Cleavable amphiphilic copolymer, nanoparticle comprising same, and uses

Abstract

The present invention pertains to an amphiphilic copolymer comprising: - a hydrophilic polymer, - a hydrophobic polymer, and - a cleavable unit located between the hydrophilic polymer and the hydrophobic polymer, characterized in that the cleavable unit has the formula (15) as defined in the present application. The present invention also relates to the nanoparticles obtained from these amphiphilic copolymers and to the uses thereof.

Description

CLEVABLE AMPHIPHILE COPOLYMER, The nanoparticle containing it, and its uses technical field

[0001] The present invention relates to amphiphilic copolymers, and more particularly to cleavable amphiphilic copolymers comprising a hydrophilic segment and a hydrophobic segment linked by a specific cleavable group, for forming stable nanoparticles, particularly in the human body, capable of releasing molecules, such as drugs, through controlled activation in response to exogenous or endogenous stimuli. The invention also relates to nanoparticles comprising amphiphilic copolymers and their uses, particularly for diagnostic or therapeutic applications. State of the art

[0002] Nanoparticle-based drug delivery systems have garnered significant interest in recent years due to their potential to enhance therapeutic efficacy and reduce side effects. These structures can encapsulate a wide variety of therapeutic molecules, both hydrophilic and hydrophobic, offering remarkable versatility for diverse biomedical applications.

[0003] Previous research has explored various strategies to improve the stability and control of nanoparticle release. Nanoparticles degradable by pH or enzymatic means have been reported. Other approaches have involved incorporating magnetic iron oxide nanoparticles coated with a hydrophobic surfactant shell into the nanoparticle membranes. While promising, these methods often depend on specific tumor microenvironment conditions, which can vary considerably between patients and even within the same tumor, or require complex formulations incorporating multiple components.

[0004] The type of nanoparticles formed (solid, micellar or vesicular) depends on various factors, including the relative lengths of the hydrophilic and hydrophobic segments, the concentration of the copolymer and the specific conditions of the self-assembly process.

[0005] Promising solutions were reported in WO 2021 / 116331, which describes amphiphilic copolymers comprising a pyridinium core substituted by a hydrophobic chain including a poly-γ-benzyl-L-glutamate (PBLG) block and a hydrophilic chain including a polyethylene oxide (PEG) block. Polymersomes, vesicles formed by the self-assembly of these amphiphilic copolymers, are capable of releasing their contents in a controlled manner in response to specific stimuli "in vivo." However, they exhibit limitations related to a lack of stability under certain conditions, particularly physiological conditions at human body temperature. This instability can lead to premature drug release or degradation of the nanoparticles before they reach their target, thus compromising their therapeutic efficacy. It therefore significantly limits their use for the controlled release of drugs in vivo.

[0006] The inventors identified that the instability of the amphiphilic copolymers described in WO 2021 / 116331 under physiological conditions can be attributed to the nature of the chemical bond linking the picolinium or quinolinium ring to the hydrophilic block. Indeed, in the copolymers of WO 2021 / 116331, the picolinium ring is linked to the polyethylene glycol block by a direct ester bond, that is, a bond in which the hydroxymethyl group of the picolinium is directly esterified with the polymer block. However, this direct ester bond in the benzyl position of the quaternized picolinium ring is particularly susceptible to hydrolysis in aqueous media, due to the electronic activation of the benzyl carbon by the positive charge of the quaternary nitrogen.This sensitivity to hydrolysis can lead to spontaneous cleavage of the copolymer under physiological conditions (37°C, pH 7.4), resulting in premature destabilization of the nanoparticles and uncontrolled release of their contents before reaching the target site.

[0007] To overcome this limitation, the present invention introduces a functional group F between the quaternized nitrogen heterocyclic ring and the hydrophilic (or hydrophobic) polymer. This functional group F, which may be, in particular, a carbamate, a carbonate, a stabilized aromatic ester, a thioester, a phos-phoester, or a quaternary amine, exhibits a bond-breaking energy greater than that of the direct benzyl ester, thereby limiting and / or preventing the spontaneous cleavage of the cleavable motif under physiological conditions. Thus, the functional group F confers upon the amphiphilic copolymer according to the invention increased stability under physiological conditions while preserving its ability to be cleaved in a controlled manner in response to exogenous or endogenous stimuli, such as ionizing radiation.

[0008] In this context, there is a significant need to develop more advanced nanoparticle systems that combine increased stability under various physiological conditions with the ability to release their contents in a controlled manner in response to specific stimuli. The goal is to develop copolymers that are stable under different conditions, including physiological ones, to enable a wide range of applications, while retaining the ability to be cleaved in a controlled manner, whether by ionizing radiation or other mechanisms (photochemical or redox activation). Such systems would not only improve therapeutic efficacy but also broaden the scope of potential applications, particularly in nanomedicine, paving the way for effective treatments for various pathologies that could be used by everyone. Summary of the invention

[0009] To address this need, the invention proposes a cleavable amphiphilic copolymer comprising a hydrophilic polymer, a hydrophobic polymer, and a cleavable motif located between the hydrophilic and hydrophobic polymers. The cleavable motif includes at least one functional group F capable of limiting and / or preventing the spontaneous cleavage of the cleavable motif under physiological conditions. Spontaneous means without exogenous or endogenous stimuli. The presence of this F motif, capable of limiting and / or preventing the cleavage of the cleavable motif under physiological conditions, allows both the stability of the amphiphilic copolymer and that of the nanoparticles comprising said amphiphilic copolymer under physiological conditions, and also enables controlled cleavage of the amphiphilic copolymer and the nanoparticles comprising said amphiphilic copolymer by exogenous or endogenous stimuli.

[0010] Thus, the presence of the F functional group confers stability to the copolymers according to the invention while also allowing them to be cleaved in response to exogenous or endogenous stimuli. This dual property enables the formation of stable nanoparticles that can maintain their integrity until triggered to release their contents in a controlled and targeted manner.

[0011] This stability under physiological conditions advantageously allows for systemic administration of the nanoparticles according to the invention, particularly intravenously. Unlike many existing drug delivery systems that require direct intratumoral injection due to their instability in the bloodstream, the nanoparticles according to the invention retain their structural integrity after intravenous injection. This property considerably expands the range of therapeutic applications, enabling the treatment of deep tumors, metastases, or disseminated tumors that would not be accessible by local injection.The preclinical studies described in the examples below (Examples 2E and 5E) demonstrate that the nanoparticles according to the invention exhibit a prolonged blood circulation time after intravenous injection, with preferential accumulation at the level of tumor tissues, while retaining their ability to release their contents in a controlled manner in response to an exogenous stimulus such as irradiation.

[0012] The functional group F can have the formula (1): in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; and - R8 is chosen between a formula group (2) and a formula group (3): R5 (2) in which: - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S; X3 (3) in which: - X3 is chosen from H and CH3; - X4 is chosen from H and CH3; - Rg is chosen from H, CH3 and an alkyl group; - n8 is an integer between 1 and 10.

[0013] This specific structure of the F functional group allows fine-tuning of the copolymer properties for various applications, including drug delivery and imaging.

[0014] According to one variant, the functional F can have the formula (4): R5 (4) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S.

[0015] According to one embodiment of this variant, the functional F can have the formula (5):O (5) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - R4 is chosen from O, NH and an aryl group; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group with 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.

[0016] According to another variant, the functional F can have the formula (6): in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - X3 is chosen from H and CH3; - X4 is chosen from H and CH3; - Rg is chosen from H, CH3 and an alkyl group; - n8 is an integer between 1 and 10.

[0017] The amphiphilic copolymer according to the invention can have various structures. The hydrophilic and hydrophobic polymers can vary, as can the cleavable motif. The overall structure of the copolymer can be modulated to optimize its physicochemical properties and its reactivity to exogenous or endogenous stimuli.

[0018] According to one representation method, the amphiphilic copolymer has a general structure that can be represented by the following formula (7): R1-R6-MF-R7-R2 (7) where Ri can be a hydrophilic polymer or a hydrophobic polymer, R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer, MF represents the cleavable motif, and R6 and R7 are optional spacers.

[0019] In the context of the present invention, the cleavable motif M designates the structural assembly comprising: - a quaternized nitrogenous heterocyclic nucleus, preferably a picolinium, acridinium, or quinolinium nucleus; - at least one functional group F bonded to said heterocyclic nucleus; and - the linking groups connecting said cleavable motif to the hydrophilic polymer on the one hand and to the hydrophobic polymer on the other hand.

[0020] This structure allows for great flexibility in copolymer design, offering the possibility of adjusting its physicochemical properties and reactivity to stimuli. The specific arrangement of hydrophilic and hydrophobic components, separated by the cleavable motif, gives the copolymer its ability to self-assemble into stable nanoparticles while maintaining the possibility of controlled release of the molecules encapsulated by these nanoparticles. This structure, with formula (7), thus enables the design of a wide range of amphiphilic copolymers with customized properties, allowing the creation of nanoparticles with specific sizes, stabilities, and release profiles suited to various biomedical applications.

[0021] Regardless of the formulation of the amphiphilic copolymer according to the invention, it can be used as such or in the form of a pharmaceutically acceptable salt, solvate and / or stereoisomer.

[0022] The amphiphilic copolymer according to the invention is stable under physiological conditions for at least 4 hours, preferably for 4 hours and 7 days, between 4 hours and 5 days, between 4 hours and 72 hours, between 4 hours and 48 hours, and between 4 hours and 24 hours. Preferably, it is stable particularly under the following conditions: - between 0 and 60°C, preferably between 20 and 50°C, advantageously between 35 and 45°C, and / or - at pH between 5 and 8, especially at pH between 6 and 8.

[0023] This stability under physiological conditions, including physiological temperatures and pH, ensures that the nanoparticles formed from these copolymers maintain their integrity in the bloodstream, allowing for prolonged circulation times and improved targeting efficiency.

[0024] The invention also relates to a process for preparing the amphiphilic copolymer involving steps of synthesizing the cleavable unit, functionalizing the hydrophilic polymer, synthesizing the hydrophobic polymer, optionally functionalizing the hydrophobic polymer, and coupling the blocks. The process may include ring-opening polymerization techniques or click chemistry, for example.

[0025] According to one variant, the process for preparing an amphiphilic copolymer according to the invention comprises carrying out the following steps: - Synthesis of the cleavable motif, the hydrophilic polymer and the hydrophobic polymer, - Functionalization of the hydrophilic polymer with the cleavable motif to obtain a hydrophilic block, - Coupling of the two blocks (hydrophilic block and hydrophobic polymer) to obtain the amphiphilic copolymer.

[0026] According to another variant, the process for preparing an amphiphilic copolymer according to the invention comprises carrying out the following steps: - Synthesis of the cleavable motif, the hydrophilic polymer, and the hydrophobic polymer, - Functionalization of the hydrophobic polymer with the cleavable motif to obtain a hydrophobic block, - Coupling of the two blocks (hydrophobic block and hydrophilic polymer) to obtain the amphiphilic copolymer.

[0027] This synthetic approach allows the controlled assembly of the amphiphilic copolymer, ensuring precise incorporation of the cleavable motif and enabling customization of the hydrophilic and hydrophobic segments.

[0028] The invention also relates to nanoparticles comprising at least one amphiphilic copolymer according to the invention. These nanoparticles are stable under physiological conditions and can encapsulate various molecules, including pharmaceutical or cosmetic active ingredients, while being cleavable in response to one or more exogenous or endogenous stimuli.

[0029] The nanoparticles according to the invention comprise at least one amphiphilic copolymer. According to one embodiment, the invention relates to nanoparticles formed from several amphiphilic copolymers of the subject of this application.

[0030] The nanoparticles according to the invention can be solid (solid) nanoparticles or hollow nanoparticles with an outer membrane and a hollow inner part.

[0031] Nanoparticles can include inorganic and / or organic elements, which broaden their application in imaging and therapy according to the invention. These are then referred to as functional nanoparticles. The incorporation of these elements can confer additional functionalities such as imaging capabilities or reactivity to exogenous stimuli, enhancing the theranostic potential of the nanoparticles according to the invention.

[0032] According to one embodiment, the nanoparticles according to the invention include inorganic elements selected in particular from mineral, metallic or semi-metallic particles. These elements can be integrated into the nanoparticles by grafting or nucleation or they can be encapsulated in the nanoparticle according to the invention.

[0033] In another embodiment, the nanoparticles can incorporate one or more biological or chemical elements for biological or chemical functionalization of the nanoparticles according to the invention, particularly for the purpose of biological targeting. These biological elements can be selected from among antibodies, peptides, nucleic acids, and sugars. This biological or chemical functionalization allows the nanoparticle to interact specifically with target cells, tissues, or molecules in a living organism. This approach is particularly advantageous in the context of targeted therapy (for example, to treat diseases such as cancer) or molecular diagnostics. The objective may be, in particular, to direct the nanoparticle to a specific site in the body, such as a cancer cell, a specific receptor, or a local pathology, while minimizing side effects on healthy cells.

[0034] The hydrodynamic diameter of nanoparticles and incorporated elements, as well as encapsulation efficiencies, can be optimized for specific applications.

[0035] The nanoparticles according to the invention exhibit advantageous characteristics in terms of stability, particularly under physiological conditions. They can efficiently encapsulate various molecules of interest while retaining the ability to release them in a controlled manner.

[0036] The nanoparticles according to the invention are preferentially stable under physiological conditions for at least 4 hours, preferably for 4 hours and 7 days, between 4 hours and 5 days, between 4 hours and 72 hours, between 4 hours and 48 hours, and between 4 hours and 24 hours. Preferably, they are stable particularly under the following conditions: - between 0 and 60°C, preferably between 20 and 50°C, advantageously between 35 and 45°C, and / or - at pH between 5 and 8, especially at pH between 6 and 8.

[0037] This prolonged stability under physiological conditions allows for extended circulation times and improved accumulation at target sites, increasing the therapeutic efficacy of encapsulated drugs. This unique combination of properties opens new perspectives for targeted drug delivery and medical imaging. The potential applications of this technology cover a wide range of therapeutic and diagnostic fields.

[0038] The nanoparticles according to the invention can be used, in particular, as contrast agents for various bioimaging modalities, for the controlled delivery of drugs for the treatment of various pathologies. They exhibit particular potential for the vectorization of active ingredients and therapeutic molecules, photodynamic therapy, theranostics, and image-guided radiotherapy.

[0039] The nanoparticles according to the invention are particularly interesting for use in oncology, especially when the cleavable motif of the amphiphilic copolymer is cleavable under ionizing radiation. Indeed, many cancer cases require radiotherapy, which acts on cancer cells using ionizing radiation. Ionizing radiation, particularly X-rays and gamma rays, has high tissue penetration and low diffusion. Recent radiotherapy techniques allow for tumor targeting with high spatial and temporal resolution. This clinical relevance, tissue penetration, and tumor targeting of radiotherapy make X-rays (or gamma rays) a particularly suitable exogenous stimulus for controlling the release of chemotherapy drugs contained in nanoparticles according to the invention that are cleavable under ionizing radiation.The nanoparticles according to the invention, containing one or more therapeutic molecules, constitute nanomedicines that are stable before irradiation and activatable by irradiation. The invention thus provides a means of increasing the effectiveness of chemotherapy molecules in cancer treatment due to their controlled release in time and space at the level of cancer cells, while simultaneously reducing their systemic toxicity.

[0040] Uses are also possible in diagnostics if the nanoparticle encapsulates a fluorescent molecule, for example, which would be released in a controlled manner at a particular site, such as a tumor site, for example, in the case of cancer diagnosis.

[0041] According to a particular embodiment, the invention also specifically relates to a nanomedicine compound. This is a nanoparticle according to the invention encapsulating at least one therapeutic molecule and / or conjugated to at least one therapeutic molecule. This compound is stable under physiological conditions, and the therapeutic molecule is inactive under these conditions, as long as it remains encapsulated within the nanoparticle. The compound according to the invention can be cleaved by an endogenous or exogenous stimulus, such as ionizing radiation, and is capable of selectively releasing the therapeutic molecule contained within the nanoparticle.

[0042] The invention also relates to methods for preparing nanoparticles comprising at least one amphiphilic copolymer according to the invention.

[0043] In one variant, the process is carried out by self-assembly of copolymers to form a nanoparticle. This could involve, for example, self-assembly through a nanoprecipitation and purification process.

[0044] The invention also relates to compositions, particularly pharmaceuticals, and medical devices comprising these nanoparticles, as well as their use in treatment methods involving the activation of the cleavable motif by specific stimuli.

[0045] In particular, the invention relates to a pharmaceutical composition comprising a nanoparticle according to the invention and a pharmaceutically acceptable excipient. The incorporation of these nanoparticles into pharmaceutical compositions facilitates their use in clinical applications, providing a means of administering encapsulated drugs or diagnostic agents in a controlled and targeted manner.

[0046] Finally, the invention proposes methods using these nanoparticles, where the activation of the cleavable motif can be achieved allowing spatiotemporal control of the release of molecule(s) transported by the nanoparticle according to the invention.

[0047] The invention also relates to a method of activating nanoparticles according to the invention, in particular using ionizing radiation(s).

[0048] According to another aspect, the invention also relates to a method of treating a disease, comprising administering to a subject in need of it, a nanoparticle according to the invention comprising at least one therapeutic molecule, and the release of this therapeutic molecule in a controlled manner by a stimulus, preferably an exogenous stimulus such as one or more ionizing radiation(s).

[0049] According to one variant, the invention also relates to a method of treating or diagnosing a disease, comprising administering to a subject in need of it, a nanoparticle according to the invention comprising at least one molecule useful for diagnosis, such as a contrast agent for example, and the release of this molecule in a controlled manner by a stimulus, preferably an exogenous stimulus such as one or more ionizing radiation(s).

[0050] Thus, the invention offers a versatile and effective solution to address the challenges of controlled drug delivery and medical imaging, with significant potential, in particular, for improving treatments in various therapeutic areas. It notably provides a versatile and innovative platform for the development of more effective and safer treatments in nanomedicine.

[0051] Other features and advantages will become apparent from the detailed description of the invention, the examples and figures that follow, these being purely illustrative and in no way limiting the scope of the invention.

[0052] Brief description of the figures [Fig. 1] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PBLG 21 obtained via SPAAC. [Fig. 2] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PBLG 21 . [Fig. 3] represents the chemical structure of the PEG copolymer 44 -carbamate-p-pico-PBLG 21 . [Fig. 4] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PBLG 15 . [Fig. 5] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PBLG 25 . [Fig. 6] represents the chemical structure of the PEG copolymer 44-carbamate-o-pico-PBLG 35 . [Fig. 7] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PBLG 42 . [Fig. 8] represents the chemical structure of the PEG copolymer 44 -carbamate-pico-o-PLL5-co-PBLG 15 . [Fig. 9] represents the chemical structure of the PEG copolymer 44 -carbamate-pico-o-PTyr5-co-PBLG 15 . [Fig. 10] represents the chemical structure of the PSar copolymer 42 -carbamate-o-pico-PBLG 21 . [Fig. 11] represents the chemical structure of the PSar copolymer 29 -carbamate-o-pico-PLA 45 [Fig. 12] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PLA 120 . [Fig. 13] represents the chemical structure of the PEG copolymer 44 -carbamate-o-pico-PLA 45 . [Fig. 14] represents the chemical structure of the PEG copolymer 44 -carbonate-o-pico-PBLG25 . [Fig. 15] represents the chemical structure of the PEG copolymer 44 -aromatic ester-o-pico-PBLG 21 . [Fig. 16a] represents a cryogenic transmission electron microscopy (Cryo-TEM) image of nanoparticles formed from the PEG copolymer 44 -carbamate-o-pico-PBLG 21 at 1 mg / mL. [Fig. 16b] represents a Cryo-TEM image of nanoparticles formed from the PEG copolymer 44 -carba-mate-o-pico-PBLG 21 at 1 mg / mL, showing vesicular morphology. [Fig. 17a] represents a Cryo-TEM image of nanoparticles formed from the PEG copolymer 44 -aromatic ester-o-pico-PBLG 21 at 3 mg / mL. [Fig. 17b] represents a Cryo-TEM image of nanoparticles formed from the PEG copolymer 44 -aromatic ester-o-pico-PBLG 21 at 3 mg / mL, showing vesicular morphology. [Fig. 18a] represents a TEM image of nanoparticles formed from the PEG copolymer 44 -carbamate-o-pico-PBLG 25 at 5 mg / mL, showing vesicles. [Fig. 18b] represents a TEM image of nanoparticles formed from the PEG copolymer 44 -carbamate-o-pico-PBLG 25 at 5 mg / mL, showing micelles. [Fig. 19] represents a TEM image of nanoparticles formed from the PSar copolymer 42 -carbamate-o-pico-PBLG 25 at 3 mg / mL. [Fig. 20] represents whole-body in vivo fluorescence imaging at different time points (T0, 1h, 2h30, 5h, 24h and 48h) after intravenous injection of Cy7-labeled nanoparticles (PEG 44 -carbamate-o-pico-PBLG 21 ) in mice bearing subcutaneous CT26 tumors. [Fig. 21] represents ex vivo fluorescence in isolated organs at 5h, 24h and 48h after intravenous injection of Cy7-labeled nanoparticles in mice bearing CT26 subcutaneous tumors. [Fig. 22] represents the quantification of ex vivo fluorescence in isolated organs at 5h, 24h and 48h after intravenous injection of Cy7-labeled nanoparticles in mice bearing CT26 subcutaneous tumors. [Fig. 23] represents the ex vivo fluorescence ratios tumor / muscle, tumor / liver and tumor / spleen at 5h, 24h and 48h after intravenous injection of Cy7-labeled nanoparticles in mice bearing CT26 subcutaneous tumors. [Fig. 24] represents the ex vivo fluorescence in isolated organs at 5h, 24h and 48h after intravenous injection of Cy7-labeled nanoparticles in mice bearing KPC subcutaneous tumors (pancreatic cancer). [Fig. 25] represents the ex vivo fluorescence ratios tumor / muscle, tumor / liver and tumor / spleen at 5h, 24h and 48h after intravenous injection of Cy7-labeled nanoparticles in mice bearing KPC subcutaneous tumors (pancreatic cancer). [Fig. 26] represents the ex vivo fluorescence ratios tumor / muscle, tumor / liver and tumor / spleen at 5h, 24h and 48h after intravenous injection of Cy7-labeled nanoparticles in mice bearing subcutaneous KPC tumors (pancreatic cancer). [Fig. 27a], [Fig. 27b] and [Fig. 27c] represent the individual tumor growth curves for groups G1 (control), G2 (1 single injection of doxorubicin-loaded DXN-1, 10 days after implantation of cancer cells without irradiation), G3 (a single irradiation at 8 GY 11 days after implantation of cancer cells), and G4 (1 single injection of doxorubicin-loaded DXN-1, 10 days after implantation of cancer cells that underwent irradiation 11 days after implantation) in CT26 subcutaneous tumor mice. Detailed description of the invention

[0053] Definitions

[0054] For the purposes of this invention, "contrast agent" means a substance used to enhance the visibility of internal body structures in medical imaging.

[0055] For the purposes of this invention, "alkyl" or "alkyl group" refers to saturated carbon chains, which may be either linear or branched. Preferably, the chain contains between 1 and 10 carbon atoms, preferably from 1 to 6 carbon atoms, and more specifically from 1 to 3 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl (n-propyl and isopropyl), butyl (n-butyl, isobutyl, tert-butyl), as well as other chains such as pentyl, hexyl, and octyl. These groups may be substituted or unsubstituted.

[0056] For the purposes of this invention, "aryl" or "aryl group" means an aromatic monocycle comprising six carbon atoms. It may also be an aromatic bicyclic system comprising ten carbon atoms or an aromatic tricyclic system comprising fourteen carbon atoms. Typical examples of aryl groups include the phenyl, naphthyl, and anthracene groups.

[0057] For the purposes of this invention, "self-assembly" refers to the process by which amphiphilic copolymers spontaneously organize themselves into ordered structures in the form of nanoparticles, such as micelles or polymersomes, in aqueous solution.

