**4.2 Polymer and biopolymers**

Among organic and inorganic compounds that have been processed with SAS process, polymers have remarkable interest and significance. Yeo and Kiran (Yeo & Kiran, 2005) and Tomasko et al. (Tomasko et al., 2003) presented extensive reviews of the supercritical processing of polymers. Because most of polymers are not soluble in supercritical fluids, this antisolvent process is especially suitable for their recrystallization or precipitation in form of microparticles. The polymer is firstly dissolved in a liquid organic solvent and a supercritical fluid is employed as an antisolvent for the polymer. Polymers in form of small particles are useful for several applications like stationary phases in chromatography, adsorbents and catalyst supports, as well as drug delivery systems (Dixon et al., 1993). The polymers must fulfil several requisites: its biocompatibility, non toxicity, providing a suitable medium for preserving the properties and activity of the active substance and easy to process with the selected precipitation technique.

It is particularly important for polymer processing with supercritical processes is the glass transition and the melting point temperature depressions induced by the supercritical fluid. In particular, the dissolution of SC-CO2 into the polymer can reduce the glass transition temperature of amorphous polymers (Tomasko et al., 2003), an effect that is caused by intermolecular interactions between the dissolved CO2 and the polymer. The melting point depression caused by the dissolution of CO2 is less noticeable in magnitude.

A number of RESS processes for the encapsulation of particles with polymer (polylactic acid (PLA), polyethylene glycol (PEG), Eudragit) or composite particle formation for the controlled release of drugs have been reported as it was referenced before.

However, the potential application of RESS for particle coating or encapsulation is limited because the solubility of polymers in SC-CO2 is generally very poor (O'Neill et al., 1998)

Compared to RESS, the SAS process offers much more flexibility in terms of choosing suitable solvents. Furthermore, SAS has advantages over RESS because SAS is usually operated under mild conditions compared with those of RESS, which is associated to relatively high temperature and high pressure. Therefore RESS is also less attractive from the perspectives of safety and cost. The SAS process has been carried out for many particles precipitation and polymeric encapsulation of particles of active ingredients.

In order to obtain polymer-drug composites several researches have been carried out at our laboratory. Ethyl cellulose (EC) is a biocompatible and non biodegradable polymer. Ethyl cellulose is commonly used as drug carrier in controlled delivery systems. For instance, ethyl cellulose microcapsules has been used as a drug-delivery device for protecting folic acid from release and degradation in the undesirable environmental conditions of the stomach, whilst allowing its release in the intestinal tract to make it available for absorption. In the same way, ethyl cellulose and antibiotic microcapsules have been developed to use as drug delivery protecting antibiotic of conditions of the stomach.

At University of Cádiz, ethyl cellulose microparticles were successfully precipitated from dichloromethane (DCM) by SAS process (Gordillo et al., 2008) and particles were reduced from 50-100 to 3-5 µm (Figure 5). The concentration was the factor that had the greatest

Particles Formation Using Supercritical Fluids 471

Fig. 6. Schematic diagram of the pilot plant

influence on the PS and PSD. An increase in the initial concentration of the solution led to larger particles sizes with a wider distribution.

Fig. 5. SEM images of a) raw ethyl cellulose and b) precipitated ethyl cellulose

A pilot plant, developed by Thar Technologies® (model SAS 200) was used to carry out all the experiments. A schematic diagram of this plant is shown in Figure 6. The SAS 200 system comprises the following components: two high-pressure pumps, one for the CO2 (P1) and the other for the solution (P2), which incorporate a low-dead-volume head and check valves to provide efficient pumping of CO2 and many solvents; a stainless steel precipitator vessel (V1) with a 2-L volume consisting of two parts, the main body and the frit, all surrounded by an electrical heating jacket (V1-HJ1); an automated back-pressure regulator (ABPR1) of high precision, attached to a motor controller with a position indicator; and a jacketed (CS1-HJ1) stainless steel cyclone separator (CS1) with 0.5-L volume, to separate the solvent and CO2 once the pressure was released by the manual back-pressure regulator (MBPR1).The following auxiliary elements were also necessary: a low pressure heat exchanger (HE1), cooling lines, and a cooling bath (CWB1) to keep the CO2 inlet pump cold and to chill the pump heads; an electric high-pressure heat exchanger (HE2) to preheat the CO2 in the precipitator vessel to the required temperature quickly; safety devices (rupture discs and safety valve MV2); pressure gauges for measuring the pump outlet pressure (P1, PG1), the precipitator vessel pressure (V1, PG1), and the cyclone separator pressure (CS1, PG1); thermocouples placed inside (V1-TS2) and outside (V1-TS1) the precipitator vessel, inside the cyclone separator (CS1-TS1), and on the electric high pressure heat exchanger to obtain continuous temperature measurements; and a FlexCOR coriolis mass flowmeter (FM1) to measure the CO2 mass flow rate and another parameters such as total mass, density, temperature, volumetric flow rate, and total volume.

