PARTICULATE STRUCTURES MADE FROM GOLD NANOPARTICLES, METHODS FOR PREPARING SAME AND USES THEREOF FOR TREATING SOLID TUMOURS

Abstract

A particulate structure that includes a/ a biodegradable polymer particle, b/ gold nanoparticles covered on their surface with macrocyclic chelators complexing at least one ion of interest and/or a radionuclide for medical imaging, c/ a polycation having a positive charge over a pH range from 5 to 11, the gold nanoparticles b/ being encapsulated in the polymer particle a/ and/or adsorbed at the surface of the polymer particle a/. Also, a method for preparing the particulate structures. Further, the use of the particulate structures for radiotherapy or chemotherapy in the context of cancer treatment.

Claims

1.-18. (canceled)

19. A particulate structure, comprising: a/ a biodegradable polymer particle, b/ gold nanoparticles covered on their surface with macrocyclic chelating agents complexing at least one ion of interest and/or a radionuclide for medical imaging, c/ a polycation having a positive charge over a pH range from 5 to 11, the gold nanoparticles b/ being encapsulated in the polymer particle a/ and/or adsorbed on the surface of the polymer particle a/.

20. The particulate structure as claimed in claim 19, further comprising a surfactant adsorbed on the surface of the polymer particle a/, said surfactant preferably being polyvinyl alcohol (PVA) and/or a poloxamer.

21. The particulate structure as claimed in claim 19, further comprising at least one active principle encapsulated in the polymer particle a/, said active principle preferably being a chemotherapeutic agent and/or a fluorophor.

22. The particulate structure as claimed in claim 19, wherein the macrocyclic chelating agents that cover the gold nanoparticles each comprise: an anchoring function that comprises at least one sulfur atom for attaching the macrocyclic chelating agent to the gold nanoparticle, and which preferably comprises two sulfur atoms forming an endocyclic disulfide bond, at least one complexation site of ions of interest and/or of radionuclides for medical imaging, said complexation site comprising at least one carboxylic acid function and/or an amine function, a spacer arm located between the anchoring function and the complexation site or sites, optionally a functionalization site allowing grafting of the chelating agent with an agent for targeting cancer cells.

23. The particulate structure as claimed in claim 22, wherein: the anchoring function of the macrocyclic chelating agent is a radical selected from the group comprising: ##STR00002## *—N—(CH.sub.2—CH.sub.2—SH)2, *—C(═O)—(CH.sub.2)n-SH with n being an integer from 2 to 5 and mixtures thereof; the spacer arm of the macrocyclic chelating agent is a radical selected from the group comprising: *—(CH.sub.2)2-CO—NH—(CH.sub.2)2-NH—*, *—NH—(CH.sub.2—CH.sub.2—O)m-CH.sub.2—CH.sub.2—NH—* with m an integer equal to 0, 4 or 11, and mixtures thereof; the functionalization site of the macrocyclic chelating agent, if present, is a radical derived from an amino acid, selected from the group comprising: *—NH—CH((CH.sub.2).sub.4—NH.sub.2)—CO—*, *—NH—CH(CH.sub.2—OH)—CO—*, *—NH—CH(CH—OH—CH.sub.3)—CO—*, *—NH—CH(CH.sub.2—C.sub.6H.sub.4—OH)—CO—*, *—NH—CH((CH.sub.2).sub.n—NH—*)—CO—* with n from 2 to 5, and mixtures thereof.

24. The particulate structure as claimed in claim 19, wherein the macrocyclic chelating agent is selected from the group comprising: TADOTAGA, TANODAGA, TADFO, TA[DOTAGA-lys-NH.sub.2], TA[NODAGA-lys-NH.sub.2], TA[DOTAGA-lys-NODAGA]and mixtures thereof.

25. The particulate structure as claimed in claim 19, wherein: the ion of interest for medical imaging, and more particularly magnetic resonance imaging (MRI), is selected from the group comprising Gd3+, Ho3+, Dy3+ and mixtures thereof; the radionuclide for medical imaging, and more particularly nuclear imaging (SPET or PET), is selected from the group comprising .sup.64Cu, .sup.89Zr, .sup.88Ga, .sup.111In and mixtures thereof.

