Article pubs.acs.org/JPCB
Biocolloids Based on Amphiphilic Block Copolymers as a Medium for Enzyme Encapsulation Victoria Sereti,† Maria Zoumpanioti,† Vassiliki Papadimitriou,† Stergios Pispas,‡ and Aristotelis Xenakis*,† †
Institute of Biology, Medicinal Chemistry & Biotechnology and ‡Theoretical & Physical Chemistry Institute, National Hellenic Research Foundation, 48, Vassileos Constantinou Avenue, Athens 11635, Greece ABSTRACT: The ability of two biocompatible amphiphilic block copolymers consisting of hydrophilic poly(ethylene oxide) and hydrophobic poly(εcaprolactone) with different hydrophilic/hydrophobic block ratio to act as stabilizers of water-in-oil (w/o) microemulsions and enzyme encapsulation therein has been tested. Phase diagrams of the two block copolymers in mixtures of chloroform/isopropanol/water were constructed, revealing that the systems can incorporate important amounts of aqueous phase. The w/o microemulsions were then used to encapsulate R. miehei lipase. Empty as well as lipase-loaded systems were characterized by DLS as well as EPR spectroscopy. It was found that the incorporated lipase was preferably localized in the interior of the droplets. The apparent hydrodynamic radii of the droplets were found to vary from 86 to 3000 nm and from 66 to 2140 nm for empty PEO−PCL 30 and PEO−PCL 53 stabilized systems, respectively. In the presence of the lipase, the hydrodynamic radii were considerably decreased. The catalytic activity of the encapsulated lipase was successfully tested via a model esterification reaction. The effect of temperature on the catalytic behavior of the encapsulated R. miehei lipase was investigated, revealing that the initial rate of the esterification reaction depended on the type of the block copolymer used.
■
w) nanoemulsions10−12 for the encapsulation of hydrophobic molecules, such as lipophilic drugs. However, they have not yet been studied as stabilizers of water-in-oil (w/o) micro- or nanoemulsions that could serve as nanobioreactors, in which enzymes could be encapsulated and used for the biocatalytical transformation of nonpolar substrates. The present study is the first report that deals with the formulation and structural characterization of w/o microemulsions stabilized by block copolymers of the type poly(ethylene oxide)−poly(caprolactone), PEO−PCL, of different bock ratio. The hydrophobic PCL block is a semicrystalline polyester, biodegradable, and biocompatible.13 The hydrophilic PEO block is biocompatible but hardly biodegradable. Nevertheless, it is accepted and used as the hydrophilic moiety in many nonionic surfactants such as polysorbates (Tween) and alkyl ethers of PEO (Brij). The parameter that controls the properties of such copolymers is the polymerization degree of the PEO and the PCL blocks. In the present study for the formation of w/o microemulsions, the generation of a large curvature is needed, and consequently the hydrophilic head should be smaller than the PCL tail. Therefore, two PEO−PCL block copolymers have been studied, having different hydrophilic/hydrophobic block ratios. The structural characteristics of the proposed microemulsions in relation to their composition have been
INTRODUCTION During the past few years, there has been a growing interest in the formulation of nontoxic, biocompatible, and safe micro- and nanoemulsions with potential industrial applications. Despite the increased interest, the application of microemulsions in industry is still limited, mainly because of the surfactants presence. To overcome this barrier, the surfactants should be biodegradable, biocompatible, nontoxic, and not irritant to be used in pharmaceutical, cosmetics, or even in the food industry. As a consequence and according to this scheme, the choice of surfactant is a quite difficult task. Among amphiphilic molecules, polymeric surfactants present obvious advantages.1 More specifically, the structure of block copolymers having a hydrophilic and a hydrophobic moiety is similar to the structure of classic surfactants. Furthermore, the properties of each block are enhanced2,3 as the hydrophobicity of the nonpolar block is strengthened whereas the hydrophilic part is being much more polar than in classic surfactants. Therefore, block copolymers show excellent surface properties even at low concentrations.2 The ability to form microemulsions with fewer quantities than surfactants can lower the cost of the industrial procedures and also limit side effects such as foaming and adsorption at interfaces, thus making it easier to apply the microemulsions in procedures that involve cell membranes or require a compound isolation. Polymers based on poly(ethylene oxide) and related copolymers are often used for encapsulation of an active substance and its protection.4−9 They have already been used in various applications, such as the stabilization of oil-in-water (o/ © 2014 American Chemical Society
Received: May 6, 2014 Revised: July 25, 2014 Published: July 25, 2014 9808
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
oxazolidin) stearic acid] (5-DSA) was obtained from SigmaAldrich, Germany. High-purity water was obtained from a Millipore Milli Q Plus water purification system. All solvents used chloroform and propan-2-ol (isopropanol) from SDS and ethanol absolute from Sigma-Aldrich (Germany) were of highest available purity. The stabilizers used in this study were block copolymers and more specifically PEO−PCL 53 and PEO−PCL 30, both consisting of the biocompatible, hydrophilic block poly(ethylene oxide), and the biocompatible and biodegradable hydrophobic block poly-ε-caprolactone. They have relatively low-molecular-weight polydispersity and different hydrophilic/ hydrophobic component ratios. The copolymers were synthesized by ring-opening polymerization of caprolactone monomer using a monohydroxy functional PEG as the macroinitiator in the presence of stannous octoate as the catalyst.22 Their molecular characteristics are listed in Table 1.
investigated using both scattering and spectroscopic techniques, namely, dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. DLS measurements were carried out to evaluate the size and the polydispersity of the aqueous droplets in the presence and in the absence of lipase. EPR spectroscopy using the spin-probing technique was employed to study the interfacial properties of the membrane that the block copolymers form to stabilize the aqueous phase in the oily phase consisting of chloroform and isopropanol. This technique requires an unpaired electron, which was provided in the system by the amphiphilic spin probe 5-doxyl stearic acid (5-DSA).When added to the w/o microemulsions, 5-DSA preferably locates itself in the copolymer monolayer giving EPR spectra, reflecting the rigidity/flexibility of its environment.14 Water-in-oil microemulsions have been associated with the idea of microreactors for enzymatically catalyzed reactions, where substrates or products are lipophilic and low water content is required. Enzymes in w/o microemulsions offer numerous advantages when considered as reaction media used for biocatalytic transformations. Most importantly, they can solubilize both hydrophilic and hydrophobic solutes in high concentrations, whereas hydrolytic reactions can be easily reversed to synthetic ones by adjusting the water content of the system.15 Furthermore, microemulsions provide an enormous interfacial area through which the conversion of hydrophobic substrates can be catalyzed. Such a large interfacial area is of great technological interest because it results in the increase in the number of substrate molecules available to react. Moreover, the increased interfacial area makes these systems suitable for the particular case of lipases that are almost exclusively acting near interfaces. Lipases (EC 3.1.1.3) represent a group of enzymes that are most widely used for various biotransformation reactions due to several factors such as low cost and availability, ease in handling, and their regio- and enantioselectivity toward a wide range of substrates. They are thus important industrial enzymes, catalyzing both the hydrolysis of fats and oils and the synthesis of various useful compounds. Because the activity of these hydrolytic enzymes is greatly increased at the lipid−water interface, a phenomenon known as interfacial activation,16,17 reverse micelles are considered as a great medium for hosting these reactions due to the large interfacial area promoting contact between enzyme and substrates.18,19 In this respect, in the present study, a lipase, namely, lipase from Rhizomucor miehei, has been encapsulated in the systems. This lipase was chosen as a typical lipase showing interfacial activation, which is very well-described in literature.20 The activity of the encapsulated lipase has been studied based on the simple model esterification reaction of propyl laurate synthesis.21
Table 1. Molecular Characteristics of Block Copolymers PEO−PCL 53 and PEO−PCL 30 are Presented
a
amphiphilic block copolymer
Mw (g mol−1)
Mw PEO (g mol−1)
PCL wt %a
PEO−PCL 53 PEO−PCL 30
10 600 7100
5000 5000
53 30
By 1H NMR.
Experimental Methods. Preparation of Water-in-Oil Microemulsions. Each block copolymer was solubilized in chloroform, followed by the addition of the proper amount of isopropanol. The final concentration of the block copolymers in the mixture of chloroform/isopropanol was 7.5 mg mL−1. Finally, the aqueous phase was added, and the system was strongly shaken using a vortex until it became homogeneous. Pseudoternary Phase Diagrams. The phase behavior of the systems consisting of chloroform, isopropanol, water, and the block copolymers, at constant temperature 18 °C, was described on pseudoternary phase diagrams. The pseudoternary phase diagrams were constructed in the following way: First, stock solutions of the oily phase were prepared. For this purpose, each block copolymer was solubilized in a mixture of chloroform: isopropanol with varying volume ratios. The concentration of block copolymer was kept stable at 7.5 mg mL−1 of sample. Finally, the mixtures were titrated with the aqueous phase to the solubilization limit, which was detected visually by the appearance of cloudiness or sharply defined separated phases. Electrical Conductivity Measurements. The electrical conductivity measurements were carried out with a Metrohm 644 conductometer. The samples were prepared as previously described, and the measurements were possible due to the presence of Tris/HCl buffer in the systems instead of deionized water. The experiments took place at 25 °C. The cell constant, c, was equal to 0.1 cm−1. Dynamic Light Scattering Measurements. DLS measurements were performed in the angular range from 20 to 150° by a ALV/CGS-3 Compact Goniometer System (ALV, Germany) using a JDS Uniphase22 mWHe-Ne laser operating at 632.8 nm and an avalanche photodiode detector interfaced with a ALV5000/EPP multitau digital correlator with 288 channels and a ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. Autocorrelation functions from DLS measurements were collected five times at each
■
EXPERIMENTAL SECTION Materials. Lipase from Rhizomucor miehei (R. miehei) was supplied by Fluka. The enzyme preparation had a specific activity of 146.88 U mg−1 of protein. (1 U corresponds to the amount of enzyme that liberates 1 μmol of butyric acid per minute at pH 7.5 and 40 °C using tributyrin as substrate.) The lipase was solubilized in 0.2 M Tris/HCl buffer, pH 7.5, or deionized water and stored frozen. The substrates, dodecanoic acid (lauric acid) and 1-propanol of purity higher than 99%, were both supplied by Merck (Germany). 5-Doxyl stearic acid [5-(1-oxyl-2,2-dimethyl9809
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
stabilizers of w/o microemulsions in the aqueous core of which bioactive molecules such as enzymes can be encapsulated. The phase diagram approach to w/o microemulsions can provide useful information on the role that each component is playing in defining the properties of these systems at any composition. The systems used in the present study consist of four components, which are water, an organic solvent (chloroform), a block copolymer, and a cosolvent that is also oily. The copolymers used are easily solubilized in chloroform, but it is not possible to disperse an adequate amount of aqueous phase in such systems. So, the use of a cosolvent is necessary. Several cosolvents, such as long chain hydrocarbons and short chain secondary and tertiary alcohols, were tested. It was concluded that isopropanol (propan-2-ol) is the most suitable cosolvent for these systems, as it significantly increases the amount of aqueous phase that can be incorporated in the examined w/o microemulsions. The pseudoternary phase diagrams prepared for systems based on both copolymers are shown in Figure 1.
