FR3073750A1 - LIPID NANOPARTICLE MARKED TO BE MONITORED BY ELECTRONIC MICROSCOPY IN TRANSMISSION - Google Patents

Abstract

Lipid nanoparticle labeled to be observed by transmission electron microscopy, the lipid nanoparticle comprising a lipid core (10) surrounded by a lipid shell (20), the lipid shell (20) comprising at least one layer of amphiphilic lipid surfactants (21) ), and functionalized hydrophilic surfactants (22) by an SH group (50), the lipid nanoparticle being covered at least locally by a metal layer (60) bonded to the SH groups (50) of functionalized hydrophilic surfactants (22) via a link metal coordination.

Description

LIPID NANOPARTICLE MARKED TO BE ABLE TO BE OBSERVED

BY TRANSMISSION ELECTRON MICROSCOPY

DESCRIPTION

TECHNICAL AREA AND PRIOR ART

The present invention relates to a lipid nanoparticle labeled to be able to be observed by transmission electron microscopy, to a method for characterizing lipid nanoparticles, and to a method for labeling lipid nanoparticles.

Lipid nanoparticles (or LNP for “Lipid NanoParticles”) have been used for many years to transport molecules of interest, such as active principles, or imaging agents, in the human body for therapeutic purposes or to carry out diagnostics.

These nanoparticles have a core-shell structure, the shell mainly comprising lipid surfactants. Lipids are the building blocks of the fat that makes up all living things. Thus, because of their physiological origin, they are naturally assimilated by the human body and therefore biocompatible, biodegradable and non-toxic. Lipid nanoparticles therefore, naturally, have the capacity to pass certain physico-chemical barriers of the human body and can be transported to their destination site without being previously degraded or assimilated. They allow controlled and / or prolonged release of molecules of interest at their site of action.

There are different types of lipid nanoparticles.

In the 1980s, nanoparticles from oil-in-water (O / W) emulsions were developed. To produce these emulsions, oil nanodroplets are dispersed in an aqueous phase, the whole being stabilized by a surfactant. Although this type of particle can dissolve a wide range of lipophilic molecules, their stability remains very fragile over time.

Another type of LNP corresponds to liposomes. They are spherical vesicles comprising at least one bilayer of phospholipids and an aqueous core. Their size can vary from a few tens of nanometers to several microns depending on the type of liposomes synthesized. These are the first lipid particles to have passed clinical trials and therefore to have been marketed as part of chemotherapy treatments for certain cancers, such as breast cancer. By virtue of their structure, both hydrophilic and hydrophobic active principles can be respectively encapsulated in the heart or else inserted into the lipid bilayer.

Finally, recently research has turned to lipid nanoparticles, comprising a more or less solid lipid phase, surrounded by a monolayer shell generally comprising an amphiphilic lipid surfactant and a hydrophilic surfactant. These lipid nanoparticles have good stability over time and are very promising vectoring elements, not only for the delivery of drugs and the diagnosis of cancers, but also in the context of gene or cell therapy.

In general, LNP are intended to cross different barriers of the human body to reach target areas for delivering drugs or diagnosing diseases, and it is therefore important to be able to characterize their sizes and their dispersion but also their shape to understand how the active ingredient will be internalized in the LNP.

LNPs can be characterized by transmission electron microscopy (MET or TEM for “Transmission Electron Microscopy”). Several TEM characterization techniques exist: dry, with or without negative staining, cryogenics, or liquid.

To observe the lipid nanoparticles with the TEM by a dry route, a drop of sample containing the LNPs dispersed in solution is deposited on a MET grid, after evaporation of the solution, only the LNPs remain. Without coloring, the LNPs are generally too little contrasted to be observed, which is why negative staining must be used. Conventionally, the coloring is carried out with uranyl acetate which is deposited around the LNPs. It is uranyl acetate which is observed. However, as shown in FIG. 1A, with such a technique, an agglomeration of the LNPs is observed. The agglomeration may be due to the drying step of the sample on the grid and / or due to a natural agglomeration of the LNPs. It is not possible to know when the LNPs gathered or to determine their exact size. In addition, the dye masks the core / shell structure of the LNP.

It is also possible to observe the LNP by cryogenic microscopy (Figure IB). For this, a drop of sample is placed on the MET grid, the excess liquid is absorbed with filter paper and the LNPs are cryogenized. The dispersion of the LNPs remains identical to that in solution, but they are generally deformed, which could come from the drainage stage during preparation for freezing. It is therefore impossible to observe the actual shape and size of the LNP with this method.

