Surface-modified gadolinium terbium fluoride nanoparticles: their preparation and use in imaging and tumor treatment
Surface-modified gadolinium and terbium fluoride nanoparticles with biocompatibility ligands and photosensitizers address aggregation issues, enhancing stability and dispersibility for effective tumor imaging and treatment by X-ray excitation.
Patent Information
- Application Number
- FR2024008117
- Authority / Receiving Office
- FR · FR
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2024-07-23
- Publication Date
- 2026-01-30
AI Technical Summary
Existing gadolinium and terbium fluoride nanoparticles used in photodynamic therapy face challenges with aggregation and settling, necessitating high concentrations to be effective, which compromises stability and dispersibility.
Surface-modified gadolinium and terbium fluoride nanoparticles with biocompatibility ligands and photosensitizers, specifically PEG-phosphonate and PEG-silane, are prepared to enhance stability and dispersibility, allowing efficient energy transfer and ROS generation under X-ray excitation.
The modified nanoparticles exhibit improved stability and dispersibility, enabling efficient ROS generation and tumor imaging/treatment with enhanced X-ray doses, outperforming previous nanoparticle technologies in stability and size.
Smart Images
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Abstract
Description
Title of the invention: Surface-modified gadolinium and terbium fluoride nanoparticles, their preparation and use in imaging and tumor treatment
[0001] The present invention relates to gadolinium and terbium fluoride nanoparticles, surface modified by at least one biocompatibility ligand and by at least one photosensitizer, as well as their preparation process and their use in imaging and treatment of a tumor.
[0002] Zhang et al. Bioconjugate Chem. 2019, 30, 2191-2200 described terbium-doped mixed gadolinium and sodium fluoride nanoparticles with a rose Bengal photosensitizer for use in X-ray excitation photodynamic therapy.
[0003] For such particles to be used as effectively as possible in therapy, their concentration in the solution to be injected into the patient must be as high as possible. It is therefore important to obtain a stable suspension without the nanoparticles aggregating and settling within it.
[0004] For this purpose, the present invention relates to GdaTbbF3 nanoparticles, where a = 0.50 to 0.95, b = 0.05 to 0.50 and a+b = 1, modified on the surface by at least one biocompatibility ligand and by at least one photosensitizer absorbing in the wavelength band of 520 to 560 nm under excitation of the nanoparticles by X-rays.
[0005] When the nanoparticles are subjected to X-rays, the organic core of gadolinium fluoride and terbium will emit scintillation radiation in response. This scintillation radiation will then be absorbed by the photosensitizer, which will subsequently cause the generation of reactive oxygen species (ROS).
[0006] In particular, a = 0.80 to 0.95, b = 0.05 to 0.20, in particular a = 0.90 and b = 0.10.
[0007] A biocompatibility ligand may be chosen from: • polymers whose chain is chosen from: r, with n = 10 to 80, in particular 19 or 20; , with p = 6 to 50, , with q = 5 to 25, in particular 25, and R representing hydrogen or a alkyl radical in Ci-C3, such as methyl, ethyl or propyl, r -v , with r = 8 to 60,
[0008] and which carries a functionality allowing its direct or indirect grafting onto GdaTbbF3 nanoparticles; • bovine serum albumin; • sodium triphosphate (TPP) with the formula
[0009]
[0010]
[0011]
[0012]
[0013]
[0014] In one particular embodiment, the photosensitizer is rose bengal. In another particular embodiment, the photosensitizer is merocyanine 540, which absorbs in the green. The nanoparticles according to the invention can be modified on the surface both by grafting a PEG-phosphonate as a biocompatibility ligand of formula: in which m = 10 to 80, in particular 19 or 20, and by grafting aminomethylphosphonic acid (AMPA), rose bengal being grafted by its reaction with an available function, such as an amine function, of PAMPA. The molar ratio of PEG-phosphonate to PAMPA can range from 20:80 to 90:10, and particularly from 50:50 to 80:20. The nanoparticles according to the invention can be surrounded by a silica layer in which the photosensitizer(s) is / are trapped, and then grafted onto said silica layer by a PEG-silane as a biocompatibility ligand, PEG-silane being represented by the formula:
[0015] in which u = 20 to 50, in particular 45.
