WO2016170010A1

WO2016170010A1 - Lipid based nanocarrier compositions loaded with metal nanoparticles and therapeutic agent

Info

Publication number: WO2016170010A1
Application number: PCT/EP2016/058805
Authority: WO
Inventors: Philippe Barthelemy; Khalid OUMZIL; Gisèle CLOFENT-SANCHEZ; Marie-Josée Jacobin-Valat; Jeanny Laroche-Traineau; Stéphane MORNET; Karen Gaudin; Abdelmajid NOUBHANI; Xavier-François Santarelli
Original assignee: Universite de Bordeaux; Institut National De La Sante Et De La Recherche Medicale (Inserm); Centre National De La Recherche Scientifique (Cnrs); Institut Polytechnique De Bordeaux (Ipb)
Priority date: 2015-04-20
Filing date: 2016-04-20
Publication date: 2016-10-27
Also published as: JP2018513195A; EP3085360A1; US20180116972A1; EP3285741A1; CA2983079A1

Abstract

The invention relates to non-polymeric lipid-based nanocarrier compositions loaded with metal nanoparticles and at least one therapeutic agent, useful.as agents for transportation, vectorization, cellular delivery cellular targeting or cellular localization of at least one therapeutic agent.

Description

Lipid based nanocarrier compositions loaded with metal nanoparticles and therapeutic agent

The invention relates to non-polymeric lipid-based nanocarrier compositions loaded with metal nanoparticles and at least one therapeutic agent.

The nanoparticle technology has allowed the implementation of novel multifunctional nanoparticles for disease, detection, therapy and treatment monitoring. Their numerous advantages include minimizing the amount of drug needed, increasing the bioavailability, in particular for hydrophobic drugs, and reducing the drug toxicity.

Magnetic Resonance Imaging (MRI) is a worldwide-used non-invasive imaging and diagnostic technique, which is very efficient for imaging soft tissues and provides detailed anatomical images of the body. Ultrasmall superparamagnetic iron oxide nanoparticles are currently used as contrast agent in magnetic resonance imaging.

The theranostic approach refers to molecular/macromolecular targeting vectors and nano-platform technologies that incorporate both diagnostic and therapeutic functionalities. These entities can be used for simultaneous targeted drug delivery and release, as well as diagnosis, which includes monitoring disease progression and response to therapy.

In addition, this approach can be used for selecting a safe and efficient dose of therapeutic agent, recognizing adverse effects at an early stage of therapy and real-time monitoring of therapy, making it possible to adapt the treatment to the individual patients as a function of the inter-individual variability in their therapeutic responses. Quantum dot lipid oligonucleotide bioconjugates are studied in A. Aime et al., Bioconjugate Chem., 2013, 24, 1345-1355. These bioconjugates consist of fluorescent semiconductor nanocrystals, named quantum dots, encapsulated by nucleolipids and lipid oligonucleotide conjugates, with a view to providing functionalized quantum dots with recognition and detection properties.

It has now been found that lipid-based theranostic systems loaded with nanoparticles and a therapeutic agent, which are non-polymeric systems, could be used, in particular, for drug targeting, drug delivery and treatment monitoring, while avoiding the side-effects usually related to the use of toxic active ingredients, in particular in cancer therapy.

Advantageously, the use of nucleoside-lipids (also called nucleolipids) in these systems allows the formation of stable supramolecular constructs.

Without wishing to be bound by theory, it can be hypothesized that the metal nanoparticles are associated to each other as clusters by hydrophilic/hydrophobic interactions with the nucleolipids, while such clusters are not formed when using lipids instead of nucleolipids. Said nucleolipids also serve to entrap the therapeutic agent(s), through hydrophilic/hydrophobic interactions and are present at the interface with the outer aqueous medium.

Advantageously, it has also been found that the lipid-based nanocarrier composition according to the invention allows a high content in active principle and the formation of nanoparticles which can be used for imaging purpose.

These nanoparticles are solid lipid nanoparticles loaded with metal nanoparticles and a therapeutic agent, and stabilized by the nucleolipids.

Actually, while the use of a lipid, such as, for example 1 ,2-Dioleyl-sn- Glycero-3-phosphocholine (DOPC) in association with metal nanoparticles does not result in a formulation having the desired colloidal stability, the inventors have found, surprisingly, that such stability could be provided by the nucleolipids of formula (I) below. In addition, the formation of clusters of metal nanoparticles creates a larger hydrophobic reservoir, which makes it advantageously possible to entrap a higher amount of therapeutic agent.

On the other hand, the magnetic sensitivity of the lipid-based nanocarrier composition is enhanced through these stable supramolecular constructs, thereby also increasing the magnetic resonance signal, and thus the detection of the lipid-based nanocarrier composition of the invention, which plays a decisive role in theranostic approaches.

The invention thus relates to a lipid-based nanocarrier composition comprising a) at least one compound of formula (I)

in which

- X is an oxygen atom, a sulfur atom or a methylene group,

B is a purine or pyrimidine base, or else a non-natural mono- or bi-cyclic heterocyclic base, each ring of which comprises 4 to 7 members, optionally substituted ;

- I_i and L₂, identical or different, represent hydrogen, an oxycarbonyl -O-C(O)- group, a thiocarbamate -O-C(S)-NH- group, a carbonate -O-C(O)-O- group, a carbamate -O-C(O)-NH- group, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched, saturated or unsaturated C2-C30 hydrocarbon chain,

- or also, l_i and L₂, together, form a ketal group of formula

- or also or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched C2-C3o alkyl chain_,

- Ri and R₂, identical or different, represent

- a linear or branched C₂-C₃o hydrocarbon chain, saturated or partially unsaturated, or