#### **4.3 Pharmaceuticals**

Pharmaceutical preparations are the final product of a technological process that gives the drugs the characteristics appropriate for easy administration, proper dosage and enhancement of the therapeutic efficacy. Among several kinds of development of modified release preparation, the design of pharmaceutical preparations in nanoparticulate form has emerged as a new strategy for drug delivery (Pasquali et al., 2006). Particle size and particle size distribution are critical parameters that determine the rate of dissolution of the drug in

influence on the PS and PSD. An increase in the initial concentration of the solution led to

Fig. 5. SEM images of a) raw ethyl cellulose and b) precipitated ethyl cellulose

total mass, density, temperature, volumetric flow rate, and total volume.

Pharmaceutical preparations are the final product of a technological process that gives the drugs the characteristics appropriate for easy administration, proper dosage and enhancement of the therapeutic efficacy. Among several kinds of development of modified release preparation, the design of pharmaceutical preparations in nanoparticulate form has emerged as a new strategy for drug delivery (Pasquali et al., 2006). Particle size and particle size distribution are critical parameters that determine the rate of dissolution of the drug in

**4.3 Pharmaceuticals** 

A pilot plant, developed by Thar Technologies® (model SAS 200) was used to carry out all the experiments. A schematic diagram of this plant is shown in Figure 6. The SAS 200 system comprises the following components: two high-pressure pumps, one for the CO2 (P1) and the other for the solution (P2), which incorporate a low-dead-volume head and check valves to provide efficient pumping of CO2 and many solvents; a stainless steel precipitator vessel (V1) with a 2-L volume consisting of two parts, the main body and the frit, all surrounded by an electrical heating jacket (V1-HJ1); an automated back-pressure regulator (ABPR1) of high precision, attached to a motor controller with a position indicator; and a jacketed (CS1-HJ1) stainless steel cyclone separator (CS1) with 0.5-L volume, to separate the solvent and CO2 once the pressure was released by the manual back-pressure regulator (MBPR1).The following auxiliary elements were also necessary: a low pressure heat exchanger (HE1), cooling lines, and a cooling bath (CWB1) to keep the CO2 inlet pump cold and to chill the pump heads; an electric high-pressure heat exchanger (HE2) to preheat the CO2 in the precipitator vessel to the required temperature quickly; safety devices (rupture discs and safety valve MV2); pressure gauges for measuring the pump outlet pressure (P1, PG1), the precipitator vessel pressure (V1, PG1), and the cyclone separator pressure (CS1, PG1); thermocouples placed inside (V1-TS2) and outside (V1-TS1) the precipitator vessel, inside the cyclone separator (CS1-TS1), and on the electric high pressure heat exchanger to obtain continuous temperature measurements; and a FlexCOR coriolis mass flowmeter (FM1) to measure the CO2 mass flow rate and another parameters such as

larger particles sizes with a wider distribution.

**a) b)**

Fig. 6. Schematic diagram of the pilot plant

Particles Formation Using Supercritical Fluids 473

final formulation of drug could be adjusted well by a change in the initial concentration of the solution. An increase in initial concentration of the solution has two opposite effects: On one hand, with a higher concentration, it is possible to achieve higher supersaturations, which tends to diminish the particle size. On the other hand, condensation is directly proportional to the concentration of solute, and the increase of the condensation rate at higher concentrations tends to increase the particle size (Martin & Cocero, 2004). In our case, an increase in the initial concentration of the solution led to larger particles sizes with a

Thus, the second effect (condensation rate) prevailed under the operating conditions used in this work; that is, the higher the initial concentration of the solution, the higher the condensation rate, and thus, the greater the particle sizes produced. This result is consistent with those obtained by Reverchon et al., which were also explained in terms of competition

