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Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide
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Journal of Industrial and Engineering Chemistry xxx (2016) xxx–xxx
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Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide Huangying Guo, Jin-Chul Kim* Department of Medical Biomaterials Engineering, College of Biomedical Science and Institute of Bioscience and Biotechnology, Kangwon National University, 192-1, Hyoja 2 dong, Chuncheon, Kangwon-do 200-701, Republic of Korea
A R T I C L E I N F O
Article history: Received 21 May 2016 Received in revised form 17 August 2016 Accepted 20 August 2016 Available online xxx Keywords: Phosphatidylcholine Benzyl disulfide Liposome Reduction Release
A B S T R A C T
Reduction-responsive liposome was prepared by incorporating benzyl disulfide in egg phosphatidylcholine (egg PC) liposomal bilayer. The fluorescence quenching degree of calcein enveloped in egg PC liposome bearing benzyl disulfide decreased from 62.7% to 49.2% and the mean hydrodynamic diameter of the liposome decreased from 276 nm to 206 nm when the egg PC to benzyl disulfide weight ratio increased from 20:0 to 20:10. The liposomes bearing benzyl disulfide were multi-lamellar vesicles on TEM photos. It was confirmed by Raman spectroscopy that benzyl disulfide was loaded in the liposomal bilayer. The release degree of calcein enveloped in liposome incorporating benzyl disulfide was investigated for 24 h at 25 C, 37 C, and 45 C, when the concentration of DL-dithiothreitol (DTT) was 0 mM, 5 mM, 10 mM and 30 mM. At all the DTT concentrations tested, the release degree was more extensive as the temperature was higher. At all the temperatures tested, the release degree became more extensive as the DTT concentration increased. For example, the maximum release degree at 37 C of calcein loaded in liposome of which egg PC to benzyl disulfide weight ratio was 20:5 increased from 2.9% to 14.8% when DTT concentration increased from 0 mM to 10 mM. DTT could reduce benzyl disulfide to benzyl thiols and the fragments of benzyl disulfide would re-orient due to its polarity change in the liposomal membrane, resulting in the membrane fluctuation and the promoted release. At all the temperatures and all the DTT concentrations tested, the release was more sensitive to the DTT concentration as the benzyl disulfide content in liposome was higher, indicating that the reduction of the disulfide compound was responsible for the promoted release. ã 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Reduction-sensitive carriers can release their content in a reducing environment. They have been claimed to be used as carriers for intracellular drug delivery because the intracellular glutathione concentration is 100–1000 times higher than the extracellular one [1,2]. A key component to fabricate reductionsensitive carriers is a compound having disulfide bond. If they are placed in a reducing environment (e.g., intracellular space), the disulfide compound can be reduced to thiols and the carriers can be destabilized, resulting in an enhanced release. A reductionsensitive micelle was prepared using a polymeric amphiphile composed of an alkyl chain-attached monomer (bridged by disulfide bond) and a hydrophilic monomer. The micelles can be
* Corresponding author. Fax: +82 33 259 5645. E-mail address: [email protected] (J.-C. Kim).
disintegrated in a reducing environment, because the amphiphile loses its amphiphilicity by the cleavage of the disulfide bond [3–5]. Poly(ethylene imine) cross-linked by disulfide bond was used as a vector for DNA delivery to increase the transfection efficiency [6– 9]. Nanoparticles were fabricated using the copolymer composed of poly(e-caprolactone) and poly(ethyl ethylene phosphate) linked through disulfide bond to deliver doxorubicin (an anti-cancer agent) intracellularly [10–12]. Polymersome was developed using the copolymer composed of poly(ethylene glycol) and poly (propylene sulfide) linked through disulfide bond as a building block [13,14]. Upon the cleavage of disulfide bond, the packing parameter of the polymeric building block changes and the vesicles are disintegrated, leading to a promoted release. Recently, a reduction-responsive monoolein cubic phase was developed by electrostatically cross-linking poly(ethylene imine) contained in the water channel of the cubic phase using dithiodipropionic acid (DTPA, a disulfide compound). The cumulative release amount of a water-soluble dye was relatively low because the DTPA-mediated
http://dx.doi.org/10.1016/j.jiec.2016.08.018 1226-086X/ã 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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Fig. 1. Schematic representation of reduction-responsive liposome incorporating benzyl disulfide. Benzyl disulfide would be intercalated into the bilayer of liposomal membrane due to its nonpolar property (A). If the liposome incorporating benzyl disulfide is exposed to a reducing environment, benzyl disulfide could be reduced to benzyl thiols and the liposomal membrane would be fluctuated because benzyl thiols will re-orient due to the polarity change (B). As a result, the liposome could release its content in response to a reducing agent.