26. The particulate structure as claimed in claim 19, wherein the polycation is selected from the group comprising polyethyleneimine (PEI), polylysine, polyarginine, polyamidoamine (PANAM), a poly(O-amino ester), chitosan and mixtures thereof, and is preferably polyethyleneimine.

27. The particulate structure as claimed in claim 19, wherein the biodegradable polymer is selected from the group comprising poly(lactic-co-glycolic) acid (PLGA), poly(lactic) acid (PLA), poly(glycolic) acid (PGA), polycaprolactone (PCL), a polyanhydride, the copolymers of each of said polymers with polyethylene glycol (PEG) and mixtures thereof, and is preferably poly(lactic-co-glycolic) acid or [poly(lactic-co-glycolic) acid-polyethylene glycol] copolymer.

28. The particulate structure as claimed in claim 19, wherein the gold nanoparticles b/ are covered on their surface with a macrocyclic chelating agent bound to an active agent targeting the integrins α.sub.Vβ.sub.III overexpressed on the tumor neovasculature, said targeting agent preferably being cyclic RGD peptide.

29. The particulate structure as claimed in claim 19, wherein: the hydrodynamic diameter of the polymer particle a/ is from 50 to 200 nm, preferably from 70 to 160 nm, the hydrodynamic diameter of the gold nanoparticles b/ is from 3 to 15 nm, preferably from 6 to 10 nm.

30. The particulate structure as claimed in claim 19, wherein the gold nanoparticles b/ and optionally the active principle are encapsulated in the polymer particle a/, and said gold nanoparticles b/ may moreover optionally be adsorbed on the surface of the polymer particle a/.

31. The particulate structure as claimed in claim 19, wherein the gold nanoparticles b/ are adsorbed on the surface of the polymer particle a/, and the active principle, if present, is encapsulated in the polymer particle a/.

32. A method for preparing a particulate structure as claimed in claim 19, comprising the following steps: contacting an aqueous suspension of gold nanoparticles b/ with an aqueous solution of polycation, in order to obtain an assembly of gold nanoparticles b/ and polycation; contacting the assembly of gold nanoparticles b/ and polycation as defined in the preceding step with a mixture of biodegradable polymer and water-miscible organic solvent, said organic solvent optionally being mixed beforehand with at least one active principle, in order to obtain a mixture of gold nanoparticles b/, polycation, biodegradable polymer and optionally active principle, contacting the mixture of gold nanoparticles b/, polycation, polymer and optionally active principle as defined in the preceding step, with water, optionally with an added surfactant, in order to precipitate the polymer a/ in the form of particles around the gold nanoparticles b/ and optionally the active principle, the encapsulation yield of the gold nanoparticles b/ and optionally of the active principle in the polymer particles a/ is at least 75%, preferably at least 90%, and even more preferably at least 95%.

33. A method for preparing a particulate structure as claimed in claim 19, comprising the following steps: contacting an aqueous solution of polycation with a mixture of biodegradable polymer and water-miscible organic solvent, said organic solvent optionally being mixed beforehand with at least one active principle, contacting the assembly of polycation with the mixture of biodegradable polymer and organic solvent as defined in the preceding step, with the aqueous suspension of gold nanoparticles b/ in order to obtain a mixture of gold nanoparticles b/, polycation, biodegradable polymer and optionally active principle, contacting the mixture of gold nanoparticles b/, polycation, polymer and optionally active principle as defined in the preceding step, with water, optionally with an added surfactant, in order to precipitate the biodegradable polymer in the form of particles around the gold nanoparticles b/ and optionally the active principle, the encapsulation yield of the gold nanoparticles b/ and optionally of the active principle in the polymer particles a/ is at least 75%, preferably at least 90%, and even more preferably at least 95%.

34. A method for preparing a particulate structure as claimed in claim 19, comprising the following steps: contacting a mixture of biodegradable polymer and water-miscible organic solvent, said organic solvent optionally being mixed beforehand with at least one active principle, with water, optionally with an added surfactant, in order to precipitate the biodegradable polymer in the form of particles, on the surface of which the surfactant is adsorbed if it is present, contacting the polymer particles a/ as defined in the preceding step with an aqueous solution of a polycation, in order to obtain polymer particles a/, on the surface of which the polycation is adsorbed, said biodegradable polymer particles additionally encapsulating the active principle if it is present, contacting the polymer particles a/, on the surface of which the polycation as defined in the preceding step is adsorbed, with an aqueous suspension of gold nanoparticles b/, in order to lead to adsorption of the gold nanoparticles b/ on the surface of the polymer particles a/, the adsorption yield of the gold nanoparticles b/ on the surface of the polymer particle a/ is from 30 to 70%, preferably from 40 to 60%.

