Pharmaceutical composition combining at least two distinct nanoparticles and a pharmaceutical compound, preparation and uses thereof

Abstract

The present invention relates to a pharmaceutical composition comprising the combination of (i) at least two distinct biocompatible nanoparticles and (ii) at least one compound of interest, typically at least one pharmaceutical compound, to be administered to a subject in need of such at least one compound of interest, wherein the at least two distinct biocompatible nanoparticles potentiate the compound(s) of interest efficiency. The at least two biocompatible nanoparticles can be administered sequentially or simultaneously to the subject but are to be administered separately, typically with an interval of between more than about 5 minutes and about 72 hours, from the at least one compound of interest, preferably before the administration of the at least one compound of interest, to said subject. The longest dimension of the at least two biocompatible nanoparticles is typically between about 4 nm and about 500 nm. The absolute surface charge value of a first biocompatible nanoparticle is of at least |10 mV| and the absolute surface charge value of the second biocompatible nanoparticle, or of any additional biocompatible nanoparticle, has a difference of at least 10 mV with the absolute surface charge value of the first biocompatible nanoparticle.

Claims

1. A method of treating a subject having a disease comprising a step of administering at least one pharmaceutical compound to a subject in need thereof and a distinct step of administering at least two distinct biocompatible nanoparticles to said subject, wherein the longest dimension of each of the at least two biocompatible nanoparticles is between about 4 nm and about 500 nm, the surface charge value of a first biocompatible nanoparticle is a negative charge below −10 mV, the surface charge value of a second biocompatible nanoparticle, or of any additional biocompatible nanoparticle, is negative and has a difference of at least 10 mV to the negative surface charge value of the first biocompatible nanoparticle, each of the at least two biocompatible nanoparticles is not used as the at least one pharmaceutical compound, and said at least two distinct biocompatible nanoparticles are administered to the subject separately from the at least one pharmaceutical compound between 5 minutes and about 24 hours before said at least one pharmaceutical compound.

2. The method according to claim 1, wherein the at least two distinct biocompatible nanoparticles are administered separately from each other to the subject in need of treatment with said pharmaceutical compound.

3. The method according to claim 1, wherein the at least two distinct nanoparticles are organic nanoparticles.

4. The method according to claim 3, wherein the at least two distinct nanoparticles are selected from lipid-based nanoparticles, protein-based nanoparticles, polymer-based nanoparticles, co-polymer-based nanoparticles, carbon-based nanoparticles, virus-like nanoparticles and any mixture thereof.

5. The method according to claim 1, wherein the at least two distinct nanoparticles are inorganic nanoparticles and the longest dimension of each of said nanoparticles is below about 7 nm.

6. The method according to claim 1, wherein the at least two nanoparticles are inorganic nanoparticles, the longest dimension of each of said nanoparticles is of at least 10 nm, and the inorganic material of each of the nanoparticles is selected from (i) one or more divalent metallic elements, (ii) one or more trivalent metallic elements, and (iii) one or more tetravalent metallic elements comprising Si.

7. The method according to claim 6, wherein the inorganic material is selected from calcium carbonate (CaCO.sub.3), magnesium carbonate (MgCO.sub.3), magnesium hydroxide (Mg(OH).sub.2), iron hydroxide (Fe(OH).sub.2), iron oxyhydroxide (FeOOH), iron(II, III) oxide (Fe.sub.3O.sub.4), iron(III) oxide (Fe.sub.2O.sub.3), aluminium oxide (Al.sub.3O.sub.4), aluminium hydroxide (Al(OH).sub.3), aluminium oxyhydroxide (AlOOH) and silicium oxide (SiO.sub.2).

8. The method according to claim 1, wherein both organic and inorganic nanoparticles are administered to the subject.

9. The method according to claim 1, wherein each of the at least two nanoparticles is further covered with a biocompatible coating.

10. The method according to claim 1, wherein the administration of the at least two biocompatible nanoparticles and of the at least one pharmaceutical compound maintains the therapeutic benefit of said at least one pharmaceutical compound and reduces toxicity, or increases the therapeutic benefit of said at least one pharmaceutical compound for an equivalent or reduced toxicity, for the subject, when compared to therapeutic benefit and toxicity induced by the standard therapeutic dose(s) of said at least one pharmaceutical compound.