cross-linked PEI chains filled the water channel and they could hinder the dye diffusion in the water channel. The release was promoted in a reducing environment because the disulfide of DTPA could be cleaved and the cross-linkage of PEI chains broke down. In this study, a reduction-responsive liposome was prepared by incorporating benzyl disulfide in the membrane of egg phosphatidylcholine (egg PC) liposome. Benzyl disulfide has two benzyl groups connected by a disulfide bond and it is a small non-polar molecule. It would be intercalated into the bilayer of liposomal membrane because the bilayer interior is composed of non-polar acyl chains. If the liposome incorporating benzyl disulfide is exposed to a reducing environment, benzyl disulfide could be reduced to benzyl thiols and the liposomal membrane would be fluctuated because benzyl thiols can re-orient due to the polarity change. As a result, the liposome would release its content in response to a reducing agent (Fig. 1). The liposome bearing benzyl disulfide developed in this study would be used as a carrier for intracellular drug delivery because the intracellular concentration of a reducing agent (i.e., glutathione) is much higher than the extracellular one. Egg PC liposomes incorporating various amounts of benzyl disulfide were prepared by a film hydration and sonication method. The cumulative release amount of a fluorescence dye (calcein) enveloped in the liposome was investigated for 24 h at different temperatures (25 C, 37 C, and 42 C) and at different concentrations of a reducing agent (DLdithiothreitol). Materials and methods Materials L-a-Phosphatidylcholine (egg PC, average Mw: 768), benzyl disulfide (Mw: 246.3), DL-dithiothreitol (DTT, Mw: 154.25), calcein (Mw: 622.5), Triton X-100 (Mw: 647), phosphotungstic acid (PTA, Mw: 2,880.2), chloroform (Mw: 119.4), sodium hydroxide (Mw: 40.0), potassium hydroxide (Mw: 56.1) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Sephadex G-100 was provided by GE Healthcare (Sweden). N-(2-Hydroxyethyl)piperazine-n0 -(2-ethanesulfonic acid) (HEPES) was obtained from USB corporation (Cleveland, OH, USA). Water was doubly distilled in a
Milli-Q water purification system (Millipore Corp, MA, USA). All other reagents were in analytical grade. Preparation of liposome incorporating benzyl disulfide Liposome incorporating benzyl disulfide was prepared by a film hydration and sonication method [15,16]. Egg PC was dissolved in chloroform so that the concentration was 10 mg/ml. In parallel, benzyl disulfide was dissolved in the same organic solvent so that the concentration was 10 mg/ml. 2 ml of egg PC solution and variable amounts of benzyl disulfide solution were mixed in a 50 ml round bottom flask so that the egg PC to benzyl disulfide weight ratio was 20:0, 20:1, 20:2, 20:5 and 20:10. A rotary evaporator was installed with the flask containing the mixture solution of egg PC and benzyl disulfide, and it was operated at 100 rpm under reduced pressure to remove the organic solvent and obtain the dry mixed film of egg PC and benzyl disulfide on the flask wall. Calcein (a fluorescence dye) was dissolved in HEPES buffer (30 mM, pH 7.0) so that the concentration was 50 mM. 2 ml of calcein solution was put in the flask and it was whirled by a hand until the dry mixed film was completely detached from the glassware wall. The suspension was sonicated at room temperature (25 C) in a bath type sonicator (Sonics & Materials, USA) with pulse-on for 10 s and pulse-off for 10 s. The suspension was left at room temperature for 6 h under dark condition to anneal the liposomal membrane. Free calcein was removed from the liposome by a gel permeation chromatography using a column filled with Sephadex G-100 (1.6 cm 38 cm). Liposome prepared from the mixture solution whose the egg PC to benzyl disulfide weight ratio was 20:0, 20:1, 20:2, 20:5 and 20:10 was called liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5) and liposome (20:10), respectively. Determination of fluorescence quenching degree The fluorescence quenching degree of calcein loaded in egg PC liposome incorporating benzyl disulfide was determined by the following equation [17–19]. % Quenching = (1 Fi/Ft) 100