observation angle for each sample, and they were analyzed by the cumulants method and the CONTIN routine. Fits to the correlation functions were made using the software provided by the manufacturer. Apparent hydrodynamic radii, Rh, at different water concentrations were calculated by aid of the Stokes− Einstein law: RH = kBT/(6πη0Dapp), where RH, kB, T, η0, and Dapp are the hydrodynamic radius of the droplet, Boltzmann’s constant, temperature in Kelvin, viscosity of the system, and diffusion coefficient calculated from the analysis of the correlation functions, respectively. Intensity-weighted size distributions determined through CONTIN analysis were also evaluated. All w/o microemulsions were filtered through 0.45 μm filters before light-scattering measurements and contained in a quartz-type cuvette. The composition of the solvent for the DLS measurements was isopropanol/chloroform 70:30. The viscosity of the mixed dispersion medium (solvent) was determined by capillary viscometry in the temperature range investigated. Electron Paramagnetic Resonance Spectroscopy. EPR spectra were recorded at constant room temperature 25 °C using a Bruker EMX EPR spectrometer operating at the XBand. Samples were contained in a WG-813-Q Wilmad (Buena, NJ) Suprasil flat cell. Typical instrument setting were: center field, 0.348 T; scan range, 0.01 T; gain, 2.83 × 103; time constant, 163.84 ms; conversion time, 5 ms; modulation amplitude 0.4 mT; frequency, 9.81 GHz. Data collection and analysis were performed using the Bruker WinEPR acquisition and processing program. Experimental results were analyzed in terms of rotational correlation time (τR) and order parameter (S) of the spin probe 5-DSA, as described elsewhere.23,24 To obtain the desired concentration of the spin probe in the microemulsions, we added 1 mL of each microemulsion to a tube into which the appropriate amount of 5-DSA had been previously deposited. This was done by placing 10 μL of a stock 5-DSA solution in ethanol (7.5 mM) in the tube and by further evaporating the ethanol. In the present study, w/o microemulsions at different aqueous contents, in the absence and in the presence of lipase, were investigated. Enzymic Activity. To evaluate the enzymic activity of R. miehei lipase in the studied microemulsions, we followed the model esterification reaction of 1-propanol with lauric acid. Solutions of each copolymer in the selected solvents were prepared. Then, the substrates of the esterification reaction were added in these systems, at a final concentration of 100 mM, each. The reaction was initiated with the addition of R. miehei lipase stock solution. Twelve μL of lipase solution was added in the w/o microemulsions stabilized by PEO−PCL 53, and 20 μL of the same solution was added to the w/o microemulsions stabilized by PEO−PCL 30. The reaction solutions were incubated at the desired temperature. At specific time intervals a small amount of the reaction mixture was withdrawn and analyzed by gas chromatography to determine the amount of ester that was produced. Gas Chromatography. At fixed time intervals, samples of 10 μL were withdrawn from the reaction solution and analyzed using HP-5 capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness) mounted on a Hewlett-Packard (HP) model GC-6890C. Injector temperature, 270 °C; FID temperature, 290 °C; oven temperature constant at 200 °C.
Figure 1. Pseudoternary phase diagram for the systems consisting of (□) PEO−PCL 53/chloroform/isopropanol/water and (■) PEO− PCL 30/chloroform/isopropanol/water. Clear area is the monophasic area. A: System with isopropanol: chloroform volume ratio 70:30, which has been chosen to correspond to w/o microemulsion area and was used in further studies.
As can be seen from Figure 1, the monophasic area of the phase diagram of the system based on PEO−PCL 30 is wider than the one of the system based on PEO−PCL 53. More specifically, for isopropanol/chloroform volume ratio 70:30 (point A in Figure 1) the system stabilized by PEO−PCL 53 can incorporate water up to 4.3% v/v, while the system stabilized by PEO−PCL 30 can incorporate water up to 8% v/ v. This observation can be attributed to the fact that this block copolymer is more hydrophilic than PEO−PCL 53 because its hydrophobic block is shorter. Moreover, it can be noticed that the increase in isopropanol alongside the decrease in chloroform in the systems stabilized by both copolymers increases the total amount of aqueous phase that can be incorporated in each system. For example, for systems stabilized by PEO−PCL 53 when isopropanol/chloroform volume ratio is shifted from 70:30 to 85:15 the maximum amount of aqueous phase that can be incorporated increases from 4.3 to 9.5% v/v, while the respective increase for the systems with PEO−PCL 30 is even higher (from 8 to 19%). This can be explained by the fact that isopropanol is soluble to chloroform and miscible with water, so the higher the amount of propanol in the system, the higher the amount of aqueous
■
RESULTS AND DISCUSSION Phase Behavior. The aim of this study was to investigate the ability of amphiphilic block copolymers to be used as 9810
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
diagrams for both systems in relation to temperature are presented in Figure 3. It is well known that nonionic surfactants
solution that can be incorporated. GC analysis conducted to clarify whether isopropanol is in the aqueous phase or in the oil phase showed that when isopropanol was added in a mixture of chloroform and water it was preferably located in the aqueous phase in a percentage of 60%, while 20% was solubilized in chloroform and 20% was located in the interface. To determine the type of microemulsion prepared and detect the phase inversion phenomenon, we carried out electrical conductivity measurements for the microemulsions with isopropanol/chloroform volume ratio 70:30 for systems stabilized by both copolymers. A sharp increase in conductivity in water-in-oil microemulsions at low-volume fractions is an indication of a “percolative behavior” or exchange of ions between droplets before the formation of bicontinuous phase. Because nonionic surfactants were used in the preparation of the microemulsion systems, the aqueous phase was replaced with a Tris/HCl 0.2 M buffer. The choice of the salt was based on the fact that it is the buffer used for the enzymatically catalyzed reactions given its minimal influence on the lipase. Figure 2 depicts the effect of the increased aqueous phase volume percentage on the electrical conductivity of the system
Figure 3. Effect of temperature on the phase behavior of w/o microemulsions stabilized by PEO−PCL 53 (empty rhombus) and PEO−PCL 30 (filled triangles) in mixtures of chloroform and isopropanol. Dashed line, the temperature in which the ternary phase diagrams were constructed; dotted line, phase diagram’s point that was further studied.
are sensitive to temperature changes. Specifically, at low temperatures nonionic surfactants become more hydrophilic, while at higher temperatures their lipophilicity increases.26 This phenomenon also extends to nonionic amphiphilic block copolymers.27 In the case of surfactants consisting of poly(ethylene oxide), such as the ones used in this study, this dependence of the surfactant’s lipophilicity on temperature is attributed to the different conformation of the −OCHCO− part, which depends on temperature.28 The change of the surfactant’s lipophilicity due to temperature changes can induce instability to the systems and lead to phase separation. To study the effect of temperature on the phase diagram of the systems, we prepared and heated samples of certain compositions until phase separation occurs. The two phases coexisting after phase separation (Biphasic area of Figure 3) correspond to a phase composed of isopropanol rich in chloroform (upper phase) and a phase composed of isopropanol and water (lower phase). It can be seen in Figure 3 that w/o microemulsions stabilized by block copolymer PEO−PCL 30 remain monophasic (hence are stable) in a wider range of temperatures than the ones stabilized by PEO−PCL 53. In the latter case, any composition tested led to biphasic systems at temperatures >20 °C, except for the system with isopropanol/chloroform volume ratio 1:9, which becomes biphasic above 27 °C. As far as w/o microemulsions stabilized by PEO−PCL 30 are concerned, the temperature in which phase separation occurs is higher when the molar fraction of isopropanol in the system increases and chloroform, respectively, decreases. For most of these systems, phase separation occurs between 27 and 30 °C. Interestingly, for systems with isopropanol/chloroform ratio higher than 70:30, phase separation occurs at quite high temperatures, higher than 50 °C. This area corresponds to bicontinuous microemulsions as the conductivity study has shown (Figure 2). To summarize, systems stabilized by PEO−PCL 30, which has a shorter nonpolar chain than PEO−PCL 53, are stable under higher temperatures, especially at high isopropanol/ chloroform ratios where bicontinuous microemulsions are detected.
Figure 2. Electrical conductivity of microemulsion stabilized by PEO− PCL 30 as a function of aqueous phase volume content for isopropanol: chloroform ratio 70:30.
stabilized by PEO−PCL 30. By increasing the water content, it can be observed that the electrical conductivity of the microemulsion systems was increased. The increase in the aqueous content results in increased interactions between the surfactant and water molecules. When water is introduced to the core, the micelle swells and more surfactant and cosurfactant participate at the interface.25 In the present investigation, the conductivity profile of the microemulsion reveals the existence of two regions manifested by different slopes (Figure 2). These regions correspond to the formulation of w/o and bicontinuous microemulsions, respectively. The transition observed, namely, from w/o to the bicontinuous phase occurred without phase separation at 5% aqueous phase volume content. For the microemulsion stabilized by PEO−PCL 53, the electrical conductivity profile showed again an increase upon aqueous content increase (data not shown). In this case, the conductivity increase was much lower and a change in slope could not be detected. This profile indicates that the type of microemulsion is w/o. Effect of Temperature. The effect of temperature on the phase diagram of the microemulsions composed of chloroform/isopropanol/water based on PEO−PCL 53 and PEO− PCL 30 block copolymers has been examined. The phase 9811
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
formation of two populations of droplets, one population with smaller and one population with larger hydrodynamic radius. The sizes of these populations of droplets are presented in Table 2.
The systems were further studied and structurally characterized by means of dynamic and static light scattering and EPR spectroscopy. Structural Study. Dynamic Light Scattering. The aim of this part of the study was to determine the mean droplet radius (hydrodynamic) and the polydispersity index (PDI) of w/o microemulsions stabilized by PEO−PCL 53 and w/o microemulsions stabilized by PEO−PCL 30 in the presence of deionized water (empty systems) or R. miehei lipase in buffer (loaded systems). Empty systems. In Figure 4, the mean droplet radius of empty w/o microemulsions stabilized by PEO−PCL 53 and PEO−PCL 30 block copolymers as determined by DLS is presented.
Table 2. Evolution of the Mean Droplet Radius of Loaded w/o Microemulsions Stabilized by Block Copolymer PEO− PCL 53 aqueous phase % v/v
R. miehei lipase (mg mL−1)
Rh (nm) small droplets
Rh (nm) large droplets
0.5 0.8 1.2 0.5 0.8 1.2
0.05 0.08 0.12 0.05 0.05 0.05
40 54 220 40 280 237
200 397 1450 200 1140 1180
In Table 2, two different cases of aqueous phase increase are presented. In the first case, the lipase-containing aqueous phase in the system increases (water and lipase concentration is increased), while in the second case the aqueous phase in the system increases with the lipase concentration remaining constant (0.05 mg mL−1). It can be observed that when the aqueous phase increases from 0.5 to 1.2% v/v, the Rh of both populations of droplets increases from 40 to 230 nm for the small radius population and from 200 to 1400 nm for the large radius population, in both cases studied. An explanation for the existence of two droplet populations could be the coexistence of spherical (the smaller ones) as well as elongated (the larger ones) droplets, which may be produced by coalescence of the smaller ones.29 Of course, although the systems are considered as microemulsions, Ostwald ripening cannot be excluded. The same study was made for the w/o microemulsions stabilized by PEO−PCL 30. In contrast with the systems stabilized with PEO−PCL 53, the increase in the aqueous phase of w/o microemulsions stabilized by PEO−PCL 30 leads to the formation of one population of droplets. As can be seen from Figure 5, the system where the lipase concentration is kept constant (0.05 mg mL−1), while the water content increases appears to have similar hydrodynamic radius of the droplets in comparison with the empty system. More specifically, the increase in the aqueous phase from 0.5 to 2% v/v leads to Rh increase from 86 to 542 nm for the empty and
Figure 4. Comparison of the evolution of mean droplet radius in w/o microemulsions stabilized by block copolymer PEO−PCL 53 (empty rhombus) and PEO−PCL 30 (filled triangles) as determined by DLS. The line is a guide to the eye.