11. The method according to claim 1, wherein the administration of the at least two biocompatible nanoparticles and of the at least one pharmaceutical compound allows a reduction of at least 10% of the administered at least one pharmaceutical compound therapeutic dose when compared to the standard therapeutic dose of said at least one pharmaceutical compound while maintaining the same therapeutic benefit for an equivalent toxicity or a reduced toxicity for the subject or while increasing the therapeutic benefit for an equivalent or reduced toxicity for the subject.

12. The method according to claim 1, wherein the at least two nanoparticles are cleared from the subject to whom it has been administered within one hour and six weeks after their administration to said subject.

13. The method according to claim 1, wherein the at least one pharmaceutical compound is an organic compound selected from a biological compound, a small molecule targeted therapeutic, an oncolytic virus and a cytotoxic compound.

14. The method according to claim 13, wherein the at least one pharmaceutical compound is selected from an antibody, an oligonucleotide, and a synthesized peptide.

15. The method according to claim 1, wherein the at least one pharmaceutical compound is an inorganic compound selected from a metallic nanoparticle, a metal oxide nanoparticle, a metal sulfide nanoparticle, and any mixture thereof.

16. The method according to claim 1, wherein the at least one pharmaceutical compound is encapsulated in, or bound to, a carrier.

17. The method according to claim 6, wherein said one or more divalent metallic elements, are selected from Mg, Ca, Ba and Sr and said one or more trivalent metallic elements are selected from Fe and Al.

18. The method according to claim 1, wherein the disease is selected from cardiovascular diseases, Central Nervous System (CNS) diseases, gastrointestinal diseases, genetic disorders, hematological disorders, hormonal disorders, immunology, infectious diseases, metabolic disorders, musculoskeletal disorders, oncology, respiratory diseases and toxicology.

19. The method according to claim 1, wherein the at least two distinct biocompatible nanoparticles are administered simultaneously to the subject in need of treatment with said pharmaceutical compound.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematic view of possible routes for therapeutic compounds removal from blood circulation depending on the compound's size (longest dimension).

(2) FIG. 2: Schematic representation of the treatment schedule for the pharmaceutical composition comprising (i) the at least two biocompatible nanoparticles of example 2 and (ii) the Dox-NP® in MDA-MB-23 1-lucD3H2LN xenografts.

(3) FIG. 3: Chemical formula of L-Glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA-lipid).

EXAMPLES

Example 1: Synthesis of Liposomes as the “First” and/or “Second” Biocompatible Nanoparticles

(4) Liposomes are prepared using the lipid film re-hydration method.

(5) Synthesis of “First” and/or “Second” Biocompatible Nanoparticles with an Absolute Surface Charge Value of at Least 10 mV (|10 mV|):

(6) a) lipids were solubilized in chloroform. Chloroform was finally evaporated under a nitrogen flow. Re-hydration of the lipid film with HEPES 20 mM and NaCl 140 mM at pH 7.4 was performed at 60° C., so that the lipid concentration was 25 mM.

(7) The following lipid composition was used to prepare charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine) 62% mol; HSPC (Hydrogenated Soybean PhosphatidylCholine) 20% mol; CHOL (Cholesterol) 16% mol; POPS (1-Palmitoyl-2-Oleoyl Phosphatidyl Serine) 1% mol; D SPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 1% mol.

(8) b) freeze-thaw cycles were then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C.

(9) c) a thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) was used to calibrate the size of the liposomes under controlled temperature and pressure. In all cases, extrusion was performed at 60° C. First, 5 passages through a polyethersulfone (PES) 0.45 μm pores-sized membrane were performed at a pressure of 5 bars, then 12 passages through a PES 0.22 μm pores-sized membrane at 10 bars, and finally 12 passages through a 0.1 μm polyvinylidene fluoride (PVDF) membrane at 15 bars.

