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(Ethyl lactate)-gel high pressure CO2 extraction for the processing of mesoporous gelatine particles
J. of Supercritical Fluids 83 (2013) 35–40
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The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
(Ethyl lactate)-gel high pressure CO2 extraction for the processing of mesoporous gelatine particles A.B. Paninho a , C. Barbosa a , I.D. Nogueira b , V. Najdanovic-Visak c , A.V.M. Nunes d,∗ a
Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, Oeiras 2781-901, Portugal Instituto de Ciência e Engenharia de Materiais e Superfcies IST, Lisbon, Portugal Energy Lancaster, Engineering Department, Lancaster University, Lancaster LA1 4YR, UK d Requimte/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, Caparica 2829-516, Portugal b c
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 31 July 2013 Accepted 1 August 2013 Keywords: CO2 Gelatin Microparticles Mesoporous Extraction Drying Supercritical control
a b s t r a c t (Ethyl lactate)-gel high pressure CO2 extraction was successfully used as final step for mesoporous gelatine particles preparation. Gelatine spherical microparticles were produced by the water in oil (W/O) emulsion method and further cross-linked with vanillin, to increase its biodegradation resistance. A multi-step solvent exchange of water by ethyl lactate was performed and the gel particles were dried using a semi-continuous high pressure CO2 extraction process. Ethyl lactate was used in this work as an alternative solvent due to high affinity to CO2 and its benign and green nature. The effect of different parameters, such as solvent exchange temperature and the CO2 extraction operating conditions were investigated. The (CO2 + ethyl lactate) binary mixture composition at the beginning of supercritical extraction process has proven to be an important parameter, considerably influencing textural properties of final dried microspheres. Surface areas of 10 to 300 cm2 g−1 and pore diameters from 10 to 17 nm were obtained as the quantity of CO2 in the mixture decreased. (Ethyl lactate)-gel high pressure CO2 drying revealed to be a feasible alternative, enabling a “supercritical-control” approach of gelatine microspheres textural properties. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Gelatine particles have been extensively investigated for pharmaceutical and medical uses [1,2]. Main reported applications describe the use of gelatine particles as drug carriers for various administration routes (e.g. gastrointestinal and nasal) [3,4], as growth factors delivery vehicles [5,6] and as 3D scaffolding systems [7]. Most publications in the field, strengthens the fact that gelatine stands for a natural choice between a wide range of possibilities due to its inherent biocompatibility and biodegradability, high water adsorbing capacity, high availability and low price. On the other hand, due to high solubility at physiologic conditions, gelatine needs to be chemically cross-linked, in order to decrease the solubization rate [8]. An important issue in this regard is to ensure that reagents used are both effective and non-toxic. Chemical cross-linking is generally induced by a chemical compound such as gluteraldehyde, although the use of benign alternatives as glyceraldehydes, genipin and vanillin has been reported [9–11]. Surface functionalization is another important feature, due to
∗ Corresponding author. Fax: +351 212948550. E-mail address: [email protected] (A.V.M. Nunes). 0896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2013.08.002
gelatine’s high content of functional groups. In this context, particles presenting mesoporous morphologies are attractive, namely for application in target drug delivery, due to larger pores and high surface areas, although with an increase risk of pore collapse [12]. A common method to produce gelatine microspheres involves the formation of small droplets of a heated aqueous gelatine solution in an immiscible medium forming a water-in-oil (W/O) emulsion, which is then cooled allowing the aqueous phase droplets to gel. The recent study of Peng et al. [13] has shown the importance of the type of post-treatment on gelatine microspheres morphology. The authors obtained different morphologies going from absence of pores on the surface to a wrinkled surface and a completely open pore structure, by using different post-treatments consisting of acetone/water solution, solely acetone and by common freeze-drying technique. Alternatively, solvent removal by freeze-drying can be performed using high pressure CO2 , with reported successful results for the production of porous particles from hydrocolloid materials [14–19]. High pressure CO2 technology has been widely reported for application in drug delivery, not only as an alternative particle formation technique [20,21], but also as a final drying step for solvent removal of particles produced by conventional techniques [15]. In this latter strategy and in the case of hydrocolloid materials, a previous preparation of hydrogel particles
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A.B. Paninho et al. / J. of Supercritical Fluids 83 (2013) 35–40
is required, followed by a high pressure CO2 drying of the gel. Due to the low affinity between water and CO2 , a solvent exchange has to be performed usually with ethanol, which is then dried at supercritical conditions. It is claimed that the presence of a supercritical fluid in the pores, instead of a liquid phase, leads to the absence of surface tension, avoiding pore collapse during solvent elimination [15]. In this work, the water present in the gel structure was substituted alternatively by ethyl lactate instead of ethanol. The possibility of increasing the variety of solvents that can be used in the drying process, open the possibility to manipulate morphologies, due to different interactions between the solvent and the material. Ethyl lactate is a benign green solvent approved by the US Food and Drug Administration (FDA) as pharmaceutical and food additive and a generally recognized as a safe (GRAS) solvent [22]. There are three main reasons which make ethyl lactate a potential alternative to ethanol: (i) Ethyl lactate is also totally water soluble, with lower polarity values than ethanol, but density values closer to water; (ii) Ethyl lactate is a non-volatile solvent, making mixture of CO2 -ethyl lactate more easier to separate than CO2 -ethanol, foreseeing less waste in industrial processes; (iii) Like ethanol, ethyl lactate has a high affinity to CO2 . In order to design the high pressure CO2 drying process, phase behavior knowledge of the binary system (ethyl lactate + CO2 ) is essential. Villanueva