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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where Fi is the fluorescence intensity of the liposome suspension without unentrapped calcein, obtained by column chromatography, and Ft was the fluorescence intensity after the liposome was disintegrated by Triton X-100. The phospholipid concentration of liposomal suspension was adjusted to 0.01% (w/v) when the fluorescence intensity was measured. The fluorescence intensity was measured at 524 nm on a fluorescence spectrophotometer (Hitachi F2500, Japan) using the excitation wavelength of 494 nm. Measurement of mean diameter of liposome The mean hydrodynamic diameter of liposome free of benzyl disulfide (liposome(20:0)) and liposome incorporating benzyl disulfide (liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10)) were determined by a light scattering method [18–21]. The light scattering intensity was adjusted to 50–200 kcps by diluting the liposomal suspension with HEPES buffer (30 mM, pH 7.0), then the mean diameter was measured on a dynamic light scattering equipment (ZetaPlus 90, Brookhaven Instrument Co., USA). Transmission electron microscopy of liposome The structure of liposomes incorporating benzyl disulfide was investigated by transmission electron microscopy [18,19]. Liposome suspension obtained after column chromatography was directly used for the negative staining without dilution. Each suspension of liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) was dropped onto Parafilm of which surface was hydrophobic. PTA was dissolved in distilled water so that the concentration was 2% (w/v), then the pH value of PTA solution was adjusted to pH 6.8 using potassium hydroxide solution (1 M). PTA solution was filtered through a syringe filter (0.2 mm) and it was dropped onto each droplet of the liposome suspension on the Parafilm. After the droplets were mixed using pipette tip for a few minutes, they were left at room temperature (20–23 C) for 3 h under dust free condition. The stained liposome suspensions were transferred onto formvar/carbon-coated grids (200 mesh) by dipping carefully them into the droplets. The grids were air-dried at room temperature for 12 h under dust-free and dark condition.
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Raman spectroscopy The presence of disulfide compound (i.e., benzyl disulfide) in liposome(20:1) was confirmed using Raman spectrometer (JobinYvon/HORIBA LabRam ARAMIS Raman spectrometer (Villeneuved'Ascq, France)). 3 ml of the liposome suspension (0.67 mg/ml) in HEPES buffer (30 mM, pH 7.0) was put in a glass cuvette and the Raman spectrum was taken in the range of 300–800 cm1. In parallel, the spectrum was also taken after the liposome was solubilized using Triton X-100. The radiation wavelength was 523 nm, the grating was 1800 lines/mm, the exposure time was 60 s, and the accumulation number was 3. Observation of reduction-responsive release 0.2 ml of each suspension of liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) was put in 3.8 ml of HEPES buffer (10 mM, pH 7.4) containing various amounts of DTT, contained in a 10 ml glass vial. The final concentration of DTT was 0 mM, 5 mM, 10 mM, and 30 mM. The glass vial was tightly sealed, wrapped with an aluminum foil, and it was rolled on a rolled mixture at room temperature for 24 h. The fluorescence intensity was measured at 524 nm with the excitation wavelength of 494 nm at a given time. The release degree was calculated by the following equation [18,19]. % release = (Ff Fi)/(Ft Fi) 100 where Ff was the fluorescence intensity at a given time and Fi was the initial fluorescence intensity. Ft was the fluorescence intensity after the liposome was completely solubilized by Triton X-100. Results and discussion Determination of fluorescence quenching degree The fluorescence quenching degree of calcein enveloped in egg PC liposome incorporating benzyl disulfide decreased with increasing the benzyl disulfide content. For example, the fluorescence quenching degree of the fluorescence dye loaded in liposome (20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) was 67.2%, 66.5%, 52.5%, 50.1%, and 49.2%,
Fig. 2. TEM photos of liposome free of benzyl disulfide (liposome(20:0) (A)), and liposomes incorporating benzyl disulfide (liposome(20:1) (B), liposome(20:2) (C), liposome (20:5) (D), and liposome (20:10) (E)).
