Nanoparticles comprising at least one metal salt and at least one peptide

Abstract

The present invention relates to nanoparticles comprising:-an association of at least one metal salt and at least one peptide; and-at least one biocompatible polymer.

Description

Field of the Invention

[0001] The present invention relates to nanoparticles and their use as pharmaceuticals or diagnostic agents. The present invention also relates to a method for preparing nanoparticles. Technical Background

[0002] Hybrid nanoparticles that combine metal moieties with biocompatible polymers and therapeutic molecules are promising nanomedicines and theranostic agents, particularly for cancer treatment. These nanoparticles can both transport therapeutic molecules to target cells and, due to their metal moieties, can serve as photothermal therapeutic agents and / or in vivo imaging markers.

[0003] Thus, Emami et al. (2019) Mol.Pharmaceutics 16:1184-1199 describe the use of hybrid nanoparticles that combine gold nanoparticles, biocompatible polymers, polyethylene glycol (PEG), therapeutic molecules, doxorubicin, and anti-PD-L1 antibodies to target colorectal cancer cells that overexpress PD-L1, thereby destroying cells of the CT-26 colorectal cancer cell line. The authors showed that these nanoparticles promoted the entry of doxorubicin into CT-26 cells, of which 66% underwent apoptosis, and that doxorubicin treatment combined with near-infrared irradiation significantly and synergistically eliminated the in vitro proliferation of CT-26 cells by enhancing apoptosis and cell cycle arrest. However, the photothermal efficacy of these nanoparticles was not optimal. In fact, the same nanoparticles without doxorubicin only induced about 20% cell death after near-infrared irradiation. In addition, the combination of doxorubicin and near-infrared irradiation only reduced the survival rate of CT-26 cells from about 20% to about 10% compared to the same treatment without irradiation.

[0004] Therefore, the full therapeutic potential of this type of hybrid nanoparticles has not yet been realized, and it would be useful to have hybrid nanoparticles that can deliver therapeutic molecules with enhanced photothermal therapeutic properties. SUMMARY OF THE INVENTION

[0006] The present invention arises from the unexpected discovery of the inventors that hybrid nanoparticles (NFL-BIOTINE-PEG-AuNPs, TAT-BIOTINE-PEG-AuNPs, VIM-BIOTINE-PEG-AuNPs) obtained by chelating chloroauric acid with a cell-penetrating peptide (particularly one of the NFL, TAT, or VIM peptides) functionalized with polyethylene glycol end-capped with biotin and a dicarboxylic acid exhibit high stability and biocompatibility and are particularly suitable for cancer treatment.

[0007] Advantageously, the process for obtaining the nanoparticles according to the present invention, in particular NFL-BIOTINE-PEG-AuNP, TAT-BIOTINE-PEG-AuNP and VIM-BIOTINE-PEG-AuNP nanoparticles, is a simple process comprising a few steps.

[0008] The inventors have demonstrated that NFL-BIOTINE-PEG-AuNP hybrid nanoparticles at concentrations of 500 μmol / L and 1000 μmol / L significantly reduced the cell viability of MiaPaCa-2 cells (a human pancreatic cancer cell line) and F98 cells (a rat glioblastoma cell line). 100 μmol / L NFL-BIOTINE-PEG-AuNP hybrid nanoparticles also significantly reduced the cell viability of irradiated MiaPaCa-2 cells and F98 cells after nanoparticle administration.

[0009] The inventors also demonstrated that TAT-BIOTINE-PEG-AuNP and VIM-BIOTINE-PEG-AuNP hybrid nanoparticles significantly reduced the cell viability of F98 cells (a rat glioblastoma cell line).

[0010] Therefore, the present invention relates to nanoparticles comprising:

[0011] - an association of at least one metal salt and at least one peptide; and

[0012] - At least one biocompatible polymer.

[0013] The present invention also relates to nanoparticles as defined above for use as pharmaceutical and / or diagnostic agents.

[0014] The present invention also relates to a pharmaceutical composition comprising as active ingredient at least one nanoparticle as defined above, optionally in combination with a pharmaceutically acceptable carrier.

[0015] The present invention also relates to a diagnostic composition comprising at least one nanoparticle as defined above.

[0016] The present invention also relates to a medical device comprising at least one nanoparticle as defined above.

[0017] The present invention also relates to a method for preparing nanoparticles, the nanoparticles comprising:

[0018] - an association of at least one metal salt and at least one peptide; and

[0019] - at least one biocompatible polymer,

[0020] The method includes:

[0021] - a step wherein the metal salt is chelated to the peptide;

[0022] - a step of mixing with said biocompatible polymer; and

[0023] - a step of reducing said mixture with a reducing agent. Detailed Description of the Invention

[0025] As used herein, the term "comprising" is synonymous with "including," "containing," or "encompassing," i.e., when an object "comprising" one or more elements, elements other than those mentioned may also be included in the object. In contrast, "consisting of" means "composed of," i.e., when an object "consists of one or more elements," the object may not include elements other than those mentioned.

[0026] As used herein, "biocompatibility" in the sense of the present invention refers to the ability of a material to be compatible with biological media (particularly tissues or fluids of a living body). Thus, biocompatible materials in the sense of the present invention can be used in pharmaceutical compositions, diagnostic compositions, and medical devices, for example, for diagnostic or therapeutic purposes.

[0027] As used herein, "biopolymers" or "natural polymers" in the sense of the present invention are polymers that can be produced by living organisms, such as animals, plants or fungi.

[0028] Metal salts

[0029] For the purposes of the present invention, metal salts are defined as metal oxides and salts produced by the action of an acid on a metal. Preferably, the metal salt according to the present invention originates from the action of an acid on a metal.

[0030] Also preferably, the metal salt according to the present invention is selected from transition metal salts, lanthanide salts, alkaline earth metal salts and mixtures thereof.

[0031] More preferably, the metal salt according to the present invention is selected from gold (Au), selenium (Se), gadolinium (Gd), cobalt (Co), europium (Eu), terbium (Tb), cerium (Ce), manganese (Mn), iron (Fe), zinc (Zn) and copper (Cu).

[0032] Also preferably, the metal salt according to the present invention is an inorganic acid metal salt.Examples of the inorganic acid metal salt according to the present invention include sulfates, chlorides, nitrates, phosphates and carbonates.

[0033] More preferably, the metal salt according to the present invention is chloroauric acid HAuCl4.

[0034] According to a particular embodiment of the present invention, the metal salt according to the invention is not an iron salt or an iron oxide.

[0035] peptides

[0036] Preferably, the peptide according to the present invention is an isolated amino acid sequence comprising no more than 100 amino acids. Also preferably, the peptide according to the present invention comprises 2 to 100 amino acids, such as 2 to 90, 2 to 80, 2 to 70, 2 to 60, 2 to 50 and 2 to 40 amino acids. Preferably, the peptide according to the present invention comprises at least 2 amino acids, such as at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 and at least 10 amino acids. More preferably, the peptide according to the present invention comprises 2 to 40 amino acids, such as 10 to 40 amino acids or 10 to 30 amino acids.

[0037] The peptides according to the present invention may be selected from any biocompatible peptides known to those skilled in the art.Preferably, the peptides according to the present invention are selected from chimeric peptides, synthetic peptides and peptides derived from proteins.

[0038] Preferably, the peptide according to the present invention is a peptide capable of penetrating cells, in particular the plasma membrane of cells. More preferably, the peptide according to the present invention is a cell penetrating peptide (CPP).

[0039] Also preferably, the peptide according to the present invention is a peptide that is able to cross the blood-brain barrier.

[0040] Preferably, the peptide according to the present invention is a cell penetrating peptide selected from the group consisting of polycationic peptides having a sequence rich in positively charged amino acid residues such as lysine or arginine, amphipathic peptides having a sequence of alternating charged polar residues and hydrophobic non-polar residues, and hydrophobic peptides containing only weakly charged non-polar or hydrophobic residues.