Liquid in-situ microscopy seems to be the most promising avenue. The sample is observed directly in the liquid state and does not require going through a phase change step. The particles are not deformed or agglomerated after the preparation of the sample. However, since LNPs are composed of light elements such as oxygen and carbon, their observation by in-situ TEM is almost impossible, due to the lack of electronic contrast (Figure IC).

In order to improve the contrast, one solution consists in functionalizing the LNPs in a non-covalent manner with gold nanoparticles. However, as shown in FIG. 1D, the gold nanoparticles are randomly dispersed in the solution and do not necessarily position themselves around the LNPs and few gold nanoparticles bind to the LNPs. For example, Xia et al. (“Construction of thermal- and lightresponsive liposomes noncovalently decorated with gold nanoparticles” RCS Adv., 2014, 4, 44568-44574) have also succeeded in non-covalently functionalizing liposomes with gold nanoparticles. However, a single gold nanoparticle functionalizes a liposome.

Another solution is to surround the liposome with a gold complex. For this, Jin et al. (“Spectrally Tunable Leakage-Free Gold Nanocontainers”, J. Am. Chem. Soc. 2009, 131, 17774-17776) functionalized liposomes with poly-L-histine (PLH). The liposomes are then placed in the presence of a solution containing Au ³⁺ ions, which are chelated by PLH at basic pH in the presence of a reducing agent NH ₂ OH. HCl. However, the connections involved are not very solid, which makes the functionalization less solid and less stable over time. In addition, the process involves reaction conditions and / or reagents, which can damage the LNPs.

STATEMENT OF THE INVENTION

It is therefore an object of the present invention to provide labeled lipid nanoparticles which can be easily observed by transmission electron microscopy, the labeling having to be stable over time.

This object is achieved by a lipid nanoparticle marked to be able to be observed by transmission electron microscopy, the lipid nanoparticle comprising a lipid core surrounded by a lipid shell, the lipid shell comprising at least one layer of amphiphilic lipid surfactants, and hydrophilic surfactants functionalized by an SH group, the lipid nanoparticle being covered at least locally by a metal layer linked to the SH groups of functionalized hydrophilic surfactants, via a metal coordination bond.

The invention is fundamentally distinguished from the prior art by the labeling of lipid nanoparticles with a metal, via a strong metallic coordination bond. This marking is stable and makes it possible to easily visualize the nanoparticles by transmission electron microscopy (TEM).

Advantageously, the metal layer is made of gold, silver, copper, nickel, cobalt, iridium, or a mixture of these. These elements are biocompatible and / or have a high atomic number in order to have a better contrast to the MET.

Advantageously, the metal layer forms a continuous layer around the lipid nanoparticles. The outline of the lipid nanoparticles is more visible.

Advantageously, the lipid core of the lipid nanoparticles comprises one or more molecules of interest, such as an active principle, an imaging agent or a drug.

Advantageously, the layer also comprises additional hydrophilic surfactants.

Advantageously, targeting agents are grafted onto the additional hydrophilic surfactants and / or onto the functionalized hydrophilic surfactants, the targeting agents being, for example, biological targeting ligands such as antibodies or peptides.

Advantageously, the additional hydrophilic surfactants are polyethylene glycols and / or the functionalized hydrophilic surfactants are functionalized polyethylene glycols.

Advantageously, the core of the nanoparticle is a mixture of a wax and a vegetable oil, such as soybean oil, and the lipid shell comprises a monolayer of phospholipids.

The invention also relates to a method for characterizing lipid nanoparticles, comprising the following successive steps:

- supply of an aqueous solution containing labeled lipid nanoparticles as defined above,

- placement of the aqueous solution in an observation chamber of an electron microscope object holder in transmission, and closure of the observation chamber,

- characterization of the labeled lipid nanoparticles in the aqueous solution by transmission electron microscopy.

It is another object of the invention to propose a method for labeling lipid nanoparticles, effective, inexpensive, and not damaging the lipid nanoparticles.