The ability to tune polymer and drug simultaneously can be used to control the nature and extent of drug loading. In order to obtain polymer-drug composites several researches have been carried out. Composites are frequently produced by the simultaneous precipitation of the core and coating materials, leading to a dispersion of particles of the core material into a matrix of coating material while encapsulates are produced when the coating material is precipitated as a thin shell over a previously existing core material particle. These systems let achieve a controlled delivery of the active ingredients into its targeted media. In addition to oral administration, these particulate carriers can also be injected intramuscularly or intravenously as long as their particle size is within physiologically acceptable range to

In the pharmaceutical compounds encapsulation, the coating material must be biocompatible and non toxic, providing a suitable medium for preserving the properties and the activity of

In our research group, ethyl cellulose amoxicillin and ampicillin co-precipitation has been carried out. For that, commercials EC and AMC and AMP have been dissolved in a mixture of DCM and dimethylsulfoxide DMSO and this solution has been pumped by the high pressure pump of the SAS equipment. A temperature increase from 308 to 328 K, independently of pressure, is traduced to particle size increase but an agglomeration of particles formed by irregular block is observed when the temperature is increased to 333 K. However, at three temperatures, an increase of pressure leads to a smaller particle size. This fact can also be explained on the basis of the numerical modelling of mass transfer proposed by Werling and Debenedetti (Werling& Debenedetti, 2000). An increase of pressure brings the system to miscible conditions. These conditions result in faster mass transfer, causing a higher degree of supersaturation that results in higher nucleation rates, thus producing

To study the ability of ethyl cellulose to encapsulate amoxicillin, a suspension of AMC microparticles in a solution of EC in DCM has been used. This suspension is sprayed by a nozzle using a KD410 Syringe Pump instead of the solvent pump of the SAS equipment to avoid blocking the pump. The supercritical CO2 acts as an antisolvent for the DCM. A rapid mutual diffusion between the supercritical CO2 and the organic solvent causes supersaturation of the polymer solution, leading to nucleation and precipitation of the

between nucleation and growth processes (Reverchon et al., 2000).

achieve a controlled dissolution of the active substance (Cocero et al., 2009).

the active substance and easy to process with the precipitation technique.

wider distribution.

smaller particle size.

**4.4 Composites and encapsulates** 

the biological fluids and, hence, have a significant effect on the bioavailability of those drugs (Perrut et al., 2005; Van Nijlen et al., 2003).

Methods used in the past for the manufacture of drug nanoparticles usually do not allow very accurate control of the particle size, and so broad particle size distributions are obtained. Supercritical antisolvent (SAS) processes have been widely used for the last ten years to precipitate Active Pharmaceutical Ingredients (APIs) (Chattopadhyay& Gupta, 2001; Rehman et al., 2001; Velaga et al., 2002; Yeo & Lee, 2004).

Supercritical antisolvent techniques overcome the main drawbacks of conventional techniques, such as the degradation of the active ingredients because of the high profiles of temperatures and tensile stresses reached and the large amount of organic solvent used, resulting in the need to remove the solvent from the final product.

Amoxicillin and ampicillin micronization have been carried out by SAS process in our laboratory (Montes et al., 2010, 2011; Tenorio et al., 2007a, 2007b, 2008). Several experiments designs to evaluate the operating conditions influences on the PS and PSD have been made. Pressures till 275 bar and temperatures till 338K have been used and antibiotic particle sizes have been reduced from 5-60 µm (raw material) to 200-500 nm (precipitated particles) (Figure 7).

Fig. 7. SEM images of commercial a) amoxicillin and b) ampicillin, c)precipitated amoxicillin and d) ampicillin

The initial concentration of the drug solution pumped into the vessel, is the factor that has the greatest influence on both PS and PSD. Therefore, both PS and the PSD required for the

the biological fluids and, hence, have a significant effect on the bioavailability of those drugs

Methods used in the past for the manufacture of drug nanoparticles usually do not allow very accurate control of the particle size, and so broad particle size distributions are obtained. Supercritical antisolvent (SAS) processes have been widely used for the last ten years to precipitate Active Pharmaceutical Ingredients (APIs) (Chattopadhyay& Gupta,