- a C2-C3o acyl chain,

- a diacyl chain in which each acyl chain is C2-C30,

- a diacylglycerol in which each acyl chain is C2-C30, or

- a sphingosine or ceramide group in which each acyl chain is C₂-C₃o, or

- when l_i or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, Ri and R₂ do not exist;

- R3 represents

- a hydroxy, amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate, phosphonium, N H₂-R₄, NH R-^Rs or NR R₅R₆ group in which R , R₅ and R6, identical or different, represent a hydrogen atom or a linear or branched C1-C6 alkyl chain or linear or branched Ci -C₆ hydroxyalkyl, or

- a linear or branched C2-C30 alkyl chain, optionally substituted by a hydroxy group, or

- a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a C₂-C₃o alkyl, optionally substituted by a hydroxy group; or by a (CH₂)m-O-(CH₂)p-R9 group in which m = 1 to 6 and p = 0 to 20 and Rg represents hydrogen or a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched C₂-C3o alkyl, or by a sterol radical,

or

- a -O-C(O)-(CH₂)_q-C(O)-O [(CH₂)₂-O]_rH group in which q is an integer from 2 to 6 and r is an integer from 4 to 30, preferably from 10 to 20, or also - R₃ is bound by a covalent bond to another substituent R₃, identical or different, of another compound of formula (I), identical or different, in order to form a compound of formula (I) in the form of a dimer,

b) at least one metal nanoparticle, and

c) at least one therapeutic agent.

By "nanocarrier" is meant that the theranostic system of the invention has an overall size (average diameter) of approximately 20 to 300 nm.

X preferably represents an oxygen atom.

By « straight or branched C1-C6 alkyl » is understood, for example, a methyl, ethyl, propyl, i-propyl, n-butyl, i-butyl, tert-butyl, preferably methyl or ethyl.

Preferred C₂-C₃o hydrocarbon chains are C₈-C₃₀, preferably C₈-C₂6, more preferably C16-C20 hydrocarbon chains.

Preferred linear or branched C₂-C₃₀ alkyl chains are C₈-C₃₀, preferably C8-C26, more preferably C16-C20 linear or branched alkyl chains.

Preferred C2-C₃o acyl chains are C₈-C₃o, preferably C8-C26, more preferably Ci6-C2o acyl chains.

In the definitions of l_i, L₂, Ri or R₂, the alkyl, acyl or hydrocarbon chain is preferably a C₈-C₃₀ alkyl, acyl or hydrocarbon chain.

Preferably, l_i and L₂ are not simultaneously hydrogen.

The invention relates, in particular, to a lipid-based nanocarrier composition comprising

a) at least one compound of formula (I)

in which

X is an oxygen atom, a sulfur atom or a methylene group,

Li and L₂, identical or different, represent hydrogen, an oxycarbonyl -O-C(O)- group, a thiocarbamate -O-C(S)-NH- group, a carbonate -O-C(O)-O- group, a carbamate -O-C(O)-NH- group, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched, saturated or unsaturated C8-C30 hydrocarbon chain, wherein l_i and L₂ are not simultaneously hydrogen, or also, l_i and L₂, together, form a ketal roup of formula

or also l_i or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched C8-C30, alkyl chain,

Ri and R₂, identical or different, represent

a linear or branched C8-C30 hydrocarbon chain, saturated or partially unsaturated, or

a C8-C30 acyl chain,

a diacyl chain in which each acyl chain is C8-C30,

a diacylglycerol in which each acyl chain is C8-C30, or

a sphingosine or ceramide group in which each acyl chain is C₈-C₃o, or when l_i or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, Ri and R₂ do not exist;

R3 represents

a hydroxy, amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate, phosphonium, NH₂-R , NHR-^Rs or NR R₅R₆ group in which R , R₅ and R6, identical or different, represent a hydrogen atom or a linear or branched C1-C6 alkyl chain or linear or branched Ci-C₆ hydroxyalkyl, or

- a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a C₂-C₃o alkyl, optionally substituted by a hydroxy group; or by a (CH₂)m-O-(CH₂)p-R9 group in which m = 1 to 6 and p = 0 to 20 and F¾ represents hydrogen or a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched C2-C30 alkyl, or by a sterol radical,

or

- a -O-C(O)-(CH₂)_q-C(O)-O [(CH₂)₂-O]_rH group in which q is an integer from 2 to 6 and r is an integer from 4 to 30, preferably from 10 to 20, or also

- R₃ is bound by a covalent bond to another substituent R₃, identical or different, of another compound of formula (I), identical or different, in order to form a compound of formula (I) in the form of a dimer,

b) at least one metal nanoparticle, and

c) at least one therapeutic agent.

The purine or pyrimidine base can be, for example, selected from, adenine, guanine, cytosine, xanthine, hypoxanthine, uric acid, caffeine, theobromine, uracile, thymine, dihydrouridine, and their derivatives.

Thymine and uracile are preferred.

Also, in formula (I) above, the purine or pyrimidine base can be substituted by at least one substituent selected from, for example, a halogen, an amino group, a carboxy group, a carbonyl group, a carbonylamino group, a hydroxy, azido, cyano, thiol, a C1-C6 straight or branched alkyl, cycloalkyl, perfluoroalkyl, alkyloxy (for example, methoxy), oxycarbonyl, vinyl, ethynyl, propynyl, acyl group etc.

By "derivatives of a purine or pyrimidine base" is meant, for example, a non-natural mono- or bi-cyclic heterocyclic base in which each cycle has 4 to 7 members, optionally substituted as stated above for the purine or pyrimidine base.

By « non-natural heterocyclic base » is meant a universal base, such as, for example, 3-nitropyrrole, 4-nitroimidazole or 5-nitroindole, which do not exist in nature.