[0041] Preferably, the peptide according to the present invention is selected from the group consisting of an NFL peptide having the following amino acid sequence: YSSYSAPVSSSLSVRRSYSSSSGS (SEQ ID NO: 1), a TAT peptide having the following amino acid sequence: GRKKRRQRRRPPQ (SEQ ID NO: 2), a VIM peptide having the following amino acid sequence: GGAYVTRSSAVRLRSSVPGVRLLQ (SEQ ID NO: 3), a peptide transporter having the following amino acid sequence: GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 4), a penetrating peptide having the following amino acid sequence: RQIKIWFQNRRMKWKK (SEQ ID NO: 5), a MAP peptide having the following amino acid sequence: KLALKLALKALKAALKLA (SEQ ID NO: 6), a Pep-1 peptide having the following amino acid sequence: KETWWETWWTEWSQPKKKRKV (SEQ ID NO: 7), a Pept 1 peptide having the following amino acid sequence: PLILLRLLRGQF (SEQ ID NO: 8). NO:8), Pept 2 peptide having the following amino acid sequence: PLIYLRLLRGQF (SEQ ID NO:9), IVV-14 peptide having the following amino acid sequence: KLWMRWYSPTTRRYG (SEQ ID NO:10), Ig(v) peptide having the following amino acid sequence: MGLGLHLLVLAAALQGAKKKRKV (SEQ ID NO:11), pVEC peptide having the following amino acid sequence: LLIILRRRIRKQAHAHSK (SEQ ID NO:12), ppTG20 peptide having the following amino acid sequence: GLFRALLRLLRSLWRLLLRA (SEQ ID NO:13), APP peptide having the following amino acid sequence: APP(GLARALTRLLRQLTRQLTRA) (SEQ ID NO:14), peptide having the following amino acid sequence: RLWMRWYSPRTRAYGC (SEQ ID NO:15); peptide having the following amino acid sequence: WRWYCR (SEQ ID NO:16). NO: 16); a PepFect 14 peptide having the following amino acid sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2 (SEQ ID NO: 17); a peptide having the following amino acid sequence: GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 18), a HRSV peptide having the following amino acid sequence: RRIPNRRPRR (SEQ ID NO: 19) or a derivative thereof.

[0042] The peptide according to the invention may also be a cyclic peptide, such as a cyclic RGD peptide.

[0043] More preferably, the peptide according to the present invention is selected from: an NFL peptide having the following amino acid sequence: YSSYSAPVSSSLSVRRSYSSSSGS (SEQ ID NO: 1), a TAT peptide having the following amino acid sequence: GRKKRRQRRRPPQ (SEQ ID NO: 2), a VIM peptide having the following amino acid sequence: GGAYVTRSSAVRLRSSVPGVRLLQ (SEQ ID NO: 3) or a derivative thereof.

[0044] According to an embodiment of the present invention, the peptide according to the present invention is not a peptide targeting the EGFR receptor, in particular an α-lysine-azidoacetic acid peptide, or a peptide targeting bone.

[0045] As used herein, peptide derivatives in the sense of the present invention refer to variants and fragments of the peptides to which they refer. Preferably, the derivatives (particularly variants or fragments) of the peptides according to the present invention are biologically active.

[0046] Biologically active derivatives of the peptides according to the present invention are variants and fragments that retain the biological activity and specificity of the parent peptide.

[0047] Variants of the peptides according to the invention may be selected from any variants known to the person skilled in the art. Examples of variants according to the invention are allelic variants of the peptides and peptidomimetic variants of the peptides.

[0048] Allelic variants of the peptides according to the present invention have the same amino acid sequence as the peptides according to the present invention (particularly SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19), except that one or more amino acids have been substituted or deleted by other amino acids, and the final peptide retains the specificity of the parent peptide.

[0049] Preferably, the peptides according to the invention have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity with the parent peptide (in particular SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 19).

[0050] It is also preferred that the variant according to the invention has the same number of amino acids as the peptide from which it is derived.

[0051] The percent identity between two peptide sequences is well known to those skilled in the art. For example, the percent identity between two peptide sequences can be determined by aligning the sequences along the entire length of the similar sequences and determining the number of positions where the amino acids are identical in each sequence, and dividing this number by the total number of amino acids in the longer sequence.

[0052] Peptoid variants according to the present invention are preferably organic molecules that simulate certain properties of the parent peptide. Preferred peptoid variants according to the present invention are obtained by structural modification of the peptide according to the present invention (e.g., using non-natural amino acids such as D-amino acids to replace L-amino acids, conformational restrictions, isosteric substitutions, cyclization or other modifications).

[0053] Examples of other modifications include the following: replacing one or more amide bonds with non-amide bonds; replacing one or more amino acid side chains with different chemical moieties; protecting one or more N-termini, C-termini, or side chains with protecting groups; introducing double bonds or rings into the backbone. Advantageously, these modifications have the effect of increasing the rigidity, binding affinity, resistance to enzymatic degradation, bioavailability of the peptides of the invention, and more generally have the effect of improving their pharmacokinetic properties.

[0054] Therefore, taking into account the peptide sequences of the peptides according to the present invention, those skilled in the art are able to design and produce peptide mimetic variants having similar or superior biological properties to the peptides according to the present invention (particularly cell penetrating peptides, more particularly NFL, TAT and VIM peptides according to the present invention).

[0055] The NFL peptide (hereinafter also referred to as NFL-TBS40-63) is a 24-amino acid polypeptide having the following sequence: YSSYSAPVSSSLSVRRSYSSSSGS (SEQ ID NO: 1). This peptide is well known to those skilled in the art and corresponds to the second tubulin binding site of the light neurofilament subunit (amino acids 40 to 63 of the TBS site of the NFL protein).

[0056] In an embodiment of the present invention, the peptide according to the present invention is a derivative of the NFL peptide, in particular a variant or fragment thereof. Preferably, the fragments and variants of the NFL peptide are biologically active.

[0057] Preferably, the NFL peptide fragment according to the present invention comprises at least 12 consecutive amino acids of the parent NFL peptide, preferably at least 16, more preferably at least 18 amino acids.

[0058] Preferably, the variant according to the invention comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to the NLF peptide. An example of an allelic variant of the NFL peptide is the TBS motif of the quail neurofilament light subunit, which retains 20 of the 24 amino acids of the NFL-TBS40-63 peptide.

[0059] The TAT peptide (hereinafter also referred to as TAT48-60) is a 13-amino acid peptide having the following sequence: GRKKRRQRRRPPQ (SEQ ID NO: 2). As is well known to those skilled in the art, this peptide corresponds to residues 48 to 60 of the TAT protein involved in the replication of human immunodeficiency virus type 1 (HIV-1).

[0060] In one embodiment of the present invention, the peptide according to the present invention is a derivative of the TAT peptide, in particular a variant or fragment thereof. Preferably, the TAT peptide derivative according to the present invention is biologically active.

[0061] Preferably, the TAT peptide fragment according to the present invention comprises at least 5 consecutive amino acids of the TAT parent peptide, preferably at least 6, at least 7, at least 8 amino acids.

[0062] Preferably, the variant according to the invention comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to the TAT peptide.

[0063] The VIM peptide is a 24 amino acid peptide having the following sequence: GGAYVTRSSAVRLRSSVPGVRLLQ (SEQ ID NO: 3).

[0064] In one embodiment of the present invention, the peptide according to the present invention is a derivative of the VIM peptide, in particular a variant or fragment thereof. Preferably, the VIM peptide derivative according to the present invention is biologically active.

[0065] Preferably, the VIM peptide fragment according to the present invention comprises at least 12 consecutive amino acids of the parent VIM peptide, preferably at least 14, at least 16 and more preferably at least 18 amino acids.

[0066] Preferably, the VIM peptide variant according to the present invention comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to the VIM peptide.

[0067] The penetrating peptide is a 16-residue long fragment having the following amino acid sequence: RQIKIWFQNRRMKWKK (SEQ ID NO: 5). As is well known to those skilled in the art, this peptide is derived from the homeodomain of the Drosophila Antennapedia transcription factor.

[0068] In one embodiment of the present invention, the peptide according to the present invention is a derivative of a penetrating peptide, in particular a variant or fragment thereof. Preferably, the penetrating peptide derivative according to the present invention is biologically active.

[0069] Preferably, the penetrating peptide fragment according to the present invention comprises at least 5 consecutive amino acids, preferably at least 6, at least 7, at least 8 amino acids of the penetrating parent peptide.

[0070] Preferably, the variant according to the invention comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% identity to the penetrating peptide.

[0071] The peptides according to the invention may be prepared by any technique known to those skilled in the art, in particular by chemical synthesis.

[0072] According to one embodiment, the peptide as defined above is coupled to at least one coupling agent. As understood herein, the terms "coupling agent" and "conjugating agent" are interchangeable. As understood herein, the terms "coupling," "conjugation," "functionalization," and "modification" are used interchangeably.

[0073] The coupling agent according to the present invention can be selected from any coupling agent known to those skilled in the art.

[0074] Preferably, the coupling agent according to the invention is non-peptidic.