Supercritical antisolvent techniques overcome the main drawbacks of conventional techniques, such as the degradation of the active ingredients because of the high profiles of temperatures and tensile stresses reached and the large amount of organic solvent used,

Amoxicillin and ampicillin micronization have been carried out by SAS process in our laboratory (Montes et al., 2010, 2011; Tenorio et al., 2007a, 2007b, 2008). Several experiments designs to evaluate the operating conditions influences on the PS and PSD have been made. Pressures till 275 bar and temperatures till 338K have been used and antibiotic particle sizes have been reduced from 5-60 µm (raw material) to 200-500 nm (precipitated particles)

Fig. 7. SEM images of commercial a) amoxicillin and b) ampicillin, c)precipitated amoxicillin

**d)**

The initial concentration of the drug solution pumped into the vessel, is the factor that has the greatest influence on both PS and PSD. Therefore, both PS and the PSD required for the

(Perrut et al., 2005; Van Nijlen et al., 2003).

(Figure 7).

and d) ampicillin

**b)**

2001; Rehman et al., 2001; Velaga et al., 2002; Yeo & Lee, 2004).

resulting in the need to remove the solvent from the final product.

**a) c)**

final formulation of drug could be adjusted well by a change in the initial concentration of the solution. An increase in initial concentration of the solution has two opposite effects: On one hand, with a higher concentration, it is possible to achieve higher supersaturations, which tends to diminish the particle size. On the other hand, condensation is directly proportional to the concentration of solute, and the increase of the condensation rate at higher concentrations tends to increase the particle size (Martin & Cocero, 2004). In our case, an increase in the initial concentration of the solution led to larger particles sizes with a wider distribution.

Thus, the second effect (condensation rate) prevailed under the operating conditions used in this work; that is, the higher the initial concentration of the solution, the higher the condensation rate, and thus, the greater the particle sizes produced. This result is consistent with those obtained by Reverchon et al., which were also explained in terms of competition between nucleation and growth processes (Reverchon et al., 2000).

#### **4.4 Composites and encapsulates**

The ability to tune polymer and drug simultaneously can be used to control the nature and extent of drug loading. In order to obtain polymer-drug composites several researches have been carried out. Composites are frequently produced by the simultaneous precipitation of the core and coating materials, leading to a dispersion of particles of the core material into a matrix of coating material while encapsulates are produced when the coating material is precipitated as a thin shell over a previously existing core material particle. These systems let achieve a controlled delivery of the active ingredients into its targeted media. In addition to oral administration, these particulate carriers can also be injected intramuscularly or intravenously as long as their particle size is within physiologically acceptable range to achieve a controlled dissolution of the active substance (Cocero et al., 2009).

In the pharmaceutical compounds encapsulation, the coating material must be biocompatible and non toxic, providing a suitable medium for preserving the properties and the activity of the active substance and easy to process with the precipitation technique.

In our research group, ethyl cellulose amoxicillin and ampicillin co-precipitation has been carried out. For that, commercials EC and AMC and AMP have been dissolved in a mixture of DCM and dimethylsulfoxide DMSO and this solution has been pumped by the high pressure pump of the SAS equipment. A temperature increase from 308 to 328 K, independently of pressure, is traduced to particle size increase but an agglomeration of particles formed by irregular block is observed when the temperature is increased to 333 K. However, at three temperatures, an increase of pressure leads to a smaller particle size. This fact can also be explained on the basis of the numerical modelling of mass transfer proposed by Werling and Debenedetti (Werling& Debenedetti, 2000). An increase of pressure brings the system to miscible conditions. These conditions result in faster mass transfer, causing a higher degree of supersaturation that results in higher nucleation rates, thus producing smaller particle size.