As can be seen in Figure 4, the increase in the aqueous phase for both systems induces an increase in the mean Rh of the water droplets. For systems stabilized by PEO−PCL 53, the increase of droplets’ mean Rh is more pronounced, leading to high Rh for even low water contents. This is not the case for systems stabilized by PEO−PCL 30, where the increase in the Rh of the droplets is considerably lower. For this system, the sharp increase in Rh observed for water contents higher than 5% v/v can be explained by the formation of bicontinuous microemulsions, which is in accordance with the findings of the electrical conductivity study (Figure 2). Comparing these two systems, we can conclude that at low water contents amphiphilic block copolymer PEO−PCL 53 leads to systems with larger dispersions, thus reaching to higher droplet radii. Moreover, the PDI of empty w/o microemulsions stabilized by both block copolymers increases with the increase in the aqueous phase (data not shown). It should be noted here that although the particle sizes are large the systems are optically clear. This could be explained by the fact that the main component is isopropanol, which is present in both continuous oil phase and dispersed polar phase, leading to similar refractive index for the two immiscible phases. According to the study of the loaded with lipase systems that follows it was obvious that the addition of even small amounts of protein to the microemulsions leads to slightly turbid systems. Apparently, the protein changes the refractive index of the dispersed phase, which no longer matches the one of the continuous phase, thus causing the visible turbidity. Loaded Systems. DLS measurements revealed that the encapsulation of R. miehei lipase in the aqueous phase of w/o microemulsions stabilized by PEO−PCL 53 leads to the
Figure 5. Evolution of the mean droplet radius of w/o microemulsions stabilized by block copolymer PEO−PCL 30 due to the increase in the aqueous phase. Empty system, empty triangles; loaded system with 0.05 mg mL−1 R. miehei lipase, striped triangles; loaded system with increasing concentration of R. miehei lipase, filled triangles. 9812
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
the order parameter, S, were calculated from the corresponding EPR spectra (Figure 7), as described elsewhere.23,24
from 80 to 462 nm for the loaded system, respectively. The same pattern was also observed for the PDI. The increase in the aqueous phase of the system leads to a similar increase in the PDI for both empty and loaded systems (data not shown). When the concentration of the lipase in the system is not constant but increases from 0.05 to 0.2 mg mL−1 with respective increase in the aqueous phase from 0.5 to 2% v/v, the increase in the Rh observed is much higher (Figure 5). Namely, it increases from 80 to 1740 nm. The same pattern is followed by the PDI of the system (data not shown). It should also be noted that it is the first time that PEO−PCL block copolymers have been used to stabilize water in oil microemulsions; therefore, there are no available data in the literature regarding the hydrodynamic radii of the droplets in such systems. Nevertheless, conventional w/o microemulsions based on surfactants present lower radii than the microemulsions studied here.30 Effect of Temperature. Finally, the effect of temperature on the mean droplet radius and PDI of w/o microemulsions stabilized by block copolymer PEO−PCL 30, which presents only one population of droplets, was examined. The results are presented in Figure 6. Systems with 2% v/v aqueous phase in the absence and presence of 0.05 mg mL−1 R. miehei lipase were examined in the temperature range from 18 to 26 °C.
Figure 7. EPR spectra of 5-DSA in w/o microemulsions based on PEO−PCL 30 in the presence and in the absence of lipase.
Figure 7 presents the EPR spectra of 5-DSA in empty w/o microemulsions based on PEO−PCL 30 at aqueous phase contents 2 and 6% v/v and also w/o microemulsions loaded with lipase at 2% v/v aqueous content. In all cases, three-line EPR spectra, characteristic of nitroxides, were obtained, although some small changes in spectral characteristics could be observed. In general, EPR spectra of unequal heights and widths are indicative of a restrictive motion of the spin probe in the membrane where it is located. In the present study, the signal intensity ratios of the center-to-high-field peak (h0/h−1), and center-to-low-field peak (h0/h+1) were higher when the water content of the microemulsions was increased from 2 to 6% v/v (Figure 7). The observed increase indicated a more restrictive movement of the spin probe on the surfactants monolayer at higher water content. On the contrary, in the presence of lipase, the signal intensity ratios of the spin probe were not altered compared with these of the empty system at water content 2% v/v. Tables 3 and 4 show the rotational correlation times, τR, and order parameters, S, of the spin probe 5-DSA in amphiphilic
Figure 6. Effect of temperature on the mean droplet radius of w/o microemulsions stabilized by block copolymer PEO−PCL 30 in the absence and presence of 0.05 mg mL−1 R. miehei lipase. Empty system, empty triangles; loaded system, filled triangles.
Table 3. Rotational Correlation Times, τR, and Order Parameters, S, of 5-DSA in PEO−PCL 30/Chloroform/ Isopropanol/Water w/o Microemulsions with Different Amounts of Aqueous Phase
It was observed that the increase in temperature from 18 to 26 °C does not significantly affect the hydrodynamic radius of empty w/o microemulsions (Figure 6) or the respective PDI (data not shown). On the contrary, when constant concentration of R. miehei lipase is present in the aqueous phase, increase in the incubation temperature from 18 to 26 °C leads to a significant increase in the hydrodynamic radius of the droplets (Figure 6) as well as of the PDI (data not shown). An interpretation of these observations may be the increase in enzyme hydrophobicity with temperature31 and also changes in the hydration of the PEO block32 that result in a less effective stabilization of the aqueous phase within the microemulsion droplets. Above 26 °C, phase separation occurs in these systems. Electron Paramagnetic Resonance Spectroscopy. Interfacial properties of the copolymers monolayer in free and loaded w/o microemulsions were studied by EPR spectroscopy using the spin-probing technique. To express the mobility of the probe and the rigidity of the interface quantitatively, the rotational correlation time, τR, and
aqueous phase (water % v/v)
τR (ns)
S
0.5 2.0 4.0 6.0
0.23 0.26 0.27 0.29
0.11 0.11 0.13 0.13
block copolymer-based microemulsions in the absence and presence of R. miehei lipase, respectively. The increase in the aqueous phase of the w/o microemulsions from 0.5 to 6% v/v causes relatively small but detectable changes to the rotational correlation time τR of the spin probe 5-DSA and to the rigidity of the membrane, as it is reflected by the order parameter S (Table 3). More specifically, the increase in the aqueous phase provokes an increase to the rotational correlation time of the spin probe, from 0.23 to 0.29 ns, and a small increase in the order parameter, from 0.11 to 9813
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
Table 4. Rotational Correlation Time, τR, and Order Parameter, S, of 5-DSA in PEO−PCL 30/Chloroform/ Isopropanol/R. miehei Lipase/Water w/o Microemulsions with Different Amounts of Aqueous Phase and Increasing Lipase Concentrations aqueous phase % v/v
R. miehei lipase (mg mL−1)
τR (ns)
S
2.0 2.0 2.0 2.0
0.00 0.05 0.12 0.20
0.26 0.26 0.26 0.27
0.11 0.11 0.11 0.11
by following the model esterification reaction of 1-propanol with lauric acid. It was observed that the lipase retains its catalytic activity in several temperatures, namely, at 18, 25, 35, and 42 °C. The profile of the esterification reaction of 1-propanol with lauric acid catalyzed by R. miehei lipase entrapped in w/o microemulsions for different temperatures is presented in Figure 8a for systems stabilized by PEO−PCL 53 and in Figure 8b for systems stabilized by PEO−PCL 30.
0.13. This means that in w/o microemulsions with higher aqueous content the spin probe’s movement is more restricted than that in w/o microemulsions with lower aqueous content. One possible explanation of this phenomenon could be that the increase in the water content of the microemulsions causes the droplets to “swell”, which in turn leads to a less curved PEO− PCL 30 monolayer. This structural change of the stabilizer layer results in an obstruction of the spin probe’s movement, which is detected as an increase in the rotational correlation time of the probe. This phenomenon has been also observed at comparable water contents in w/o microemulsions consisting of nonionic surfactants, naturally occurring oils and alcohols,33 as well as in tertiary microemulsion-like systems without surfactant.34 In addition, the increase in the order parameter due to the increase in the water content indicates a decrease in the flexibility of the interface, which is in agreement with the decreased spin probe’s mobility previously mentioned. The swelling of the droplets implied by the EPR findings is in accordance with the findings of the DLS study, where it was revealed that the hydrodynamic radii of the droplets increases with the increase in the aqueous phase in the microemulsions. Interestingly, in the presence of lipase from R. miehei, the observed effect of the droplets swelling on the rigidity of the membrane was neutralized. More specifically, when the overall concentration of the enzyme in the system was increased from 0 to 0.20 mg mL−1, at constant aqueous phase content, 2% v/v, both τR and S values remained practically invariable. As it has been previously demonstrated (Figure 5), the size of the dispersed aqueous droplets was not considerably affected by the presence of lipase at low concentration. On the contrary, a remarkable increase in the hydrodynamic diameter was detected as the lipase concentration was increased to 0.20 mg mL−1. Notably, membrane dynamics were not affected upon droplet swelling induced by lipase incorporation. This unexpected result could possibly reflect a sphere to rod transition of the droplets upon enzyme incorporation. Swelling and elongation of the lipase containing micelles may result to micelles with less swollen cores compared with the empty ones. From another point of view, the obtained EPR results could possibly indicate lipase localization in the aqueous cores of the w/o microemulsions, having minimum interaction with the copolymer monolayer. Nevertheless, the fact that EPR spin probing is a local technique reflecting membrane dynamics of the solubilization site exclusively should be taken into consideration when investigating enzyme location in a compartmentalized system. In this respect, the spin probe could be solubilized in an interfacial region where the lipase is not present. Enzymic Activity Study. The catalytic activity of R. miehei lipase entrapped in w/o microemulsions stabilized by block copolymers PEO−PCL 53 and PEO−PCL 30 has been tested
Figure 8. Profile of the esterification reaction of 1-propanol with lauric acid catalyzed by R. miehei lipase in w/o microemulsions stabilized by block copolymer PEO−PCL 53 (a) and PEO−PCL 30 (b) incubated in various temperatures. 18 °C, empty rhombus and triangles; 25 °C, dotted rhombus and triangles; 35 °C, striped rhombus and triangles; 42 °C, filled rhombus and triangles. Composition of systems as described in text. Ester concentrations determined by gas chromatography, as described in the Experimental Methods.
As can be seen from Figure 8a, the profile of the reaction is similar when the w/o microemulsions are incubated at 18, 25, and 42 °C giving similar final conversion yield (23%) as well as initial rates, namely, 21, 18, and 21 mM d−1 mg−1 of enzyme, respectively. However, when the reaction mixtures were incubated at 35 °C, the initial rate of the reaction was lower, namely, 6.5 mM d−1 mg−1 of lipase as well as the final conversion yield (10%). A different effect of the increase in temperature on the reaction profile could be observed for the systems stabilized by PEO−PCL 30. As can be seen from Figure 8b, at 35 °C, the highest and not the lowest initial rate was observed, being 28 mM d−1 mg−1 of lipase. The lowest initial rate of the esterification reaction has been observed for the reaction taking place at 42 °C. These results are presented in Figure 9, where the effect of temperature on initial rates of propyl laurate synthesis is depicted. This behavior is not typical for enzymic catalysis in microemulsions. In other studies involving lipases encapsulated in conventional microemulsions based on surfactants, increase in the temperature leads to increase in the lipase activity with the maximum activity appearing at the enzyme’s optimum 9814
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
could be observed as they remained clear and stable during the 5 days period that the experiments lasted.
■
CONCLUSIONS Amphiphilic block copolymers consisting of hydrophilic poly(ethylene oxide) and hydrophobic poly(ε-caprolactone) differing on the hydrophilic/hydrophobic block ratio were successfully used to stabilize water-in-oil (w/o) microemulsions. The constructed phase diagrams of the two block copolymers with mixtures of chloroform/isopropanol/water revealed that the systems can incorporate important amounts of aqueous phase showing an expanded single phase region. Such systems were used for the encapsulation of R. miehei lipase and the effectuation of a lipase catalyzed esterification. The catalytic activity of the encapsulated enzyme was successfully tested visa-vis the model esterification reaction of 1-propanol with lauric acid. Structural characterization of the systems with both scattering and spectroscopic techniques clearly showed that solubilized lipase influenced the properties of the loaded microemulsions.