By « heteroaryl comprising 1 to 4 nitrogen atoms » is meant a mono-or bi-cyclic carbocyclic group, aromatic or partially unsaturated, comprising 5 to 12 atoms in total, interrupted by 1 to 4 nitrogen atoms, which can be, for example, selected from furane, pyrrole, oxazole, oxadiazole, isoxazole, pyrazole, triazole, tetrazole, imidazole, pyridine, pyrimidine, pyridazine, pyrazine, benzofurane, indole, quinoleine, isoquinoleine, chromane, naphtyridine and benzodiazine groups, triazole being preferred.

The following compounds of formula (I), in which at least one condition is fulfilled, are preferred: - X is an oxygen atom;

- B is thymine or uracile;

- L-Ϊ and L₂ are oxycarbonyl -O-C(O)- groups which are substituted by a linear or branched C2-C30, preferably C8-C30, hydrocarbon chain, preferably Cs- C26, more preferably C16-C20, saturated or partially unsaturated; or - l_i is a phosphate group which is substituted by diacylglycerol in which each acyl group is C2-C30, preferably C8-C30, more preferably C8-C26, even more preferably C16-C20, and L₂ is hydrogen;

- R3 is hydroxy, a NR R₅R₆ group in which R , R₅ and R6 represent a hydrogen atom or a -O-C(O)-(CH₂)_q-C(O)-O [(CH₂)₂-O]_rH group in which q is an integer from 2 to 6 and r is an integer from 4 to 30, preferably from 10 to 20.

Particularly preferred compounds of formula (I) are selected from - N-[5'-(2\3'-dioleoyl)uridine]-N\N',N'-tnmethylammonium (DOTAL!) (CAS Registry Number: 868226-06-6),

- Thymidine 3'-(1 ,2-dipalmitoyl-sn-glycero-3-phosphate), also called diC16dT(CAS Registry Number: 1 160002-70-9), and - Poly(oxy-1 ,2-ethanediyl), a-hydro-co-methoxy-, ester with uridine

5'-(hydrogen butanedioate) 2',3'-di-(9Z)-9-octadecenoate (DOU-PEG) (CAS Registry Number: 1353570-75-8).

In a preferred embodiment, the lipid-based nanocarrier composition according to the invention comprises at least 2 different compounds of formula (I).

In particular, the lipid-based nanocarrier composition can comprise at least one co-lipid in addition to the compound(s) of formula (I).

By "co-lipid", is meant a compound used in combination with the compound of formula (I), which contributes to the production of the structure of the lipid-based nanocarrier composition.

Preferably, a zwitterionic co-lipid will be used.

Said co-lipid can be, for example, chosen from dioleylphosphatidylcholine (DOPC), dioleylphosphatidyluridinephosphatidyl- choline (DOUPC) or dioleylphosphatidylethanolamine (DOPE).

These compounds can play the role of co-lipid when they are used in a mixture with a compound of formula (I). Alternatively, they can be included in formula (I), such as, for example, dioleylphosphatidyluridinephosphatidylcholine (DOUPC). In this case, they will either play the role of a compound of formula (I) or, in combination with another compound of formula (I), the role of co-lipid.

For the preparation of the compounds of formula (I), reference can be made to WO 2005/1 16043, which describes different access routes to this type of compounds (see in particular pp. 8-17 and the examples), as well as to WO2009/098404 or WO 2010/136676. Preferably, the lipid-based nanocarrier composition according to the invention contains a plurality of metal nanopartides, in particular 10 to 90 % (w/w) of metal nanopartides, preferably 50 to 80%.

The metal nanopartides contain, preferably, metal oxides which can be, for example, selected from iron oxide (magnetite Fe3O₄, maghemite y-Fe₂O3, or other Co ferrite or Ni ferrite) which is known for its magnetic properties while metals (Au, Ag, Cu, Si, Ge, Fe, Co etc), metal alloys (FeCo, FePt, CoPt, FeBi etc.) and metal chalcogenides (CdS, CdSe, CdSe, ZnS...etc.) can be used for their plasmonic, luminescent or magnetic properties which are interesting in the aim to the development of a contrast agent or a labelling tool for bioimaging, tacking or sensoring.

Preferably, the metal nanopartides have an overall size (average diameter) of approximately from 2 nm to 20 nm, preferably 5 to 10 nm.

The metal nanopartides contained in the lipid-based nanocarrier compositions according to the invention may be mono-disperse (for example, they consist of a population having an average diameter centered around 10 nm), or mono- and/or poly-disperse (such as, for example, two populations having an average diameter centered around 4 nm and 10 nm), or else poly-disperse (different populations having different average diameters ranging from 2 nm and 20 nm).

Iron oxide nanopartides are preferred.

The metal nanopartides contained in the lipid-based nanocarrier compositions according to the invention can be, for instance, surface functionalized nanopartides, or any type of nanopartide presenting surface reactive groups, with a size comprised between 2 nm and 20 nm, preferably 5 to 10 nm, having a surface modified by grafting of amphiphilic or fatty ligand composed of an anchoring function - mainly carboxylates, phosphonate groups used for metal oxide and amine, thiol functions or other strong nucleophilic functions for metal nanopartides such as gold nanopartides - and a hydrophobic moiety which could be saturated or unsaturated hydrocarbon chains or peril uorinated carbon chains.

For example, coprecipited hydrophobic iron oxide nanoparticles have been prepared as previously published (G.A. van Ewijk, G.J. Vroege, A. P. Philipse, Convenient preparation methods for magnetic colloids, J. Magn. Magn. Mater. 201 (1999) 31 -33) using stearic acid as fatty acid.

