WO2008149215A2

WO2008149215A2 - Method for the preparation of solid lipid micro and nanoparticles

Info

Publication number: WO2008149215A2
Application number: PCT/IB2008/001463
Authority: WO
Inventors: Luigi Sebastiano Battaglia; Michele Trotta; Roberta Cavalli
Original assignee: Universita degli Studi di Torino
Current assignee: Universita degli Studi di Torino
Priority date: 2007-06-08
Filing date: 2008-06-06
Publication date: 2008-12-11
Anticipated expiration: 2009-12-08
Also published as: WO2008149215A3; ITTO20070411A1

Abstract

Method for the preparation of solid lipid micro and nanoparticles comprising the phase of mixing an acid solution with a micellar aqueous solution comprising at least one salt soluble in water of a fatty acid in the presence of a non- ionic and biocompatible polymeric stabilising agent. The micro and nanoparticles can furthermore incorporate an active ingredient, in particular, a thermosensitive active ingredient.

Description

"METHOD FOR THE PREPARATION OF SOLID LIPID MICRO AND NANOPARTICLES"

TECHNICAL FIELD The present invention concerns a new method for the preparation of solid lipid micro and nanoparticles .

BACKGROUND ART

Solid lipid micro and nanoparticles are widely used in the pharmaceutical field as a vehicle for active ingredients, due to their low toxicity and the fact that they are simple to prepare .

In the field of pharmaceutical technique, numerous methods are known for their preparation, for example the method of cold homogenisation, the method of hot homogenisation (Muller et al . , EP0605497) , the method of dilution of the microemulsion

(Gasco, EP0526666) , the method of cooling of the microemulsion

(Mumper et al., US2006/0292183) , the method of evaporation of the solvent from the emulsion (Siekmann et al . , European

Journal of Pharmaceutics and Biopharmaceuticals 1996; 43: 104-

109) , the method of dilution of the solvent from the emulsion

(Trotta et al . , International Journal of Pharmaceutics 2003;

257(1-2): 153-60) and the method of injection of solvent (Schubert et al., European Journal of Pharmaceutics and

Bi©pharmaceutics 2003; 5 5(1): 125-31).

All these methods, with the exception of the method of cold homogenisation which is only a grinding method and produces microparticles with large dimensions, permit the production of small nanoparticles with a reduced size distribution.

All these methods have their drawbacks, however. For example the method of hot homogenisation requires the use of complex and costly equipment and high temperatures; the method of dilution and the method of cooling microemulsions require the use of large quantities of surface-active agents and cosurface-active agents and high temperatures which do not permit the incorporation of thermosensitiye drugs ; the methods involving the use of solvents are not able to guarantee complete elimination of the particles ^• of the solvent ^"used, which can sometimes be toxic . . .

The precipitation of lipids by acidification of the salts of the fatty acids is generally used for their purification.

The purification consists in separation of the fatty acid from a solution by the addition of a strong acid at high^' temperatures, generally concentrated sulphuric acid.

The precipitate consists of lipid crystals with an irregular shape, in particular needle-shaped.

Disadvantageously, said purification technique does not permit control of the form and dimensions of the precipitate.

Currently therefore, a new method is being searched for without the drawbacks of the known methods for the preparation of spherical solid lipid micro and nanoparticles .

DISCLOSURE OF INVENTION

The object of the present invention is therefore to find a method for the production of solid micro- and nanoparticles of fatty acids of controlled form, with a reduced size distribution, which provides easily reproducible results and which permits the incorporation of active ingredients, including thermosensitive active ingredients .

The invention furthermore aims to find a method which does not require the use of complex equipment and toxic solvents and which is therefore inexpensive not only for laboratory application but also for industrial application. According to the present invention said object is achieved by means of a method as claimed in claim 1.

Said method is based on a process of acidification of salts of fatty acids in the presence of specific stabilising agents, which overcomes the problems of the methods known in the art .

Hereinafter the term "micellar aqueous solution" indicates a solution comprising lipid aggregates in a colloidal phase; the term "nanoparticles" indicates particles with dimensions from 10 to 1000 nm, the term "microparticles" indicates particles with dimensions from 1 to 100 μm, the term "biocompatible" indicates a substance biologically compatible with tissues, organs and functions of the organism and which does not cause toxic or immunological responses in the organism; the term "acid solution" indicates a solution of a biocompatible acid with pH between 1 and 7.

Advantageously the method according to the present invention permits the preparation of micro and nanoparticles of solid fatty acids, the form and dimensions of which can be controlled by control of the reaction conditions .