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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respectively. This indicates that the efficiency of liposome formation decreased with increasing the benzyl disulfide content because the fluorescence quenching degree is a measure of the liposome formation. Benzyl disulfide would be intercalated into the phospholipid bilayer due to its non-polar property. The amount of benzyl disulfide the phospholipid bilayer could accommodate without disintegration would be limited, and the bilayer vesicle could be more readily disintegrated into a non-bilayer structure (e.g., mixed micelle) as the content of benzyl disulfide increased. This would explain why the fluorescence quenching degree decreased with increasing the benzyl disulfide content. Measurement of mean diameter of liposome The mean diameter of liposome incorporating benzyl disulfide decreased as the benzyl disulfide content increased. For example, the mean diameter of liposome(20:0), liposome(20:1), liposome (20:2), liposome(20:5), and liposome(20:10) was 276 nm, 267 nm, 254 nm, 226 nm, and 206 nm, respectively. As described previously, benzyl disulfide is a non-polar compound and it would be solubilized in the phospholipid bilayer. Upon the intercalation of benzyl disulfide into the bilayer, the curvature of the liposomal membrane could increase, leading to a decrease in the mean diameter. Since the size measured by light scattering method was hydrodynamic diameter, the diameter reported here would be larger than the real diameter. If the sonication intensity had been stronger and the sonication duration had been longer, the diameter could have been smaller. But more intensive sonication would have chemically deteriorated egg PC and benzyl disulfide. Transmission electron microscopy of liposome Fig. 2 shows the TEM photos of liposome free of benzyl disulfide (liposome(20:0)) and liposomes incorporating benzyl disulfide (liposome(20:1), liposome(20:2), liposome(20:5), and liposome (20:10)). Multi lamellar vesicles were observed regardless of whether the liposome was incorporating benzyl disulfide. Benzyl disulfide would be incorporated in the bilayer of liposome owing to its non-polar property. Non-polar compounds were reported to be solubilized in the liposomal bilayer membrane without affecting the lamellarity [22,23]. The major factors affecting the lamellartiy of liposome are known to be the preparation method and the homogenization energy (e.g., sonication energy) [24–26]. For example, multi-lamellar vesicles are obtained by hand-shaking method and uni-lamellar vesicles are obtained by reversed-phase evaporation method and sonication method. The liposome in present study was prepared by hand-shaking and sonicating the aqueous mixture suspension of egg PC/benzyl disulfide. The sonication intensity and the sonication duration were thought not to high enough to convert multi-lamellar vesicle to uni-lamellar one. Raman spectroscopy Fig. 3(A) shows the Raman spectrum before liposome(20:1) was solubilized by Triton X-100. The disulfide signal is known to appear in the range of 425–550 cm1 [27–29]. There seemed to be a weak signal in the range but it was not apparent. Fig. 3(B) shows the Raman spectrum after liposome(20:1) was solubilized by Triton X100. An evident signal was found between 425 and 550 cm1 and it could be ascribed to the disulfide bond of benzyl disulfide [30,28]. Benzyl disulfide is a non-polar compound and it could be intercalated in the liposomal membrane [22]. Moreover, the liposome was multi-lamellar on the TEM photo (Fig. 2). In this circumstance, a small amount of laser photons could reach benzyl disulfide and a small amount of Raman scattered light photons
Fig. 3. Raman spectrum before (a) and after (b) liposome (20:1) was solubilized by Triton X-100.
could emit due to the light-shielding effect of the liposomal bilayer [31,32]. This could be one of reasons why no appreciable signal of disulfide bond was found even if benzyl disulfide was in the liposomal sample. Furthermore, laser light is scattered by liposomal particles and the scattered light intensity would be much higher than Raman scattered light intensity produced by the disulfide bond [33–35]. Thus, the signal of Raman scattered light would hardly appear due to the strong background signal. On the other hand, liposome can be disintegrated into mixed micelle in the presence of Triton X-100 (a surfactant). Due to the smaller size of micelle, the incident light-shielding effect would be less and the background signal would be weaker [31,32]. This could elucidate the reason why an evident signal of disulfide bond could be obtained after the liposome was solubilized. Based on the Raman spectrum obtained before and after liposome (20:1) was solubilized, it could be said the benzyl disulfide was loaded in the liposomal bilayer. Observation of reduction-responsive release Fig. 4 shows the release profile of calcein enveloped in liposome (20:1) at 25 C, 37 C, and 42 C when the concentration of DTT was 0 mM, 5 mM, 10 mM and 30 mM. The release degree increased rapidly in early stage and the release was almost completed in 8 h. The release degree was higher as the DTT concentration was higher. The maximum release degree at 25 C was 2%, 3.5%, 5% and 11%, respectively, when the DTT concentration was 0 mM, 5 mM, 10 mM, and 30 mM (Fig. 4(A)). DTT is a reducing agent and it can break down the disulfide bond of benzyl disulfide in the liposomal membrane, leading to the formation of benzyl thiols. Due to the reduction-caused polarity change, the fragments of benzyl disulfide (i.e., benzyl thiols) would re-orient in the liposomal membrane. Thiol groups would face water phase with benzyl groups embedded in the interior of phospholipid bilayer. During the re-orientation, the liposomal membrane could be subjected to fluctuation, leading to a promoted release. This could account for why the release increased with increasing the DTT concentration. However, the release was not promoted markedly by the reducing agent. DTT is water-soluble and it would not readily penetrate into the nonpolar interior of the liposomal membrane where benzyl disulfide exists. Accordingly, only a small amount of benzyl disulfide would undergo the reduction and the membrane fluctuation would be weak, resulting in a small increase in the release. The maximum release degree at 37 C was 2.5%, 5.1%, 7.2%, and 12.5%, respectively, when the concentration of DTT was 0 mM,
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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Fig. 4. Release profile of calcein enveloped in liposome (20:1) at 25 C (A), 37 C (B), and 42 C (C) when the concentration of DTT was 0 mM (*), 5 mM (), 10 mM (!) and 30 mM (D).