In a preferred embodiment the metal nanoparticles have a surface functionalization which provides them with hydrophobic properties. For instance they bear fatty acid residues on their surface. By « therapeutic agent » is meant, for example, a natural or synthetic molecule used for preventing or treating a pathological condition, or restoring a biological function, in vitro or in vivo, in particular in animals, in particular in human beings, or else in isolated cells.

Advantageously, said therapeutic agent can be selected, for example, from anti-tumoral agents, antibiotic agents, anti-microbial agents, analgesic agents, anti-histaminic agents, bronchodilators agents, agents which are active on the central nervous system, anti-hypertension agents or agents which are active on the cardiovascular system (in particular vasodilator agents, anti- atherosclerosis agents such as agents having a platelet anti-aggregating activity), nucleic acids and their fragments; peptides, oligopeptides, proteins, antigens, antibodies or else stem cells etc.

Anti-cancer agents are of particular interest, such as, for example, sorafenib, sunitinib, taxanes (docetaxel, paclitaxel, cabazitaxel...), doxorubicine, adriamycin, daunomycin, melphalan, gemcitabine, cisplatin, derivatives of gemcitabine, or derivative of cisplatin, imatinib (Gleevec®), 5-fluorouracil, 9-aminocamptothecin, amine-modified geldanamycin, Taxol®, procarbazine or hydroxyurea.

Anti-atherosclerosis agents, such as, for example, tocopherols, resveratrol, anthocyanes, succinobucol, prostacycline, terutroban, picotamide, varespladib, darapladib, fumagilline, chemokins (CXCR3, IL10), aliskiren, lisinopril, losartan, irbesartan, telmisartan, epleronone, apolipoproteine A1 (APOA-1 ) or glutathion are also of particular interest.

The invention also relates to a process for preparing a lipid-based nanocarrier composition comprising metal nanoparticles and at least one therapeutic agent, as described above, which comprises the following steps:

- preparing a solution containing at least one compound of formula (I), at least one metal nanoparticle and at least one therapeutic agent in an organic solvent

- adding this solution to an aqueous medium while stirring,

- sonicating the resulting aqueous solution and evaporating the organic solvent, and

- recovering the lipid-based nanocarrier composition thus obtained.

Preferred reaction conditions are as follows:

- the organic solvent can be selected, for example, from ether, choloroform, methylene chloride, ethyl acetate, butanol, propanol, ethanol and methanol;

- the aqueous medium is water;

- the evaporation of the organic solvent is carried out under reduced pressure, namely, for example, the pressure is reduced from 1 bar to 5 mbar;

- the lipid-based nanocarrier composition is recovered by centrifugation, for example at 14000 rpm during 15 to 30 min.

After centrifugation, the pellet containing the lipid-based nanocarrier composition may be re-suspended in water.

Preferably, when contained in the lipid-based nanocarrier compositions according to the invention as defined above, the therapeutic agent will be present at a concentration of about of 0.1 ng/mL to 10 mg/mL, and metal nanoparticles at a concentration of about of 0.1 ng/mL to 10 img/mL, in the aqueous phase.

The invention also relates to the use of a lipid-based nanocarrier composition as an agent for transportation, vectorization, cellular delivery cellular targeting or cellular localization of at least one therapeutic agent, in particular for imaging guided therapy.

For example, the lipid-based nanocarrier composition according to the invention may be used in various therapeutic fields relating to diseases and/or disorders such as cancers, atherosclerosis, viral and non-viral infections, immunity disorders, inflammation etc.

The invention also concerns pharmaceutical compositions containing a lipid-based nanocarrier composition as described above and a pharmaceutically acceptable carrier.

The invention is illustrated non-limitatively by the examples below. All commercially available reagents and solvents (Fluka, Sigma-Aldrich,

Alfa-Aesar) were used without further purification.

Column chromatography was performed with flash silica gel (0.04-0.063 mm, Merck) or LH20 size-exclusion column (Sephadex LH-20)

The following abbreviations are used : - 1 ,2-Dioleyl-sn-Glycero-3-phosphocholine (DOPC) (CAS Registry

Number: 4235-95-4)

- N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium (DOTAL!) (CAS Registry Number: 868226-06-6)

- Thymidine 3'-(1 ,2-dipalmitoyl-sn-glycero-3-phosphate) (also called diC16dT), CAS Registry Number: 1 160002-70-9 and

- Poly(oxy-1 ,2-ethanediyl), a-hydro-co-methoxy-, ester with uridine 5'-(hydrogen butanedioate) 2',3'-di-(9Z)-9-octadecenoate (DOU-PEG) (CAS Registry Number: 1353570-75-8) Figure 1 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (figure 1A) and the zeta potential distribution (figure 1 B) of DOTAL! nanoparticles (control).

Figure 2 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (figure 2A) and the Zeta potential distribution (figure 2B) of nanoparticles of diC16dT (control).

Figure 3 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (figure 3A) and the Zeta potential distribution (figure 3B) of a lipid-based (DOTAL!) nanocarrier composition containing a-tocopherol. Figure 4 shows the size distribution by intensity measured by Dynamic

Light Scattering (DLS) (figure 4A) and the Zeta potential distribution (figure 4B) of a lipid-based (diC16dT, DOPC (co-lipid) and DOU-PEG2000) nanocarrier composition containing prostacycline (PGI2.Na).

Figure 5 shows a SDS Page gel experiment of a lipid-based (DOTAL!) nanocarrier composition containing Apolipoprotein-A1 (APOA1 ).