In particular, the precipitation of the micro and nanoparticles is performed by mixing an acid solution with a micellar aqueous solution comprising at least one salt soluble in water of a fatty acid in the presence of an amphiphilic polymeric stabilising agent which is non-ionic and biocompatible .

Preferably the salt of the fatty acid is selected from alkaline salt, ammonium salt and amine salt.

Advantageously, the fatty acid is a solid fatty acid at room temperature, more preferably selected from the group consisting of stearic acid, palmitic acid, myristic acid, lauric acid, arachidonic acid, behenic acid.

The salt of the fatty acid is preferably present in a concentration of between 0.1 and 30% w/w, more preferably between 1 arid 5% w/w, and is selected from • the group consisting of sodium stearate, sodium palmitate, sodium myristate, sodium laurate, sodium arachidonate , sodium behenate .

The stabilising agent, present in a concentration of between 0.1 and 30% w/w, preferably between 1 and 5% w/w, is a non- ionic amphiphilic polymer, preferably selected from the group consisting of partially hydrolysed polyvinyl acetate, polyoxyethylene/polyoxy-propylene copolymers, polyacrylamides , polyvinyl pyrrolidone and its derivatives, stabilising agents derived from polysaccharides, for example derivatives of dextran and agarose of various molecular weights, derivatives of cellulose, non-ionic gums, cyclodextrins and their derivatives .

The acid solution used in the present method preferably comprises at least one acid selected from hydrochloric acid and an acid with pKa between 2 and 6.

Even more preferably, the acid is selected from the group consisting of acetic acid, carbonic acid, lactic acid, glycolic acid, tartaric acid, maleic acid, pyruvic acid, malic acid, succinic acid, citric acid, hydrochloric acid, phosphoric acid, acid polyphosphates, acid salts of ammonium and their derivatives, amino acids, polyamino acids, polymers containing acid groups, for example alginic acid and chitosan.

Preferably, the acid solution comprises an acid in a concentration from 0.01M to 5M. The micellar solution is mixed with the acid solution at a temperature below the melting temperature of the fatty acid.

Preferably, the mixing temperature varies from 25°C to 80⁰C, more preferably between 4O⁰C and 50⁰C.

To further reduce the mixing^' temperature it is possible to add to the reaction mixture a biocompatible co-solvent selected from the group consisting of ethanol, propylene glycol, glycerol and butyl lactate up to a concentration of 30% w/w of the aqueous phase.

Advantageously the method of the present invention permits the incorporation or superficial adsorption by the micro or nanoparticles of an active ingredient for therapeutic, diagnostic, cosmetic and alimentary use. Preferably the active ingredient is selected from the group consisting of antitumoral molecules, antioxidant molecules, antibiotics, metals, immunosuppressors . Even more preferably, the active ingredient is selected from azulene, amphotericin B, cisplatin, tocopherol, cyclosporin, retinol.

For said purpose an active ingredient can be added to the micellar aqueous solution.

The active ingredient can be dissolved directly in the micellar solution or alternatively can be dissolved in a small volume of biodegradable solvent miscible with water. The solution thus obtained is then mixed with the micellar aqueous solution of the fatty acid.

The active ingredient is incorporated in the solid particles when the acid solution is mixed.

If the active ingredient is soluble in water with basic pH, it can be dissolved in a small volume of basic aqueous solution and then added to the micellar aqueous solution of the fatty- acid. In this way, the active ingredient co-precipitates with the particles of fatty acid as the pH is lowered.

Alternatively, if the active . ingredient forms salts or complexes that are insoluble with ^■ the components of the micelles, it cannot be added directly to ^' the micellar solution. It can, however, be dissolved in the acid solution for combination with the micellar aqueous solution. In this way the insoluble salts or complexes of the active ingredient with the fatty acids will precipitate with the lipids.

Further characteristics of the present invention will emerge from the following description of some merely illustrative non-limiting examples.

Example 1

Preparation of nanoparticles of 1% stearic acid

The sodium stearate is dissolved in water at a concentration of 1%. 1% of PVA 9000 hydrolysed to 80% under agitation at 47⁰C is added to the resulting solution.

The nanoparticles of stearic acid are precipitated by adding lactic acid IM dropwise under agitation and the suspension is then cooled slowly at room temperature.

Particles are obtained with dimensions equal to 290 nm with a close size range (polydispersion = 0.05).

The TEM microphotograph of the nanoparticles of 1% stearic acid obtained according to the method illustrated in the present example is provided in Figure 1 and shows the formation of spherical particles .

