Microencapsulation of Coenzyme Q10 in Poly(ethylene glycol) and

Departamento de Ingeniería y Ciencias Químicas, Universidad Iberoamericana, Prol. Paseo de la Reforma 880, Lomas de Santa Fe, México D. F. 01219, ...
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Microencapsulation of Coenzyme Q10 in Poly(ethylene glycol) and Poly(lactic acid) with Supercritical Carbon Dioxide María del Socorro Vergara-Mendoza,† Ciro-Humberto Ortiz-Estrada,*,† Juana Gonzalez-Martínez,† and Jesus-Alberto Quezada-Gallo† †

Departamento de Ingeniería y Ciencias Químicas, Universidad Iberoamericana, Prol. Paseo de la Reforma 880, Lomas de Santa Fe, Mexico D. F. 01219, Mexico ABSTRACT: The rapid expansion of supercritical solutions (RESS) process was applied to produce polymeric microparticles loaded with coenzyme Q10 (coQ10). Mixtures of poly(lactic acid) (PLA)/(coQ10) and poly(ethylene glycol) (PEG)/(coQ10) containing a cosolvent were dissolved in supercritical CO2 and sprayed through a nozzle to atmospheric pressure. The effect of process parameters such as encapsulating material/coQ10 ratio and cosolvent on the properties of the microcapsules were investigated. The characterization of microcapsules was carried out through Scanning Electronic Microscopy (SEM), Infrared Spectrophotometry (IR), Differential Scanning Calorimetry (DSC), and solubility kinetics. More important effect of the ratio encapsulating material/coQ10, compared with the effect of type of cosolvent, on studied properties was found.

1. INTRODUCTION Microencapsulation technology has been used over 70 years, mainly in pharmaceutical industry, with the purpose of protecting active components from degradation and to release them in a controlled way.1 3 This controlled liberation has been a subject of scientific interest the last two decades, requiring the formations of particles having a diameter of approximately 10 μm, obtained by conventional methods such as coacervation and interfacial polymerization. However, these methods have some disadvantages like the use of toxic organic solvents and the difficulty to control the particle size.4,5 An alternative to eliminate those disadvantages is the use of a supercritical fluid (SCF), which possess low viscosity and diffusivity, comparable with those of a gas, as well as a low superficial tension, conferring them excellent mass transfer properties. Supercritical carbon dioxide (CO2SC) is the fluid widely used due to its relatively low critical temperature and moderated critical pressure, besides the fact that it is not toxic, not flammable and with low cost.3 6 Rapid expansion of supercritical solutions (RESS) is the simplest technique using a SCF, consisting on a solution of a solute in CO2SC, followed by an instantaneous depressurization through a nozzle, provoking a rapid nucleation and the formations of small particles.3 A disadvantage of RESS is the fact that a large number of molecules are not soluble in CO2 which is caused by the absence of dipolar momentum and its small density of cohesive energy, nevertheless, this problem may be eliminated by using a cosolvent such as ethanol or acetone, increasing solute solubility.4,7,8 This method has been progressing in the past few years; however, some of the important aspects in the process are not yet clear, i.e. the influence of operation parameters on produced particles characteristics (morphology and size), the nucleation phenomenon and particles agglomeration, turning the study of this parameters an important matter.7,8 In this work, coenzyme Q10 (coQ10) is encapsulated in poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) by the r 2011 American Chemical Society

application of RESS technique, to analyze the effect of the ratio encapsulating material/coQ10 and the cosolvent on microcapsules properties. CoQ10 has been used in several medical treatments, such as the prevention of cancer and neurodegenerative diseases, to name a few examples.9,10 PLA is a biodegradable polymer. Its monomer unit is lactic acid, which is produced in the human organism during normal metabolism. PLA can thus be fully absorbed by the human organism after its degradation to its monomer units.5 PEG, although it is not a biodegradable polymer, is used as materials for drug delivery systems due to its biocompatibility. Before the application of the RESS technique, it was necessary to better know the equilibrium of the implied system and, therefore, to determine experimentally the phases behavior of systems encapsulating material/coQ10 in the presence of a supercritical fluid and a cosolvent, and separately. The characterization of microcapsules has been carried out through Scanning Electronic Microscopy (SEM), Infrared Spectrophotometry (IR), Differential Scanning Calorimetry (DSC), and solubility kinetics.

2. EXPERIMENTAL SECTION 2.1. Materials. CoQ10 was donated by Nano Nutrition S. de R.L. de C.V. (Mexico); PEG-4000 (99% purity) and PLA (finished ester) were purchased from Sigma-Aldrich; carbon dioxide (CO2), highly pure (99.99%), was acquired from Infra S.A. de C. V. (Mexico); ethanol and acetone (99.8%) and dichloromethane (DCM) were obtained from J.T. Baker purity degree for HPLC (Mexico). Special Issue: AMIDIQ 2011 Received: July 12, 2011 Accepted: December 13, 2011 Revised: December 4, 2011 Published: December 13, 2011 5840

dx.doi.org/10.1021/ie2014839 | Ind. Eng. Chem. Res. 2012, 51, 5840–5846

Industrial & Engineering Chemistry Research

ARTICLE

Figure 1. Diagram of the high pressure equipment.