(10) Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 90° C. The liposomes suspension was diluted 100 times in HEPES 20 mM and NaCl 140 mM at pH 7.4. Liposome size (i.e. hydrodynamic diameter) was equal to about 145 nm (distribution by intensity) with a polydispersity index (PDI) equal to about 0.1.

(11) As understandable by the skilled person, the desired surface charge was obtained thanks to the selected lipidic composition, and its value was confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument).

(12) The liposomes were diluted 100 times in a sodium chloride solution at 1 mM and the pH of the resulting suspension was adjusted to pH 7.4. The liposomes surface charge was equal to about −25 mV at pH 7.4, NaCl 1 mM.

(13) Synthesis of “Second” and/or “First” Biocompatible Nanoparticles:

(14) a) lipids were solubilized in chloroform. Chloroform was finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 was performed at 60° C., so that the lipid concentration was 50 mM.

(15) The following lipid composition was used: DPPC (DiPalmitoylPhosphatidylCholine) 57% mol; HSPC (Hydrogenated Soybean PhosphatidylCholine) 21% mol; CHOL (Cholesterol) 16% mol; POPS (1-Palmitoyl-2-Oleoyl Phosphatidyl Serine) 5% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 1% mol.

(16) b) freeze-thaw cycles were then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C.

(17) c) the liposomes solution was then ultra-sonicated with a probe at a power of 230 W during 30s.

(18) d) a thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) was used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion was performed at 60° C. First, 10 passages were applied through a 0.1 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 10 bars, then 10 passages through a 0.08 μm pores size polycarbonate (PC) membrane under a pressure of 15 bars, and finally 10 passages through a 0.05 μm pores size PC membrane under a pressure of 20 bars.

(19) e) the liposomes solution was then concentrated twice by membrane ultrafiltration on Vivaspin concentrators, with polyethylene sulfone (PES) membrane with a cut-off 300 KDa.

(20) Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution was diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) was equal to about 70 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.1.

(21) The surface charge of the liposomes was determined by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution was diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution was adjusted to pH 7. The liposomes surface charge was equal to about −40 mV at pH 7, NaCl 1 mM.

Example 2: Synthesis of Liposomes as the “First” and/or “Second” Biocompatible Nanoparticles

(22) Liposomes are prepared using the lipid film re-hydration method.

(23) —Synthesis of “First” and/or “Second” Biocompatible Nanoparticles with Absolute Surface Charge Value of at Least 10 mV (|10 mV|):

(24) a) lipids were solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60° C., so that the lipid concentration is 50 mM.

(25) The following lipid composition is used: HSPC (Hydrogenated Soybean PhosphatidylCholine) 59% mol; CHOL (Cholesterol) 38% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 3% mol.

(26) b) freeze-thaw cycles were then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C.

(27) c) the liposomes solution was then ultra-sonicated with a probe at a power of 230 W during 30s.

(28) d) a thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) was used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion was performed at 60° C. First, 10 passages were applied through a 0.1 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 15 bars, then 18 passages through a 0.08 μm pores size polycarbonate (PC) membrane under a pressure of 20 bars.

(29) e) the liposomes solution was then concentrated twice by membrane ultrafiltration on Vivaspin concentrators, with polyethylene sulfone (PES) membrane with a cut-off 300 KDa.

(30) Size distribution of the as-prepared liposomes was determined by DLS using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution was diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) was equal to about 90 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.1.

(31) The surface charge of the liposomes was determined by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution was diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution was adjusted to pH 7. The liposomes surface charge was equal to about −25 mV at pH 7, NaCl 1 mM.

(32) The final lipid concentration of the liposomes solution was measured by a colorimetric assay: phospholipase D cuts the phosphaticylcholine molecules, thus relieving the choline group which is going to form a blue pigment by reacting with the chromogenic substrate. Lipids concentration was found at 100 mM.

(33) —Synthesis of “Second” and/or “First” Biocompatible Nanoparticles:

(34) a) lipids were solubilized in chloroform. Chloroform was finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 was performed at 60° C., so that the lipid concentration is 50 mM.

