**3. Drug encapsulation using scCO2**

In the last decade, drugs loaded in porous biodegradable polymeric foams have found to have important applications in tissue engineering and delivery systems. These polymeric porous scaffolds with an open-pore structure can ensure and increase seeding, attachment, growth of cells, extracellular matrix production, vascularization, and tissue growth. Supercritical CO2 is an excellent choice to produce impregnated polyester foams in a one-step process creating porosity, without residual solvent in the products. After polymer scaffold degradation, obtained tissue would not contain synthetic polymer. The rate degradation of the scaffold should be similar or slower than the rate of tissue formation; therefore, it is important to elucidate the mechanism of this degradation process.

Some examples of polymers used as porous scaffolds were poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA), respectively, and poly(methyl methacrylate), PMMA, PMMA/poly(ε-caprolactone), PCL, etc. Such polymers are harmless to the growing cells and can be removed from the organism by normal metabolic pathways. They also can be used in other in vivo applications, such as resorbable sutures [16].

Encapsulation of drugs within colloidal-sized polymeric matrix is largely used to improve the sustained release, reduce the side effect of the drugs, and increase the bioavailability of the drug from the pharmaceutical formulations. Polymeric beads for the controlled release of the drugs are most often used as the best solution due to easy preparation procedure and high drug-loading efficiency.

The special properties of scCO2 made it a good transport vector for solid matrix impregnation. This process depends on the partition of interest substance between the supercritical fluid phase and matrix (such as porous polymers) used for impregnation. First the substance is mixed with high-pressure carbon dioxide and then is

placed in contact with matrix used as support. Because scCO2 has a good diffusion capacity into porous scaffolds, inducing a swelling and/or plasticization, impregnation of a lot of materials could be possible. Among them were polymers as swellable matrices or silica as non-swellable matrices [17].

The controlled release of the very hydrophilic drugs is still a challenge due to the difficulties to encapsulate the drug in a suitable drug delivery system able to control the rate of the release and to ensure a minimum retention of the active substance inside the pharmaceutical vehicles, but with a good control of the particle size and surface properties, nanoparticles may be directed to specific sites for targeted drug delivery.

The encapsulation of active substances can be improved by impregnation in the presence of compressed fluids like carbon dioxide [18], at temperatures and pressures near or above critical point (CO2, *pcr* = 7.382 MPa; *tcr* = 31.04°C).

In a typical experiment in our laboratory, 2-pyridinealdoxime methochloride (PAM) was chosen as a hydrophilic drug model, with a high solubility in water. PAM is an acetylcholinesterase reactivator used as an antidote in poisoning with organophosphoric substances characterized by high solubility in water. Due to its hydrophilicity, PAM is rapidly eliminated from the body. Consequently, PAM is relatively short acting, and repeated doses may be needed. The encapsulation of PAM in alginate beads (used as sustained drug release system) was tested.

Alginate is a water-soluble linear polysaccharide extracted from brown seaweed, and it is composed of alternating blocks of 1–4 linked α-L-guluronic (G) and β-D-mannuronic (M) acid fragments. Alginate is a biocompatible and a hydrophilic biopolymer. These properties and its relatively low cost have recommended it for pharmaceutical applications [19]. The anionic biopolymer has the ability to bind multivalent cations, leading to the formation of insoluble hydrogels with "egg box" type structure [19]. In the formation of water-insoluble gels, a specific interaction occurs between calcium ions (Ca2+) and -COO<sup>−</sup> and -OH groups of the guluronic acid fragments in a simple procedure of dripping a sodium alginate aqueous solution into a calcium chloride solution [20]. The drug can be encapsulated in alginate beads by two methods. A first method consists in dripping the aqueous solution of alginate/PAM mixture in calcium chloride solution. The deficiency of this method is the elimination of PAM outside the beads during the ionically cross-linking of alginate with calcium ions. A second method consists in immersing and soaking the polymeric beads into the solution of drug. In this case the impregnation efficiency is low.