5 mM, 10 mM, and 30 mM (Fig. 4(B)). At all the DTT concentrations tested, the release degree at 37 C (Fig. 4(B)) was higher than the release degree at 25 C (Fig. 4(A)). The maximum release degree at 42 C was 2.7%, 6.7%, 8.5%, and 13.3%, respectively, when the DTT concentration was 0 mM, 5 mM, 10 mM, and 30 mM (Fig. 4(C)). At all the DTT concentrations tested, the release degree at 42 C (Fig. 4(C)) was higher than the release degree observed at the lower temperatures (Fig. 4(A) and (B)). The fluidity of liposomal membrane is higher at a higher temperature [36,37], thus calcein could release out of liposome more readily at a higher temperature. The diffusivity of a solute (calcein) itself would also increase with increasing the temperature. According to Stoke–Einstein’s equation, the diffusion coefficient is proportional to the root square of absolute temperature [38,39]. In addition, DDT would also penetrate into the liposomal membrane and reduce benzyl disulfide more easily at a higher temperature. Thus, the reduction of benzyl disulfide and the fluctuation of the liposomal membrane would be more favorable at a higher temperature. These could account for why the release was more extensive as the temperature was higher. Fig. 5 shows the release degree in 24 h of calcein enveloped in liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) at 25 C, 37 C, and 42 C with the DTT concentration. At all the DTT concentrations tested, the release degree of dye enveloped in liposome(20:0) increased a little as the temperature of release medium increased (Fig. 5(A)). For example, the release degree at the DTT concentration of 30 mM was 5%, 6.3%, and 6.5%, respectively, when the temperature of release medium was 25 C, 37 C, and 42 C. On the other hand, at all the temperatures tested, the DTT concentration had no
significant effect on the release degree. For example, the release degree increased only 2–3% when the DTT concentration increased from 0 to 30 mM. The slight increase in the release degree was possibly because the osmotic pressure difference between the aqueous core of liposome and the release medium could be developed by DTT existing in the release medium. Like the release degree of calcein loaded in liposome(20:0), the release degree of calcein loaded in liposome(20:1) somewhat increased with increasing the temperature at all the DTT concentrations tested (Fig. 5(B)). For example, the release degree at the DTT concentration of 30 mM was 11%, 12.5%, and 13.3%, respectively, when the temperature of release medium was 25 C, 37 C, and 42 C. Unlike the release degree of calcein loaded in liposome(20:0), the release degree of calcein loaded in liposome(20:1) seemed to be affected by the DTT concentration at all the temperatures tested. For example, the release degree at 25 C increased from 2% to 11% when the DTT concentration increased from 0 to 30 mM. Benzyl disulfide was contained in liposome(20:1) and it could be reduced to benzyl thiols by the reducing agent (DTT). Since the polarity of the fragment of benzyl disulfide (i.e., benzyl thiol) is different from that of the disulfide compound, the fragment would re-orient in the membrane. It was thought that thiol groups would orient toward water phase with benzyl groups in the interior of liposomal bilayer. The liposomal membrane would be stressed and fluctuated upon the re-orientation and it could promote the release. The release degree of calcein enveloped in liposome(20:2) was also proportional to the temperature at all the DTT concentrations tested (Fig. 5(C)). The DTT concentration seemed to have an effect on the release degree of calcein enveloped in liposome(20:2) at all
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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Fig. 5. Release degree in 24 h of calcein enveloped in liposome (20:0) (A), liposome (20:1) (B), liposome (20:2) (C), liposome (20:5) (D), and liposome (20:10) (E) at 25 C (*), 37 C (), and 42 C (!) with DTT concentration.