et al. [23] have recently reported data on solubility of CO2 in the ethyl lactate liquid phase at temperatures of (311, 318, and 323) K and pressures ranging from (1 to 8.1) MPa. Earlier, Chylinski and Gregorowicz [24] reported data on solubility of ethyl lactate in CO2 at the same temperatures. Moreover, Cho et al. [25] studied high-pressure equilibrium of the binary system (ethyl lactate + CO2 ) from 323.2 to 363.2 K. In this work, high pressure CO2 drying of (ethyl lactate)-gel was performed at two different temperatures, 313.15 and 333.15 K, using a semi-continuous extraction apparatus. In order to assure the supercritical conditions of the extraction process for both temperatures, phase behavior of the binary system (ethyl lactate + CO2 ) was studied at 333.15 K, due to a considerable deviation between experimental data available in the literature. High pressure CO2 drying of (ethyl lactate)-gel enabled the production of porous gelatine microparticles and proved to be a feasible alternative to the use of ethanol. 2. Materials and methods 2.1. Materials High purity carbon dioxide 99.998 mol% was supplied by Air Liquide and was used as received. Bacteriological gelatine, type A “Cultimed” was supplied from Panreac, Spain while vanillin (99%), was supplied by Sigma-Aldrich. Ethyl lactate was purchased from Sigma-Aldrich and dried on 3 A˚ molecular sieves for at least 48 h. The water content after drying was determined regularly by KarlFischer Coulometric titration (Metrohm 831 KF Coulometer) to less than 130 ppm. 2.2. Methods 2.2.1. Phase equilibrium measurements Phase equilibrium measurements were performed using a highpressure apparatus (New Ways of Analytics GmbH, Germany), described in detail elsewhere [25]. The apparatus is composed by a cell, equipped with two sapphire windows positioned at the front and at the back of the cell, allowing the visual observation of phase transitions. The back sapphire acted as a piston, moving inside and along the stainless steel cylinder by means of a hydraulic
fluid pump, varying the internal volume of the cell (between 38 and 70 ml). The apparatus operates between ambient temperature and 345 K and pressures between atmospheric up to 70 MPa. The temperature control is achieved by means of a PID controller (Eurotherm 2216e), connected to a temperature sensor in direct contact with the fluid mixture inside the cell body, and two electrical band heaters. Pressure is measured by an Omega DP41-E230 transducer. Each cloud point was determined using the same procedure as follows. Depending on the desired composition, known amounts of ethyl lactate and CO2 were loaded into the equilibrium cell. The addition of CO2 was performed using a manual screw injector and calculated by the variation of volume per rotation as described by Podilla et al. [26]. Briefly, the mixture inside the cell was vigorously stirred using a magnetic drive propeller. After attaining the desired temperature, the cell pressure was increased by applying pressure on the back sapphire piston with the hydraulic pump. When a single phase was reached, the system was stirred for more 30 min and then the cell pressure was decreased very slowly, until visual observation of a new phase formation. Two different phase transition patterns were clearly observed. At the dew point, the falling of dew generated a down flow pattern and increase in the liquid level inside the cell. At the bubble point, ascending bubbles provoked an up flow pattern and a corresponding decrease in the liquid level. The critical composition and corresponding critical pressure was assigned when the dew point and the bubble point were undistinguishable. All vapour liquid experiments were performed at least twice and the average result was taken into account. The reproducibility was ±0.01 mole fraction of CO2 and ±0.1 MPa for pressure. 2.2.2. Gelatine ethyl lactate-gel particles preparation Gelatine particles were prepared by the water in oil (W/O) emulsion method following a similar procedure to the one reported by Peng et al. [11] Gelatine (1 g) aqueous solutions (10% w/w) was prepared at 60 ◦ C and slowly added to vegetable oil also heated to 60 ◦ C, while stirring, with the formation of a W/O emulsion. The emulsion was stirred for 10 min using mechanical stirring (IKA, model RW 20) at 600 rpm and then placed on ice to induce aqueous phase droplets to gel. To cross-link the gelatine hydrogel particles, a 0.8 M solution of vanillin in acetone was added and the emulsion was left stirring for another 40 min. Finally, the microspheres were filtered and a multistep solvent exchange using solutions of ethyl lactate/water with different volume ratios was performed as described by Carlos Garcia et al. [13]. Solvent exchange of water by ethyl lactate was performed at two different temperatures, 293 K (ambient temperature) and 278 K, in order to find out it influence on particles final morphological properties. 2.2.3. (Ethyl lactate)-gel high pressure CO2 drying Supercritical fluid extraction of ethyl lactate was carried out using a semi-continuous high pressure apparatus, described in detail elsewhere [27], with few modifications. Briefly, the high pressure apparatus is composed of a cylindrical extractor of 28 cm3 , positioned vertically in a thermostated water bath, heated to the desired temperature by means of a controller that maintained the temperature within ±1 K (JP Selecta Termotronic). The gel particles, were placed at the bottom of the extractor sole, or immersed in 5 ml of ethyl lactate, in order to study the influence of different binary mixture (CO2 + ethyl lactate) initial compositions. The addition of CO2 was made using a pneumatic compressor (Electrolux) connected to the top of the reactor, through a 1/16 stainless steel tubing, which passes through the sealing system, releasing the CO2 directly at the bottom of the reactor. The pressure in the system was measured with a pressure transducer Setra Datum 2000 TM
A.B. Paninho et al. / J. of Supercritical Fluids 83 (2013) 35–40
14
37
323 K (Villanueva Bermejo et al.) BP + (Chylinski and Gregorowicz) DP
318 K (Villanueva Bermejo et al.) BP + (Chylinski and Gregorowicz) DP
12
311 K (Villanueva Bermejo et al.) BP + (Chylinski and Gregorowicz) DP 343 K (Cho et al.) BP, DP and CP 333 K (Cho et al.) BP and DP
10
P(MPa)
333 K (Cho et al.) CP
323 K (Cho et al.) BP and CP
8
333 K (this work) BP and DP 333 K (this work) CP
6
4
2
0 0
0.1
0.2
0.3
0.4 0.5 0.6 CO2 mol fraction
0.7
0.8
0.9
1
Fig. 1. Comparison of vapour-liquid equilibrium data published in literature, for the binary system ethyl lactate + CO2 at different temperatures, with results obtained in this work at 333.15 K. BP = Bubble point; DP = Dew point; CP = Critical point.
