FR2885538A1 - Preparing solid lipid nanoparticles comprises providing membrane reactor, where aqueous phase is circulated, maintaining temperature higher than melting point of lipid phase, forming droplets and solidifying - Google Patents

Abstract

Preparing particles, particularly solid lipid nanoparticles from a lipid liquid phase A comprising a lipid or lipids mixture and a second liquid phase B in which the phase A is insoluble or partially soluble comprises: (a) implementing a membrane reactor comprising a porous membrane in which the phase B is circulated tangentially with the membrane and the phase A; (b) maintaining a temperature higher than the melting point of the phase A before passing through the membrane, forming droplets by passing through the pores that contacts the phase B; and (c) solidifying optionally after cooling.

Description

The present invention relates to a new process n- being in

  A membrane reactor is used, which makes it possible to prepare solid lipid particles, and in particular particles of nanometric size.

  In recent years, colloidal systems are increasingly used in agrochemistry, cosmetology and especially in pharmacy. In this area, sub-micron sized systems are used as therapeutic systems, such as liposomes, microemulsions and nanoparticles. These different forms have been developed to improve the performance of drugs and convey the active ingredients in the body to target cells to improve the therapeutic effect and / or reduce the toxic effect.

  Solid lipid nanoparticles were developed in the 1990s, as an alternative to nanoparticles made of polymers or natural macromolecules. Solid lipid nanoparticles are particles composed of a solid lipid matrix. In aqueous dispersion, they are stabilized by surfactants or polymers. They combine the advantages of polymeric nanoparticles, emulsions and liposomes. Like the polymeric particles, they consist of a solid matrix protecting the active ingredients incorporated in the chemical degradations and offer great flexibility to modify the release profile of the incorporated active ingredients. Like emulsions and liposomes, they consist of well tolerated and biodegradable lipids. The aqueous dispersion can be converted into dried product by spray drying or lyophilization.

  The present invention relates to the preparation of solid lipid particles and in particular nanoparticles with a diameter of less than 1000 nm.

  The main method of preparation of solid lipid nanoparticles is high pressure homogenization. The principle of a homogenizer is to push the liquid at high pressure (100-200 bar) through a narrow conduit (of the order of a few microns in diameter). The fluid accelerates over a very short distance at high speed (over 1000 km / h). A very high shear rate and the cavitation forces then penalize the formation of submicron-sized particles.

  Two homogenization techniques exist: hot homogenization and cold homogenization. The hot homogenization is carried out at a temperature above the melting point of the lipid and can therefore be considered as the homogenization of an emulsion. A pre-emulsion of the lipid mixture containing the active ingredient and the aqueous phase is made with a high shear mixer. The high pressure homogenization is then carried out at a temperature above the melting point of the lipid. A nanoemulsion is thus obtained which solidifies at room temperature and forms solid lipid nanoparticles.

  As regards the cold homogenization, the lipid mixture containing the active principle is cooled rapidly (for example by ice or liquid nitrogen). Rapid cooling promotes the homogeneous distribution of the active ingredient in the solid lipid matrix. The lipid-solid mixture is then milled to obtain microparticles and then dispersed in an aqueous solution. The presuspension is then subjected to high pressure homogenization at a temperature equal to or lower than the ambient temperature.

  For example, the patent application EP 0506197 describes the preparation of an aqueous solution of solid lipid nanoparticles, comprising at least one lipid and preferably also at least one surfactant. The solid lipid nanoparticles thus obtained have a mean diameter of between 50 and 1000 nm and their concentration in the solution is between 0.01 and 60% by weight. An appropriate amount of a solid lipid or a mixture of solid lipids is added in a heated liquid aqueous phase. A vigorous stirring makes it possible to obtain droplets of molten lipids whose diameter is between 50 and 1000 nm.

  Finally, the dispersion is cooled until the dispersed lipid droplets solidify and a suspension of solid lipid nanoparticles is obtained.

  US Patent 5,250,236 describes the preparation of solid lipid microspheres whose diameter is less than one micron. A molten lipid, which may contain an active ingredient, is brought into contact with a mixture of water, a surfactant, and if possible a co-surfactant, preheated to a temperature at least equal to the melting temperature of the lipid. The microemulsion thus obtained is dispersed in water at a temperature of between 2 and 10 degrees C. The lipid microspheres obtained can be washed with water by diafiltration, and lyophilized.