[0058] For the purposes of this invention, "copolymer cleavage" refers to the copolymer's ability to break its chemical bonds at the cleavable unit under the influence of an endogenous or exogenous stimulus. This cleavage is designed and controlled to occur under specific conditions. Cleavage is therefore a compromise between the copolymer's stability under physiological conditions and its ability to decompose or destabilize under an exogenous or endogenous stimulus. This cleavage of the amphiphilic copolymer allows the nanoparticle to release its contents or decompose after activation by one or more exogenous and / or endogenous stimuli.

[0059] For the purposes of this invention, "cleavage" refers to the breaking of at least one chemical bond within the copolymer, resulting in the separation of its constituent units. A "cleavable motif" is a chemical unit integrated into the copolymer structure that enables this cleavage under specific conditions, such as ionizing radiation, radiation, redox reactions, or other environmental stimuli. Cleavage by ionizing radiation occurs when the polymer is exposed to ionizing radiation, such as X-rays or gamma rays. The energy of the ionizing radiation breaks the chemical bonds of the cleavable motif, which can lead to the degradation of the polymer into smaller fragments. This cleavage can occur, in particular, through a local electron transfer reaction generated by the interaction of X-rays / gamma rays, water, and the cleavable motif of the copolymer according to the invention.This indirect mechanism, involving the radiolysis of water by ionizing radiation, leads to the generation of reactive species, particularly aqueous electrons, which interact with the cleavable motif, causing bond breaking through electron transfer. Photocleavage is induced by ultraviolet (UV) or visible radiation. A photocleavable motif can break when exposed to light of a specific wavelength. Redox cleavage is based on the modification of the cleavable motif in response to changes in the redox state of one or more parts of the polymer. Redox reactions, which involve the loss or gain of electrons, can lead to the breaking of chemical bonds.

[0060] For the purposes of this invention, "physiological conditions" refers to the physiological conditions of a living being, in particular the physiological conditions of a human being, that is to say, non-pathological conditions. Preferably, "physiological conditions" refers to the physiological parameters of a living being in a non-pathological state, preferably a human being, and in particular the body temperature of the living being and / or the body pH.

[0061] The term "degree of polymerization" or "DP" in the context of this invention refers to the average number of monomer units per polymer chain. The degree of polymerization is related to the number-average molar mass (Mn) by the equation DP = Mn / M0, where M o is the molar mass of the repeating monomer unit.

[0062] For the purposes of this invention, a "derivative" is defined as a compound whose skeletal structure is identical to that of the parent compound, but which may contain various substituents. These substituents may include, for example, a ring atom replaced by an alkyl, alkenyl, alkoxy, halogen, cyano, amino, alkylamino, nitro, or hydroxyl (-OH) group. This modified structure retains the basic framework while introducing functional diversity.

[0063] By "hydrodynamic diameter" in the sense of the invention, we mean the diameter of a hypothetical sphere which would have the same diffusion coefficient as the measured particle, taking into account the hydration layer around the particle.

[0064] For the purposes of this invention, "encapsulation efficiency" means a percentage of the amount of active substance encapsulated in a system relative to the amount initially used, indicating the system's ability to retain the active substance during the encapsulation process.

[0065] For the purposes of this invention, "encapsulation" refers to the physical trapping of one or more molecules within the structure of a nanoparticle, whether solid or hollow. In the case of a solid nanoparticle (solid or micellar), encapsulation refers to the trapping of molecules within the hydrophobic matrix formed by the hydrophobic segments of amphiphilic copolymers, or through electrostatic or complexation interactions within the nanoparticle structure. In the case of a hollow nanoparticle (polymersome), encapsulation refers to the trapping of molecules either in the internal aqueous compartment (for hydrophilic molecules), in the hydrophobic bilayer membrane (for hydrophobic molecules), or at the membrane / aqueous compartment interface.The term "encapsulation" thus encompasses all mechanisms of physical retention of molecules within nanoparticles, regardless of their morphology (solid, micellar, or vesicular), and is distinct from covalent conjugation, in which the molecule is chemically bonded to the copolymer. "Theranostics," as used in this invention, refers to an approach combining diagnostic and therapeutic capabilities within a single entity, enabling diagnosis, targeted drug delivery, and monitoring of treatment response using an imaging technique.

[0066] For the purposes of this invention, "polydispersity index" means a measure of the homogeneity of the particle size distribution in a suspension, where a value close to 0 indicates a monodisperse distribution.

[0067] For the purposes of this invention, a "heterocyclic group" or "heterocycle" is defined as a group containing one or more non-aromatic or aromatic rings, comprising 5, 6, or 7 members, in which one or more carbon atoms of the ring are replaced by heteroatoms (oxygen, sulfur, or nitrogen). This group may be monocyclic, bicyclic, or tricyclic, and includes structures such as pyrrolidine, imidazole, piperazine, dioxane, and others. The heteroatoms may be oxidized or quaternized, depending on the nature of the group.

[0068] "Controlled release" in the context of the invention refers to the ability to modulate the release of the content encapsulated in the nanoparticles in response to specific stimuli, allowing for targeted and time-defined delivery.

[0069] For the purposes of this invention, "cleavable motif" means a part of the copolymer structure designed to break under the influence of specific stimuli, thereby enabling the controlled release of the encapsulated contents.

[0070] For the purposes of this invention, "nanoparticle" means any object, regardless of its shape, having at least one dimension between 1 and 400 nanometers, preferably between 10 and 400 nm, between 40 and 200 nm, and in particular between 80 and 120 nm. The nanoparticles according to this invention can take various forms. These may include, in particular: - solid (filled) nanoparticles: these structures generally form when the hydrophobic segment of the copolymer is significantly longer than the hydrophilic segment. - Micelles: These structures are formed by the self-assembly of amphiphilic copolymers, in which hydrophobic segments form a compact core surrounded by a ring of hydrophilic segments. Micelles can encapsulate hydrophobic molecules within their core. - Hollow nanoparticles: including polymersomes, these structures have an outer membrane and a hollow inner portion. Polymersomes, in particular, offer a biomimetic structure similar to liposomes, but with improved stability and versatility.

[0071] For the purposes of this invention, a "functional nanoparticle" is defined as a nanoparticle comprising at least one inorganic element and / or at least one organic element (biological or chemical). These incorporated elements are integrated into the nanoparticle by grafting or nucleation, or are encapsulated within the nanoparticle. A nanoparticle can be considered functional when a drug is encapsulated within it.

[0072] For the purposes of this invention, "nanoprecipitation" means a method for preparing nanoparticles by self-assembly based on solvent exchange, i.e. a solvent displacement process leading to the transfer of a portion of the polymer from a good to a bad solvent when added to an aqueous medium or vice versa.

[0073] For the purposes of this invention, "pharmaceutically acceptable" means a compound or composition that is chemically and / or toxicologically compatible with the other components of the compound or composition, as well as with living beings, including humans, who use it to prevent or treat diseases or conditions.

[0074] For the purposes of this invention, "polymer" means any polymer in the broadest sense, including copolymers. Thus, the hydrophobic polymer covered by this application may be a hydrophobic copolymer, and the hydrophilic polymer covered by this application may be a hydrophilic copolymer. Similarly, the term polymer also includes functionalized polymers onto which one or more inorganic, biological, or chemical molecules are grafted.

[0075] For the purposes of this invention, "biodegradable polymer" means a polymer that will degrade or be absorbed naturally in the body of a subject.

[0076] For the purposes of this invention, "polymersome" means an artificial vesicle formed by the self-assembly of amphiphilic copolymers, exhibiting a bilayer structure similar to that of liposomes.

[0077] For the purposes of this invention, "micelles" are nanoparticles formed by the self-assembly of amphiphilic copolymers in an aqueous medium, in which the hydrophobic segments aggregate to form a compact core while the hydrophilic segments form an outer ring in contact with the aqueous phase. Micelles differ from polymersomes in that they do not have a bilayer membrane structure or an internal aqueous cavity. The micelles according to this invention can encapsulate hydrophobic molecules in their core, as well as hydrophilic or charged molecules, such as nucleic acids, through electrostatic interactions or complexation within the micelle structure.

[0078] For the purposes of this invention, "pharmaceutical or cosmetic active ingredient" means any substance or mixture of substances intended for use in the diagnosis, treatment or prevention of disease, or in modifying physiological functions in humans or animals.

[0079] "Image-guided radiotherapy" in the context of the invention means a treatment technique using ionizing radiation, the accuracy of which is improved by the simultaneous use of medical imaging techniques.

[0080] For the purposes of this invention, "radiation" or "ionizing radiation" means non-visible or ultraviolet light radiation, in particular any radiation used in radiotherapy, preferably an X-ray or a gamma ray.

[0081] For the purposes of this invention, a "pharmaceutically acceptable salt" is defined as a non-toxic addition salt formed from the compound of the invention. Pharmaceutically acceptable salts of the amphiphilic copolymers of the invention may be, for example, salts formed with inorganic acids such as hydrochloric acid, sulfuric acid, phosphoric acid, or organic acids such as acetic acid, formic acid, propionic acid, lauric acid, benzoic acid, salicylic acid, citric acid, malonic acid, succinic acid, glutamic acid, or other similar organic and inorganic acids. These salts may also be formed with physiologically acceptable amino acids or organic bases, such as lysine, serine, creatine, glucosamine, or derivatives such as N-methylglucosamine or dimethylglucosamine.Furthermore, pharmaceutically acceptable salts can also be obtained with alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (such as calcium or magnesium), or ammonium salts. These salts can be prepared by conventional methods of reaction between the compound of the invention and the appropriate acid or base. It is also possible to form quaternary salts from nitrogen-containing basic groups using reagents such as alkyl halides, dialkyl sulfates, or other reagents known for quaternization. Those skilled in the art know that these addition salts can be prepared by well-established methods by reacting the compounds of the invention with appropriate acids or bases, whether inorganic or organic.The present invention also covers all possible salts of the disclosed compounds, whether individual salts or mixtures of salts in different proportions.

[0082] For the purposes of this invention, "solvate" means a compound formed by the physical and / or chemical interaction (such as solvation or hydrogen bonding) between the copolymer according to the invention and a solvent molecule. A solvate may be a simple solvate, a disolvated, or a semi-solvated, and may be formed in specific molecular ratios, such as 2:1, 1:1, or 1:2, respectively. This bonding may be ionic or covalent, and in some cases, for example, when one or more solvents are bonded to a solid crystal lattice, this solvate may be separated into two distinct phases: a solution phase and a separable solvent. The copolymers according to the invention may be solvated with pharmaceutically acceptable solvents, such as water, methanol, or ethanol. This application covers both solvated and unsolvated forms of the copolymers according to the invention. In particular, a solvate can be a hydrate.

[0083] For the purposes of this invention, "endogenous stimulus" means a factor that is present only or amplified (overexpressed) in a pathological environment compared to non-pathological physiological conditions, such as pH, reactive oxygen species (ROS) or enzymes, for example, capable of inducing copolymer cleavage.

[0084] For the purposes of this invention, "exogenous stimulus" means a controlled (external) environmental factor, such as light, ultrasound, or ionizing radiation, capable of triggering copolymer cleavage.

[0085] The term "stable amphiphilic copolymer" or "stable copolymer" in the context of this invention refers to the ability of the amphiphilic copolymer to maintain its chemical and structural integrity under physiological conditions, i.e., in the absence of specific exogenous stimuli. This implies the strength of the chemical bonds within the copolymer, particularly in a physiological environment. Thus, copolymer stability designates the copolymer's resistance to degradation, hydrolysis, or other processes that could alter its structure under physiological conditions, including temperature and / or pH and / or the presence of certain ions and / or enzymes. Therefore, a stable amphiphilic copolymer is an amphiphilic copolymer that maintains its chemical structure and physicochemical properties under physiological conditions for a given period of time.

[0086] The term "nanoparticle stability" or "stable nanoparticle" as used in this invention refers to the nanoparticle's ability to maintain its structure and integrity over time, particularly under physiological conditions. This means maintaining its shape, size, and composition under physiological conditions. This stability is essential to ensure that the nanoparticle retains its shape and functionality until a specific endogenous or exogenous stimulus causes its cleavage, allowing the release of its contents and / or the degradation of the nanoparticle's structure. This guarantees that it remains effective in its role of transporting and delivering molecules without prematurely degrading. Thus, a stable nanoparticle as used in this invention is defined as a nanoparticle that retains its size, shape, and functional properties under physiological conditions for a given period of time.

[0087] For the purposes of this invention, "stereoisomer" means a compound having the same chemical structure, but whose atoms or groups are arranged differently in space. Stereoisomers include enantiomers, diastereoisomers, conformational isomers (rotamers), geometric isomers (cis / trans), trans-resistant isomers, etc.

[0088] For the purposes of this invention, "particulate structure" means, in particular, a polymer particle within which nanoparticles are encapsulated and / or on the surface of which nanoparticles are adsorbed.

[0089] Unless otherwise stated, each value mentioned to describe a range of values ​​in this application, including any sub-range of values ​​within that range, is included in the range. For example, when the range of values ​​is stated as being from 1 to 5, this includes all values ​​within that interval, including the values ​​1 and 5, as well as all possible sub-ranges such as 1-4, 1-3, and 1-2.

[0090] This disclosure should be interpreted in accordance with generally accepted chemical principles. In some cases, it may be possible to remove a hydrogen atom to incorporate a substituent at a specific position.

[0091] Amphiphilic copolymer according to the invention:

[0092] According to a first object, the invention relates to an amphiphilic copolymer comprising a hydrophilic polymer, a hydrophobic polymer, and a cleavable motif located between the hydrophilic polymer and the hydrophobic polymer.

[0093] The amphiphilic copolymers of this disclosure can be used in various forms, including as pharmaceutically acceptable salts, solvates and / or stereoisomers.

[0094] The cleavable motif, positioned between the hydrophilic segment and the hydrophobic segment, is preferentially designed to be sensitive to exogenous stimuli such as light, ionizing radiation or ultrasound, or to endogenous stimuli such as changes in pH or the presence of specific enzymes.

[0095] According to one embodiment, the cleavable motif according to the invention comprises a redox-sensitive group, such as, for example, an N-alkyl heterocycle derivative, such as picolinium, acri-dinium or quinolinium derivatives.

[0096] According to one embodiment, the cleavable motif is a photocleavable motif and / or a cleavable motif under ionizing radiation.

[0097] The cleavable motif includes a functional group F that plays a role in controlling spontaneous cleavage under physiological conditions. The cleavable motif comprises at least one functional group F capable of limiting and / or preventing spontaneous cleavage of the motif under physiological conditions. Spontaneous cleavage is defined as occurring without exogenous or endogenous stimuli. The cleavable motif thus includes a functional group that limits spontaneous cleavage under physiological conditions while allowing controlled cleavage in response to specific stimuli. Functional group F is capable of preventing spontaneous cleavage of the motif under physiological conditions for at least 4 hours, preferably for 4 hours to 7 days, and even more preferably for at least 10 hours, at least 24 hours, at least 48 hours, at least 72 hours, and at least 5 days.

[0098] Preferably, functional group F is capable of preventing spontaneous cleavage of the cleavable motif under the following conditions: - between 0 and 60°C, preferably between 20 and 50°C, advantageously between 35 and 45°C, and / or - at pH between 5 and 8, especially at pH between 6 and 8.

[0099] The functional group F preferentially exhibits a bond breaking energy (i.e., the energy required to break a specific chemical bond within the cleavable motif of the copolymer) that allows both the stability of the cleavable motif (and consequently that of the amphiphilic copolymer comprising said cleavable motif, and that of the nanoparticle comprising said amphiphilic copolymer) under physiological conditions, and its cleavage in response to exogenous and / or endogenous stimuli. Thus, the functional group F can have a specific bond breaking energy range that allows stability under physiological conditions while also permitting controlled cleavage in response to specific stimuli.

[0100] The functional group F can have the formula (1): (1) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; and - R8 is chosen between a group of formula (2) and a group of formula (3): R 5 (2) in which: - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S. in which: - X3 is chosen from H and CH3; - X4 is chosen from H and CH3; - R9 is chosen from H, CH3 and an alkyl group; - n8 is an integer between 1 and 10.

[0101] This specific structure of the F functional group allows fine-tuning of the copolymer properties for various applications, including drug delivery and imaging.

[0102] According to one variant, the functional F can have the formula (4): R5 (4) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S.

[0103] According to one embodiment of this variant, the functional F can have the formula (5): O (5) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - R4 is chosen from O, NH and an aryl group; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group with 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.

[0104] According to another variant, the functional F can have the formula (6): in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - X3 is chosen from H and CHA; - X4 is chosen from H and CHA; - Rg is chosen from H, CHa and an alkyl group; - n8 is an integer between 1 and 10.

[0105] The cleavable motif can be represented by the following formula (8): MF (8)

[0106] According to one variant, the cleavable motif may have the following formula (9): O)

[0107] According to one variant, the cleavable motif can have the following formula (10): (10)

[0108] According to one variant, the cleavable motif can have the following formula (11): O (11)

[0109] According to one variant, the cleavable motif may have the following formula (12): (12)

[0110] According to a preferred embodiment, the copolymer according to the invention has the following formula (7): R1-R6-MF-R7-R2 (7) where Ri can be a hydrophilic polymer or a hydrophobic polymer, R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer, MF represents the cleavable motif, and R6 and R7 are optional spacers.

[0111] According to a particular embodiment, the copolymer according to the invention has the formula 4 in which: - Ri can be a hydrophilic polymer or a hydrophobic polymer; - R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer; - MF represents the cleavable pattern; - R6 presents the following formula (13): in which: * n2 and n4 are identical or different integers between 0 and 5; * n3 is 0 or 1; - R7 presents the following formula (14): (14) in which: * n5 and n7 are identical or different integers between 0 and 5; * n6est 0 or 1.

[0112] The unique structure and properties of these amphiphilic copolymers offer potential advantages in areas such as targeted drug delivery, controlled release of therapeutic agents, and the development of advanced diagnostic tools. The ability to fine-tune the copolymer composition and the nature of the cleavable motif provides a flexible platform for designing nanoparticles with tailored characteristics for diverse biomedical applications.

[0113] M can present the following formula (15): R3' (15) in which: - N + represents a positively charged nitrogen atom; -Y -is an anion, preferentially chosen from among the halogens (Cl - , F - , Br - ), acetate, OTs, OTf, N-acetyl type amino acid; - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * Or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure.

[0114] Thus, according to one embodiment, the cleavable motif MF can have the following formula (16): R3' in which: - N + represents a positively charged nitrogen atom; - Y - is an anion, preferentially chosen from among the halogens (Cl - , F - , Br -), acetate, OTs, OTf, N-acetyl type amino acid; - R1', R2', R3', R4' and R5' are substituents which can be:* H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * Or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure. - F being located at any position of R1', R2', R3', R4' and R5', where said R1', R2', R3', R4' and R5' is then absent.

[0115] When F has formula (1), the cleavable motif can have the following formula (17): (17) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - N + represents a positively charged nitrogen atom; - Y - is an anion, preferably chosen from among halogens (Cl', F', Br), acetate, OTs, OTf, N-acetyl type amino acids; - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure; the lateral chain comprising R3 being located at any position of R1', R2', R3', R4' and R5', where said R1', R2', R3', R4' and R5' is then absent, and - R8 is chosen between a group of formula (2) and a group of formula (3): R5 II X1-X2-R4 (2) in which: - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S. in which: - X3 is chosen from H and CH3; - X4 is chosen from H and CH3; - Rg is chosen from H, CH3 and an alkyl group; - n8 is an integer between 1 and 10.

[0116] When F has formula (4), the cleavable motif can have the following formula (18): R3 Rs (18) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S; - N + represents a positively charged nitrogen atom; - Y - is an anion, preferably chosen from among the halogens (Cl', F", Brj, acetate, OTs, OTf, N-acetyl type amino acids; - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure; the lateral chain comprising R3, R4 and R5, being located at any position of Rr, R2', R3 1 , R4 1 and R5', where said Rr, R2', R3 1 , R4 1 and R5' is then absent.

[0117] When F has formula (5), the cleavable motif can have the following formula (19): R3' (19) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - R4 is chosen from O, NH and an aryl group; - N +represents a positively charged nitrogen atom; - Y - is an anion, preferentially chosen from among the halogens (Cl - , F - , Br - ), acetate, OTs, OTf, N-acetyl type amino acid; - Rr, R2 1 Ra 1 , R4 1 and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure; the lateral chain comprising R3, R4 and R5, being located at any position of R1', R2', R3', R4' and R5', where said R1', R2', R3', R4' and R5' is then absent.

[0118] When F has formula (6), the cleavable motif can have the following formula (20): (20) in which: - R3 is chosen from H, CH3 and an alkyl group; - n1 is an integer between 1 and 10; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - Xa is chosen from H and CHA; - X4 is chosen from H and CHA; - Rg is chosen from H, CHa and an alkyl group; - n8 is an integer between 1 and 10; - N + represents a positively charged nitrogen atom; Tl - Y - is an anion, preferentially chosen from among the halogens (Cl - , F - , Br - ), acetate, OTs, OTf, N-acetyl type amino acid; - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure; the lateral chain comprising R3, being located at any position of Rr, R2', R3 1 , R4 1 and R5', where said Rr, R2 1 , R3 1 , R4 1 and R5' is then absent.

[0119] Other formulas and structures for M known to those skilled in the art to be cleavable may replace the variants described in this application.

[0120] Advantageously, the structure of the cleavable motif according to the invention contributes to the properties of the copolymer to enable its use in nanoparticles for the controlled release of molecules in several ways, including: - Stability under physiological conditions: The F functional group limits spontaneous cleavage in normal physiological environments. This stability ensures that nanoparticles formed from amphiphilic copolymers maintain their integrity during circulation and accumulation at target sites. - Reactivity to specific stimuli: The structure of the cleavable pattern can vary to respond to exogenous stimuli such as light, ionizing radiation or ultrasound, or to endogenous stimuli such as changes in pH or the presence of specific enzymes. - Adjustable release kinetics: By adjusting the chemical structure of the cleavable motif, the speed and extent of cleavage can be finely tuned. This allows for customization of release profiles for different therapeutic applications. - Charge-mediated interactions: The positively charged nitrogen atom in the cleavable motif can interact with negatively charged molecules or surfaces, potentially influencing the self-assembly behavior and stability of the resulting nanoparticles. - Potential for further functionalization: The different substituents in the copolymer offer opportunities for further modification, such as the attachment of targeting ligands or imaging agents for example.

[0121] Furthermore, the cleavable motif allows for a balance between stability and reactivity, enabling the development of nanoparticles that can maintain their integrity under physiological conditions while providing controlled release of encapsulated molecules in response to specific stimuli. This controlled release mechanism can improve the efficacy and safety of therapeutic agents delivered using these amphiphilic copolymer-based nanoparticles.

[0122] According to a particular embodiment, the amphiphilic copolymer according to the invention has the following formula (21): (21) in which: - Ri can be a hydrophilic polymer or a hydrophobic polymer, - R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer; - R3 is chosen from H, CH3 and an alkyl group; - Rg is a group of formula (13); - R7 is a group of formula (14); - n1 is an integer between 1 and 10; - N + represents a positively charged nitrogen atom; - Y - is an anion, preferentially chosen from among the halogens (Cl - , F - , Br - ), acetate, OTs, OTf, N-acetyl type amino acid; - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure; the lateral chain comprising R3 being located at any position of R1', R2', R3', R4' and R5', where said R1', R2', R3', R4' and R5' is then absent, and - Rg is chosen between a group of formula (2) and a group of formula (3).

[0123] According to a particular embodiment, the amphiphilic copolymer according to the invention has the following formula (22): (22)

[0124] According to a particular embodiment, the amphiphilic copolymer according to the invention has the following formula (23): R6 R, (23)

[0125] According to a particular embodiment, the amphiphilic copolymer according to the invention has the following formula (24), (25), (26), (27), (28), (29), (30), (31): (30) (31) in which: - Ri can be a hydrophilic polymer or a hydrophobic polymer; - R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer; - R3 is an H, a CH3 is an alkyl group; - R4 is chosen from NH, an aryl group, an O; - n1 is an integer between 1 and 10; - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane; - R4 is chosen from O, NH and an aryl group; - if Xi is not an O then R5 is O, if Xi is an O then R5 is chosen from O or S; - N + represents a positively charged nitrogen atom; - Y -is an anion, preferentially chosen from among the halogens (Cl - , F - , Br - ), acetate, OTs, OTf, N-acetyl type amino acid; - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OMe (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure; the lateral chain comprising R3, being located at any position of Rr, R2', R3 1 , R4 1 and R5', where said Rr, R2 1 , R3 1 , R4 1 and R5' is then absent; - n2, n4, n5 and n7 are identical or different integers between 0 and 5; - n3 and ng are identical or different integers chosen from 0 and 1.