calibrated between 0 and 34.3 MPa (with a precision of ±0.1% at the lowest pressure). CO2 exit the system through two high-pressure valves, connected directly to the sealing system at the top of the extractor, and further connected to a gas flow meter. The two high-pressure valves are slowly released and manipulated to control the CO2 flow and maintain constant the pressure inside the system. The process was carried out during two hours, with a constant CO2 flow rate of 3 g min−1 . Different temperature and pressure conditions were experimented in order to determine their influence on final particles morphological properties. The validity of the experimental method for the drying was confirmed by carrying out replicas of the assays at random conditions, resulting in the same textural properties. The formation of a white free flowing powder was observed in all experiments. 2.2.4. Gelatine microparticles characterization Particle morphology was analyzed by Field Emission Scanning Microscopy (FE-SEM JEOL 7001F). Before analysis particles were covered with approximately 300 A˚ of gold by a sputter-coater in argon atmosphere (Polaron). Furthermore, gelatine particles textural properties, namely, surface area, pore size and pore size distribution were determined by N2 adsorption–desorption technique at 77 K with a Micromeritics ASAP 2010 apparatus. 3. Results and discussion Gelatine hydrogel microspheres were prepared by conventional (W/O) emulsion technique and further processed in order to obtain dried particles with high surface areas and porosity. Furthermore, with the intention of removing water from gel particles, maintaining undamaged the porous texture of the wet material, a multistep solvent exchange to ethyl lactate was performed, followed by a high pressure CO2 extraction of the solvent. Ethyl lactate was explored as an alternative solvent to replace water in the gel structure due to its high affinity to CO2 and its benign and green nature. Two moderate temperatures were selected to perform the supercritical fluid extraction step, 313.15 and 333.15 K. To assure supercritical conditions of extraction process for both temperatures and since experimental data available in literature exhibits a
considerable deviation, phase behavior of the binary system (ethyl lactate + CO2 ) was studied for 333.15 K. Fig. 1 compares experimental data on the binary system (ethyl lactate + CO2 ) published by different authors at the temperature range of interest, with experimental results obtained in this work at 333.15 K. As expected, solubilities were enhanced by pressure increase and by temperature decrease. Furthermore, experimental data obtained in this work, shows consistency with data reported by Cho et al. [25] using a similar synthetic visual method. On the other hand, data reported by Villanueva Bermejo et al. [24] using a different analytical method presents a considerable deviation, which is very likely due to the difference in the methodology used. Mixture critical pressure and composition were estimated to be 10.81 MPa and 0.964 CO2 mol fraction, respectively. Based on literature data [23–25] and experimental results obtained in this work, a pressure of 14 MPa was selected to perform ethyl lactate extraction from the gel. This pressure is sufficiently above mixture critical pressure, to assure supercritical conditions of the extraction process for both working temperatures of 313.15 and 333.15 K and still allowing to further decrease to 9.5 MPa to study the pressure effect at 313.15 K. Table 1 summarizes different operating conditions investigated and textural properties of gelatine microparticles obtained. Gelatine microparticles in the form of a white free flowing powder were obtained in all experiments. The temperature at which solvent exchange was performed had the most influence on particles physical appearance. In fact, when this step was performed at ambient temperature, final particles were more aggregated, presenting much more irregular surfaces. On the other hand, for experiments in which solvent exchange was performed at 278 K, nice, non-aggregated spherical particles with smooth surfaces were obtained. Fig. 2 shows the SEM images corresponding to gelatine microparticles obtained from the first two experiments presented in Table 1, in which only the temperature of solvent exchange was varied. With regard to the influence of temperature and pressure during CO2 extraction process (third and fourth entry of Table 1), either temperature increase and pressure decrease, significantly increased particles surface areas (270 and 300 m2 g−1 , respectively), although with a relatively similar impact. Fig. 3 shows the SEM images corresponding to gelatine microparticles obtained
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Table 1 The effect of different operating conditions investigated on the textural properties of gelatine microparticles. Solvent exchange
Supercritical extraction
T (K)
Ethyl lactate (ml)
P (MPa)
CO2 (g/cm3 )
T (K)
Textural properties
278 293 278 278 278
5 5 5 5 ∼0
14 14 9.5 14 14
763.27 763.27 580.01 561.37 763.27
313.15 313.15 313.15 333.15 313.15
Surface area (BET) (m2 g−1 ) 85 140 270 300 10
Pore volume (cm3 g−1 )
Pore diameter (nm)
0.39 0.62 1.30 1.96 0.08
19 11 13 17 10
Fig. 2. SEM images of gelatine microparticles obtained from the two first experiments presented in Table 1 varying only the solvent exchange temperature (a) T = 278 K; (b) T = 293 K.