[0075] The coupling agent can be a bifunctional reagent, preferably a heterobifunctional reagent such as N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS) and sulfo-GMBS derivatives, m-maleimidobenzoyl-n-hydroxysuccinimide ester (MBS) and sulfo-MBS derivatives, 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimidyl ester (SMCC), ... Sulfo-succinimidyl ester (sulfo-SMCC), carbodiimide, bis(diazobenzidine) (BDB) or glutaraldehyde, biotin, sulfo-NHS-bitoin or fluorophores (also known as fluorescent dyes) such as hydroxycoumarin, aminocoumarin, methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer Yellow, NBD, R phycoerythrin, PE-Cy5 conjugate, R phycoerythrin, PE-Cy5 conjugate, PE-Cy7 conjugate, Red 613, PerCP, TruRed, FluorX, fluorescein, BODIPY-FL, TRITC, X-rhodamine, lissamine rhodamine B, Texas Red, Allophycocyanine, APC-Cy7 conjugate, 5-carboxytetramethylrhodamine, 5,6-carboxytetramethylrhodamine, 6-carboxytetramethylrhodamine, 5-carboxyfluorescein, 5,6-carboxyfluorescein, 6-carboxyfluorescein, and dimethylaminonaphthylpyridinium.

[0076] The use of coupling agents is described in detail in the chapter "Production of Antisera Using Peptide Conjugates" in the reference "The Protein Protocols Handbook" (2002), which is incorporated herein by reference.

[0077] When GMBS, MBS, SMCC or sulfo-SMCC are used, they are preferably linked to a cysteine ​​(C), which may be added if not present in the peptide sequence, particularly at its N- or C-terminus.

[0078] The coupling agent according to the present invention can also be selected from any functional group that can be combined with peptides well known to those skilled in the art. Examples of functional groups according to the present invention include fluorine, phosphate, acetyl, alkyl, glutamic acid residues, carboxyl, glycine residues, glycosyl, hydroxyl, amide and sulfate groups.

[0079] Preferably, the coupling agent according to the present invention is selected from biotin and luciferin.

[0080] In one embodiment, the peptide according to the invention is functionalized with at least one biotin, for example with one biotin, two biotins or three biotins.

[0081] In another embodiment, the peptide according to the invention is functionalized with at least one luciferin, for example with one luciferin, two luciferins or three luciferins.

[0082] The peptides according to the present invention may be modified by any conventional peptide coupling method well known to those skilled in the art (e.g. chemical or enzymatic). Examples of methods for modifying the peptides according to the present invention include acetylation, alkylation, biotinylation, glutamylation, glycation, glycosylation, hydroxylation, phosphorylation, sulfation, amidation, and pegylation.

[0083] Preferably, the coupling agent according to the invention, in particular fluorescein or biotin, is covalently bound to the peptide according to the invention.

[0084] Functionalization of the peptides according to the invention with at least one coupling agent, in particular fluorescein or biotin, can be performed directly on primary amines, secondary amines or both.

[0085] In one embodiment of the invention, at least one coupling agent, in particular fluorescein or biotin, is bound to the N-terminus or C-terminus of the peptide according to the invention.

[0086] In one embodiment of the invention, the functionalization of the peptide with at least one coupling agent, in particular fluorescein or biotin, is carried out at the primary carboxyl function or the secondary carboxyl function or both using intermediates well known to those skilled in the art. Examples of bonding intermediates are carbodiimides, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0087] Biocompatible polymers

[0088] The biocompatible polymer according to the present invention may be a natural or synthetic polymer and may be selected from any biocompatible polymer known to those skilled in the art.

[0089] Preferably, the biocompatible polymer according to the present invention is selected from the group consisting of synthetic biocompatible polymers, biopolymers and thermosensitive biocompatible polymers.

[0090] More preferably, the biocompatible polymer according to the present invention is selected from polyethylene glycol (PEG), polyethylene glycol diacid (PEG-diacid), polyethylene glycol diamine (PEG-diamine), collagen, alginate, elastin, hyaluronic acid, cellulose, gelatin, polylactic acid, maltodextrin, glucose polymers, lactose polymers, chitosan, dextran, thermosensitive polymers, poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethylmethylmethacrylate] (pDMAEMA), hydroxypropylcellulose, poly(vinylcaprolactam) and polyvinyl methyl ether) or a combination thereof.

[0091] More preferably, the biocompatible polymer is dicarboxypolyethylene glycol.

[0092] Nanoparticles

[0093] Preferably, the nanoparticles according to the present invention have a diameter between 5 nm and 500 nm, more preferably between 5 nm and 200 nm, for example between 5 nm and 150 nm, between 5 nm and 100 nm, or between 5 nm and 90 nm. More preferably, the nanoparticles according to the present invention have a diameter between 20 nm and 200 nm, for example between 20 nm and 150 nm, or between 20 nm and 100 nm.

[0094] Preferably, the peptide according to the invention is located inside the nanoparticle according to the invention. Thus, advantageously, the peptide adopts a spatial configuration such that the therapeutic activity of the peptide is retained and / or enhanced without affecting the stability of the nanoparticle.

[0095] Preferably, the metal salt is chelated by the peptide according to the invention.

[0096] Preferably, the metal salt and the peptide are encapsulated by a polymer.

[0097] Preferably, the nanoparticles according to the present invention are prepared according to a method comprising the following steps:

[0098] - a step of chelating the metal salt with the peptide;

[0099] - a mixing step with a biocompatible polymer; and

[0100] - a step of reducing the mixture with a reducing agent.

[0101] Preferably, the step of chelating the metal salt to the peptide comprises dissolving the metal salt, preferably in an aqueous solution, and adding the peptide to the metal salt solution.

[0102] Advantageously, a biocompatible polymer is used as stabilizer.

[0103] Preferably, the nanoparticle preparation method according to the present invention is performed in the absence of any surfactant other than the biocompatible polymer and in the absence of any chemical binder.

[0104] Preferably, the reducing agent according to the present invention can be any reducing agent known to those skilled in the art. Examples of reducing agents include LiAlH4, NaBH4, NaHg, B2H6, SO2. Preferably, the reducing agent according to the present invention is NaBH4.

[0105] The method for preparing nanoparticles according to the present invention may further comprise a step of centrifuging the nanoparticle solution, preferably after the reduction step, in order to remove excess biocompatible polymer.

[0106] Advantageously, the nanoparticles according to the present invention are stable. Preferably, the nanoparticles according to the present invention are stable and their biological activity, in particular the targeting and cellular internalization properties of the peptides, are maintained for at least 2 months, 4 months, 6 months, 8 months, 10 months, 12 months, 14 months, 16 months, 18 months, 20 months, 22 months or 24 months.

[0107] use

[0108] The nanoparticles according to the present invention are particularly suitable for use as pharmaceutical and / or diagnostic agents in individuals.

[0109] The nanoparticles according to the invention are particularly suitable for use as theranostic agents.

[0110] The nanoparticles according to the invention are also particularly suitable for use in medical devices intended for use by individuals.

[0111] Therefore, one embodiment of the present invention also relates to a pharmaceutical composition comprising as active ingredient at least one nanoparticle according to the present invention, optionally in combination with a pharmaceutically acceptable carrier or excipient.

[0112] The present invention also relates to the use of at least one nanoparticle according to the present invention for the preparation of a medicament, in particular a medicament for the prevention or treatment of cancer, especially for the prevention or treatment of glioblastoma or pancreatic cancer.

[0113] The present invention also relates to the use of at least one nanoparticle according to the invention for targeting, detecting and destroying cancer cells.

[0114] The present invention also relates to a method for treating cancer, in particular glioblastoma or pancreatic cancer, in an individual, comprising administering to the individual an effective amount of at least one nanoparticle or pharmaceutical composition or medicament according to the present invention.

[0115] The present invention also relates to a diagnostic composition comprising as active ingredient at least one nanoparticle according to the present invention, optionally in combination with a pharmaceutically acceptable carrier or excipient.

[0116] The medical device according to the present invention preferably comprises at least one nanoparticle according to the present invention. Preferably, the medical device according to the present invention is used for the diagnosis, prevention or treatment of cancer, in particular glioblastoma or pancreatic cancer. The medical device according to the present invention can be selected from any medical device known to the person skilled in the art. Preferably, the medical device according to the present invention is selected from a medical imaging device or an implantable device such as an implant, a stent or a catheter. By implantable device we mean any device intended to be introduced in whole or in part into the human or animal body by medical intervention.