This object is achieved by a method of labeling lipid nanoparticles, in order to obtain lipid nanoparticles as defined above, comprising the following successive steps:

a) supply of an aqueous solution comprising lipid nanoparticles, the lipid nanoparticles comprising a lipid core surrounded by a lipid shell, the lipid shell comprising at least one layer of amphiphilic lipid surfactants, and hydrophilic surfactants functionalized by an SR group with R a group protecting the thiol function,

b) adding a deprotection molecule to the aqueous solution, so as to deprotect the S-R group in order to obtain hydrophilic surfactants functionalized with an S-H group,

c) dissolving a metal salt in the solution so as to at least locally cover the lipid nanoparticles with a metal layer via a metal coordination bond with the S-H group of functionalized hydrophilic surfactants.

The process used is efficient, inexpensive, with mild reaction conditions, which makes it possible to preserve the lipid nanoparticles which are very sensitive to temperature, pH and chemicals.

The method makes it possible to label the lipid nanoparticles stably without modifying their sizes and / or their chemical compositions.

Advantageously, the metal salt is added in excess relative to the S-H group of hydrophilic surfactants functionalized so as to form a sufficiently thick metal layer and / or covering the largest surface of lipid nanoparticles.

Advantageously, the metal salt is a salt of gold, silver, copper, nickel, cobalt, iridium or a mixture of these salts.

Advantageously, the deprotection molecule is dithiothreitol. This compound is particularly reactive.

Advantageously, the aqueous solution supplied in step a) is obtained according to the following successive steps:

al) preparation of an oily phase comprising one or more lipid compounds to form the lipid core of the lipid nanoparticles, and the amphiphilic lipid surfactants, a2) preparation of an aqueous phase, a3) dispersion of the oily phase in the aqueous phase, for example under the action of a shear, such as by sonication, to form lipid nanoparticles, the hydrophilic surfactants functionalized by an SR group being added during step a2) or a3).

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood on the basis of the description which follows and of the appended drawings in which:

FIGS. 1A to 1D, previously described, are photographs obtained by transmission electron microscopy corresponding to samples of lipid nanoparticles, according to the prior art,

FIG. 2 is a diagrammatic representation, in section, of a lipid nanoparticle marked according to a first embodiment of the invention,

FIG. 3 is a schematic representation, in section, of a lipid nanoparticle marked according to a second embodiment of the invention,

FIG. 4 represents a reaction diagram of the method for labeling a lipid nanoparticle according to a particular embodiment of the invention,

FIG. 5 is a photograph obtained with an electron microscope in transmission of a sample of lipid nanoparticles marked with gold according to the invention, observed by the dry route,

FIG. 6 is a photograph obtained with an electron microscope in transmission of a sample of lipid nanoparticles marked with gold according to the invention, observed in cryogenics,

FIG. 7 is a photograph obtained with an electron microscope in transmission of a sample of lipid nanoparticles marked with gold according to the invention, observed in situ in the liquid channel,

FIG. 8 is a photograph obtained with an electron microscope in transmission of a sample of lipid nanoparticles marked with silver according to the invention, observed in cryogenics,

- Figure 9 is a photograph obtained with an electron microscope in transmission of a sample of lipid nanoparticles marked with silver according to the invention, observed in situ in the liquid channel.

The different parts shown in the figures are not necessarily shown on a uniform scale, to make the figures more readable.

The different possibilities (variants and embodiments) must be understood as not being mutually exclusive and capable of being combined with one another.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

Labeled lipid nanoparticles:

As shown in Figures 2 and 3, the labeled lipid nanoparticles (or LNPs "Lipid NanoParticles") have a heart-shell structure.

Core of lipid nanoparticles:

The heart 10 of the lipid nanoparticles is a lipid heart.

By a lipid core is meant a core comprising one or more lipids, that is to say fatty substances or substances containing fatty acids (ie aliphatic carboxylic acids having a carbon chain of at least 4 carbon atoms , for example from 4 to 28 carbon atoms) originating from fats of animal origin and / or oils of vegetable origin. These are hydrophobic or amphiphilic molecules formed mainly of carbon, hydrogen and oxygen. The oils can be chosen from biocompatible oils, and in particular from oils of natural (vegetable or animal) or synthetic origin. The oils of natural vegetable origin are, for example, soybean, linseed, palm, peanut, olive, grape seed and sunflower oils. Synthetic oils are, for example, triglycerides, di glycerides and mono glycerides. The lipid (s) of the heart 10 preferably have an affinity with the amphiphilic lipid surfactant to allow its solubilization.