  Finally, US Pat. No. 5,188,837 describes the preparation of solid lipid nanoparticles with a phospholipid layer attached to their surface. The core of the lipid particle is a solid substance to be delivered, or a substance to be delivered which is dispersed in a solid inert compound, such as a wax. The substance to be delivered is mixed, dissolved or dispersed in a molten compound to form a liquid substance that can solidify without changing the substance to be delivered. The phospholipid is then added to an aqueous medium to the mixed substance at a temperature above the melting temperature of the substance to form an emulsion. It is then necessary to homogenize the emulsion at a temperature above the melting temperature until a homogeneous preparation is obtained, and then rapidly cool the preparation to room temperature or below the melting point of the molten compound. Dispersed in an aqueous solution, the final product is a dispersion of solid microparticles covered with a layer of phospholipid particles, the hydrophobic side of which is attached to the surface of the hydrophobic solid core and the hydrophilic side is interfaced with the aqueous solution.

  One of the essential objectives of the present invention is to provide a method for preparing solid lipid particles, and in particular nanoparticles, which is easy and cost-effective to implement on an industrial scale.

  An object of the present invention is to provide a new method for preparing solid lipid nanoparticles which implements a main step and which is simpler than the methods of the prior art.

  Another essential objective of the present invention is to provide a process for the preparation of solid lipid particles, and in particular of solid lipid nanoparticles, which is industrializable and which allows a coni: role of the size of the nanoparticles obtained, and in particular which allows obtaining nanoparticles with a diameter of less than 800 nm, preferably 600 nm, and preferably between 100 and 500 nm.

  In this context, the subject of the present invention is a process for preparing particles, and in particular nanoparticles, solid lipids, from a first liquid lipid phase A comprising a lipid or a mixture of lipids, and a second phase B liquid in which the lipid or the mixture of lipids of the first phase A is insoluble or poorly soluble, characterized in that it uses a membrane reactor comprising a porous membrane, in which the second phase B circulates tangentially to the membrane and the first phase A, maintained before passing through the membrane at a temperature above the melting point of the lipid or lipid mixture, passes through the pores of the membrane to form droplets which meet the second phase B, and solidify, possibly after cooling.

  The description which follows, with reference to the appended figures will allow to better understand the invention.

  Figure 1 illustrates, schematically, the method according to ivention at the porous membrane.

  FIG. 2 represents a device for implementing the method according to the invention.

  The process according to the invention makes it possible to manufacture, in large quantities, particles, and in particular nanoparticles of essentially lipidic nature. By nanoparticles is meant particles of generally spherical shape, of diameter less than 1 micron, in particular less than 800 nm, preferably 600 nm, and preferably between 100 and 500 nm. In the remainder of the description, the term "particle" or "nanoparticle" will be used interchangeably, in the knowledge that the invention is more particularly intended for the production of nanoparticles as defined above.

  By solid lipid nanoparticles is meant nano 3articles solid at room temperature, that is to say at a temperature of 25 C.

  In the context of the invention, to say that a lipid or melanp lipid is poorly soluble in the second phase B means that when the lipid or mixture of lipids in question is introduced into the second phase B at 25 C, less 10% of the total mass of lipid (s) introduced (s) is solubilized in the second phase B. Advantageously, the second phase B and the lipid or lipid mixtures will be chosen so that the lipid or the lipid mixtures are insoluble at 25 ° C. in the second phase B. The concept of solubility referred to is, for example, defined in the EUROPEAN PHARMACOPOEIA Seventh edition.

  The solid lipid nanoparticles of the invention may comprise a single solid lipid or a solid mixture of lipids. Therefore, the lipid or lipid mixture intended to constitute the matrix of the particle, which corresponds to lipid phase A, preferably has a melting point greater than or equal to 30 ° C., preferably between 30 ° C. and 100 ° C. and preferably between 40 and 95 C, so that the particles formed are solid at room temperature. When a lipid mixture is used, it may contain one or more lipids with a melting temperature of less than or equal to 100 ° C., the essential point being that the lipid mixture has a melting temperature which is notch; these limits.