(35) The following lipid composition was used: DPPC (DiPalmitoylPhosphatidylCholine) 57% mol; HSPC (Hydrogenated Soybean PhosphatidylCholine) 21% mol; CHOL (Cholesterol) 16% mol; POPS (1-Palmitoyl-2-Oleoyl Phosphatidyl Serine) 5% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 1% mol.

(36) b) freeze-thaw cycles were then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C.

(37) c) a thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) was used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion was performed at 60° C. First, 5 passages through a polyethersulfone (PES) 0.45 μm pores-sized membrane were performed at a pressure of 5 bars, then 12 passages through a PES 0.22 μm pores-sized membrane at 10 bars, and finally 12 passages through a 0.1 μm polyvinylidene fluoride (PVDF) membrane at 15 bars.

(38) d) the liposomes solution was then concentrated twice by membrane ultrafiltration on Vivaspin concentrators, with polyethylene sulfone (PES) membrane with a cut-off 300 KDa.

(39) Size distribution of the as-prepared liposomes was determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution was diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) was equal to about 145 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.1.

(40) The surface charge of the liposomes was determined by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution was diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution was adjusted to pH 7. The liposomes surface charge was equal to about −40 mV at pH 7, NaCl 1 mM.

(41) The final lipid concentration of the liposomes solution was measured by a colorimetric assay: phospholipase D cuts the phosphaticylcholine molecules, thus relieving the choline group which is going to form a blue pigment by reacting with the chromogenic substrate. Lipids concentration was found at 100 mM.

Example 3: Method for the Evaluation of the Efficacy and Toxicity of a Pharmaceutical Composition Comprising the at Least Two Biocompatible Nanoparticles Suspensions of Example 2 and the Dox-NP® in MDA-MB-231-lucD3H2LN Xenografts (Cf. FIG. 2)

(42) The pharmaceutical composition comprising (i) the at least two distinct “first” and “second” biocompatible nanoparticles from example 2 and (ii) Dox-NP® (Liposomal Encapsulated Doxorubicin) as the therapeutic compound of interest, is administered in nude mice bearing MDA-MB-231-lucD3H2LN xenografted tumor in the following manner:

(43) a) the Dox-NP® and the biocompatible nanoparticles from example 2 are administered by intravenous injection (IV) via lateral tail vein.

(44) The Dox-NP® (Avanti Polar lipids—Liposomal formulation of 2 mg/ml doxorubicin HCl at pH 6.5-6.8, in 10 mM histidine buffer, with 10% w/v sucrose) are injected without additional dilution at the volume required to obtain 3 mg/kg of injected doxorubicin.

(45) The biocompatible nanoparticles suspensions from example 2 are used without any additional dilution.

(46) b) four groups of mice are treated as illustrated on FIG. 2:

(47) —Group 1: sterile glucose 5% (control (vehicle) group).

(48) Mice are intravenously (IV) injected with a sterile glucose 5% solution on day 1, day 7 and day 14.

(49) —Group 2: “First” and “second” biocompatible nanoparticles from example 2 (control group).

(50) Mice are intravenously (IV) injected with the “first” and “second” biocompatible nanoparticles from example 2 (10 ml/kg) on day 1, day 7 and day 14. Each time (day), the injection of the first biocompatible nanoparticles is performed 4 hours before injection of the second biocompatible nanoparticles.

(51) —Group 3: Dox-NP® (3 mg/kg doxorubicin) (treatment group).

(52) Mice are intravenously (IV) injected with Dox-NP® (3 mg/kg doxorubicin) on day 1, day 7 and day 14.

(53) —Group 4: pharmaceutical composition, i.e. the combination of (i) the at least two distinct (“first” and “second”) biocompatible nanoparticles from example 2 and of (ii) Dox-NP® (3 mg/kg doxorubicin) (treatment group).

(54) Mice are intravenously (IV) injected with the biocompatible nanoparticles from example 2 (10 ml/kg) and with the Dox-NP® (3 mg/kg doxorubicin) on day 1, day 7 and day 14. Each time (day), the simultaneous injection of the “first” and “second” biocompatible nanoparticles from example 2 is performed 4 hours before the injection of Dox-NP® (3 mg/kg doxorubicin).

