WO2007028421A1

WO2007028421A1 - Process for the production of nano-particles

Info

Publication number: WO2007028421A1
Application number: PCT/EP2005/054489
Authority: WO
Inventors: Alberto Bertucco; Paolo Caliceti; Nicola Elvassore
Original assignee: Universita' Degli Studi Di Padova
Priority date: 2005-09-09
Filing date: 2005-09-09
Publication date: 2007-03-15

Abstract

Process for the preparation of solid micro- or nano-particles, comprising the following steps: melting a solid component; solution, under pressure, of a first compressed gas into the molten solid component, to create a pressurised semisolid or liquid mixture; generating a dispersion of the semisolid or liquid mixture with a second compressed gas, which does not completely dissolve into the semisolid or liquid mixture; expansion of the dispersion obtained at the previous step with evaporation of the first gas and formation of micro or nanosized particles of the solid component. An apparatus for performing said process is also provided.

Description

PROCESS FOR THE PRODUCTION OF NANO-PARTICLES Technical field

The present invention relates to a process for the preparation of solid particles, more in particular solid nano-particles. Background art

Micro- and nano-particles find a variety of technical applications, for example in the field of pigments, or in the preparation of pharmaceutical, cosmetic or chemical products.

Processes are known for the preparation of micro-particles of solid substances, such as lipids, fats, polymers, based on the dissolution of a compressed or nearly critical or supercritical fluid, such as carbon dioxide, into the solid component to yield melted or softened substances, and on the subsequent atomisation of the material thus obtained by means of nozzles into a spray tower, whereto a stream of washing gas may be fed. The solution of the gas into the solid contributes to decrease its viscosity. The cooling effect due to the expansion and vaporisation of the gas into the precipitation chamber generates solid particles, which are collected from the gas stream leaving the spray tower, for example by means of mechanical filters, cyclones or electrofilters. The choice of carbon dioxide is generally advantageous because of its low cost, non-toxicity, non-inflammability and its easy removal from the solid component into which it has been previously dissolved, due to its gaseous state under ambient conditions. Other compressed gases, suitable to be dissolved into solid components to be micronized, may be employed. Actually, the use of the carbon dioxide as compressed, nearly critical or supercritical fluid is limited by the fact that it does not posses a dipole moment and then it can be dissolved in a relatively few solid substances. Nevertheless, carbon dioxide is highly soluble in non-polar or poorly polar or organic compounds.

A problem connected to the existing processes resides in that the efficiency of the atomisation step is limited by the physical characteristics of the processed material (melting temperature and pressure, viscosity, density) to be micronised. These characteristics are, in turn, bond to the solubility of the gas into the solid substances and to the operating conditions of the dissolution step (pressure, temperature). This limits the choice of the operative conditions of the process as well as the kind of solid substances that can be treated. Consequently, the results in terms of particle size and morphology of the obtained product are not always satisfactory. In order to overcome the above explained limitations, it has been proposed to atomise solutions of solid substances in organic or other kinds of solvents into streams of carrier gases and then to evaporate the solvent by heating; a subsequent cooling is then necessary. Other processes foresee the dissolution of the supercritical compressed gas into a solution of the solid to be treated. These processes bring about the use of organic toxic solvents, which must be removed from the product by specific process steps. The use of solvents may be undesirable because it entails the risk of inactivation of the active ingredients, incomplete solvent removal may achieve toxic effect, solvent elimination is expensive and can provoke environmental pollution . It would be desirable a process for the preparation of micro- and nano-particles allowing a wider freedom of choice of the operative conditions and of the solid component that may undergo the process, without the need of adding liquid solvents to the solid to be micronised/atomised. Therefore, it is one purpose of the present invention to provide a process for the preparation of solid particles, being said process suitable to produce micro- and preferably nano-sized particles. Another purpose of the invention is to provide a process for the preparation of solid particles avoiding the use of any solvent and in particular organic solvents. It is a further purpose of the invention to provide a process for the preparation of solid particles operative at low temperature in order to be implemented also with thermolabile substances.

In addition the process is easily controllable by a separation of melting and atomization process and is easily scalable at high. High yield of dry product without further down-stream treatment Summary of the invention The above exposed problems have been solved and the purposes of the invention are fulfilled by a process where for the pulverisation of substances to be treated, consisting in one single substance or also in a mixture of substances and forming a solid component, compressed gas or nearly critical compressed or supercritical fluid are used.