The lipids can be in the solid state at room temperature (25 ° C), as in waxes, and / or in the liquid state as in the case of oils. The lipid heart can therefore be more or less solid.

There are four variants of nanoparticles with a lipid core. For these four variants, the shell 20 is a monolayer of amphiphilic lipids 21 (Figures 2 and 3).

According to a first variant, the lipid phase of the heart 10 is solid at room temperature. The lipid phase can comprise waxes and / or glycerides. This type of lipid nanoparticle is called a solid lipid nanoparticle (SLN "Solid Lipid Nanoparticle").

According to a second variant, the core 10 of the particle comprises a mixture of solid lipids and lipids liquid at room temperature. With such a structure, the core of the nanoparticle remains solid but it has an imperfect amorphous or crystalline structure (polycrystalline). Thus, the molecules of interest can be inserted more freely between the chains of solid lipids. This type of lipid nanoparticle is a nanostructured lipid transporter (NLC "Nanostructured Lipid Carrier").

According to a third variant, a hydrophilic molecule of interest (active principle) is conjugated to a lipid insoluble in water. This lipid compound is called a lipid-active ingredient conjugate (LDC "Lipid-Drug Conjugate") and may be mixed with other lipids to form the core of the nanoparticles.

According to a fourth variant, the heart 10 comprises a liquid or semi-liquid oil and a shell of lipids solid at room temperature. The lipid nanoparticle is called a lipid nanocapsule (LNC).

As shown in FIG. 3, molecules of interest 41 can be encapsulated in the heart 10 of the lipid nanoparticles. The expression molecule of interest 41 means a therapeutic or diagnostic agent. It can be an active principle acting chemically, biologically or physically, or an imaging agent in particular for MRI (magnetic resonance imaging), PET (in English “Positron Emission Tomography”) ), SPECT (“Single Photon Emission Computed Tomography”), ultrasound ultrasound, radiography, X-ray tomography and optical imaging (fluorescence, bioluminescence, scattering ...).

Shell of lipid nanoparticles:

The core 10 of the lipid nanoparticles is coated in a lipid shell 20. The lipid shell comprises amphiphilic lipid surfactants 21, hydrophilic surfactants functionalized 22 by an S-H group and optionally additional hydrophilic surfactants 23.

By amphiphilic lipid surfactant 21 is meant compounds having a structure, conferring a specific affinity for the oil / water and water / oil interfaces, which makes it possible to reduce the free energy of these interfaces and to stabilize the dispersed systems. The amphiphilic lipid surfactant is, for example, a phospholipid, a cholesterol, a lysolipid, or a glucolipid.

By phospholipid is meant lipids having a phosphate group, in particular phosphoglycerides. Most often, the phospholipids comprise a hydrophilic end formed by the optionally substituted phosphate group and two hydrophobic ends formed by chains of fatty acids. By way of illustration, mention may be made of phosphatidylcholine (also called lecithin), phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine and sphingomyelin. For example, lecithin is a lipid formed from a choline, a phosphate, a glycerol and two fatty acids.

By hydrophilic surfactant is meant a molecule making it possible to lower the energy of the water / oil interface and to ensure better dispersion of the lipid nanoparticles in the aqueous phase. The hydrophilic surfactant is preferably alkoxylated and preferably comprises at least one chain composed of units of ethylene oxide (PEO or PEG) or of ethylene oxide and propylene oxide.

The hydrophilic functionalized surfactants 22 of the labeled LNP have an S-H group. They are preferably functionalized PEGs.

As shown in FIGS. 2 and 3, the shell 20 may also comprise additional hydrophilic surfactants 23.

According to a particular embodiment, all or at least part of the additional hydrophilic surfactants 23 may have a reactive function, such as a maleimide, amine, ester, oxyamine or aldehyde group. The presence of reactive functions allows the grafting of functional compounds.

The functionalized hydrophilic surfactants 22 can be interposed between the additional hydrophilic surfactants 23.