35. The method of preparation as claimed in claim 32, wherein: the aqueous solution of gold nanoparticles b/ is at a concentration from 8 to 12 grams of gold nanoparticles per liter of water, the aqueous solution of polycation is at a concentration from 30 to 70 grams of polycation per liter of water, the mixture of biodegradable polymer with the water-miscible organic solvent is at a concentration from 10 to 20 grams of polymer per liter of solvent, said organic solvent is selected from the group comprising dimethylsulfoxide (DMSO), dimethylformamide (DMF) and N-methyl-pyrrolidone, the amount of active principle, if present, in the organic solvent is at a concentration from 0.1 to 0.75 grams of active principle per liter of solvent, the amount of surfactant, if present, in water is from 5 to 10 grams of surfactant per liter of water.

36. A method of treating cancerous solid tumors in a subject, comprising administering to a subject in need thereof a therapeutically effect amount of at least one particulate structure as claimed in claim 19.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0119] Other features, details and advantages will become clearer on reading the following detailed description, and on examining the appended drawings.

[0120] FIG. 1a is a schematic representation of the functionalized gold nanoparticles b/ in which the macrocyclic chelating agents are complexed to ions of interest.

[0121] FIG. 1b is a schematic representation of the functionalized gold nanoparticles b/ in which the macrocyclic chelating agents are complexed to a radionuclide.

[0122] FIG. 1c is a schematic representation of the functionalized gold nanoparticles b/ in which the macrocyclic chelating agents are complexed to ions of interest and a radionuclide.

[0123] FIG. 2a is a schematic representation of a particulate structure of the invention comprising a biodegradable polymer particle a/ in which 100% of the gold nanoparticles b/ are encapsulated. The gold nanoparticles form electrostatic interactions with the polycation.

[0124] FIG. 2b is a schematic representation of a particulate structure of the invention comprising gold nanoparticles b/ encapsulated in the biodegradable polymer particle a/ and adsorbed on the surface of the polymer particle a/.

[0125] FIG. 2c is a schematic representation of a particulate structure of the invention in which 100% of the gold nanoparticles b/ are adsorbed on the surface of the polymer particle a/.

A surfactant, adsorbed on the surface of the polymer particle a/, is represented in each of FIGS. 2a, 2b and 2c. The presence of the latter is optional, however, and each of these figures could also be represented without the surfactant.
The polycation is not shown with its positive charge in each of the particulate structures of the invention, so as not to complicate each of FIGS. 2a, 2b and 2c.

[0126] FIG. 3a is a schematic representation of a particulate structure of the invention corresponding to that in FIG. 2a but which further comprises an active principle encapsulated in the polymer particle a/.

[0127] FIG. 3b is a schematic representation of a particulate structure of the invention corresponding to that in FIG. 2b but which further comprises an active principle encapsulated in the polymer particle a/.

[0128] FIG. 3c is a schematic representation of a particulate structure of the invention corresponding to that in FIG. 2c but which further comprises an active principle encapsulated in the polymer particle a/.

[0129] The active principle is represented by a star in each of FIGS. 3a, 3b and 3c.

[0130] FIG. 4a shows the structural formulas of the macrocyclic chelating agents (L).

[0131] FIG. 4b shows the structural formulas of the polycations.

[0132] FIG. 4c shows the structural formulas of the biodegradable polymers.

[0133] FIG. 5a shows the method for preparing the particulate structures of the invention in which the gold nanoparticles b/ are encapsulated in the polymer particle a/, some of the gold nanoparticles b/ also being adsorbed on the surface of the polymer particle a/ (encapsulation process according to method 2).

[0134] FIG. 5b shows the method for preparing the particulate structures of the invention in which the gold nanoparticles b/ are adsorbed on the surface of the polymer particle a/ (adsorption process).