[0117] According to one embodiment of the present invention, the nanoparticles, pharmaceutical compositions, drugs or medical devices according to the present invention are used to prevent or treat cancer, in particular glioblastoma or pancreatic cancer.

[0118] According to one embodiment of the present invention, the nanoparticle, diagnostic composition or medical device according to the present invention is used for detecting cancer cells or tumors, for diagnosing cancer, for monitoring the progression of cancer and / or for monitoring therapeutic treatment of cancer.

[0119] According to one embodiment of the present invention, the nanoparticles, pharmaceutical compositions, diagnostic compositions, drugs or medical devices are used as contrast agents for diagnostic imaging (e.g., radiography, magnetic resonance imaging (MRI), Raman spectroscopy, optical imaging, optical coherence tomography, X-ray, computed tomography, positron emission tomography or a combination thereof).

[0120] One embodiment of the present invention relates to monitoring the administration of nanoparticles of a pharmaceutical composition, medicament or diagnostic composition according to the present invention to an individual, its biodistribution and bioavailability by means of a diagnostic device and diagnosing the individual.

[0121] Preferably, the nanoparticles, medicaments, pharmaceutical compositions or diagnostic compositions, medical devices according to the present invention are used in at least one of the following situations:

[0122] - Diagnosis of cancer, particularly glioblastoma or pancreatic cancer;

[0123] - determine the status or evolution of cancer (particularly glioblastoma or pancreatic cancer),

[0124] - Treatment of cancer, especially glioblastoma or pancreatic cancer,

[0125] -Monitoring therapeutic treatment of cancer (particularly glioblastoma or pancreatic cancer).

[0126] cancer

[0127] The cancer prevented or treated according to the present invention may be any cancer known to those skilled in the art.

[0128] The cancer prevented or treated according to the present invention may be stage 1, stage 2, stage 3, or stage 4.

[0129] Preferably, the cancer to be prevented or treated according to the present invention is selected from cancers of the central nervous system, endocrine system and digestive system.

[0130] Preferably, the cancer prevented or treated according to the present invention is selected from thyroid cancer, parathyroid cancer, thymic cancer, ovarian cancer, testicular cancer, pancreatic islet cancer, gallbladder cancer, gastric cancer, esophageal cancer, colon cancer, glioblastoma, liver cancer, pancreatic cancer, rectal cancer, intestinal cancer, small intestine cancer. More preferably, the cancer prevented or treated according to the present invention is selected from glioblastoma and pancreatic cancer.

[0131] According to one embodiment of the present invention, the nanoparticles, pharmaceutical compositions, drugs, diagnostic compositions or medical devices according to the present invention are particularly suitable for use in photothermal therapy, in particular for treating cancer by photothermal therapy.

[0132] Photothermal therapy is a cancer treatment method well known to those skilled in the art. Preferably, the nanoparticles, pharmaceutical compositions, or medicaments according to the present invention are injected into the individual's bloodstream, preferably intravenously. The nanoparticles then penetrate the tumor or cancer cells to be treated and expose them to infrared radiation to destroy them.

[0133] Pharmaceutical composition

[0134] As used herein, "pharmaceutically acceptable carrier, vehicle, or excipient" refers to any material suitable for use in a pharmaceutical composition.

[0135] Preferably, the pharmaceutically acceptable carrier or excipient according to the present invention is suitable for use with the nanoparticles according to the present invention, preferably in liquid form.

[0136] Preferably, the pharmaceutically acceptable carrier or excipient according to the present invention is suitable for oral, parenteral, intradermal, intravenous, intraarterial, intramuscular, nasal, rectal or subcutaneous administration.

[0137] Preferably, the pharmaceutically acceptable carriers or excipients according to the present invention include, but are not limited to, any standard carriers or excipients well known to those skilled in the art, such as water, glycerol, alcohol, oil emulsions, water emulsions, physiological saline, buffers, preservatives, stabilizers, and the like.

[0138] individual

[0139] Preferably, the subject according to the present invention is an animal, preferably a mammal such as a human, a canine, in particular a dog, a feline, in particular a cat, a horse, a cow, a pig, a goat, in particular a goat or sheep, a camelid, a rodent, in particular a mouse or a rat. More preferably, the subject according to the present invention is a human.

[0140] Preferably, the individual according to the present invention suffers from cancer, in particular cancer of the central nervous system, endocrine system and digestive system.

[0141] More preferably, the individual according to the present invention suffers from glioblastoma or pancreatic cancer.

[0142] Also preferably, the individual according to the present invention is suspected of having cancer, in particular glioblastoma or pancreatic cancer.

[0143] Drug administration

[0144] Preferably, the nanoparticles according to the present invention, the pharmaceutical composition, the diagnostic composition, the medicament or the medical device according to the present invention are administered in a prophylactically or therapeutically effective amount for the detection, prevention or treatment of cancer as defined above.

[0145] Preferably, the nanoparticles according to the present invention, the pharmaceutical composition, the medicament, the diagnostic composition or the medical device according to the present invention can be administered orally, parenterally, intradermally, intravenously, intraarterially, intramuscularly, intranasally, rectally or subcutaneously.

[0146] The nanoparticles, pharmaceutical compositions, drugs, diagnostic compositions or medical devices according to the present invention can be formulated as injectable suspensions, gels, oils, suppositories, capsules and the like.

[0147] The nanoparticles, pharmaceutical compositions, drugs, diagnostic compositions or medical devices according to the present invention can be administered to an individual at a dose of 10 μmol / L to 2000 μmol / L, preferably 50 μmol / L to 1500 μmol / L, more preferably 100 μmol / L to 1000 μmol / L, for example 200 μmol / L to 1000 μmol / L, 300 μmol / L to 1000 μmol / L, 400 μmol / L to 1000 μmol / L, 500 μmol / L to 1000 μmol / L.

[0148] One skilled in the art will be able to adjust the dosage of the nanoparticles or pharmaceutical composition, diagnostic composition or drug according to the body weight of the individual to be treated.

[0149] Other compounds

[0150] The nanoparticles, pharmaceutical compositions, drugs, diagnostic compositions or medical devices according to the present invention may be administered together with additional compounds to prevent or treat cancer.

[0151] Nanoparticles according to the present invention can be used as carriers for additional compounds for the prevention or treatment of cancer. Specifically, nanoparticles according to the present invention can be used to deliver additional compounds for the intracellular prevention or treatment of cancer. Advantageously, nanoparticles according to the present invention can be used to transport therapeutic molecules to target cells, particularly cancer cells of the tumor to be treated.

[0152] According to one embodiment, the present invention relates to a product comprising:

[0153] - at least one nanoparticle as defined above;

[0154] - at least one additional compound for use in the prevention or treatment of cancer, in particular glioblastoma and pancreatic cancer

[0155] As a combined preparation, it is particularly used simultaneously, separately or sequentially for preventing or treating cancer, particularly glioblastoma or pancreatic cancer.

[0156] As understood herein, "combined" or "combination" means that the nanoparticles according to the present invention are administered simultaneously or together (i.e. at the same administration site) with another compound, or are administered separately or at different times, provided that the time period during which the nanoparticles according to the present invention exert their effect in the individual and the time period during which the additional compound produces its pharmacological effect in the individual overlap at least in part.

[0157] The additional compound used to prevent or treat cancer may be any compound known to those skilled in the art.

[0158] Preferably, the additional compound is selected from cyclophosphamide, docetaxel, doxorubicin, epirubicin, fluorouracil, methotrexate, paclitaxel, everolimus, alectinib, melphalan, brigatinib, nilutamide, cyproterone acetate, anastrozole, exemestane, lomustine, bosutinib, encorafenib, cabozantinib, vandenatib, bicalutamide, etoposide, chlorambucil, cobimetinib, enzalutamide, idarubicie, capecitabine, tamoxifen, dexamethasone, disulfiram, lenvatinib, gemcitabine, 5-FU, irinotecan, temozolomide, oxaliplatin, nab-paclitaxel, and combinations thereof.

[0159] The invention will be further explained in a non-limiting manner with the aid of the following figures and examples. BRIEF DESCRIPTION OF THE DRAWINGS

[0160] Figure 1

[0161] Figure 1 The UV-visible spectrum of the synthesized NFL-BIOTINE-PEG-AuNP nanoparticles is shown, where the relationship between wavelength (nm) and absorbance (au) is shown.

[0162] Figure 2

[0163] Figure 2 Shown are the UV-visible spectra of NFL-BIOTINE-PEG-AuNP nanoparticles at T0 and T0+11 months (Test 1 and Test 2).