The DSC analysis (Figure 2) shows an endothermic transition at 53⁰C (Tp_eak) and the X-ray analysis (Figure 3) reveals a crystalline structure, a characteristic of the form B of stearic acid.

Example 2

Preparation of nanoparticles of palmitic, acid 1%

The sodium palmitate is dissolved in water at a concentration of 1%. 1% of PVA 120000 hydrolysed to 89% under agitation at 47⁰C is added to the solution obtained.

The nanoparticles of palmitic acid are precipitated by adding lactic acid IM dropwise under agitation and the suspension is then cooled slowly at room temperature .

Particles with dimensions of 700 nm (polydispersion = 0.25) are obtained.

The TEM microphotograph of the nanoparticles of 1% stearic acid obtained according to the method illustrated in the present example is provided in Figure 4 and shows the formation of spherical particles .

The DSC analysis (Figure 5) shows an endothermic transition at 58⁰C (Tpi_CCO) and the X-ray analysis (Figure 6) reveals a crystalline structure similar to the one obtained for the nanoparticles of 1% stearic acid.

Example 3 Preparation of nanoparticles of 1% stearic acid -cetomacrogol 1000

The sodium stearate is dissolved in water at a concentration of 1% with 0.1% cetomacrogol 1000. 1% of PVA 9000 hydrolysed to 80% under agitation at 47⁰C is added to the solution obtained. The lipid nanoparticles are precipitated by adding lactic acid IM dropwise under agitation and the suspension is then cooled slowly at room temperature.

Particles with dimensions of 450 nm are obtained with a close size interval (polydispersion = 0.1) .

Example 4 Production of nanoparticles of 1% stearic acid with lactic acid or citric acid

The sodium stearate is dissolved in water at a concentration of 1%. 1% of PVA 14000 hydrolysed to 89% under agitation at 47⁰C is added to the solution obtained.

The sample is then divided into two solutions.

In one, the nanoparticles of stearic acid are precipitated by adding lactic acid IM dropwise under agitation, while in the other the same operation is performed by adding citric acid IM.

The suspensions are then cooled slowly at room temperature.

Particles with dimensions of 600 nm with a close size interval

(polydispersion = 0.15) are obtained from the solution to which lactic acid has been added, while aggregated microparticles with larger dimensions with a wider size distribution (dimensions of 1 micron, polydispersion = 0.25) are obtained from the solution to which citric acid has been added.

The optical microscope microphotographs of the particles of stearic acid obtained with lactic acid and citric acid are shown in Figures 7(A) and 7 (B) respectively. The DSC (Figure 8) shows only a slight variation in the melting temperature, without variation in the enthalpy.

Example 5

Production of particles of 1%, 2% and 5% stearic acid

Three different micellar solutions are prepared containing sodium stearate at a concentration of 1%, 2% and 5% respectively.

PVA 9000 hydrolysed to 80% is added to each micellar solution obtained in a concentration of 1%, 2% and 5% respectively under agitation at 47⁰C.

The nanoparticles of stearic acid are precipitated by adding lactic acid IM, 2M and 5M respectively dropwise under agitation and the suspensions are then cooled slowly at room temperature .

The mean diameters and the size distributions increase as the concentration of lipids increases, shifting from nanoparticles and microparticles as indicated in table I (following page) . Table I

The optical microscope microphotographs of the particles of 1%, 2% and 5% stearic acid are shown respectively in Figures 9 (A) , 9 (B) and 9 (C) .

The DSC analysis (Figure 10) shows the influence of the lipid concentration on the melting temperature and enthalpy. Example 6

Production of nanoparticles of 2% stearic acid with 2% and

4% of PVA 9000 hydrolysed to 80%

The sodium stearate is dissolved in water at a concentration of 2%. The solution obtained is divided into two parts and PVA 9000 hydrolysed to 80% is added under agitation at a concentration of 2% and 4% respectively at 47⁰C.

The nanoparticles of stearic acid are precipitated by adding lactic acid 2M dropwise under agitation and the suspensions are then cooied slowly at room temperature.

The mean diameters and the size distributions increase as the concentration of stabilising agent increases (from 380 nm with polydispersion = 0.1 to 425 nm with polydispersion = 0.2.

The DSC (Figure 11) shows a slight change in the melting temperature, but not in the relative enthalpy.