Fig. 3. SEM images of gelatine microparticles obtained from the third and fourth experiments presented in Table 1 in which the influence of the supercritical extraction conditions were investigated (a) T = 333 K and P = 14 MPa; (b) T = 313 K and P = 9.5 MPa.
A.B. Paninho et al. / J. of Supercritical Fluids 83 (2013) 35–40
from the conditions of third and fourth entry in Table 1 and in which the influence of temperature and pressure of supercritical extraction process was investigated. This result suggests an influence of binary mixture (CO2 + ethyl lactate) initial composition (the starting point of the extraction process). In fact, for both experiments, the supercritical mixture will be less rich in CO2 either by temperature increase or pressure decrease. This possibility was investigated in this work, through an experiment (fifth entry of Table 1), in which no ethyl lactate was added to the CO2 extractor, drastically decreasing ethyl lactate-CO2 ratio. Since ethyl lactate is a non-volatile solvent, it was possible to perform this experiment without the risk of solvent evaporation from the gel, before being in contact with scCO2 atmosphere. In this way, the quantity of ethyl lactate that entered the extractor was already embedded into the gel structure, which was significantly less when compared with the five mL introduced for all other experiments. The N2 adsorption–desorption analysis revealed a significantly decrease in the surface area, which was actually visible by the physical appearance of particles with naked eye (clear and light versus opaque and dense). Curiously, micrographs obtained by SEM, do not let to guess the 30 times increase in the surface area obtained by changing operational conditions, as determined by ASAP. Furthermore, a direct comparison with results from literature for different materials using ethanol instead of ethyl lactate, could not be made, since there are other factors influencing the process, such as CO2 flow rate, reactor size, quantity of particles used, hydrogel particles preparation method, and others, that are not comparable. Nevertheless, surface areas, as well as pore diameters obtained, are about the same order of magnitude as ones reported, using ethanol for different materials [14]. From results obtained and presented in Table 1, it is clear that the variation of (CO2 + ethyl lactate) binary mixture composition at the beginning of supercritical extraction process had a marked effect in the gel textural properties, which resulted in a surface area increase from 10 to 300 g cm−2 and a pore diameter increase from 10 to 17 nm, as CO2 composition in the mixture decreased. This result provides an additional important mean to control textural properties of high pressure CO2 dried particles. In fact, at the beginning of the supercritical extraction process, after supercritical conditions are attained, when the material is still very flexible, different binary (CO2 + ethyl lactate) mixture compositions are able to control pores volume through mixture density and degree of swelling. Furthermore, along the extraction process, the binary (CO2 + ethyl lactate) mixture composition will vary at a constant temperature and pressure until all solvent is extracted, but maintaining gel initial structure. The path along the phase diagram may be quite different depending on the quantity of solvent introduced into the reactor together with the gel particles to be dried. In limit conditions even the transition from two phases (liquid–gas) to one supercritical phase can occur nearby particles surface if a very low amount of liquid solvent is present comparing to CO2 .
4. Conclusion In this work, a significant influence of (CO2 + ethyl lactate) binary mixture composition at the beginning of supercritical extraction process, on the textural properties of final dried particles, was found out. This observation results from a combined effect of ethyl lactate degree of swelling/mixture density that, at this stage, due to gel high physical flexibility, is able to control the matrix pores. This ability is a promising tool regarding “morphology control” and open for new opportunities on the utilization of CO2 as a final drying step of hydrocolloid highly porous structures.