Figure 6 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (figure 6A) and the Zeta potential distribution (figure 6B) of a lipid-based (DOTAL!) nanocarrier composition containing Apolipoprotein-A1 (APOA1 ). Figure 7 shows the particule size of iron oxide nanoparticles clusters encapsulated by DOTAL! or diC16dT (preparations 1 and 2) or of a DOTAU- based nanocarrier composition comprising iron oxide nanoparticles and a-tocopherol (example 1 ) determined with a Zetasizer 3000 HAS MALVERN.

Figure 8 shows the detection standard curve for DOTAL! (figure 8A) and α-tocopherol (figure 8B).

Figure 9 shows the HPLC analysis of iron oxide nanoparticles clusters encapsulated by DOTAL! prepared in preparation 1 and of a DOTAU-based nanocarrier composition comprising iron oxide nanoparticles and a-tocopherol prepared in example 1 .

Figure 10 shows a stability comparison of iron oxide nanoparticles clusters encapsulated by a lipid (DOPC) or by a nucleolipid (DOTAU). Figure 1 1 shows the Magnetic Resonance Relaxometry study of preparations of cationic or anionic metal nanoparticles.

Figure 12 shows the platelet anti-aggregating activity of the nanocarrier composition of example 2.

Preparation 1 : Encapsulation of iron oxide nanoparticles clusters by

DOTAU (control)

100 μΙ_ of stock solution of positively charged nucleolipid (DOTAU), (50 mg/mL in ether) and 20 μΙ_ of stock solution of iron oxide nanoparticles (10 mg/mL in ether) were mixed. DOTAU was prepared according to P. Chabaud et al., Bioconjugate Chem., 2006, 17, 466-472. The organic phase was added dropwise into the aqueous phase (2 mL of Milli-Q Water) placed in glass tube under stirring by vortex. Then the mixture was placed in glass flask.

Ether was removed under vacuum and the resulting crude material solution was sonicated for 3x15 min time and purified on LS columns to give pure solution of nanoparticles.

The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d=80 nm) and the Zeta potential distribution measured with a MalvernNanoZS device (Zeta potential = +55 mV) are shown on figures 1A and 1 B. Preparation 2: Encapsulation of iron oxide nanoparticles clusters by diC16dT (control)

75 μΙ_ of stock solution of negatively charged nucleolipid (diC16dT) (50 img/mL in chlorofornn), 25 μΙ_ of stock solution of 1 ,2-Dioleoyl-sn-Glycero-3- Phosphocholine (DOPC) (Avanti Polar lipids, 50 img/mL in chlorofornn), 30 μΙ_ of stock solution of neutral nucleolipid (DOU-PEG2000) (10 mg/mL in chlorofornn) and 20 μΙ_ of stock solution of iron oxide nanoparticles (10 mg/mL in chloroform) were mixed. DIC16dT was prepared as disclosed in WO2010/13676. DOU-PEG2000 was prepared according to K.Oumzil et al., 201 1 , J. Control. Release, doi:10.1016/j.jconrel.201 1 .02.008.

The organic phase was added dropwise into the aqueous phase (2 ml of Milli-Q Water) placed in glass tube under stirring by vortex. Then the mixture was placed in glass flask.

Chloroform was removed under vacuum and the resulting crude material solution was sonicated for 3x15 min and purified on LS columns to give pure solution of nanoparticles.

The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d= 92 nm) and the Zeta potential distribution measured with a Malvern NanoZS device (Zeta potential = -23.6 mV) are shown on figures 2A and 2B.

Example 1 : Preparation of a lipid-based (DOTAL!) nanocarrier composition containing iron oxide nanoparticles and a-tocopherol

100 μί of stock solution of positively charged nucleolipid (DOTAL!) (50 mg/mL in ether), 10 μί of stock solution of a-tocopherol (Sigma Aldrich, 50 mg/mL in ether) and 20 μί of stock solution of iron oxide nanoparticles (10 mg/mL in ether) were mixed. The organic phase was added dropwise into the aqueous phase (2 mL of Milli-Q Water) placed in glass tube under stirring by vortex. Then the mixture was placed in glass flask. Ether was removed under vacuum and the resulting crude material solution was sonicated for 3x15 min and purified on LS columns to give pure solution of nanoparticles.

The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d= 108 nm) and the Zeta potential distribution measured with a Malvern NanoZS device (Zeta potential = +49,2 mV) are shown on figures 3A and 3B.

Example 2: Preparation of a lipid-based (diC16dT, DOPC and DOU-PEG2000) nanocarrier composition containing iron oxide nanoparticles and prostacycline (PGI2.Na)

75 μΙ_ of stock solution of negatively charged nucleolipid (diC16dT), (50 mg/mL in chloroform + 2% Et₃N), 25 μί of stock solution of DOPC as co- lipid (50 mg/mL in chloroform + 2% Et₃N), 30 μί of stock solution of neutral nucleolipid (DOU-PEG2000) (10 mg/mL in chloroform + 2% Et₃N), 1 mg of PGI2.Na (Sigma Aldrich) and 20 μί of stock solution of iron oxide nanoparticles (10 mg/mL in chloroform + 2% Et₃N) were mixed. The organic phase was added dropwise into the aqueous phase (2 ml of carbonate-bicarbonate buffer, pH 9.6 at 25 C) placed in glass tube under stirring by vortex. Then the mixture was placed in glass flask.

Chloroform was removed under vacuum and the resulting crude material solution was sonicated for 3x15 min and purified on LS column to give pure solution of nanoparticles.