Therefore it is an object of the present invention a process for the preparation of solid micro- and, preferably, nano-particles, comprising the following steps: - melting a solid component

- solution under pressure of a first compressed gas into the molten solid or solid component, to create a pressurised semisolid or liquid mixture;

- dispersion of the semisolid or liquid mixture in a stream of a second compressed gas, which does not completely dissolve into the semisolid or liquid mixture; - expansion of the dispersion obtained at the previous step with evaporation of the first gas and formation of micro- or nano-sized particles of the solid component.

The steps of melting the solid component and of solution of the first compressed gas may preferably take place at least in part contemporaneously, more preferably in the same reactor. The solution of the gas into the component, actually lowers its melting point, thus facilitating the whole operation.

Further objects of the present invention are an apparatus suitable to perform the process above described and the solid micro- and nano-particles obtained thereby. Brief description of the drawings.

The invention will now be better illustrated with the description of preferred, but not exclusive, embodiments with the aid of appended drawings, of which:

- figure 1 schematically represents a layout a process according to which the present invention is performed; - figure 2 schematically represents a more detailed layout of an apparatus in which a process according to the present invention is performed;

-figures 3a, 3b and 3c schematically represents a dispersing device in top view in figure 3c, and in the longitudinal section along section planes A-A and B-B of figure 3c in figures 3a and 3b respectively; -figure 3d shows an expanding device comprising an atomisation nozzle in longitudinal section;

-figure 3e shows a perforated annular plate to be connected to the lower surface of the device of figures 3a, 3b and 3c, to act as a diffusor for a washing gas;

- figure 4 represents the release profile of estradiol expressed in percentage over time in hours from solid particles prepared by the sample 2 of example 1 with the process; - figure 5 represents the release profile of insulin expressed in percentage over time in hours from solid particles by the sample 2 example 1 with the process. Detailed description of preferred embodiments

The objects as well as features and advantages of the present invention will be better understood from the detailed description and from the preferred embodiments, which are given for illustration and have no limiting purposes, set forth below.

The present invention provides a process for the preparation of solid nano-sized particles wherein one component, substantially solid, is the substance or substances to be treated and other components are compressed gases or nearly critical compressed or supercritical fluids used in two different steps. Firstly a mixture is prepared by dissolution of the compressed gas or nearly critical compressed or supercritical fluid in the substance or mixture of substances to be treated. Afterwards, a compressed gas is used to assist the atomisation of the sprayed material in a precipitation chamber. The solid component may consist in a single substance to be treated or in a mixture of substances having different properties and/or functions and may be charged for example with one or more active ingredients before of the second compressed gas. According to an embodiment of the invention, the active ingredients are added before the melting of the solid. However, the active ingredient can be added continuously into the liquid stream by a high pressure pump. This step is preferably before the atomization and the addition of the second compressed gas. Melting of the solid component may be performed by mild heating; preferred solid components are those solid at ambient temperature and having a melting point up to 60⁰C. The first compressed gas or supercritical or nearly critical compressed fluid has to be soluble into the substance or mixture of substances to be treated and has preferably a solubility into said component of at least 0.4 molar fraction and preferably at least 0.9 molar fraction. According to a preferred embodiment it is a supercritical or nearly critical fluid ("nearly critical" commonly designates a fluid whose pressure and absolute temperature fall within 10% of its critical pressure and temperature). The mixture consisting of the molten substance or mixture of substances to be treated and the first compressed gas may be prepared either in batch or in continuous. The first compressed gas is dissolved, preferably at saturation, in the molten solid under pressure preferably under stirring in batch preparation or in a static mixer under flow in continuous preparation.

Therefore, the mixture so prepared is dispersed with a second compressed gas or nearly critical compressed or supercritical fluid. This second compressed gas is at least partly insoluble into the mixture created in the previous step. It is preferably a compressed nearly critical or supercritical fluid, under process conditions, as well and it may be preferably insoluble into the solid component (i.e. having a solubility lower than molar fraction of 0.1). This gas is introduced into the semisolid/liquid mixture formed by the molten solid and the first gas, and is preferably different from the first gas. In the case the first and the second gas are the same one, it is introduced into the semisolid/liquid mixture in a quantity above its solubility in the molten solid. The semisolid/liquid material is sprayed through a nozzle by rapid de- pressurization and the atomisation is assisted by the flow stream of the second gas.