  Examples of solid lipids at such temperatures are: highly saturated alcohols, particularly aliphatic alcohols having from 14 to 30 carbon atoms, such as cetostearyl alcohol; waxes, like carnauba wax; hydrocarbons, such as solid paraffins; synthetic esters, such as cetyl palmitate; fatty acids having between 12 and 30 carbon atoms such as stearic acid; saturated mono-, di- and triglycerides of fatty acids having from 10 to 30 carbon atoms, such as trilaurate glyceryl or castor oil.

  The particles according to the invention consist of a lipid matrix in which at least one substance S may be distributed. The lipid matrix may also be associated with at least one surfactant.

  The principle of the invention is as follows. A first lipid phase A, consisting of a lipid or a mixture of lipids to which is added, optionally a substance of interest S and / or a surfactant, is heated to a temperature above its melting point, to be in the liquid state. The first lipid phase A therefore consists essentially of a lipid or mixture of lipids which corresponds to a value of between 50% and 99% of the total volume, preferably 70% to 98% and preferably 85% to 95% of the total volume. the lipid phase A. It is then pressurized to pass through the pores of a porous membrane M, as illustrated in FIG. 1. A second phase B optionally comprising a surfactant, circulates tangentially along the membrane M. After passing through the membrane M, the lipid phase A, in the form of droplets formed by passage through the pores of the membrane, meets the second phase B. Either the second phase B is at a temperature below the melting point of the lipid or lipids constituting the first phase A, in this case, the formed droplets solidify to directly form solid lipid nanoparticles. This variant is particularly advantageous because the second phase B can be at room temperature, that is to say at a temperature of about 25 C.

  Either the second phase B is at a temperature above the melting point of the lipid or lipids, in this case the droplets formed, by pz ssage of the lipid phase A through the pores of the membrane, form an emulsion in the second phase B In this case, the solid lipid nanoparticles are formed upon further cooling of the emulsion A in B at a temperature below the lipid melting temperature (e.g., at room temperature) or the lipid mixture. forming the droplets.

  The method for manufacturing solid lipid particles, and in particular solid lipid nanoparticles, according to the invention therefore uses a porous membrane for the introduction under pressure of the lipid phase: A, brought to a temperature above the melting point of the lipid or lipids it contains in a second phase B which flows on the other side of the membrane, along the latter as shown in FIG. 1. The lipid phase A is for example placed in a thermostatically controlled container or container ensuring its passage through the pores of the membrane at the pressure and at the desired temperature. Advantageously, the second phase B is also maintained at a given temperature by a thermostatically controlled system.

  By membrane is preferably meant a porous element homogeneous throughout its thickness, this is particularly the case of polymeric membranes and ceramic supports. It could also be envisaged to use a non-homogeneous ceramic membrane on its thickness, consisting of a support and a separating layer.

  The maximum average diameter of pores adapted for the preparation of particles in accordance with the invention is preferably in the range of 1 nm to 10 μm, and preferably in the range of 10 nm to 1 μm, in the case of the preparation of nanoparticles.

  In the case of a membrane comprising a suppo -t and a separating layer, it is the pore diameter of the separating layer which is decisive.

  This separating layer will be in contact with the second phase B which will circulate tangentially to the surface of the latter.

  Any form of membrane can be envisaged: flat merr branes, tubular membranes comprising one or more circulation channels, along which circulates the second phase B. Although there are solid lipids for which a preparation according to the invention can be carried out without adding surfactant, in most cases it will be preferable to use a surfactant. Also, advantageously. the lipid phase A and / or the second phase B, will contain a surfactant.

  The concentration of surfactant (s) may vary with the type of lipids and surfactants used. The mass percentage of surfactants in the final suspension of nanoparticles obtained can be between 0.01 and 20%, preferably between 0.1 and 10%, more preferably between 1 and 5% of the total mass of the nanoparticle suspension. obtained.