The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d= 154 nm) and the Zeta potential distribution measured with a Malvern NanoZS device (Zeta potential = +-22.6 mV) are shown on figures 4A and 4B. Example 3: Preparation of a lipid-based (DOTAL!) nanocarrier composition containing Apolipoprotein-A1 (APOA1 )

100 μΙ_ of stock solution of positively charged nucleolipid (DOTAL!) (50 mg/mL in ether) and 20 μί of stock solution of iron oxide nanoparticles (10 mg/mL in ether) were mixed. The organic phase was added dropwise into the aqueous phase (100 pg of APOA1 (Sigma Aldrich) in 2 ml of Milli-Q Water) placed in glass tube under stirring by vortex. Then the mixture was placed in glass flask. Ether was removed under vacuum and the resulting crude material solution was sonicated for 3x15 min and purified on magnetic LS columns to give pure solution of nanoparticles. To demonstrate the presence of the APOA1 protein in the nanoparticles a SDS Page gel experiment was realised. The gel shows that the protein is present in the nanoparticles (two experiments, Figure 5).

The size distribution by number measured by Dynamic Light Scattering (DLS) measured with a Malvern NanoZS device of APOA1 (d= 5.01 nm) and of the lipid-based (DOTAL!) nanocarrier composition containing APOA1 (d= 105 nm) and the size distribution by number measured by intensity are shown on figures 6A and 6B.

Example 4: Stability study

Iron oxide nanoparticles clusters encapsulated by DOTAL! or diC16dT (preparations 1 and 2) and DOTAU-based nanocarrier composition comprising iron oxide nanoparticles and a-tocopherol (example 1 ) in 500 μί of Milli-Q water were incubated at 37°C under a 500rpm stirring. For different times (0, 1 , 3, 6, 24 , 48h), particle sizes were determined using a Zetasizer 3000 HAS MALVERN.

The results are shown on figure 7.

The results show that the overall sizes, either in absence or presence of therapeutic agents, and either positively or negatively charged, are not modified as a function of time (more than 2 days), which indicates colloidal stability both at room temperature and at 37°C.

Example 5: Preparation of samples for HPLC analysis and dosage of DOTAU and a-tocopherol

Pure suspensions of cationic nanoparticles prepared in preparation 1 and example 1 were centrifuged at 14000 rpm for 15 min in order to remove the supernatant. Cationic nanoparticles (in the form of pellet) were suspended in ethanol. The resulting solution was mixed for 15 min at RT and centrifuged at 14000 rpm for 5 min. Then the supernatant (ethanol) was analyzed by HPLC.

A reverse phase U-HPLC method was developed for nucleolipid (DOTAU) and α-tocopherol quantification from the lipid-based nanocarrier composition containing iron oxide nanoparticles. This method allows the separation of the DOTAU and lipid-based (DOTAU) nanocarrier composition within 5 min.

The separation was carried out with a column Syncronis C18 50x2.1 mm, 1 .7 μιτι with a mobile phase composed of MeOH + 0.1 % HCOOH. The flow rate was set to 0.2 mL/min. The detection was performed at 293 nm and 260 nm for α-tocopherol and DOTAU, respectively. The injected volume was 1 .0 μί, which allows the detection of DOTAU and α-tocopherol at thresholds of 5 ng and 15 ng, respectively.

Standard curves for DOTAU and α-tocopherol, as shown on figures 8A and 8B, were generated by determining the intensity of signal versus concentrations. The HPLC analysis is shown on figures 9A (260 nm) and 9B (293 nm).

Quantification of both DOTAU and α-tocopherol was then possible, which led to encapsulated recovery and determination of loading ratio values. Loading ratio was 38% that obtained in the case of a DOTAU/ a-tocopherol (example 1 ) and the encapsulated drug recovery was around 10%, as shown in Table 1 below.

Table 1

This method has the advantage of using a low amount for analysis and a short analysis time, and has compatibility with mass spectrometry, which can be useful for biological analysis.

Example 6 : stability of a suspension of iron oxide nanoparticles clusters encapsulated by a lipid (DOPC) (control) in comparison to a nucleolipid (DOTAU)

In order to show the role of nucleolipids of formula (I) in the stabilisation of encapsulated metal nanoparticles, a control experiment was achieved with DiOleylPhosphatidylCholine (DOPC) only, in the absence of nucleolipid.

75 μΙ_ of stock solution of (DOPC) (Avanti Polar lipids, 50 mg/mL in chloroform) and 20 μΙ_ of stock solution of iron oxide nanoparticles (10 mg/mL in chloroform) were mixed. The organic phase was added dropwise into the aqueous phase (2 ml of Milli-Q Water) placed in glass tube under stirring by vortex. Then the mixture was placed in a glass flask. Chloroform was removed under vacuum and the resulting crude material solution was sonicated 3 times (3 x 15 minutes) to give a precipitate.

The same protocol was followed, except that the nucleolipid N-[5'- (2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium (DOTAL!) was used instead of DOPC. The sonication did not give a precipitate. The photographs of figure 10 show that the nucleolipid DOTAL! allows the formation of a stable colloidal suspension (photograph A), whereas in similar conditions with the lipid DOPC a precipitate is formed, which evidences that DOPC does not provide this colloidal stabilization (photograph B).

Example 7 : Magnetic Resonance relaxometry study

A total number of 8 different concentrations ranging from 0 to 0.5 mM Fe of iron oxide nanoparticles clusters encapsulated by DOTAL! or diC16dT (preparations 1 and 2) were prepared in Eppendorf PCR Tubes (0.5 mL).

Transverse images passing through the 8 tubes were acquired on a 4.7 T Bruker Biospin (Billerica, MA) magnetic resonance imaging (MRI) system with a 1 H whole body radiofrequency (RF) volume coil of 35 mm inner diameter and the relaxation rate (R_n) maps were computed using the Paravision 6.0 software. Samples were scanned at 21 °C with a 256 x 192 matrix and a field of view (FOV) = 40 x 30 mm. Ri measurements were acquired using the Bruker Ti map Rapid Acquisition with Relaxation Enhancement (RARE) method (Repetition time (TR) = 5000, 3000, 1500, 800, 400, 200 ms; Echo time (TE) = 6 ms; RARE factor = 2).