(55) —Group 5: pharmaceutical composition, i.e. the combination of (i) the at least two distinct biocompatible nanoparticles from example 2 and of (ii) Dox-NP® (3 mg/kg doxorubicin) (treatment group).

(56) Mice are intravenously (IV) injected with the biocompatible nanoparticles from example 2 (10 ml/kg) and with the Dox-NP® (3 mg/kg doxorubicin) on day 1, day 7 and day 14. Each time (day), the injection of the “first” and “second” biocompatible nanoparticles from example 2 is performed 4 hours and 1 hours respectively before the injection of Dox-NP® (3 mg/kg doxorubicin).

(57) c) any clinical sign of toxicity is assessed after the administration of the pharmaceutical composition; and

(58) d) the tumor volume is measured from two dimensional tumor volume measurements with a digital caliper using the following formula:

(59) $\text{Tumor volume}\left({\mathrm{mm}}^3\right)=\frac{\text{length (mm)}\times {\left(\mathrm{width}\right)}^2\left({\mathrm{mm}}^2\right)}{2}$

Example 4: Synthesis of Liposomes as the “First” or “Second” Biocompatible Nanoparticles

(60) Liposomes are prepared using the lipid film re-hydration method:

(61) a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60° C., so that the lipid concentration is 50 mM.

(62) The following lipid composition was used to prepare charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine) 58% mol; HSPC (Hydrogenated Soybean PhosphatidylCholine) 21% mol; CHOL (Cholesterol) 16% mol; POPS (1-Palmitoyl-2-Oleoyl PhosphatidylSerine) 5% mol.

(63) b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C. Ultra-sonication of the liposomes solution is performed during 30s every 3 freeze-thaw cycles and just before extrusion.

(64) c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) is used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion is performed at 60° C. Ten passages are applied through a 0.1 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 10 bars.

(65) Size distribution of the as-prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution is diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) is equal to about 170 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.2.

(66) As understandable by the skilled person, the desired surface charge is obtained thanks to the selected lipid composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes are diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution is adjusted to pH 7. The liposomes surface charge is equal to about −40 mV at pH 7, NaCl 1 mM.

(67) The final lipid concentration of the liposomes solution is measured by a colorimetric assay (Bartlett method). The method is based on total phosphorus determination through an acidic digestion of phospholipid. The released inorganic phosphate is reacted with ammonium molybdate, the complex giving a strong blue color. Lipids concentration is equal to about 50 mM.

Example 5: Synthesis of Liposomes as the “First” or “Second” Biocompatible Nanoparticles

(68) Liposomes are prepared using the lipid film re-hydration method:

(69) a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60° C., so that the lipid concentration is 50 mM.

(70) The following lipid composition was used to prepare the charged liposomes: DPPC (DiPalmitoylPhosphatidylCholine) 45.15% mol; CHOL (Cholesterol) 45.15% mol; DSPE-PEG (DiStearylPhosphatidylEthanolamine-[methoxy(PolyElthyleneGlycol)-2000]) 0.60% mol; L-Glutamic acid, N-(3-carboxy-1-oxopropyl)-, 1,5-dihexadecyl ester (SA-lipid) 9.10% mol. The SA-lipid brings COOH groups on the liposomes surface.

(71) b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C.

(72) c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) is used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion is performed at 60° C. Seven passages are applied through a 0.45 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 3 bars and ten passages through a 0.22 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 10 bars. Size distribution of the as-prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution is diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) is equal to about 230 nm (distribution by intensity) with a polydispersity index (PdI) equal to about 0.2.

(73) As understandable by the skilled person, the desired surface charge is obtained thanks to the selected lipid composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution is diluted 200 times in a sodium chloride solution at 1 mM and the pH of the solution is adjusted to pH 7. The liposomes surface charge is equal to about −60 mV at pH 7, NaCl 1 mM.

(74) The final lipid concentration of the liposomes solution is measured by a colorimetric assay (Bartlett method). The method is based on total phosphorus determination through an acidic digestion of phospholipid. The released inorganic phosphate is reacted with ammonium molybdate and the complex giving a strong blue color. Lipids concentration is equal to about 50 mM.