In the case when an active principle is added, the latter is mixed with the organic solvent and the biodegradable polymer, whether in the encapsulation process or in the adsorption process.
In these figures “Gold Np” denotes the gold nanoparticles.

[0135] FIG. 6 shows a transmission electron micrograph of a particulate structure of the invention, in which we can see a polymer particle a/ comprising several encapsulated and/or adsorbed gold nanoparticles.

[0136] FIG. 7 shows a blood kinetics graph showing the variation of the injected gold dose (as a percentage) per gram of blood as a function of time, for the gold nanoparticles alone (denoted by “Gold Np”), the gold nanoparticles encapsulated in PLGA particles (denoted by “NP3”) or in PLGA-PEG particles (denoted by “NP3-PEG”).

DESCRIPTION OF THE EMBODIMENTS

Examples

[0137] Preparation of particulate structures according to the invention in which: [0138] the biodegradable polymer a/ is poly(lactic-co-glycolic) acid (PLGA) or a conjugate of poly(lactic-co-glycolic) acid and polyethylene glycol (PLGA-PEG), [0139] the macrocyclic chelating agent is TADOTAGA and the ion of interest is gadolinium (Gd3+), [0140] the polycation is polyethyleneimine (PEI).
The surfactant is polyvinyl alcohol (PVA) and the water-miscible organic solvent is dimethylsulfoxide (DMSO).
The gold nanoparticles b/, covered on their surface with the chelating agent TADOTAGA complexing the gadolinium ion, are represented hereinafter by:

“Au@TADOTAGA(Gd)”.

[0141] Materials

[0142] More particularly, PLGA 50:50 (MW 7000-17000 Da) (marketed under the name Resomer® RG 502H) is obtained from Evonik Industries (Evonik Röhm GmbH) and PLGA-PEG 50:50 (PLGA: MW 25000 Da, PEG: MW 5000 Da) is obtained from Sigma Aldrich (St Louis, United States).

Chloroauric acid (HAuCl.sub.4.3H.sub.2O), sodium borohydride (NaBH.sub.4), PVA (MW 30000-70000 Da), branched polyethyleneimine (PEI) (MW 25000 Da), gadolinium chloride (GdCl.sub.3.6H.sub.2O) and dimethylsulfoxide (DMSO) are obtained from Sigma Aldrich (Saint Louis, United States). The ligand TADOTAGA is obtained from Chematech (Dijon, France).

[0143] Synthesis of the Au@TADOTAGA(Gd) Nanoparticles

[0144] Synthesis of the gold nanoparticles is adapted from the single-phase protocol developed by Brust et al. (6). The gold nanoparticles are obtained by reduction of the gold salt (HAuCl.sub.4.3H.sub.2O) with NaBH.sub.4 in the presence of the ligand TADOTAGA. Adsorption of TADOTAGA on the surface of the gold nanoparticles makes it possible to control the size and colloidal stability and allows immobilization of the gadolinium. More particularly, HAuCl.sub.4.3H.sub.2O (50 mg, 1.22×10−4 mol), dissolved in methanol (20 mL), is placed in a 250-mL round-bottomed flask. The ligand TADOTAGA (86 mg, 1.22×10.sup.−4 mol) in water (10 mL) is added to the solution of gold salt, with stirring. The mixture changes from yellow to orange. After some minutes, NaBH.sub.4 (48 mg, 12.7×10−4 mol) dissolved in water (3 mL) is added to the mixture while stirring vigorously at room temperature. Stirring is maintained for 1 h. Then the mixture is dialyzed using a 6000-8000 kDa MWCO membrane.

[0145] To obtain the Au@TADOTAGA(Gd) final suspension ([Au]=51 mM, [Gd]=5 mM) before the process of encapsulation in the polymer particles, the gold suspension is concentrated and the gadolinium is trapped in the TADOTAGA chelating agent, stirring the suspension overnight with GdCl.sub.3.6H.sub.2O (370 μL at 135 mM for an Au@TADOTAGA(Gd) suspension at 10 mL). A gadolinium concentration of 5 mM guarantees stability of the suspension and an optimal MRI signal.