[0126] Advantageously, the cleavable motif of the amphiphilic copolymers according to the invention, when used to form nanoparticles containing a molecule, allows controlled release of the molecule from the nanoparticles in response to specific stimuli, thus providing precise control over the delivery of said molecule.

[0127] The hydrophilic polymer segment of the amphiphilic copolymer plays a role in determining the properties and behavior of the resulting nanoparticles, which must be adapted according to the molecules to be encapsulated and the intended applications, based on the knowledge of those skilled in the art. Different types of hydrophilic polymers can be used, each offering distinct characteristics that can be exploited for specific applications.

[0128] The hydrophilic polymer or hydrophilic segment of the amphiphilic copolymer according to the invention can be selected in particular from polyethylene glycol (PEG), polyacrylamide, poly(N-vinylpyrrolidone), poly(N-isopropylacrylamide), poly(N,N-dimethylacrylamide), poly(N-hydroxypropylmethacrylamide), poly(2-oxazoline), hydrophilic polysaccharides (dextran, chitosan, hyaluronic acid), elastin-based polymers (ELP), poly(L-glutamic acid) (in unprotected / hydrophilic form), poly(acrylic acid), poly(methacrylic acid), DNA and its derivatives, functionalized oligonucleotides and polysar-cosine.

[0129] According to another embodiment, the hydrophilic polymer or hydrophilic segment of the amphiphilic copolymer according to the invention can be chosen in particular from polyethylene glycol (PEG), polyacrylamide, poly(N-vinylpyrrolidone), poly(N-isopropylacrylamide), poly(N,N-dimethylacrylamide), poly(N-hydroxypropylmethacrylamide), poly(2-oxazoline), and polysarcosine.

[0130] Polyethylene glycol (PEG) is a hydrophilic polymer commonly used due to its biocompatibility and its ability to reduce protein adsorption and opsonization. PEG can be synthesized with varying molecular weights, typically ranging from 2000 to 20000 Da, which affects the hydrophilicity and steric stabilization of the nanoparticles. Higher molecular weight PEG generally provides increased stability and longer circulation times in vivo.

[0131] Polyacrylamide offers good water solubility and can be easily functionalized. The degree of polymerization can be adjusted to control the hydrophilicity and size of the resulting nanoparticles.

[0132] Poly(N-vinylpyrrolidone) (PVP) is known for its excellent biocompatibility and its ability to form stable complexes with various substances. PVP can improve the solubility of hydrophobic drugs and enhance the stability of nanoparticles in biological environments.

[0133] Poly(N-isopropylacrylamide) (PNIPAM) exhibits temperature-sensitive behavior, undergoing a phase transition at its lower critical temperature of solution (LCST). This property can be exploited for the temperature-triggered release of molecules encapsulated from nanoparticles.

[0134] Poly(N,N-dimethylacrylamide) (PDMA) offers good water solubility and biocompatibility. PDMA-based nanoparticles may exhibit reduced protein adsorption compared to some other hydrophilic polymers.

[0135] Poly(N-hydroxypropyl methacrylate) (PHPMA) is a hydrophilic polymer with good biocompatibility and low immunogenicity. PHPMA can be functionalized with various side groups, allowing for customized properties and the conjugation of targeting groups or drugs.

[0136] Poly(2-oxazoline)s represent a class of polymers with properties that can be adjusted depending on the selection of the side chain. These polymers can offer good biocompatibility and may exhibit thermoreactive behavior depending on the specific composition.

[0137] Polysarcosin is a polypeptoid that demonstrates excellent biocompatibility and resistance to protein adsorption. Polysarcosin-based nanoparticles can exhibit prolonged circulation times in vivo.

[0138] Hydrophilic polysaccharides, such as dextran, chitosan, and hyaluronic acid, constitute a class of naturally occurring hydrophilic polymers with excellent biocompatibility and biodegradability. Dextran is a branched polysaccharide composed of glucose units, widely used as a plasma substitute and drug delivery vehicle. Chitosan, obtained by deacetylation of chitin, possesses mucoadhesive properties and can facilitate transport across biological barriers. Hyaluronic acid is a glycosaminoglycan naturally present in connective tissues, which can specifically target the CD44 receptor, overexpressed on the surface of many tumor cells, thus providing intrinsic active targeting.

[0139] Elastin-like polypeptides (ELPs) are recombinant polypeptides composed of repetitive sequences derived from elastin, typically the pentapeptide (Val-Pro-Gly-Xaa-Gly) where Xaa can be any amino acid except proline. ELPs exhibit thermoreactive behavior characterized by a reverse transition temperature (RTT), below which they are soluble in water and above which they reversibly aggregate. This property can be exploited to modulate the self-assembly of nanoparticles as a function of temperature.

[0140] Poly(L-glutamic acid) (PGA), in its deprotonated and deprotected form at physiological pH, is a biodegradable and biocompatible hydrophilic polymer. PGA has pendant carboxylate groups that impart a negative charge at physiological pH, promoting aqueous solubility and allowing the conjugation of therapeutic molecules or targeting ligands.

[0141] Poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA) are pH-sensitive, hydrophilic polymers that exhibit pH-dependent swelling behavior. At physiological pH, the carboxyl groups are ionized, conferring strong hydrophilicity. This pH sensitivity can be exploited to modulate the properties of nanoparticles according to the biological environment.

[0142] Functionalized DNA derivatives and oligonucleotides can also be used as hydrophilic segments in the amphiphilic copolymers according to the invention. DNA-polymer conjugates can self-assemble into nanostructures thanks to the complementary hybridization properties of DNA, offering possibilities for structural programming and molecular targeting. DNA aptamers, in particular, can simultaneously serve as a hydrophilic segment and a targeting ligand, enabling intrinsic active targeting of the nanoparticles to specific cells or tissues.

[0143] By selecting a suitable hydrophilic polymer and optimizing its properties, nanoparticles can be tailored for specific applications in drug delivery, diagnostics, and other biomedical fields. In particular, the length and composition of the hydrophilic polymer can be adjusted to optimize self-assembly properties, the stability of nanoparticles formed with copolymers, and their in vivo behavior.

[0144] According to one embodiment of the invention, the hydrophilic polymer as defined above has a degree of polymerization between 5 and 500, preferably between 5 and 300, preferably between 15 and 150 and even more preferably between 15 and 120.

[0145] The hydrophobic polymer segment of the amphiphilic copolymer plays a role in determining the properties and behavior of the resulting nanoparticles, which must be adapted according to the molecules to be encapsulated and the intended applications, based on the knowledge of those skilled in the art. Different types of hydrophobic polymers can be used, each offering distinct characteristics that can be exploited for specific applications.

[0146] The hydrophobic polymer or hydrophobic segment of the amphiphilic copolymer according to the invention can be selected in particular from poly(γ-benzyl-L-glutamate) (PBLG), poly(ε-caprolactone) (PCL), poly(lac-tide) (PLA), poly(glycolide) (PGA), poly(lactide-co-glycolide) (PLGA), poly(orthoesters) (POE), poly(anhydrides), poly(propylene fumarate) (PPF), polycarbonate (PC), poly(alkyl cyanoacrylate), poly(trimethylene carbonate) (PTMC), poly(siloxanes) (Silicones), poly(urethane), aromatic polyanhydrides, poly(alkyl acrylates), poly(alkyl methacrylates), poly(P-aminoesters), poly(styrene-alt-maleic anhydride), poly(L-leucine) (pLeu), poly(L-phenylalanine) (pPhe), protected Poly( L-lysine) (e.g. poly(e-Boc-L-Lysine) or poly(Ne-trifluoroacetyl-L-lysine)), Poly(O-benzyl-L-tyrosine) (pTyr) and polysulfones.

[0147] According to an alternative embodiment, the hydrophobic polymer is selected from poly(y-benzyl-L-glutamate) (PBLG), poly(e-caprolactone) (PCL), Poly(lactide) (PLA), Poly(glycolide) (PGA), Poly(lactide-co-gly-colide) (PLGA), Poly(y-benzyl-L-glutamate) (PBLG), Poly(orthoesters) (POE), Poly(anhydrides), Poly(propylene fumarate) (PPF), Polycarbonate (PC), Poly(alkyl cyanoacrylate), Poly(trimethylene carbonate) (PTMC), Poly(siloxanes) (Silicones), Poly(urethane), Aromatic polyanhydrides, Poly(alkyl acrylates), Poly(alkyl methacrylates), Poly(P-aminoesters), Poly(styrene-alt-maleic anhydride) and Polysulfones.

[0148] Poly(γ-benzyl-L-glutamate) (PBLG) is a synthetic polypeptide that forms stable α-helical structures in aqueous environments. PBLG can be synthesized with varying molecular weights, typically ranging from 5000 to 50000 Da. The rigid, rod-like structure of PBLG contributes to the formation of stable vesicles and influences the mechanical properties of the resulting nanoparticles.

[0149] Poly(ε-caprolactone) (PCL) is a biodegradable polyester with a low glass transition temperature. PCL exhibits good biocompatibility and can be used to form nanoparticles with controlled degradation rates. The molecular weight of PCL can be varied to adjust the hydrophobicity and degradation rate of the resulting nanoparticles.

[0150] Poly(lactide) (PLA) and poly(glycolide) (PGA) are biodegradable aliphatic polyesters commonly used in biomedical applications. These polymers can be used individually or as copolymers (PLGA) to form nanoparticles with adjustable degradation rates. The lactide / glycolide ratio in PLGA copolymers affects the hydrophobicity and degradation rate of the resulting nanoparticles.

[0151] Poly(orthoesters) (POEs) are hydrophobic, surface-eroding polymers that can be used to form nanoparticles with controlled-release properties. Hydrolysis of POEs occurs primarily at the surface of the nanoparticles, allowing for more predictable release kinetics of the encapsulated molecules.

[0152] Poly(anhydrides) are another class of surface erosion polymers that can be used as hydrophobic segments. These polymers degrade rapidly in aqueous environments, making them suitable for applications requiring the rapid release of encapsulated molecules.

[0153] Poly(propylene fumarate) (PPF) is a linear unsaturated polyester that can be crosslinked to form biodegradable networks. PPF-based nanoparticles can exhibit improved mechanical properties and controlled degradation rates.

[0154] Polycarbonate (PC) is a thermoplastic polymer with good mechanical properties and biocompatibility. PC-based nanoparticles may exhibit improved stability and controlled release properties.

[0155] Poly(alkyl cyanoacrylate) polymers can form nanoparticles with rapid degradation rates.

[0156] Poly(trimethylene carbonate) (PTMC) is a biodegradable polymer that exhibits surface erosion behavior. PTMC-based nanoparticles can provide controlled release of encapsulated molecules with minimal initial release.

[0157] Polysiloxanes, also known as silicones, are hydrophobic polymers with high flexibility and biocompatibility. Silicone-based nanoparticles can exhibit unique surface properties and controlled release characteristics.

[0158] Polyurethane polymers can be synthesized with varying degrees of hydrophobicity and biodegradability. Polyurethane-based nanoparticles can offer adjustable mechanical properties and degradation rates.

[0159] Aromatic polyanhydrides are hydrophobic polymers that exhibit surface erosion behavior. These polymers can be used to form nanoparticles with controlled release properties and improved stability in aqueous environments.

[0160] Poly(alkyl acrylates) and poly(alkyl methacrylates) are versatile hydrophobic polymers that can be synthesized with various alkyl chain lengths. The alkyl chain length affects the hydrophobicity and glass transition temperature of the resulting nanoparticles.

[0161] Poly(P-aminoesters) are biodegradable polymers that can be synthesized with varying degrees of hydrophobicity. These polymers can form nanoparticles with pH-sensitive release properties.

[0162] Poly(styrene-alt-maleic anhydride) is a hydrophobic copolymer that can be used to form nanoparticles with unique surface properties. The anhydride groups can be further functionalized to modify the properties of the resulting nanoparticles.

[0163] Polysulfones are thermoplastic polymers with high chemical and thermal stability. Polysulfone-based nanoparticles can exhibit improved mechanical properties and resistance to degradation in harsh environments.

[0164] By selecting a suitable hydrophobic polymer and optimizing its properties, nanoparticles can be tailored for specific applications in drug delivery, diagnostics, and other biomedical fields. In particular, the nature and length of the hydrophobic polymer can be modulated to control encapsulation properties, nanoparticle stability, and in vivo behavior.

[0165] According to one embodiment of the invention, the hydrophobic polymer as defined above has a degree of polymerization between 5 and 200, preferably between 5 and 100, preferably between 5 and 60 and even more preferably between 10 and 50.

[0166] The amphiphilic copolymer can include additional functional groups on the polymer chains for further functionalization. These additional functional groups can be incorporated into the hydrophilic or hydrophobic polymer segments (including the chain ends), preferably at the chain ends. The presence of these functional groups allows for the attachment of targeting groups, imaging agents, or other molecules that can enhance the functionality of the amphiphilic copolymer or of nanoparticles formed from the amphiphilic copolymer. Examples of such functional groups include, but are not limited to, carboxyl, amine, thiol, azide, or alkyne groups. The functional groups can also be one or more inorganic elements and / or biological or chemical molecules such as, for example, mineral, metallic, or semi-metallic particles, antibodies, peptides, nucleic acids, or sugars.

[0167] The incorporation of these additional functional groups provides a versatile platform for customizing the amphiphilic copolymer for specific applications. For example, the attachment of targeting ligands can enable selective binding to specific cell types or tissues. The conjugation of imaging agents can allow for the tracking and monitoring of nanoparticles formed from the amphiphilic copolymer in vivo. Furthermore, these functional groups can be used for crosslinking or other modifications that can alter the properties of the resulting nanoparticles, such as their stability, size, or release characteristics.

[0168] The copolymer can exhibit various structural variants, including: - variations in the nature and position of the substituents on the cleavable motif M, allowing adjustment of its sensitivity to stimuli. - modifications in the length of hydrophilic and hydrophobic polymer chains, influencing self-assembly properties and the size of the nanoparticles formed. - the incorporation of additional functional groups on the polymer chains for further functionalizations.

[0169] These variants offer great flexibility to adapt the properties of the copolymer to specific applications.

[0170] The structure of the copolymer can be modified in several ways: - different combinations of hydrophilic and hydrophobic polymers can be used to adjust the physico-chemical and biological properties of nanoparticles; - the position and number of cleavable motifs can be varied, for example by incorporating several cleavable motifs along the polymer chain for a more gradual release of the contents; - Additional components can be integrated, such as targeting agents or markers for imaging, thus expanding the application possibilities of the copolymer.

[0171] These variations allow the copolymer to be optimized for various therapeutic and diagnostic applications, providing a versatile platform for the development of advanced drug delivery systems and contrast agents.

[0172] Preferably, the amphiphilic copolymer according to the invention is stable under physiological conditions for at least 4 hours, preferably from 4 hours to 7 days, and even more preferably for at least 10 hours, at least 24 hours, at least 48 hours, at least 72 hours, and at least 5 days. Preferably, the amphiphilic copolymer according to the invention is stable under the following conditions: - between 0 and 60°C, preferably between 20 and 50°C, advantageously between 35 and 45°C, and / or - at pH between 5 and 8, particularly at pH between 6 and 8.

[0173] Specific, non-limiting examples of amphiphilic copolymers according to the invention are given below:

[0174] - Formula copolymer (32) (PEG 44 -Carbamate-Pico)-b-PBLG 21 ): (32)

[0175] In this example, the carbamate of the F group can be ortho or para.

[0176] - Copolymer of formula (33): (33) In this example, the carbamate of the F group can be ortho or para.

[0177] - Formula copolymer (34) (mPEG 44 -aromatic ester-Pico)-b-PBLG 21 ): 0 0 (34)

[0178] In this example, the stabilized aromatic ester of the F group can be ortho or para.

[0179] - Copolymer of formula (35): (35)

[0180] - Copolymer of formula (36) (PBLG 21 -(carbamate-Pico)-b-PEG 44 ): (36)

[0181] - Copolymer of formula (37) (pSar 30 -(carbamate-Pico)-b-PBLG 21 ) (37)

[0182] - Formula copolymer (38) (mPEG 44 -(carbamate-Pico)-b-PLA 34 )

[0183] - Copolymer with formula (39 (mPEG 44 -(carbamate-PICO)-b-pLeu 34 (39)

[0184] - Copolymer of formula (40) (PMeOx 26 -(carbamate-PICO)-b-PBLG 21 ) (40)

[0185] - Formula copolymer (41) (mPEG 44 -(aromatic thioester-Pico)-b-PBLG 21 )

[0186] - Formula copolymer (42) (mPEG 44 -(carbonate-PICO)-b-PBLG 21 )

[0187] - Formula copolymer (43) (mPEG 44 -(carbamate-QUINO)-b-PBLG 21 )

[0188] - Formula copolymer (44) (mPEG 44 -(carbamate-Acr)-b-PBLG 21 )

[0189] - Formula copolymer (45) (mPEG 44 -(phosphoester-Acr)-b-PBLG 21 ) (45)

[0190] - Formula copolymer (46) (mPEG 44 -(Quat amine-Acr)-b-PBLG 21 ) (46)

[0191] In general, all amphiphilic copolymers described in this application may include one or more chiral centers (asymmetric carbons), allowing these copolymers to exist in various stereochemical forms, such as enantiomers, diastereomers, or any combination thereof, including racemic mixtures. All such forms, whether optically active or inactive, and their mixtures, are considered to be part of the invention. Methods known in the art for the preparation and separation of these stereochemical forms, such as racemic mixture resolution, chiral chromatography, preferential salt formation, recrystallization, and other standard techniques, may be used to obtain the desired forms.Furthermore, these stereochemical forms can be obtained by the direct synthesis of chiral centers or by the use of chiral raw materials.

[0192] According to certain embodiments, the amphiphilic copolymer of the invention may comprise a plurality of cleavable motifs. This configuration makes it possible to increase the copolymer's sensitivity to stimuli and / or to modulate the cleavage and release kinetics of the encapsulated molecules.

[0193] In one embodiment, the amphiphilic copolymer comprises at least two cleavable units located between the hydrophilic polymer and the hydrophobic polymer. The cleavable units may be identical or different.

[0194] According to another embodiment, the amphiphilic copolymer comprises at least two cleavable motifs, at least three cleavable motifs, or at least four cleavable motifs.

[0195] When the amphiphilic copolymer comprises several cleavable units, these may be: - arranged in series (one after the other) between the hydrophilic polymer and the hydrophobic polymer, - arranged in parallel (connected to the same junction point), - or a combination of both configurations.

[0196] In one particular embodiment, the amphiphilic copolymer comprises several identical cleavable motifs, each cleavable motif including a functional group F capable of limiting and / or preventing the spontaneous cleavage of the cleavable motif under physiological conditions. This configuration increases the number of cleavage sites and thus improves the efficiency of copolymer degradation in response to a stimulus.

[0197] According to another embodiment, the amphiphilic copolymer comprises several different cleavable units, for example: - a first cleavable motif sensitive to a first type of stimulus (for example, ionizing radiation), - a second cleavable motif sensitive to a second type of stimulus (e.g., pH, enzymes, light or ultrasound).

[0198] This configuration allows for the design of multi-stimulus release systems, offering greater flexibility in controlling the release of encapsulated molecules.

[0199] According to one embodiment, when the amphiphilic copolymer comprises several cleavable motifs, each cleavable motif independently comprises: a redox-sensitive M group, such as a derivative of picolinium, acridinium or quinolinium, and a functional F group selected from a carbamate, a carbonate, a stabilized aromatic ester, a urea, a thiocarbamate, a thiocarbonate, a thioester or a thiourea.

[0200] According to one embodiment, the amphiphilic copolymer comprises n cleavable motifs, where n is an integer between 2 and 10, preferably between 2 and 5, more preferably between 2 and 3.

[0201] The presence of multiple cleavable motifs may allow: - to increase the copolymer's sensitivity to exogenous or endogenous stimuli, - to modulate the degradation kinetics of the copolymer, - to allow the sequential or simultaneous release of different encapsulated molecules, - to combine different cleavage mechanisms for a more controlled release.

[0202] According to a particular embodiment, the amphiphilic copolymer has a structure of formula (48): R₁-R₆[MF]R₇-R₂ J n (48) in which: - R₁ is a hydrophilic polymer or a hydrophobic polymer; - R₂ is a hydrophobic polymer if R₁ is a hydrophilic polymer, or R₂ is a hydrophilic polymer if R₁ is a hydrophobic polymer. - MF represents the cleavable motif, and - R₆ and R₇ are optional spacers, - and n is a natural number from 2 to 10.

[0203] In one embodiment, the amphiphilic copolymer is a multi-block copolymer comprising alternating hydrophilic segments, hydrophobic segments, and cleavable units. For example, the copolymer may have a structure of the type: R₁-[MF]-R₂-[MF]-R₁ or R₁-[MF]-R₂-[M'-F']-R₂'-[MF]-R₁ Or: - R₁ represents a hydrophilic segment; - R₂, R₂' represent hydrophobic segments (identical or different); - MF, M'-F' represent cleavable patterns (identical or different).

[0204] This architecture makes it possible to create nanoparticles with tunable degradation properties, where the sequential cleavage of the different motifs can lead to a progressive release of the encapsulated molecules.

[0205] According to one embodiment, the amphiphilic copolymer can comprise several cleavable MF motifs arranged in series between R₁ and R₂, separated by spacers R₆ and / or R₇.

[0206] The general structure of such a copolymer can be schematically represented by: R₁ – [R₆ – MF – R₇]n – R₂ where R₁ can be a hydrophilic or hydrophobic polymer, R₂ can be a hydrophobic polymer if R₁ is a hydrophilic polymer, or R₂ can be a hydrophilic polymer if R₁ is a hydrophobic polymer, MF represents the cleavable motif, R6 and R7 are optional spacers, and n is an integer greater than or equal to 2, e.g. between 2 and 10, preferably between 2 and 5.

[0207] For example, a copolymer comprising three cleavable units may have the following structure: R₁ – R₆ – MF – R₇ – R₆ – MF – R₇ – R₆ – MF – R₇ – R₂

[0208] The incorporation of multiple cleavable motifs along the polymer chain can allow for a more gradual release of the encapsulated contents. Indeed, each cleavage event can lead to partial destabilization of the nanoparticle, resulting in a fractionated and controlled release of the encapsulated molecules. The number of cleavable motifs can thus be adjusted to modulate the release profile according to the intended therapeutic application.

[0209] According to one variant, the multiple cleavable motifs may be identical, responding to the same type of stimulus. According to another variant, the multiple cleavable motifs may be different, each being sensitive to a distinct stimulus (for example, one motif cleavable under ionizing radiation and one motif cleavable via redox), thus allowing for sequential or combined activation of release.

[0210] In some embodiments, the amphiphilic copolymer according to the invention may further comprise at least one targeting ligand covalently linked to the hydrophilic polymer, preferably at the terminal end of the hydrophilic polymer opposite the cleavable motif.

[0211] The targeting ligand can be chosen from: - targeting peptides, such as RGD (Arg-Gly-Asp) peptides, NGR peptides, cell penetration peptides (CPP); - small targeting molecules, such as folic acid, biotin, mannose, galactose; - aptamers, such as anti-nucleolin, anti-PSMA, anti-EGFR aptamers; - antibodies or antibody fragments, such as anti-HER2, anti-EGFR, anti-PD-Ll antibodies, Fab fragments, nanobodies, scFv; - target proteins, such as transferrin, EGF, VEGF; - sugars or glycans targeting specific receptors.

