Lipid based nanocarrier compositions loaded with metal nanoparticles and therapeutic agent

Abstract

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

Claims

1. A lipid-based nanocarrier composition comprising a) at least one compound of formula (I) ##STR00005## 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; L.sub.1 and L.sub.2, identical or different, represent hydrogen, an oxycarbonyl OC(O) group, a thiocarbamate OC(S)NH group, a carbonate OC(O)O group, a carbamate OC(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 C.sub.2-C.sub.30 hydrocarbon chain, or also, L.sub.1 and L.sub.2, together, form a ketal group of formula ##STR00006## or also L.sub.1 or L.sub.2 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 C.sub.2-C.sub.30 alkyl chain, R.sub.1 and R.sub.2, identical or different, represent a linear or branched C.sub.2-C.sub.30 hydrocarbon chain, saturated or partially unsaturated, or a C.sub.2-C.sub.30 acyl chain, a diacyl chain in which each acyl chain is C.sub.2-C.sub.30, a diacylglycerol in which each acyl chain is C.sub.2-C.sub.30, or a sphingosine or ceramide group in which each acyl chain is C.sub.2-C.sub.30, or when L.sub.1 or L.sub.2 represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, R.sub.1 and R.sub.2 do not exist; R.sub.3 represents a hydroxy, amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate, phosphonium, NH.sub.2R.sub.4, NHR.sub.4R.sub.5 or NR.sub.4R.sub.5R.sub.6 group in which R.sub.4, R.sub.5 and R.sub.6, identical or different, represent a hydrogen atom or a linear or branched C.sub.1-C.sub.6 alkyl chain or linear or branched C.sub.1-C.sub.6 hydroxyalkyl, or a linear or branched C.sub.2-C.sub.30 alkyl chain, optionally substituted by a hydroxy group, or a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a C.sub.2-C.sub.30 alkyl, optionally substituted by a hydroxy group; or by a (CH.sub.2).sub.mO(CH.sub.2).sub.pR.sub.9 group in which m=1 to 6 and p=0 to 20 and R.sub.9 represents hydrogen or a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched C.sub.2-C.sub.30 alkyl, or by a sterol radical, or a OC(O)(CH.sub.2).sub.qC(O)O [(CH.sub.2).sub.2O].sub.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 R.sub.3 is bound by a covalent bond to another substituent R.sub.3, 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) ##STR00007## 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; -L.sub.1 and L.sub.2, identical or different, represent hydrogen, an oxycarbonyl OC(O) group, a thiocarbamate OC(S)NH group, a carbonate OC(O)O group, a carbamate OC(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 C.sub.8-C.sub.30 hydrocarbon chain, wherein L.sub.1 and L.sub.2 are not simultaneously hydrogen, or also, L.sub.1 and L.sub.2, together, form a ketal group of formula ##STR00008## or also L.sub.1 or L.sub.2 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 C.sub.8-C.sub.30, alkyl chain, R.sub.1 and R.sub.2, identical or different, represent a linear or branched C.sub.8-C.sub.30 hydrocarbon chain, saturated or partially unsaturated, or a C.sub.8-C.sub.30 acyl chain, a diacyl chain in which each acyl chain is C.sub.8-C.sub.30, a diacylglycerol in which each acyl chain is C.sub.8-C.sub.30, or a sphingosine or ceramide group in which each acyl chain is C.sub.8-C.sub.30, or when L.sub.1 or L.sub.2 represents hydrogen, and the other represents a hydroxy group or a heteroaryl group comprising 1 to 4 nitrogen atoms, R.sub.1 and R.sub.2 do not exist; R.sub.3 represents a hydroxy, amino, phosphate, phosphonate, phosphatidylcholine, O-alkyl phosphatidylcholine, thiophosphate, phosphonium, NH.sub.2R.sub.4, NHR.sub.4R.sub.5 or NR.sub.4R.sub.5R.sub.6 group in which R.sub.4, R.sub.5 and R.sub.6, identical or different, represent a hydrogen atom or a linear or branched C.sub.1-C.sub.6 alkyl chain or linear or branched C.sub.1-C.sub.6 hydroxyalkyl, or a linear or branched C.sub.2-C.sub.30 alkyl chain, optionally substituted by a hydroxy group, or a heteroaryl group containing 1 to 4 nitrogen atoms, unsubstituted or substituted by a C.sub.2-C.sub.30 alkyl, optionally substituted by a hydroxy group; or by a (CH.sub.2).sub.mO(CH.sub.2).sub.pR.sub.9 group in which m=1 to 6 and p=0 to 20 and R.sub.9 represents hydrogen or a cyclic ketal group containing 5 to 7 carbon atoms, unsubstituted or substituted by at least one linear or branched C.sub.2-C.sub.30 alkyl, or by a sterol radical, or a OC(O)(CH.sub.2).sub.qC(O)O[(CH.sub.2).sub.2O].sub.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 R.sub.3 is bound by a covalent bond to another substituent R.sub.3, 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.

