# US 2019/0167818 A1 · Fluorescent solid lipid nanoparticles composition and preparation thereof

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- Number: US 2019/0167818 A1
- Document ID: US20190167818A1
- Status: Published application
- Kind: A1
- Application number: 16/263,769
- Filed: 2019-01-31
- Published: 2019-06-06
- Earliest claimed: 2013-05-30
- Assignees: Bracco Imaging S.P.A.
- Applicants: Bracco Imaging S.P.A.
- Inventors: Simona GHIANI; Alessandro MAIOCCHI; Lara CAMINITI; Luigi MIRAGOLI
- Web page: https://patentgrep.com/patent/US20190167818A1

## CPC

- A61K 49/0093: {Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle}
- A61K 49/0034: {Indocyanine green, i.e. ICG, cardiogreen}
- A61K 49/0052: {Small organic molecules (oligomers, polymers, dendrimers A61K49/0054)}
- A61K 49/0054: {Macromolecular compounds, i.e. oligomers, polymers, dendrimers}
- A61K 49/0078: {microemulsion, nanoemulsion}
- A61K 9/10: Dispersions; Emulsions
- A61K 9/5123: {Organic compounds, e.g. fats, sugars}
- A61K 49/0017: {Fluorescence in vivo}
- A61K 49/0032: {Methine dyes, e.g. cyanine dyes}
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)

## Abstract

The present invention discloses a solid lipid nanoparticle (SLN) comprising: a) a solid lipid core comprising at least a glyceride and/or at least a fatty add; b) a mixture of amphiphilic components forming a shell around said core a); c) an alkaline-earth complex with a compound of formula I and/or II: d) at least a fluorescent dye selected from: a cyanine fluorescent dye and/or a polyetherocyclic compound selected from: coumarin, pyrano, quinoline, pyranoquinoline, indole and pyranoindele derivates in acid form or a pharmaceutically acceptable salt thereof. These nanoparticles allow a prolonged blood circulation half-life, show enhanced photostability and improved fluorescence signal. The dye is preserved from degradation and improves the fluorescent quantum yield.

## Claims

### Claim 1

A solid lipid nanoparticle comprising:
a) a solid lipid core comprising at least a glyceride and/or at least a fatty acid;
b) a mixture of amphiphilic components forming a shell around said core a);
c) an alkaline-earth complex with a compound of formula II:
wherein:
Y is a group of formula (Y′)2—N—, wherein Y′, which —can be the same or different, is a linear or branched, saturated or unsaturated C12-C20 alkyl group;
L is a bivalent linker selected from the group consisting of: aliphatic C3-C10, or linear or branched C1-C6 alkanediyl, alkenediyl, alkynediyl, optionally interrupted with one or more atoms or atom groups selected from the group consisting of: —C═O—, —C═S—, —NR1—, —COO—, —OCO—, —NR1CO—, —CONR1—, —O— and —S—, wherein R1 is selected from the group consisting of hydrogen H and a linear or branched C1-C4 alkyl group:
R′-′″are each, independently, a —R2—COOR3, wherein R2 is a C1-C6 linear or branched alkyl, R3 is H or a pharmaceutically acceptable cation;
d) at least a fluorescent dye selected from the group consisting of: a cyanine fluorescent dye and/or a polyetherocyclic compound selected from: coumarin, pyrano, quinoline, pyranoquinoline, indole and pyranoindole derivates in acid form or a pharmaceutically acceptable salt thereof.

### Claim 2 (depends on 1)

The nanoparticle according to claim 1, wherein said alkaline-earth complex compound of formula II is as defined in claim 1 and wherein:
Y′ is a linear or branched, saturated C16-C18 alky group;
L is a bivalent linker selected from the group consisting of: methylcarbonyl, ethylcarbonyl, propylcarbonyl, butylcarbonyl, pentylcarbonyl and hexylcarbonyl;
and R′-′″ is as defined above.

### Claim 3

(canceled)

### Claim 4 (depends on 1)

The nanoparticle according to claim 1, further comprising:
e) a hydrophilic polymeric function covalently linked to said shell b) having the function of stealth agent.

### Claim 5 (depends on 4)

The nanoparticle according to claim 4, further comprising:
f) a molecular targeting moiety for the specific binding towards one pathology-related biological marker, said moiety being linked either to said core shell b) or said hydrophilic polymeric function e).

### Claim 6 (depends on 1)

The nanoparticle according to claim 1, wherein said solid lipid core a) comprises at least one glyceride, and/or at least one fatty acid or a mixture thereof, said at least one glyceride or fatty acid or an ester thereof is or are in the solid form at room temperature and at body temperature.

### Claim 7 (depends on 6)

The nanoparticle according to claim 6, wherein, said glyceride is selected from the group consisting of a monoglyceride, a diglyceride and a triglyceride having a saturated or unsaturated, linear or branched C12-C24 acyl chain, wherein in case of di- and triglyceride, the acyl chains can be the same or different; said fatty acid, or an ester thereof, has a saturated or unsaturated, linear or branched C12-C24 carbon chain; and said core optionally comprises esters C12-C24 fatty acids with C12-C-24 fatty alcohols.