[0016] The present invention also relates to a method for preparing nanoparticles having a PEG-phosphonate as a biocompatibility ligand as defined above, characterized in that it comprises the following steps: • heat for 1-24h, at a temperature of 25 to 80°C, a mixture in aqueous medium of PEG-phosphonate and AMPA, and add GdaTbbF3 nanoparticles to obtain an aqueous suspension of these nanoparticles modified on the surface by PEG-phosphonate and AMPA; • Add to this suspension rose bengal in solution in an organic solvent, and react for 1–24 hours at a temperature of 25 °C; and • purify the obtained nanoparticles by dialysis, and if necessary, freeze-dry them.
[0017] The present invention also relates to a method for preparing nanoparticles containing PEG-silane as a biocompatibility ligand as defined above, characterized in that it comprises the following steps: • add sodium silicate in aqueous solution to a suspension of GdaTbbF3 nanoparticles and then heat at a temperature of 25 to 80°C for 1 to 24 h to obtain a suspension of GdaTbbF3 particles modified by sodium silicate; • disperse these particles in a water / alcohol / photosensitizer(s) mixture, successive additions of an ethanolic solution of tetraethyl orthosilicate (TEOS) being carried out to lead to GdaTbbF 3 particles coated with a layer of silica trapping the photosensitizer(s); • modify the surface of the particles thus obtained by grafting PEG chains through the addition of an ethanolic solution of PEG-silane in a basic medium to obtain the desired nanoparticles; and • purify the obtained nanoparticles by dialysis, and if necessary, freeze-dry them.
[0018] The present invention also relates to the nanoparticles defined above for their use in a method of imaging a tumor or of imaging a tumor followed by treatment thereof.
[0019] In particular, it is possible to follow the following steps using the nanoparticles according to the invention: • inject nanoparticles into a patient in need; • place the patient in an X-ray machine; • subject the patient to an initial dose of X-rays, to perform a imaging of a tumor; • subject the patient to a second dose of X-rays, of higher intensity than the first dose, to treat the tumor; • subject the patient to the first dose of X-rays again to perform imaging of the tumor.
[0020] The following examples illustrate the present invention without however limiting its scope.
[0021] In these Examples, the following abbreviations have been used:
[0022] NP: nanoparticles of Gdo, 9oTbo, ioF3
[0023] PEG-phosphonate: j- x \ J ® *
[0024] with n = 19-20, Me = methyl, and M = H, methyl or ethyl
[0025] AMPA: aminomethylphosphonic acid
[0026] RB: Bengal rose
[0027] PEG-silane: _
[0028] with p = 20-50, Me = methyl, Et = ethyl
[0029] TEOS: tetraethyl orthosilicate
[0030] NP@xPEG-phosphonate / y AMPA: NP nanoparticles modified on the surface by grafting PEG-phosphonate and AMPA in a molar ratio x / y, x+y = 100.
[0031] NP@xPEG-phosphonate / y AMPA @ RB: NP@xPEG-phosphonate / y AMPA nanoparticles with RB grafted onto AMPA
[0032] DMSO: dimethyl sulfoxide
[0033] EDC: l-ethyl-3-(3-dimethylaminopropyl)carbodiimide
[0034] NP@ silicate: NPs functionalized by silicate
[0035] NP@SiO2@RB: NP@ silicate having reacted with RB
[0036] NP@SiO2@RB@PEG-silane: NP@SiO2@RB grafted by PEG-silane
[0037] The following examples illustrate the present invention without however limiting its scope.