In detail, the process of the invention may comprise the following steps: a - melting the substance or mixture of substances to be treated by heating b - selection of a first compressed gas having a low boiling point, preferably below 20⁰C, more preferably below 0⁰C, vapour tension at 35°C under 400 bar and high vaporisation enthalpy; c - addition of the first compressed gas selected to the molten substance or mixture of substances to be treated obtaining a pressurised semisolid or liquid mixture, preferably contemporaneously with step a; d - control of the temperature of mixture previously obtained, said temperature being as lowest as possible but over the solidification temperature of said mixture; e - optionally addition of one or more actives principle by means of a high pressure injection system; f - transfer by a high pressure pump or equivalent means olthe pressurized mixture to a pre-atomisation system; g - selection of the second compressed gas for the atomisation step; h - transfer of the atomisation gas to the injection system through suitable means; i - control of the operative conditions, in particular with reference to pressure, temperature and fluid load in the atomisation system; j - dispersion of the mixture in the atomisation gas flow in a pre-atomisation chamber; k - atomisation of the dispersion previously obtained by one or more nozzles or equivalent means suitable to produce micro- or nano-particles;

I — solidification of the particles obtained through expansion and evaporation of the first compressed gas; m - collection of the solid particles by any suitable means (for example, cyclones, filters, electrofilters), and ; n - optionally separation of the coarse material by centrifugation or filtration of the product suspended in a suitable liquid, namely water or physiological solution. With reference to figure 1 , molten solid from a source 1 and a first compressed gas, is fed to a saturator 4, which can be a mixing device, working under pressure, that may be static or may have moving parts to improve mixing. The first gas may come from a source 3, for example a gas bottle and may, if necessary, be fed at a suitable pressure by means of a compressor 2; after a suitable residence time in the saturator, the molten mixture, containing the dissolved first gas is sent to a dispersing device 5, for example a dispersing chamber, a coaxial pipe, a static or dynamic mixer, a device equipped with ultrasound dispersing means, whereto a second compressed gas, from a source 7 (ex.: gas bottle) is also fed; the dispersion thus obtained is expanded through an expanding device 9, for example a nozzle o plurality of nozzles able to pulverise the mixture in order to obtain particles of micro, or preferably nano-metric dimensions. Upon evaporation of the first gas, and, possibly, thanks to the cooling effect due to the expansion of the second gas (Joule-Thomson effect) temperature is reduced and the particles solidify. The particles are separated from the gases, and collected by means of any suitable separator of known type. It has been found that the addition of a second gas, to create a heterogeneous dispersion, before expansion, allows to better select the physical characteristics of the fluid to be expanded, in order to obtain smaller size of the particles, and an improved morphology, without the need to use liquid solvents.

The solid that may be processed is preferably a solid with a melting point comprised between 0 and 100⁰C, preferably is solid at ambient temperature and has melting point up to 60 ⁰C. This includes solid components generally used in the preparation of micro- or nano-particies; examples are pigments, polymers including polyethylenglycol (PEG) and polymethylmethacriiate (PMMA), lipids, fats, polyols, mono or diglycerids, pharmaceutical compounds (for example nifedipine, lidocaine, chloralio hydrate). It may be a mixture of substances in order to give a solid with the desired characteristics.

The processed solid is constituted by a carrier with active ingredient(s) dissolved therein, which is common in the pharmaceutical field; for example lipidic nano- particles are commonly used to vehiculate active molecules or diagnostics into the body, due to their suitability to prepare stable colloidal solutions. The active ingredients may be dissolved into the solid before or during the process according to the present invention. Other ingredients may also be introduced, such as surfactants, co-surfactants, stabilisers.