  A wide variety of surfactants can be used, the hydrophilic / lipophilic HLB (HLB: hydrophilic / lipopailic balance) is between 2 and 80. Preferably, the HLB is between 8 and 40. The choice of surfactant depends on the solid lipid phase used. For example, the following surfactants may be used: cationic surfactants, such as cetyltriethylammonium bromide; anionic surfactants, such as sodium lauryl sulfate; amphoteric surfactants, such as imidazoline hydroxyethyl (VARINE); copolymers, such as polyoxyethylenepolyoxypropylene alkyl ether (e.g. PLURONIC F68); nonionic surfactants, such as polyoxyethylene sorbitan esters (eg TWEEN 20), polyoxyethylene alkyl ether (eg BRIJ 97 and CETOMACROGOL 1000), polyoxyethylene esters (eg MYRJ 52), sorbitan esters (eg SPAN 80) sucrose esters (eg WASAG ESTER 7); and other suitable surfactants, such as lecithins, silicone surfactants, and polyglycerol esters.

  Preferably, nonionic surfactants are used. More preferably, the surfactants are selected from the group consisting of polyoxyethylene alkyl ethers and sorbitan esters.

  In the lipid phase A, the surfactants whose HLB is less than or equal to 7, and in the second phase B, which is preferably aqueous surfactants whose HLB is greater than or equal to 7, will be used.

  A mixture of different surfactants can also be used.

  Advantageously, the second phase B, intended to constitute the continuous phase of the suspension of solid lipid particles prepared according to the present invention, consists essentially of water, or a mixture of water and one or more solvents. nonaqueous polar liquid, for example chosen from alcohols, such as ethyl alcohol, glycerol, propylene glycol; and pyrrolidones, such as N-methyl pyrrolidone and 2-pyrrolidone.

  The lipid phase A contains, advantageously, a substance S in solution or in suspension in the lipid or mixture of lipids. The substance to be incorporated S can be any substance soluble or dispersible in the lipid or the mixture of lipids chosen. The substance S can be a biologically active substance, for example a molecule that can be used as a drug active ingredient or as a precursor of a drug active ingredient, or a contrast agent or a biological reagent. The substance S can also be a pigment, an ink, a lubricant, a surface treatment agent. It is also possible to use as substance S a mixture of the above substances. The amount of substance S is a function of its solubility in the lipid phase. As substances which can be incorporated in the lipid phase A, there may be mentioned as an example of vitamin, vitamin A, as an example of anti-inflammatory agent, indomethacin; as an example of antioxidant agent vitamin E. Finally, the concentration of solid lipid nanoparticles obtained in the solution, according to the method of the invention, is between 0.02%, preferably between 5 and 45%, more preferably between 10 and 30% (mass percentage of the nanoparticles relative to the total mass nanoparticles + continuous phase).

  The particle size obtained is controlled by appropriate choice of membrane-bound parameters and process parameters. In particular, those skilled in the art will be able to choose, depending on the desired size, on the one hand the material of the membrane used and on the other hand its porosity and its pore diameter. Different types of membranes can be used: ceramic membranes, mineral or organic membranes, for example. Also, a wide range of pore diameter may be used: nanofiltration membranes which have a pore diameter of less than 1 nm, ultrafiltration membranes which have a pore diameter of between 1 and 100 nm or microfiltration membranes which have a pore diameter of between 0.1 and 10 m. The choice of the membranes used will affect the size (d) of the particles and the flow (.1) of the lipid phase A. Of course, in order to obtain homogeneously sized particles, membranes will be used whose Pore diameter is as homogeneous as possible.

  It is also possible, to control the size of the particles obtained, to play on the process parameters which are the pressure (P) of the lipid phase A and the tangential velocity (U) of the second phase B. A wide range of pressure and tangential velocity can be considered. Typically, a pressure of 0.1 bar to bar, preferably 0.3 to 15 bar, for the lipid phase will be used. In general, a tangential velocity of 0.001 to 20 m.sup.-1, preferably 0.01 to 10 m.s.sup.-1, for the second phase B will be used.

  Moreover, it is possible to play along the length of the membrane, or more generally on the membrane surface crossed by the lipid phase A, to increase the production yield.

  It is also possible to have several reactors or membrane modules in parallel.