Figure 9. Initial rates of the esterification reaction of 1-propanol with lauric acid catalyzed by R. miehei lipase encapsulated in w/o microemulsions stabilized by block copolymers PEO−PCL 53 (white columns) and PEO−PCL 30 (gray columns).
temperature and further decreasing.18,35−39 The different profile observed for the systems studied here may be attributed to interactions between the encapsulated lipase and the block copolymers stabilizing the microemulsions. It should be noted that caprolactone (the building block of PCL) contains a carboxylic bond and may act as a possible substrate to the lipase. Such a reaction would lead to reduced system’s stability due to block copolymer decomposition that may take place in a different scale depending on the temperature. To clarify whether there is polymer degradation or other changes induced by the lipase, we used DLS to measure droplet sizes during propyl laurate synthesis catalyzed in the microemulsions for five consecutive days. For this purpose, both empty and loaded-with-lipase systems were prepared, containing lauric acid and 1-propanol. The results of the measurements of all systems showed two populations of droplets; for the smaller of which, the observed Rh was ∼2 nm. This value corresponds to the backbone of lipids, as recently shown.40 The second population showed Rh of ∼500 nm for microemulsions stabilized by PEO−PCL 53 and ∼700 nm for microemulsions stabilized by PEO−PCL 30, in both cases for empty and loaded systems. As can be observed by comparing these results to the ones obtained for the systems formed in the absence of substrates (and products), during the reaction the observed Rh is lower. In empty systems, the difference is not large and thus can be explained by the presence of lauric acid, which contains a hydrophobic tale but also a carboxyl group and may act as a surfactant, interfering and influencing the droplets. In the case of loaded microemulsions, the difference in the observed Rh is larger. For systems stabilized by PEO−PCL 30 in the absence of substrates, the observed Rh is 1600 nm, similar to the one observed for this system in the beginning of the reaction. However, during the reaction it becomes 700 nm. This can be explained by the degradation of the copolymer, which may occur by partial cleavage of the caprolactone’s ester bonds. This leads to copolymer with shorter hydrophobic block and smaller, thus mean droplet radii. The droplet radii decrease was more obvious for systems stabilized by PEO−PCL 53, which has a longer caprolactone block, where the observed Rh was shifted from 1450 to 500 nm. After the second day, no more changes can be observed. It should be also noted here that during the first 2 days of reaction a phase separation occurs in systems stabilized by both copolymers, with the formation of a lower blurry phase, which does not contain chloroform. DLS measurements took place in the upper clear phase. This was not the case for the empty systems, where no phase separation
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel: +302107273762. Fax: +302107273758. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank the EU COST Action CM 1101 scientific program on Colloidal Aspects of Nanoscience for Innovative Processes and Materials for offering the possibility to carry out useful discussions on the subject.
■
ABREVIATIONS PEO, poly(ethylene oxide); PCL, poly(ε-caprolactone); DLS, dynamic light scattering; EPR, electron paramagnetic resonance; PDI, polydispersity index; 5-DSA, 5-doxyl stearic acid
■
REFERENCES
(1) Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C. Formation and Stability of Nano-Emulsions. Adv. Colloid Interface Sc. 2004, 108−109, 303−318. (2) Riess, G. Micellization of Block Copolymers. Prog. Polym. Sci. 2003, 28, 1107−1170. (3) Chausson, M.; Fluchère, A. S.; Landreau, E.; Aguni, Y.; Chevalier, Y.; Hamaide, T.; Abdul-Malak, N.; Bonnet, I. Block Copolymers of the Type poly(caprolactone)-b-poly(ethylene oxide) for the Preparation and Stabilization of Nanoemulsions. Int. J. Pharm. 2008, 362, 153− 162. (4) Allen, C.; Han, J.; Yu, Y.; Maysinger, D.; Eisenberg, A. Polycaprolactone-b-poly(ethylene oxide) Copolymer Micelles as a Delivery Vehicle for Dihydrotestosterone. J. Controlled Release 2000, 63, 275−286. (5) Soo, P. L.; Luo, L.; Maysinger, D.; Eisenberg, A. Incorporation and Release of Hydrophobic Probes in Biocompatible Polycaprolactone-block-poly(ethylene oxide) Micelles: Implications for Drug Delivery. Langmuir 2002, 18, 9996−10004. (6) Rösler, A.; Vendermeulen, G. W. M.; Klok, H. A. Advanced Drug Delivery Devices via Self-assembly of Amphiphilic Block Copolymers. Adv. Drug Delivery Rev. 2001, 53, 95−108. (7) Meier, M. A. R.; Aerts, S. H. N.; Staal, B. B. P.; Rasa, M.; Schubert, U. S. PEO-b-PCL Block Copolymers: Synthesis, Detailed 9815
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
Structure, Phase Behaviour and Mechanism. J. Phys.: Condens. Matter. 2001, 13, 9055−9074. (28) Lindman, B.; Carlsson, A.; Karlström, G.; Malmsten, M. Nonionic Polymers and Surfactants − Some Anomalies in Temperature Dependence and in Interactions with Ionic Surfactants. Adv. Colloid Interface Sci. 1990, 32, 183−203. (29) Ivanov, I. B.; Danov, K. D.; Kralchevsky, P. A. Flocculation and Coalescence of Micron-Size Emulsion Droplets. Colloids Surf., A 1999, 152, 161−182. (30) Kalaitzaki, A.; Poulopoulou, M.; Xenakis, A.; Papadimitriou, V. Surfactant-Rich Biocompatible Microemulsions as Effective Carriers of Methylxanthine Drugs. Colloids Surf., A 2014, 442, 80−87. (31) Baldwin, R. L. Temperature Dependence of the Hydrophobic Interaction in Protein Folding. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8069−8072. (32) Kjellander, R.; Florin, E. Water Structure and Changes in Thermal Stability of the System Polyethylene oxide-Water. J. Chem. Soc., Faraday Trans. 1981, 77, 2053−2077. (33) Kalaitzaki, A.; Emo, M.; Stébé, M. J.; Xenakis, A.; Papadimitriou, V. Biocompatible Nanodispersions as Delivery Systems of Food Additives: A Structural Study. Food Res. Int. 2013, 54, 1448−1454. (34) Zoumpanioti, M.; Stamatis, H.; Papadimitriou, V.; Xenakis, A. Spectroscopic and Catalytic Studies of Lipases in Ternary Hexane−1propanol−water Surfactantless Microemulsion Systems. Colloids Surf., B 2006, 47, 1−9. (35) Valis, T. P.; Xenakis, A.; Kolisis, F. N. Comparative Studies of Lipase from Rhizopus delemar in Various Microemulsion Systems. Biocatalysis 1992, 6, 267−279. (36) Chen, J. P.; Chang, K. C. Lipase Catalyzed Hydrolysis of Milk Fat in Lecithin Reverse Micelles. J. Ferment. Bioeng. 1993, 76, 98−104. (37) Fukumoto, J.; Iwai, M.l; Tsujisaka, Y. Studies on Lipase IV. Purification and Properties of a Lipase Secreted by Rhizopus delemar. J. Gen. Appl. Microbiol. 1964, 10, 257−265. (38) Crooks, G. E.; Rees, G. D.; Robinson, B. H.; Svensson, M.; Stephenson, G. R. Comparison of Hydrolysis and Esterification of Humicola lanuginose and Rhizomucor miehei Lipases in AOT Stabilized water-in-oil Microemulsions. II. Effect of Temperature on Reaction Kinetics and General Considerations of Stability and Productivity. Biotechnol. Bioeng. 1995, 48, 190−196. (39) Oliveira, A. C.; Cabral, J. M. Kinetic Studies of Mucor miehei Lipase in Phosphatidylcholine Microemulsions. J. Chem. Technol. Biotechnol. 1993, 56, 247−252. (40) Xenakis, A.; Papadimitriou, V.; Sotiroudis, T. G. Colloidal Structures in Natural Oils. Curr. Opin. Colloid Interface Sci. 2010, 15, 55−60.
Characterization, and Selected Micellar Drug Encapsulation Behavior. Macromol. Rapid Commun. 2005, 26, 1918−1924. (8) Shuai, X.; Ai, H.; Nasongkla, N.; Kim, S.; Gao, J. Micellar Carriers Based on Block Copolymers of Poly(ε-caprolactone) and Poly(ethylene glycol) for Doxorubicin Delivery. J. Controlled Release 2004, 98, 415−426. (9) Shi, B.; Fang, C.; You, M. X.; Zhang, Y.; Fu, S.; Pei, Y. Y. Stealth MePEG-PCL Micelles: Effect of Polymer Composition on Micelle Physicochemical Characteristics, in Vitro Drug Release, in Vivo Pharmacokinetics in Rats and Biodistribution in S180 Tumor Bearing Mice. Colloid Polym. Sci. 2005, 283, 954−967. (10) Jones, M.; Leroux, J. Polymeric Micelles - a New Generation of Colloidal Drug Carriers. Eur. J. Pharm. Biopharm. 1999, 48, 101−111. (11) Riess, G.; Nervo, J.; Rogez, D. Emulsifying Properties of Block Copolymers. Oil-Water Emulsions and Microemulsions. Polym. Eng. Sc. 1977, 17, 634−638. (12) Nam, Y. S.; Kim, J. W.; Han, S. H.; Kim, H. K. Nanosized Emulsions Stabilized by Semisolid Polymer Interface. Langmuir 2010, 26, 13038−13043. (13) Wang, S.; Cai, Q.; Bei, J. An Important Biodegradable PolymerPolylactone Family Polymer. Macromol. Symp. 2003, 195, 263−268. (14) Griffith, O. H.; Jost, P. C. Lipid Spin Labels in Biological Membrane. In Spin Labeling, Theory and Applications; Berliner, L. J., Eds.; Academic Press: New York, 1976; pp 454−523. (15) Xenakis, A.; Papadimitriou, V.; Stamatis, H.; Kolisis, F. N. Biocatalysis in Microemulsions. In Microemulsions: Properties and Applications; Fanun, M., Ed.; Surfactant Science Series,CRC Press: Boca Raton, FL, 2009; Vol.144, pp 349−385. (16) Verger, R. Enzyme Kinetics of Lipolysis. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1980; Vol. 64B, pp 340−392. (17) Derewenda, Z. S.; Sharp, A. M. News from the Interface: The Molecular Structures of Triacylglyceride Lipases. Trends Biochem. Sci. 1993, 205, 20−25. (18) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Bioorganic Reactions in Microemulsions: the Case of Lipases. Biotechnol. Adv. 1999, 17, 293− 318. (19) Carvalho, C. M. L.; Cabral, J. M. S. Reverse Micelles as Reaction Media for Lipases. Biochimie 2000, 82, 1063−1085. (20) Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; et al. A Model for Interfacial Activation in Lipases from the Structure of a Fungal Lipase-Inhibitor Complex. Nature 1991, 351, 491−494. (21) Delimitsou, C.; Zoumpanioti, M.; Xenakis, A.; Stamatis, H. Activity and Stability Studies of Mucor miehei Lipase Immobilized in Novel Microemulsion-Based Organogels. Biocatal. Biotransform. 2002, 20, 319−327. (22) Pippa, N.; Kaditi, E.; Pispas, S.; Demetzos, C. PEO-b-PCL− DPPC Chimeric Nanocarriers: Self-Assembly Aspects in Aqueous and Biological Media and Drug Incorporation. Soft Mater. 2013, 9, 4073− 4082. (23) Papadimitriou, V.; Sotiroudis, T. G.; Xenakis, A. Olive Oil Microemulsions: Enzymatic Activities and Structural Characteristics. Langmuir 2007, 23, 2071−2077. (24) Papadimitriou, V.; Pispas, S.; Syriou, S.; Pournara, A.; Zoumpanioti, M.; Sotiroudis, T. G.; Xenakis, A. Biocompatible Microemulsions Based on Limonene: Formulation, Structure, and Applications. Langmuir 2008, 24, 3380−3386. (25) Yaghmur, A.; Aserin, A.; Tiunova, I.; Garti, N. Sub-zero Temperature Behaviour of Non-Ionic Microemulsions in the Presence of Propylene Glycol by DSC. J. Therm. Anal. Calorim. 2002, 69, 163− 177. (26) Shinoda, K.; Saito, H. The Stability of O/W Type Emulsions as Functions of Temperature and the HLB of Emulsifiers: The Emulsification by PIT-method. J. Colloid Interface Sci. 1969, 30, 258−263. (27) Gompper, G.; Richter, D.; Strey, R. Amphiphilic Block Copolymers in Oil−Water−Surfactant Mixtures: Efficiency Boosting, 9816
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
Biocolloids Based on Amphiphilic Block Copolymers as a Medium for Enzyme Encapsulation Victoria Sereti,† Maria Zoumpanioti,† Vassiliki Papadimitriou,† Stergios Pispas,‡ and Aristotelis Xenakis*,† †
Institute of Biology, Medicinal Chemistry & Biotechnology and ‡Theoretical & Physical Chemistry Institute, National Hellenic Research Foundation, 48, Vassileos Constantinou Avenue, Athens 11635, Greece ABSTRACT: The ability of two biocompatible amphiphilic block copolymers consisting of hydrophilic poly(ethylene oxide) and hydrophobic poly(εcaprolactone) with different hydrophilic/hydrophobic block ratio to act as stabilizers of water-in-oil (w/o) microemulsions and enzyme encapsulation therein has been tested. Phase diagrams of the two block copolymers in mixtures of chloroform/isopropanol/water were constructed, revealing that the systems can incorporate important amounts of aqueous phase. The w/o microemulsions were then used to encapsulate R. miehei lipase. Empty as well as lipase-loaded systems were characterized by DLS as well as EPR spectroscopy. It was found that the incorporated lipase was preferably localized in the interior of the droplets. The apparent hydrodynamic radii of the droplets were found to vary from 86 to 3000 nm and from 66 to 2140 nm for empty PEO−PCL 30 and PEO−PCL 53 stabilized systems, respectively. In the presence of the lipase, the hydrodynamic radii were considerably decreased. The catalytic activity of the encapsulated lipase was successfully tested via a model esterification reaction. The effect of temperature on the catalytic behavior of the encapsulated R. miehei lipase was investigated, revealing that the initial rate of the esterification reaction depended on the type of the block copolymer used.