As shown in FIGS. 2 and 3, the shell 20 can optionally be functionalized with targeting agents 42. The targeting agents can be fixed to the surface of the nanoparticles, by adsorption or by covalent bonding, for example at the level of hydrophilic surfactants 23 and / or at the level of the functionalized hydrophilic surfactants 22. The hydrophilic surfactants 22 and / or 23 can act as a spacer to distance the targeting agents from the core of the lipid nanoparticle and make them more accessible. By way of illustration, the targeting agents 42 can be chosen from: biological targeting ligands, to allow specific recognition of certain cells or of certain organs, such as antibodies, peptides, saccharides, aptamers, oligonucleotides or compounds like folic acid; a stealth agent to give the lipid nanoparticle a stealth vis-à-vis the immune system, which increases its circulation time in the body, and slows its elimination.

The lipid nanoparticles have, for example, a diameter ranging from 10 nm to 500 nm, and preferably from 30 to 300 nm.

According to a particularly advantageous embodiment, the lipid nanoparticle is a Lipidot ™, that is to say a structure comprising:

a lipid core 10, a mixture of mono-, di- and triglycerides with long carbon chains (typically a C ₆ -C ₂ 2 carbon chain, more particularly Ci ₆ -Ci ₈ ), the mixture being for example a mixture of a wax and a vegetable oil such as soybean oil, the lipid heart possibly comprising, in addition, a molecule of interest 41 to be delivered into the body,

a shell 20 comprising a monolayer of phospholides 21, for example soy lecithin (also known under the trade name Lecithin S75), preferably combined with PEGylated surfactants 23, such as PEG2000 stearate, also known under the trade name Myrj s40, and functionalized PEGylated surfactants 22, with an SH group.

PEGylated surfactants 23 such as Myrj s50 or Myrj slOO could also be used.

Preferably, the Lipidot ™ have a diameter ranging from 30 nm to 120 nm.

Metallic labeling of lipid nanoparticles:

As shown in Figures 2 and 3, the lipid nanoparticles are covered by a metallic layer 60. The layer 60 forms a continuous or discontinuous layer around the lipid nanoparticles.

By discontinuous, it is meant that it can be located only at the level of the S-H groups, or even at the level of the S-H groups and between certain S-H groups (FIG. 2).

By continuous, it is meant that the external metallic layer 60 completely covers the lipid nanoparticle (FIG. 3).

The marking of nanoparticles is a strong marking via a metallic coordination bond of the S-M type, with M a metal.

The metallic layer 60 is made of gold, silver, copper, nickel, cobalt, iridium or a mixture of these

Preferably, it is continuous and in gold or silver.

The metallic marking of lipid nanoparticles, in addition to facilitating their characterization, can find therapeutic applications in particular with regard to photo-thermal (destruction of tumor cells by irradiation of the grafted nanoparticles, taking into account the thickness of the metallic layer 60 for the irradiation wavelength).

TEM characterization process for labeled lipid nanoparticles:

To characterize the lipid nanoparticles at TEM, a sample of the solution of labeled lipid nanoparticles as defined above is taken.

The sample is, for example, placed in a holder. The object-holder comprises an observation chamber, preferably sealed, intended to receive the liquid sample. The observation chamber comprises two main faces, parallel to each other, intended to be crossed by the electron beam and a lateral face closing the observation chamber. The main faces are for example two SiN plates. The lateral face has, for example, a thickness ranging from 50 nm to lpm. The chamber may include a fluid system for circulating a fluid through the chamber. The marking process can also be carried out in the chamber of the object-holder. The formation of the external metal layer can thus be monitored at TEM.

The labeled LNPs can then be characterized in situ by the liquid route. TEM observations show that the nanoparticles labeled according to the invention are clearly visible with TEM in the liquid channel.

Process for the preparation of lipid nanoparticles:

We will now describe the process for preparing lipid nanoparticles. It is a process for preparing a nanoemulsion, comprising a continuous aqueous phase and at least one dispersed oily phase.

Solid lipid transporters (SLN), nanostructured lipid transporters (NLC), and LNC lipid nanoparticles can be synthesized either by ultrasound for small quantities or by high pressure homogenization (HPH) for industrial quantities.

Lipid nanocapsules (LNC) can also be synthesized using a phase inversion with temperature (PIT) process or by ultrasound.

We will describe more particularly the process for preparing a solution by sonication.

The process includes the following successive steps:

al) preparation of an oily phase comprising the lipid compound (s) of the lipid heart, the amphiphilic lipid surfactant 21, optionally the molecules of interest 41, a2) preparation of an aqueous phase optionally comprising the additional hydrophilic surfactants 23, a3 ) dispersing the oily phase in the aqueous phase, for example under the action of shearing, such as by sonication, to form lipid nanoparticles having a lipid core surrounded by a lipid shell.