With regard to the first gas it is a gas soluble into the molten solid, at process conditions, preferably it has a solubility of at least of 0.4 molar fraction and preferably at least 0.9 is actually solved in this solution step. The conditions under which solution is performed may vary due to the characteristics of the substances involved; typical pressures may range between 10 and 20 MPa and temperatures from 298K and 398K, preferably below the melting point of the pure solid substance, and more preferably in the range from 40K below the melting point of the pure solid component and the melting point of the pure solid substance. Pressure and temperature upon the expansion, i.e. right before the expanding device, for example a nozzle, may substantially be in the same ranges. As above explained, the gas is preferably nearly critical or supercritical. Examples of preferred gases are carbon dioxide, fluorocarbons, fluorinated hydrocarbons, chlorofluorocarbons. The choice can depend on the characteristics of the solid and thus the operative conditions of the process. For example, the use of certains fluorocarbons, hydrofiuorocarbons, chlorofluorocarbons, hydrochlorofluorocarbons may lower the melting point (at a fixed pressure) of substances commonly used for producing micro or nano particles (for example tristearin and mixtures thereof), or in alternative allows to perform solution at lower pressures. The lowering of the melting temperature with the pressure increase may also be more intense with such gases with respect to carbon dioxide. Moreover, chemical inertia may also be a choice criterion: carbon dioxide may have acid reaction in presence of water, and thus may be unsuitable for sensitive components. On the other hand, carbon dioxide may be preferred for its low cost and its physical properties. Preferably, the second gas is different from the first gas and it is insoluble into the molten solid, or at least into the mixture produced in the solution step (for example if a saturated solution is produced and the second gas is the same as the first one). Preferred gases for this purpose are low cost gases such as air, nitrogen, oxygen or carbon dioxide. The dispersion may be performed at similar pressure and temperature conditions than those of the solution step, or may be varied according to particular needs, bearing in mind that the dispersion step is performed before the expansion, and the components of the dispersion must be kept in a fluid state. Parameters to be taken into account are the viscosity of the fluid (semisolid/liquid) to be expanded, the surface tension, the density of the gas, and any other parameter having influence on the size and on the morphology of the particles generated.

The process can be carried out as batch or continuous process; it is particularly suitable to be carried out as a continuous process also in large size production plants.