■
w) nanoemulsions10−12 for the encapsulation of hydrophobic molecules, such as lipophilic drugs. However, they have not yet been studied as stabilizers of water-in-oil (w/o) micro- or nanoemulsions that could serve as nanobioreactors, in which enzymes could be encapsulated and used for the biocatalytical transformation of nonpolar substrates. The present study is the first report that deals with the formulation and structural characterization of w/o microemulsions stabilized by block copolymers of the type poly(ethylene oxide)−poly(caprolactone), PEO−PCL, of different bock ratio. The hydrophobic PCL block is a semicrystalline polyester, biodegradable, and biocompatible.13 The hydrophilic PEO block is biocompatible but hardly biodegradable. Nevertheless, it is accepted and used as the hydrophilic moiety in many nonionic surfactants such as polysorbates (Tween) and alkyl ethers of PEO (Brij). The parameter that controls the properties of such copolymers is the polymerization degree of the PEO and the PCL blocks. In the present study for the formation of w/o microemulsions, the generation of a large curvature is needed, and consequently the hydrophilic head should be smaller than the PCL tail. Therefore, two PEO−PCL block copolymers have been studied, having different hydrophilic/hydrophobic block ratios. The structural characteristics of the proposed microemulsions in relation to their composition have been
INTRODUCTION During the past few years, there has been a growing interest in the formulation of nontoxic, biocompatible, and safe micro- and nanoemulsions with potential industrial applications. Despite the increased interest, the application of microemulsions in industry is still limited, mainly because of the surfactants presence. To overcome this barrier, the surfactants should be biodegradable, biocompatible, nontoxic, and not irritant to be used in pharmaceutical, cosmetics, or even in the food industry. As a consequence and according to this scheme, the choice of surfactant is a quite difficult task. Among amphiphilic molecules, polymeric surfactants present obvious advantages.1 More specifically, the structure of block copolymers having a hydrophilic and a hydrophobic moiety is similar to the structure of classic surfactants. Furthermore, the properties of each block are enhanced2,3 as the hydrophobicity of the nonpolar block is strengthened whereas the hydrophilic part is being much more polar than in classic surfactants. Therefore, block copolymers show excellent surface properties even at low concentrations.2 The ability to form microemulsions with fewer quantities than surfactants can lower the cost of the industrial procedures and also limit side effects such as foaming and adsorption at interfaces, thus making it easier to apply the microemulsions in procedures that involve cell membranes or require a compound isolation. Polymers based on poly(ethylene oxide) and related copolymers are often used for encapsulation of an active substance and its protection.4−9 They have already been used in various applications, such as the stabilization of oil-in-water (o/ © 2014 American Chemical Society
Received: May 6, 2014 Revised: July 25, 2014 Published: July 25, 2014 9808
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
oxazolidin) stearic acid] (5-DSA) was obtained from SigmaAldrich, Germany. High-purity water was obtained from a Millipore Milli Q Plus water purification system. All solvents used chloroform and propan-2-ol (isopropanol) from SDS and ethanol absolute from Sigma-Aldrich (Germany) were of highest available purity. The stabilizers used in this study were block copolymers and more specifically PEO−PCL 53 and PEO−PCL 30, both consisting of the biocompatible, hydrophilic block poly(ethylene oxide), and the biocompatible and biodegradable hydrophobic block poly-ε-caprolactone. They have relatively low-molecular-weight polydispersity and different hydrophilic/ hydrophobic component ratios. The copolymers were synthesized by ring-opening polymerization of caprolactone monomer using a monohydroxy functional PEG as the macroinitiator in the presence of stannous octoate as the catalyst.22 Their molecular characteristics are listed in Table 1.
investigated using both scattering and spectroscopic techniques, namely, dynamic light scattering (DLS) and electron paramagnetic resonance (EPR) spectroscopy. DLS measurements were carried out to evaluate the size and the polydispersity of the aqueous droplets in the presence and in the absence of lipase. EPR spectroscopy using the spin-probing technique was employed to study the interfacial properties of the membrane that the block copolymers form to stabilize the aqueous phase in the oily phase consisting of chloroform and isopropanol. This technique requires an unpaired electron, which was provided in the system by the amphiphilic spin probe 5-doxyl stearic acid (5-DSA).When added to the w/o microemulsions, 5-DSA preferably locates itself in the copolymer monolayer giving EPR spectra, reflecting the rigidity/flexibility of its environment.14 Water-in-oil microemulsions have been associated with the idea of microreactors for enzymatically catalyzed reactions, where substrates or products are lipophilic and low water content is required. Enzymes in w/o microemulsions offer numerous advantages when considered as reaction media used for biocatalytic transformations. Most importantly, they can solubilize both hydrophilic and hydrophobic solutes in high concentrations, whereas hydrolytic reactions can be easily reversed to synthetic ones by adjusting the water content of the system.15 Furthermore, microemulsions provide an enormous interfacial area through which the conversion of hydrophobic substrates can be catalyzed. Such a large interfacial area is of great technological interest because it results in the increase in the number of substrate molecules available to react. Moreover, the increased interfacial area makes these systems suitable for the particular case of lipases that are almost exclusively acting near interfaces. Lipases (EC 3.1.1.3) represent a group of enzymes that are most widely used for various biotransformation reactions due to several factors such as low cost and availability, ease in handling, and their regio- and enantioselectivity toward a wide range of substrates. They are thus important industrial enzymes, catalyzing both the hydrolysis of fats and oils and the synthesis of various useful compounds. Because the activity of these hydrolytic enzymes is greatly increased at the lipid−water interface, a phenomenon known as interfacial activation,16,17 reverse micelles are considered as a great medium for hosting these reactions due to the large interfacial area promoting contact between enzyme and substrates.18,19 In this respect, in the present study, a lipase, namely, lipase from Rhizomucor miehei, has been encapsulated in the systems. This lipase was chosen as a typical lipase showing interfacial activation, which is very well-described in literature.20 The activity of the encapsulated lipase has been studied based on the simple model esterification reaction of propyl laurate synthesis.21
Table 1. Molecular Characteristics of Block Copolymers PEO−PCL 53 and PEO−PCL 30 are Presented
a
amphiphilic block copolymer
Mw (g mol−1)
Mw PEO (g mol−1)
PCL wt %a
PEO−PCL 53 PEO−PCL 30
10 600 7100
5000 5000
53 30
By 1H NMR.
Experimental Methods. Preparation of Water-in-Oil Microemulsions. Each block copolymer was solubilized in chloroform, followed by the addition of the proper amount of isopropanol. The final concentration of the block copolymers in the mixture of chloroform/isopropanol was 7.5 mg mL−1. Finally, the aqueous phase was added, and the system was strongly shaken using a vortex until it became homogeneous. Pseudoternary Phase Diagrams. The phase behavior of the systems consisting of chloroform, isopropanol, water, and the block copolymers, at constant temperature 18 °C, was described on pseudoternary phase diagrams. The pseudoternary phase diagrams were constructed in the following way: First, stock solutions of the oily phase were prepared. For this purpose, each block copolymer was solubilized in a mixture of chloroform: isopropanol with varying volume ratios. The concentration of block copolymer was kept stable at 7.5 mg mL−1 of sample. Finally, the mixtures were titrated with the aqueous phase to the solubilization limit, which was detected visually by the appearance of cloudiness or sharply defined separated phases. Electrical Conductivity Measurements. The electrical conductivity measurements were carried out with a Metrohm 644 conductometer. The samples were prepared as previously described, and the measurements were possible due to the presence of Tris/HCl buffer in the systems instead of deionized water. The experiments took place at 25 °C. The cell constant, c, was equal to 0.1 cm−1. Dynamic Light Scattering Measurements. DLS measurements were performed in the angular range from 20 to 150° by a ALV/CGS-3 Compact Goniometer System (ALV, Germany) using a JDS Uniphase22 mWHe-Ne laser operating at 632.8 nm and an avalanche photodiode detector interfaced with a ALV5000/EPP multitau digital correlator with 288 channels and a ALV/LSE-5003 light scattering electronics unit for stepper motor drive and limit switch control. Autocorrelation functions from DLS measurements were collected five times at each
■
EXPERIMENTAL SECTION Materials. Lipase from Rhizomucor miehei (R. miehei) was supplied by Fluka. The enzyme preparation had a specific activity of 146.88 U mg−1 of protein. (1 U corresponds to the amount of enzyme that liberates 1 μmol of butyric acid per minute at pH 7.5 and 40 °C using tributyrin as substrate.) The lipase was solubilized in 0.2 M Tris/HCl buffer, pH 7.5, or deionized water and stored frozen. The substrates, dodecanoic acid (lauric acid) and 1-propanol of purity higher than 99%, were both supplied by Merck (Germany). 5-Doxyl stearic acid [5-(1-oxyl-2,2-dimethyl9809
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
stabilizers of w/o microemulsions in the aqueous core of which bioactive molecules such as enzymes can be encapsulated. The phase diagram approach to w/o microemulsions can provide useful information on the role that each component is playing in defining the properties of these systems at any composition. The systems used in the present study consist of four components, which are water, an organic solvent (chloroform), a block copolymer, and a cosolvent that is also oily. The copolymers used are easily solubilized in chloroform, but it is not possible to disperse an adequate amount of aqueous phase in such systems. So, the use of a cosolvent is necessary. Several cosolvents, such as long chain hydrocarbons and short chain secondary and tertiary alcohols, were tested. It was concluded that isopropanol (propan-2-ol) is the most suitable cosolvent for these systems, as it significantly increases the amount of aqueous phase that can be incorporated in the examined w/o microemulsions. The pseudoternary phase diagrams prepared for systems based on both copolymers are shown in Figure 1.