Example 7

Production of nanoparticles of 1% stearic acid with different types of stabilising agents: 1% PVA 9000 hydrolysed to 80%, 1% PVA 14000 hydrolysed to 89%, 1% PVA 120000 hydrolysed to 89%, Pluronic^*8 F-68 and Pluronic^® F-127

The sodium stearate is dissolved in water at a concentration of 1%. The solution obtained is divided into five parts and a different stabilising agent is added to each one, 1% PVA 9000 hydrolysed to 80%, 1% PVA 14000 hydrolysed to 89%, 1% PVA 120000 hydrolysed to 89%, 1% Pluronic^® F- 68 and 1% Pluronic^® F- 127 respectively at 47°C.

The nanoparticles of stearic acid are precipitated by adding lactic acid IM dropwise under agitation and the suspensions are then cooled slowly at room temperature. The mean diameters and size distributions are illustrated in Table II.

TABLE II

The DSC analysis (Figure 12) shows the influence of the type of stabilising agent used on the melting temperature and on the form of the peak and relative enthalpy, since the interaction between the stabilising agent and the lipid matrix changes .

Example 8

Lyophilisation of nanoparticles of 1% stearic acid

The sodium stearate is dissolved in water at a concentration of 1% and 1% PVA 9000 hydrolysed to 80% is added.

The nanoparticles of stearic acid are precipitated by adding lactic acid IM dropwise under agitation and the suspensions are then cooled slowly at room temperature .

Said nanoparticles are then lyophilised for one night with and without trehalose 5% as cryoprotector. Table III below shows the data relative to lyophilised samples dispersed again in water by simple mechanical agitation (following page) .

TABLE III

As can be seen, the samples can be easily dispersed again, especially when the cryoprotector is present .

Example 9

Long-term stability of nanoparticles of 1% stearic acid stabilised with PVA 9000 80% hydrolysed

The sodium stearate is dissolved in water at a concentration of 1%. 1% PVA 9000 80% hydrolysed under agitation at 47⁰C is added to the solution obtained.

The mean diameters and the size distributions are maintained practically unchanged for over 1 month (295 nm and polydispersion = 0.05 just after preparation as against 305 nm and polydispersion 0.05 after 1 month).

Example 10

Production of nanoparticles of stearic acid loaded with tocopherol

The sodium stearate is dissolved in water at a concentration of 1%.

Subsequently 0.05% of tocopherol is added under agitation at 47⁰C, permitting solubilisation of the active ingredient. Lastly, 1% PVA 9000 hydrolysed to 80% is added under agitation.

The nanoparticles of stearic acid are precipitated by adding lactic acid .INT.,dropwise _ under agitation and the suspension is then cooled slowly at room temperature.

Particles are obtained with diameter of 350 nm with a close size distribution (polydispersion = 0.1).

The preparation carried out at low temperatures protects the tocopherol from thermal degradation and its incorporation in nanoparticles is useful for increasing its stability in both cosmetic and pharmaceutical products .

Example 11

Production of nanoparticles of stearic acid loaded with azulene

The sodium stearate is dissolved in water at a concentration of 2%.

Subsequently 0.1% of azulene is added to the solution under agitation at 47⁰C, permitting solubilisation.

Lastly, 2% PVA 9000 hydrolysed to 80% is added under agitation.

The nanoparticles of stearic acid are precipitated by adding lactic acid 2M dropwise under agitation and the suspension is then cooled slowly at room temperature.

Particles with diameter of 480 nm are obtained with a good size distribution (polydispersion = 0.15).

The incorporation of azulene into nanoparticles is useful to increase its long-term photostability in cosmetic products.

Example 12

Production of nanoparticles of stearic acid loaded with ^' amphotericin B

The sodium stearate is dissolved in water at a concentration of 2%.

Subsequently 0.05% of amphotericin B is added to the solution under agitation at 47⁰C, permitting solubilisation.

Lastly, 2% PVA 9000 hydrolysed to 80% is added under agitation.

Particles with diameter of 350 nm are obtained with a good size distribution (polydispersion = 0.17).

The DSC analysis (Figure 13) reveals a slight change in the melting temperature and enthalpy with respect to the white nanopartic1es .

The preparation at low temperatures protects the amphotericin B from thermal degradation.

The high incorporation of amphotericin B (>85% of the dose) into nanoparticles is useful for vehiculation of the drug.

Example 13

Production of nanoparticles of stearic acid loaded with AOT- Cisplatin as ion pair The sodium stearate is dissolved in water at a concentration of 1%.

Subsequently 0.05% of AOT-cisplatin, dissolved in a small -quantity of ethanol, is added to the solution at 47⁰C and then 1%. Pluronic F-68 under agitation.

Particles with diameter of 400 nm are obtained with a size distribution (polydispersion = 0.18).

The DSC analysis (Figure 14) shows a slight change in the enthalpy and in the transition temperature with loading of the drug.