[0164] Figure 3

[0165] Figure 3 Shown are the temperature rises (in °C) over time (in minutes) for water (H2O), a solution of NFL peptide functionalized with biotin (NFL-BIOT), a solution of a gold salt complex with a biotin-functionalized peptide (AuNP), and a solution of NFL-biotin-PEG-AuNP nanoparticles.

[0166] Figure 4

[0167] Figure 4 Mitochondrial activity of MiaPaCa-2 cells, colchicine-treated MiaPaCa-2 cells, and MiaPaCa-2 cells treated with 50, 100, 250, 500, and 1000 μmol / L PEG-AuNP nanoparticles complexed or not with NFL-biotin peptide for 24 hours is shown. Mitochondrial activity is expressed as the average percentage relative to the activity of control MiaPaCa-2 cells.

[0168] Figure 5

[0169] Figure 5 Mitochondrial activity of MiaPaCa-2 cells, colchicine-treated MiaPaCa-2 cells, and MiaPaCa-2 cells treated with 50, 100, 250, 500, and 1000 μmol / L PEG-AuNP nanoparticles complexed or not with NFL-biotin peptide for 72 hours is shown. Mitochondrial activity is expressed as the average percentage relative to the activity of control MiaPaCa-2 cells.

[0170] Figure 6

[0171] Figure 6 Mitochondrial activity of F98 cells, colchicine-treated F98 cells, and F98 cells treated with 50, 100, 250, 500, and 1000 μmol / L PEG-AuNP nanoparticles complexed or not with NFL-biotin peptide for 24 hours is shown. Mitochondrial activity is expressed as the average percentage relative to the activity of control F98 cells.

[0172] Figure 7

[0173] Figure 7 Mitochondrial activity of F98 cells, colchicine-treated F98 cells, and F98 cells treated with 50, 100, 250, 500, and 1000 μmol / L PEG-AuNP nanoparticles complexed or not with NFL-biotin peptide for 72 hours is shown. Mitochondrial activity is expressed as the average percentage relative to the activity of control F98 cells.

[0174] Figure 8

[0175] Figure 8 Figure 2 shows the cell viability of MiaPaCa-2 cells treated with 0.1 μM peptide (NFL-BIOT) alone, 100 μM PEG-AuNP nanoparticles (AuNPs), and 100 μM NFL-BIOTINE-Au-NP nanoparticles, 24 hours after irradiation and nanoparticle internalization. Cell viability is expressed as a percentage of the cell viability of untreated cells.

[0176] Figure 9

[0177] Figure 9 Figure 48: Cell viability of MiaPaCa-2 cells treated with 0.1 μM peptide (NFL-BIOT) alone, 100 μM PEG-AuNP nanoparticles (AuNPs), and 100 μM NFL-BIOTINE-Au-NP nanoparticles, respectively, 48 hours after irradiation and nanoparticle internalization. Cell viability is expressed as a percentage of cell viability of untreated cells.

[0178] Figure 10

[0179] Figure 10 Figure 2 shows the cell viability of F98 cells treated with 0.1 μM of the peptide (NFL-BIOT) alone, 100 μM of PEG-AuNP nanoparticles (AuNPs), and 100 μM of NFL-BIOTINE-Au-NPs nanoparticles 24 hours after irradiation and internalization of the nanoparticles. Cell viability is expressed as a percentage of the cell viability of untreated cells.

[0180] Figure 11

[0181] Figure 11 Figure 48 shows the cell viability of F98 cells treated with 0.1 μM peptide (NFL-BIOT) alone, 100 μM PEG-AuNP nanoparticles (AuNPs), and 100 μM NFL-BIOTINE-Au-NP nanoparticles, respectively, 48 hours after irradiation and internalization of nanoparticles. Cell viability is expressed as a percentage of cell viability relative to untreated cells.

[0182] Figure 12

[0183] Figure 12The in vitro mitochondrial activity of rat glioblastoma cells (F98 cells) as a control, F98 cells treated with colchicine (Col, 1 μg / mL) as a positive control, and F98 cells treated with PEG-AuNP, BIOT-TAT-PEGAuNP, and BIOT-VIM-PEG-AuNP nanoparticles at different concentrations (0, 50, 100, 250, 500, or 1000 μmol / L) for 24 hours was shown. Mitochondrial activity was assessed by MTS assay. The experiment was performed at least three times. The symbol # indicates that there were no cells at the end of the incubation treatment. The data represent the mean ± standard error relative to the activity of the control F98 cells. Statistical analysis was performed by Student's t-test (*p < 0.05 and ***p < 0.001).

[0184] Figure 13

[0185] Figure 13 The in vitro mitochondrial activity of rat glioblastoma cells (F98 cells) as a control, F98 cells treated with colchicine (Col, 1 μg / mL) as a positive control, and F98 cells treated with PEG-AuNP, BIOT-TAT-PEGAuNP, and BIOT-VIM-PEG-AuNP nanoparticles at different concentrations (0, 50, 100, 250, 500, or 1000 μmol / L) for 72 hours was shown. Mitochondrial activity was assessed by MTS assay. The experiment was performed at least three times. The symbol # indicates that there were no cells at the end of the incubation treatment. The data represent the mean ± standard error relative to the activity of the control F98 cells. Statistical analysis was performed by Student's t-test (*p < 0.05 and ***p < 0.001).

[0186] Figure 14

[0187] Figure 14 Transmission electron microscopy images are shown, illustrating the internalization of gold nanoparticles (PEG-AuNPs) in rat glioblastoma cells (F98). F98 cells were treated with 250 μmol / L nanoparticles for 24 hours. N represents nucleus, and V represents vacuole. Scale bar (left): 2 μm. Scale bar (right): 50 nm.

[0188] Figure 15

[0189] Figure 15Transmission electron microscopy images are shown, illustrating the internalization of gold nanoparticles (PEG-AuNPs) conjugated to the peptide TAT-biotin (BIOT-TAT-PEGAuNPs) in rat glioblastoma (F98) cells. F98 cells were treated with 250 μmol / L of nanoparticles for 24 hours. N represents nucleus, and V represents vacuole. Scale bar (left): 2 μm. Scale bar (right): 50 nm.

[0190] Figure 16

[0191] Figure 16 Transmission electron microscopy images are shown, illustrating the internalization of gold nanoparticles (PEG-AuNPs) conjugated to the peptide TAT-biotin (BIOT-TAT-PEGAuNPs) in rat glioblastoma (F98) cells. F98 cells were treated with 250 μmol / L of nanoparticles for 24 hours. N represents nucleus, and V represents vacuole. Scale bar (left): 2 μm. Scale bar (right): 50 nm.

[0192] Figure 17

[0193] Figure 17 Shown are the average number of vacuoles containing more than 20 gold nanoparticles in F98 cells incubated for 24 hours with 250 μmol / L PEG-AUNP nanoparticles (black bars), BIOT-TAT-PEG-AUNP nanoparticles (dark gray bars), and BIOT-VIM-PEG-AuNP nanoparticles (light gray bars). Nanoparticles were manually counted on electron microscopy images.

[0194] Example 1

[0195] The inventors have synthesized and characterized the NFL-biotin-PEG-AuNP gold nanoparticles according to the present invention according to the following method.

[0196] A. Materials and Methods

[0197] 1. Preparation of NFL-biotin-PEG-AuNP nanoparticles

[0198] Gold nanoparticles were mainly prepared by reduction of 1 mmol / L chloroauric acid (HAuCl4, Sigma-Aldrich).

[0199] After the gold salt is dissolved, the solution is shaken vigorously and then 0.08 mL of biotinylated NFL peptide (NFL-biotin) at a concentration of 1 mg / mL is added. The NFL peptide is pre-diluted with 10% ethanol. The complexation of the peptide with the HAuCl4 gold salt is achieved by chelation.

[0200] Then, 250 μL of the stabilizer PEG-COOH 600 (polyethylene glycol dicarboxylic acid, Sigma-Aldrich) was added at room temperature.

[0201] Finally, 1.2 mL of 3 mg / mL reducing agent NaBH4 (sodium tetrahydroborate, Sigma-Aldrich) was added to 3+ ions are reduced to neutral gold atoms (Au 0 ).

[0202] After adding the reducing agent, the formation of NFL-biotin-PEG-AuNP nanoparticles was observed by the solution color changing from light yellow to bright pink purple.

[0203] The products of each synthesis stage were stored at 27–29 °C and characterized by UV–visible spectroscopy, Raman spectroscopy, and transmission electron microscopy (TEM).