Figure 2 shows a layout of an apparatus for performing the method according to the present invention. A source, such as a gas bottle 3 of the first gas is provided, and a compressor 2 is used to circulate it and feed it, through gas feed line, to the saturator 4, which may be provided with mixing means, such as a stirrer 10, for example a magnetically driven stirrer, to dissolve a desired amount of the first gas. The introduction of the solid as well as that of the gas may be performed continuously or according to a batch operation. The amount of the gas amount can reach the saturation limit, if this is desired and must impart the physical characteristics to the solution required by an engine 11. The solid to be micronised is also fed to the saturator 4, where it dissolves the desired amount of the first gas the amount may reach, if desired, the saturation limit, and must impart to the mixture the desired physical and rheological characteristics. Means are preferably provided to keep the temperature inside the saturator 4 at a desired level, for example electric resistors, or a line 13 for a thermostatic fluid, circulating around the walls of the saturator 4 and being heated in the thermostatic tank 14, for example by electric resistors 15. The liquid mixture obtained in the saturator is sent through discharge line 16 to dispersing device 5, by means of suitable circulating and dosing apparatus, for example by means of a high pressure syringe pump 22, capable of aspirating an amount of mixture from saturator 4 and injecting it to the dispersing device 5, with the aid of the valves 50 and 50'. The syringe pump may be a dual syringe pump and may be capable to deliver a constant quantity of the mixture, without interruption; suitable syringe pumps are manufactured for example by ISCO and can be also dual pumps, capable of sustaining continuous processes. The mixture reaches the dispersing device 5, whereto the second gas, from source 7, e.g. a gas bottle, is also sent through feed line 18, possibly by means of a compressor 8. The dispersing device may be provided with means for temperature control 24. The liquid-gas dispersion created in dispersing device 5 is expanded to a pressure close to the atmospheric pressure into spray tower 19, through expanding device 9 that may comprise a nozzle or a structure with a plurality of nozzles or holes, for example a plate with a series of holes. For example, the nozzles or holes may have diameters between 50 and 500 μm, for example 150-200 μm, to produce nano-particles. The differential pressure across the expanding device 9, as well as other parameters, such as dimensions and shape of the expanding device, and hence fluid velocity through the device, are such that a pulverisation of the liquid carried out by the dispersion is provoked in order to obtain, by expansion and evaporation of the gases, solid particles having the desired equivalent diameters. The process of the invention allows to obtain micro-particles (up to several hundreds μm) or preferably nano-particles, for example diameters in the range 100-900 nm. The solid may be collected in any known way, for example a collecting chamber 20 may be provided on the bottom of the spray tower 19. The expanded product may be subjected to removal of the expanded first and second gas, for example by means of a stream of washing gas; for example may be fed to the spray tower 19 by means of a circulating compressor 21. Figure 3a, 3b and 3c show a dispersing device 5 suitable for use in a process according to the invention. The dispersing device presents a main duct 26 for the passage of the liquid mixture, fed by all line connecting the device to the saturator 4. A secondary duct 27 is provided to feed the second gas into the main duct, and thus into the stream of liquid or semisolid mixture. The main duct is preferably provided with a needle 29 placed coaxially with the duct, preferably along its whole length. The needle imparts an annular section to the duct and, while allowing a sufficient cross section, permits to obtain a more uniform velocity profile and turbulent flow. Both the ducts may be provided with suitable means to connect them with the lines upstream for supplying the liquid and the second compressed gas, such as threaded inlet parts 28 and 30. The main duct 26 leads to the expanding device 9. A possible implementation of the latter is represented in figure 3d. The expanding device can be connected to the disperding device in any suitable manner. For example it can be screwed into the seat 32 purposely provided at the end of the main duct, by its threaded part 33. Its head 34 can be engaged for example by a tubular key, and can be placed into the bore 43. The disperding device 5 ( see figure 2) may be connected to the top of the spray tower 19, for example screwed by the thread 35, so that the gas-liquid mixture is sprayed into the tower. The disperding device 5 may also be provided with means for feeding a washing gas into the tower 19. The washing gas, preferably an inert gas, such as air has also the aim to avoid that the atomised mixture or the formed particles impinge on the tower's walls. The means for feeding said washing gas may comprise a tertiary duct 31 , provided with means for connecting it to the source of gas, which leads to an annular groove 36 on the bottom surface 37 of the dispersing device 5, surface overlooking the inside of the spray tower. The groove surrounds the seat of the expanding device, and can be closed by the perforated (having a series of preferably equidistanced holes 38) annular plate 39 of figure 3e, thus acting therewith as a distributor, distributing the gas all around the atomised stream, thus preventing impingement on the walls. The expanding device 9 comprises preferably, as already said, a nozzle 46. The latter can be suitably placed in a channel 38 passing through the device, and connecting the duct 26 to the spray tower. The channel may end with a cone shaped opening downstream of the nozzle, to facilitate the widening of the atomised stream. The dispersing device 5 may comprise, as above explained, means for temperature control. These can comprise, for example, one or more (for example three) electric resistors 40, 40', conveniently placed around the main duct 26, for example each in a hole 41 , 41' provided in the top surface of the dispersing device and parallel to the main duct 26. As previously said, the solid to be micronised may comprise other components dissolved; for example may comprise active pharmaceutical ingredients. Such components may be dissolved into the solid before it is subject to the process described, in any known way, or they can be fed to the molten solid or to the solution comprising the molten solid or to the dispersion of said solution in the second gas at any point of the process it is found suitable. For example it can be fed into the saturator 4, or it can be added to the first gas, for example by bubbling the latter through the component or a solution thereof. Such components may also be fed into the liquid or semisolid mixture after it has been formed, before the introduction of the second gas. This embodiment is particularly suitable when thermolabile components must be added, since it may reduce the time during which the component must be kept at temperatures needed to perform the various process step, or it allows to increase the temperature in the saturator 4 to accelerate the melting of the solid, temperature that may be decreased appropriately after the solution step. Figure 2 shows other temperature control devices, such as thermostatic jackets or electric resistors that can be provided to control the temperature of the various line. Such control devices, as well as other control devices (e.g. pressure control devices) can be provided according to the knowledge of the skilled in the art and do not need more detailed description. Also a series of flow regulating valves and devices (such as compressors or volumetric pumps can be provided). The invention relates also to an apparatus as described above. The preparation of particles from solid components consisting in a mixture of substances hereinafter described are provided to demonstrate that the process according to the present invention fulfils the purposes of the present invention. Further implementation or adaptations as well as embodiments readily apparent to those skilled in the art are to be considered within the scope of the present invention.

Experimental part

Example 1 : preparation of samples of the solid component to be treated

Different samples to be micronised were prepared. These samples were obtained by mixing substances with different physico-chemical characteristics either in the presence and absence of suitable organic solvents. The substances employed were mixed in different weight/weight ratios and were selected from lipids, amphiphiles, surfactants, a variety of model molecules in order to evaluate the loading degree and release rate from the particles prepared with the process. In the case of use of organic solvents the selected substances were dissolved in the organic solvent and the mixture kept under stirring in order to obtain a homogeneous mass and eliminate the solvent by heating. Then the mixture was cooled under stirring.