observation angle for each sample, and they were analyzed by the cumulants method and the CONTIN routine. Fits to the correlation functions were made using the software provided by the manufacturer. Apparent hydrodynamic radii, Rh, at different water concentrations were calculated by aid of the Stokes− Einstein law: RH = kBT/(6πη0Dapp), where RH, kB, T, η0, and Dapp are the hydrodynamic radius of the droplet, Boltzmann’s constant, temperature in Kelvin, viscosity of the system, and diffusion coefficient calculated from the analysis of the correlation functions, respectively. Intensity-weighted size distributions determined through CONTIN analysis were also evaluated. All w/o microemulsions were filtered through 0.45 μm filters before light-scattering measurements and contained in a quartz-type cuvette. The composition of the solvent for the DLS measurements was isopropanol/chloroform 70:30. The viscosity of the mixed dispersion medium (solvent) was determined by capillary viscometry in the temperature range investigated. Electron Paramagnetic Resonance Spectroscopy. EPR spectra were recorded at constant room temperature 25 °C using a Bruker EMX EPR spectrometer operating at the XBand. Samples were contained in a WG-813-Q Wilmad (Buena, NJ) Suprasil flat cell. Typical instrument setting were: center field, 0.348 T; scan range, 0.01 T; gain, 2.83 × 103; time constant, 163.84 ms; conversion time, 5 ms; modulation amplitude 0.4 mT; frequency, 9.81 GHz. Data collection and analysis were performed using the Bruker WinEPR acquisition and processing program. Experimental results were analyzed in terms of rotational correlation time (τR) and order parameter (S) of the spin probe 5-DSA, as described elsewhere.23,24 To obtain the desired concentration of the spin probe in the microemulsions, we added 1 mL of each microemulsion to a tube into which the appropriate amount of 5-DSA had been previously deposited. This was done by placing 10 μL of a stock 5-DSA solution in ethanol (7.5 mM) in the tube and by further evaporating the ethanol. In the present study, w/o microemulsions at different aqueous contents, in the absence and in the presence of lipase, were investigated. Enzymic Activity. To evaluate the enzymic activity of R. miehei lipase in the studied microemulsions, we followed the model esterification reaction of 1-propanol with lauric acid. Solutions of each copolymer in the selected solvents were prepared. Then, the substrates of the esterification reaction were added in these systems, at a final concentration of 100 mM, each. The reaction was initiated with the addition of R. miehei lipase stock solution. Twelve μL of lipase solution was added in the w/o microemulsions stabilized by PEO−PCL 53, and 20 μL of the same solution was added to the w/o microemulsions stabilized by PEO−PCL 30. The reaction solutions were incubated at the desired temperature. At specific time intervals a small amount of the reaction mixture was withdrawn and analyzed by gas chromatography to determine the amount of ester that was produced. Gas Chromatography. At fixed time intervals, samples of 10 μL were withdrawn from the reaction solution and analyzed using HP-5 capillary column (30 m × 0.32 mm i.d., 0.25 μm film thickness) mounted on a Hewlett-Packard (HP) model GC-6890C. Injector temperature, 270 °C; FID temperature, 290 °C; oven temperature constant at 200 °C.
Figure 1. Pseudoternary phase diagram for the systems consisting of (□) PEO−PCL 53/chloroform/isopropanol/water and (■) PEO− PCL 30/chloroform/isopropanol/water. Clear area is the monophasic area. A: System with isopropanol: chloroform volume ratio 70:30, which has been chosen to correspond to w/o microemulsion area and was used in further studies.
As can be seen from Figure 1, the monophasic area of the phase diagram of the system based on PEO−PCL 30 is wider than the one of the system based on PEO−PCL 53. More specifically, for isopropanol/chloroform volume ratio 70:30 (point A in Figure 1) the system stabilized by PEO−PCL 53 can incorporate water up to 4.3% v/v, while the system stabilized by PEO−PCL 30 can incorporate water up to 8% v/ v. This observation can be attributed to the fact that this block copolymer is more hydrophilic than PEO−PCL 53 because its hydrophobic block is shorter. Moreover, it can be noticed that the increase in isopropanol alongside the decrease in chloroform in the systems stabilized by both copolymers increases the total amount of aqueous phase that can be incorporated in each system. For example, for systems stabilized by PEO−PCL 53 when isopropanol/chloroform volume ratio is shifted from 70:30 to 85:15 the maximum amount of aqueous phase that can be incorporated increases from 4.3 to 9.5% v/v, while the respective increase for the systems with PEO−PCL 30 is even higher (from 8 to 19%). This can be explained by the fact that isopropanol is soluble to chloroform and miscible with water, so the higher the amount of propanol in the system, the higher the amount of aqueous
■
RESULTS AND DISCUSSION Phase Behavior. The aim of this study was to investigate the ability of amphiphilic block copolymers to be used as 9810
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
diagrams for both systems in relation to temperature are presented in Figure 3. It is well known that nonionic surfactants
solution that can be incorporated. GC analysis conducted to clarify whether isopropanol is in the aqueous phase or in the oil phase showed that when isopropanol was added in a mixture of chloroform and water it was preferably located in the aqueous phase in a percentage of 60%, while 20% was solubilized in chloroform and 20% was located in the interface. To determine the type of microemulsion prepared and detect the phase inversion phenomenon, we carried out electrical conductivity measurements for the microemulsions with isopropanol/chloroform volume ratio 70:30 for systems stabilized by both copolymers. A sharp increase in conductivity in water-in-oil microemulsions at low-volume fractions is an indication of a “percolative behavior” or exchange of ions between droplets before the formation of bicontinuous phase. Because nonionic surfactants were used in the preparation of the microemulsion systems, the aqueous phase was replaced with a Tris/HCl 0.2 M buffer. The choice of the salt was based on the fact that it is the buffer used for the enzymatically catalyzed reactions given its minimal influence on the lipase. Figure 2 depicts the effect of the increased aqueous phase volume percentage on the electrical conductivity of the system
Figure 3. Effect of temperature on the phase behavior of w/o microemulsions stabilized by PEO−PCL 53 (empty rhombus) and PEO−PCL 30 (filled triangles) in mixtures of chloroform and isopropanol. Dashed line, the temperature in which the ternary phase diagrams were constructed; dotted line, phase diagram’s point that was further studied.
are sensitive to temperature changes. Specifically, at low temperatures nonionic surfactants become more hydrophilic, while at higher temperatures their lipophilicity increases.26 This phenomenon also extends to nonionic amphiphilic block copolymers.27 In the case of surfactants consisting of poly(ethylene oxide), such as the ones used in this study, this dependence of the surfactant’s lipophilicity on temperature is attributed to the different conformation of the −OCHCO− part, which depends on temperature.28 The change of the surfactant’s lipophilicity due to temperature changes can induce instability to the systems and lead to phase separation. To study the effect of temperature on the phase diagram of the systems, we prepared and heated samples of certain compositions until phase separation occurs. The two phases coexisting after phase separation (Biphasic area of Figure 3) correspond to a phase composed of isopropanol rich in chloroform (upper phase) and a phase composed of isopropanol and water (lower phase). It can be seen in Figure 3 that w/o microemulsions stabilized by block copolymer PEO−PCL 30 remain monophasic (hence are stable) in a wider range of temperatures than the ones stabilized by PEO−PCL 53. In the latter case, any composition tested led to biphasic systems at temperatures >20 °C, except for the system with isopropanol/chloroform volume ratio 1:9, which becomes biphasic above 27 °C. As far as w/o microemulsions stabilized by PEO−PCL 30 are concerned, the temperature in which phase separation occurs is higher when the molar fraction of isopropanol in the system increases and chloroform, respectively, decreases. For most of these systems, phase separation occurs between 27 and 30 °C. Interestingly, for systems with isopropanol/chloroform ratio higher than 70:30, phase separation occurs at quite high temperatures, higher than 50 °C. This area corresponds to bicontinuous microemulsions as the conductivity study has shown (Figure 2). To summarize, systems stabilized by PEO−PCL 30, which has a shorter nonpolar chain than PEO−PCL 53, are stable under higher temperatures, especially at high isopropanol/ chloroform ratios where bicontinuous microemulsions are detected.
Figure 2. Electrical conductivity of microemulsion stabilized by PEO− PCL 30 as a function of aqueous phase volume content for isopropanol: chloroform ratio 70:30.
stabilized by PEO−PCL 30. By increasing the water content, it can be observed that the electrical conductivity of the microemulsion systems was increased. The increase in the aqueous content results in increased interactions between the surfactant and water molecules. When water is introduced to the core, the micelle swells and more surfactant and cosurfactant participate at the interface.25 In the present investigation, the conductivity profile of the microemulsion reveals the existence of two regions manifested by different slopes (Figure 2). These regions correspond to the formulation of w/o and bicontinuous microemulsions, respectively. The transition observed, namely, from w/o to the bicontinuous phase occurred without phase separation at 5% aqueous phase volume content. For the microemulsion stabilized by PEO−PCL 53, the electrical conductivity profile showed again an increase upon aqueous content increase (data not shown). In this case, the conductivity increase was much lower and a change in slope could not be detected. This profile indicates that the type of microemulsion is w/o. Effect of Temperature. The effect of temperature on the phase diagram of the microemulsions composed of chloroform/isopropanol/water based on PEO−PCL 53 and PEO− PCL 30 block copolymers has been examined. The phase 9811
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
formation of two populations of droplets, one population with smaller and one population with larger hydrodynamic radius. The sizes of these populations of droplets are presented in Table 2.
The systems were further studied and structurally characterized by means of dynamic and static light scattering and EPR spectroscopy. Structural Study. Dynamic Light Scattering. The aim of this part of the study was to determine the mean droplet radius (hydrodynamic) and the polydispersity index (PDI) of w/o microemulsions stabilized by PEO−PCL 53 and w/o microemulsions stabilized by PEO−PCL 30 in the presence of deionized water (empty systems) or R. miehei lipase in buffer (loaded systems). Empty systems. In Figure 4, the mean droplet radius of empty w/o microemulsions stabilized by PEO−PCL 53 and PEO−PCL 30 block copolymers as determined by DLS is presented.
Table 2. Evolution of the Mean Droplet Radius of Loaded w/o Microemulsions Stabilized by Block Copolymer PEO− PCL 53 aqueous phase % v/v
R. miehei lipase (mg mL−1)
Rh (nm) small droplets
Rh (nm) large droplets
0.5 0.8 1.2 0.5 0.8 1.2
0.05 0.08 0.12 0.05 0.05 0.05
40 54 220 40 280 237
200 397 1450 200 1140 1180
In Table 2, two different cases of aqueous phase increase are presented. In the first case, the lipase-containing aqueous phase in the system increases (water and lipase concentration is increased), while in the second case the aqueous phase in the system increases with the lipase concentration remaining constant (0.05 mg mL−1). It can be observed that when the aqueous phase increases from 0.5 to 1.2% v/v, the Rh of both populations of droplets increases from 40 to 230 nm for the small radius population and from 200 to 1400 nm for the large radius population, in both cases studied. An explanation for the existence of two droplet populations could be the coexistence of spherical (the smaller ones) as well as elongated (the larger ones) droplets, which may be produced by coalescence of the smaller ones.29 Of course, although the systems are considered as microemulsions, Ostwald ripening cannot be excluded. The same study was made for the w/o microemulsions stabilized by PEO−PCL 30. In contrast with the systems stabilized with PEO−PCL 53, the increase in the aqueous phase of w/o microemulsions stabilized by PEO−PCL 30 leads to the formation of one population of droplets. As can be seen from Figure 5, the system where the lipase concentration is kept constant (0.05 mg mL−1), while the water content increases appears to have similar hydrodynamic radius of the droplets in comparison with the empty system. More specifically, the increase in the aqueous phase from 0.5 to 2% v/v leads to Rh increase from 86 to 542 nm for the empty and
Figure 4. Comparison of the evolution of mean droplet radius in w/o microemulsions stabilized by block copolymer PEO−PCL 53 (empty rhombus) and PEO−PCL 30 (filled triangles) as determined by DLS. The line is a guide to the eye.