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Acknowledgements This work was supported by Fundac¸ão para a Ciência e a Tecnologia–FCT (Portugal) through project PTDC/EQUEQU/104552/2008 and through PEst-C/EQB/LA0006/2011. A.V.M. Nunes is thankful to FCT for the post-doctoral fellowship SFRH/BPD/74994/2010. References [1] S. Young, M. Wong, Y. Tabata, A.G. Mikos, Gelatin as a delivery vehicle for the controlled release of bioactive molecules, Journal of Controlled Release 109 (2005) 256–274. [2] Y. Tabata, Y. Ikada, Protein release from gelatin matrices, Advanced Drug Delivery Reviews 31 (1998) 287–301. [3] R. Narayani, K.P. Rao, Biodegradable microspheres using two different gelatin drug conjugates for the controlled delivery of methotrexate, International Journal of Pharmaceutics 128 (1996) 261–268. [4] Y.-Z. Zhao, X. Li, C.-T. Lu, Y.-Y. Xu, H.-F. Lv, D.-D. Dai, L. Zhang, C.-Z. Sun, W. Yang, X.-K. Li, Y.-P. Zhao, H.-X. Fu, L. Cai, M. Lin, L.-J. Chen, M. 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Contents lists available at ScienceDirect
The Journal of Supercritical Fluids journal homepage: www.elsevier.com/locate/supflu
(Ethyl lactate)-gel high pressure CO2 extraction for the processing of mesoporous gelatine particles A.B. Paninho a , C. Barbosa a , I.D. Nogueira b , V. Najdanovic-Visak c , A.V.M. Nunes d,∗ a
Instituto de Biologia Experimental e Tecnológica (IBET), Apartado 12, Oeiras 2781-901, Portugal Instituto de Ciência e Engenharia de Materiais e Superfcies IST, Lisbon, Portugal Energy Lancaster, Engineering Department, Lancaster University, Lancaster LA1 4YR, UK d Requimte/CQFB, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, Campus de Caparica, Caparica 2829-516, Portugal b c
a r t i c l e
i n f o
Article history: Received 31 May 2013 Received in revised form 31 July 2013 Accepted 1 August 2013 Keywords: CO2 Gelatin Microparticles Mesoporous Extraction Drying Supercritical control
a b s t r a c t (Ethyl lactate)-gel high pressure CO2 extraction was successfully used as final step for mesoporous gelatine particles preparation. Gelatine spherical microparticles were produced by the water in oil (W/O) emulsion method and further cross-linked with vanillin, to increase its biodegradation resistance. A multi-step solvent exchange of water by ethyl lactate was performed and the gel particles were dried using a semi-continuous high pressure CO2 extraction process. Ethyl lactate was used in this work as an alternative solvent due to high affinity to CO2 and its benign and green nature. The effect of different parameters, such as solvent exchange temperature and the CO2 extraction operating conditions were investigated. The (CO2 + ethyl lactate) binary mixture composition at the beginning of supercritical extraction process has proven to be an important parameter, considerably influencing textural properties of final dried microspheres. Surface areas of 10 to 300 cm2 g−1 and pore diameters from 10 to 17 nm were obtained as the quantity of CO2 in the mixture decreased. (Ethyl lactate)-gel high pressure CO2 drying revealed to be a feasible alternative, enabling a “supercritical-control” approach of gelatine microspheres textural properties. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Gelatine particles have been extensively investigated for pharmaceutical and medical uses [1,2]. Main reported applications describe the use of gelatine particles as drug carriers for various administration routes (e.g. gastrointestinal and nasal) [3,4], as growth factors delivery vehicles [5,6] and as 3D scaffolding systems [7]. Most publications in the field, strengthens the fact that gelatine stands for a natural choice between a wide range of possibilities due to its inherent biocompatibility and biodegradability, high water adsorbing capacity, high availability and low price. On the other hand, due to high solubility at physiologic conditions, gelatine needs to be chemically cross-linked, in order to decrease the solubization rate [8]. An important issue in this regard is to ensure that reagents used are both effective and non-toxic. Chemical cross-linking is generally induced by a chemical compound such as gluteraldehyde, although the use of benign alternatives as glyceraldehydes, genipin and vanillin has been reported [9–11]. Surface functionalization is another important feature, due to
∗ Corresponding author. Fax: +351 212948550. E-mail address: [email protected] (A.V.M. Nunes). 0896-8446/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.supflu.2013.08.002
gelatine’s high content of functional groups. In this context, particles presenting mesoporous morphologies are attractive, namely for application in target drug delivery, due to larger pores and high surface areas, although with an increase risk of pore collapse [12]. A common method to produce gelatine microspheres involves the formation of small droplets of a heated aqueous gelatine solution in an immiscible medium forming a water-in-oil (W/O) emulsion, which is then cooled allowing the aqueous phase droplets to gel. The recent study of Peng et al. [13] has shown the importance of the type of post-treatment on gelatine microspheres morphology. The authors obtained different morphologies going from absence of pores on the surface to a wrinkled surface and a completely open pore structure, by using different post-treatments consisting of acetone/water solution, solely acetone and by common freeze-drying technique. Alternatively, solvent removal by freeze-drying can be performed using high pressure CO2 , with reported successful results for the production of porous particles from hydrocolloid materials [14–19]. High pressure CO2 technology has been widely reported for application in drug delivery, not only as an alternative particle formation technique [20,21], but also as a final drying step for solvent removal of particles produced by conventional techniques [15]. In this latter strategy and in the case of hydrocolloid materials, a previous preparation of hydrogel particles
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is required, followed by a high pressure CO2 drying of the gel. Due to the low affinity between water and CO2 , a solvent exchange has to be performed usually with ethanol, which is then dried at supercritical conditions. It is claimed that the presence of a supercritical fluid in the pores, instead of a liquid phase, leads to the absence of surface tension, avoiding pore collapse during solvent elimination [15]. In this work, the water present in the gel structure was substituted alternatively by ethyl lactate instead of ethanol. The possibility of increasing the variety of solvents that can be used in the drying process, open the possibility to manipulate morphologies, due to different interactions between the solvent and the material. Ethyl lactate is a benign green solvent approved by the US Food and Drug Administration (FDA) as pharmaceutical and food additive and a generally recognized as a safe (GRAS) solvent [22]. There are three main reasons which make ethyl lactate a potential alternative to ethanol: (i) Ethyl lactate is also totally water soluble, with lower polarity