the temperatures tested. The release sensitivity of liposome(20:2) with respect to the DTT concentration was not markedly different from the sensitivity of liposome(20:1). The release degree of calcein enveloped in liposome(20:5) was also dependent on the temperature at all the DTT concentrations tested (Fig. 5(D)). DTT had a significant effect on the release degree of calcein enveloped in liposome(20:5) at all the temperatures tested. For example, the release degree at 25 C increased from 2.3% to 15.5% when the DTT concentration increased from 0 to 30 mM. The release sensitivity of liposome(20:5) with respect to the DTT concentration was significantly higher than the sensitivity of liposome(20:1) and that of liposome(20:2). The benzyl disulfide content of liposome (20:5) was much higher than that of liposome(20:1) and that of liposome(20:2). Accordingly, upon the reduction of the disulfide compound, the membrane of former liposome would be fluctuated more than the membrane of latter one. This could explain why liposome(20:5) was more sensitive to the DTT concentration. Like the other liposomes described above, liposome(20:10) released its content more at a higher temperature (Fig. 5(E)). Liposome(20:10) also released its content in a DTT concentration-dependent manner at all the temperatures tested, and its release sensitivity to the DTT concentration was the highest among the liposomes tested. For example, the release degree at 25 C increased from 3.1% to 17.9% when the DTT concentration increased from 0 to 30 mM. The highest content of disulfide compound would be responsible for the highest sensitivity with respect to the DTT concentration. Conclusions Reduction-responsive liposome bearing benzyl disulfide was prepared by hydrating the dry mixed film of egg PC and the disulfide compound and sonicating the suspension. The fluorescence quenching degree decreased from 62.7% to 49.2% as the egg
PC to benzyl disulfide weight ratio increased from 20:0 to 20:10, possibly due to the disintegration of the liposome by the intercalation of benzyl disulfide into the bilayer. Increase in the egg PC to benzyl disulfide weight ratio (from 20:0 to 20:10) led to decrease in the mean diameter of the liposome (from 276 nm to 206 nm), probably because the curvature of the liposomal membrane could increase upon the intercalation of benzyl disulfide into the liposomal bilayer. Multi-lamellar vesicles were found on the TEM photos of the liposomes bearing benzyl disulfide. Benzyl disulfide was confirmed to be loaded in the liposomal bilayer by Raman spectroscopy. At all the DTT concentrations tested (0 mM, 5 mM, 10 mM, 30 mM), the release degree in 24 h was more extensive as the temperature was higher, possibly because the membrane fluidity and the dye diffusivity could be greater at a higher temperature. At all the temperatures tested (25 C, 37 C, and 45 C), the release degree became more extensive as the DTT concentration increased. It was thought that benzyl thiol, produced by the reduction of benzyl disulfide, would re-orient in the liposomal membrane due to its polarity change and it could cause the liposomal membrane fluctuation and the promoted release. At all the temperatures and all the DTT concentrations tested, the release sensitivity with respect to the DTT concentration was higher as the benzyl disulfide content in liposome was higher. This means that the promoted release was due to the reduction of the disulfide compound in the liposomal membrane. The liposome bearing benzyl disulfide developed in this study could be simply prepared by hydrating the dry mixed film of egg PC and the disulfide compound at room temperature. It could release its content more extensively at a higher DTT concentration. The liposome bearing benzyl disulfide is thought to deliver a pharmaceutical drug to intracellular space in a selective manner because the intracellular concentration of a reducing agent (i.e., glutathione) is much higher than the extracellular one.