### Claim 8 (depends on 7)

The nanoparticle according to claim 7, wherein said core a) comprises tripalmitin and/or stearic acid.

### Claim 9 (depends on 1)

The nanoparticle according to claim 1, wherein said mixture of amphiphilic components b) comprises
a component selected from the group consisting of phospholipids, lysolipids and sphingolipids having linear or branched, saturated or unsaturated C6-C24 hydrocarbon chains; and
optionally at least one of cholesterol and steroid derivatives, glycolipids, fatty acids, fatty alcohols and dialkyl ethers, non-ionic surfactant selected from sorbitan derivatives, di- and tri-esters of saturated and unsaturated fatty acid derivatives from C6-C24 carbon atoms and ethoxylated analogue thereof, mono or oligo-glycosides and ethoxylated analogues thereof, and glycerol mono, di- and tri-esters soluble at room and at body temperature.

### Claim 10 (depends on 9)

The nanoparticle, according to claim 9, wherein said mixture of amphiphilic components comprises phosphatidylcholine from soy lecithin.

### Claim 11 (depends on 9)

The nanoparticle according to claim 9, wherein said non-ionic surfactant is polyoxyethylene sorbitan monooleate.

### Claim 12 (depends on 4)

The nanoparticle according to claim 4, wherein said hydrophilic polymer e) is selected from the group consisting of functionalized poloxamer, polysiloxanes, polyalkyl polyether, polyglycerine, polyvinilalcohol and a polyethyleneglycol, optionally covalently linked to a phospholipidic moiety.

### Claim 13 (depends on 1)

The nanoparticle according to claim 1, wherein said fluorescent dye is Indocyanine Green of formula

### Claim 14 (depends on 5)

The nanoparticle according to claim 5, wherein said molecular targeting moiety f) is a tumor targeting ligand selected from the group consisting of cell surface receptor, proteins, aptamers, peptides and polypeptides, vitamins, antibodies or fragments thereof and carbohydrates.

### Claim 15 (depends on 1)

The nanoparticle of claim 1 wherein the alkaline-earth complex with a compound of formula II is selected from the group consisting of:
and

### Claim 16

(canceled)

### Claim 17

(canceled)

### Claim 18 (depends on 1)

A pharmaceutical composition comprising the nanoparticle of claim 1.

### Claim 19 (depends on 1)

A process for the preparation of the nanoparticle of claim 1 comprising the following steps:
i. preparing au organic phase (O) by dissolving in a water immiscible or low-miscible organic solvent, a lipid substance or substances which will form the solid lipid crystalline core a), the amphiphilic component b) and an alkaline-earth complex with a compound of formula II, c), as defined in claim 1;
the fluorescent dye d);
ii. preparing i first aqueous solution (W) by dissolving one or more hydrophilic surfactants and optionally co-surfactants components;
iii. mixing said organic phase (O) of step i) with said first aqueous solution (W) of step ii) and stirring until a stable W/O micro-emulsion is formed;
iv. said W/O micro-emulsion obtained in step iii) is subsequently added to a second aqueous solution (W1) which can contain at least a surfactant, to provide a W/O/W1 multiple emulsion;
v. stripping said organic solvent from the multiple emulsion by evaporation to provide a suspension;
vi. cooling down the suspension obtained in step v) to provide the complete crystallization of said solid core a);
vii. washing said dispersion obtained in step vi) from the excess of the components;
viii. optionally storing said dispersion obtained in step vii) in aqueous phase or as a solid phase after water removal.

### Claim 20 (depends on 19)

The process according to claim 19, wherein said organic solvent in step i) has a boiling point from 20° C. to 70° C.

### Claim 21 (depends on 20)

The process according to claim 20, wherein said solvent is selected from the group consisting of methylene chloride, 1,2-dichloroethane, chloroform, diethyl ether, ethylacetate, methylacetate, ethyl formate and a mixture thereof.

### Claim 22 (depends on 21)

The process according to claim 21 wherein in said step ii)
the hydrophilic surfactant is selected from the group consisting of cholic acid, taurocholic acid, taurodeoxycholic acid, a salt thereof, and a derivative thereof; and
the co-surfactant is selected in the group consisting of 1-butanol and 1-hexanol.

### Claim 23 (depends on 19)

The process according to claim 19 wherein in said step iv) the surfactant is a sorbitan derivative.

### Claim 24 (depends on 18)

A method of diagnostic imaging comprising;
administering to a subject an effective amount of the pharmaceutical composition of claim 18; and
subjecting said subject to a suitable diagnostic imaging method.

### Claim 25 (depends on 24)

The method of claim 24, wherein the diagnostic imaging method is selected from the group consisting of
real-time imaging guided surgery; and
tumor detection and/or lymph nodes mapping in clinical fluorescence imaging applications.