[0212] The targeting ligand can be linked to the hydrophilic polymer via a linker group comprising a reactive function chosen from: - azide (-N₃) functions for coupling by click chemistry CuAAC or SPAAC; - terminal alkyne or cyclooctyne functions (DBCO; BCN); - maleimide functions for coupling with thiols; - NHS-ester or activated carboxyl functions for coupling with amines; - aldehyde functions for coupling with hydrazides or aminooxy. - tetrazine-norbornene pairs for ultrafast bioorthogonal click-type reactions (iEDDA).

[0213] Method for manufacturing an amphiphilic copolymer according to the invention

[0214] The invention also relates to a process for preparing the amphiphilic copolymer involving steps of synthesizing the cleavable unit and / or synthesizing the hydrophobic and / or hydrophilic polymers, functionalizing the hydrophilic polymer, functionalizing the hydrophobic polymer, synthesizing the hydrophobic polymer, and coupling the blocks. The process may include ring-opening polymerization techniques or click chemistry, for example.

[0215] The process for preparing the amphiphilic copolymer according to the invention comprises several key steps, allowing precise control of the structure and properties of the final copolymer. This process can be carried out in several variations, including two main variations, offering flexibility in the synthesis depending on the desired characteristics of the copolymer.

[0216] According to a first variant, the process for preparing an amphiphilic copolymer according to the invention comprises carrying out the following steps: - Synthesis of the cleavable motif, the hydrophilic polymer, and the hydrophobic polymer, - Functionalization of the hydrophilic polymer with the cleavable motif to obtain a hydrophilic block, - Coupling of the two blocks (hydrophilic block and hydrophobic polymer) to obtain the amphiphilic copolymer.

[0217] According to a second variant, the process for preparing an amphiphilic copolymer according to the invention comprises carrying out the following steps: - Synthesis of the cleavable motif, the hydrophilic polymer, and the hydrophobic polymer, - Functionalization of the hydrophobic polymer with the cleavable motif to obtain a hydrophobic block, - Coupling of the two blocks (hydrophobic block and hydrophilic polymer) to obtain the amphiphilic copolymer.

[0218] The synthesis step of the cleavable motif involves its preparation, which plays a crucial role in the stimulus response properties of the final copolymer. The synthesis may involve complex organic reactions, potentially with the use of protecting groups to prevent undesirable side reactions. The reaction conditions (temperature, solvent, catalyst, concentration, stirring conditions) are carefully controlled to optimize the yield and purity of the cleavable motif.

[0219] The hydrophilic and hydrophobic segments of the amphiphilic copolymer according to the invention can be synthesized, in particular, by controlled polymerization techniques such as: - ionic polymerization (anionic or cationic), - radical polymerization, such as atom transfer radical polymerization (ATRP), nitroxide-controlled radical polymerization (NMP), or reversible addition-fragmentation polymerization by chain transfer (RAFT), - ring-opening polymerization (ROP) which allows excellent control of the polymer structure and its properties. ROP can be implemented for the polymerization of cyclic esters (such as lactide or caprolactone), cyclic anhydrides, cyclic carbonates, N-carboxyanhydrides (NCA) for obtaining polypeptide blocks, or lactams (such as caprolactam); - Polymerization by polycondensation.

[0220] The step of coupling the blocks to form the amphiphilic copolymer according to the invention can be carried out in particular by techniques such as: - ring-opening polymerization (ROP), in which one of the blocks functionalized by the cleavable motif serves as a macro-initiator for the polymerization of the other block. ROP can be implemented for the polymerization of cyclic esters (such as lactide or caprolactone), cyclic anhydrides, cyclic carbonates, N-carboxyanhydrides (NCA) for obtaining polypeptide blocks, or lactams (such as caprolactam); - click chemistry reactions, such as copper-catalyzed azide-alkyne cycloaddition (CuAAC) or stress-promoted azide-alkyne cycloaddition (SPAAC), - peptide coupling or amidation reactions, - esterification or transesterification reactions, - reactions that form carbamate, carbonate, or thioester, - nucleophilic substitution reactions, - or any other coupling reaction known to a person skilled in the art that allows a covalent bond to form between the blocks.

[0221] These methods allow precise control of chain length and molar mass distribution.

[0222] The step of functionalizing the hydrophilic polymer with the cleavable motif to form a hydrophilic block may involve coupling reactions such as click chemistry reactions (e.g., copper-catalyzed azide-alkyne cycloaddition) or esterification reactions.

[0223] In the first variant, functionalization is carried out under conditions that preserve the integrity of the hydrophilic polymer and the cleavable motif. The functionalized hydrophilic block is then coupled to the hydrophobic polymer. This step can also utilize click chemistry techniques or other efficient coupling methods. The reaction conditions are optimized to ensure complete coupling and avoid the formation of unwanted byproducts.

[0224] In the second variant, the hydrophobic polymer is functionalized with the cleavable motif. This approach may be preferred depending on the chemical nature of the polymers and the cleavable motif, potentially offering better coupling efficiency or different final copolymer properties. The functionalized hydrophobic block is then coupled to the hydrophilic polymer, using methods similar to those described in the first variant.

[0225] Throughout the process, it may be necessary to protect certain reactive functional groups (hydroxy, amino, imino, thio, carboxy) to prevent undesirable side reactions. Conventional protecting groups (Pg) can be used according to standard organic chemistry practices.

[0226] The reactions are generally carried out in suitable solvents (hexane, cyclohexane, benzene, toluene, xylene, dimethylformamide, ethanol, methanol, diethyl ether, tetrahydrofuran) at temperatures between 0°C and 150°C, preferably between room temperature and 100°C. The duration of the reactions can vary from 3 to 20 hours depending on the specific conditions.

[0227] Intermediate and final products are preferentially purified, for example by standard techniques such as distillation, liquid-liquid extraction, recrystallization, precipitation, or various chromatographic methods (column, preparative thin layer).

[0228] Each step of the process is preferably monitored by appropriate analytical techniques (NMR, mass spectrometry, size exclusion chromatography) to confirm the structure and purity of the intermediate products and the final copolymer.

[0229] This synthesis process offers great flexibility to adjust the properties of the final amphiphilic copolymer, allowing optimization of its ability to form stable nanoparticles with controlled release characteristics in response to specific stimuli.

[0230] Nanoparticle according to the invention

[0231] The amphiphilic copolymers according to the invention can self-assemble into different types of nanoparticles.

[0232] The nanoparticles according to the invention can take various forms. These may include: - solid (solid) nanoparticles: these structures generally form when the hydrophobic segment of the copolymer is significantly longer than the hydrophilic segment. - Micelles: these structures are formed by the self-assembly of amphiphilic copolymers, in which the hydrophobic segments form a compact core surrounded by a ring of hydrophilic segments. Micelles can encapsulate hydrophobic molecules within their core. - Hollow nanoparticles: including polymersomes, these structures have an outer membrane and a hollow inner portion. Polymersomes, in particular, offer a biomimetic structure similar to liposomes, but with improved stability and versatility.

[0233] Polymersomes are vesicular structures with an aqueous core surrounded by a bilayer membrane formed by amphiphilic copolymers. These structures are particularly useful for encapsulating both hydrophilic and hydrophobic molecules, as hydrophilic compounds can be loaded into the aqueous core while hydrophobic compounds can be incorporated into the membrane.

[0234] The type of nanoparticle formed (solid or hollow) depends on various factors, including the relative lengths of the hydrophilic and hydrophobic segments, the copolymer concentration, and the specific conditions of the self-assembly process.

[0235] The nanoparticles according to the invention, formed from the amphiphilic copolymers according to the invention, can have a hydrodynamic diameter ranging from 1 to 400 nanometers. Preferably, the hydrodynamic diameter of the nanoparticles according to the invention is between 10 and 400 nanometers, between 40 and 200 nanometers, or between 80 and 120 nanometers.

[0236] The size of the nanoparticles can be controlled by adjusting various parameters of the nanoprecipitation process, such as the concentration of the copolymer solution, the rate of addition to the aqueous phase, the stirring speed, and the temperature. Furthermore, post-formation techniques such as extrusion or sonication can be used to further refine the size distribution of the nanoparticles.

[0237] Nanoparticles formed from these amphiphilic copolymers exhibit unique properties due to the presence of the cleavable motif comprising the F group. Although stable under physiological conditions, these nanoparticles can undergo controlled disassembly or structural changes in response to specific stimuli, allowing targeted release of encapsulated molecules.

[0238] The nanoparticles according to the invention exhibit remarkable stability under physiological conditions: - stable under physiological conditions for at least 4 hours, preferably for 4 hours to 7 days, even more preferably for at least 10 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, and / or - stability maintained between 0 and 60°C, preferably between 20 and 50°C, and ideally between 35 and 45°C, and / or - stability at pH between 5 and 8, especially between 6 and 8.

[0239] Nanoparticles formed from amphiphilic copolymers can be functionalized to enhance their capabilities for various imaging and therapeutic applications. Functionalization can be achieved by incorporating inorganic and / or organic (chemical or biological) components.

[0240] Inorganic elements can be incorporated into nanoparticles to extend their functionality. These inorganic elements can include mineral, metallic, or semi-metallic particles. The incorporation of inorganic elements can be achieved by grafting, nucleation, or encapsulation within the nanoparticle structure. The inorganic elements that can be incorporated into the nanoparticles according to the invention can, for example, be selected from ultrasmall superparamagnetic iron oxide nanoparticles (USPIOs), superparamagnetic iron oxide nanoparticles (SPIONs), very small iron oxide nanoparticles (VSIONs), hafnium oxide nanoparticles, iron-bismuth or iron-platinum alloy nanoparticles, gadolinium oxide nanoparticles, silver nanoparticles, platinum nanoparticles, and gold nanoparticles or clusters.

[0241] The incorporation of these inorganic elements can confer additional properties to nanoparticles, such as magnetic reactivity for magnetic resonance imaging (MRI) applications or enhanced X-ray contrast for computed tomography (CT) imaging.

[0242] Organic elements can also be incorporated into nanoparticles for biological or chemical functionalization. This biological or chemical functionalization allows the nanoparticles to interact specifically with target cells, tissues, or molecules in a living organism. Examples of biological elements that can be used for functionalization include antibodies, peptides, nucleic acids, and sugars.

[0243] These organic elements can be attached to the surface of the nanoparticles or incorporated into their structure during the self-assembly process. The choice of biological or chemical element depends in particular on the specific targeting requirements of the application.

[0244] Antibodies can be used for highly specific targeting of nanoparticles to particular cell types or tissues. For example, antibodies against tumor-specific antigens can be used to direct nanoparticles to cancer cells.

[0245] Peptides can serve as targeting ligands, binding to specific receptors on the cell surface. Short peptide sequences can be designed to recognize and bind to particular cellular targets.

[0246] Nucleic acids such as aptamers or oligonucleotides can be used for molecular recognition and targeting. These nucleic acid sequences can be selected to bind specifically to target molecules or cell surface receptors.

[0247] Sugars can be used to target specific carbohydrate-binding proteins (lectins) on the cell surface. Different sugar groups can be used to target different cell or tissue types.

[0248] Functionalizing nanoparticles with organic elements can improve their specificity for targeted drug delivery, enhance their accumulation in specific tissues, or enable their use as targeted imaging agents.

[0249] Combining inorganic and organic components in nanoparticle functionalization can create multifunctional systems capable of both imaging and therapeutic applications. For example, nanoparticles incorporating magnetic iron oxide particles and functionalized with tumor-targeting antibodies can serve as both MRI contrast agents and targeted drug delivery vectors.

[0250] Nanoparticle functionalization methods can vary depending on the nature of the functional element and the desired application. These methods may include: - covalent bonding: Functional groups on the surface of nanoparticles can be used to form covalent bonds with functional elements. - Electrostatic interactions: Charged functional elements can be adsorbed onto the surfaces of nanoparticles of opposite charge. - Hydrophobic Interactions: Hydrophobic functional elements can be incorporated into the hydrophobic regions of nanoparticles. - nucleation. - Encapsulation: Functional elements can be encapsulated within the structure of nanoparticles during the self-assembly process.

[0251] The functionalization of nanoparticles expands their potential applications in various fields, including targeted drug delivery, molecular imaging, and theranostics. By carefully selecting and combining different functional elements, nanoparticles can be tailored to specific biomedical applications, improving their efficacy and versatility in both diagnostic and therapeutic contexts.

[0252] Method for manufacturing a nanoparticle according to the invention

[0253] The manufacturing process for nanoparticles according to the invention relies on the ability of amphiphilic copolymers to self-assemble in an aqueous environment. This self-assembly can lead to the formation of various structures, including polymersomes, which are vesicles formed by a bilayer of amphiphilic copolymers.

[0254] The process of nanoparticle formation generally involves self-assembly techniques, with nanoprecipitation being a commonly used method.

[0255] In the nanoprecipitation process, amphiphilic copolymers are first dissolved in a water-miscible organic solvent. This solution is then added dropwise to an aqueous phase while stirring. As the organic solvent diffuses into the aqueous phase, the amphiphilic copolymers spontaneously self-assemble into nanoparticles. The hydrophobic segments of the copolymers aggregate to form the core of the nanoparticles, while the hydrophilic segments orient themselves toward the aqueous environment, forming the exogenous shell.

[0256] The process may include the following steps: - 1. Preparation of the copolymer solution: The amphiphilic copolymer is first dissolved in a water-miscible organic solvent, such as tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), acetone, or various alcohols. This solvent must be suitable for both blocks of the copolymer. - 2. Nanoprecipitation: The copolymer solution is then added to a larger volume of water or buffered aqueous solution. This step, also called solvent displacement, causes the copolymers to self-assemble into nanoparticles. Water acts as a selective solvent, being a good solvent for the hydrophilic block but a poor solvent for the hydrophobic block. - 3. Removal of the organic solvent: After the formation of the nanoparticles, the excess organic solvent is removed, either by gentle heating (e.g., at 40°C overnight), or by dialysis in a semi-permeable membrane tube against a suitable aqueous solution. -4. Characterization of nanoparticles:

[0257] The nanoparticles obtained can be characterized by various techniques, including: - Dynamic light scattering (DLS) to determine size and size distribution; - Electron microscopy (TEM, SEM, cryo-TEM) to visualize the morphology; - Small angle X-ray scattering (SAXS) or small angle neutron scattering (SANS) to analyze the internal structure; - The measurement of the zeta potential to evaluate the surface charge and colloidal stability of nanoparticles.

[0258] The size and morphology of nanoparticles can be controlled by adjusting various parameters, such as: - The chemical composition and length of the copolymer blocks - The concentration of the initial copolymer solution - The temperature and type of solvent - The pH and salinity of the aqueous phase - Mixing conditions (stirring speed, addition rate, order of addition)

[0259] For applications requiring functionalized nanoparticles, the process may include the incorporation of inorganic nanoparticles (metals, metal oxides, chalcogenides) into the polymersome membrane. These inorganic nanoparticles can represent from 5% to 50% of the total polymer weight, preferably between 10% and 20%.

[0260] The process also allows the encapsulation of active pharmaceutical ingredients (APIs) either in the aqueous compartment or in the hydrophobic membrane of the nanoparticles of the invention, depending on the solubility of the API.

[0261] The preparation method can be adapted to produce aqueous suspensions of nanoparticles, which can then be used in pharmaceutical compositions or medical devices.

[0262] This manufacturing process offers great flexibility to adjust the properties of nanoparticles according to the intended applications.

[0263] The manufacturing process for nanoparticles according to the invention makes it possible to obtain a suspension of nanoparticles in water, with a concentration that can reach hundreds of millions, or even billions of nanoparticles per microliter, measurable with an instrument such as the qNano counter (iZON Science Inc.).

[0264] The process allows fine control of the size (from a few tens of nm to less than 10 pm in diameter), morphology, and composition of the nanoparticles, offering great flexibility for various biomedical applications.

[0265] Functional nanoparticles:

[0266] The invention also relates to functional nanoparticles comprising a nanoparticle according to the invention and at least one molecule, preferably a therapeutic molecule. These functional nanoparticles can be obtained either by encapsulating the molecule inside the nanoparticle or by covalently conjugating the molecule to the surface of the nanoparticle.

[0267] In encapsulation, the molecule is physically trapped inside the nanoparticle structure (without covalent bonds), typically in the hydrophobic core for hydrophobic molecules or in the internal aqueous compartment for hydrophilic molecules. Encapsulation can be achieved during the nanoparticle self-assembly process by incorporating the molecule into the amphiphilic copolymer solution before nanoparticle formation.

[0268] According to another embodiment, the amphiphilic copolymer of the invention can be covalently conjugated to at least one active pharmaceutical ingredient, thus forming a polymer-drug conjugate, also called a polymer prodrug. In this configuration, the active ingredient is covalently linked to the amphiphilic copolymer, for example to the hydrophobic segment, the hydrophilic segment, or the cleavable motif, via a cleavable bond such as an imine, hydrazone, oxime, ester, amide, carbamate, carbonate, disulfide, or any other bond known to those skilled in the art to be cleavable under pathological physiological conditions or in response to an exogenous or endogenous stimulus. The active ingredient can thus constitute an integral part of the structure of the amphiphilic copolymer.In this polymer prodrug approach, the active ingredient is maintained in an inactive or reduced activity state as long as it is covalently bound to the copolymer.

[0269] Cleavage of the cleavable motif of the amphiphilic copolymer and / or of the bond between the active ingredient and the copolymer, in response to an exogenous stimulus (such as ionizing radiation, light irradiation, or ultrasound) or an endogenous stimulus (such as a change in pH, the presence of specific enzymes, or particular redox conditions), allows the release of the active ingredient in its active form. In the case of covalent conjugation, the molecule is chemically bound to the surface of the nanoparticle, generally via functional groups present on the hydrophilic segment of the amphiphilic copolymer. This conjugation can be achieved through various chemical reactions, such as the formation of amide or ester bonds, or by click chemistry.

[0270] The functional nanoparticles according to the invention offer several advantages: - Protection of the therapeutic molecule: The nanoparticle acts as a shield, protecting the therapeutic molecule from enzymatic degradation and rapid elimination from the bloodstream. - Improved solubility: For hydrophobic therapeutic molecules, encapsulation in nanoparticles can significantly improve their apparent solubility in aqueous media. - Passive targeting: Nanoparticles can preferentially accumulate in tumor tissues due to the effect of increased permeability and retention (EPR), allowing passive targeting. - Controlled release: The cleavable structure of the amphiphilic copolymer allows controlled release of the therapeutic molecule in response to specific stimuli. - Strong accumulation in the tumor: The combination of nanometric size, colloidal stability and amphiphilic behavior promotes a high concentration of nanoparticles within the tumor microenvironment, enhancing therapeutic efficacy while limiting systemic exposure.

[0271] A particularly interesting variant of the functional nanoparticles according to the invention is the nanomedicine type. In this configuration, the therapeutic molecule is maintained in an inactive state as long as it is associated with the nanoparticle, whether by encapsulation or covalent conjugation.

[0272] Advantageously, the functional nanoparticles according to the invention are stable under physiological conditions: they remain intact and stable under physiological conditions, particularly in the bloodstream. This stability is ensured by the design of the amphiphilic copolymer, specifically thanks to the F functional group present in the cleavable motif. The therapeutic molecule is maintained in an inactive state as long as it is bound to the nanoparticle. It can be selectively activated by a specific stimulus, which can be endogenous, such as a change in pH, the presence of specific enzymes, or particular redox conditions, or exogenous, such as ionizing radiation, light irradiation, or the application of ultrasound, for example, depending on the structure of the cleavable motif constituting the nanoparticle. Following activation, the therapeutic molecule is released in a controlled manner, allowing it to act at the target site.This release occurs through the total or partial disassembly of the nanoparticle following the cleavage of the amphiphilic copolymer.

[0273] A specific example of a functional nanoparticle according to the invention is one that is activatable by ionizing radiation. In this case, the cleavable motif of the amphiphilic copolymer is designed to respond specifically to ionizing radiation, such as X-rays or gamma rays used in radiotherapy. Exposure to the radiation triggers the cleavage of the copolymer, leading to the disintegration of the nanoparticle and the subsequent release of the therapeutic molecule.

[0274] This type of functional nanoparticle is of particular interest for oncology applications, where it can be used in combination with radiotherapy. Advantageously, this approach allows: - targeted release of the drug specifically in the irradiated area, thus reducing systemic side effects; - a potential synergy between the effect of radiation and the action of the released drug; - the possibility of using higher doses of the drug, which would otherwise be too toxic in free form; - the possibility of injecting much lower systemic doses while obtaining significantly higher local concentrations in the tumor, thanks to the preferential accumulation of nanoparticles compared to the same drug injected in free form and the precise targeting of release in the tumor by a stimulus.

[0275] The functional nanoparticles according to the invention thus offer a versatile platform for the development of targeted and controlled therapies, with particular potential to improve the efficacy and safety of treatments in oncology and other therapeutic areas.

[0276] The therapeutic molecule is preferentially chosen from the group consisting of anticancer agents, immunomodulators, anti-inflammatory agents, antidiabetic agents, antibacterial agents, antiviral agents, agents for the treatment of pediatric or age-related diseases, and agents for the treatment of diseases of the nervous or cardiovascular system and their mixtures.

[0277] Compositions

[0278] The present invention also relates to compositions comprising one or more nanoparticles according to the invention, in particular functional nanoparticles comprising at least one molecule, notably a therapeutic molecule. These compositions can take various forms and be adapted to different routes of administration and therapeutic applications.

[0279] The compositions according to the invention may be pharmaceutical compositions.

[0280] The pharmaceutical compositions according to the invention may comprise several nanoparticles according to the invention, one or more pharmaceutically acceptable excipients, and optionally other active ingredients. These compositions may be formulated for different routes of administration, including parenteral, oral, topical, or pulmonary.

[0281] For the parenteral route, the compositions according to the invention may be presented in particular in the form of injectable solutions or suspensions for intravenous, intramuscular or subcutaneous administration.

[0282] For oral administration, the compositions according to the invention may be presented in particular in the form of tablets, capsules, soft capsules, suspensions or oral solutions, or modified release formulation.

[0283] For topical use, the compositions according to the invention can be in the form of creams, ointments, gels or transdermal patches.

[0284] For the pulmonary route, the compositions according to the invention can be in the form of aerosols or powders for inhalation.

[0285] For the ophthalmic route, the compositions according to the invention can be presented in the form of eye drops or ocular inserts.

[0286] The excipients used in these compositions may include stabilizing agents (to maintain the integrity of the nanoparticles), cryoprotecting agents (for lyophilization), viscosity modifying agents, buffers, isotonicizing agents, preservatives, etc.

[0287] The compositions according to the invention can also be diagnostic compositions. In particular, they may include compositions comprising nanoparticles according to the invention conjugated to contrast agents or fluorescent markers. These compositions can be used for medical imaging (MRI, CT scans, optical imaging) or for in vitro diagnostic tests.

[0288] The compositions according to the invention can also be cosmetic compositions. Indeed, the invention also relates to compositions comprising nanoparticles according to the invention formulated for the controlled delivery of cosmetic active ingredients. These compositions can take the form of creams, serums, lotions, or masks.

[0289] The nanoparticles according to the invention can also be incorporated into medical device-type compositions, such as drug-eluting stents, advanced wound-healing dressings or controlled-release implants of active ingredients, for example.

[0290] The invention also relates to complex compositions, such as, in particular, compositions comprising: -different populations of functional nanoparticles according to the invention, each conjugated to a different therapeutic molecule, allowing for combination therapy with controlled release of each agent, or - mixture of functional therapeutic and diagnostic nanoparticles, for theranostic applications, - or functional nanoparticles conjugated to both therapeutic molecules and targeting ligands, for targeted and controlled delivery.

[0291] Use of nanoparticles and compositions comprising them according to the invention

[0292] Amphiphilic copolymers, nanoparticles and compositions containing them according to the invention have potential applications in various fields, including medical, particularly for drug delivery, imaging and theranostics.

[0293] The unique properties of these materials, particularly their stability under physiological conditions and their controlled release capabilities, make them suitable for a wide range of biomedical applications.

[0294] In the field of drug delivery, nanoparticles formed from these amphiphilic copolymers can be used to encapsulate and deliver therapeutic agents to specific target sites in the body. The controlled-release mechanism, triggered by specific stimuli, allows for precise temporal and spatial control of drug release. This approach can improve treatment efficacy while reducing systemic side effects.