3. A lipid-based nanocarrier composition according to claim 1, 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.sub.1 and L.sub.2 are oxycarbonyl OC(O) groups which are substituted by a linear or branched C.sub.2-C.sub.30, preferably C.sub.8-C.sub.30, hydrocarbon chain, saturated or partially unsaturated; L.sub.1 is a phosphate group which is substituted by diacylglycerol in which each acyl group is C.sub.2-C.sub.30, preferably C.sub.8-C.sub.30, and L.sub.2 is hydrogen; R.sub.3 is hydroxy, a NR.sub.4R.sub.5R.sub.6 group in which R.sub.4, R.sub.5 and R.sub.6 represent a hydrogen atom or a OC(O)(CH.sub.2).sub.qC(O)O[(CH.sub.2).sub.2O].sub.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 claim 1, wherein in formula (I), in the definitions of L.sub.1, L.sub.2, R.sub.1 R.sub.2 or R.sub.3, the linear or branched alkyl chain is C.sub.8-C.sub.26, preferably C.sub.16-C.sub.20; and/or the linear or branched C.sub.2-C.sub.30 hydrocarbon chain is C.sub.8-C.sub.26, preferably C.sub.16-C.sub.20; and/or the C.sub.2-C.sub.30 acyl chain is C.sub.8-C.sub.26, preferably C.sub.16-C.sub.20.

5. A lipid-based nanocarrier composition according to claim 1, comprising at least one compound of formula (I), selected from N-[5-(2,3-dioleoy)uridine]N,N,N-trimethylammonium Thymidine 3-(1,2-dipalmitoyl-sn-glycero-3-phosphate), and Poly(oxy-1,2-ethanediyl), -hydro--methoxy-, ester with uridine 5-(hydrogen butanedioate) 2,3-di-(9Z)-9-octadecenoate.

6. A lipid-based nanocarrier composition according to claim 1, which comprises at least 2 different compounds of formula (I).

7. A lipid-based nanocarrier composition according to claim 1, which comprises a plurality of metal nanoparticles.

8. A lipid-based nanocarrier composition according to claim 1, in which the metal nanoparticle contains metal oxides, metals, metal alloys or metal chalcogenides.

9. A lipid-based nanocarrier composition according to claim 1, in which the metal nanoparticle contains iron oxide.

10. A lipid-based nanocarrier composition according to claim 1, in which the metal nanoparticles have an overall size of 2 nm to 20 nm.

11. A lipid-based nanocarrier composition according to claim 1, 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 claim 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 claim 1, 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 claim 1, 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.

15. A lipid-based nanocarrier composition according to claim 1, 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 claim 1 and a pharmaceutically acceptable carrier.

Description

[0129] FIG. 1 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (FIG. 1A) and the zeta potential distribution (FIG. 1B) of DOTAU nanoparticles (control).

[0130] FIG. 2 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (FIG. 2A) and the Zeta potential distribution (FIG. 2B) of nanoparticles of diC16dT (control).

[0131] FIG. 3 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (FIG. 3A) and the Zeta potential distribution (FIG. 3B) of a lipid-based (DOTAU) nanocarrier composition containing -tocopherol.

[0132] FIG. 4 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (FIG. 4A) and the Zeta potential distribution (FIG. 4B) of a lipid-based (diC16dT, DOPC (co-lipid) and DOU-PEG2000) nanocarrier composition containing prostacycline (PGl2.Na).

[0133] FIG. 5 shows a SDS Page gel experiment of a lipid-based (DOTAU) nanocarrier composition containing Apolipoprotein-A1 (APOA1).

[0134] FIG. 6 shows the size distribution by intensity measured by Dynamic Light Scattering (DLS) (FIG. 6A) and the Zeta potential distribution (FIG. 6B) of a lipid-based (DOTAU) nanocarrier composition containing Apolipoprotein-A1 (APOA1).

[0135] FIG. 7 shows the particule size of iron oxide nanoparticles clusters encapsulated by DOTAU or diC16dT (preparations 1 and 2) or of a DOTAU-based nanocarrier composition comprising iron oxide nanoparticles and -tocopherol (example 1) determined with a Zetasizer 3000 HAS MALVERN.

[0136] FIG. 8 shows the detection standard curve for DOTAU (FIG. 8A) and -tocopherol (FIG. 8B).