[0146] Synthesis of the PLGA or PLGA-PEG Polymer Particles Encapsulatinq Au@TADOTAGA(Gd)

[0147] The method for preparing the polymer particles encapsulating the gold nanoparticles b/ (Au@TADOTAGA(Gd)) is based on the method of nanoprecipitation by solvent displacement (13), but with the novel feature of using PEI. The inventors found that the size of the polymer particles can be modulated as a function of the PEI/ gold ratio and of the pH of the aqueous solution of PEI.

[0148] The inventors found in particular in the course of their research that a PEI/ gold ratio of 5 and a pH of about 10.8 were suitable for obtaining polymer particles having a hydrodynamic diameter of about 160 nm. In fact, a size of 160 nm±15 nm is advantageous in that it makes it possible to encapsulate a satisfactory amount of gold nanoparticles b/ while allowing a satisfactory manufacturing yield.

[0149] An aqueous solution of PEI (25 μL, 5% w/w) is mixed with 1 mL of solution of PLGA or of solution of PLGA-PEG in DMSO at 15 mg/mL and 18 mg/mL respectively.

[0150] 1 N HCl is added to the aqueous solution of PEI beforehand in order to obtain a hydrodynamic diameter of the PLGA particles close to 160 nm±15 nm.

[0151] To modulate the PEI/ gold ratio for preparing different particles, only the concentration of the PEI is adjusted. The same volume of HCl is added to the solution as for preparation of the PLGA particles with a diameter of about 160 nm, independently of the concentration of PEI.

[0152] A suspension of Au@TADOTAGA(Gd) (25 μL, 10 mg/mL (i.e. 51 mM)) is added to the preceding solution comprising PEI and PLGA.

[0153] Then 4 mL of PVA dissolved in water at 0.75% is added gradually to the mixture, vortexed beforehand.

[0154] For preparing the PLGA particles by adsorption of the gold nanoparticles, the PLGA particles are formed beforehand according to the same protocol as the conventional PLGA particles.

[0155] Then a 5% solution of PEI (25 μL) is transferred to the suspension of PLGA particles with stirring. After incubation for 5 minutes, a suspension of Au@TADOTAGA(Gd) (25 μL, 10 mg/mL (i.e. 51 mM)) is finally added to the PLGA particles coated with PEI.

[0156] The various preparations are washed three times by ultracentrifugation at 30 000 g for 1 h, at 4° C. to remove the free gold nanoparticles. Finally the preparations are lyophilized using sucrose as cryoprotective, except in the batches used for determining the production yield, encapsulation yield and encapsulation rate.

[0157] These parameters are determined as follows:

[00001]$\begin{array}{cc}\mathrm{Production}\mathrm{yield}\left(\%\right)=\frac{\mathrm{Quantity}\mathrm{of}\mathrm{PLGA}\mathrm{particles}\mathrm{formed}}{\mathrm{Quantity}\mathrm{of}\mathrm{PLGA}\mathrm{used}}\times 100& \left(1\right)\end{array}$$\begin{array}{cc}\mathrm{Encapsulation}\mathrm{yield}\left(\%\right)=\frac{\mathrm{Quantity}\mathrm{of}\mathrm{gold}\mathrm{encapsulated}\mathrm{and}\mathrm{optionally}\mathrm{adsorbed}}{\mathrm{Quanitity}\mathrm{of}\mathrm{gold}\mathrm{used}}\times 100& \left(2\right)\end{array}$$\begin{array}{cc}\mathrm{Encapsulation}\mathrm{rate}\left(\%\right)=\frac{\mathrm{Quantity}\mathrm{of}\mathrm{gold}\mathrm{encapsulated}\mathrm{and}\mathrm{optionally}\mathrm{adsorbed}}{\mathrm{Quantity}\mathrm{of}\mathrm{PLGA}\mathrm{particles}\mathrm{formed}}\times 100& \left(3\right)\end{array}$

[0158] The various characteristics of the particles obtained in accordance with this protocol by varying the PEI/Gold ratio are described in Table 1 hereunder:

TABLE-US-00001 TABLE 1 NP3 Formulation NP1 NP2 NP3 NP4 adsorbed NP3-PEG PEI/Gold 0 6 5 4 5 5 ratio Hydrodynamic 136 ± 4  135 ± 19  159 ± 14 196 ± 11 153 ± 3  198 ± 5  diameter (nm) Polydispersity 0.05 ± 0.02 0.16 ± 0.03  0.16 ± 0.01 0.017 ± 0.03 0.007 ± 0.01 0.17 ± 0.03 index Production 35 ± 5  54 ± 9  71 ± 7 82 ± 6 64 ± 3 54 ± 5  yield Encapsulation 2 ± 2 102 ± 5  95 ± 8 88 ± 7 52 ± 7 86 ± 6  yield Encapsulation 0.0 ± 0.0 1.5 ± 0.1  1.4 ± 0.2  1.3 ± 0.1  0.7 ± 0.1 1.1 ± 0.0 rate

[0159] We thus obtain particles having a hydrodynamic diameter from 130 nm to 200 nm (their size may be reduced further by adjusting the PEI/ gold ratio) with an encapsulation rate of about 1.4. The reduction in size leads inevitably to a reduction in production yield owing to the washing by centrifugation.

[0160] The NP3 particles (PEI/ gold ratio of 5) are selected for the tests in vivo. These particles represent a good compromise between size and production yield. The encapsulation rate is half as much in the case of the adsorption protocol (NP3 adsorbed) than the encapsulation protocol (NP3), which does indeed indicate encapsulation of the gold nanoparticles. The presence of gold is confirmed by imaging by transmission electron microscopy (see FIG. 6).

[0161] Image-Guided Therapy

[0162] The particulate structures of the invention are promising candidates for image-guided therapy if they display suitable behavior after intravenous injection: accumulation in the zone to be treated, absence of nanoparticles in the surrounding healthy tissues, preferential renal elimination (relative to the hepatobiliary route) and if the plasma half-life is increased relative to the gold nanoparticles.

[0163] Thus, a blood kinetic study was carried out on rats by injecting 500 μL of the NP3 suspension (or NP3-PEG) at 100 mg/mL in PLGA or an equivalent amount of gold of gold nanoparticles “alone” (Gold Np) by the intravenous route (penile vein) after isoflurane anesthesia. A blood sample was taken from the tail at different times and then the amount of gold present in the samples was measured by atomic absorption spectroscopy.

[0164] The results obtained are shown in FIG. 7.

CONCLUSION

[0165] Encapsulation, whether carried out with PLGA or PLGA-PEG, increases the plasma half-life of the gold nanoparticles.

The encapsulation process of the invention advantageously allows encapsulation of gold nanoparticles within particles of reduced size (between 100 and 200 nm) with a yield close to 100% while maintaining a low polydispersity index. The particulate structure thus obtained makes it possible to increase the plasma half-life of the gold nanoparticles, and therefore has considerable, promising potential for improving the therapeutic effect of said gold nanoparticles.

[0166] The present invention is not limited to the examples described in the foregoing, only as examples, but includes all the variants that a person skilled in the art might envisage within the scope of protection sought.

LIST OF DOCUMENTS CITED

Nonpatent Literature

[0167] To all intents and purposes, the following nonpatent elements are cited: [0168] (1) J. F. Hainfeld et al., Phys. Med. Biol., 49 (2004) N309-315; [0169] (2) Gautier Laurent et al, Nanoscale, 8(2016) 12054-65; [0170] (3) A. M. Gobin et al, Nano Lett., 7 (2007) 1929-1934; [0171] (4) J. F. Hainfeld et al, Br. J. Radiol., 79 (2006) 248-253; [0172] (5) K. T. Butterworth et al, Nanoscale, 4 (2012) 4830-4838; [0173] (6) M. Brust et al, J. Chem. Soc. Chem, Commun., (1995) 0, 1655-1656; [0174] (7) P. C. S. John Turkevich, Discuss Faraday Soc, 11 (n.d.) 55-75; [0175] (8) Thesis of G. Laurent, Synthesis of multifunctional nanoparticles for image-guided radiotherapy. Organic chemistry. Franche-Comté University, 2014; [0176] (9) T. Butterworth et al., Nanoscale, 2012; 4, 4830-4838; [0177] (10) M. Yu et al., ACS nano, 2015, 9, 6655-6674; [0178] (11) Wang Y et al., Biomed Opt Express, 2016, 7, 4125-4138; [0179] (12) Luque-Michel et al., Nanoscale, 2016, 8, 6495-6506; [0180] (13) H. Fessi et al., International Journal of Pharmaceutics, 1989, 55, R1-R4.