[0038] Example 1: Preparation of an aqueous suspension of NP nanocrystals
[0039] 3.64 g of GdCl3·6H2O (9.8 mmol) and 0.41 g of TbCl3·6H2O (1.1 mmol) were dissolved in 5g of ethylene glycol. The resulting solution was added to a 100mL Teflon insert.
[0040] In parallel, 875 µl of an aqueous solution of 50% by mass of hydrofluoric acid (25 mmol) were added to a container containing 20 g of pyrrolidinone.
[0041] The resulting solution was slowly added to the Teflon insert containing the salts and the container was rinsed with 45g of pyrrolidinone.
[0042] The mixture was then heated in an autoclave at 170°C for 1h30.
[0043] The product obtained was treated with 100 mL of acetone to precipitate the nanoparticles, which were recovered by centrifugation.
[0044] The nanoparticles thus recovered were purified by centrifugation-redispersion cycles in methanol, then redispersed in ultrapure water.
[0045] The mass fraction of the aqueous suspension is approximately 15% by mass of NP.
[0046] An aqueous suspension of 10% by mass of NP was obtained.
[0047] Examples 2 to 4: Obtaining NP@xPEG-phosphonate / yAMPA@RB General operating procedure
[0048] First step: Obtaining NP@xPEG-phosphonate / yAMPA
[0049] A mg of AMPA-HCl and B mg of PEG-phosphonate were introduced into 20 mL of water. To this mixture, 25 mL of the aqueous suspension obtained in Example 1 (at 1.2 x 10² mol of Gd+Tb) was added dropwise. This mixture was then heated at 80°C with stirring for 2 hours. The nanoparticles referred to in the title of this step were purified by dialysis.
[0050] Second step: Obtaining the NPs from the titles of these Examples 2 to 4
[0051] 50 mL of an aqueous suspension of NP obtained in the first step was sonicated for 10 minutes. 5 mL of a solution of RB (10 mg / mL) in DMSO were added to this suspension.
[0052] After a few minutes of stirring, 570 µL of EDC were added dropwise to the reaction mixture, which became cloudy. After stirring at room temperature for about ten minutes, the mixture became clear again.
[0053] The reaction medium was left under stirring at room temperature for 24 hours.
[0054] The NPs obtained were then purified by dialysis until the dialysis waters remained colorless, then they were lyophilized and stored in the form of a powdery pink solid.
[0055] In the following Table 1, the parameters A and B (see first step) which led to the NPs of the title are indicated with x and y values.
[0056] During the synthesis of NP@PEG, the molar proportions of ligands are such that there are 3 moles of lanthanide per ligand, i.e., 0.3 ligand equivalents. When typically using 50%PEG and 50%EMPA, this means that 0.15 equivalents of PEG and 0.15 equivalents of AMPA will be introduced.
[0057] [Tables 1] Example A (mg) B (mg) 3mol) 80 20
[0058] Example 5: Preparation of NP@SiO2 @RB @PEG-silane
[0059] First step: Obtaining NP@ silicate
[0060] 50g of an aqueous suspension obtained in Example 1 (with 1.39x103 mol of Gd+Tb) have were placed under ultrasound.
[0061] Under ultrasound, 20.8 mL of an aqueous sodium silicate solution with 3% by mass of SiO2 were added (1.35x10 2mol of Si).
[0062] The mixture became viscous white, then, as sonication progressed, became a stable colloidal suspension with a whitish tint.
[0063] The mixture was then heated for 1h at 80°C, and then purified by dialysis. 100 pL of ammonia at 30% by mass in water was then added to maintain the suspension's stability over time.
[0064] Second step: Obtaining NP@SiO2 @RB
[0065] 407.2 mg of RB were dissolved in a mixture containing 8.4 mL of water, 3.15 mL of absolute ethanol and 114 pL of ammonia at 30% by mass in water.
[0066] After sonication, 14.1 mL of an aqueous suspension of nanoparticles obtained in the first step (71 mg / mL) were added to the mixture.