The incorporation of cisplatin (>60%) into nanoparticles is useful for vehiculation of the drug and for the production of modified-release pharmaceutical forms.

Example 14

Production of nanoparticles of stearic acid loaded with cyclosporin

The sodium stearate is dissolved in water at a concentration of 1%.

Subsequently 0.05% cyclosporin, dissolved in a small quantity of ethanol, is added to the solution under agitation at 47⁰C and then 1% PVA 9000 hydrolysed to 80%.

The nanoparticles of stearic acid are precipitated by adding lactic acid IM dropwise under agitation and the suspension is then cooled slowly at room temperature. Particles with a diameter of 480 ran are obtained with a size distribution (polydispersion = 0.14).

The DSC analysis (Figure 15) shows a slight change in the enthalpy and transition temperature with loading of the drug.

Incorporation of the cyclosporin into nanoparticles is useful for vehiculation of the drug.

Claims

1. Method for the preparation of solid lipid micro and nanoparticles comprising the phase of mixing an acid solution with a micellar aqueous solution comprising at least one water soluble salt of a fatty acid in the presence of a non-ionic and biocompatible amphiphilic stabilising agent.

2. Method as claimed in claim 1, characterised in that said salt is selected from alkaline salt, ammonium salt and amine salt .

3. Method as claimed in claim 1, characterised in that said fatty acid is a fatty acid solid at room temperature.

4. Method as claimed in claim 3, characterised in that said fatty acid is selected from the group consisting of stearic acid, palmitic acid, myristic acid, lauric acid, arachidonic acid, behenic acid.

5. Method as claimed in any one of the preceding claims characterised in that said fatty acid salt is selected from the group consisting of sodium stearate and sodium palmitate, sodium myristate, sodium laurate, sodium arachidonate , sodium behenate .

6. Method as claimed in any one of the preceding claims, characterised in that said fatty acid salt is present in a concentration of between 0.1 and 30% w/w.

7. Method as claimed in claim 6, characterised in that said fatty acid salt is present in a concentration of between 1 and 5% w/w.

8. Method as claimed in claim 1, characterised in that said stabilising agent is selected from the group consisting of partially hydrolysed polyvinyl acetate, polyoxyethylene/polyoxy-propylene copolymers , polyacrylamides , ^' polyvinyl pyrrolidone and its derivatives, stabilising agents derived from polysaccharides, derivatives of cellulose, non-ionic gums, cyclodextrins and their derivatives.

9. Method as claimed in claim 8, characterised in that said stabilising agent is present in a concentration of between 0.1 and 30% w/w.

10. Method as claimed in claim 9, characterised in that said stabilising agent is present in a concentration of between 1 and 5%.

11. Method as claimed in claim 1, characterised in that said acid solution comprises at least one acid selected from the group consisting of hydrochloric acid and an acid with pKa between 2 and 6.

12. Method as claimed in claim 11, characterised in that said acid solution comprises at least one acid selected from the group consisting of acetic acid, carbonic acid, acrylic acid, lactic acid, glycolic acid, tartaric acid, maleic acid, pyruvic acid, malic acid, succinic acid, citric acid, hydrochloric acid, phosphoric acid, acid polyphosphates, acid salts of ammonium and their derivatives, amino acids, polyamino acids, polymers containing acid groups .

13. Method as claimed in claim 11 or 12, characterised in that said acid solution comprises an acid in a concentration from 0.01M to 5M.

14. Method as claimed in any one of the preceding claims, characterised in that the temperature varies between 25°C and 80⁰C.

15. Method as claimed in claim 14 , characterised in that the temperature varies between 40⁰C and 5O⁰C.

16. Method as claimed in claim 1, characterised in that said micellar aqueous solution comprises a biocompatible co- solvent selected from the group consisting of ethanol, propylene glycol, glycerol and butyl lactate.

17. Method as claimed in claim 16, characterised in that said co-solvent is present in a concentration of up to 30% w/w of the aqueous phase .

18. Method as claimed in claim 1, characterised in that said micellar aqueous solution comprises an active ingredient.

19. Method as claimed in claim 1, characterised in that said acid solution comprises an active ingredient.

20. Method as claimed in any one of the claims from 17 to 19, characterised in that said active ingredient is selected from the group consisting of antitumoral molecules, antioxidant molecules, antibiotics, metals, immunosuppressors .

21. Method as claimed in any one of the claims from 18 to 20, characterised in that said active ingredient is selected from the group consisting of azulene, amphotericin B, cisplatin, tocopherol, cyclosporin, retinol.