In the case of preparation of samples in absence of organic solvents, the substances were melted at the melting point and then kept under stirring in order to obtain a homogeneous mass. Any insoluble substance was added after dissolution in a minimum volume of auxiliary solvent subsequently eliminate by heating. The homogeneous mass was then cooled under stirring. The compositions of the samples prepared are summarized in the following table 1 : Table 1

The samples of 2 g. of lipidic mass were prepared as follow: a - samples 1-8: phosphatidyl choline was dissolved in 5 ml of CH₂CI₂ and then the remaining substances (triglycerides, Tween 80 and dioctyl sulfosuccinate) were added to the organic solution. The mixture was kept under stirring at 45⁰C to eliminate the solvent CH₂CI₂. A 100-500 μl solution of selected model molecules (estradiol in methanol in samples 1-4, 9, 10; fluorescein in dimethylformamide in samples 1-4, 9, 10; p-nitroaniline in acetone in samples 1-4, 9, 10; a protein such as insulin in dimethylsuloxide in samples 1-4, 9, 10 or dimethylformamide in samples 1-4, 9, 10; 7-hydroxycoumarin in samples 1-4, 9, 10) were added to the molten mass. The ratio from model molecules and lipidic mass was 5:100 w/w (5% w/w). The mixture was kept under stirring in order to obtain a homogeneous mass and let to solidify slowly always under stirring. b - samples 1-8 : triglycerides, Tween 80 or dioctyl sulfosuccinate were mixed at the melting point. Then phosphatidylcholine was added in 2 ml of CH₂CI₂ and the mixture was kept under stirring up to complete elimination of the solvent. A solution of the model molecules was added as before and the mixture was kept under stirring in order to obtain a homogeneous mass and let to solidify slowly always under stirring. c - samples 9-10 : triglycerides, Tween 80 and dioctyl sulfosuccinate were mixed at the melting point. Similarly to example 1 a, a solution of the model molecules was added as before and the mixture was kept under stirring in order to obtain a homogeneous mass and let to solidify slowly always under stirring.

Example 2: preparation of particles of the solid component of the samples 1-10 Particles of the solid components prepared in example 1 were obtained by the following process. The process was performed in an apparatus like the one of figure 2, equipped with the dispersing and expanding devices like those of figure 3a, 3b, 3c, 3d and 3e. The solid was introduced into the saturator 4, after it was brought to the temperature, under which the preparation of the liquid mixture was performed. The stirrer 10 was started. By suitable means, such as lines for the heat exchanging liquid of the thermostatic tank 14 and suitably placed electrical resistors, the saturator the lines connecting it to the dispersing device, the syringe pump 22, the dispersing device 5, as well as the lines for feeding the gases, were brought and kept to the operative temperature. By means of compressor 2 the first gas, coming from gas bottle 3, was fed to the saturator to pressurise it. The solid was left in contact with the first gas for 1 hour under the mixing conditions thus created, to allow the solid met and the liquid to be saturated. After this time, feeding of the second gas and of the washing gas was started by means of the compressors 8 and 21 , to the dispersing device. The injection lines for the molten mixture were kept at the same temperature of the nozzle of the dispersing device. An amount of the mixture was aspirated by the syringe pump 22 and then fed to the dispersing device under constant pressure and flow rate. This is performed also by suitably operating the valves provided on the lines connecting the saturator, the syringe pump and the dispersing device. After the amount of mixture aspirated by the syringe pump is fed to the dispersing device, the syringe pump may be reloaded, or the apparatus may be depressurised. The solid particles were collected in chamber 22 at the bottom of the spray tower 19. Operative parameters were as follow:

Nozzle temperature: 50⁰C

Saturator temperature: 45 ⁰C

Saturator pressure: 15 MPa Stirrer speed: 200rpm

Saturation and mixing time 1 h

Pressure of atomisation and of the second gas: 16 Mpa

Temperature of the second gas: 45 ⁰C

Pressure of the washing gas: 0.2 MPa Temperature of the washing gas: 30 ⁰C

Flow ratio of the liquid mixture to the nozzle 0.2 mL/min

First gas: carbon dioxide

Second gas: air

Washing gas : air. Nozzle diameter: 0.18 mm

Nozzle length (restricted section): 3mm.