A certain number of calcium alginate beads were immersed in the 3 × 10<sup>−</sup><sup>3</sup> wt% solution of PAM. The impregnation of PAM in calcium alginate beads was carried out in a high-pressure cell, in the presence of compressed carbon dioxide. The impregnation was performed at different pressures (2.5, 5.0, 7.5 MPa) and temperatures (20, 40, 60°C) and also at atmospheric conditions (0.1 MPa, 20°C). The impregnation time was 30 minutes. The samples are designated *p/t*, where *p* and *t* represent the pressure and the temperature of impregnation.

The efficiency of PAM impregnation at various temperatures and pressures is presented in **Figure 5**.

The results showed that the efficiency of PAM impregnation increases with temperature at 2.5 MPa and the interaction between the cationic drug and active sites of biopolymer are favored by high pressure. Both state parameters influence the encapsulation of the ionic drug in the polymeric beads. The encapsulation of PAM in calcium alginate beads at 2.5 MPa depends on temperature. Adsorption or/ and absorption of PAM are the main processes which take place between 20 and 40°C. At 60°C, the dissociation constants of polyelectrolyte and ionic drug increase and electrostatic interactions between the polymer and drug molecules are promoted. At higher pressure (5.0 and 7.5 MPa), the impregnation efficiency varies in the same way like ratio of CO2 density to viscosity versus temperature and pressure.

**167**

**3.1 In vitro release studies**

**Figure 6.**

**Figure 5.**

processed for establishing the release kinetics.

The release curves for sample 1/20 and for samples with higher encapsulation efficiency are presented in **Figure 6**. The release curves for samples 0.1/20, 7.5/20, and 5.0/20 show a "burst effect"; the encapsulated PAM is released in the first 15 minutes. The sustained release of PAM is observed for samples 7.5/40 and 2.5/60. The maximum amount of released PAM is lower than 50% for samples 7.5/40, 2.5/60, and 5.0/20. The observed initial burst release profiles may be an indication that the impregnated drug was mainly located at the polymer surface for the sample impregnated with PAM at 20°C and different pressures. The kinetic data were fitted by first-order, Weibull, and Korsmeyer-Peppas kinetic equations. The experimental concentration values of the active substance released as a function of time were

*PAM release profiles from impregnated alginate beads; inset, the release curves in the first 30 minutes.*

The kinetic parameters offer information about the encapsulation mechanism which can take place by adsorption on the surface of the beads or by interactions

*Synthesis and Functionalization of Nanoparticles in Supercritical CO2*

*Efficiency of encapsulation of PAM in alginate microbeads at different pressures and temperatures.*

*DOI: http://dx.doi.org/10.5772/intechopen.89353*

*Synthesis and Functionalization of Nanoparticles in Supercritical CO2 DOI: http://dx.doi.org/10.5772/intechopen.89353*

*Advanced Supercritical Fluids Technologies*

matrices or silica as non-swellable matrices [17].

placed in contact with matrix used as support. Because scCO2 has a good diffusion capacity into porous scaffolds, inducing a swelling and/or plasticization, impregnation of a lot of materials could be possible. Among them were polymers as swellable

The controlled release of the very hydrophilic drugs is still a challenge due to the difficulties to encapsulate the drug in a suitable drug delivery system able to control the rate of the release and to ensure a minimum retention of the active substance inside the pharmaceutical vehicles, but with a good control of the particle size and surface properties, nanoparticles may be directed to specific sites for targeted drug delivery. The encapsulation of active substances can be improved by impregnation in the presence of compressed fluids like carbon dioxide [18], at temperatures and pres-

In a typical experiment in our laboratory, 2-pyridinealdoxime methochloride (PAM) was chosen as a hydrophilic drug model, with a high solubility in water. PAM is an acetylcholinesterase reactivator used as an antidote in poisoning with organophosphoric substances characterized by high solubility in water. Due to its hydrophilicity, PAM is rapidly eliminated from the body. Consequently, PAM is relatively short acting, and repeated doses may be needed. The encapsulation of PAM in alginate beads (used as sustained drug release system) was tested.