  In view of the above, it appears that the method according to the invention, despite its simplicity, is suitable for the preparation of different types of solid lipid particles. In addition, it allows to prepare large volumes continuously and is therefore ideally suited to industrialization. The method according to the invention has a very good adaptability, it allows to adjust the size of the particles. For example, to change the size of the particles obtained, it will be possible to modify the formulation, and / or to change the pressure to be applied to the lipid phase A, and / or to change the tangential flow rate of the second phase B. The following examples allow to illustrate the invention but have no limiting character.

Example 1

  The experimental setup used in this example is shown in FIG. 2. It comprises: a first pressurized container 1 containing the lipid phase A, and connected to a nitrogen bottle 2 and equipped with a manometer 3; this container 1 is heated to a higher temperature, at the melting point of the lipid, for example by means of a bath 11 whose temperature is regulated by a thermostat 12, - a second container 4, containing the second phase B, and equipped with an agitator 5 and connected to a pump 6; this second container 4 is kept at a constant temperature, for example by means of a bath 41 whose temperature is regulated by a thermostat 4 :, - a tangential filtration module 7 equipped with its pressure gauges 8 and 9 and a valve 10 , connected to the first and second containers so that the second phase B flows tangentially to the membrane and the lipid phase A passes through the membrane to join the second phase B. The assembly is carried out in a closed circuit, that is, that is to say that at the outlet of the membrane, the colloidal suspension is remixed to the second phase B. It could also be envisaged to carry out the assembly in open circuit and to put at the outlet of the membrane an evacuation circuit to another container, the flow can be controlled by a valve.

  The membrane used is a Kerasep membrane (Orelis, France) with a pore diameter of 0.1 m. The effective membrane area is 7.5-10-3 m. The tangential velocity is equal to 1.68 ms-1 for the second phase B and the transmembrane pressure equal to 6 bar for the lipid phase A. The preparation is evaluated in terms of average particle diameter, measured on a Zetas zer (France) , and lipid phase flow.

  Different formulations were tested. For these different formulations, the composition of the lipid phase A and the second aqueous phase B are as follows: Formulation 1 Lipid phase A = 300 g of Gelucire at the temperature of 65 C aqueous phase B = 1.2 L water + 2 , 04 g Tween 20 at the temperature ce 60 C Formulation 2 Lipid phase A = 300 g Gelucire at the temperature of 65 C Aqueous phase B = 1.2 L water +2.04 g Tween 20 +2.04 g 13pikuron 200 to the temperature of 60 C Formulation 3 Lipid phase A = 300 g Gelucire + 3 g Span 80 at the temperature of 65 C Aqueous phase B = 1.2 L water +2.04 g Tween 20 at the temperature of 60 C Formulation 4 Phase lipid A = 300 g Gelucire +31.25 g Lipoid S100 (2%) at a temperature of 65 C Aqueous phase B = 1.2 L water +31.25 g Tyloxapol (2%) at a temperature of 60 C The results The lipid phase flow is high (up to 0.27 m 3 / hr 2), which confirms the potentialities of this process for industrial application. The average diameter of the particles can be modified by the choice of the formulation (mean diameter between 175 and 260 nm) .Tablet 1 Particle Size (nm) Phase.ipidic Flux Formulation 1 175 0.2 Formulation 2 180 0.25 Formulation 3,215 0.25 Formulation 4,260 0.27

Example 2

  The experimental set-up is represented on the Figur (2. The membrane used is a Kerasep membrane (Orelis, France) with a diameter of 0.1 m, the useful membrane surface is 7.5 10 m 2. The tangential velocity is equal to 1 , 68 ms-1 for the aqueous phase B and the transmembrane pressure é at 6 bar for the lipid phase A. The lipid phase contains 300 g of Gelucire at the temperature of 65 C, and the aqueous phase 1.2 L water and 2.04 g Tween 20 at a temperature of 60 ° C. The preparation is evaluated in terms of the average zzeta portion diameter, measured on a Zetasizer (France), and the lipid phase flow.

