**6. Supercritical fluid based methods**

Supercritical fluid (SCF) technology has gained increasing interest in the last years for nanoparticle production. SCF is obtained above its critical pressure and temperature: above this fluid's critical point, the solubility of a substance in the fluid can be modulated by a relatively small change in pressure. Due to its low critical point at 31 °C and 74 bar, and its low cost and non toxicity, carbon dioxide (CO2) is the most widely used SCF [37].

The four main SCF processes used to produce nano-or microparticles are:


#### **6.1. RESS**

an acid solution (coacervating solution) in the presence of different amphiphilic polymeric stabilizing agents: fatty acid nanoparticles precipitate as proton exchange occurs between the

In this method the precursor for SLN preparation is a soap micellar solution, obtained at a temperature above its Krafft point (that is the solubilisation temperature of the soap in water): drug can be dissolved directly in the micellar solution, or pre-dissolved in a small amount of ethanol, in order to enhance micellisation. As for microemulsion templates, the good solubil‐ ising properties of micellar solutions allow an advantageous drug loading within SLN for

The salt of fatty acid is chosen from the group consisting from sodium stearate, sodium palmitate, sodium myristate, sodium arachidate and sodium behenate in a concentration preferably between 1 and 5% w/w. The stabilizing agent is selected from the group of surface active non ionic polymers: polyvynilacetate/polyvynilalcohol and polyoxyethylene/polyoxypropylene copolymers, dextrans, hydroxypropylmethylcellulose. Normally the acidification takes place at a temperature between 40 and 50 °C, above the Krafft point of the fatty acid sodium salt; sodium arachidate and behenate need higher temperatures. Then the obtained

A very important feature of this method is the possibility to control the size of SLN changing the reaction conditions. In order to obtain a homogeneous and stable nanoparticle suspension, a key role is played by the right coupling between the fatty acid alkaline salt and the proper coacervating solution. Particle size is highly influenced by the lipid concentration: increasing the micellar solution concentration the SLN size increases. Also the type and the grade of the

Solvent based methods have been proposed in order to encapsulate molecules with stability and bioavailability problems, despite toxicological issues of the solvent are a limiting aspect. One of the main advantages of solvent based methods is the mild operating temperature, which can be useful for the encapsulation of thermosensitive drugs. According to the different

In solvent injection (or solvent displacement) method the lipid and the drug are dissolved in a water-miscible organic solvent (ethanol, acetone, isopropanol) and this solution is injected through a syringe needle in water under stirring: lipid precipitates as nanoparticles while

coacervating solution and the soap solution [27,28].

58 Application of Nanotechnology in Drug Delivery

suspension is rapidly cooled to 15 °C [27,28].

**5. Solvent based methods**

**•** solvent evaporation from emulsions;

**•** solvent diffusion from emulsions.

**5.1. Solvent injection method**

**•** solvent injection;

many drugs, especially for poorly water soluble drugs [29, 30].

polymer used as stabiliser influence the SLN mean particle size [28].

precursor/method used, solvent based methods can be divided in:

RESS, which is also called supercritical fluid nucleation (SFN), is based on a simple principle: the matrix is dissolved in SCF, which is then expanded through a nozzle, in order to form the particles [38]. This major limitation of RESS lies in the too low solubility of compounds in SCF, that precludes production at acceptable costs. In fact its applications to lipid particles is very limited.

A modified RESS process has been used for lipid coating of bovine serum albumin (BSA) microcrystals [39]. The lipid coated microparticles are prepared as follows: coating material and BSA crystals are placed in an autoclave, equipped with a rotating impeller, heated and pressurised with CO2 (temperature typically ranging between 35 and 45 °C and pressure about 200 bar). The system is allowed to equilibrate at these conditions, so as to solubilise the coating material. Then, cooling the autoclave induced a pressure decrease and a phase change from SCF to liquid state, therefore insolubilising the coating material that precipitated upon the insoluble BSA crystals dispersed in the medium. Afterwards, the autoclave is vented to ambient conditions and the coated particles are collected from the bottom of the autoclave.

further expanded through a nozzle with the formation of solid particles or droplets. The

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In literature some examples of PGSS applied to lipid nano-and microparticles can be recovered. The most important is the so called Gas Assisted Melting Atomisation (GAMA) [40,41,42].