Multi-spin-echo (ΔΤΕ = 8.45; number of echoes = 20; TR = 2 s) and gradient-echo (flip angle = 60°; number of echo = 8; TE initial = 3.5 ms; ΔΤΕ = 5 ms; TR = 800 ms; flyback) sequences were employed to compute an R2 map and R2^*map, respectively. The mean relaxation rates, Rn, of each dilution were calculated from regions of interest (ROIs) encompassing each tube and plotted versus their corresponding Fe concentrations. A linear regression was used to extract the relaxivity (r_n) of each sample, given as the slope of the resulting line in units of s^"1 mM^"1 of Fe, as shown on figure 1 1 .

The results show that the iron oxide nanoparticles clusters encapsulated by DOTAU (represented by the line with x signs) or diC16dT (represented by the line with · signs), have improved magnetic properties in comparison to contrast agents commonly used in the medical field, such as Feridex ® (dotted line). Feridex particles ® are described in D. Patel et al., Biomaterials, 201 1 , 32, 1 167-1 176.

Accordingly, the iron oxide nanoparticles clusters encapsulated by either cationic or anionic nucleolipids of formula (I) (preparations 1 and 2), which are part of the lipid based-nanocarrier composition of the invention, allow an improved MRI detection, due to an increased sensitivity for the imaging system.

These results were published in K. Oumzil et al., Bioconjugate Chem., 2016, 27, 569-575.

Example 8 : Analysis of Bioactivity of Encapsulated API.

Blood was obtained in 1/10th volume of 3.8% sodium citrate from healthy volunteers who had not taken any drugs known to affect platelet function for 2 weeks prior to the study. Platelet rich plasma (PRP) was prepared by centrifugation at 20 °C for 10-15 min at 150-200g and stored at room temperature. Platelet-poor plasma (PPP) was prepared by further centrifugation of the remaining plasma at 2700g for 15 min and was used to calibrate the 100% light transmission of the aggregometer.

The percentage of aggregation was followed versus time. PRP (207 μΙ_) was stirred in cuvette at 37 °C and agonists adenosine 5'-diphosphate (ADP) or thrombin receptor-activating peptide -6 (TRAP-6) were added at 5 s to promote platelet aggregation, and was incubated either with the DOU- PEG2000-based nanocarrier composition containing iron oxide nanoparticles and prostacyclin (PGI2) of example 2, or with the iron oxide nanoparticles clusters encapsulated by diC16dT of preparation 2 (used as negative control) or with the agonist(s) only (negative control) or with the agonist(s) and PGI2 (positive control) for 15 min and 3h for evaluating PGI2 activity. The in vitro platelet aggregation was determined using a four-channel light transmission aggregometer (APACT 4004, ELITech, France).

The results are shown on figure 12, where the curves numbered 1 to 7 represent the following experimental conditions:

Curve No.1 : PRP + TRAP-6

Curve No.2 : PRP + ADP + preparation 2

Curve No.3 : PRP + ADP

Curve No.4 : PRP + TRAP-6 + example 2 (3h incubation) Curve No.5 : PRP + TRAP-6 + PGI2 Curve No.6 : PRP + ADP + PGI2

Curve No.7 : PRP + ADP + example 2 (15 min incubation)

The results show that the DOU-PEG2000-based nanocarrier composition containing iron oxide nanoparticles and PGI2 of example 2 as well as free PGI2 totally inhibit the platelet aggregation induced by both ADP (15 min incubation) and TRAP-6 (3 h incubation) agonist, whereas the iron oxide nanopartides dusters encapsulated by diC16dT of preparation 2, used as negative control (curve No.2), show complete ADP aggregation.

Claims

in which

X is an oxygen atom, a sulfur atom or a methylene group,

- or also, and L_2, together, form a ketal roup of formula

- or also l_i or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched C2-C3o alkyl chain_,

- Ri and R₂, identical or different, represent

- a linear or branched C2-C30 hydrocarbon chain, saturated or partially unsaturated, or - a C₂-C₃o acyl chain,

- a diacyl chain in which each acyl chain is C2-C30,

- a diacylglycerol in which each acyl chain is C2-C30, or

- a sphingosine or ceramide group in which each acyl chain is C2-C30, or - when Li or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, Ri and R₂ do not exist;

- R3 represents

- a hydroxy, amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate, phosphonium, NH₂-R₄, NHR₄R₅ or NR R₅R₆ group in which R , R₅ and R6, identical or different, represent a hydrogen atom or a linear or branched C1-C6 alkyl chain or linear or branched C1-C6 hydroxyalkyl, or

- a linear or branched C₂-C₃o alkyl chain, optionally substituted by a hydroxy group, or

- a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a C2-C30 alkyl, optionally substituted by a hydroxy group; or by a (CH₂)m-O-(CH₂)p-R9 group in which m = 1 to 6 and p = 0 to 20 and R₉ represents hydrogen or a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched C₂-C3o alkyl, or by a sterol radical,

or

- R3 is bound by a covalent bond to another substituent R₃, identical or different, of another compound of formula (I), identical or different, in order to form a compound of formula (I) in the form of a dimer,

b) at least one metal nanoparticle, and

c) at least one therapeutic agent.