Example 3: assessment of particle size and distribution

The analysis of the size and distribution of the particles obtained in example 2 and the analysis of the zeta potential were performed with a Nicomp 380. The samples for the analysis were prepared suspending 1 mg of particles in 5 ml of distilled water. The suspension was dispersed through a vortex and 5 min of sonication in ice bath. The evaluation was conducted on the suspension as a such or after suitable dilution with distilled water or after treatment of the suspension as follow: a - further sonication in ice bath for 10, 20, 30, 40, 50 and 60 min.; b - filtration of the suspensions cooled at 4°C with filters of different exclusion size

(41 μm, 5μm, 1.2μm, 0.45μm, 0.22μm); c - centrifugation for 5, 10, 20, 30, 40, 50, 60 min. at 2000 rpm at 4°C

The results obtained at 5 min. of sonication and 5 min. of centrifugation expressed particle diameter (nm) are reported in the following table 2: Table 2

After sonication for times above 10 min the size and distribution of the particles remained substantially unmodified.

After centrifugation for times above 10 min the size and distribution of the particles remained substantially unmodified.

After filtration with filters of exclusion size 5 μm and 1.2 μm, the size and distribution of the particles remained substantially unmodified, The evaluation of zeta potential performed on all the samples was found to be in the range of -13/-28mV.

Example 4: assessment of particle suspension

The suspendability of the particles obtained in example 2 was evaluated by a turbidimetric analysis after sonication. Ten milligrams of particles were dispersed in 5 ml of deionized water through a vortex; then the suspension was placed in a sonicator in ice bath for defined periods of time (15, 30, 45, 60 min) and after further 5 min the absorbance at 600 nm of the suspension was measured. The results obtained expressed in % OD At 600 nm are reported in the following table 3:

Table 3

Example 5: assessment of particle physical stability

Stability studies were carried out subjecting the particle suspensions in distilled water to a centrifugation and evaluating at defined times the sedimentation degree by turbidimetric analysis. Two milligrams of particles obtained in example 2 were dispersed in 10ml of in distilled water with a vortex and then sonicated for 10 min in an ice bath. The suspensions were centrifuged at 5000 rpm for increasing centrifugation times and the absorbance at 600 nm was evaluated.

The results obtained expressed in % OD at 600 nm are reported in the following table 4:

Table 4

Example 6: assessment of model molecule loading

The loading capacity of the particles was conducted on particles as prepared in example 2 by extraction of the model molecules loaded. Fifty milligrams of fluorescein and insulin loaded particles were melted by heating and 500 μl of dimethylsulfoxide and then centrifuged. Two hundred microliters of supernatant were taken and fluorescein and insulin were extracted in of 1 ml of water and 1 ml of water containing 0.05% trifluoroacetic acid, respectively and the aqueous solutions were analysed by UV-Vis and RP-HPLC analysis. Fifty milligrams of p- nitroanilin loaded particles were dissolved in 1 ml methylene chloride and p- nitroaniline in the organic solution was determined by UV-Vis analysis. Fifty milligrams of estradiol loaded particles were dissolved in 500 μl of methanol and 4500 μl of acetonitrile and the hormone was determined in the organic solution by RP-HPLC analysis. The content of any model molecule was determined with a known specific analytical protocol.

The results obtained expressed in % of loading are reported in the following table 5: Table 5

Example 7: assessment of model molecule release

Twenty milligrams of loaded particles as prepared in example 2 were dispersed in 1 ml of phosphate buffer 20 mM, NaCI 0.15 M at pH 7.2.

The samples were sonicated for 10min in an ice bath and then placed in a dialysis membrane (cut off 100 kDa) and dialysed in 10 ml of the same buffer. Five hundred microliters of the dialysis buffer were taken at fixed times until complete release of the model molecules which was determined by chromatography. In the case of estradiol the used buffers were added with 10 mg/ml of β-cyclodextrin.

The release rate of the model molecules, estradiol and insulin, was slow and complete. Estradiol entrapped into the nano-particles was completely released throughout 100 hours and the release rate was found to approach a zero-order kinetic. Insulin release displayed a burst release which corresponded to non- loaded protein (about 15% of the loaded hormone). Afterwards insulin was constantly released throughout the time up to about 70 hours. As example in fig. 4 and 5 the release profiles of two model molecules (i.e. estradiol and insulin) are reported.