values than ethanol, but density values closer to water; (ii) Ethyl lactate is a non-volatile solvent, making mixture of CO2 -ethyl lactate more easier to separate than CO2 -ethanol, foreseeing less waste in industrial processes; (iii) Like ethanol, ethyl lactate has a high affinity to CO2 . In order to design the high pressure CO2 drying process, phase behavior knowledge of the binary system (ethyl lactate + CO2 ) is essential. Villanueva et al. [23] have recently reported data on solubility of CO2 in the ethyl lactate liquid phase at temperatures of (311, 318, and 323) K and pressures ranging from (1 to 8.1) MPa. Earlier, Chylinski and Gregorowicz [24] reported data on solubility of ethyl lactate in CO2 at the same temperatures. Moreover, Cho et al. [25] studied high-pressure equilibrium of the binary system (ethyl lactate + CO2 ) from 323.2 to 363.2 K. In this work, high pressure CO2 drying of (ethyl lactate)-gel was performed at two different temperatures, 313.15 and 333.15 K, using a semi-continuous extraction apparatus. In order to assure the supercritical conditions of the extraction process for both temperatures, phase behavior of the binary system (ethyl lactate + CO2 ) was studied at 333.15 K, due to a considerable deviation between experimental data available in the literature. High pressure CO2 drying of (ethyl lactate)-gel enabled the production of porous gelatine microparticles and proved to be a feasible alternative to the use of ethanol. 2. Materials and methods 2.1. Materials High purity carbon dioxide 99.998 mol% was supplied by Air Liquide and was used as received. Bacteriological gelatine, type A “Cultimed” was supplied from Panreac, Spain while vanillin (99%), was supplied by Sigma-Aldrich. Ethyl lactate was purchased from Sigma-Aldrich and dried on 3 A˚ molecular sieves for at least 48 h. The water content after drying was determined regularly by KarlFischer Coulometric titration (Metrohm 831 KF Coulometer) to less than 130 ppm. 2.2. Methods 2.2.1. Phase equilibrium measurements Phase equilibrium measurements were performed using a highpressure apparatus (New Ways of Analytics GmbH, Germany), described in detail elsewhere [25]. The apparatus is composed by a cell, equipped with two sapphire windows positioned at the front and at the back of the cell, allowing the visual observation of phase transitions. The back sapphire acted as a piston, moving inside and along the stainless steel cylinder by means of a hydraulic
fluid pump, varying the internal volume of the cell (between 38 and 70 ml). The apparatus operates between ambient temperature and 345 K and pressures between atmospheric up to 70 MPa. The temperature control is achieved by means of a PID controller (Eurotherm 2216e), connected to a temperature sensor in direct contact with the fluid mixture inside the cell body, and two electrical band heaters. Pressure is measured by an Omega DP41-E230 transducer. Each cloud point was determined using the same procedure as follows. Depending on the desired composition, known amounts of ethyl lactate and CO2 were loaded into the equilibrium cell. The addition of CO2 was performed using a manual screw injector and calculated by the variation of volume per rotation as described by Podilla et al. [26]. Briefly, the mixture inside the cell was vigorously stirred using a magnetic drive propeller. After attaining the desired temperature, the cell pressure was increased by applying pressure on the back sapphire piston with the hydraulic pump. When a single phase was reached, the system was stirred for more 30 min and then the cell pressure was decreased very slowly, until visual observation of a new phase formation. Two different phase transition patterns were clearly observed. At the dew point, the falling of dew generated a down flow pattern and increase in the liquid level inside the cell. At the bubble point, ascending bubbles provoked an up flow pattern and a corresponding decrease in the liquid level. The critical composition and corresponding critical pressure was assigned when the dew point and the bubble point were undistinguishable. All vapour liquid experiments were performed at least twice and the average result was taken into account. The reproducibility was ±0.01 mole fraction of CO2 and ±0.1 MPa for pressure. 2.2.2. Gelatine ethyl lactate-gel particles preparation Gelatine particles were prepared by the water in oil (W/O) emulsion method following a similar procedure to the one reported by Peng et al. [11] Gelatine (1 g) aqueous solutions (10% w/w) was prepared at 60 ◦ C and slowly added to vegetable oil also heated to 60 ◦ C, while stirring, with the formation of a W/O emulsion. The emulsion was stirred for 10 min using mechanical stirring (IKA, model RW 20) at 600 rpm and then placed on ice to induce aqueous phase droplets to gel. To cross-link the gelatine hydrogel particles, a 0.8 M solution of vanillin in acetone was added and the emulsion was left stirring for another 40 min. Finally, the microspheres were filtered and a multistep solvent exchange using solutions of ethyl lactate/water with different volume ratios was performed as described by Carlos Garcia et al. [13]. Solvent exchange of water by ethyl lactate was performed at two different temperatures, 293 K (ambient temperature) and 278 K, in order to find out it influence on particles final morphological properties. 2.2.3. (Ethyl lactate)-gel high pressure CO2 drying Supercritical fluid extraction of ethyl lactate was carried out using a semi-continuous high pressure apparatus, described in detail elsewhere [27], with few modifications. Briefly, the high pressure apparatus is composed of a cylindrical extractor of 28 cm3 , positioned vertically in a thermostated water bath, heated to the desired temperature by means of a controller that maintained the temperature within ±1 K (JP Selecta Termotronic). The gel particles, were placed at the bottom of the extractor sole, or immersed in 5 ml of ethyl lactate, in order to study the influence of different binary mixture (CO2 + ethyl lactate) initial compositions. The addition of CO2 was made using a pneumatic compressor (Electrolux) connected to the top of the reactor, through a 1/16 stainless steel tubing, which passes through the sealing system, releasing the CO2 directly at the bottom of the reactor. The pressure in the system was measured with a pressure transducer Setra Datum 2000 TM
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14
37
323 K (Villanueva Bermejo et al.) BP + (Chylinski and Gregorowicz) DP
318 K (Villanueva Bermejo et al.) BP + (Chylinski and Gregorowicz) DP
12
311 K (Villanueva Bermejo et al.) BP + (Chylinski and Gregorowicz) DP 343 K (Cho et al.) BP, DP and CP 333 K (Cho et al.) BP and DP
10
P(MPa)
333 K (Cho et al.) CP
323 K (Cho et al.) BP and CP
8
333 K (this work) BP and DP 333 K (this work) CP
6
4
2
0 0
0.1
0.2
0.3
0.4 0.5 0.6 CO2 mol fraction
0.7
0.8
0.9
1
Fig. 1. Comparison of vapour-liquid equilibrium data published in literature, for the binary system ethyl lactate + CO2 at different temperatures, with results obtained in this work at 333.15 K. BP = Bubble point; DP = Dew point; CP = Critical point.