[0137] FIG. 9 shows the HPLC analysis of iron oxide nanoparticles clusters encapsulated by DOTAU prepared in preparation 1 and of a DOTAU-based nanocarrier composition comprising iron oxide nanoparticles and -tocopherol prepared in example 1.

[0138] FIG. 10 shows a stability comparison of iron oxide nanoparticles clusters encapsulated by a lipid (DOPC) or by a nucleolipid (DOTAU).

[0139] FIG. 11 shows the Magnetic Resonance Relaxometry study of preparations of cationic or anionic metal nanoparticles.

[0140] FIG. 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)

[0141] 100 L of stock solution of positively charged nucleolipid (DOTAU), (50 mg/mL in ether) and 20 L 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.

[0142] Ether was removed under vacuum and the resulting crude material solution was sonicated for 315 min time and purified on LS columns to give pure solution of nanoparticles.

[0143] 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 FIGS. 1A and 1B.

[0144] PREPARATION 2: ENCAPSULATION OF IRON OXIDE NANOPARTICLES CLUSTERS BY diC16dT (CONTROL)

[0145] 75 L of stock solution of negatively charged nucleolipid (diC16dT) (50 mg/mL in chloroform), 25 L of stock solution of 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC) (Avanti Polar lipids, 50 mg/mL in chloroform), 30 L of stock solution of neutral nucleolipid (DOU-PEG2000) (10 mg/mL in chloroform) and 20 L 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., 2011, J. Control. Release, doi:10.1016/j. j conrel.2011.02.008.

[0146] 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.

[0147] Chloroform was removed under vacuum and the resulting crude material solution was sonicated for 315 min and purified on LS columns to give pure solution of nanoparticles.

[0148] The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d=92 nm) and the Zeta potential distribution measured with a MalvernNanoZS device (Zeta potential=23.6 mV) are shown on FIGS. 2A and 2B.

EXAMPLE 1

Preparation of a Lipid-Based (DOTAU) Nanocarrier Composition Containing Iron Oxide Nanoparticles and -Tocopherol

[0149] 100 L of stock solution of positively charged nucleolipid (DOTAU) (50 mg/mL in ether), 10 L of stock solution of -tocopherol (Sigma Aldrich, 50 mg/mL in ether) and 20 L 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.

[0150] Ether was removed under vacuum and the resulting crude material solution was sonicated for 315 min and purified on LS columns to give pure solution of nanoparticles.

[0151] The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d=108 nm) and the Zeta potential distribution measured with a MalvernNanoZS device (Zeta potential=+49,2 mV) are shown on FIGS. 3A and 3B.

EXAMPLE 2

Preparation of a Lipid-Based (diC16dT, DOPC and DOU-PEG2000) Nanocarrier Composition Containing Iron Oxide Nanoparticles and Prostacycline (PGl2.Na)

[0152] 75 L of stock solution of negatively charged nucleolipid (diC16dT), (50 mg/mL in chloroform+2% Et.sub.3N), 25 L of stock solution of DOPC as co-lipid (50 mg/mL in chloroform+2% Et.sub.3N), 30 L of stock solution of neutral nucleolipid (DOU-PEG2000) (10 mg/mL in chloroform+2% Et.sub.3N), 1 mg of PGI2.Na (Sigma Aldrich) and 20 L of stock solution of iron oxide nanoparticles (10 mg/mL in chloroform+2% Et.sub.3N) 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.

[0153] Chloroform was removed under vacuum and the resulting crude material solution was sonicated for 315 min and purified on LS column to give pure solution of nanoparticles.

[0154] The size distribution by intensity measured by Dynamic Light Scattering (DLS) (d=154 nm) and the Zeta potential distribution measured with a MalvernNanoZS device (Zeta potential=+22.6 my) are shown on FIGS. 4A and 4B.

EXAMPLE 3

Preparation of a Lipid-Based (DOTAU) Nanocarrier Composition Containing Apolipoprotein-A1 (APOA1)

[0155] 100 L of stock solution of positively charged nucleolipid (DOTAU) (50 mg/mL in ether) and 20 L 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 g 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 315 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, FIG. 5).

[0156] The size distribution by number measured by Dynamic Light Scattering (DLS) measured with a MalvernNanoZS device of APOA1 (d=5.01 nm) and of the lipid-based (DOTAU) nanocarrier composition containing APOA1 (d=105 nm) and the size distribution by number measured by intensity are shown on FIGS. 6A and 6B.

EXAMPLE 4

Stability Study

[0157] Iron oxide nanoparticles clusters encapsulated by DOTAU or diC16dT (preparations 1 and 2) and DOTAU-based nanocarrier composition comprising iron oxide nanoparticles and -tocopherol (example 1) in 500 L of Milli-Q water were incubated at 37 C. under a 506 rpm stirring. For different times (0, 1, 3, 6, 24, 48 h), particle sizes were determined using a Zetasizer 3000 HAS MALVERN.