To study the ability of ethyl cellulose to encapsulate amoxicillin, a suspension of AMC microparticles in a solution of EC in DCM has been used. This suspension is sprayed by a nozzle using a KD410 Syringe Pump instead of the solvent pump of the SAS equipment to avoid blocking the pump. The supercritical CO2 acts as an antisolvent for the DCM. A rapid mutual diffusion between the supercritical CO2 and the organic solvent causes supersaturation of the polymer solution, leading to nucleation and precipitation of the

Particles Formation Using Supercritical Fluids 475

cellulose-ampicillin systems have been obtained successfully in our laboratory. A temperature increase of the experiments is traduced to particle size increase. An agglomeration of particles formed by irregular block is observed when the temperature is

We are grateful to the Spanish Ministry of Education and Science (Project No. CTQ2007-

Ai-Zheng C. , Yi, L. , Foo-Tim, C. , Tsui-Yan, L. , Jun-Yan, H., Zheng, Z. ,Daniel Kam-wah,

Bush, J. R., Akgerman, A., Hall, K. R. (2007). Synthesis of controlled release device with supercritical CO2 and co-solvent. *J. Supercrit. Fluids*, 41, pp. 311–316. Cardoso, M. A. T.; Monteiro, G. A.; Cardoso, J. P.; Prazeres, T. J. V.; Figueiredo, J.M. F.;

Chen, Y.-M., Tang, M., Chen. Y.-P. (2010). Recrystallization and micronization of

Chong, G.H., Spotar, S.Y., Yunus, R. (2009). Numerical Modeling of Mass Transfer for

Chong, G.H., Yunus, R., Abdullah, N., Choong, T.S.Y., Spotar, S. (2009). Coating and

Cocero, M. J., Martin, A., Mattea, F., Varona, S. (2009). Encapsulation and co-precipitation

Corazza, M. L., Filho, C. L., Dariva, C. (2006). Modeling and simulation of rapid expansion of supercritical solutions. *Brazilian J. of Chem. Eng.*, 23(3), pp. 417-425. Dixon, D.J., Johnston, K.P., Bodmeier, R.A. (1993).Polymeric materials formed by precipitation with a compressed fluid antisolvent, *AIChE J.*, 39 (1), pp. 127–139. Dukhin, S.S., Zhu, C., Pfeffer, R., Luo, J.J., Chavez, F., Shen, Y. (2003). Dynamic interfacial

Dukhin, S. S., Shen, Y., Dave, R., Pfeffer, R. (2005). Droplet mass transfer, intradroplet

supercritical antisolvent emulsion. *Colloids Surf. A*, 261, pp. 163-176.

stabilization, *Physicochem. Eng. Aspects*, 229, pp. 181-199.

supercritical CO2 process. *Acta Biomaterialia* , 5, pp. 2913-2919.

M. (2009). Microencapsulation of puerarin nanoparticles by poly(L-lactide) in a

Martinho, J. M. G.; Cabral, J. M. S.; Palavra, A. M. F. (2008). Supercritical antisolvent micronization of minocycline hydrochloride. *J. Supercrit. Fluids*, 44, pp. 238–244. Chattopadhyay, P., Gupta, R. B. (2001). Production of antibiotic nanoparticles using

supercritical CO2 as antisolvent with enhanced mass transfer, *Ind. Eng. Chem. Res.*,

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processes with supercritical fluids: Fundamentals and applications. *J. Supercrit.* 

tension near critical point of a solvent–antisolvent mixture and laminar jet

nucleation and submicron particle production in two-phase flow of solvent-

increased to 333 K. However, an increase of pressure leads to a smaller particle size.

**6. Acknowledgment** 

**7. References** 

67622) for financial support.

40, pp. 3530-3539.

165, pp. 358–364.

9(17), pp. 3055-3061.

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*Fluids*, 47, pp. 546-555.

polymer to encapsulate the AMC particles. In the precipitation over a suspension of particles, the particles behave as nuclei for the precipitation of the polymer, and a polymer matrix of encapsulated particles is produced by agglomeration (Cocero et al., 2009).

SEM images of these microparticles are shown in Figure 8. SEM images are not accurate enough to observe the distribution of both compounds because all the active substance could be situated on the surface of these microspheres and/or into the core. Thus, X-ray photoelectron spectroscopy (XPS) is one of the main techniques used to determine the success of the encapsulation process by the chemical analysis of the particles on the precipitated surface (Morales et al, 2006). In this case, the elements that differentiate amoxicillin from ethyl cellulose are sulphur (S) and nitrogen (N) atoms. Therefore, these elements can indicate the location of the drug in the precipitated powders. In the coprecipitated the sulphur peak can be identified but there is an absence of this peak in the encapsulate. Moreover, an elemental analysis of the encapsulate is needed to confirm that the drug was situated into the core.

Fig. 8. SEM images of amoxicillin ethyl cellulose a) encapsulates and b) co-precipitated