[0204] The NFL-biotin-PEG-AuNP solution was centrifuged three times at 10,000 rpm for 10 minutes, and the supernatant was discarded. The pellet was redispersed in an equal amount of water. This process was repeated twice to remove excess dicarboxy PEG that was not conjugated to the NFL-biotin peptide.

[0205] 2. Nanoparticle characterization

[0206] UV-visible absorption spectra

[0207] Absorption spectra were measured using a UV-visible spectrophotometer (Uvikon 941 from Kontron Instruments) controlled by Thermalys Uvikon 900 software. Absorption spectra were recorded in the spectral range between 200 and 900 nm. The solutions were placed in 1 cm quartz cuvettes.

[0208] Raman spectroscopy

[0209] Raman spectra were recorded using an Xplora Raman microspectrometer (Horiba Scientifics) equipped with a 660 nm HeNe laser (Helium-Neon), a CCD camera for data acquisition, and an optical filter set to 100% (laser power 8 mW and 600 line network) and processed using Labspec software.

[0210] 2.3 Zeta potential measurement

[0211] The zeta potential of NFL-biotin-PEG-AuNPs dispersed in water was measured using the electrophoresis mode of a Zetasizer NanoZS (Malvern Instruments Ltd, Malvern, UK).

[0212] Dynamic light scattering (DLS)

[0213] Nanoparticle tracking analysis was used to characterize the average hydrodynamic diameter of NFL-biotin-PEG-AuNP nanoparticles dispersed in water. The device, developed by NanoSight (Malvern Instruments Ltd), uses the properties of light scattering and Brownian motion to obtain the particle size distribution of samples in liquid suspensions. Measurements were performed using an NS500 system equipped with a 405nm laser (version 3.0). Six 60-second videos were recorded at a nanoparticle concentration sufficient to obtain at least 200 terminated tracks per video to achieve statistical significance. Data were recorded in ± standard deviation mode.

[0214] Transmission electron microscopy

[0215] Gold nanoparticles, either complexed or uncomplexed with the NFL-biotin peptide, were observed by transmission electron microscopy. To this end, 2 μL of sample was deposited onto a 150-mesh copper grid, which was itself coated with a Formvar film for 1 minute. The grid was then contrasted with 2% uranyl acetate for 1 minute. The samples were observed using a Jeol microscope (JEM-1400, accelerating voltage 120 kV; Japan) equipped with a Gatan 832 Orius SC-1000 camera.

[0216] B. Results

[0217] 1. UV-Vis characterization

[0218] Complexation of HAuCl4 with NFL-biotin peptide was observed, where the band at 300 nm represented HAuCl4 and the band at 280 nm represented NFL-biotin peptide (see Figure 1 A UV-visible band at 525 nm, representing the plasmon band of the NFL-biotin-PEG-AuNP nanoparticles, was also observed. These results confirmed the formation of NFL-biotin-PEG-AuNP nanoparticles. The 280 nm band (representing the peptide control) was present at all stages of the synthesis, confirming that the peptide had been properly grafted.

[0219] DLS and zeta potential results showed that the diameter of NFL-biotin-PEG-AuNP nanoparticles was approximately 91±2 nm and the zeta potential was -24±1 mV.

[0220] 2. Raman spectroscopy characterization

[0221] Raman spectroscopy also confirmed the successful peptide grafting. The Raman spectrum of the free NFL-biotin peptide (control) showed a band of the amino acid (tyrosine) that constitutes the peptide. -1 The band represents the CH2 group of biotin, 1656 cm -1 The band belongs to amide I. At 1600 cm -1 The broad band observed nearby is attributed to water. It was observed that with the formation of NFL-biotin-PEG-AuNP nanoparticles, a clearer peptide signature fingerprint was obtained compared to the complex. These results confirm the formation of NFL-biotin-PEG-AuNP nanoparticles, as nanoparticle formation enhances this signal.

[0222] 3. Observing nanoparticles using transmission electron microscopy

[0223] Transmission electron microscopy was used to observe PEGylated gold nanoparticles (PEG-AuNPs) with and without complexing with NFL-biotin peptide. These observations revealed differences in nanoparticle shape in the absence and presence of NFL-biotin peptide. PEG-AuNP nanoparticles were round and relatively uniform in size.

[0224] When these same particles were complexed with the NFL-biotin peptide, different particle shapes (round, rod-shaped, hexagonal) were observed, with considerable heterogeneity in size.

[0225] 4. stability

[0226] The bright red-violet and UV-visible spectra of the nanoparticles remained unchanged after 11 months of storage at room temperature, confirming that the formation of the nanoparticle suspension remained stable (see Figure 2 ).

[0227] Example 2

[0228] The inventors evaluated the effects of NFL-biotin-PEG-AuNP gold nanoparticles on two cancer cell lines.

[0229] A. Materials and Methods

[0230] 1. Cell lines used

[0231] Two cell lines obtained from the American Tissue Culture Collection (ATCC) were used in this study:

[0232] -MiaPaCa-2 cells, a human pancreatic cancer cell line, and

[0233] -F98 cells, a rat glioblastoma cell line.

[0234] Both cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium; Gibco, Bio-Sciences Ltd, Ireland) supplemented with 10% FBS (fetal bovine serum; Sigma-Aldrich), 1% antibiotics (penicillin 50 IU / mL and streptomycin 50 μg / mL), and L-glutamine (2 mmol / L).

[0235] 2. Measurement of cellular mitochondrial activity

[0236] To investigate the effects of gold nanoparticles complexed or not with NFL-biotin peptide on cell viability, an MTS cell viability assay (ab197010; Abcam, Paris, France) that measures mitochondrial activity in cells was performed.

[0237] These tests were performed on two cell types. Briefly, cells were seeded in 96-well plates at 1000 or 3000 cells per well (cell number varied depending on treatment duration) and incubated at 37°C and 5% CO2 for 24 hours. The cells were then subjected to various treatments: colchicine (1 μg / mL, C9754; Sigma-Aldrich) or gold nanoparticle concentrations (50 to 1000 μmol / L) complexed or not with NFL-biotin peptide, for 24 or 72 hours at 37°C and 5% CO2. At the end of treatment (24 or 72 hours), 20 μL of MTS reagent was added to each well for 4 hours. The absorbance at 490 nm was measured using a Spectra Max M2 spectrophotometer (Molecular Devices, San Jose, California, USA).

[0238] 3. Observation of nanoparticle internalization by transmission electron microscopy

[0239] Cells (MiaPaCa-2 and F98) were seeded in 6-well plates at 400,000 cells per well and incubated at 37°C and 5% CO2 for 24 hours. 100 or 500 μmol / L of nanoparticles (without or with 0.08 mmol / L of peptide) were then incubated with the cells for 24 or 72 hours. After incubation, the cells were washed with 0.1 mol / L phosphate buffer (pH 7.4) and fixed overnight at 4°C with a solution of 2.5% glutaraldehyde in 0.1 mol / L phosphate buffer (pH 7.4). The cells were then rinsed with 0.1 mol / L phosphate buffer. The cells were then rinsed with distilled water and post-fixed in a 1% osmium tetroxide solution for 1 hour. They were then rinsed with water for 3 x 5 minutes and incubated in 50° ethanol for 15 minutes, 70° ethanol for 15 minutes, 95° ethanol for 15 minutes, and 100° ethanol for 3 x 30 minutes. They were then placed in a mixture of 50% ethanol 100° / 50% Epon resin (volume / volume) and incubated overnight. The next day, the remaining diluted Epon resin was removed and replaced with a pure Epon bath for 4 hours, which was then replaced with another pure Epon bath. The plates containing cells in Epon were placed at 37°C for 24 hours, then at 45°C for 24 hours, and finally at 60°C for 72 hours. Once the resin was polymerized at 60°C, 60nm thick ultrathin sections were cut using a UC7 ultramicrotome (Leica, Wetzlar, Germany) and deposited on 150-mesh copper grids. The sections were then contrasted with 3% uranyl acetate in 50° ethanol for 15 minutes and then rinsed with ultrapure water. The sample was then observed using a Jeol microscope (JEM-1400, accelerating voltage 120 kV; Japan) equipped with a Gatan brand 832 Orius SC-1000 camera.