Example 8: effect of atomisation pressure and nozzle temperature on particle dimensions.

Tests were performed to produce stearic acid particles with the same apparatus and the same process described under example 2 except that the nozzle temperature and the atomisation pressure were changed. As no surfactants were introduced into the solid component composition, the solid was suspended in liquid, and particle agglomerates floating on the liquid surface were eliminated before evaluation. The results are shown in table 6.

Table 6

Mean weight Standard

Temperature Pressure diameter deviation

(⁰C) (MPa) (nm) (nm)

50 10 383.8 233.4

50 15 697.5 490.4

50 20 821.7 693.6

55 10 225.4 116.1

55 15 443.1 365.1

55 20 530.1 393.8

60 10 183.3 116.0

60 15 518.3 411.0

60 20 236.8 133.1

Claims

1. Process for the preparation of solid micro- or nano-particles, comprising the following steps:

- melting a solid component; - solution, under pressure, of a first compressed gas into the molten solid component, to create a pressurised semisolid or liquid mixture;

- generating a dispersion of the semisolid or liquid mixture with a second compressed gas, which does not completely dissolve into the semisolid or liquid mixture; - expansion of the dispersion obtained at the previous step with evaporation of the first gas and formation of micro or nanosized particles of the solid component.

2. Process according to claim 1 , wherein the steps of melting the solid component and of solution of the first gas take place at least partly contemporaneously.

3. Process according to any preceding claim, wherein nano-particles with equivalent diameter between 100 and 900 nm are prepared. 4. Process according to any preceding claim, wherein the first gas has a solubility into the solid component of at least 0.

4 molar fraction, preferably of at least 0.9. under process conditions.

5. Process according to any preceding claim, wherein the second gas has a solubility Into the semisolid or liquid mixture of less than 0.1 molar fraction, under process conditions.

6. Process according to any preceding claim, wherein the first gas is chosen among carbon dioxide, fluorocarbons, fluorinated hydrocarbons, chlorofluorocarbons, hydrochlorofluorocarbons or mixtures thereof.

7. Process according to any preceding claim, wherein the second gas is chosen among air, nitrogen, oxygen, carbon dioxide or mixtures thereof.

8. Process according to any preceding claim, wherein the solid component comprises at least a substance among polymers, lipids, fats, polyols, mono or diglycerids.

9. Process according to claim 8, wherein further substances are introduced into the solid component or in the liquid or semisolid mixture, before or after solution of the first gas.

10. Process according to claim 8 or 9, wherein said further substances are pharmaceutical compounds or pigments.

11. Process according to any preceding claim, wherein the pressure upon which solution step is performed is from10 to 20 Mpa.

12. Process according to any preceding claim, wherein the temperature upon which solution step is performed is from 298K to 398K.

13. Process according to any preceding claim, wherein the temperature upon which solution step is performed is in the range from 40K below the melting point of the pure solid component and the melting point of the pure solid component.

14. Process according to any preceding claim, wherein the expansion is performed through a nozzle with diameter between 50 and 500 μm.

15. Process according to claim 14, wherein the expansion is performed through a nozzle with diameter between 150-200 μm.

16. Apparatus for performing a process according to any of the preceding claims comprising: a saturator (4) adapted to perform the melting and solution steps; means (2, 3) for feeding the first gas to the saturator; a dispersing device (5) adapted to perform the generation of the dispersion a line connecting the saturator to the dispersing device; means (7, 8, 27) for feeding the second gas to the saturator; an expanding device (9).

17. Apparatus according to claim 16, further comprising a syringe pump (22) for aspirating a part of the semisolid or liquid mixture from the saturator and feeding it to the disperding device.

18. Apparatus according to claim 16 or 17, wherein the expanding device comprises a nozzle (46) having a diameter between 150-200 μm.

19. Apparatus according to any claim from 16 to 18, comprising means (14,15) for thermostating the saturator.

20. Apparatus according to any claim from 16 to 19, comprising means (24, 40, 41) for thermostating the disperding device.

21. Apparatus according to any claim from 16 to 20, wherein the disperding device comprises a main duct (26) for the passage of the liquid or semisolid mixture and a needle (29) placed inside and coaxially with said main duct.

22. Micro- or nano-particles obtainable with the process according to any claim from 1 to 15.