[0067] A solution of TEOS in absolute ethanol was prepared by adding 841.6 pL of TEOS to 1248 pL of absolute ethanol. Every hour, for 4 h, 521.6 pL of this solution were added to the reaction medium.
[0068] The NPs obtained were then purified by dialysis for 48 hours, with a pink color appearing in the dialysis waters.
[0069] Third step: Obtaining NP@SiO2 @RB @PEG-silane
[0070] To 20.8 mL of the suspension obtained in the second step (25 mg / mL of NP), 150 pL of ammonia at 30% by mass in water was added. Then, 1.52 mL of an ethanolic solution of PEG-silane was added dropwise under vigorous stirring.
[0071] The medium was left under agitation for 48 hours at room temperature, protected from light.
[0072] The final suspension was purified by dialysis until the dialysis waters were no longer colored. The NPs were then lyophilized and stored as a powdered solid protected from light.
[0073] Example 6: X-ray irradiation
[0074] The scintillation of RB-functionalized NP suspensions was studied by X-ray excitation of a micro-computed tomography (micro-CT) system.
[0075] Fig. 1 shows the results obtained for an aqueous suspension of 0.1 mol / L of Gdo.9Tbo.1F3 (column A), and an aqueous suspension of 0.1 mol / L of nanoparticles from Example 2 (column B), Example 3 (column C) and Example 4 (column D).
[0076] The luminescence of Tb3+ under X-ray radiation is not detected for the aqueous suspensions of Examples 2 to 4. This confirms an efficient energy transfer between Tb3+ and RB present on the surface.
[0077] Fig. 2 shows the results obtained for an aqueous suspension of 0.1 mol / L of Gdo.9Tbo.1F3 (column A), and an aqueous suspension of 0.1 mol / L of nanoparticles of Example 5 (column B).
[0078] The nanoparticle suspension of Example 5 still exhibits scintillation. However, the intensity of this scintillation is significantly lower than that of the Gdo.9Tbo.1F3 nanoparticles, indicating energy transfer between Tb3+ and RB.
[0079] Example 7: Stability of nanoparticles
[0080] A chemical stability test (solubilization) was carried out in water for 3 days at 37°C to compare the stabilities of the nanoparticles of Example 1 with those of the nanoparticles described by Zhang et al. After removal of the remaining nanoparticles, the liquid media were analyzed by ICP to quantify the main rare earth elements. Figure 3 shows the concentrations obtained.
[0081] Compared to the terbium-doped mixed gadolinium and sodium fluoride nanoparticles described by Zhang et al., the terbium-doped gadolinium fluoride nanoparticles are extremely stable in dilute media.
[0082] The nanoparticles of Examples 2 to 4 have a hydrodynamic diameter in the range of 20 to 25 nm and the nanoparticles of Example 5 have a hydrodynamic diameter of 50 nm, as measured by dynamic light scattering. The particles described by Zhang et al. have a hydrodynamic diameter well above 100 nm.
[0083] Compared to the particles described by Zhang et al., the nanoparticles according to the invention have a smaller size and much better dispersibility.
[0084] Example 8: X-ray doses used
[0085] The nanoparticles according to the invention are capable of generating reactive oxidizing species (ROS) under X-ray radiation.
[0086] The generation efficiency of these species is measured by the decomposition of 1,3-diphenylisobenzofuran (DPBF). The protocol used is as follows: 21 qg of DPBF are solubilized with a suspension of 4.7 mg of nanoparticles from each of Examples 2 to 5 in 2 mL of a suitable solvent and placed in a 10 mm x 10 mm quartz cuvette. The degradation of DPBF is measured by absorbance. The results are shown in [Fig. 4].
[0087] Upon X-ray irradiation, the nanoparticles of Example 5 are capable of decomposing all of the DPBF (21 qg) after 40 seconds. Zhang et al. present nanoparticles requiring an irradiation time of more than one minute for a similar result in the best-case scenario. Similarly, after 40 seconds of irradiation, the nanoparticles described by Zhang et al. degraded barely 10 qg of DPBF.