Table 2

  Temperature of Particle Size Flux phase lipid aqueous phase (C) (nrn) (m3.IL.-I.m2) 125 0.26 175 0.21 190 0.20 70 0.20 80 0, 20 The results obtained are reported in Table 2. lipid phase flow are again high (up to 0.26 m3 / h.m2). The average diameter of the particles can be modified by the choice of the temperature of the aqueous phase (mean diameter between 70 and 190 nm).

  DBF 05 04 883 Reply to the notification of 03.08.05

Claims (9)

CLAIMS:   1 - Process for preparing particles, and in particular nanoparticles, solid lipids, from a first liquid lipid phase A comprising a lipid or a mixture of lipids, and a second liquid phase B in which the lipid or mixture of lipids of the first phase A is insoluble or poorly soluble, characterized in that it uses a membrane reactor comprising a porous membrane, in which the second phase B circulates tangentially to the membrane, and the first phase A, maintained before passing through the membrane at a temperature above the melting point of the lipid-bu of the lipid mixture, passes through the pores of the membrane, to form droplets that meet the second phase B, and solidify, optionally, after cooling.   2 - Preparation process according to claim 1 characterized in that the lipid or the lipid mixture constituting the lipid phase A has a melting point greater than or equal to 30 C, preferably a melting point between 30 and 100 C and preferably between 40 and 95 C.   3 - Preparation process according to claim 2 characterized in that the lipid or lipids of the lipid phase A are chosen from: aliphatic alcohols having between 14 and 30 carbon atoms, such as cetostearyl alcohol; waxes, like carnauba wax; hydrocarbons, such as solid paraffins; synthetic esters, such as cetyl palmitate; fatty acids having from 12 to 30 carbon atoms, such as stearic acid; saturated mono-, di- and triglycerides of fatty acids having from 10 to 30 carbon atoms, such as trilaurate glyceryl or castor oil.   4 - Preparation process according to one of the preceding claims characterized in that the second phase B is aqueous.   - Method according to one of claims 1 to 3, characterized in that the second phase B consists essentially of water, or a mixture of water and one or more polar nonaqueous liquid solvents, for example selected from alcohols, such as ethyl alcohol, glycerol, propylene glycol; and pyrrolidones, such as N-methyl pyrrolidone and 2-pyrrolidone.   DBF 05 04 883 Reply to the notification of 03.08.05 6 - Preparation process according to one of the preceding claims characterized in that the second phase B is maintained at a temperature below the melting point of the lipid or mixture of constituent lipids of the lipid phase A, so that the droplets of lipid phase A formed solidify in contact with the second phase B. 7 -Process preparation according to one of claims 1 to 5, characterized in that the temperature of the second phase B corresponds to the ambient temperature, of the order of 25 C.   8 - Preparation process according to one of claims 1 to 5, characterized in that the second phase B is maintained at a temperature above the melting temperature of the lipid or mixture of lipids constituting the lipid phase A, so as to the lipid phase droplets A formed form an emulsion in the second phase B and in that the formed droplets solidify in a subsequent cooling step of the formed emulsion.   9 - Preparation process according to one of the preceding claims characterized in that at least one surfactant is contained in the lipid phase A and / or the second phase B. - Preparation process according to the preceding claim characterized in that at least a nonionic surfactant, for example selected from the group of polyoxyethylene alkyl ethers and sorbitan esters, is used.   11 - A method of preparation according to one of the preceding claims characterized in that the lipid phase A contains a substance S in solution or in suspension in the lipid or mixture of lipids, preferably selected from a drug active ingredient, a product of contrast, a biological reagent, a pigment, an ink, a lubricant, a surface treatment agent.   12 - Preparation process according to one of the preceding claims characterized in that the particles formed have a diameter of less than 600 nm, preferably 500 nm, and preferably between 200 and 350 nm 13 - Preparation process according to one of the preceding claims characterized in that the membrane used has a pore diameter of between 1 nm and 10 m, preferably between 10 nm and 1 m.   14 - Preparation process according to one of the preceding claims characterized in that a pressure of 0.1 bar at 50 bar, preferably from 0.3 to 15 bar, is applied to the lipid phase A. -Procedure according to one of the preceding claims, characterized in that the second phase B flows along the membrane, at a tangential speed of 0.001 to 20 ms -1, preferably of 0.01 to 10 ms -1.