The functionalized hydrophilic surfactants 22 can be added during step a2) or a3).

During step a1), the mixing can optionally be facilitated by dissolving one of the constituents or the complete mixture in an appropriate organic solvent. The organic solvent is then evaporated, to obtain a homogeneous oily mixture. Advantageously, step a1) is carried out at a temperature at which all of the different compounds are liquid.

The aqueous phase, prepared in step a2), comprises, for example, water and / or a buffer such as a phosphate buffer, such as PBS (“Phosphate Butter Saline”) or a saline solution, for example sodium chloride. The aqueous phase could also include a thickening agent, such as glycerol, a saccharide, a polysaccharide or a protein. A non-saline aqueous solution is preferably chosen to avoid precipitation of the salts during the TEM characterization.

The process may include a step in which targeting agents are grafted onto the hydrophilic surfactants 23 or onto the additional hydrophilic surfactants 22. The coupling can be carried out before or after the emulsification (before or after step a3). Preferably, the pH during the coupling reaction is between 5 and 11, and preferably between 6 and 8.

Advantageously, during step a3), the oily phase is dispersed in the aqueous phase in the liquid state. It is preferable to carry out the mixing at a temperature greater than or equal to the melting point of the various reactants.

The proportion of oily phase and aqueous phase is very variable. One can choose from 1 to 50%, preferably from 5 to 40% by weight of oily phase and from 50 to 99%, and preferably from 60 to 95% by weight of aqueous phase.

The emulsification is preferably carried out using a sonicator or a microfluidizer. Preferably, the aqueous phase and then the oily phase are introduced in the desired proportions into an appropriate cylindrical container, then the sonicator is immersed in the medium and started up for a sufficient time to obtain a nanoemulsion, usually a few minutes. The emulsion is then purified by dialysis using membranes, typically with a cutoff threshold of more than 10 kDaltons (for example Roth Zellu Trans membrane 12-14 kDa). A homogeneous nanoemulsion is then obtained in which the average diameter of the oil droplets ranges from 10 nm to 500 nm, and preferably from 30 to 300 nm.

The nanoemulsion can then be packaged. Before packaging, the nanoemulsion can be diluted and / or sterilized, for example by filtration and / or dialysis. This step eliminates any aggregates that may have formed during the preparation of the emulsion. The emulsion thus obtained is ready for use, if necessary after dilution.

Method for labeling lipid nanoparticles:

The method of labeling lipid nanoparticles, with a view to their characterization by scanning electron microscopy, will now be described. It is shown diagrammatically in FIG. 4. The marking process comprises the following successive steps:

a) supply of an aqueous solution comprising lipid nanoparticles, the lipid nanoparticles comprising a lipid core 10 surrounded by a lipid shell, the lipid shell comprising amphiphilic lipid surfactants 21, optionally hydrophilic surfactants 23, and functionalized hydrophilic surfactants 22 by a group SR with R a group protecting the thiol function,

b) deprotection of the S-R function, by adding a deprotection molecule, having, for example, a thiol function, to the aqueous solution, so as to obtain hydrophilic surfactants functionalized 22 with an S-H 50 group,

c) dissolving a metal salt in the solution so as to cover at least locally the lipid nanoparticles with a metal layer 60, linked via metal coordination bonds to the S-H groups 50 of the functionalized hydrophilic surfactants 22.

Preferably, the group for protecting the thiol SR function is chosen from Spyridine, and a triphenylmethyl group, also called trityl, ie C (C ₆ H ₅ ) ₃ .

Preferably, S-R corresponds to the S-Spyridine group. For example, the functionalized hydrophilic surfactant 23 is a PEG having on one side a stearic acid chain and on the other the S-Spyridine function, that is to say a thiopyridine. The group R protects the thiol from the functionalized hydrophilic surfactant 23. Preferably, for R, Spyridine will be chosen, which can be easily removed by reaction with a molecule functionalized with a thiol. The protection group for the thiol S-R function is positioned, preferably at the end of the PEG chain.

Preferably, the molecule having a thiol function is dithiothreitol.

By metal salt is meant one or more metal salts of the same metal and / or one or more salts of different metals. Preferably, the metal salt is a salt of gold, silver, copper, nickel, cobalt, or iridium.