As can be seen in Figure 4, the increase in the aqueous phase for both systems induces an increase in the mean Rh of the water droplets. For systems stabilized by PEO−PCL 53, the increase of droplets’ mean Rh is more pronounced, leading to high Rh for even low water contents. This is not the case for systems stabilized by PEO−PCL 30, where the increase in the Rh of the droplets is considerably lower. For this system, the sharp increase in Rh observed for water contents higher than 5% v/v can be explained by the formation of bicontinuous microemulsions, which is in accordance with the findings of the electrical conductivity study (Figure 2). Comparing these two systems, we can conclude that at low water contents amphiphilic block copolymer PEO−PCL 53 leads to systems with larger dispersions, thus reaching to higher droplet radii. Moreover, the PDI of empty w/o microemulsions stabilized by both block copolymers increases with the increase in the aqueous phase (data not shown). It should be noted here that although the particle sizes are large the systems are optically clear. This could be explained by the fact that the main component is isopropanol, which is present in both continuous oil phase and dispersed polar phase, leading to similar refractive index for the two immiscible phases. According to the study of the loaded with lipase systems that follows it was obvious that the addition of even small amounts of protein to the microemulsions leads to slightly turbid systems. Apparently, the protein changes the refractive index of the dispersed phase, which no longer matches the one of the continuous phase, thus causing the visible turbidity. Loaded Systems. DLS measurements revealed that the encapsulation of R. miehei lipase in the aqueous phase of w/o microemulsions stabilized by PEO−PCL 53 leads to the
Figure 5. Evolution of the mean droplet radius of w/o microemulsions stabilized by block copolymer PEO−PCL 30 due to the increase in the aqueous phase. Empty system, empty triangles; loaded system with 0.05 mg mL−1 R. miehei lipase, striped triangles; loaded system with increasing concentration of R. miehei lipase, filled triangles. 9812
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
the order parameter, S, were calculated from the corresponding EPR spectra (Figure 7), as described elsewhere.23,24
from 80 to 462 nm for the loaded system, respectively. The same pattern was also observed for the PDI. The increase in the aqueous phase of the system leads to a similar increase in the PDI for both empty and loaded systems (data not shown). When the concentration of the lipase in the system is not constant but increases from 0.05 to 0.2 mg mL−1 with respective increase in the aqueous phase from 0.5 to 2% v/v, the increase in the Rh observed is much higher (Figure 5). Namely, it increases from 80 to 1740 nm. The same pattern is followed by the PDI of the system (data not shown). It should also be noted that it is the first time that PEO−PCL block copolymers have been used to stabilize water in oil microemulsions; therefore, there are no available data in the literature regarding the hydrodynamic radii of the droplets in such systems. Nevertheless, conventional w/o microemulsions based on surfactants present lower radii than the microemulsions studied here.30 Effect of Temperature. Finally, the effect of temperature on the mean droplet radius and PDI of w/o microemulsions stabilized by block copolymer PEO−PCL 30, which presents only one population of droplets, was examined. The results are presented in Figure 6. Systems with 2% v/v aqueous phase in the absence and presence of 0.05 mg mL−1 R. miehei lipase were examined in the temperature range from 18 to 26 °C.
Figure 7. EPR spectra of 5-DSA in w/o microemulsions based on PEO−PCL 30 in the presence and in the absence of lipase.
Figure 7 presents the EPR spectra of 5-DSA in empty w/o microemulsions based on PEO−PCL 30 at aqueous phase contents 2 and 6% v/v and also w/o microemulsions loaded with lipase at 2% v/v aqueous content. In all cases, three-line EPR spectra, characteristic of nitroxides, were obtained, although some small changes in spectral characteristics could be observed. In general, EPR spectra of unequal heights and widths are indicative of a restrictive motion of the spin probe in the membrane where it is located. In the present study, the signal intensity ratios of the center-to-high-field peak (h0/h−1), and center-to-low-field peak (h0/h+1) were higher when the water content of the microemulsions was increased from 2 to 6% v/v (Figure 7). The observed increase indicated a more restrictive movement of the spin probe on the surfactants monolayer at higher water content. On the contrary, in the presence of lipase, the signal intensity ratios of the spin probe were not altered compared with these of the empty system at water content 2% v/v. Tables 3 and 4 show the rotational correlation times, τR, and order parameters, S, of the spin probe 5-DSA in amphiphilic
Figure 6. Effect of temperature on the mean droplet radius of w/o microemulsions stabilized by block copolymer PEO−PCL 30 in the absence and presence of 0.05 mg mL−1 R. miehei lipase. Empty system, empty triangles; loaded system, filled triangles.
Table 3. Rotational Correlation Times, τR, and Order Parameters, S, of 5-DSA in PEO−PCL 30/Chloroform/ Isopropanol/Water w/o Microemulsions with Different Amounts of Aqueous Phase
It was observed that the increase in temperature from 18 to 26 °C does not significantly affect the hydrodynamic radius of empty w/o microemulsions (Figure 6) or the respective PDI (data not shown). On the contrary, when constant concentration of R. miehei lipase is present in the aqueous phase, increase in the incubation temperature from 18 to 26 °C leads to a significant increase in the hydrodynamic radius of the droplets (Figure 6) as well as of the PDI (data not shown). An interpretation of these observations may be the increase in enzyme hydrophobicity with temperature31 and also changes in the hydration of the PEO block32 that result in a less effective stabilization of the aqueous phase within the microemulsion droplets. Above 26 °C, phase separation occurs in these systems. Electron Paramagnetic Resonance Spectroscopy. Interfacial properties of the copolymers monolayer in free and loaded w/o microemulsions were studied by EPR spectroscopy using the spin-probing technique. To express the mobility of the probe and the rigidity of the interface quantitatively, the rotational correlation time, τR, and
aqueous phase (water % v/v)
τR (ns)
S
0.5 2.0 4.0 6.0
0.23 0.26 0.27 0.29
0.11 0.11 0.13 0.13
block copolymer-based microemulsions in the absence and presence of R. miehei lipase, respectively. The increase in the aqueous phase of the w/o microemulsions from 0.5 to 6% v/v causes relatively small but detectable changes to the rotational correlation time τR of the spin probe 5-DSA and to the rigidity of the membrane, as it is reflected by the order parameter S (Table 3). More specifically, the increase in the aqueous phase provokes an increase to the rotational correlation time of the spin probe, from 0.23 to 0.29 ns, and a small increase in the order parameter, from 0.11 to 9813
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
Table 4. Rotational Correlation Time, τR, and Order Parameter, S, of 5-DSA in PEO−PCL 30/Chloroform/ Isopropanol/R. miehei Lipase/Water w/o Microemulsions with Different Amounts of Aqueous Phase and Increasing Lipase Concentrations aqueous phase % v/v
R. miehei lipase (mg mL−1)
τR (ns)
S
2.0 2.0 2.0 2.0
0.00 0.05 0.12 0.20
0.26 0.26 0.26 0.27
0.11 0.11 0.11 0.11
by following the model esterification reaction of 1-propanol with lauric acid. It was observed that the lipase retains its catalytic activity in several temperatures, namely, at 18, 25, 35, and 42 °C. The profile of the esterification reaction of 1-propanol with lauric acid catalyzed by R. miehei lipase entrapped in w/o microemulsions for different temperatures is presented in Figure 8a for systems stabilized by PEO−PCL 53 and in Figure 8b for systems stabilized by PEO−PCL 30.
0.13. This means that in w/o microemulsions with higher aqueous content the spin probe’s movement is more restricted than that in w/o microemulsions with lower aqueous content. One possible explanation of this phenomenon could be that the increase in the water content of the microemulsions causes the droplets to “swell”, which in turn leads to a less curved PEO− PCL 30 monolayer. This structural change of the stabilizer layer results in an obstruction of the spin probe’s movement, which is detected as an increase in the rotational correlation time of the probe. This phenomenon has been also observed at comparable water contents in w/o microemulsions consisting of nonionic surfactants, naturally occurring oils and alcohols,33 as well as in tertiary microemulsion-like systems without surfactant.34 In addition, the increase in the order parameter due to the increase in the water content indicates a decrease in the flexibility of the interface, which is in agreement with the decreased spin probe’s mobility previously mentioned. The swelling of the droplets implied by the EPR findings is in accordance with the findings of the DLS study, where it was revealed that the hydrodynamic radii of the droplets increases with the increase in the aqueous phase in the microemulsions. Interestingly, in the presence of lipase from R. miehei, the observed effect of the droplets swelling on the rigidity of the membrane was neutralized. More specifically, when the overall concentration of the enzyme in the system was increased from 0 to 0.20 mg mL−1, at constant aqueous phase content, 2% v/v, both τR and S values remained practically invariable. As it has been previously demonstrated (Figure 5), the size of the dispersed aqueous droplets was not considerably affected by the presence of lipase at low concentration. On the contrary, a remarkable increase in the hydrodynamic diameter was detected as the lipase concentration was increased to 0.20 mg mL−1. Notably, membrane dynamics were not affected upon droplet swelling induced by lipase incorporation. This unexpected result could possibly reflect a sphere to rod transition of the droplets upon enzyme incorporation. Swelling and elongation of the lipase containing micelles may result to micelles with less swollen cores compared with the empty ones. From another point of view, the obtained EPR results could possibly indicate lipase localization in the aqueous cores of the w/o microemulsions, having minimum interaction with the copolymer monolayer. Nevertheless, the fact that EPR spin probing is a local technique reflecting membrane dynamics of the solubilization site exclusively should be taken into consideration when investigating enzyme location in a compartmentalized system. In this respect, the spin probe could be solubilized in an interfacial region where the lipase is not present. Enzymic Activity Study. The catalytic activity of R. miehei lipase entrapped in w/o microemulsions stabilized by block copolymers PEO−PCL 53 and PEO−PCL 30 has been tested
Figure 8. Profile of the esterification reaction of 1-propanol with lauric acid catalyzed by R. miehei lipase in w/o microemulsions stabilized by block copolymer PEO−PCL 53 (a) and PEO−PCL 30 (b) incubated in various temperatures. 18 °C, empty rhombus and triangles; 25 °C, dotted rhombus and triangles; 35 °C, striped rhombus and triangles; 42 °C, filled rhombus and triangles. Composition of systems as described in text. Ester concentrations determined by gas chromatography, as described in the Experimental Methods.
As can be seen from Figure 8a, the profile of the reaction is similar when the w/o microemulsions are incubated at 18, 25, and 42 °C giving similar final conversion yield (23%) as well as initial rates, namely, 21, 18, and 21 mM d−1 mg−1 of enzyme, respectively. However, when the reaction mixtures were incubated at 35 °C, the initial rate of the reaction was lower, namely, 6.5 mM d−1 mg−1 of lipase as well as the final conversion yield (10%). A different effect of the increase in temperature on the reaction profile could be observed for the systems stabilized by PEO−PCL 30. As can be seen from Figure 8b, at 35 °C, the highest and not the lowest initial rate was observed, being 28 mM d−1 mg−1 of lipase. The lowest initial rate of the esterification reaction has been observed for the reaction taking place at 42 °C. These results are presented in Figure 9, where the effect of temperature on initial rates of propyl laurate synthesis is depicted. This behavior is not typical for enzymic catalysis in microemulsions. In other studies involving lipases encapsulated in conventional microemulsions based on surfactants, increase in the temperature leads to increase in the lipase activity with the maximum activity appearing at the enzyme’s optimum 9814
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
could be observed as they remained clear and stable during the 5 days period that the experiments lasted.
■
CONCLUSIONS Amphiphilic block copolymers consisting of hydrophilic poly(ethylene oxide) and hydrophobic poly(ε-caprolactone) differing on the hydrophilic/hydrophobic block ratio were successfully used to stabilize water-in-oil (w/o) microemulsions. The constructed phase diagrams of the two block copolymers with mixtures of chloroform/isopropanol/water revealed that the systems can incorporate important amounts of aqueous phase showing an expanded single phase region. Such systems were used for the encapsulation of R. miehei lipase and the effectuation of a lipase catalyzed esterification. The catalytic activity of the encapsulated enzyme was successfully tested visa-vis the model esterification reaction of 1-propanol with lauric acid. Structural characterization of the systems with both scattering and spectroscopic techniques clearly showed that solubilized lipase influenced the properties of the loaded microemulsions.