2. A lipid-based nanocarrier composition according to claim 1 , comprising a) at least one compound of formula (I)

in which

X is an oxygen atom, a sulfur atom or a methylene group,

Li and L₂, identical or different, represent hydrogen, an oxycarbonyl -O-C(O)- group, a thiocarbamate -O-C(S)-NH- group, a carbonate -O-C(O)-O- group, a carbamate -O-C(O)-NH- group, an oxygen atom, a phosphate group, a phosphonate group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched, saturated or unsaturated C8-C30 hydrocarbon chain, wherein l_i and L₂ are not simultaneously hydrogen, or also, l_i and L_2, together, form a ketal roup of formula

- or also l_i or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, unsubstituted or substituted by a linear or branched C8-C30, alkyl chain_,

- Ri and R₂, identical or different, represent

- a linear or branched C8-C30 hydrocarbon chain, saturated or partially unsaturated, or

- a C₈-C₃o acyl chain,

- a diacyl chain in which each acyl chain is C8-C30,

- a diacylglycerol in which each acyl chain is C8-C30, or

- a sphingosine or ceramide group in which each acyl chain is C8-C30, or - when l_i or L₂ represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, Ri and R₂ do not exist;

- F¾ represents

- a hydroxy, amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate, phosphonium, NH₂-R₄, NHR-^Rs or NR₄R₅R₆ group in which R₄, R₅ and R₆, identical or different, represent a hydrogen atom or a linear or branched C1-C6 alkyl chain or linear or branched C1-C6 hydroxyalkyl, or

- a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a C2-C30 alkyl, optionally substituted by a hydroxy group; or by a (CH₂)m-O-(CH₂)p-R9 group in which m = 1 to 6 and p = 0 to 20 and Rg represents hydrogen or a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched C2-C30 alkyl, or by a sterol radical,

or

b) at least one metal nanoparticle, and

c) at least one therapeutic agent.

3. A lipid-based nanocarrier composition according to claim 1 or 2, comprising at least one compound of formula (I) in which at least one condition is fulfilled:

- X is an oxygen atom;

- B is thymine or uracile; - L-Ϊ and L₂ are oxycarbonyl -O-C(O)- groups which are substituted by a linear or branched C2-C30, preferably C8-C30, hydrocarbon chain, saturated or partially unsaturated;

- Li is a phosphate group which is substituted by diacylglycerol in which each acyl group is C2-C30, preferably C8-C30, and L₂ is hydrogen;

- R3 is hydroxy, a NR R₅R₆ group in which R , R₅ and R6 represent a hydrogen atom or a -O— C(O)-(CH₂)_q-C(O)-O [(CH₂)₂-O]_rH group in which q is 2 to 6 and r is an integer from 4 to 30, preferably from 10 to 20.

4. A lipid-based nanocarrier composition according to any one of claims 1 , 2 or 3, wherein in formula (I), in the definitions of l_i , L₂, Ri R₂ or R₃, the linear or branched alkyl chain is Cs-C₂6, preferably Ci6-C₂o; and/or the linear or branched C₂-C3o hydrocarbon chain is Cs-C₂6, preferably Ci6-C₂o; and/or the C₂-C3o acyl chain is Cs-C₂6, preferably Ci6-C₂o.

5. A lipid-based nanocarrier composition according to any one of claims 1 to

4, comprising at least one compound of formula (I), selected from

- N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium

- Thymidine 3'-(1 ,2-dipalmitoyl-sn-glycero-3-phosphate), and

- Poly(oxy-1 ,2-ethanediyl), a-hydro-co-methoxy-, ester with uridine 5'-(hydrogen butanedioate) 2',3'-di-(9Z)-9-octadecenoate.

6. A lipid-based nanocarrier composition according to any one of claims 1 to

5, which comprises at least 2 different compounds of formula (I).

7. A lipid-based nanocarrier composition according to any one of claims 1 to

6, which comprises a plurality of metal nanoparticles.

8. A lipid-based nanocarrier composition according to any one of claims 1 to 7, in which the metal nanopartide contains metal oxides, metals, metal alloys or metal chalcogenides.

9. A lipid-based nanocarrier composition according to any one of claims 1 to 8, in which the metal nanopartide contains iron oxide.

10. A lipid-based nanocarrier composition according to any one of claims 1 to

9, in which the metal nanoparticles have an overall size of 2nm to 20nm.

1 1 . A lipid-based nanocarrier composition according to any one of claims 1 to

10, in which the population(s) of metal nanoparticles is/are either monodisperse, or monodisperse and/or polydisperse, or else polydisperse.

12. A lipid-based nanocarrier composition according to any one of claims 1 to

1 1 , in which the therapeutic agent is selected from anti-tumoral agents, antibiotic agents, anti-microbial agents, analgesic agents, anti-histaminic agents, bronchodilators agents, agents which are active on the central nervous system, anti-hypertension agents or agents which are active on the cardiovascular system, anti-atherosclerosis agents, nucleic acids and their fragments; peptides, oligopeptides, proteins, antigens, antibodies or stem cells.

13. A lipid-based nanocarrier composition according to any one of claims 1 to

12, in which the therapeutic agent is selected from anti-tumoral agents and anti-atherosclerosis agents.

14. A process for preparing a lipid-based nanocarrier composition comprising metal nanoparticles according to any one of claims 1 to 13, which comprises the following steps:

- preparing a solution containing at least one compound of formula (I), at least one metal nanopartide and at least one therapeutic agent in an organic solvent

- adding this solution to an aqueous medium while stirring,

- sonicating the resulting aqueous solution and evaporating the organic solvent, and - recovering the lipid-based nanocarrier composition thus obtained.

15. A lipid-based nanocarrier composition according to any one of claims 1 to 13 for use as an agent for transportation, vectorization, cellular delivery, cellular targeting or cellular localization of at least one therapeutic agent.

16. Pharmaceutical compositions containing a lipid-based nanocarrier composition according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.