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Journal of Industrial and Engineering Chemistry xxx (2016) xxx–xxx
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Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide Huangying Guo, Jin-Chul Kim* Department of Medical Biomaterials Engineering, College of Biomedical Science and Institute of Bioscience and Biotechnology, Kangwon National University, 192-1, Hyoja 2 dong, Chuncheon, Kangwon-do 200-701, Republic of Korea
A R T I C L E I N F O
Article history: Received 21 May 2016 Received in revised form 17 August 2016 Accepted 20 August 2016 Available online xxx Keywords: Phosphatidylcholine Benzyl disulfide Liposome Reduction Release
A B S T R A C T
Reduction-responsive liposome was prepared by incorporating benzyl disulfide in egg phosphatidylcholine (egg PC) liposomal bilayer. The fluorescence quenching degree of calcein enveloped in egg PC liposome bearing benzyl disulfide decreased from 62.7% to 49.2% and the mean hydrodynamic diameter of the liposome decreased from 276 nm to 206 nm when the egg PC to benzyl disulfide weight ratio increased from 20:0 to 20:10. The liposomes bearing benzyl disulfide were multi-lamellar vesicles on TEM photos. It was confirmed by Raman spectroscopy that benzyl disulfide was loaded in the liposomal bilayer. The release degree of calcein enveloped in liposome incorporating benzyl disulfide was investigated for 24 h at 25 C, 37 C, and 45 C, when the concentration of DL-dithiothreitol (DTT) was 0 mM, 5 mM, 10 mM and 30 mM. At all the DTT concentrations tested, the release degree was more extensive as the temperature was higher. At all the temperatures tested, the release degree became more extensive as the DTT concentration increased. For example, the maximum release degree at 37 C of calcein loaded in liposome of which egg PC to benzyl disulfide weight ratio was 20:5 increased from 2.9% to 14.8% when DTT concentration increased from 0 mM to 10 mM. DTT could reduce benzyl disulfide to benzyl thiols and the fragments of benzyl disulfide would re-orient due to its polarity change in the liposomal membrane, resulting in the membrane fluctuation and the promoted release. At all the temperatures and all the DTT concentrations tested, the release was more sensitive to the DTT concentration as the benzyl disulfide content in liposome was higher, indicating that the reduction of the disulfide compound was responsible for the promoted release. ã 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Introduction Reduction-sensitive carriers can release their content in a reducing environment. They have been claimed to be used as carriers for intracellular drug delivery because the intracellular glutathione concentration is 100–1000 times higher than the extracellular one [1,2]. A key component to fabricate reductionsensitive carriers is a compound having disulfide bond. If they are placed in a reducing environment (e.g., intracellular space), the disulfide compound can be reduced to thiols and the carriers can be destabilized, resulting in an enhanced release. A reductionsensitive micelle was prepared using a polymeric amphiphile composed of an alkyl chain-attached monomer (bridged by disulfide bond) and a hydrophilic monomer. The micelles can be
* Corresponding author. Fax: +82 33 259 5645. E-mail address: [email protected] (J.-C. Kim).
disintegrated in a reducing environment, because the amphiphile loses its amphiphilicity by the cleavage of the disulfide bond [3–5]. Poly(ethylene imine) cross-linked by disulfide bond was used as a vector for DNA delivery to increase the transfection efficiency [6– 9]. Nanoparticles were fabricated using the copolymer composed of poly(e-caprolactone) and poly(ethyl ethylene phosphate) linked through disulfide bond to deliver doxorubicin (an anti-cancer agent) intracellularly [10–12]. Polymersome was developed using the copolymer composed of poly(ethylene glycol) and poly (propylene sulfide) linked through disulfide bond as a building block [13,14]. Upon the cleavage of disulfide bond, the packing parameter of the polymeric building block changes and the vesicles are disintegrated, leading to a promoted release. Recently, a reduction-responsive monoolein cubic phase was developed by electrostatically cross-linking poly(ethylene imine) contained in the water channel of the cubic phase using dithiodipropionic acid (DTPA, a disulfide compound). The cumulative release amount of a water-soluble dye was relatively low because the DTPA-mediated
http://dx.doi.org/10.1016/j.jiec.2016.08.018 1226-086X/ã 2016 The Korean Society of Industrial and Engineering Chemistry. Published by Elsevier B.V. All rights reserved.