[0158] The results are shown on FIG. 7.

[0159] 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 -Tocopherol

[0160] 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.

[0161] A reverse phase U-HPLC method was developed for nucleolipid (DOTAU) and -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.

[0162] The separation was carried out with a column Syncronis C18 502.1 mm, 1.7 m 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 -tocopherol and DOTAU, respectively. The injected volume was 1.0 L, which allows the detection of DOTAU and -tocopherol at thresholds of 5 ng and 15 ng, respectively.

[0163] Standard curves for DOTAU and -tocopherol, as shown on FIGS. 8A and 8B, were generated by determining the intensity of signal versus concentrations.

[0164] The HPLC analysis is shown on FIGS. 9A (260 nm) and 9B (293 nm).

[0165] Quantification of both DOTAU and -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/-tocopherol (example 1) and the encapsulated drug recovery was around 10%, as shown in Table 1 below.

TABLE-US-00001 TABLE 1 Mass in the Mass in supernatant the pellet Loading Recovery Sample No Molecule (mg) (mg) ratio ratio 1 DOTAU 2.80 0.12 5 mg 2 DOTAU 4.02 0.12 30% 10% 5 mg alpha- 0.52 0.05 tocopherol 0.5 mg 3 DOTAU 3.61 0.11 38% 6% 5 mg alpha- 0.93 0.06 tocopherol 1 mg

[0166] 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)

[0167] 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.

[0168] 75 L of stock solution of (DOPC) (Avanti Polar ipids, 50 mg/mL in chloroform) and 20 L 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 (315 minutes) to give a precipitate.

[0169] The same protocol was followed, except that the nucleolipid N-[5-(2,3-dioleoyl)uridine]-N,N,N-trimethylammonium (DOTAU) was used instead of DOPC. The sonication did not give a precipitate.

[0170] The photographs of FIG. 10 show that the nucleolipid DOTAU allows the formation of a stable collodal 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

[0171] A total number of 8 different concentrations ranging from 0 to 0.5 mM Fe of iron oxide nanoparticles clusters encapsulated by DOTAU or diC16dT (preparations 1 and 2) were prepared in Eppendorf PCR Tubes (0.5 mL).

[0172] Transverse images passing through the 8 tubes were acquired on a 4.7 T Bruker Biospin (Billerica, Mass.) 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.sub.n) maps were computed using the Paravision 6.0 software. Samples were scanned at 21 C. with a 256192 matrix and a field of view (FOV)=4030 mm. R.sub.1 measurements were acquired using the Bruker T.sub.1 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).

[0173] Multi-spin-echo (TE=8.45; number of echoes=20; TR=2 s) and gradient-echo (flip angle=60; number of echo=8; TE initial=3.5 ms; TE=5 ms; TR=800 ms; flyback) sequences were employed to compute an R.sub.2 map and R.sub.2*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.

[0174] A linear regression was used to extract the relaxivity (r.sub.n) of each sample, given as the slope of the resulting line in units of s.sup.1 mM.sup.1 of Fe, as shown on FIG. 11.

[0175] 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, 2011, 32, 1167-1176.

[0176] 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.

[0177] These results were published in K. Oumzil et al., Bioconjugate Chem., 2016, 27, 569-575.

EXAMPLE 8

Analysis of Bioactivity of Encapsulated API.

[0178] 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 150-200 g and stored at room temperature.

[0179] Platelet-poor plasma (PPP) was prepared by further centrifugation of the remaining plasma at 2700 g for 15 min and was used to calibrate the 100% light transmission of the aggregometer.

[0180] The percentage of aggregation was followed versus time. PRP (207 L) 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 3 h for evaluating PGI2 activity. The in vitro platelet aggregation was determined using a four-channel light transmission aggregometer (APACT 4004, ELITech, France).

[0181] The results are shown on FIG. 12, where the curves numbered 1 to 7 represent the following experimental conditions:

PRP+TRAP-6 Curve No. 1

PRP+ADP+preparation 2 Curve No. 2

PRP+ADP Curve No. 3

PRP+TRAP6+example 2 (3 h incubation) Curve No. 4

PRP+TRAP6+PGI2 Curve No.5

PRP+ADP+PGI2 Curve No.6

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

[0182] 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 nanoparticles clusters encapsulated by diC16dT of preparation 2, used as negative control (curve No.2), show complete ADP aggregation.

[0183] These results were published in K. Oumzil et al., Bioconjugate Chem., 2016, 27, 569-575.