Alginate is a water-soluble linear polysaccharide extracted from brown seaweed,

wt%

and it is composed of alternating blocks of 1–4 linked α-L-guluronic (G) and β-D-mannuronic (M) acid fragments. Alginate is a biocompatible and a hydrophilic biopolymer. These properties and its relatively low cost have recommended it for pharmaceutical applications [19]. The anionic biopolymer has the ability to bind multivalent cations, leading to the formation of insoluble hydrogels with "egg box" type structure [19]. In the formation of water-insoluble gels, a specific interaction occurs between calcium ions (Ca2+) and -COO<sup>−</sup> and -OH groups of the guluronic acid fragments in a simple procedure of dripping a sodium alginate aqueous solution into a calcium chloride solution [20]. The drug can be encapsulated in alginate beads by two methods. A first method consists in dripping the aqueous solution of alginate/PAM mixture in calcium chloride solution. The deficiency of this method is the elimination of PAM outside the beads during the ionically cross-linking of alginate with calcium ions. A second method consists in immersing and soaking the polymeric beads into the solution of drug. In this case the impregnation efficiency is low. A certain number of calcium alginate beads were immersed in the 3 × 10<sup>−</sup><sup>3</sup>

solution of PAM. The impregnation of PAM in calcium alginate beads was carried out in a high-pressure cell, in the presence of compressed carbon dioxide. The impregnation was performed at different pressures (2.5, 5.0, 7.5 MPa) and temperatures (20, 40, 60°C) and also at atmospheric conditions (0.1 MPa, 20°C). The impregnation time was 30 minutes. The samples are designated *p/t*, where *p* and *t*

The efficiency of PAM impregnation at various temperatures and pressures is

The results showed that the efficiency of PAM impregnation increases with temperature at 2.5 MPa and the interaction between the cationic drug and active sites of biopolymer are favored by high pressure. Both state parameters influence the encapsulation of the ionic drug in the polymeric beads. The encapsulation of PAM in calcium alginate beads at 2.5 MPa depends on temperature. Adsorption or/ and absorption of PAM are the main processes which take place between 20 and 40°C. At 60°C, the dissociation constants of polyelectrolyte and ionic drug increase and electrostatic interactions between the polymer and drug molecules are promoted. At higher pressure (5.0 and 7.5 MPa), the impregnation efficiency varies in the same way like ratio of CO2 density to viscosity versus temperature and pressure.

represent the pressure and the temperature of impregnation.

sures near or above critical point (CO2, *pcr* = 7.382 MPa; *tcr* = 31.04°C).

**166**

presented in **Figure 5**.

**Figure 5.** *Efficiency of encapsulation of PAM in alginate microbeads at different pressures and temperatures.*

**Figure 6.** *PAM release profiles from impregnated alginate beads; inset, the release curves in the first 30 minutes.*

### **3.1 In vitro release studies**

The release curves for sample 1/20 and for samples with higher encapsulation efficiency are presented in **Figure 6**. The release curves for samples 0.1/20, 7.5/20, and 5.0/20 show a "burst effect"; the encapsulated PAM is released in the first 15 minutes. The sustained release of PAM is observed for samples 7.5/40 and 2.5/60. The maximum amount of released PAM is lower than 50% for samples 7.5/40, 2.5/60, and 5.0/20. The observed initial burst release profiles may be an indication that the impregnated drug was mainly located at the polymer surface for the sample impregnated with PAM at 20°C and different pressures. The kinetic data were fitted by first-order, Weibull, and Korsmeyer-Peppas kinetic equations. The experimental concentration values of the active substance released as a function of time were processed for establishing the release kinetics.

The kinetic parameters offer information about the encapsulation mechanism which can take place by adsorption on the surface of the beads or by interactions

between drug and polymer inside the alginate beads. The PAM release curves show that the release mechanism depends on the drug impregnation conditions.

The encapsulation of PAM in alginate beads was improved by impregnation in the presence of compressed carbon dioxide at high temperature. The release of ionic drug from drug delivery beads obtained by impregnation of PAM in supercritical CO2 (7.5 MPa, 40°C) is controlled by diffusion of active substances. The alginate beads with encapsulated PAM in subcritical conditions (7.5 MPa, 40°C) release PAM according to swelling and erosion of an amorphous biopolymer.

The release of PAM from alginate beads can be controlled by changing the initial conditions of impregnation in the presence of CO2.