Preferably, the metal salt is added in excess relative to the S-H 50 group. For example, an excess of 30 equivalents maximum is chosen, ie 3000%.

Illustrative and nonlimiting examples of particular embodiments:

Lipid nanoparticles (LNP) and their synthesis The lipid core 10 of LNP comprises 75% by weight of solid lipid (semi-synthetic glycerides, sold under the trade name Suppocire® NC (Gattefossé)) and 25% by weight of liquid lipid (oil of soy, enriched with 75% phosphatidylcholine, sold by the company Lipoïd under the trade name Lipoïd® S75)).

The shell 20 comprises a monolayer of additional hydrophilic surfactants 23 (PEG) and of amphiphilic lipid surfactants 21 (lecithin) which stabilizes the heart of the LNPs. The shell also comprises PEGs functionalized 22 by S-Spyridine groups, interposed between the additional hydrophilic PEGs 23.

By way of illustration, the weights of the various reagents used are: water / PBS mixture: 1220 mg, internal lipids (heart): 410 mg, lecithin: 50 mg, and PEG: 300 mg.

To prepare the LNPs, an oily phase is mixed, containing the lipids forming the lipid core and the amphiphilic lipid surfactants 21, with an aqueous phase, containing the hydrophilic surfactants 23, and the functionalized hydrophilic surfactants 22. After homogenization at 45 ° C., the mixture is subjected to a sonification step at 45 ° C., with an AV505® sonicator equipped with a conical probe of 3mm in diameter (Sonies, Newtown).

The emulsion obtained can be dialyzed with a dialysis membrane so as to remove the unreacted reagents.

Deprotection of the S-R group of lipid nanoparticles and formation of an S-H group:

In order to be able to strongly functionalize the LNPs with a metal, it is necessary to deprotect the thiol (SH) functions of the LNPs, that is to say to break the disulfide bridge of the S-Spyridine grafted onto them. For this, DTT (Dithiothreitol) is used up to 20 equivalents which are added directly to a dialysis membrane into which the LNPs have been placed beforehand. The solvent used for dialysis is ultrapure water. The dialysis lasts approximately 24 hours and the water should be changed as often as possible in order to eliminate the SPyridine group and the excess DTT.

Labeling of lipid nanoparticles with gold via a metallic coordination bond:

LNP lots include 3.55.10 ¹⁴ particles / mL. 400 μL of LNP are removed using a micropipette, which represents 1.42 × 10 ¹⁴ particles. Knowing that there are approximately 1300 thiol groups per particle, there are 1.85.10 ¹⁷ SH groups in the 400 μL sampled, ie 3.07.10 ⁷ moles of SH.

1.2 mg of gold chloride salt are added (HAuCI ₄ .3H ₂ O, CAS number: 1696125-4, M = 393.83 g / mol). The salt is in excess of 10% relative to the number of moles of SH.

The reaction is carried out in an Eppendorf tube. Leave to react for 24 hours. The whole is then dialyzed for a period of 24 hours.

Characterization of lipid nanoparticles by transmission electron microscopy:

The labeled LNPs were recovered and observed in dry TEM, without negative staining, in cryogenics and in liquid in situ.

It appears that in the dry process (Figure 5), the LNPs marked with gold are barely visible at all. The particles may still be too sensitive to be seen when dried. The same trend is found cryogenic (Figure 6): the particles are visible but deformed.

LNPs with a metallic layer of gold are clearly visible when observed in liquid in-situ TEM (Figure 7). The contrasts obtained were compared with LNPs grafted with gold nanoparticles, stabilized in citrate. The contrasts with a shell are greater than those obtained with the gold nanoparticles. The improvement in contrast may be due to better grafting of the gold ions onto the thiols and / or to a greater quantity of gold.

The results were compared with LNPs comprising PEGs lacking S-R groups. It has been confirmed that the grafting of gold on these LNPs did not work. No LNP is observable at TEM with this sample. You can only see gold particles.

Labeling of lipid nanoparticles with silver via a metal coordination link and observation by transmission electron microscopy:

In this example, 0.52 mg of silver nitrate salt (AgNO ₃ , CAS #: 7761-88-8, M = 169.87 g / mol) was added to the LNPs in an Eppendorf tube, which 5 represents an excess of 10 mol%.

The whole is left to react for 24 hours, then dialysis is carried out for 22 hours.