Example 6: Synthesis of Liposomes as the “First” or “Second” Biocompatible Nanoparticles

(75) Liposomes are prepared using the lipid film re-hydration method:

(76) a) Lipids are solubilized in chloroform. Chloroform is finally evaporated under a nitrogen flow to form a lipid film on the Pyrex tube walls. Re-hydration of the lipid film with HEPES 25 mM and NaCl 150 mM at pH 7.4 is performed at 60° C. and the lipid concentration is 50 mM. The following lipid composition was used to prepare the charge liposomes: DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) 60% mol, CHOL (Cholesterol) 35% mol; and Succinyl PE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-succinyl) 5% mol.

(77) b) Freeze-thaw cycles are then performed 6 times, by successively plunging the sample into liquid nitrogen and into a water bath regulated at 60° C. Ultra-sonication of the liposomes solution is performed during 30s, every 3 freeze-thaw cycles and just before extrusion.

(78) c) A thermobarrel extruder (LIPEX™ Extruder, Northern Lipids) is used to calibrate the size of the liposomes under controlled temperature and pressure. Extrusion is performed at 60° C. Twelve passages are applied through a 0.22 μm pores size polyvinylidene fluoride (PVDF) membrane under a pressure of 12 bars.

(79) d) Conjugation of p-aminophenyl-α-D-mannopyranoside (MAN) to Succinyl PE liposome: The succinyl PE liposome surface are modified with a mannose derived ligand p-aminophenyl-α-D-mannopyranoside (MAN), using carbodiimide coupling to develop mannose conjugated liposome. MAN is covalently coupled by its amino group to the carboxylic acid group of Succinyl PE, present on the surface of preformed Succinyl PE liposome. Briefly, to the preformed Succinyl PE liposome solution are added EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride), (Succinyl PE/EDC 1:10 molar ratio) and N-hydroxysuccinimide (NHS) (NHS/EDC 1:2.5 molar ratio). The pH of the suspension is then adjusted at 6 with NaOH 1M and the resulting suspension is stirred for 15 minutes at room temperature. Subsequently, the pH of the solution is adjusted at 7 with NaOH 1M and the aqueous MAN solution is added (Succinyl PE/MAN 1:2 molar ratio) to the solution. pH is readjusted at 7 using NaOH 1M and the suspension is stirred for 2 additional hours at room temperature. Excessive unbound MAN, EDC and NHS molecules are removed by 3 steps of dialysis with dilution factor (×500; ×500; ×500) using a 50 KDa cellulose membrane.

(80) Of note, due to possible dilution upon dialysis, the liposomes solution can be concentrated by centrifugation (typically a Sigma 3-15K centrifuge at 5° C.; 1,200 rpm) using membrane ultrafiltration on Vivaspin concentrators with a polyethylene sulfone (PES) membrane and a cut-off 300 KDa.

(81) Size distribution of the as-prepared liposomes is determined by dynamic light scattering (DLS) using a Zetasizer NanoZS (Malvern instrument) with a 633 nm HeNe laser at an angle of 173° C. The liposomes solution is diluted 200 times in HEPES 25 mM and NaCl 150 mM at pH 7.4. Liposomes size (i.e. hydrodynamic diameter) is about 230 nm (distribution by intensity) with a polydispersity index (PDI) around 0.2.

(82) As understandable by the skilled person, the desired surface charge is obtained thanks to the selected lipid composition, and its value is confirmed by zeta potential measurement using a Zetasizer NanoZS (Malvern instrument). The liposomes solution is diluted 200 times in a sodium chloride solution at 1 mM and at pH 7. The liposomes surface charge is around −70 mV at NaCl 1 mM, pH 7.

(83) The final lipid concentration of the liposomes solution is measured by a colorimetric assay (Bartlett method). The method is based on total phosphorus determination through an acidic digestion of phospholipid. The released inorganic phosphate is reacted with ammonium molybdate and the complex giving a strong blue color. Lipids concentration is equal to about 50 mM.