Lipids are placed in a thermostated mixing chamber (CM), where they are melted and kept in contact with supercritical CO2 at selected temperature and pressure conditions (Figure 2). Then, the lipid saturated fluid is forced through the nozzle by opening the valve at the bottom of the CM, in order to produce microparticles. A CO2 reservoir is allowed to keep constant the pressure in the CM, and the flow rate through the nozzle as well. Particles are gathered by a collection system and dispersed in water by vortexing and sonicating, in order to obtain suspensions. Polyethylenglycol (PEG) can be added to the formulation in order to increase the

**Figure 2.** GAMA apparatus for micro and nanoparticles production: supercritical CO2 supplier (1), on–off valves (2,6,7,11,13), heater exchanger (3), pump (4), electrical resistances (5,12,16,19), pressure indicator (PI), temperature indicator (TI), temperature controller (TC), mixing device (8,9), melting chamber (10), heating system (14), spraying pump (15), air supplier (17), pressure reducer (18), 100 mm diameter nozzle (20), compressor for tangential air flux

SFEE is based on a simple principle, whereby the lipid nanosuspensions are produced by supercritical fluid extraction of the organic solvent from O/W emulsions. O/W emulsion is prepared by dissolving lipid and drug in a volatile solvent (i.e. chloroform), dispersing this solution into an aqueous phase of a surfactant and passing the mixture through a high pressure

advantage of this process is that the substances need not be soluble in CO2 [38].

rate of dispersion in water.

(21), precipitation vessel (22), filter (23), flowmeter (24).

**6.4. SFEE**

**Figure 1.** Schematic representation of the coating process. (A) Filling step: BSA crystals represented in white, coating material in black. (B) Solubilisation of the coating material in the SCF CO2. Insoluble BSA crystals are dispersed in the medium. (C) Decompression phase: insolubilisation of the coating material. (D) Coated particles are harvested after autoclave has been vented.

#### **6.2. GAS**

GAS process has been developed in order to achieve nanosizing of the hydrophobic materials that can not be processed by RESS technique owing to their poor solubility in SCF. The origin of GAS process is based on the fact that when a solution is expanded sufficiently by a gas, the liquid phase is no longer a good solvent for the solute and nucleation occurs [38]. Up to now GAS technique has very limited applications for lipid particles.

#### **6.3. PGSS**

PGSS involves melting of the material to be processed, which then dissolves the SCF under pressure. The saturated solution is then expanded across a nozzle where the SCF, which is more volatile, escapes, leaving dry fine particles. As the solubilities of compressed gases in liquids and solids are usually high, and much higher than the solubilities of such liquids and solids in the compressed gas phase, the process consists in solubilizing CO2 in melted or liquidsuspended substance(s), leading to a so-called gas-saturated solution/suspension that is further expanded through a nozzle with the formation of solid particles or droplets. The advantage of this process is that the substances need not be soluble in CO2 [38].

In literature some examples of PGSS applied to lipid nano-and microparticles can be recovered. The most important is the so called Gas Assisted Melting Atomisation (GAMA) [40,41,42].

Lipids are placed in a thermostated mixing chamber (CM), where they are melted and kept in contact with supercritical CO2 at selected temperature and pressure conditions (Figure 2). Then, the lipid saturated fluid is forced through the nozzle by opening the valve at the bottom of the CM, in order to produce microparticles. A CO2 reservoir is allowed to keep constant the pressure in the CM, and the flow rate through the nozzle as well. Particles are gathered by a collection system and dispersed in water by vortexing and sonicating, in order to obtain suspensions. Polyethylenglycol (PEG) can be added to the formulation in order to increase the rate of dispersion in water.

**Figure 2.** GAMA apparatus for micro and nanoparticles production: supercritical CO2 supplier (1), on–off valves (2,6,7,11,13), heater exchanger (3), pump (4), electrical resistances (5,12,16,19), pressure indicator (PI), temperature indicator (TI), temperature controller (TC), mixing device (8,9), melting chamber (10), heating system (14), spraying pump (15), air supplier (17), pressure reducer (18), 100 mm diameter nozzle (20), compressor for tangential air flux (21), precipitation vessel (22), filter (23), flowmeter (24).

#### **6.4. SFEE**

A modified RESS process has been used for lipid coating of bovine serum albumin (BSA) microcrystals [39]. The lipid coated microparticles are prepared as follows: coating material and BSA crystals are placed in an autoclave, equipped with a rotating impeller, heated and pressurised with CO2 (temperature typically ranging between 35 and 45 °C and pressure about 200 bar). The system is allowed to equilibrate at these conditions, so as to solubilise the coating material. Then, cooling the autoclave induced a pressure decrease and a phase change from SCF to liquid state, therefore insolubilising the coating material that precipitated upon the insoluble BSA crystals dispersed in the medium. Afterwards, the autoclave is vented to ambient conditions and the coated particles are collected from the bottom of the autoclave.