Claims
Demands
1. - GdaTbbF3 nanoparticles, where a = 0.50 to 0.95, b = 0.05 to 0.50 and a+b = 1, surface modified by at least one biocompatibility ligand and by at least one photosensitizer absorbing in the wavelength band of 520 to 560 nm under X-ray excitation of the nanoparticles.
2. - Nanoparticles according to claim 1, characterized by the fact that a = 0.80 to 0.95, b = 0.05 to 0.20, in particular a = 0.90 and b = 0.
10.
3. - Nanoparticles according to any one of claims 1 and 2, characterized in that a biocompatibility ligand is selected from: • polymers whose chain is selected from: r_, with n = 10 to 80, in particular 19 or 20; oj, with p = 6 to 50; r, with q = 5 to 25, in particular 25, and R representing hydrogen or a C1-C3 alkyl radical, such as methyl, ethyl, or propyl; r'y., with r = 8 to 60; and which carries a functionality allowing its direct or indirect grafting onto GdaTbbF3 nanoparticles; • bovine serum albumin; • sodium triphosphate (TPP) of formula
4.
5.
6. - Nanoparticles according to any one of claims 1 to 3, characterized by the fact that the photosensitizer is rose bengal. - Nanoparticles according to any one of claims 1 to 3, characterized in that the photosensitizer is merocyanine 540 which absorbs in the green. - Nanoparticles according to any one of claims 3 and 4, characterized in that they consist of GdaTbbF3 nanoparticles modified on the surface by grafting a PEG-phosphonate as a biocompatibility ligand of formula:
7.
8. in which m = 10 to 80, in particular 19 or 20, and by grafting aminomethylphosphonic acid (AMPA), rose bengal being grafted by its reaction with an available function, such as an amine function, of PAMPA. - Nanoparticles according to claim 6, characterized by the fact that the molar ratio of PEG-phosphonate to PAMPA is 20:80 to 90:10, in particular 50:50 to 80:
20. - Nanoparticles according to any one of claims 1 to 5, characterized in that they consist of GdaTbbF 3 nanoparticles surrounded by a silica layer in which the photosensitizer(s) is / are trapped, and then grafted onto said silica layer by a PEG-silane as a biocompatibility ligand, PEG-silane being represented by the formula: in which u = 20 to 50, in particular 45.
9. - A process for preparing nanoparticles as defined in any one of claims 6 and 7, characterized in that it comprises the following steps: • heating for 1-24 h, at a temperature of 25 to 80°C, a mixture in aqueous medium of PEG-phosphonate and AMPA, and adding GdaTbbF3 nanoparticles to obtain an aqueous suspension of these nanoparticles modified on the surface by PEG-phosphonate and AMPA; • adding to this suspension rose bengal in solution in an organic solvent, and reacting for 1-24 h, at a temperature of 25°C; and • purifying the nanoparticles obtained by dialysis, and where appropriate lyophilizing them.
10. - A method for preparing nanoparticles as defined in claim 8, characterized in that it comprises the following steps: • adding sodium silicate in aqueous solution to a suspension of GdaTbbF3 nanoparticles and then heating at a temperature of 25 to 80°C for 1 to 24 hours to obtain a suspension of GdaTbbF3 particles modified with sodium silicate; • dispersing these particles in a water / alcohol / photosensitizer(s) mixture, successive additions of an ethanolic solution of tetraethyl orthosilicate (TEOS) being made to produce GdaTbbF3 particles coated with a silica layer trapping the photosensitizer(s); • modifying the surface of the particles thus obtained by grafting PEG chains by adding an ethanolic solution of PEG-silane in a basic medium to obtain the desired nanoparticles;and • purify the nanoparticles obtained by dialysis, and if necessary, freeze-dry them.
11. - Nanoparticles according to any one of claims 1 to 7 for their use in a method of imaging a tumor or of imaging a tumor followed by treatment thereof.