macrophages (10.sup.6 per well, 6-well plate), with 2 mL of a suspension of nanoparticles in DMEM serum-free culture medium (1 mg/mL) for 6 h. After incubation, the cells were washed several times in the culture medium to remove excess nanoparticles, taken up in the same medium and concentrated by centrifugation at 900 rpm for mouse injection (300 L). Efficient marking of the cells can be verified by exciting persistent luminescence with the LED system (FIG. 8).

(40) In vivo study was performed by comparing the biodistribution of amino nanoparticles, 2 mg in suspension in the serum-free culture medium, injected into the tail vein, with the biodistribution of the macrophages tagged with the nanoparticles (10.sup.6 cells) in suspension in the same medium. The results are given in FIG. 9.

(41) A distinct difference in biodistribution is seen between the particles alone and the tagged macrophages. The particles alone are concentrated at the liver for the same reasons as those given above. The tagged cells are attached to the lungs.

(42) Re-excitation through the tissues also allows ex vivo quantification after organ sampling (the excitation conditions are the same as previously with the LED system). The luminescence images obtained are given in FIG. 10.

7Example of Application of ZnGa2(1-x-y)Cr2xGd2yO4 for Multimodal Imaging

(43) It has already been pointed out above that the adding of gadolinium to the zinc gallate structure does not alter the nature of the optical properties. In particular, the phenomenon of persistent luminescence, less intense it is true, is maintained as are the excitability properties of the material above 600 nm (LED system, FIG. 2.A).

(44) The nanoparticles used for measuring relaxation times (T1 and T2) in vitro were prepared following the protocol described in the two first parts. The nanoparticles have a diameter of about 80 nm.

(45) It can be seen in FIGS. 11 and 12 (left pictures) that the nanoparticles non-doped with gadolinium do not display any concentration-dependent contrast effect (same contrast at the circles) and show relaxivity values similar to those of the control (without nanoparticles). On the other hand, the relaxivity measurements R1 and R2, and the images obtained with 7T MRI (FIGS. 11, 12, on the right; then FIG. 13), indicate a T1 and T2 effect of the nanoparticles related to the addition of gadolinium. This effect is indeed dependent on nanoparticle concentration: the greater the concentration the greater the contrast. Comparisons with T1 and T2 relaxivity values of commercial agents (Dotarem and Endorem), in the light of the relaxivities calculated for the nanoparticles of ZnGa.sub.2(1-x-y)Cr.sub.2xGd.sub.2yO.sub.4, place us in a position to say that the T2 effect is preponderant (supported by calculation of the ratio R2/R1>1).

(46) Example of In Vivo Application to Small Animals:

(47) At an initial step, optical imaging allowed locating of the particles after intravenous injection. The accumulation of persistent luminescence nanoparticles at the liver was monitored by optical imaging 24 hours after injection (FIG. 14, 2 mg of ZnGa.sub.1.955Cr.sub.0.005Gd.sub.0.04O.sub.4 diameter of 80 nm, in suspension in 5% glucose for injection). MRI acquisition with T2 contrast allowed confirmed presence of the nanoparticles distributed homogeneously in the bulk of the liver (as compared with the control liver not having received any particle). This type of diffuse negative contrast at the liver is characteristic of capture by the organ's macrophages, Kupffer cells.

8Synthesis of Mesoporous Persistent Luminescence Nanoparticles (MPLNP)

(48) The principle underlying the use of mesoporous persistent luminescence nanoparticles for theranostic applications is summarised in FIG. 15.

(49) Example of Synthesis with ZnGa.sub.1.99Cr.sub.0.005O.sub.4:

(50) The mesoporous layer was formed by condensation of tetraethoxysilane (TEOS) around the nanoparticles in the presence of a cationic surfactant: cetyltrimethylammonium bromide (CTAB).

(51) The nanoparticles were suspended in a CTAB solution (4 mg/mL) in 5 mM sodium hydroxide at a concentration of 1 mg/mL. The mixture was well dispersed in an ultrasound bath and left at 45 C. under strong agitation. TEOS was then added dropwise to the suspension of nanoparticles to obtain a final concentration of 1% by volume (ex.: 10 L of TEOS per 1 mL of suspension). After an agitation time of 3 hours at 45 C., the suspension was transferred to a Teflon reactor to undergo maturing under pressure for 24 h at 100 C. The suspension was finally washed several times in water and ethanol to remove excess surfactant.

(52) The porous structure was obtained by extraction of the surfactant (CTAB) from the silica layer coating the persistent luminescence nanoparticles. This extraction was performed in a solution of NaCl in methanol (1% by weight). The nanoparticles are suspended in this saline solution of methanol and left under agitation for 3 h. After extraction, the nanoparticles were washed several times in ethanol. This extraction step was repeated 3 times to ensure that all the surfactant had effectively been removed.

(53) FIG. 16 gives an electron microscope image of the core-shell structure obtained after encapsulation of the gallate crystals in a layer of mesoporous silica. The crystal at the core of the structure can clearly be seen being of greater density since less transparent to the electrons, and the layer of amorphous silica that is light-coloured in the image on account of its transparency to electrons. The porosity parameters were determined by nitrogen adsorption at 77 K.

(54) The results in FIG. 17 indicate an accessible specific surface area of 400 m.sup.2/g of compound. The pore diameter is about 2 nm.

9Example of Application of MPLNPs to Deliver Cytotoxic Molecules

(55) The idea was to use this structure to administer active ingredients and we therefore conducted a first proof of concept study with doxorubicin (Dox), used in the clinical treatment of some cancers. This molecule distinctly absorbs light at around 480 nm. For this reason the loading of Dox into the porous structure was followed by measurement of absorbance at 480 nm. We evaluated the amount of Dox (unit weight of particles) at 150 g per mg of mesoporous nanoparticles. The same assay technique was used to evaluate the release kinetics of the compound in a phosphate buffer (FIG. 18).

(56) With a view to evaluating the possibility of using these nanoparticles to convey and release a cytotoxic molecule we compared the toxicity of the nanoparticles loaded with Dox (MPLNP-Dox) with that of non-loaded nanoparticles (MPLNP) on several cell lines (CT26 and U87MG).

(57) Assay protocol: The cells were cultured and placed in 96-well plates at a concentration of 10000 cells/well. The toxicity assay (MTT) was performed 24 hours after depositing the nanoparticles at different concentrations on the cells. The results are given in FIG. 19. The clearly-defined toxicity can be seen of the nanoparticles loaded with doxorubicin compared with the control nanoparticles (non-loaded).

(58) Finally, we demonstrated in vivo that the formation of the mesoporous silica layer on these persistent luminescence nanoparticles does not prevent excitation of persistent luminescence through the tissues. The picture in FIG. 20 shows the persistent luminescence signal obtained after excitation in the visible range (LED system, see FIG. 2.A) in a mouse injected with the MPLNPs.

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