**Figure 1.** Schematic representation of the coating process. (A) Filling step: BSA crystals represented in white, coating material in black. (B) Solubilisation of the coating material in the SCF CO2. Insoluble BSA crystals are dispersed in the medium. (C) Decompression phase: insolubilisation of the coating material. (D) Coated particles are harvested after

GAS process has been developed in order to achieve nanosizing of the hydrophobic materials that can not be processed by RESS technique owing to their poor solubility in SCF. The origin of GAS process is based on the fact that when a solution is expanded sufficiently by a gas, the liquid phase is no longer a good solvent for the solute and nucleation occurs [38]. Up to now

PGSS involves melting of the material to be processed, which then dissolves the SCF under pressure. The saturated solution is then expanded across a nozzle where the SCF, which is more volatile, escapes, leaving dry fine particles. As the solubilities of compressed gases in liquids and solids are usually high, and much higher than the solubilities of such liquids and solids in the compressed gas phase, the process consists in solubilizing CO2 in melted or liquidsuspended substance(s), leading to a so-called gas-saturated solution/suspension that is

GAS technique has very limited applications for lipid particles.

autoclave has been vented.

60 Application of Nanotechnology in Drug Delivery

**6.2. GAS**

**6.3. PGSS**

SFEE is based on a simple principle, whereby the lipid nanosuspensions are produced by supercritical fluid extraction of the organic solvent from O/W emulsions. O/W emulsion is prepared by dissolving lipid and drug in a volatile solvent (i.e. chloroform), dispersing this solution into an aqueous phase of a surfactant and passing the mixture through a high pressure homogeniser in order to form fine emulsions with a mean droplet size ranging between 30– 100 nm.

One of the advantages of this technique is that the solvent extraction efficiency using super‐ critical CO2 is much higher than for the conventional methods, such as solvent evaporation/ diffusion from emulsions. It therefore provides for a fast and complete removal of the solvent and more uniform particle size distribution. Supercritical CO2 also tends to extract other lowmolecular weight impurities, purifying the lipids. The size of SLN obtained in the SFEE process is directly related to the emulsion droplet size and it is therefore dependent upon the method

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SLN can be produced by using a membrane contactor [45]: a proper module has been realised (Figure 4), including a Kerasep ceramic membrane (0.1, 0.2, 0.45 μm pore size), which separates the water phase, allowed to circulate tangentially to the membrane surface, and the lipid phase; the lipid phase is heated in a pressurised vessel above its melting point, conveyed through a tube to the module (Figure 5) and pressed through the membrane pores, allowing the forma‐ tion of small droplets, which are detached from the membrane pores by tangential water flow.

SLN particle size depends on many process parameters: larger sizes are obtained with higher lipid phase content, reduced lipid phase pressure and aqueous cross-flow velocity. If the temperature of the aqueous phase is below the melting point of the lipid, smaller SLN are obtained: this is due to the fact that the lipid phase solidifies suddenly in the aqueous phase. Instead the SLN size decreases when the lipid temperature increases. Particle size is also highly

**Figure 4.** Module for membrane contactor. A (lipid phase), B (water phase), M (porous membrane), 7 (tangential flow

influenced by type and concentration of surfactants added to the formulation [47].

of formulation; a mean particle diameter between 20–90 nm can be obtained [44].

SLN are formed after cooling of the obtained water dispersion [46].

**7. Membrane contactor technique**

filtration unit).

SLN suspensions are obtained using a continuous extraction method. The O/W emulsions are introduced into an extraction column from the top; simultaneously, supercritical CO2 (at constant pressure of 80 bar and temperature 35 °C) is introduced counter-currently from the bottom [43,44] (Figure 3).

**Figure 3.** Extraction column for emulsion with SCF in counter-current

The operating pressure and temperature conditions have to be selected to minimise losses of product due to lipid and drug dissolution into the CO2 phase at maximum extraction efficiency. The residence time required for producing pure aqueous suspensions of SLN is approximately two minutes, with the product continuously removed from the bottom of the extraction column.

When the O/W emulsion containing the lipid and the drug is introduced into the supercritical CO2 phase, parallel processes of solvent extraction into the supercritical CO2 phase, and the inverse flux of CO2 into the emulsion droplets occurs, leading to expansion of the organic phase of the emulsion. This in turn leads to precipitation of lipid-drug material dissolved in the organic phase as composite particles [44].

One of the advantages of this technique is that the solvent extraction efficiency using super‐ critical CO2 is much higher than for the conventional methods, such as solvent evaporation/ diffusion from emulsions. It therefore provides for a fast and complete removal of the solvent and more uniform particle size distribution. Supercritical CO2 also tends to extract other lowmolecular weight impurities, purifying the lipids. The size of SLN obtained in the SFEE process is directly related to the emulsion droplet size and it is therefore dependent upon the method of formulation; a mean particle diameter between 20–90 nm can be obtained [44].