The labeled LNPs are recovered and observed in TEM in cryogenics (Figure 8) and in liquid in-situ (Figure 9). LNPs cannot be characterized by cryogenics, only the silver nanoparticles are visible. The marked LNPs are, however, particularly clearly visible when observed in liquid in-situ TEM.

Claims (14)

1. Lipid nanoparticle marked to be able to be observed by transmission electron microscopy, the lipid nanoparticle comprising a lipid core (10) surrounded by a lipid shell (20), the lipid shell (20) comprising at least one layer of amphiphilic lipid surfactants (21), and hydrophilic surfactants functionalized (22) by an SH group (50), the lipid nanoparticle being covered at least locally by a metallic layer (60) linked to the SH groups (50) of the hydrophilic surfactants functionalized (22) a metallic coordination link. 2. Nanoparticle according to claim 1, characterized in that the metallic layer (60) is gold, silver, copper, nickel, cobalt, iridium or a mixture of these. 3. Nanoparticle according to one of claims 1 and 2, characterized in that the metal layer (60) forms a continuous layer around the lipid nanoparticles. 4. Nanoparticle according to one of claims 1 to 3, characterized in that the lipid core of the lipid nanoparticles comprises one or more molecules of interest (41), such as an active principle, an imaging agent or a drug . 5. Nanoparticle according to any one of the preceding claims, characterized in that the shell (20) further comprises additional hydrophilic surfactants (23). 6. Nanoparticle according to the preceding claim, characterized in that targeting agents (42) are grafted onto the additional hydrophilic surfactants (23) and / or onto the functionalized hydrophilic surfactants (22), the targeting agents (42) being, for example, targeting biological ligands such as antibodies or peptides. 7. Nanoparticle according to one of claims 5 and 6, characterized in that the additional hydrophilic surfactants (23) are polyethylene glycols and / or in that the functionalized hydrophilic surfactants (22) are functionalized polyethylene glycols. 8. Nanoparticle according to any one of the preceding claims, characterized in that the core (10) of the nanoparticle is a mixture of a wax and a vegetable oil, such as soybean oil, and in that the lipid shell (20) comprises a monolayer of phospholipids (21). 9. Method for characterizing lipid nanoparticles, comprising the following successive steps: - supply of an aqueous solution containing labeled lipid nanoparticles as defined in any one of claims 1 to 8, - placement of the aqueous solution in an observation chamber of an electron microscope object holder in transmission, and closure of the observation chamber, - characterization of the labeled lipid nanoparticles in the aqueous solution by transmission electron microscopy. 10. Method for labeling lipid nanoparticles, to obtain lipid nanoparticles as defined in any one of claims 1 to 8, comprising the following successive steps: a) supply of an aqueous solution comprising lipid nanoparticles, the lipid nanoparticles comprising a lipid core (10) surrounded by a lipid shell (20), the lipid shell (20) comprising at least one layer of amphiphilic lipid surfactants (21 ), and hydrophilic surfactants functionalized (22) by a group SR with R a group protecting the thiol function, b) adding a deprotection molecule to the aqueous solution, so as to deprotect the S-R group in order to obtain functionalized hydrophilic surfactants (22) with an S-H group (50), c) dissolving a metal salt in the solution so as to cover at least locally the lipid nanoparticles with a metal layer (60) via a metal coordination bond with the S-H group (50) of the functionalized hydrophilic surfactants (22). 11. The method of claim 10, characterized in that the metal salt is added in excess relative to the S-H group of functionalized hydrophilic surfactants (22). 12. Method according to one of claims 10 and 11, characterized in that the metal salt is a salt of gold, silver, copper, nickel, cobalt, iridium or a mixture of these salts. 13. Method according to any one of claims 10 to 12, characterized in that the deprotection molecule is dithiothreitol. 14. Method according to any one of claims 10 to 13, characterized in that the aqueous solution supplied in step a) is obtained according to the following successive steps: al) preparation of an oily phase comprising one or more lipid compounds to form the lipid core (10) of the lipid nanoparticles, and the amphiphilic lipid surfactants (21), a2) preparation of an aqueous phase, a3) dispersion of the phase oily in the aqueous phase, for example under the action of a shear, such as by sonication, to form lipid nanoparticles, the hydrophilic surfactants functionalized (22) by an SR group, 5 being added during step a2) or a3).