Figure 9. Initial rates of the esterification reaction of 1-propanol with lauric acid catalyzed by R. miehei lipase encapsulated in w/o microemulsions stabilized by block copolymers PEO−PCL 53 (white columns) and PEO−PCL 30 (gray columns).
temperature and further decreasing.18,35−39 The different profile observed for the systems studied here may be attributed to interactions between the encapsulated lipase and the block copolymers stabilizing the microemulsions. It should be noted that caprolactone (the building block of PCL) contains a carboxylic bond and may act as a possible substrate to the lipase. Such a reaction would lead to reduced system’s stability due to block copolymer decomposition that may take place in a different scale depending on the temperature. To clarify whether there is polymer degradation or other changes induced by the lipase, we used DLS to measure droplet sizes during propyl laurate synthesis catalyzed in the microemulsions for five consecutive days. For this purpose, both empty and loaded-with-lipase systems were prepared, containing lauric acid and 1-propanol. The results of the measurements of all systems showed two populations of droplets; for the smaller of which, the observed Rh was ∼2 nm. This value corresponds to the backbone of lipids, as recently shown.40 The second population showed Rh of ∼500 nm for microemulsions stabilized by PEO−PCL 53 and ∼700 nm for microemulsions stabilized by PEO−PCL 30, in both cases for empty and loaded systems. As can be observed by comparing these results to the ones obtained for the systems formed in the absence of substrates (and products), during the reaction the observed Rh is lower. In empty systems, the difference is not large and thus can be explained by the presence of lauric acid, which contains a hydrophobic tale but also a carboxyl group and may act as a surfactant, interfering and influencing the droplets. In the case of loaded microemulsions, the difference in the observed Rh is larger. For systems stabilized by PEO−PCL 30 in the absence of substrates, the observed Rh is 1600 nm, similar to the one observed for this system in the beginning of the reaction. However, during the reaction it becomes 700 nm. This can be explained by the degradation of the copolymer, which may occur by partial cleavage of the caprolactone’s ester bonds. This leads to copolymer with shorter hydrophobic block and smaller, thus mean droplet radii. The droplet radii decrease was more obvious for systems stabilized by PEO−PCL 53, which has a longer caprolactone block, where the observed Rh was shifted from 1450 to 500 nm. After the second day, no more changes can be observed. It should be also noted here that during the first 2 days of reaction a phase separation occurs in systems stabilized by both copolymers, with the formation of a lower blurry phase, which does not contain chloroform. DLS measurements took place in the upper clear phase. This was not the case for the empty systems, where no phase separation
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: [email protected]. Tel: +302107273762. Fax: +302107273758. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS The authors thank the EU COST Action CM 1101 scientific program on Colloidal Aspects of Nanoscience for Innovative Processes and Materials for offering the possibility to carry out useful discussions on the subject.
■
ABREVIATIONS PEO, poly(ethylene oxide); PCL, poly(ε-caprolactone); DLS, dynamic light scattering; EPR, electron paramagnetic resonance; PDI, polydispersity index; 5-DSA, 5-doxyl stearic acid
■
REFERENCES
(1) Tadros, T.; Izquierdo, P.; Esquena, J.; Solans, C. Formation and Stability of Nano-Emulsions. Adv. Colloid Interface Sc. 2004, 108−109, 303−318. (2) Riess, G. Micellization of Block Copolymers. Prog. Polym. Sci. 2003, 28, 1107−1170. (3) Chausson, M.; Fluchère, A. S.; Landreau, E.; Aguni, Y.; Chevalier, Y.; Hamaide, T.; Abdul-Malak, N.; Bonnet, I. Block Copolymers of the Type poly(caprolactone)-b-poly(ethylene oxide) for the Preparation and Stabilization of Nanoemulsions. Int. J. Pharm. 2008, 362, 153− 162. (4) Allen, C.; Han, J.; Yu, Y.; Maysinger, D.; Eisenberg, A. Polycaprolactone-b-poly(ethylene oxide) Copolymer Micelles as a Delivery Vehicle for Dihydrotestosterone. J. Controlled Release 2000, 63, 275−286. (5) Soo, P. L.; Luo, L.; Maysinger, D.; Eisenberg, A. Incorporation and Release of Hydrophobic Probes in Biocompatible Polycaprolactone-block-poly(ethylene oxide) Micelles: Implications for Drug Delivery. Langmuir 2002, 18, 9996−10004. (6) Rösler, A.; Vendermeulen, G. W. M.; Klok, H. A. Advanced Drug Delivery Devices via Self-assembly of Amphiphilic Block Copolymers. Adv. Drug Delivery Rev. 2001, 53, 95−108. (7) Meier, M. A. R.; Aerts, S. H. N.; Staal, B. B. P.; Rasa, M.; Schubert, U. S. PEO-b-PCL Block Copolymers: Synthesis, Detailed 9815
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
The Journal of Physical Chemistry B
Article
Structure, Phase Behaviour and Mechanism. J. Phys.: Condens. Matter. 2001, 13, 9055−9074. (28) Lindman, B.; Carlsson, A.; Karlström, G.; Malmsten, M. Nonionic Polymers and Surfactants − Some Anomalies in Temperature Dependence and in Interactions with Ionic Surfactants. Adv. Colloid Interface Sci. 1990, 32, 183−203. (29) Ivanov, I. B.; Danov, K. D.; Kralchevsky, P. A. Flocculation and Coalescence of Micron-Size Emulsion Droplets. Colloids Surf., A 1999, 152, 161−182. (30) Kalaitzaki, A.; Poulopoulou, M.; Xenakis, A.; Papadimitriou, V. Surfactant-Rich Biocompatible Microemulsions as Effective Carriers of Methylxanthine Drugs. Colloids Surf., A 2014, 442, 80−87. (31) Baldwin, R. L. Temperature Dependence of the Hydrophobic Interaction in Protein Folding. Proc. Natl. Acad. Sci. U.S.A. 1986, 83, 8069−8072. (32) Kjellander, R.; Florin, E. Water Structure and Changes in Thermal Stability of the System Polyethylene oxide-Water. J. Chem. Soc., Faraday Trans. 1981, 77, 2053−2077. (33) Kalaitzaki, A.; Emo, M.; Stébé, M. J.; Xenakis, A.; Papadimitriou, V. Biocompatible Nanodispersions as Delivery Systems of Food Additives: A Structural Study. Food Res. Int. 2013, 54, 1448−1454. (34) Zoumpanioti, M.; Stamatis, H.; Papadimitriou, V.; Xenakis, A. Spectroscopic and Catalytic Studies of Lipases in Ternary Hexane−1propanol−water Surfactantless Microemulsion Systems. Colloids Surf., B 2006, 47, 1−9. (35) Valis, T. P.; Xenakis, A.; Kolisis, F. N. Comparative Studies of Lipase from Rhizopus delemar in Various Microemulsion Systems. Biocatalysis 1992, 6, 267−279. (36) Chen, J. P.; Chang, K. C. Lipase Catalyzed Hydrolysis of Milk Fat in Lecithin Reverse Micelles. J. Ferment. Bioeng. 1993, 76, 98−104. (37) Fukumoto, J.; Iwai, M.l; Tsujisaka, Y. Studies on Lipase IV. Purification and Properties of a Lipase Secreted by Rhizopus delemar. J. Gen. Appl. Microbiol. 1964, 10, 257−265. (38) Crooks, G. E.; Rees, G. D.; Robinson, B. H.; Svensson, M.; Stephenson, G. R. Comparison of Hydrolysis and Esterification of Humicola lanuginose and Rhizomucor miehei Lipases in AOT Stabilized water-in-oil Microemulsions. II. Effect of Temperature on Reaction Kinetics and General Considerations of Stability and Productivity. Biotechnol. Bioeng. 1995, 48, 190−196. (39) Oliveira, A. C.; Cabral, J. M. Kinetic Studies of Mucor miehei Lipase in Phosphatidylcholine Microemulsions. J. Chem. Technol. Biotechnol. 1993, 56, 247−252. (40) Xenakis, A.; Papadimitriou, V.; Sotiroudis, T. G. Colloidal Structures in Natural Oils. Curr. Opin. Colloid Interface Sci. 2010, 15, 55−60.
Characterization, and Selected Micellar Drug Encapsulation Behavior. Macromol. Rapid Commun. 2005, 26, 1918−1924. (8) Shuai, X.; Ai, H.; Nasongkla, N.; Kim, S.; Gao, J. Micellar Carriers Based on Block Copolymers of Poly(ε-caprolactone) and Poly(ethylene glycol) for Doxorubicin Delivery. J. Controlled Release 2004, 98, 415−426. (9) Shi, B.; Fang, C.; You, M. X.; Zhang, Y.; Fu, S.; Pei, Y. Y. Stealth MePEG-PCL Micelles: Effect of Polymer Composition on Micelle Physicochemical Characteristics, in Vitro Drug Release, in Vivo Pharmacokinetics in Rats and Biodistribution in S180 Tumor Bearing Mice. Colloid Polym. Sci. 2005, 283, 954−967. (10) Jones, M.; Leroux, J. Polymeric Micelles - a New Generation of Colloidal Drug Carriers. Eur. J. Pharm. Biopharm. 1999, 48, 101−111. (11) Riess, G.; Nervo, J.; Rogez, D. Emulsifying Properties of Block Copolymers. Oil-Water Emulsions and Microemulsions. Polym. Eng. Sc. 1977, 17, 634−638. (12) Nam, Y. S.; Kim, J. W.; Han, S. H.; Kim, H. K. Nanosized Emulsions Stabilized by Semisolid Polymer Interface. Langmuir 2010, 26, 13038−13043. (13) Wang, S.; Cai, Q.; Bei, J. An Important Biodegradable PolymerPolylactone Family Polymer. Macromol. Symp. 2003, 195, 263−268. (14) Griffith, O. H.; Jost, P. C. Lipid Spin Labels in Biological Membrane. In Spin Labeling, Theory and Applications; Berliner, L. J., Eds.; Academic Press: New York, 1976; pp 454−523. (15) Xenakis, A.; Papadimitriou, V.; Stamatis, H.; Kolisis, F. N. Biocatalysis in Microemulsions. In Microemulsions: Properties and Applications; Fanun, M., Ed.; Surfactant Science Series,CRC Press: Boca Raton, FL, 2009; Vol.144, pp 349−385. (16) Verger, R. Enzyme Kinetics of Lipolysis. In Methods in Enzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1980; Vol. 64B, pp 340−392. (17) Derewenda, Z. S.; Sharp, A. M. News from the Interface: The Molecular Structures of Triacylglyceride Lipases. Trends Biochem. Sci. 1993, 205, 20−25. (18) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Bioorganic Reactions in Microemulsions: the Case of Lipases. Biotechnol. Adv. 1999, 17, 293− 318. (19) Carvalho, C. M. L.; Cabral, J. M. S. Reverse Micelles as Reaction Media for Lipases. Biochimie 2000, 82, 1063−1085. (20) Brzozowski, A. M.; Derewenda, U.; Derewenda, Z. S.; Dodson, G. G.; Lawson, D. M.; Turkenburg, J. P.; et al. A Model for Interfacial Activation in Lipases from the Structure of a Fungal Lipase-Inhibitor Complex. Nature 1991, 351, 491−494. (21) Delimitsou, C.; Zoumpanioti, M.; Xenakis, A.; Stamatis, H. Activity and Stability Studies of Mucor miehei Lipase Immobilized in Novel Microemulsion-Based Organogels. Biocatal. Biotransform. 2002, 20, 319−327. (22) Pippa, N.; Kaditi, E.; Pispas, S.; Demetzos, C. PEO-b-PCL− DPPC Chimeric Nanocarriers: Self-Assembly Aspects in Aqueous and Biological Media and Drug Incorporation. Soft Mater. 2013, 9, 4073− 4082. (23) Papadimitriou, V.; Sotiroudis, T. G.; Xenakis, A. Olive Oil Microemulsions: Enzymatic Activities and Structural Characteristics. Langmuir 2007, 23, 2071−2077. (24) Papadimitriou, V.; Pispas, S.; Syriou, S.; Pournara, A.; Zoumpanioti, M.; Sotiroudis, T. G.; Xenakis, A. Biocompatible Microemulsions Based on Limonene: Formulation, Structure, and Applications. Langmuir 2008, 24, 3380−3386. (25) Yaghmur, A.; Aserin, A.; Tiunova, I.; Garti, N. Sub-zero Temperature Behaviour of Non-Ionic Microemulsions in the Presence of Propylene Glycol by DSC. J. Therm. Anal. Calorim. 2002, 69, 163− 177. (26) Shinoda, K.; Saito, H. The Stability of O/W Type Emulsions as Functions of Temperature and the HLB of Emulsifiers: The Emulsification by PIT-method. J. Colloid Interface Sci. 1969, 30, 258−263. (27) Gompper, G.; Richter, D.; Strey, R. Amphiphilic Block Copolymers in Oil−Water−Surfactant Mixtures: Efficiency Boosting, 9816
dx.doi.org/10.1021/jp504449y | J. Phys. Chem. B 2014, 118, 9808−9816