[0240] 4. Photothermal effects on cell lines

[0241] MiaPaCa-2 and F98 cells were seeded at a density of 200,000 cells / mL in a 25 cm 2 The cells were cultured in flasks and grown at 37°C and 5% CO2. The cells were then seeded into 96-well plates at 200 μL per well and left for 24 or 48 hours. 50 μL of culture medium per well was then removed and replaced with a nanoparticle solution and incubated for 24 hours. The culture medium was then removed and the cells were washed three times with PBS (phosphate buffered saline) to remove excess non-internalized nanoparticles. The same volume of culture medium was then added to each well. Each well of the plate was subjected to a power of 0.5 W / cm 2The cells were irradiated with an 808nm laser source. Experiments were performed beforehand to determine the optimal parameters to avoid any risk of artifacts due to laser parameters. The study was performed twice: for 5 minutes and 10 minutes, to see if the "time" parameter would affect the results. After the cells were irradiated with the 808nm near-infrared laser, the culture medium was replaced and left for 24 hours. Cell viability was then tested to check that the nanoparticles had the same effect on the cells before and after the irradiation treatment. To perform the photothermal method, 1mL of the nanoparticle solution was deposited in a tank and the nanoparticles were heated using an infrared laser. A thermal probe was then used to record the temperature rise over 15 minutes.

[0242] B. Results

[0243] 1. Photothermalization of individual nanoparticles

[0244] Figure 3 The temperature was shown to rise above the threshold of 4 °C. Therefore, NFL-biotin-PEG-AuNP nanoparticles are excellent candidates for photothermal testing.

[0245] 2. Effects of NFL-biotin-PEG-AuNPs on cell lines

[0246] 2.1 Effects of nanoparticles on mitochondrial activity

[0247] To assess mitochondrial activity, both cell lines were treated with 1 μg / mL colchicine or with 0, 50, 100, 250, 500, or 1000 μmol / L PEG-AuNP nanoparticles, either complexed or not with NFL-biotin peptide, for 24 or 72 hours. Colchicine interacts with tubulin and disrupts microtubule assembly (Bhattacharyya et al., 2008), leading to cell death. Therefore, colchicine was used as a positive control.

[0248] For MiaPaCa-2 cells treated for 24 h, the MTS assay showed no effect of PEG-AuNPs, but a decrease in mitochondrial activity was observed with 500 μmol / L NFL-biotin-PEG-AuNPs, and a very strong decrease was observed at the 1000 μmol / L dose ( Figure 4 ).

[0249] For MiaPaCa-2 cells treated for 72 h, the MTS assay showed similar results, i.e., PEG-AuNPs had no effect, and mitochondrial activity decreased starting from the treatment with 500 μmol / L NFL-biotin-PEG-AuNPs ( Figure 5 ).

[0250] The same test was performed on F98 rat glioblastoma cells. Similar results were observed, with no effect on mitochondrial activity after 24 hours of PEG-AuNP treatment, whereas mitochondrial activity was dramatically reduced when cells were treated with 1000 μmol / L NFL-biotin-PEG-Au-NPs ( Figure 6 ).

[0251] When F98 cells were treated with nanoparticles for 72 h, a decrease in mitochondrial activity was observed for both particle types at a concentration of 1000 μmol / L ( Figure 7 ).

[0252] 2.2 Cellular internalization of nanoparticles

[0253] MiaPaCa-2 and F98 cells were treated with 500 μmol / L of PEG-AuNPs or NFL-biotin-PEG-AuNPs for 72 hours. In MiaPaCa-2 cells, nanoparticles with or without peptides entered the cells, primarily into the vacuole. Some nanoparticles were trapped in the intercellular spaces. These observations also revealed diverse particle shapes and heterogeneity in particle size.

[0254] The same observations were made for F98 cells.

[0255] Photothermal therapy of MiaPaCa-2 pancreatic and F98 glioblastoma cell lines

[0256] Figures 8 to 11 The irradiation treatment showed no effect on cells alone without nanoparticles (control, 100% cell viability). For the peptides, similar effects were observed with or without irradiation, regardless of the duration of internalization. For the PEG-AuNP nanoparticles without peptides, a decrease in cell viability was observed, with cell viability of approximately 60% relative to the cell viability obtained in the control after 24 and 48 hours of internalization. For the NFL-biotin-PEG-AuNP nanoparticles, a significant decrease in cell viability was observed, with cell viability of approximately 60% for cells irradiated for 5 minutes and approximately 45% for cells irradiated for 10 minutes after 24 hours of internalization. In addition, after 48 hours of internalization, cell viability of cells irradiated for 5 minutes was approximately 40%, and cell viability of cells irradiated for 10 minutes was approximately 30%.

[0257] Example 3

[0258] The inventors have synthesized gold nanoparticles TAT-biotin-PEG-AuNP and VIM-biotin-PEG-AuNP according to the present invention according to a method similar to that described in the previous Example 1 as outlined above.

[0259] By chloroauric acidGold nanoparticles were prepared by reducing HAuCl4 (Sigma-Aldrich) to a concentration of 1 mmol / L.

[0260] After the gold salt was dissolved, biotinylated TAT peptide (TAT.48-60 peptide; TAT-biotin-peptide; BIOT-GRKKRRQRRPPQ-CONH2; Millegen, Toulouse, France) or biotinylated VIM peptide (VIM-biotin-peptide; BIOT-GGAYVTRSSAVRLRSSVPGVRLLQ-CONH2; Millegen, Toulouse, France) was added. The solution was stirred vigorously for 10 minutes. Complexation of the peptide with the HAuCl4 gold salt was achieved by chelation.

[0261] The stabilizer PEG-COOH 600 (polyethylene glycol dicarboxylic acid, Sigma-Aldrich) was then added at room temperature.

[0262] Then, the reducing agent NaBH4 (sodium tetrahydroborate, Sigma-Aldrich) was added to 3+ ions are reduced to neutral gold atoms (Au 0 ).

[0263] After adding the reducing agent, the formation of TAT-biotin-PEG-AuNP and VIM-biotin-PEG-AuNP nanoparticles was observed by the solution color changing from light yellow to bright pink purple.

[0264] Example 4

[0265] The inventors evaluated the effects of gold nanoparticles TAT-biotin-PEG-AuNP and VIM-biotin-PEG-AuNP on cancer cell lines.

[0266] A. Materials and Methods

[0267] 1. Cell lines used

[0268] For this study, the F98 cell line (rat glioblastoma cell line) obtained from the American Tissue Culture Collection (ATCC) was used.

[0269] The cell lines were cultured in DMEM (Dulbecco's modified Eagle's medium; Sigma-Aldrich) supplemented with 10% FBS (fetal bovine serum; Sigma-Aldrich), 1% antibiotics (penicillin 50 IU / mL and streptomycin 50 μg / mL), and L-glutamine (2 mmol / L).

[0270] 2. Measurement of cellular mitochondrial activity

[0271] To investigate the effects of gold nanoparticles complexed or not with TAT-biotin or VIM-biotin peptide on cell viability, an MTS cell viability assay (ab197010; Abcam, Paris, France) measuring mitochondrial activity in cells was performed.

[0272] These tests are carried out on F98 cells. Briefly, cells were seeded in 96-well plates with 1000 or 3000 cells per well (cell number varied depending on treatment duration) and incubated at 37°C and 5% CO2 for 24 hours. Cells were then treated with colchicine (1 μg / mL, C9754; Sigma-Aldrich) or with or without VIM-biotin or TAT-biotin peptide complexed gold nanoparticle concentrations (50 to 1000 μmol / L) at 37°C and 5% CO2 for 24 or 72 hours. At the end of the treatment (24 or 72 hours), 20 μL of MTS reagent was added to each well for 4 hours. The absorbance at 490 nm was measured using a Spectra Max M2 spectrophotometer (Molecular Devices, San Jose, California, USA).

[0273] 3. Observation of Nanoparticle Internalization by Transmission Electron Microscopy

[0274] F98 cells were seeded in 12-well plates at a density of 100,000 cells per well and incubated at 37°C and 5% CO2 for 24 hours. PEG-AuNP (500 μmol / L), VIM-biotin-PEG-AuNP (250 μmol / L) or TAT-biotin-AuNP (250 μmol / L) nanoparticles were then incubated with the cells for 24 or 72 hours. After incubation, the cells were washed with 0.1 mol / L phosphate buffer (pH 7.4) and fixed overnight at 4°C with a solution of 2.5% glutaraldehyde in 0.1 mol / L phosphate buffer (pH 7.4). The cells were then rinsed with 0.1 mol / L phosphate buffer. The cells were then rinsed with distilled water and post-fixed in a 1% osmium tetroxide solution for 1 hour. They were then rinsed with water 3 times for 5 minutes and incubated in 50° ethanol for 15 minutes, 70° ethanol for 15 minutes, 95° ethanol for 15 minutes, and 100° ethanol for 3 times for 30 minutes. They were then placed in a mixture of 50% ethanol 100° / 50% Epon resin (volume / volume) and incubated overnight. The next day, the remaining diluted Epon resin was removed and replaced with a pure Epon bath for 4 hours, which was then replaced with another pure Epon bath. The plates containing cells in Epon were placed at 37°C for 24 hours, then at 45°C for 24 hours, and finally at 60°C for 72 hours. Once the resin was polymerized at 60°C, 60nm thick ultrathin sections were cut using a UC7 ultramicrotome (Leica, Wetzlar, Germany) and deposited on 150-mesh copper grids. The sections were then contrasted with 3% uranyl acetate in 50° ethanol for 15 minutes and then rinsed with ultrapure water. The sample was then observed using a Jeol microscope (JEM-1400, accelerating voltage 120 kV; Japan) equipped with a Gatan brand 832 Orius SC-1000 camera.