[0295] The invention relates in particular to nanoparticles according to the invention or compositions containing them, for their use in the treatment of a pathology chosen from among cancers (preferably solid tumors), inflammatory diseases, diabetes, bacterial or viral infections and neurodegenerative diseases.

[0296] For example, in cancer therapy, nanoparticles loaded with chemotherapeutic agents can accumulate in tumor tissues through the effect of increased permeability and retention (EPR), and release their charge in response to specific stimuli present in the tumor microenvironment or applied externally.

[0297] The nanoparticles according to the invention can be used in the treatment of various diseases other than cancer. For example, in the context of inflammatory diseases, the nanoparticles can be designed to target inflamed tissues and deliver anti-inflammatory agents. The controlled-release properties can help maintain therapeutic drug concentrations at the site of inflammation while minimizing systemic exposure.

[0298] According to a preferred embodiment, the pharmaceutical active ingredient encapsulated in the nanoparticle according to the invention is doxorubicin, also known as doxorubicin hydrochloride. Doxorubicin is an anticancer agent belonging to the anthracycline family, widely used in the treatment of many solid and hematological cancers. Doxorubicin may be particularly well-suited for encapsulation in the nanoparticles according to the invention due to its amphiphilic nature, which allows its incorporation either into the hydrophobic membrane or into the aqueous compartment of the vesicular nanoparticles. Encapsulating doxorubicin in the nanoparticles according to the invention can reduce its systemic toxicity, particularly its known cardiotoxicity, while maintaining its therapeutic efficacy through controlled release at the tumor site.

[0299] In the field of infectious diseases, the nanoparticles according to the invention can be used to deliver antibiotics or antiviral agents. The ability to functionalize the surface of the nanoparticles with targeting groups can improve their accumulation at sites of infection, potentially enhancing treatment efficacy and reducing the development of drug resistance.

[0300] For neurodegenerative diseases, the nanoparticles according to the invention can be designed to cross the blood-brain barrier and deliver therapeutic agents to the central nervous system. Their controlled-release properties can help maintain consistent drug levels in the brain, which may be beneficial for the long-term management of chronic neurodegenerative conditions.

[0301] In cardiovascular medicine, these nanoparticles can be used for targeted delivery of thrombolytic agents to treat blood clots, or for delivering therapeutic agents to atherosclerotic plaques. The imaging capabilities of nanoparticles can also be exploited for diagnostic purposes, such as visualizing vascular abnormalities or assessing plaque composition.

[0302] The use of compositions containing nanoparticles according to the invention in treatment methods preferably involves administering a composition according to the invention to the patient, followed by activation of the cleavable pattern of the nanoparticles by specific stimuli. These stimuli may be applied exogenously (for example, localized irradiation for radiation-sensitive nanoparticles) or may be endogenous conditions specific to the pathological site (such as an acidic pH or the presence of particular enzymes in tumors).

[0303] This approach allows for controlled and localized release of therapeutic molecules, thus optimizing treatment efficacy while minimizing systemic side effects.

[0304] The combination of stable nanoparticle formation and controlled release properties therefore makes amphiphilic copolymers particularly advantageous for drug delivery applications.

[0305] Nanoparticles can also be used as contrast agents for various biological imaging techniques and applications. The invention therefore also relates to a method for diagnosing one or more pathologies, such as cancer, using nanoparticles according to the invention and / or compositions containing them. Depending on the specific composition and functionalization of the nanoparticles, they can be suitable for different imaging techniques, including magnetic resonance imaging (MRI), computed tomography (CT), ultrasound imaging, and optical imaging. For example, nanoparticles incorporating superparamagnetic iron oxide nanoparticles (SPIONs) can serve as MRI contrast agents, improving the visibility of specific tissues or organs. Similarly, nanoparticles containing high atomic number elements, such as gold or iodine, can provide contrast for CT imaging.In imaging applications, nanoparticles can incorporate contrast agents either through encapsulation or by functionalizing polymer segments. The stability provided by the copolymer structure ensures that the contrast agents remain concentrated within the nanoparticles, enhancing signal intensity and image quality. Controlled-release properties can be exploited to create activatable imaging agents, where the imaging signal is enhanced upon exposure to specific stimuli.

[0306] A particular application of these nanoparticles is image-guided radiotherapy. In this context, the nanoparticles can serve as both imaging agents and radiosensitizers. Their imaging capabilities allow for precise tumor localization, while their radiosensitizing properties can enhance the effectiveness of radiotherapy. When exposed to ionizing radiation, the nanoparticles can generate localized effects that increase damage to tumor cells, potentially improving therapeutic outcomes while sparing healthy tissue.

[0307] A particularly suitable application of the nanoparticles according to the invention is theranostics, which combines diagnostic and therapeutic capabilities on a single platform. Theranostic nanoparticles can be designed to simultaneously provide imaging contrast and deliver therapeutic agents. This approach enables real-time monitoring of drug delivery and treatment response. For example, nanoparticles loaded with both imaging agents and chemotherapeutic drugs can be used to visualize tumor accumulation, monitor drug release, and assess treatment efficacy in a single system.

[0308] The potential applications of the amphiphilic copolymers and nanoparticles according to the invention in the biomedical field are therefore very broad. Advantageously, their unique stability under physiological conditions and their ability to achieve controlled release, along with their versatility in functionalization, enable a wide range of applications. The cleavable motif, positioned between the hydrophilic and hydrophobic segments, plays a crucial role in controlling the stability and reactivity of the nanoparticles. The F functional group within the cleavable motif limits spontaneous cleavage under physiological conditions, ensuring that the nanoparticles remain intact during circulation and accumulation at target sites. This same functional group, however, does not prevent controlled cleavage of the cleavable motif in response to specific stimuli, allowing precise temporal and spatial control over the release of the encapsulated molecules.

[0309] The activation of the nanoparticles according to the invention can be achieved by cleaving the cleavable motif located between the hydrophilic and hydrophobic segments of the amphiphilic copolymer. This controlled cleavage leads to structural changes in the nanoparticles, allowing the release of the encapsulated molecules. The stimuli that can trigger the cleavage can be exogenous or endogenous. This cleavage can be induced by various stimuli, including UV light, visible photons, ionizing radiation (beta, X-rays, or gamma rays), focused ultrasound, or endogenous or exogenous redox activation at the target site.

[0310] The rate and extent of release can be modulated by adjusting various parameters, including: - the chemical structure of the cleavable motif, which affects its sensitivity to specific stimuli. - the position and number of cleavable motifs in the copolymer structure. - the relative lengths of the hydrophilic and hydrophobic segments, which influence the stability of the nanoparticles and their response to cleavage events. - the intensity and duration of the applied stimulus, which determine the degree of cleavage.

[0311] In a particularly suitable embodiment, activation—that is, the triggering exogenous signal—can be achieved by irradiation with beta, X-rays, or gamma rays. The cleavable motif can indeed be designed to respond to ionizing radiation, such as X-rays or gamma rays. When exposed to ionizing radiation, the cleavable motif undergoes radiolysis, resulting in the cleavage of the bonds. This mechanism is particularly useful for radiotherapy applications, where the release of therapeutic agents can be synchronized with radiation treatment.

[0312] Preferably, radiation doses can range from 0 to 80 Gy, or from 0.1 to 80 Gy, with effective activation ranges of 80 Gy or less, 60 Gy or less, 50 Gy or less, 40 Gy or less, 30 Gy or less, 20 Gy or less, 10 Gy or less, 8 Gy or less, 6 Gy or less, 4 Gy or less, and 2 Gy or less. For diagnostic or low-impact applications, doses of 0.1 to 2 Gy may be sufficient, while doses of 0.1 to 50 Gy may be preferred for therapeutic applications. Doses may be cumulative or individual.

[0313] Activation can preferably be carried out between 30 minutes and 72 hours after administration of the nanoparticle.

[0314] In certain aspects of the invention, the administration of nanoparticles conjugated to molecules, preferably in compositions, and irradiation can be carried out according to different time schedules, for example, once a week, every two weeks, every three weeks, or every four weeks. The treatment can comprise at least one, two, three, four, five, six, seven, eight, nine, or ten cycles of administration and irradiation. This flexibility in dosage and administration schedule allows for optimization of therapeutic efficacy while minimizing potential side effects.

[0315] The invention may also relate to the use of nanoparticles according to the invention whose cleavable motifs of the copolymers are activatable (cleavable) in response to endogenous signals such as redox enzymes, metalloproteinases, reactive oxygen species (ROS) such as peroxides, or reactive nitrogen species (NO).

[0316] The invention may be based on the generation of local electrons (Auger or Compton electrons) formed by the interaction of high-energy electromagnetic waves (X-rays or gamma rays) with nanoparticles. In the case of nanoparticles incorporating magnetic transition metals or magnetic lanthanides, the proton relaxivity properties can lead to high efficiency for in vivo MRI, enabling image-guided therapy.

[0317] The local release of active substances by cleaving the nanoparticles of the present invention can be an important factor in improving therapeutic efficacy and patients' quality of life, particularly by reducing toxicity to tissues and vital organs located away from the target.

[0318] Monitoring the activation and release of therapeutic agents can be achieved through various non-invasive bioimaging techniques. These techniques may include MRI with contrast agents based on magnetic metals, metal oxides or lanthanides (such as gadolinium or dysprosium), optical modalities (fiber optic endoscopy in the visible or near-infrared, or photoluminescence with quantum dots or lanthanide-doped upconversion nanoparticles), positron emission tomography (PET), single-photon emission tomography (SPECT) with a radionuclide, or X-ray tomography with a high atomic number element.

[0319] The invention also specifically relates to a method for the therapeutic treatment and / or prevention of various disorders, including cancer, immune-related diseases, inflammation, diabetes, bacterial and / or viral infections, and pediatric or age-related diseases. The nanoparticles according to the invention and the compositions containing them can indeed be used for the treatment and / or prevention of these disorders. The treatment method may include administering a composition containing the nanoparticles to a patient in need, followed by activation of the nanoparticles by an appropriate stimulus.

[0320] The doses of functional nanoparticles (such as the nanoparticles according to the invention comprising at least one therapeutic molecule) can be adjusted over a wide range from approximately 0.005 mg / m² to approximately 1000 mg / m² of body weight. More specifically, the doses can be approximately 0.05, 0.055, 0.5, 5, 10, 20, 30, 40, 50, or 100 mg / m². The choice of dose will depend on factors such as the nature of the pathology being treated, the route of administration, and the specific characteristics of the patient.

[0321] Alternatively, doses of functional nanoparticles (such as the nanoparticles according to the invention comprising at least one therapeutic molecule) can be adjusted over a wide range from approximately 0.0005 mg / kg to approximately 100 mg / kg body weight. More specifically, doses can be approximately 0.0005, 0.005, 0.05, 0.5, 5, 10, 20, 30, 40, 50, or 100 mg / kg. The choice of dose will depend on factors such as the nature of the pathology being treated, the route of administration, and the specific characteristics of the patient.

[0322] According to one embodiment, the therapeutic method of the invention uses nanoparticles of the invention whose cleavable copolymer motif is sensitive to radiation. The therapeutic treatment method of the invention may include a step of exposing the nanoparticles to ionizing radiation after their administration to the subject. The ionizing radiation may be applied at a dose between 0 and 80 Gy, for example, between 2 and 16 Gy. The radiation exposure may trigger the controlled release of the therapeutic agents encapsulated in the nanoparticles. The doses may be therapeutic in themselves (radiotherapy) to combine the effect of the radiation and the released therapeutic molecules, or sub-therapeutic only to release the therapeutic molecules contained in the nanoparticles.

[0323] The method may involve the administration of a single dose of radiation or multiple fractionated doses. For example, the method may include the administration of several doses of 0.1 to 4 Gy at predetermined intervals for a cumulative dose over several days of 10 to 80 Gy.

[0324] In some cases, the method may include a step to determine the optimal radiation dose to achieve the desired release of therapeutic agents. This determination may be based on factors such as the nature of the pathology being treated, the location of the area to be treated, and the specific characteristics of the patient.

[0325] The method may also include a step to monitor the release of therapeutic agents. This monitoring can be performed using medical imaging techniques, allowing visualization of the distribution of nanoparticles and / or the release of therapeutic agents in vivo.

[0326] In some respects, the method can be adapted for the treatment of various pathologies, including cancers. The method can be used in combination with other therapeutic modalities, such as conventional chemotherapy or immunotherapy.

[0327] The method may also include steps to personalize the treatment based on the patient's response. For example, radiation doses or the frequency of administration can be adjusted according to the observed effectiveness and the patient's tolerance to the treatment.

[0328] In some cases, the method may include the use of functionalized nanoparticles to specifically target certain tissues or cell types, thereby enabling increased targeting of target cells during the release of therapeutic agents.

[0329] The method may also include long-term follow-up steps to assess the effectiveness of the treatment and detect any potential late side effects.

[0330] According to another aspect, the invention relates to a method of treating a disease, in particular cancer, in a subject in need of it, comprising: (a) administering to the subject a nanoparticle according to the invention comprising at least one encapsulated therapeutic molecule, and (b) exposing the nanoparticle to ionizing radiation at a dose of between 0.1 and 80 Gy, preferably between 2 and 16 Gy, so as to trigger the cleavage of the cleavable motif and the controlled release of the therapeutic molecule.

[0331] In one embodiment, exposure to ionizing radiation can be performed between 30 minutes and 72 hours after administration of the nanoparticle, preferably between 1 and 48 hours after administration. This delay allows for the accumulation of nanoparticles at the tumor site before the drug release is triggered by irradiation.

[0332] In a preferred embodiment, the therapeutic molecule is doxorubicin and the disease is cancer, preferably a solid tumor, and even more preferably colon carcinoma or pancreatic cancer. The preclinical studies described in the preceding examples demonstrate the efficacy of this approach in murine models of CT26 colon carcinoma and KPC pancreatic cancer.

[0333] The ionizing radiation dose can be a single dose or fractionated. Doses of 2 to 16 Gy, including 8 Gy, can be used in combination with the release of the encapsulated therapeutic molecule. This method can create synergy between the direct effect of ionizing radiation on tumor cells and the effect of the therapeutic molecule released locally by the cleavage of nanoparticles.

[0334] In another aspect, the invention also relates to a diagnostic method using nanoparticles whose cleavable copolymer motif is sensitive to stimuli, particularly radiation. This method may involve administering to a subject a composition containing nanoparticles according to the invention, these nanoparticles encapsulating one or more contrast agents or diagnostic molecules. In some aspects, the method may involve exposing the nanoparticles to ionizing radiation after their administration, with doses ranging from 0 to 80 Gy, for example, between 1 and 4 Gy. These lower doses may be sufficient to trigger the release of the contrast agents without causing significant therapeutic effects. The controlled release of the contrast agents can enable targeted imaging of the tissues of interest.The method may involve the use of various medical imaging techniques, such as MRI, CT, or optical imaging, depending on the nature of the encapsulated contrast agents. In some cases, the method can enable dynamic monitoring of nanoparticle distribution and contrast agent release in vivo, thus providing valuable information on the location and extent of certain pathologies, particularly tumors. The method can also be adapted to assess response to ongoing treatment, allowing visualization of changes in nanoparticle distribution or accumulation over time.

[0335] EXAMPLES:

[0336] In the following examples, the following abbreviations are used:

[0337] Regarding synthesis methods: - SPAAC: Strain-Promoted Azide-Alkyne Cycloaddition. - CuAAC: Copper-Catalyzed Azide-Alkyne Cycloaddition. - NCA: N-Carboxyanhydride. Solvents, reagents: - PBS: Phosphate saline buffer, isotonic solution at pH 7.4 used for dilutions and washes under physiological conditions. -TRIS: Tris(hydroxymethyl)aminomethane, biological buffer used in the pH range 7-9. - EDTA: Ethylenediaminetetraacetic acid, a chelating agent used to remove residual metal ions (especially Cu 2+ ). - DMAP: 4-dimethylaminopyridine, nucleophilic catalyst for acylation reactions. Markers and proteins - BSA: Bovine serum albumin (~66 kDa), model protein for grafting studies. - Cy5, Cy7: Cyanine fluorophores emitting in the near infrared (Cy5: ~670 nm; Cy7: ~775 nm), used for labeling and fluorescence imaging. For characterization: - DLS: Dynamic Light Scattering - GPC: Gel Permeation Chromatography - PDI: Polydispersity Index - DCR: Derived Count Rate - MWCO: Molecular Weight Cut-Off threshold - FTIR: Fourier Transform Infrared Spectroscopy, used to monitor the consumption of NCA monomers. -TEM: Transmission electron microscopy. Cryo-TEM: variant with vitrified sample to observe nanoparticles in a hydrated state. - SARRP: Radiotherapy platform for small animals, enabling targeted irradiation with image guidance. Molar mass parameters: - Đ : Dispersity (Mw / Mn) - Mn: Average molar mass by number - Mw: Mass average molar mass - DP: Degree of polymerization Histology: - HES: Hematoxylin-eosin-saffron staining, standard technique for histopathological analysis (nuclei in blue, cytoplasm in pink, collagen in yellow). - PAS: Periodic acid-Schiff staining, used to visualize tubular basement membranes and brush borders. PART A: SYNTHESIS OF AMPHIPHILIC COPOLYMERS

[0338] Proton nuclear magnetic resonance (NMR) 1 H) was recorded on a Bruker Advance 400 spectrometer (400 MHz). The chemical shifts of the protons are expressed in parts per million (ppm) relative to tetramethylsilane (TMS) and referenced to the residual proton of the NMR solvent.

[0339] The data are represented as follows: chemical shift, integration, multiplicity, coupling constants in Hertz (Hz). The following abbreviations are used: s = singlet, bs = wide singlet, d = doublet, dd = double doublet, t = triplet, m = multiplet, br m = wide multiplet, bb = wideband.

[0340] In the following examples, different examples of amphiphilic copolymers of formula (49) R1-R6-MF-R7-R2 were synthesized: (49)

[0341] For all of these formulas: - Motif M is represented by formula (50): i 6 (50) R7 is an ethyl group: -CH2-CH2

[0342] Table 1: Summary of the amphiphilic copolymers of the present invention synthesized Amphiphilic copolymers R2 Hydrophobic Ri Hydrophilic F Figures PEG44-carbamate-o-pico-PBLG 2i(via SPAAC) PBLG2I PEG44 -OC(=O)-NH- Figure 1 PEG44-carbamate-o-pico-PBLG 2i PBLG2I PEG44 -OC(=O)-NH- Figure 2 PEG 44 -carbamate-p-pico-PBLG 2i PBLG2I PEG44 -OC(=O)-NH- Figure 3 PEG44-carbamate-o-pico-PBLGi5 PBLG15 PEG44 -OC(=O)-NH- Figure 4 PEG44-carbamate-o-pico-PBLG25 PBLG 25 PEG44 -OC(=O)-NH- Figure 5 PEG44-carbamate-o-pico-PBLG35 PBLG 35 PEG44 -OC(=O)-NH- Figure 6 PEG44-carbamate-o-pico-PBLG 42 PBLG 42 PEG44 -OC(=O)-NH- Figure 7 PEG44-carbamate-o-pico-PLL5-co-PBLGi5 PLL5-CO-PBLG15 PEG44 -OC(=O)-NH- Figure 8 PEG44-carbamate-o-pico-PTyr5-co PBLG15 PTyr5-co-PBLGi5 PEG44 -OC(=O)-NH- Figure 9 PSar 42 -carbamate-o-picPBLG 2i PBLG2I PSar 42 -OC(=O)-NH- Figure 10 PSar 29 -carbamate-o-pico-PLA45 PLA45 PSar 29 -OC(=O)-NH- Figure 11 PEG44-carbamate-o-pico-PLAi2o PLA 120PEG44 -OC(=O)-NH- Figure 12 PEG44-carbamate-o-pico-PLA45 PLA45 PEG44 -OC(=O)-NH- Figure 13 PEG44-carbonate-o-pico-PBLG25 PBLG25 PEG44 -OC(=O)-O- Figure 14 PEG44-p-ester aromatic-o-pico-PBLG2i PBLG21 PEG44 ester aroma. Figure 15

[0343] The various formulas of these copolymers of formula (7) are grouped in Table 1 above. The following copolymers are given as non-limiting examples.

[0344] Example AI: Synthesis of hydrophobic polymers R2

[0345] 1A.1 Case of homopolymers:

[0346] In a dry flask under an inert atmosphere, an NCA monomer (Z mmol), such as: - γ-benzyl-L-glutamate N-carboxyanhydride (BLG-NCA), - Ne-trifluoroacetyl-L-lysine N-carboxyanhydride (Lys(TFA)-NCA), or - O-benzyl-L-tyrosine N-carboxyanhydride (Tyr-NCA) is dissolved in anhydrous dimethylformamide (DMF) (V mL) to obtain a 1 M concentration (e.g., V = Z mL when Z is expressed in mmol). The initiator (Y mmol), such as propargylamine or 3-azidopropylamine, is added under an inert atmosphere to give a monomer / initiator ratio of Z / Y (target DP ~ Z / Y).

[0347] The mixture is stirred at 20°C for 24 hours under an inert atmosphere until monomer consumption is confirmed by FTIR (Fourier Transform Infrared Spectroscopy) (disappearance of the NCA carbonyl bands at ~1850 and ~1780 cm⁻¹). -1 ).

[0348] The polymer is isolated by precipitation in cold diethyl ether (ZxlOmL), recovered by centrifugation (4000 rpm, 10 min, 5°C), washed with diethyl ether (3 times), and dried under high vacuum at room temperature for 12-24 hours to give a white solid with a yield of 70-90%.

[0349] 1A.2 Special case of PLAs:

[0350] In a dry container under an inert atmosphere, D,L-lactide (Z mmol) is dissolved in anhydrous tetrahydrofuran (THF) (V mL) to obtain a concentration of 1 M. The initiator (Y mmol) with a monomer / initiator ratio of Z / Y and 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) (0.5xY mmol) are added under an inert atmosphere and vigorous stirring.

[0351] The reaction is left under stirring for 1 hour at room temperature. The reaction is stopped with benzoic acid (4.0 x Z mmol). The homopolymer is isolated by precipitation in cold n-propanol, recovered by centrifugation, washed with diethyl ether 3 times, and dried under vacuum to give a white solid with a yield of 40–50%.

[0352] 1A.3 Case of copolymers:

[0353] In a dry container under an inert atmosphere, an NCA A monomer (Z mmol) and an NCA B monomer (Z Bmmol) are dissolved in anhydrous DMF (VmL) to obtain a concentration of 1 M (V = Z A + Z B mL). The initiator (Y mmol) is added under an inert atmosphere to give a monomer / initiator ratio of (Z A + Z B ) / Y (DP target = (Z A + Z B ) / Y).

[0354] The mixture is stirred at 20°C for 24 hours under an inert atmosphere until monomer consumption is confirmed by FTIR. The copolymer is isolated by precipitation in diethyl ether ((Z A + Z B ) x 10 mL), recovered by centrifugation, washed with diethyl ether 3 times, and dried under vacuum to give a white solid with a yield of 70-90%.

[0355] Example 2A: Synthesis of the N3-(CH2)3-MF-R7-RI motif:

[0356] 2A.1 Case where RI = PEG44:

[0357] In a dry container under an inert atmosphere, 1-(3-azidopropyl)-2-(hydroxymethyl)pyridinium tosylate (X mmol), carbodiimidazole (X mmol), and DMAP (0.1 x X mmol) are dissolved in acetonitrile (V mL). The reaction is initiated at approximately -30°C under an inert argon atmosphere. The mixture is stirred for approximately 3 hours, during which time the temperature is gradually raised to room temperature.

[0358] After the reaction is complete, the acetonitrile is evaporated under reduced pressure, and the residue is dissolved in dichloromethane at room temperature. Then, the MeO-PEG 44 -NH2 (X mmol) is added with stirring at room temperature. The reaction mixture is allowed to react with stirring at room temperature for approximately 12 hours. The product is purified by precipitation in cold diethyl ether.