calibrated between 0 and 34.3 MPa (with a precision of ±0.1% at the lowest pressure). CO2 exit the system through two high-pressure valves, connected directly to the sealing system at the top of the extractor, and further connected to a gas flow meter. The two high-pressure valves are slowly released and manipulated to control the CO2 flow and maintain constant the pressure inside the system. The process was carried out during two hours, with a constant CO2 flow rate of 3 g min−1 . Different temperature and pressure conditions were experimented in order to determine their influence on final particles morphological properties. The validity of the experimental method for the drying was confirmed by carrying out replicas of the assays at random conditions, resulting in the same textural properties. The formation of a white free flowing powder was observed in all experiments. 2.2.4. Gelatine microparticles characterization Particle morphology was analyzed by Field Emission Scanning Microscopy (FE-SEM JEOL 7001F). Before analysis particles were covered with approximately 300 A˚ of gold by a sputter-coater in argon atmosphere (Polaron). Furthermore, gelatine particles textural properties, namely, surface area, pore size and pore size distribution were determined by N2 adsorption–desorption technique at 77 K with a Micromeritics ASAP 2010 apparatus. 3. Results and discussion Gelatine hydrogel microspheres were prepared by conventional (W/O) emulsion technique and further processed in order to obtain dried particles with high surface areas and porosity. Furthermore, with the intention of removing water from gel particles, maintaining undamaged the porous texture of the wet material, a multistep solvent exchange to ethyl lactate was performed, followed by a high pressure CO2 extraction of the solvent. Ethyl lactate was explored as an alternative solvent to replace water in the gel structure due to its high affinity to CO2 and its benign and green nature. Two moderate temperatures were selected to perform the supercritical fluid extraction step, 313.15 and 333.15 K. To assure supercritical conditions of extraction process for both temperatures and since experimental data available in literature exhibits a
considerable deviation, phase behavior of the binary system (ethyl lactate + CO2 ) was studied for 333.15 K. Fig. 1 compares experimental data on the binary system (ethyl lactate + CO2 ) published by different authors at the temperature range of interest, with experimental results obtained in this work at 333.15 K. As expected, solubilities were enhanced by pressure increase and by temperature decrease. Furthermore, experimental data obtained in this work, shows consistency with data reported by Cho et al. [25] using a similar synthetic visual method. On the other hand, data reported by Villanueva Bermejo et al. [24] using a different analytical method presents a considerable deviation, which is very likely due to the difference in the methodology used. Mixture critical pressure and composition were estimated to be 10.81 MPa and 0.964 CO2 mol fraction, respectively. Based on literature data [23–25] and experimental results obtained in this work, a pressure of 14 MPa was selected to perform ethyl lactate extraction from the gel. This pressure is sufficiently above mixture critical pressure, to assure supercritical conditions of the extraction process for both working temperatures of 313.15 and 333.15 K and still allowing to further decrease to 9.5 MPa to study the pressure effect at 313.15 K. Table 1 summarizes different operating conditions investigated and textural properties of gelatine microparticles obtained. Gelatine microparticles in the form of a white free flowing powder were obtained in all experiments. The temperature at which solvent exchange was performed had the most influence on particles physical appearance. In fact, when this step was performed at ambient temperature, final particles were more aggregated, presenting much more irregular surfaces. On the other hand, for experiments in which solvent exchange was performed at 278 K, nice, non-aggregated spherical particles with smooth surfaces were obtained. Fig. 2 shows the SEM images corresponding to gelatine microparticles obtained from the first two experiments presented in Table 1, in which only the temperature of solvent exchange was varied. With regard to the influence of temperature and pressure during CO2 extraction process (third and fourth entry of Table 1), either temperature increase and pressure decrease, significantly increased particles surface areas (270 and 300 m2 g−1 , respectively), although with a relatively similar impact. Fig. 3 shows the SEM images corresponding to gelatine microparticles obtained
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Table 1 The effect of different operating conditions investigated on the textural properties of gelatine microparticles. Solvent exchange
Supercritical extraction
T (K)
Ethyl lactate (ml)
P (MPa)
CO2 (g/cm3 )
T (K)
Textural properties
278 293 278 278 278
5 5 5 5 ∼0
14 14 9.5 14 14
763.27 763.27 580.01 561.37 763.27
313.15 313.15 313.15 333.15 313.15
Surface area (BET) (m2 g−1 ) 85 140 270 300 10
Pore volume (cm3 g−1 )
Pore diameter (nm)
0.39 0.62 1.30 1.96 0.08
19 11 13 17 10
Fig. 2. SEM images of gelatine microparticles obtained from the two first experiments presented in Table 1 varying only the solvent exchange temperature (a) T = 278 K; (b) T = 293 K.