[0275] B. Results

[0276] 1. Effects of nanoparticles on mitochondrial activity

[0277] To assess mitochondrial activity, F98 cells were treated with 1 μg / mL colchicine or with 0, 50, 100, 250, 500, or 1000 μmol / L PEG-AuNP nanoparticles complexed or not with VIM-biotin peptide or TAT-biotin peptide for 24 or 72 hours. Similar to the experiments described in Example 2 above, colchicine was used as a positive control.

[0278] For F98 cells treated for 24 hours, the MTS assay showed that PEG-AuNPs had no effect on mitochondrial activity. On the other hand, a significant decrease in mitochondrial activity was observed with 1000 μmol / L VIM-biotin-PEG-AuNPs, and very strong toxicity was observed with 500 μmol / L and 1000 μmol / L TAT-biotin-PEG-AuNPs. In fact, for both treatment concentrations of TAT-biotin-PEG-AuNP nanoparticles, no cells survived after 24 hours ( Figure 12 ).

[0279] For F98 cells treated for 72 h, the MTS assay showed similar results at lower concentrations. In fact, a significant decrease in mitochondrial activity was observed after treatment with 1000 μmol / L PEG-AuNPs, 250 μmol / L TAT-biotin-PEG-AuNPs, or 500 μmol / L VIM-biotin-PEG-AuNPs. In addition, 500 μmol / L TAT-biotin-PEG-AuNPs produced strong toxicity, as did 1000 μmol / L VIM-biotin-PEG-AuNPs ( Figure 13 ).

[0280] The functionality and bioactivity of both VIM-biotin and TAT-biotin peptides were not affected by their combination with PEG-AuNP nanoparticles.

[0281] 2. Cellular internalization of nanoparticles

[0282] F98 cells were treated with 250 μmol / L VIM-biotin-PEG-AuNP or TAT-biotin-PEG-AuNP nanoparticles for 24 hours. Transmission electron microscopy images showed that the PEG-AuNP nanoparticles ( Figure 14 )、VIM-biotin-PEG-AuNP( Figure 15 ) or TAT-biotin-PEG-AuNP ( Figure 16 )-treated F98 cells.

[0283] For cells treated with VIM-biotin-PEG-AuNP or TAT-biotin-PEG-AuNP nanoparticles, more vacuoles were detected. Nanoparticles were quantified by manual counting ( Figure 17 ). It can be seen that the average number of vacuoles containing more than 20 nanoparticles for VIM-biotin-PEG-AuNP and TAT-biotin-PEG-AuNP nanoparticles is higher than that for PEG-AuNP nanoparticles.

Claims

1. Nanoparticles comprising: - an association of at least one metal salt and at least one peptide; and - At least one biocompatible polymer.

2. Nanoparticles according to claim 1, wherein the metal salt is chelated to the peptide.

3. Nanoparticles according to claim 1 or 2, wherein the metal salt and the peptide are encapsulated by the polymer.

4. The nanoparticle according to any one of claims 1 to 3, wherein the nanoparticle has been reduced by a reducing agent.

5. The nanoparticle according to any one of claims 1 to 4, wherein the metal salt is selected from salts of gold (Au), selenium (Se), gadolinium (Gd), cobalt (Co), europium (Eu), terbium (Tb), cerium (Ce), manganese (Mn), iron (Fe), zinc (Zn) and copper (Cu).

6. Nanoparticles according to any one of claims 1 to 5, wherein the metal salt is chloroauric acid HAuCl4.

7. The nanoparticle according to any one of claims 1 to 6, wherein the peptide comprises 2 to 40 amino acids.

8. The nanoparticle according to any one of claims 1 to 7, wherein the peptide is selected from the group consisting of an NFL peptide having the following amino acid sequence: YSSYSAPVSSSLSVRRSYSSSSGS, a TAT peptide having the following amino acid sequence: GRKKRRQRRRPPQ, a VIM peptide having the following amino acid sequence: GGAYVTRSSAVRLRSSVPGVRLLQ, a peptide transporter having the following amino acid sequence: GWTLNSAGYLLGKINLKALAALAKKIL, a penetrating peptide having the following amino acid sequence: RQIKIWFQNRRMKWKK, a MAP peptide having the following amino acid sequence: KLALKLALKALKAALKLA, a Pep-1 peptide having the following amino acid sequence: KETWWETWWTEWSQPKKKRKV, a Pept 1 peptide having the following amino acid sequence: PLILLRLLRGQF, a Pept having the following amino acid sequence: 2 peptides: PLIYLRLLRGQF, IVV-14 peptide having the following amino acid sequence: KLWMRWYSPTTRRYG, Ig(v) peptide having the following amino acid sequence: MGLGLHLLVLAAALQGAKKKRKV, pVEC peptide having the following amino acid sequence: LLIILRRRIRKQAHAHSK, ppTG20 peptide having the following amino acid sequence: GLFRALLRLLRSLWRLLLRA, APP peptide having the following amino acid sequence: APP GLARALTRLLRQLTRQLTRA, peptide having the following amino acid sequence: RLWMRWYSPRTRAYGC; peptide having the following amino acid sequence: WRWYCR, PepFect 14 peptide having the following amino acid sequence: stearyl-AGYLLGKLLOOLAAAALOOLL-NH2, peptide having the following amino acid sequence: GWTLNSAGYLLGKINLKALAALAKKIL, HRSV peptide having the following amino acid sequence: RRIPNRRPRR and its derivatives.

9. The nanoparticle according to any one of claims 1 to 8, wherein the peptide is selected from the group consisting of an NFL peptide having the following amino acid sequence: YSSYSAPVSSSLSVRRSYSSSSGS, a TAT peptide having the following amino acid sequence: GRKKRRQRRRPPQ or a VIM peptide having the following amino acid sequence: GGAYVTRSSAVRLRSSVPGVRLLQ.

10. The nanoparticle according to any one of claims 1 to 9, wherein the biocompatible polymer is a thermosensitive polymer.

11. The nanoparticle according to any one of claims 1 to 10, wherein the biocompatible polymer is selected from polyethylene glycol, polyethylene glycol diacid, polyethylene glycol diamine, collagen, alginate, chitosan, dextran, poly(N-isopropylacrylamide), poly[2-(dimethylamino)ethylmethylmethacrylate] (pDMAEMA), hydroxypropylcellulose, poly(vinyl caprolactam), polyvinyl methyl ether, and combinations thereof.

12. Nanoparticles according to any one of claims 1 to 11, wherein the biocompatible polymer is dicarboxy polyethylene glycol.

13. Nanoparticles according to any one of claims 1 to 12, having a diameter between 20 and 200 nm.

14. Nanoparticles according to any one of claims 1 to 13 for use as pharmaceutical and / or diagnostic agent.

15. The nanoparticle according to any one of claims 1 to 14, for use in preventing or treating glioblastoma or pancreatic cancer.

16. A pharmaceutical composition comprising as active ingredient at least one nanoparticle according to any one of claims 1 to 13, optionally in combination with a pharmaceutically acceptable carrier.

17. A diagnostic composition comprising at least one nanoparticle according to any one of claims 1 to 13.

18. Medical device comprising at least one nanoparticle according to any one of claims 1 to 13.

19. A method for preparing the nanoparticles of any one of claims 1 to 13, the nanoparticles comprising: - an association of at least one metal salt and at least one peptide; and - at least one biocompatible polymer, The method includes: - a step wherein said metal salt is chelated to said peptide; - a step of mixing with said biocompatible polymer; and - a step of reducing said mixture with a reducing agent.

20. The method according to claim 19, characterized in that The method is performed in the absence of chemical surfactants other than the biocompatible polymer and in the absence of chemical binders.

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