[0359] 2A.2: Case where Ri = Psar x :

[0360] In a dry container under an inert atmosphere, l-(3-azidopropyl)-2-(hydroxymethyl)pyridinium to-sylate (X mmol), carbodiimidazole (X mmol) and DMAP (0.1xX mmol) were dissolved in acetonitrile (V mL, the volume of solvent V having been chosen to achieve a target concentration of 100 mg / mL; V = m / 100, where m corresponds to the mass of polymer in mg and V is expressed in mL).

[0361] The reaction was initiated at a temperature of approximately -30 °C, then allowed to continue under stirring and in an inert argon atmosphere for approximately 3 hours, during which time the temperature was allowed to gradually rise to room temperature.

[0362] At the end of the reaction, acetonitrile was removed under reduced pressure using a vacuum line, and the residue obtained was then solubilized in dichloromethane at room temperature.

[0363] Next, hexylamine-PsarDP (X mmol), where DP denotes the degree of polymerization of the polysarcosine block, was added with stirring at room temperature. The reaction mixture was kept under stirring at room temperature for approximately 12 hours.

[0364] The solvent was then removed under reduced pressure to obtain a crude residue, which was dissolved in distilled water (V mL). The resulting solution was placed in a dialysis membrane (MWCO₃ 1 kDa) and dialyzed against distilled water (5 L) for 6 h, with a water change every 2 h (3 baths in total).

[0365] The solution contained in the dialysis bag was recovered and then lyophilized to obtain the product in powder form (yield 50-70%).

[0366] 2A.3: Case where F is a carbonate (F= -C(=O)-O-):

[0367] In a dry container under an inert atmosphere, l-(3-azidopropyl)-2-(hydroxymethyl)py-ridinium tosylate (X mmol) and carbodiimidazole (X mmol) were dissolved in dichloromethane (V mL, the volume of solvent (V) having been chosen to achieve a target concentration of 100 mg / mL; V = m / 100, where m corresponds to the mass of polymer in mg and V is expressed in mL).

[0368] The reaction was initiated at a temperature of approximately -30 °C, then allowed to continue under stirring and in an inert argon atmosphere for approximately 3 hours, during which time the temperature was allowed to gradually rise to room temperature.

[0369] At the end of the reaction, the MeO-PEG 44-OH (X / 5 mmol) was added with stirring at room temperature. The reaction mixture was kept under stirring at room temperature for approximately 12 hours. The resulting reaction mixture was then subjected to aqueous treatment, consisting of three successive washes with 5 mL of deionized water and 45 mL of dichloromethane (DCM).

[0370] The organic phase was recovered and the solvent removed under reduced pressure using a rotary evaporator. The crude product was then redissolved in a minimal volume of DCM and precipitated in 45 mL of diethyl ether. The resulting solid was collected and dried under vacuum (30% yield).

[0371] Example 3A: Coupling and obtaining amphiphilic copolymers:

[0372] 3A.1.a General procedure for coupling by CuAAC click chemistry:

[0373] In a dry container under an inert atmosphere, the polymer N3-(CH2)3-MF-R7-RI_(X mmol), the hydrophobic polymer R7 comprising an alkyne function (0.95xX mmol) and the Cu + [CH3CN]4RF6“ (0.95xX mmol) are dissolved in anhydrous dichloromethane (DCM) (V mL, the solvent volume (V) is selected for a target polymer concentration of 100 mg / mL). The mixture is immediately deoxygenated by bubbling with argon for 5 minutes. The mixture is stirred at room temperature for 2 hours under an inert atmosphere.

[0374] 3A.1.b: Purification for PEG-based copolymers:

[0375] The organic solvent is evaporated under reduced pressure. The crude residue is dissolved in dichloromethane, and the resulting solution is added dropwise to a large excess of cold n-propanol (DCM:n-propanol ratio ~ 1:15). The precipitated amphiphilic copolymer is isolated by centrifugation, washed with cold n-propanol, and dried under high vacuum.

[0376] 3A.1. C: Purification for Psar-based copolymers:

[0377] The organic solvent was removed under reduced pressure. The crude residue was dissolved in aceto-nitrile (ACN), and then a 50:50 (v / v) mixture of saturated EDTA solution (pH ~7) and brine was added. The resulting solution was placed in a dialysis membrane (MWCO 10 kDa) and dialyzed against distilled water (5 L) for 6 h, with a water change every 2 h (3 baths in total). The solution in the dialysis bag was collected and then lyophilized to obtain the product as a powder.

[0378] 3A.2: Coupling procedure using SPAAC (copper-free):

[0379] In a dry container, the polymer N3-(CH2)3-MF-R7-RI (1.3XX mmol) and the hydrophobic polymer R7 functionalized with cyclooctyne (X mmol) are dissolved in anhydrous dichloromethane (V mL). The mixture is stirred at room temperature for approximately 16 hours. The product is purified by precipitation in cold n-propanol.

[0380] 3A.3: Special case of the labeled amphiphilic copolymer:

[0381] In a dry container under an inert atmosphere, the PEG 44 -carbamate-o-pico-PBLG25 (100 mg, 0.013 mmol), cyanine-5-NHS ester (Cy5-NHS ester) (8.6 mg, 0.013 mmol), and triethylamine (1.8 pL, 0.013 mmol) were dissolved and mixed in anhydrous THF at 25 °C. After 12 hours of stirring at 25 °C, the solvent was evaporated, and the dry residue was washed three times with cold n-propanol to obtain the final product (70%).

[0382] GPC (relative to PMMA, DMF standards): Đ = 1.6; Mn = 1572 g / mol; Mw = 2557 g / mol.

[0383] 3A.4: Synthesized amphiphilic copolymers:

[0384] Table 2 summarizes the obtained yields of the amphiphilic copolymers synthesized under the present invention. AmphicopolymersRendering1H NMR Method Characterization philes ment (%) 5 9.11 (m, 1H, peak), 8.75 - 8.50 (m, 1H, peak), 7.72 - 6.88 (m, PEG 44 -carbamate-o- 112H, CHarom, PBLG, DBCO), 5.53 (m, 2H, CH2-OC(O)-NH), 5.01 (s, pico-PBLG 2i (via SPAAC 82 42H, CH2-Ph, PBLG), 4.10 - 3.73 (m, 21H, (C=O)CHNH, PBLG), 3.51 SPAAC) (s, 176H, O-CH2-CH2-O (PEG)) 59.00 (d, 1H, peak), 8.59 (t, 1H, peak), 7.39–6.98 (m, 105H, CHarom PEG 44 -carbamate-o- PBLG), (5.49, s, 2H, CH2-OCONH), 5.17 - 4.87 (m, 42H, CH2Ph ​​CuAAC 86 pico-PBLG 2i PBLG), 4.06–3.79 (m, 21H, COCHNH, PBLG), 3.71–3.41 (m, 178H, CH2-TZ, PEG). 5 8.99 - 8.93 (d, 37HH =6.3 Hz, 2H, pico), 8.01 - 7.95 (d, 3 7HH =6.3 PEG 44 -carbamate-p- Hz, 2H, pico), 7.44 - 6.97 (m, 105H, arom, PBLG), 5.36 - 5.32 (s, CuAAC 74 pico-PBLG 2i 2H, CHÜ-OCONH), 5.09 -4.82 (br s, 42H, CFbPh, PBLG), 4.02 - 3.81 (s, 21H, COCHNH, PBLG), 3.53 - 3.48 (s, 172H, PEG). 5 9.00 (d, 3 J HH= 6.1 Hz, 1H, pico), 8.59 (t, 3 7HH = 7.7 Hz, 1H, pico), PEG 44 -carbamate-o- CuAAC 76 7.61 - 6.96 (m, 75H, Ph, PBLG), 5.50 (s, 2H, CH2-OC(O)-NH), 5.30 pico-PBLGis -4.76 (m, 30H, CFbPh, PBLG), 3.51 (s, 176H, O-CH2-CH2-O, PEG). 5 9.00 (d, 3 J HH= 6.1 Hz, 1H, pico), 8.59 (t, 3 7HH = 7.7 Hz, 1H, pico), PEG 44 -carbamate-o- CuAAC 92 7.61 -6.96 (m, 125H, Ph, PBLG), 5.50 (s, 2H, CH2-OC(O)-NH), 5.30 pico-PBLG 25 -4.76 (m, 50H, CFbPh, PBLG), 3.51 (s, 176H, O-CH2-CH2-O, PEG). 5 9.00 (d, 3J HH= 6.1 Hz, 1H, pico), 8.59 (t, 3 7HH = 7.7 Hz, 1H, pico), PEG 44 -carbamate-o- CuAAC 90 7.61 -6.96 (m, 175H, Ph, PBLG), 5.50 (s, 2H, CH2-OC(O)-NH), 5.30 pico-PBLG 35 -4.76 (m, 70H, CFbPh, PBLG), 3.51 (s, 176H, O-CH2-CH2-O, PEG). 5 9.00 (d, 3 J HH= 6.1 Hz, 1H, pico), 8.59 (t, 3 7HH = 7.7 Hz, 1H, pico), PEG 44 -carbamate-o- CuAAC 84 7.61 -6.96 (m, 210H, Ph, PBLG), 5.50 (s, 2H, CH2-OC(O)-NH), 5.30 pico-PBLG 42 -4.76 (m, 84H, CFbPh, PBLG), 3.51 (s, 176H, O-CH2-CH2-O, PEG). 5 9.43 - 9.23 (m, 5H, NHCOCF3, PLL), 9.00 (m, 1H, pico), 8.59 (m, PEG 44 -carbamate-o- 1H, pico), 7.39 - 7.13 (m, 75H, CHarom, PBLG), 5.49 (s, 2H, CH2- CuAAC 65 pico-PLL5-co-PBLGi5 OCONH), 4.87-4.16 (m, 30H, CH2Ph, PBLG), 3.71-3.40 (m, 176H, CH2(PEG)). Copolymères amphiRendeMéthode RMN 1H Caractéristique philes ment (%) 5 9.29 - 8.98 (m, 5H, Ar-OH, pTYR), 7.42 - 7.09 (m, 75H, CHarom, PEG44 -carbamate-o- PBLG), 7.05 - 6.78 (m, 12H, CHarom, pTyr), 6.74 - 6.42 (m, 12H, CuAAC 73 pico-PTyr5-co-PBLGi5 CHarom, Tyr), 5.18 - 4.71 (m, 40H, CH2Ph, PBLG), 3.32 (s, 176H, OCH2CH2O, PEG) 59.06 (br s, 1H, Hi), 8.62 (br s, 1H, pico), 7.66 - 6.94 (m, 105H, Ph, PSar 42 -carbamate-o- PBLG), 5.80 - 5.43 (m, 2H, CH2-OC(O)-NH), 5.27 - 4.85 (m, 42H, CuAAC 54 pico-PBLG 2i CEbPh, PBLG), 4.77 - 3.76 (br m, 88H, 2H, N-CHz-C(O), pSar), 3.18 - 2.69 (m, 126H, 3H, N-CH3, pSar) 5 9.10 (br s, 1H, Hi), 8.61 (br s, 1H, pico), 8.25 (s, 1H, Tz), 5.70 - PSar 29 -carbamate-o- 5.25 (br m, 2H, CEb-OCONH), 5.28 - 5.07 (br m, 45H, COCHCHaO, CuAAC 50 pico-PLA 45PLA), 4.48 - 3,55 (br m, 58H, -C(O)CH2NCH3, pSar), 3,0 - 2,69 (br m, 87H, NCHa, pSar), 1.60 - 1.35 (br m, 135H, CfOJCHCHsO, PLA) 5 9.06 (d, 3 JHH = 6.2 Hz, 1H, pico), 8.64 (t, 3 7HH = 7.9 Hz, 1H, pico), PEG 44 -carbamate-o- 8.21 (s, 1H, Tz), 5.59 - 5.39 (m, 4H, Tz-CEb-O, CEb-OCONH), 5.31 CuAAC 89 pico-PLAi 2O - 5.07 (m, 120H, CO-CH(CH3)-O, PLA), 3.57 - 3.40 (m, 176H, O- CH2-CH2O, PEG), 1.66 - 1.21 (m, 360H, C(O)-CH(CH3)-O, PLA). 5 9.06 (d, 3 JHH = 6.2 Hz, 1H, pico), 8.64 (t, 3 7HH = 7.9 Hz, 1H, pico), PEG 44 -carbamate-o- 5.59 - 5.39 (m, 4H, Tz-CEb-O, CEb-OCONH), 5.31 - 5.07 (m, 45H, CuAAC 50 piCO-PLA45 CO-CH(CH3)-O, PLA), 3.57 - 3.40 (m, 176H, O-CH2-CH2O, PEG), 1.66 - 1.21 (m, 135H, C(O)-CH(CH3)-O, PLA). 59.04 (d, J = 6.2 Hz, 1H, pico), 8.59 (t, 3 7HH = 7.9 Hz, 1H, pico), 7.37 PEG 44-carbonate-o- - 7.15 (m, 125H, CHarom, PBLG), 5.62 (s, 2H, CH2O(C=O)O), 5.22 - CuAAC 92 pico-PBLG 25 4.78 (m, 50H, CH2Ph, PBLG), 4.06-3.71 (m, 25H, COCHNH, PBLG), 3.51 (m, 176H, PEG). 59.03 (d, 3 JHH=7.87 HZ, 1H, pico), 8.61 (t, 3 7HH =7.87 Hz, 1H, pico), PEG44-p-ester aroma- 7.38 - 7.16 (m, 105H, CH2Ph, PBLG), 5.84 - 5.50 (s, 2H, CH2- CuAAC 73 tic-o-pico-PBLG 2i OOCPh-O), 5.12 - 4.83 (m, 42H, CH2Ph, PBLG), 3.82 - 4.05 (br s, 21H, COCHNH), 3.41 - 3.63 (s, 172H, PEG).

[0385] Example 5A: Chemical stability of copolymers:

[0386] The chemical stability of each of the aforementioned copolymers was evaluated under aqueous conditions at physiological temperature. Each copolymer was formulated at a concentration of 8 mg / mL in distilled water (total volume: 5 mL) and incubated at 37 °C with continuous stirring.

[0387] No visible aggregation or precipitation was observed during the incubation period. Aliquots were taken after 24 and 48 hours of incubation and then lyophilized. The resulting dry materials were then analyzed by NMR spectroscopy. 1 H in DMSO-d6 and by GPC using DMF as the eluent. Stability was monitored by NMR 1 H by observing the methylene resonance of 2-picolinium around 5.3–5.7 ppm. NMR 1 H showed no significant change in this signal. The GPC chromatograms recorded before and after 48 h were superimposable, with no significant measurable change in Mn, Mw, or dispersity (D), indicating the absence of degradation under these conditions. These results demonstrate that the copolymers of the present invention are chemically stable in aqueous media at physiological temperature for at least 48 hours.

[0388] PART B: FORMULATION OF NANOPARTICLES

[0389] Example IB: Nanoprecipitation and self-assembly:

[0390] 1B.1. General nanoprecipitation procedure:

[0391] The amphiphilic copolymer (mass m, mg) is dissolved in a water-miscible organic solvent (THF, acetonitrile, or a THF / ACN mixture) at the concentration indicated in Table 3ex below. The organic solution is rapidly added (all at once) to the aqueous phase (PBS pH 7.4 or distilled water) preheated to the temperature indicated in Table 3. The mixture is stirred at this temperature for 1 hour. The organic solvent is then allowed to evaporate at room temperature for 16 hours under stirring, in a fume hood.

[0392] 1B.2: Morphological characterization:

[0393] 1B.2.a Dynamic Light Scattering (DLS):

[0394] The hydrodynamic radii and size distribution of self-assembled copolymers were measured by dynamic light scattering (DLS) on a Zetasizer™ nano ZS instrument (Malvern, UK) equipped with a 633 nm helium-neon laser using backscatter detection at a fixed scattering angle of 173° on equilibrated samples at different temperatures (4°C, 20°C or 37°C).

[0395] 1B.2.b Cryo-transmission electron microscopy (Cryo-TEM):

[0396] The morphology of the nanoparticles was also observed by Cryo-TEM.

[0397] The images were recorded using electron microscopy, which allows for the observation of biological samples without prior drying or dehydration. This method involves rapidly freezing the samples at extremely low temperatures (-196°C), preserving their three-dimensional structure and enabling detailed observation at the nanoscale.

[0398] Cryo-TEM images confirm the formation of vesicles (polymersomes) for the following copolymers: - PEG 44 -carbamate(o)-pico(Cl⁻)-b-PBLG 21 at 1 mg / mL (Figures 16a and 16b) - PEG 44 -aromatic ester-pico-b-PBLG 21 at 3 mg / mL (Figures 17a and 17b)

[0399] 1B.2. C Transmission electron microscopy (TEM):

[0400] Transmission electron microscopy (TEM) images were recorded on a Hitachi H7650 microscope operating at 80 kV. A 0.1 mg-mL nanoparticle dispersion was used. 1The droplet was deposited onto a carbon-coated copper grid (200 mesh). The residual droplet was removed after 1 minute, and the resulting grids were stained negatively twice with 1.2% uranyl acetate for two consecutive 1-minute periods. The TEM images in Figures 18a, 18b, and 19 were obtained using this conventional TEM method.

[0401] TEM images confirm the formation of vesicles (polymersomes) for the following copolymer: - PSar 42 -carbamate-o-pico-PBLG 25 at 3 mg / mL (Figure 19) TEM images (Figures 18a and 18b) confirm the formation of a mixture of vesicles (polymersomes) and micelles at a concentration of 5 mg / mL for the following PEG copolymer 44 -carbamate-o-pico-PBLG 25

[0402] lB.2.d. Multi-angle light scattering (MALS) analysis:

[0403] MALS analysis was performed to confirm the vesicular structure of the nanoparticles. The Guinier plot provides information on the radius of gyration (Rg) of the nanoparticles. The hydrodynamic radius (Rh) was determined from the τq analysis. 2 allowing the measurement of the diffusion coefficient.

[0404] Results for PEG44-carbamate(o)-Pico(CI-)-b-PBLG2i at 1 mg / ml: - Rg = 49 nm - Rh = 50 nm - Rg / Rh (Guinier) = 0.98

[0405] Results for PEG^-aromatic ester-PICOj-b-PBLG i at 1 mg / ml - Rg / Rh (Guinier) = 0.86

[0406] The Rg / Rh ratio close to 1 is the mark of a hollow structure, which confirms the vesicular nature of the nanoparticles.

[0407] 1B.3 Formulation of nanoparticles:

[0408] Various amphiphilic copolymers according to the invention were formulated into nanoparticles by nanoprecipitation. The formulation conditions and characteristics of the resulting nanoparticles are presented in Table 3 below.

[0409] For each copolymer, the copolymer solution was prepared in an organic solvent (THF alone or a THF-ACN mixture in a 1:1 v / v ratio) at different concentrations. Nanoprecipitation was performed by adding the organic solution to an aqueous phase; the percentage indicated corresponds to the proportion of organic solvent in the final mixture.

[0410] The resulting nanoparticles were characterized by dynamic light scattering (DLS) to determine the mean hydrodynamic diameter Z (in nm) and the polydispersity index (PDI). Morphology was determined by DLS and / or transmission electron microscopy (TEM) when available.

[0411] Table 3 below summarizes the characterization of the nanoparticles obtained according to the present invention. Amphi-copolymers Organic solvent: Concentration Morphology Z (nm) PDI philes nature and ratio (mg / ml) (DLS or TEM) PEG44-carbamate-o-30%THF-ACN mixture pico-PBLG2i obtained via 3 231 0.15 N. D (1:1 v / v) SPAAC PEG 44 -carbamate-o- 30% THF 1 150 0.1 Pico-PBLG vesicles 2i PEG 44 -carbamate-p- 30% THF 5 110 0.05 Pico-PBLG vesicles 2i PEG44-carbamate-o-30%THF-ACN mixture 5 89 0.15 N. D pico-PBLGis (1:1 v / v) PEG44-carbamate-o- Vesicles / mi- 30% THF 5 71 0.17 pico-PBLG25 those PEG44-carbamate-o-30%THF-ACN mixture 5 59 0.13 N. D pico-PBLGas (1:1 v / v) PEG44-carbamate-o-30%THF-ACN mixture 5 76 0.14 N.D pico-PBLG42 (1:1 v / v) PEG 44-carbamate--o- 30%THF-ACN mixture 3 163 0.13 N.D pico-PLL5-co-PBLGi5 (1:1 v / v) PEG44-carbamate-o-20%THF-ACN mixture 3 160 0.10 N. D pico-PTyrs-co-PBLGis (1:1 v / v) PSar42-carbamate-o-30%THF-ACN mixture 3145 0.14 Pico-PBLG2i vesicles (1:1 v / v) PSar 29 -carbamate-o- 20% THF 5 67 0.17 N. D pico-PLA 45 PEG 44 -carbamate-o- 30% THF 0.5 88 0.10 Pico-PLAi micelles 2O PEG 44 -carbamate-o- 30% THF 5 69 0.26 piCO-PLA45 micelles PEG 44 -carbonate-o- 30% THF 5 131 0.03 Pico-PBLG2s vesicles PEG44-p-ester aromati- 30% THF 1 100 0.11 Vesicles que-o-pico-PBLG2i

[0412] The acronym N.D. in the table above means not determined because these measurements were not carried out.

[0413] These results demonstrate that the amphiphilic copolymers according to the invention can form nanoparticles of varying sizes, ranging from approximately 59 nm to 231 nm, with low polydispersity indices (PDI < 0.26), indicating homogeneous size distributions. The observed morphologies include vesicles (polymersomes) and micelles, depending on the nature and relative lengths of the hydrophilic and hydrophobic segments of the copolymers used.

[0414] Example 2B: Colloidal stability of nanoparticles:

[0415] The colloidal stability of nanoparticles formed from different amphiphilic se-Ion copolymers of the invention was evaluated at two temperatures: 4°C (storage temperature) and 37°C (physiological temperature).

[0416] After formulation by nanoprecipitation, the nanoparticle suspensions were stored at the indicated temperatures. At different time points, an aliquot of each sample was analyzed by dynamic light scattering (DLS) to determine the mean hydrodynamic diameter Z (in nm) and the polydispersity index (PDI).

[0417] The results are presented in Table 4 below.

[0418] Table 4: Stability of the nanoparticles of the present invention. 4°C 37°C Amphiphilic copolymers C* (mg / ml) Z-days (nm) PDI C* (mg / ml) Z-days (nm) PDI 0 216 0.12 0 216 0.12 PEG44-carbamate- o-pico-PBLG21 ( SPAAC) 1 1 3 216 0.16 1 224 0.12 0 159 0.10 0 159 0.10 PEG44-carbamate- o-pico- PBLG21 3 3 3 155 0.11 3 156 0.12 0 120 0.11 0 120 0.11 PEG44-carbamate- p-pico- PBLG21 3 3 3 121 0.12 3 121 0.09 0 101 0.15 0 101 0.15 PEG44-carbamate- o-pico- PBLG15 1 1 6 104 0.12 4 102 0.14 0 86 0.13 0 86 0.13 PEG44-carbamate- o-pico- PBLG25 1 1 6 84 0.12 4 88 0.11 0 90 0.12 0 90 0.12 PEG44-carbamate- o-pico- PBLG35 1 1 6 91 0.11 4 94 0.10 0 102 0.17 0 102 0.17 PEG44-carbamate- o-pico- PBLG42 1 1 6 99 0.18 4 107 0.15 0 143 0.13 0 143 0.13 PEG44 -carbamate-o-pico-PLL5-co-PBLGi5 1 1 3 147 0.13 1 140 0.13 0 145 0.03 0 164 0.09 PEG44-carbamate- o-pico- PTyrs-co-PBLGis 0.1 1 3 150 0.07 1 167 0.12 0 144 0.08 0 144 0.08 PSar42-carbamate- o-pico- PBLG21 1 1 3 143 0.08 1 144 0.08 0 67 0.17 0 67 0.17 PSar29-carbamate- o-pico- PLA45 5 5 3 65 0.21 1 66 0.21 0 88 0.10 0 88 0.10 PEG44- carbamate- o-pico- PLA120 5 5 3 89 0.09 1 81 0.08 0 131 0.03 0 127 0.07 PEG44 -carbonate-o-pico-PBLG25 5 5 5 131 0.03 1 118 0.02 0 102 0.05 0 102 0.05 PEG44-p-ester aromatique-o-pico-PBLG2i 3 3 3 103 0.05 3 104 0.07

[0419] C: concentration of the nanoparticle suspension

[0420] These results demonstrate that the nanoparticles formed from the amphiphilic copolymers according to the invention exhibit satisfactory colloidal stability. At 4°C, the nanoparticles remain stable for at least 3 to 6 days, depending on the formulation, with minimal variations in hydrodynamic diameter and polydispersity index. At 37°C (physiological temperature), the nanoparticles maintain their integrity for at least 1 to 4 days, which is consistent with the circulation times required for in vivo applications.