Fig. 3. SEM images of gelatine microparticles obtained from the third and fourth experiments presented in Table 1 in which the influence of the supercritical extraction conditions were investigated (a) T = 333 K and P = 14 MPa; (b) T = 313 K and P = 9.5 MPa.
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from the conditions of third and fourth entry in Table 1 and in which the influence of temperature and pressure of supercritical extraction process was investigated. This result suggests an influence of binary mixture (CO2 + ethyl lactate) initial composition (the starting point of the extraction process). In fact, for both experiments, the supercritical mixture will be less rich in CO2 either by temperature increase or pressure decrease. This possibility was investigated in this work, through an experiment (fifth entry of Table 1), in which no ethyl lactate was added to the CO2 extractor, drastically decreasing ethyl lactate-CO2 ratio. Since ethyl lactate is a non-volatile solvent, it was possible to perform this experiment without the risk of solvent evaporation from the gel, before being in contact with scCO2 atmosphere. In this way, the quantity of ethyl lactate that entered the extractor was already embedded into the gel structure, which was significantly less when compared with the five mL introduced for all other experiments. The N2 adsorption–desorption analysis revealed a significantly decrease in the surface area, which was actually visible by the physical appearance of particles with naked eye (clear and light versus opaque and dense). Curiously, micrographs obtained by SEM, do not let to guess the 30 times increase in the surface area obtained by changing operational conditions, as determined by ASAP. Furthermore, a direct comparison with results from literature for different materials using ethanol instead of ethyl lactate, could not be made, since there are other factors influencing the process, such as CO2 flow rate, reactor size, quantity of particles used, hydrogel particles preparation method, and others, that are not comparable. Nevertheless, surface areas, as well as pore diameters obtained, are about the same order of magnitude as ones reported, using ethanol for different materials [14]. From results obtained and presented in Table 1, it is clear that the variation of (CO2 + ethyl lactate) binary mixture composition at the beginning of supercritical extraction process had a marked effect in the gel textural properties, which resulted in a surface area increase from 10 to 300 g cm−2 and a pore diameter increase from 10 to 17 nm, as CO2 composition in the mixture decreased. This result provides an additional important mean to control textural properties of high pressure CO2 dried particles. In fact, at the beginning of the supercritical extraction process, after supercritical conditions are attained, when the material is still very flexible, different binary (CO2 + ethyl lactate) mixture compositions are able to control pores volume through mixture density and degree of swelling. Furthermore, along the extraction process, the binary (CO2 + ethyl lactate) mixture composition will vary at a constant temperature and pressure until all solvent is extracted, but maintaining gel initial structure. The path along the phase diagram may be quite different depending on the quantity of solvent introduced into the reactor together with the gel particles to be dried. In limit conditions even the transition from two phases (liquid–gas) to one supercritical phase can occur nearby particles surface if a very low amount of liquid solvent is present comparing to CO2 .
4. Conclusion In this work, a significant influence of (CO2 + ethyl lactate) binary mixture composition at the beginning of supercritical extraction process, on the textural properties of final dried particles, was found out. This observation results from a combined effect of ethyl lactate degree of swelling/mixture density that, at this stage, due to gel high physical flexibility, is able to control the matrix pores. This ability is a promising tool regarding “morphology control” and open for new opportunities on the utilization of CO2 as a final drying step of hydrocolloid highly porous structures.
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Acknowledgements This work was supported by Fundac¸ão para a Ciência e a Tecnologia–FCT (Portugal) through project PTDC/EQUEQU/104552/2008 and through PEst-C/EQB/LA0006/2011. A.V.M. Nunes is thankful to FCT for the post-doctoral fellowship SFRH/BPD/74994/2010. References [1] S. Young, M. Wong, Y. Tabata, A.G. Mikos, Gelatin as a delivery vehicle for the controlled release of bioactive molecules, Journal of Controlled Release 109 (2005) 256–274. [2] Y. Tabata, Y. Ikada, Protein release from gelatin matrices, Advanced Drug Delivery Reviews 31 (1998) 287–301. [3] R. Narayani, K.P. Rao, Biodegradable microspheres using two different gelatin drug conjugates for the controlled delivery of methotrexate, International Journal of Pharmaceutics 128 (1996) 261–268. [4] Y.-Z. Zhao, X. Li, C.-T. Lu, Y.-Y. Xu, H.-F. Lv, D.-D. Dai, L. Zhang, C.-Z. Sun, W. Yang, X.-K. Li, Y.-P. Zhao, H.-X. Fu, L. Cai, M. Lin, L.-J. Chen, M. 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