Figure 2. Solubility with 24 mol % of cosolvent A) PEG and B) coQ10.

2.2. Equipment RESS. The necessary equipment to study phases equilibrium, micronization, and microencapsulation is shown in Figure 1. It is composed of two systems, a high pressure system and a feeding and pressurization of CO2 system. The high pressure system is formed by a pressurized cylinder for CO2 and a high pressure cell (HPC) where the solution is confined. The HPC is a cylinder divided in two chambers by a piston; the supercritical solution is prepared in the front chamber and the CO2 is stored in the back chamber, having the function of maintaining the desired pressure in the front chamber. The cell possesses lateral sapphire windows and two available caps for the front chamber, one of them useful in equilibrium determination by seeing at the solution through the sapphire glass, and the other one to microencapsulate and to micronize with the RESS technique, being connected to a valve with the expansion nozzle, this one at its time inserted in the hole of the expansion chamber where the sample is collected. 2.3. Phase Equilibrium. Solubility of each material and each system encapsulating material/coQ10, in CO2SC/cosolvent was measured by determing the cloud point, described by SantoyoArreola et al.,11 defined as the pressure at which the solution loses transparency or a precipitated has been formed (turbidity is determined visually and by a decrease of the registered voltage), at a constant temperature. All the solubility measurements were replicated at least twice.

2.4. Production of Microparticles with RESS Technique. To micronize and to microencapsulate, the high pressure cell is loaded with the correspondent solution, material/cosolvent, or encapsulating material/coQ10/cosolvent, approximately 4 g and a mass of CO2 is added to complete the CO2/cosolvent ratio desired. Once the CO2 is added, the system is kept for 30 min at the process conditions (pressure 275 bar and temperature 35 °C), remaining homogeneous according to the phase equilibrium study. The expansion takes place next through a 50 μm diameter nozzle, from a high pressure to an atmospheric pressure. The material being conducted to the expansion chamber, where it is collected. 2.5. Microparticles Morphology. Scanning electron microscopy (JEOL, JSM-6390 LV) was used to study the morphology of the microparticles. Samples were coated with a thin layer of gold applied before observation by SEM at 20 KV. The measurement was made and registered by software Digital Micrograph (Version 1.71.38. Gatan, Inc.). At least 100 microparticles were observed per sample. 2.6. Interactions between Encapsulating Material and coQ10. The analysis of interaction was performed with IR spectrophotometry (IR) and thermal analysis. An IR spectrophotometer (Perkin-Elmer, model 1600) was employed with resolution of 4 cm 1 and 32 scans, applying potassium bromide to place the samples. Differential scanning calorimetry (DSC) 5841

dx.doi.org/10.1021/ie2014839 |Ind. Eng. Chem. Res. 2012, 51, 5840–5846

Industrial & Engineering Chemistry Research

ARTICLE

thermograms were obtained using DSC calorimeter (STA 1500) in an inert atmosphere. All samples were placed into aluminum pans, using an empty pan for reference. The temperature range tested was 25 250 °C with a heating rate of 10 °C min 1. With both methods all sample were recorded 3 times and had good reproducibility. 2.7. Release Study. Microparticules (4 mg) were suspended into 3 mL of ethanol and placed in the spectrophotometer cell. At regular time intervals (60 min), the coQ10 concentration was spectrophotometrically determined at 407 nm. The encapsulating

material did not interfere with coQ10 at this wavelength. The release assay was performed in triplicate for each simple.

3. RESULTS AND DISCUSSION 3.1. Phases Equilibrium. Equilibrium diagrams plotting concentration of PEG and coQ10 vs pressure are shown in Figure 2. Above these curves there is one homogeneous phase in the supercritical solution, and beneath two phases. Both diagrams show that an increase in material concentration produces a rise in the needed pressure to maintain a stable system with a homogeneous phase; this behavior was observed for PLA with dichloromethane as a cosolvent. For PEG (Figure 2A), ethanol presents a favorable effect on its solubility compared to acetone, requiring a smaller pressure to solve the same amount of PEG, while for coQ10 (Figure 2B) is acetone who increases solubility in the supercritical system, compared with ethanol. Solubility of these materials has been proved before with supercritical CO2 without the addition of a cosolvent, resulting in all cases very low compared to the results in this work. For coQ10, Matias A. A. et al.12 have reported its solubility in CO2SC,

Table 1. Phases Equilibrium of Quaternary Systems at 35 °C PEG

PLA

CoQ10

cloud point

experiment (% weight) (% weight) (% weight) cosolvent

(bar)

1

1

--

1

ethanol

2

2

--

1

ethanol

80 125

3

1

--

1

acetone

193

4

2

--

1

acetone

265

5

--

1

1

DCM