[0421] Example 2B-bis: Comparison of colloidal stability of ester vs carbamate at 37°C:

[0422] To demonstrate the advantage of the carbamate-type F functional group over a direct ester bond, the colloidal stability of nanoparticles formed from a copolymer containing an ester bond (PEG44-ester-o-pico-PBLG21) and a copolymer containing a carbamate (PEG44-carbamate-o-pico-PBLG21) was compared at 37°C. The results are presented in Table 4bis below.

[0423] Table 4bis: Comparison of colloidal stability of ester vs carbamate at 37°C 37°C Amphiphilic copolymers C* (mg / ml) hours Z (nm) PDI PEG44-ester-o-pico-PBLG21 3 0 108 0.0532h 133 0.294 24h 3200 1.0 0 150 0.10 2h 149 0.10 PEG44-carbamate-o-pico-PBLG21 3 24h 151 0.09 72h 151 0.09

[0424] These results demonstrate improved colloidal stability at physiological temperature (37°C) of the compounds of the present invention compared to those of the prior art.

[0425] These data confirm that the direct ester bond in the benzyl position of the quaternized picolinium ring is susceptible to hydrolysis in aqueous media at physiological temperature, while the carbamate-type functional group F confers increased stability to the amphiphilic copolymer under these conditions. PART C: CLIVABILITY of the nanoparticles of the invention:

[0426] Example IC: Clitivity under gamma irradiation

[0427] The ability of the nanoparticles according to the invention to degrade in a controlled manner under the effect of ionizing radiation is an essential property for their use as activatable drug delivery systems. This example demonstrates that the nanoparticles formed from the amphiphilic copolymers according to the invention can be cleaved by gamma ray irradiation, resulting in a significant modification of their colloidal structure.

[0428] After formulation at 3 mg / ml, nanoparticle solutions at concentrations of 0.05 to 0.10 mg / ml were prepared in PBS pH 7.4 from the various copolymers obtained. The 40 ml solutions were sealed in glass bottles with a rubber stopper and a metal cap. All solutions intended for irradiation experiments were degassed with argon. The samples were irradiated using a Co-60 panoramic irradiator emitting gamma rays with energies between 1.17 and 1.33 MeV.

[0429] All irradiations were performed at the same dose rate, with the samples held in a fixed position during each irradiation session. The irradiation times required to reach the target doses were adjusted accordingly. Nanoparticles were irradiated at doses of 0, 50, 75, or 100 Gy, depending on the formulation. Before and after irradiation, colloidal stability was assessed by dynamic light scattering (DLS) to determine the mean size (in nm) and the polydispersity index (PDI).

[0430] The results are presented in Table 5 below.

[0431] Table 5: Cleavage results of the nanoparticles of the present invention Irra dose Average size Amphiphilic copolymers Content (mg / mL) PDI (Gy) (nm) PEG-PBLG (control) 0.05 0 101 0.2375 103 0.23 0 94 0.24 PEG44-carbamate- o-pico- PBLG21 obtained via SPAAC 0.05 50 1830 0.65 0 111 0.14 PEG44-carbamate- o-pico- PBLG21 0.05 50 367 0.67 0 118 0.07 PEG44-carbamate- p-pico- PBLG21 0.05 75 764 0.57 0 134 0.4 PEG44-carbamate- o-pico- PBLG15 0.05 50 707 0.91 0 140 0.07 PEG44-carbamate- o-pico- PBLG25 0.05 100 912 0.61 0 118 0.2 PEG44-carbamate- o-pico- PBLG35 0.05 50 2954 0.78 0 84 0.20 PEG44 -carbamate-o-pico-PLL5-co-PBLGi5 0.05 50 1663 0.64 0 154 0.08 PSar42-carbamate-o-pico-PBLG 2i 0.05 50 1405 0.57 0 129 0.06 PEG44-carbamate-o-pico-PTyr5-co-PBLGi5 0.05 50 3076 0.55 0 82 0.12 PSar29-carbamate- o-pico- PLA45 0.1 100 1738 0.36 0 113 0.28 PEG44-carbamate-o-pico-PLA45 0.1 100 5143 0.84 0 81 0.24 PEG44- carbamate- o-pico- PLA120 0.1 100 395 0.45 0 115 0.07 PEG44-p-ester aromatique-o-pico-PBLG2i 0.05 [75 764 0.57 0432] These results demonstrate that the nanoparticles formed from the amphiphilic copolymers according to the invention degrade under gamma irradiation. The significant increase in the average particle size after irradiation (by a factor greater than 3) as well as the increase in the polydispersity index indicate destabilization and aggregation of the nanoparticle structures following the cleavage of the copolymer's cleavable motif.

[0433] This controlled degradation under ionizing radiation confirms the potential of the nanoparticles according to the invention for applications in oncology, where they can be used as radiotherapy-activated drug delivery systems. The controlled release of encapsulated therapeutic molecules can thus be synchronized with radiation treatment, enabling combined and targeted action at the level of tumor tissues.

[0434] The irradiation doses tested (50 to 100 Gy) to trigger the release of encapsulated molecules are compatible with the cumulative doses used in clinical radiotherapy.

[0435] PART D: ENCAPSULATION

[0436] Nanoparticles formed from amphiphilic copolymers according to the invention can encapsulate various therapeutic molecules, including cytotoxic agents used in chemotherapy. This example describes the preparation of functional (drug-loaded) nanoparticles, the determination of the loading rate, and the evaluation of the stability and cleavage of the functional nanoparticles under ionizing radiation.

[0437] Example 1D. Preparation of functional nanoparticles:

[0438] The functional nanoparticles were prepared by co-nanoprecipitation according to the following protocol: - an aqueous buffer (PBS or TRIS pH 7.4 - 300 mOsm) was prepared. - a copolymer solution in an organic solvent (THF) was prepared in the presence of the drug, with a copolymer / drug ratio of 10:1. The organic solvent / aqueous phase ratio can vary from 30:70 to 05:95, notably 20:80. - the aqueous phase was quickly added to the organic phase. - the mixture was heated between 25°C and 40°C. - The solvent and excess free drug were removed by Sephadex column chromatography (G-100). The formulation results are presented in Table 6.

[0439] Table 6: Encapsulation of doxorubicin by the nanoparticles of the present invention: Midsized Amphiphilic copolymers, PDI drug, Drug load, Morphology (nm) Nano vesiPEG44-carbamate-o-pico-PBLG25 Doxorobucin 128 0.19 4.2% [cules 0440] These results demonstrate that the functional nanoparticles according to the invention can efficiently encapsulate drugs such as doxorubicin (DOXO), with loading rates of approximately 4%.

[0441] Example 2D. Determining the loading rate:

[0442] The drug loading rate was determined using the following methods:

[0443] 2D.1 Determination of the loading rate for functional nanoparticles:

[0444] After purification, a dry extract of the solution was prepared by lyophilization (500 pL to 1 mL). The amphiphilic copolymer content was estimated by subtracting the weight of the salts from the total weight of the sample. The resulting solid was dissolved in an organic solvent (DMSO with one drop of 5M HCl) to quantify the drug content by absorbance measurement, with the concentration determined from a standard calibration curve.

[0445] 2. D.2 Calculation of the loading rate:

[0446] The loading rate was defined as follows:

[0447] LD50 (%) = (mass of encapsulated drug / total mass of nanoparticles) x 100

[0448] 3D Example: Stability of functional nanoparticles:

[0449] The colloidal stability of functional nanoparticles was evaluated under conditions similar to those described in Example 2B. Functional nanoparticles including doxorubicin showed stability comparable to that of empty nanoparticles, maintaining their structural integrity for several days at 4°C and 37°C.

[0450] Example 4P: Cleavage of functional nanoparticles under ionizing radiation:

[0451] The ability of functional nanoparticles, including a drug, to degrade under ionizing radiation was evaluated according to the following protocol:

[0452] After formulation at 3 mg / ml, a nanoparticle solution (Nanol) containing encapsulated doxorubicin (2% loading rate) at a concentration of 0.05 mg / ml was prepared in PBS pH 7.4. The irradiation conditions were identical to those described in Example IC. Before and after irradiation, colloidal stability was assessed by DLS.

[0453] The results are presented in Table 7:

[0454] Table 7: Cleavage of nanoparticles of the present invention loaded with a drug Sample Concentration (mg / ml) Loading rate (%) Irradiation (Gy) Average size (nm) PDI 0 133 0.12 Nanol (Doxo) 0.05 2 50 3719 0.57

[0455] These results demonstrate that the nanoparticles according to the invention can efficiently encapsulate cytotoxic agents such as doxorubicin, with loading rates satisfactory for therapeutic applications. The loaded nanoparticles retain their ability to be cleaved under ionizing radiation, as evidenced by the significant increase in average size (from 133 nm to 3719 nm) and polydispersity index after irradiation at 50 Gy.

[0456] This property of controlled cleavage of functional nanoparticles is compatible with use for oncology applications, where drug release can be triggered in a targeted manner by radiotherapy.

[0457] Encapsulating the drug in nanoparticles allows it to remain in an inactive state until activation by ionizing radiation, enabling localized release at the level of irradiated tumor tissues.

[0458] 5D Examples: Encapsulation of siRNA:

[0459] The functional nanoparticles formed from the amphiphilic copolymers according to the invention can also encapsulate nucleic acids, in particular small interfering RNAs (siRNAs). This example describes the preparation of functional nanoparticles comprising an siRNA and the determination of the encapsulation efficiency.

[0460] 5D.1 Preparation of siRNA-loaded nanoparticles:

[0461] The siRNA-eGFP was dissolved in nuclease-free water at the desired N / P ratios (0 and 2.5). The copolymer was dissolved in a water-miscible organic solvent. The two phases were temperature-equilibrated, and then the organic phase was rapidly added to the aqueous phase with moderate stirring. The organic solvent was allowed to evaporate. The resulting nanoparticles were dialyzed against Tris-buffered saline using a dialysis cassette. The formulations were then purified by centrifugal filtration (with several TBS washes to remove unencapsulated siRNA). The formulations were stored at 4 °C until characterization.

[0462] 5D.2 Characterization:

[0463] The hydrodynamic diameter and polydispersity index (PDI) of the formulations were measured by DLS. Each sample was measured in triplicate at 25 °C. The zeta potential was measured after dilution of the samples in a buffer at physiological pH (pH 7.4).

[0464] The quantification of encapsulated and free siRNA was performed using a fluorometric assay kit. For the determination of total siRNA, samples were treated with dichloromethane to extract the copolymer, which was confirmed by DLS. The percentage of encapsulated siRNA was calculated by subtracting the amount of unencapsulated siRNA from the total measured siRNA.

[0465] 5D.3 Results:

[0466] The characterization results are presented in the tables below.

[0467] The N / P ratio refers to the ratio between the number of positively charged amine groups (N, for nitrogen) on the copolymer and the number of negatively charged phosphate groups (P) on the siRNA. This ratio determines the stoichiometry of complexation between the cationic carrier and the nucleic acid: - N / P = 0: no copolymer (controls siRNA alone) - Low N / P (e.g., 2.5): relative excess of siRNA compared to the positive charges of the copolymer.

[0468] Table 8: Characterization of siRNA-loaded nanoparticles N / P Ratio Average Zeta Potential (mV) Average Size (nm) PDI 0 (control) 2.89 ± 0.52 52 0.349 2.5 (trial 1) -0.145 48 0.215 2.5 (trial 2) 1.600 49 0.264 2.5 (trial 3) 0.660 48 0.239

[0469] Table 9: siRNA encapsulation efficiency N / P ratio, encapsulated siRNA concentration (ng / pL), encapsulation efficiency 0 (control) 0 0 2.5 (trial 1) 127.618 36.1 2.5 (trial 2) 100.109 35.8 2.5 (trial 3) 121.316 41.9

[0470] These results demonstrate that the nanoparticles according to the invention can encapsulate siRNAs with sizes ranging from approximately 48 nm. The highest encapsulation efficiency was obtained at a N / P ratio of 2.5, with an efficiency between 36% and 42%. After purification, more than 90% of the siRNA present in the samples at the N / P ratio of 2.5 was encapsulated, indicating efficient removal of free siRNA. These data demonstrate the versatility of the nanoparticles according to the invention for encapsulating various types of therapeutic molecules, including not only cytotoxic agents such as doxorubicin, but also nucleic acids such as siRNAs.

[0471] PART E: PRECLINICAL STUDIES

[0472] Example 1E: Functionalized copolymers for fluorescent marking

[0473] For in vivo biodistribution and pharmacokinetic studies, the amphiphilic copolymers according to the invention can be functionalized with fluorophores, allowing their detection by fluorescence imaging. This example describes the ex vivo and in vivo biodistribution of cyanine-7 (Cy7)-labeled copolymers after intravenous injection in mice bearing subcutaneous tumors.

[0474] Fluorescence device:

[0475] IVIS Lumina III (PerkinElmer), excitation: 720 ± 10 nm, emission: 790 ± 20 nm.

[0476] Fluorescence detection of PEG44-carbamate-o-pico-PBLG21-Cy7 in solution in murine plasma was linear (coefficient of determination R 2 > 0.98) in the 1.95 x 10" range 3mg / mL < C < 1 mg / mL, which allows for its quantitative detection by measuring the fluorescence signal in this concentration range.

[0477] PEG44-carbamate-o-pico-PBLG21 was readily detectable by fluorescence imaging at ex: 720 nm / em 790 nm with sufficient sensitivity to consider in vivo and ex vivo detection in mice after intravenous injection of 200 pL at 1 mg / mL.

[0478] Example 2E: Biodistribution and blood pharmacokinetics in mice grafted with subcutaneous syngeneic tumors

[0479] 2E.1. Biodistribution study - CT26 model (murine colon carcinoma cell line)

[0480] This study evaluated the in vivo biodistribution and blood pharmacokinetics of the Cy7-labeled PEG44-carbamate-o-pico-PBLG21 copolymer after intravenous injection in mice bearing CT26 subcutaneous tumors by fluorescence imaging.

[0481] The animal experiments were carried out in accordance with the authorization for animal experimentation issued by the Grenoble Ethics Committee and the French Ministry of Higher Education and Research, under reference: APAFIS 33137.

[0482] Results of in vivo fluorescence imaging:

[0483] Following intravenous injection of Cy7-labeled PEG44-carbamate-o-pico-PBLG21, no adverse effects were observed on the appearance and behavior of the mice throughout the experiment. The fluorescence signal intensity of Cy7-labeled PEG44-carbamate-o-pico-PBLG21 was strong, thus allowing for good in vivo detection.

[0484] In the tumor, the fluorescence signal increased progressively over time until it became very well contrasted 24 to 48 hours post-injection.

[0485] Within 1 hour of injection of Cy7-labeled PEG44-carbamate-o-pico-PBLG21, moderate fluorescence signals were observed in the spleen and liver. These signals remained fairly stable for up to 48 hours.

[0486] The in vivo tumor / skin fluorescence ratio reached a maximum of 5.9 ± 0.2 at 24h post-injection and remained stable up to 48h post-injection (5.9 ± 0.5), indicating excellent tumor specificity for Cy7-labeled PEG44-carbamate-o-pico-PBLG21.

[0487] Results of ex vivo fluorescence imaging on isolated tissues (Figures 21, 22 and 23)

[0488] Quantification of ex vivo fluorescence signals showed significant signals in most organs at 5 hours post-injection.

[0489] Strong fluorescence signals were observed in the tumor. These signals increased between 5 and 24 hours and then remained stable for up to 48 hours post-injection.

[0490] Strong fluorescence signals were observed in the liver 5 to 48 hours post-injection.

[0491] Moderate fluorescence signals were observed in the spleen, intestine, kidney and skin 5 to 48h post-injection.

[0492] Fluorescence signals in the liver and kidneys indicate both renal / urinary and hepatobiliary elimination pathways. The ex vivo tumor / muscle fluorescence ratio demonstrated excellent tumor specificity for PEG. 44 -carbamate- o-pico- PBLG21 marked Cy7.

[0493] Table 10: Monitoring of fluorescence ratios as a function of post-injection time Post-injection time Tumor / Muscle ratio 5h 6.1 ± 0.2 24h 14.2 ± 2.3 48 h 10.5 ± 0.6 Not injected 1.2 ± 0.1

[0494] 2E.2 Biodistribution study - KPC model (pancreatic cancer) (Figures 24, 25 and 26)

[0495] This study evaluated the ex vivo biodistribution of Cy7-labeled PEG44-carbamate-o-pico-PBLG21 after intravenous injection in mice bearing subcutaneous KPC tumors by fluorescence imaging.

[0496] The animal experiments were carried out in accordance with the authorization for animal experimentation issued by the Grenoble Ethics Committee and the French Ministry of Higher Education and Research, under reference: APAFIS 33137.

[0497] Animal model:

[0498] 12 C57BL / 6JR mice], female, 7 weeks old (January Labs).

[0499] Day 0: Subcutaneous implantation of 2 x 10 6 KPC cells on the right side.

[0500] Clinical follow-up: observation of behavior, monitoring of animal weight and tumor volume (measurements with calipers): 3 times per week.

[0501] On day 7, the mean tumor volume was 358 ± 102 mm 3 .

[0502] Mice received an intravenous injection of 200 pL of nanoparticle comprising the Cy7-labeled PEG 44-carbamate-o-pico-PBLG21 copolymer (n=9 mice).

[0503] All mice were sacrificed at 5h (3 mice), 24h (3 mice) and 48h (3 mice) post-injection.

[0504] Results of ex vivo fluorescence imaging on isolated tissues:

[0505] Quantification of ex vivo fluorescence signals showed significant signals in most organs at 5 hours post-injection.

[0506] Strong fluorescence signals were observed in the tumor and liver. These signals increased between 5 and 24 hours and then remained stable for up to 48 hours post-injection.

[0507] Moderate fluorescence signals were observed in the spleen, uterus-ovaries and kidney 5 to 48h post-injection.

[0508] Fluorescence signals in the liver and kidneys indicate both renal / urinary and hepatobiliary elimination pathways.

[0509] Ex vivo fluorescence ratios:

[0510] The ex vivo tumor / muscle fluorescence ratio demonstrated the good tumor specificity of the nanoparticle containing Cy7-labeled PEG44-carbamate-o-pico-PBLG21. It increased between 5h and 24h, and decreased at 48h after injection (16.9 ± 3.3; 32.9 ± 2.6; 23.8 ± 1.9 at 5, 24 and 48h respectively, compared to 1.2 ± 0.1 for uninjected mice).

[0511] The ex vivo tumor / liver fluorescence ratio remained close to 1, which is rather favorable (0.6 ± 0.2; 1.1 ± 0.1; 1.0 ± 0.2 at 5, 24 and 48h respectively, compared to 0.4 ± 0.1 for uninjected mice).

[0512] The ex vivo tumor / spleen fluorescence ratio was 1.2 ± 0.4 at 5h post-injection and increased at 24 and 48h post-injection (2.9 ± 0.3; 2.3 ± 0.3 at 24 and 48h respectively, 1.1 ± 0.2 for uninjected mice).

[0513] Table 11: Ex vivo fluorescence ratios after injection of the Cy7-labeled PEG44-carbamate-o-pico-PBLG21 nanoparticle (KPC model) Post-injection time Tumor / Muscle Ratio Tumor / Spleen Ratio Tumor / Liver Ratio 5h 16.9 ± 3.3 1.2 ± 0.4 0.6 ± 0.2 24h 32.9 ± 2.6 2.9 ± 0.3 1.1 ± 0.1 48 hours 23.8 ± 1.9 2.3 ± 0.3 1.0 ± 0.2 Not injected 1.2 ± 0.1 1.1 ± 0.2 0.4 ± 0.1

[0514] The results of this study, conducted in the KPC pancreatic cancer model, showed a distribution profile superior to that observed for the CT26 colon cancer model, with good tumor specificity from 5 hours post-injection up to 48 hours post-injection.

[0515] Example 3E. Pharmacokinetics

[0516] Following intravenous injection of Cy7-labeled PEG44-carbamate-o-pico-PBLG21, no adverse effects were observed on the appearance and behavior of the mice throughout the experiment.

[0517] Blood samples taken from mice after intravenous injection of Cy7-labeled PEG44-carbamate-o-pico-PBLG21 displayed clearly detectable fluorescence signals.

[0518] Cy7-labeled PEG44-carbamate-o-pico-PBLG21 demonstrated the advantage of a long circulation time, which promotes tumor accumulation. Indeed, a fluorescent signal was still detected 24 hours post-injection but no longer at 48 hours for Cy7-labeled PEG44-carbamate-o-pico-PBLG21.

[0519] Blood fluorescence signals were fitted using a biphasic decay model. The corresponding half-lives were: PEG44-carbamate-o-pico-PBLG21 labeled Cy7-5: - 0.35h for the diffusion+elimination phase; - 6.9 hours for the slow elimination phase

[0520] Example 4E: Toxicological studies of the nanoparticles of the invention:

[0521] This preliminary toxicology study of copolymers administered as a single intravenous dose to healthy Balb / c mice was carried out to evaluate the safety of the nanoparticles according to the invention.

[0522] The animal experiments were carried out in accordance with the authorization for animal experimentation issued by the Grenoble Ethics Committee and the French Ministry of Higher Education and Research, under reference: APAFIS46890.

[0523] Animal model:

[0524] 19 Balb / c mice, 7 weeks old (January Labs).

[0525] Toxicology study:

[0526] Six mice (n=6) received an intravenous injection of 200 pL of PEG44-carbamate-o-pico-PBLG copolymer 21 at 10 mg / mL

[0527] Seven mice (n=7) received an intravenous injection of 200pL of PBS.

[0528] Clinical follow-up:

[0529] The animals were carefully examined daily for their general condition and behavior, and their weight was monitored three times a week.

[0530] Blood sampling and analysis:

[0531] For differential blood counts and hemograms: blood samples were collected by intracardiac puncture into EDTA-coated tubes just before mouse sacrifice, 1 week after injection.

[0532] These samples were analyzed for: Erythrocyte count, hemoglobin concentration, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, reticulocytes, atypical cells, leukocytes, lymphocytes, monocytes, basophils, eosinophils, segmented neutrophils, banded neutrophils.

[0533] For biochemical analysis: blood samples were collected by intracardiac puncture into tubes coated with heparin-lithium just before the sacrifice of the mice, 1 week after the injection.

[0534] Sacrifice and tissue harvesting:

[0535] All mice were sacrificed one week after injection / once ethical limits were reached.

[0536] Removal and weighing of the liver, spleen, kidney, brain, heart and lung. Photography of the organs in case of abnormalities.

[0537] Histological analysis:

[0538] n=3 mice / group were randomly selected. 3 organs (liver, kidney, lung) were taken for histological analyses to identify potential toxicity.

[0539] Each tissue fragment was embedded in paraffin and sectioned at 5 pm.

[0540] A standard hematoxylin-eosin-saffron (H&E) stain was performed on all three slides for the liver and spleen samples. For the kidneys, H&E staining was performed on two slides, and periodic acid-Schiff (PAS) staining was performed on the third.

[0541] PAS staining was used to highlight basement membranes and brush borders. The following histopathological features were systematically evaluated in each sample: integrity of the proximal tubular brush border, tubular morphology and luminal content, presence of PAS-positive casts, cytoplasmic vacuolization, tubular atrophy, mesangial expansion, segmental glomerular sclerosis.

[0542] No hematological toxicity was observed, all blood cell parameters were normal.

[0543] Clinical follow-up:

[0544] No adverse effects were observed on the body weight, appearance, or behavior of the mice during the entire monitoring period.

[0545] Necropsy:

[0546] Seven days after the injection, the mice were sacrificed and the organs were carefully observed macroscopically.

[0547] No macroscopic abnormalities were observed in any organ of the treated mice compared to untreated mice.

[0548] There was no statistically significant difference in organ weight between animals receiving the 10 mg / mL dose and control animals.

[0549] Histopathological analysis:

[0550] No relevant histopathological differences were identified between the treated and control groups in either the spleen or the kidneys.

[0551] The only minor observation in the treated animals was moderate cytoplasmic clearing in hepatocytes, without associated structural damage. Taken together, these observations are consistent with a mild and non-adverse tissue response and do not indicate organ toxicity under the tested conditions.

[0552] Conclusion

[0553] These results demonstrate that the nanoparticles according to the invention exhibit a favorable tolerability profile at the tested dose of 10 mg / mL, without significant systemic toxicity. These data support the continued preclinical development of the nanoparticles according to the invention for therapeutic applications.

[0554] Example 4E-bis: Toxicological study of functional nanoparticles including doxorubicin:

[0555] As part of the therapeutic efficacy study described below (Example 5E), a toxicological evaluation was also carried out on the groups receiving the functional nanoparticles including doxorubicin (Nanol (Doxo)).

[0556] The animal experiments were carried out in accordance with the authorization for animal experimentation issued by the Grenoble Ethics Committee and the French Ministry of Higher Education and Research, under reference: APAFIS #51830.

[0557] Animal model: Female BALB / cJRj mice, 7 weeks old (Janvier Labs), carrying CT26 subcutaneous tumors.

[0558] J:0 Subcutaneous implantation of 0.5xl0 6 CT26 cells on the right flank.

[0559] Day 9: Mice were randomly divided into 3 groups (n=6) when the mean tumor volume was 96.2 + / - 5.7 mm 3 .

[0560] Day 10: Single intravenous injection.

[0561] Jll: Irradiation 24 hours after injection. Radiotherapy was performed using the SARRP system (Xstrahl). Treatment plans were designed to deliver the precise dose to the tumor isocenter in 180° arc therapy, with the gantry rotating 90° to 90° above the mouse. A 10 mm diameter collimator was used, providing an irradiation volume of approximately 523 mm³ 3Irradiation parameters: 8 Gy at 3.06 Gy / min, 35 cm from the irradiation source, voltage: 220 kVp, current: 13 mA, filtration: 0.15 mm Cu (average energy delivered to the cells: 78.54 keV).

[0562] The groups assessed for toxicity were (Table 11bis): Treatment Group Irradiation Control Group - NaCl No. 1 Nano Group l(Doxo): Nanoparticle of the present invention (200 pL - 2.6 mg / mL) loaded with 32 Non 2 pg / mL of doxorubicin (dose corresponding to 320 pg / kg for a 20 g mouse) Nano Group l(Doxo): Nanoparticle of the present invention (200 pL - 2.6 mg / mL) loaded with 32 Yes (8 3 pg / mL of doxorubicin (dose corresponding to 320 pg / kg for a 20 g mouse) Gy)

[0563] Analyses performed: Complete blood count, blood biochemistry, necropsy with organ weighing, and histopathology (liver, spleen, kidney with H&E and PAS stains) on day 22.

[0564] Blood collection and analysis: For differential blood counts and hemograms, blood samples were collected by intracardiac puncture into EDTA-coated tubes when the untreated control group (NaCl) reached ethical limits.

[0565] For biochemical analysis, blood samples were collected by intracardiac puncture into heparin-lithium coated tubes. Plasma was isolated by centrifugation at 2000 g for 5 min at room temperature and frozen at -80°C. Statistical analysis was performed using GraphPad Prism with the Kruskal-Wallis test and Dunnett's multiple comparisons test.

[0566] Histological analysis: n=3 mice / group were randomly selected. Three organs (liver, kidney, spleen) were harvested for histological analysis. Each tissue fragment was paraffin-embedded and sectioned at 5 µm. Three slides per sample were prepared, with sections spaced 100 µm apart. Standard hematoxylin-eosin-saffron (H&E) staining was performed on all three slides for the liver and spleen samples. For the kidneys, H&E staining was performed on two slides, and periodic acid-Schiff (PAS) staining was performed on the third.

[0567] Results:

[0568] Clinical follow-up: Post-irradiation body weight monitoring (day 11) showed a significant decrease in weight for group 3 compared to group 1. No other adverse effects were observed on the appearance and behavior of the mice. Necropsy: The isolated organs were carefully examined macroscopically, and no abnormalities were found. Liver weight was significantly higher in groups 2 and 3 compared to group 1. No differences were observed for the other organs (brain, heart, lung, spleen, kidney).

[0569] Blood count: The erythrocyte parameters (erythrocytes, hemoglobin concentration, platelets, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, hematocrit, reticulocytes and atypical cells) were normal for groups 2 and 3.

[0570] Leukopenia was observed only in group 3: decrease in leukocytes (p=0.006 vs Gl), lymphocytes (p=0.0125), monocytes (p=0.0175) and segmented neutrophils (p=0.0449).

[0571] Blood biochemistry: No significant difference was observed for any of the parameters measured between the different groups.

[0572] Histopathology: No differences were identified in the spleen or kidneys between the treated and control groups. Moderate cytoplasmic clearing was observed in hepatocytes without associated structural damage, corresponding to a mild and non-adverse tissue response.

[0573] Conclusion: Functional nanoparticles containing doxorubicin exhibit a tolerability profile comparable to that of empty nanoparticles. The leukocyte changes observed in group 3 are attributable to the combined effect of radiotherapy and doxorubicin release.

[0574] The absence of biochemical and histopathological toxicity confirms the safety of the nanoparticle system according to the invention for the encapsulation and controlled delivery of doxorubicin.

[0575] Example 5E: Anticancer therapeutic efficacy (Figures 27a, 27b and 27c)

[0576] This study evaluated the preclinical anticancer therapeutic efficacy of the doxorubicin-loaded nanoparticles of the present invention (as exemplified in Part D of the present invention) controlled by radiotherapy in mice bearing CT26 subcutaneous tumors.

[0577] Materials and methods

[0578] The animal experiments were carried out in accordance with the authorization for animal experimentation issued by the Grenoble Ethics Committee and the French Ministry of Higher Education and Research, under reference: APAFIS 51830.

[0579] Animal model:

[0580] Seven-week-old female BALB / cJRj mice (Janvier Labs) received a subcutaneous implantation of 0.5 x 10 6 CT26 cells on the right flank at J0.

[0581] Clinical follow-up:

[0582] Observation of behaviour, monitoring of animal weight and tumor volume (measurements with calipers): 3 times per week.

[0583] Day 9: Random assignment: Mean tumor volume 96.2 ± 5.7 mm 3 The mice were divided into 4 groups such that the means and SEM were similar in all groups.

[0584] Day 10: Single intravenous injection of Nano l (Doxo): Nanoparticle of the present invention (200 pL - 2.6 mg / mL) loaded with 32 pg / mL of doxorubicin (corresponding dose to 320 pg / kg for a 20 g mouse) for groups 2 and 4 (n=6 per group). Day 11: Irradiation 24 h post-injection. Radiotherapy was performed using the SARRP system (Xstrahl). Treatment plans were designed to deliver the precise dose to the tumor isocenter in 180° arc therapy, with the gantry rotating 90° to 90° above the mouse. A 10 mm diameter collimator was used, providing an irradiation volume of approximately 523 mm³ 3 Irradiation parameters: 8 Gy at 3.06 Gy / min, 35 cm from the irradiation source, voltage: 220 kVp, current: 13 mA, filtration: 0.15 mm Cu (average energy delivered to the cells: 78.54 keV).

[0585] The experimental groups are listed in Table 12 below. Treatment Group Irradiation Group 1 Control - NaCl No Group 2 Nanoparticle of the present invention loaded with doxorubicin (Nano l(Doxo)) No Group 3 - Yes (8 Gy) Group 4 Nanoparticle of the present invention loaded with doxorubicin (Nano l(Doxo)) Yes (8 Gy)

[0586] Group 1 is a control group. Group 3 underwent irradiation (8GY).

[0587] Groups 2 and 4 received an intravenous injection of nanoparticles according to the invention loaded with doxorubicin.

[0588] The animals were sacrificed when the untreated group 1 reached ethical limits (at day 22).

[0589] Results

[0590] Clinical follow-up:

[0591] No adverse effects were observed on the body weight, appearance, or behavior of the mice after treatment.

[0592] Tumor growth and survival

[0593] Group 1 (control) and Group 2 (nanoparticles according to the invention without irradiation) showed rapid and similar tumor growth, confirming that the nanoparticles according to the invention retain their integrity under physiological conditions and do not release doxorubicin in the absence of irradiation.

[0594] Group 3 (8 Gy) showed a slowing of tumor growth to a lesser extent compared to group 4.

[0595] Group 4 (nanoparticles according to the invention + 8 Gy) showed a marked slowing of tumor growth compared to G1 and G2. Analysis of individual tumor growth slopes between J15 and J22 (3 to 10 days post-irradiation) revealed that this group had the lowest average slope among all the tested conditions and included the three lowest individual slopes, with tumor regressions observed in several animals.

[0596] Group 4 showed improved survival compared to the non-irradiated groups and compared to group 3.

[0597] Necropsy: The isolated organs were carefully observed macroscopically and no abnormalities were found in any group.

[0598] Conclusion:

[0599] These results indicate that: - the nanoparticles according to the invention retain their integrity in the absence of irradiation (G2 ~ Gl), confirming the stability of the system under physiological conditions and the necessity of the exogenous stimulus for the release of the drug; - Irradiation triggers the release of encapsulated doxorubicin, leading to a therapeutic response with the lowest tumor growth slopes and the best individual responses among all conditions tested.

[0600] These preclinical data support the potential of the nanoparticles according to the invention as a radiotherapy-activated drug delivery system, enabling controlled release of therapeutic agents at the level of irradiated tumor tissues.

Claims

DEMANDS

1. Amphiphilic copolymer comprising: - a hydrophilic polymer, - a hydrophobic polymer, and - a cleavable motif located between the hydrophilic polymer and the hydrophobic polymer, characterized in that the cleavable motif has the following formula (15): R3' in which: - N + represents a positively charged nitrogen atom - Y - is an anion, preferentially chosen from among the halogens (Cl', F, Br j, acetate, OTs, OTf, N-acetyl type amino acids, - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OME, CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure, the lateral chain comprising functional group F being located at any position of R1', R2', R3', R4' and R5', where said R1', R2', R3', R4' and R5' is then absent, and F is a functional group of Formula (1): in which, - R3 is chosen from H, CH3 and an alkyl group, - ni is an integer between 1 and 10, - R8 is chosen between a group of formula (2) and a group of formula (3): * Formula (2): R5 X2 xr -R4 in which: - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group comprising 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane, - R4 is chosen from O, NH and an aryl group, - if X1 is not an O then R5 is O, if X1 is an O then R5 is chosen from O or S; * Formula (3): in which: - X3 is chosen from H and CH3, - X4 is chosen from H and CH3, - Rg is chosen from H, CH3 and an alkyl group, - n8 is an integer between 1 and 10.

2. Amphibious copolymer according to claim 1, characterized in that the functional group F has the following formula (4): R 5 (4) in which: - R3 is chosen from H, CH3 and an alkyl group, - ni is an integer between 1 and 10, - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane, - R4 is chosen from O, NH and an aryl group, - if X1 is not an O then R5 is O, if X1 is an O then R5 is chosen from O or S.

3. Amphibious copolymer according to claim 1 or 2 characterized in that the functional group F has the following formula (5): O (5) in which: - R3 is chosen from H, CH3 and an alkyl group, - ni is an integer between 1 and 10, - R4 is chosen from O, NH and an aryl group, - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group with 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.

4. Amphibious copolymer according to claim 1, characterized in that the functional group F has the formula (6): in which: - R3 is chosen from H, CH3 and an alkyl group, - ni is an integer between 1 and 10, - X3 is chosen from H and CH3, - X4 is chosen from H and CH3, - Rg is chosen from H, CH3 and an alkyl group - n8 is an integer between 1 and 10.

5. Amphibious copolymer according to any one of the preceding claims, characterized in that it has the formula (7): R1-R6-MF-R7-R2 (7) in which: - R₁ is a hydrophilic polymer or a hydrophobic polymer; - R₂ is a hydrophobic polymer if R₁ is a hydrophilic polymer, or R₂ is a hydrophilic polymer if R₁ is a hydrophobic polymer. - MF represents the cleavable motif, and - Rg and R7 are optional spacers.

6. Amphibious copolymer according to the preceding claim, characterized in that: - R6 has the following formula (13): (13) in which: * n2 and n4 are identical or different integers between 0 and 5;* n3 is 0 or 1; - R7 presents the following formula (14): (14) in which * n5 and n7 are identical or different integers between 0 and 5; * n6est 0 or 1.

7. Amphibious copolymer according to any one of claims 1 to 6, characterized in that it has the following formula (21): (21) in which: - Ri can be a hydrophilic polymer or a hydrophobic polymer, - R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer. - R3 is chosen from H, CH3 and an alkyl group, - ni is an integer between 1 and 10, - R8 is chosen between a group of formula (2) and a group of formula (3), - R6 is a group of formula (13), - R7 is a group of formula (14), - N + represents a positively charged nitrogen atom - Y - is an anion, preferably chosen from among halogens (Cl', F', Br), acetate, OTs, OTf, N-acetyl type amino acids, - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OME (methoxy), CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group containing 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure, the side chain containing R3 being located at any position of R1, R2, R3, R4 1 and R5■, where said Rr, R2 1 , R3 1 , R4 1 and R5' is then absent.

8. An amphiphilic copolymer according to any one of the preceding claims, characterized in that it has one of the following formulas: - formula (24): (24) - formula (25): - formula (26): (26) - formula (27): - Formula (28): - Formula (29): - formula (30): (30) - formula (31): (31) in which: - Ri can be a hydrophilic polymer or a hydrophobic polymer, - R2 can be a hydrophobic polymer if Ri is a hydrophilic polymer, or R2 can be a hydrophilic polymer if Ri is a hydrophobic polymer. - R3 is an H, a CH3 is an alkyl group, - R4 is chosen from NH, an aryl group, an O,- ni is an integer between 1 and 10, - Xi is chosen from O, NR, S and Se, where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms; - X2 is chosen from C, PO, PO-aryl, PO-alkane, PNH-aryl and PNH-alkane, - R4 is chosen from O, NH and an aryl group, - if X1 is not an O then R5 is O, if X1 is an O then R5 is chosen from O or S, - N + represents a positively charged nitrogen atom - Y - is an anion, preferably chosen from among halogens (Cl', F', Br), acetate, OTs, OTf, N-acetyl type amino acids, - R1', R2', R3', R4' and R5' are substituents that can be: * H, OH, OME, CN, N(R)2, NO2, halogen where R represents a hydrogen atom or an alkyl group consisting of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. * or two adjacent substituents can form a fused aromatic ring to give a quinolinium or acridinium structure, the lateral chain comprising R3, being located at any position of Rr, R2', R3 1 , R4 1 and R5', where said Rr, R2 1 , R3 1 , R4 1 and R5' is then absent. - n2, n4, n5 and n7 are identical or different integers between 0 and 5; - ns and ng are identical or different integers chosen from 0 and 1.

9. An amphiphilic copolymer according to any one of the preceding claims, characterized in that the hydrophilic polymer is selected from polyethylene glycol (PEG), polyacrylamide, poly(N-vinylpyrrolidone), poly(N-isopropylacrylamide), poly(N,N-dimethylacrylamide), poly(N-hydroxypropylmethacrylamide), poly(2-oxazoline), polysarcosine, hydrophilic polysaccharides (dextran, chitosan, hyaluronic acid), elastin-based polymers (ELP), poly(L-glutamic acid) (in unprotected / hydrophilic form), poly(acrylic acid), poly(methacrylic acid), DNA and its derivatives, and functionalized oligonucleotides.

10. Copolymer according to any one of the preceding claims, characterized in that the hydrophobic polymer is selected from poly(γ-benzyl-L-glutamate) (PBLG), poly(γ-caprolactone) (PCL), poly(lactide) (PLA), poly(glycolide) (PGA), poly(lactide-co-glycolide) (PLGA), poly(orthoesters) (POE), poly(anhydrides), poly(propylene fumarate) (PPF), polycarbonate (PC), poly(alkyl cyanoacrylate), poly(trimethylene carbonate) (PTMC), poly(siloxanes) (Silicones), poly(urethane), aromatic polyanhydrides, poly(alkyl acrylates), poly(alkyl methacrylates), poly(P-aminoesters), poly(styrene-alt-maleic anhydride), poly(L-leucine) (pLeu), Poly(L-phenylalanine) (pPhe), protected Poly(L-lysine) (e.g. poly(e-Boc-L-Lysine) or poly(Ne-trifluoroacetyl-L-lysine)), Poly(O-benzyl-L-tyrosine) (pTyr) and polysulfones.

11. Amphibious copolymer according to any one of the preceding claims, characterized in that it is stable under physiological conditions for at least 4 hours, preferably between 4 hours and 7 days, between 4 hours and 5 days, between 4 hours and 72 hours, between 4 hours and 48 hours, 4 hours and 24 hours.

12. Amphibious copolymer according to any one of the preceding claims, characterized in that it is stable: - between 0 and 60°C, preferably between 20 and 50°C, between 35 and 45°C, and / or - at pH between 5 and 8, preferably at pH between 6 and 8.

13. Copolymer according to any one of the preceding claims, characterized in that the cleavable motif is cleavable under ionizing radiation.

14. Amphibious copolymer according to any one of the preceding claims, characterized in that it comprises at least two cleavable motifs, preferably at least three cleavable motifs.

15. Amphibious copolymer according to any one of the preceding claims, characterized in that the hydrophilic polymer further comprises, at its end opposite the cleavable motif, at least one covalently linked targeting ligand.

16. A cleavable amphiphilic copolymer according to any one of the preceding claims, characterized in that the targeting ligand is selected from: - an RGD peptide or a cyclic derivative thereof; - folic acid or a derivative thereof; - an aptamer; - an antibody or a fragment of an antibody; - transferrin or a fragment thereof.

17. Pharmaceutically acceptable salt, solvate or stereoisomer of an amphiphilic copolymer according to any one of the preceding claims.

18. Nanoparticle comprising at least one amphiphilic copolymer according to any one of claims 1 to 16 or a salt, solvate or stereoisomer according to claim 17.

19. Nanoparticle according to claim 18, characterized in that it is stable under physiological conditions for at least 4 hours, preferably for 4 hours and 7 days, between 4 hours and 5 days, between 4 hours and 72 hours, between 4 hours and 48 hours, 4 hours and 24 hours.

20. Nanoparticle according to any one of claims 18 or 19, characterized in that it is stable under the following conditions: - between 0 and 60°C, preferably between 20 and 50°C, between 35 and 45°C, and / or - at pH between 5 and 8, preferably at pH between 6 and 8.

21. Nanoparticle according to any one of claims 18 to 20, comprising at least one encapsulated molecule.

22. Nanoparticle according to any one of claims 18 to 21, comprising at least one encapsulated pharmaceutical active ingredient and / or cosmetic active ingredient.

23. Nanoparticle according to claim 22, wherein the pharmaceutical active ingredient is selected from the group consisting of anticancer agents, immunomodulators, anti-inflammatory agents, antidiabetic agents, antibacterial agents, antiviral agents, and agents for the treatment of pediatric or age-related diseases.

24. Nanoparticle according to any one of claims 18 to 23, wherein the encapsulated pharmaceutical active ingredient is doxorubicin.

25. Nanoparticle according to any one of claims 18 to 24, characterized in that it is a solid nanoparticle, micelles or a hollow nanoparticle with an outer membrane and a hollow inner part, in particular a polymersome.

26. Nanoparticle according to any one of claims 18 to 25, characterized in that it comprises at least one inorganic element and / or at least one organic element

27. ​​Nanoparticle according to the preceding claim, characterized in that it comprises less an inorganic element selected from mineral, metallic, semi-metallic, oxide or chalcogenide particles and mixtures thereof, preferably from ultrasmall iron oxide particles (USPIO), superparamagnetic iron oxide nanoparticles (SPION), very small iron oxide nanoparticles (VSION), hafnium oxide, an iron-bismuth or iron-platinum alloy, gadolinium oxide, dysprosium oxide, bismuth selenide or bismuth telluride, silver, platinum, or a gold nanoparticle or atom cluster, and mixtures thereof

28. Nanoparticle according to any one of claims 18 to 27, characterized in that it comprises on its surface at least one targeting ligand.

29. Nanoparticle according to any one of claims 18 to 28, characterized in that it is conjugated to both therapeutic molecules and targeting ligands.

30. A process for preparing a copolymer according to any one of claims 1 to 16, comprising carrying out the following steps: - Synthesis of the cleavable motif, the hydrophilic polymer, and the hydrophobic polymer, - Functionalization of the hydrophilic polymer with the cleavable motif to obtain a hydrophilic block, - Coupling of the two blocks (hydrophilic block and hydrophobic polymer) to obtain the amphiphilic copolymer.

31. A process for preparing a copolymer according to any one of claims 1 to 16, the process comprising carrying out the following steps: - Synthesis of the cleavable motif, the hydrophilic polymer, and the hydrophobic polymer, - Functionalization of the hydrophobic polymer with the cleavable motif to obtain a hydrophobic block, - Coupling of the two blocks (hydrophobic block and hydrophilic polymer) to obtain the amphiphilic copolymer.

32. Method for preparing a nanoparticle according to any one of claims 18 to 28, comprising a step of self-assembly of amphiphilic copolymers by nanoprecipitation.

33. Nanoparticle according to any one of claims 18 to 29, for its use as a contrast agent in a bio-imaging modality selected from optical endoscopy, ultrasound, magnetic resonance imaging (MRI), computed tomography (CT scan).

34. Nanoparticle according to any one of claims 18 to 29, for its use in the vectorization of active ingredients and therapeutic molecules and / or the controlled delivery of drugs.

35. Nanoparticle according to any one of claims 18 to 29, for its use in the treatment of a medical disorder, wherein the treatment includes the activation of the cleavable motif of the copolymer by an exogenous and / or endogenous stimulus.

36. Nanoparticle for its use according to claim 35, wherein the exogenous stimulus is selected from UV light, visible or near-infrared photons, ionizing radiation (beta rays, X-rays or gamma rays), and ultrasound.

37. Nanoparticle according to any one of claims 18 to 29, for its use in photodynamic therapy, in theranostics and / or in image-guided radiotherapy.

38. Nanoparticle according to any one of claims 18 to 29, for its use in the treatment of a pathology selected from cancers, inflammatory diseases, diabetes, bacterial or viral infections and neurodegenerative diseases.

39. Nanoparticle for its use according to any one of claims 33 to 38, wherein the activation of the cleavable motif is achieved by ionizing radiation at a dose between 0.1 and 80 Gy.

40. Nanoparticle for its use according to any one of claims 33 to 39, wherein the nanoparticle comprises at least one encapsulated anticancer agent, and wherein the activation of the cleavable motif is achieved by ionizing radiation at a dose between 0.1 and 80 Gy.

41. Method of treating a disease in a subject in need, comprising: - administering to the subject a nanoparticle according to any one of claims 18 to 29, said nanoparticle comprising at least one encapsulated therapeutic molecule; - exposure of the nanoparticle to ionizing radiation at a dose between 0.1 and 80 Gy, preferably between 2 and 16 Gy, so as to trigger the cleavage of the cleavable motif and the controlled release of the therapeutic molecule.

42. Treatment method according to claim 41, wherein exposure to ionizing radiation is carried out between 30 minutes and 72 hours after administration of the nanoparticle, preferably between 1 hour and 48 hours after administration.

43. Treatment method according to claim 41 or 42, wherein the therapeutic molecule is doxorubicin and the disease is cancer, preferably a solid tumor.

44. Pharmaceutical composition comprising at least one nanoparticle according to any one of claims 18 to 29 and a pharmaceutically acceptable excipient.

45. Medical device comprising at least one nanoparticle according to any one of claims 18 to 29.

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