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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Fig. 1. Schematic representation of reduction-responsive liposome incorporating benzyl disulfide. Benzyl disulfide would be intercalated into the bilayer of liposomal membrane due to its nonpolar property (A). If the liposome incorporating benzyl disulfide is exposed to a reducing environment, benzyl disulfide could be reduced to benzyl thiols and the liposomal membrane would be fluctuated because benzyl thiols will re-orient due to the polarity change (B). As a result, the liposome could release its content in response to a reducing agent.
cross-linked PEI chains filled the water channel and they could hinder the dye diffusion in the water channel. The release was promoted in a reducing environment because the disulfide of DTPA could be cleaved and the cross-linkage of PEI chains broke down. In this study, a reduction-responsive liposome was prepared by incorporating benzyl disulfide in the membrane of egg phosphatidylcholine (egg PC) liposome. Benzyl disulfide has two benzyl groups connected by a disulfide bond and it is a small non-polar molecule. It would be intercalated into the bilayer of liposomal membrane because the bilayer interior is composed of non-polar acyl chains. If the liposome incorporating benzyl disulfide is exposed to a reducing environment, benzyl disulfide could be reduced to benzyl thiols and the liposomal membrane would be fluctuated because benzyl thiols can re-orient due to the polarity change. As a result, the liposome would release its content in response to a reducing agent (Fig. 1). The liposome bearing benzyl disulfide developed in this study would be used as a carrier for intracellular drug delivery because the intracellular concentration of a reducing agent (i.e., glutathione) is much higher than the extracellular one. Egg PC liposomes incorporating various amounts of benzyl disulfide were prepared by a film hydration and sonication method. The cumulative release amount of a fluorescence dye (calcein) enveloped in the liposome was investigated for 24 h at different temperatures (25 C, 37 C, and 42 C) and at different concentrations of a reducing agent (DLdithiothreitol). Materials and methods Materials L-a-Phosphatidylcholine (egg PC, average Mw: 768), benzyl disulfide (Mw: 246.3), DL-dithiothreitol (DTT, Mw: 154.25), calcein (Mw: 622.5), Triton X-100 (Mw: 647), phosphotungstic acid (PTA, Mw: 2,880.2), chloroform (Mw: 119.4), sodium hydroxide (Mw: 40.0), potassium hydroxide (Mw: 56.1) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Sephadex G-100 was provided by GE Healthcare (Sweden). N-(2-Hydroxyethyl)piperazine-n0 -(2-ethanesulfonic acid) (HEPES) was obtained from USB corporation (Cleveland, OH, USA). Water was doubly distilled in a
Milli-Q water purification system (Millipore Corp, MA, USA). All other reagents were in analytical grade. Preparation of liposome incorporating benzyl disulfide Liposome incorporating benzyl disulfide was prepared by a film hydration and sonication method [15,16]. Egg PC was dissolved in chloroform so that the concentration was 10 mg/ml. In parallel, benzyl disulfide was dissolved in the same organic solvent so that the concentration was 10 mg/ml. 2 ml of egg PC solution and variable amounts of benzyl disulfide solution were mixed in a 50 ml round bottom flask so that the egg PC to benzyl disulfide weight ratio was 20:0, 20:1, 20:2, 20:5 and 20:10. A rotary evaporator was installed with the flask containing the mixture solution of egg PC and benzyl disulfide, and it was operated at 100 rpm under reduced pressure to remove the organic solvent and obtain the dry mixed film of egg PC and benzyl disulfide on the flask wall. Calcein (a fluorescence dye) was dissolved in HEPES buffer (30 mM, pH 7.0) so that the concentration was 50 mM. 2 ml of calcein solution was put in the flask and it was whirled by a hand until the dry mixed film was completely detached from the glassware wall. The suspension was sonicated at room temperature (25 C) in a bath type sonicator (Sonics & Materials, USA) with pulse-on for 10 s and pulse-off for 10 s. The suspension was left at room temperature for 6 h under dark condition to anneal the liposomal membrane. Free calcein was removed from the liposome by a gel permeation chromatography using a column filled with Sephadex G-100 (1.6 cm 38 cm). Liposome prepared from the mixture solution whose the egg PC to benzyl disulfide weight ratio was 20:0, 20:1, 20:2, 20:5 and 20:10 was called liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5) and liposome (20:10), respectively. Determination of fluorescence quenching degree The fluorescence quenching degree of calcein loaded in egg PC liposome incorporating benzyl disulfide was determined by the following equation [17–19]. % Quenching = (1 Fi/Ft) 100
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where Fi is the fluorescence intensity of the liposome suspension without unentrapped calcein, obtained by column chromatography, and Ft was the fluorescence intensity after the liposome was disintegrated by Triton X-100. The phospholipid concentration of liposomal suspension was adjusted to 0.01% (w/v) when the fluorescence intensity was measured. The fluorescence intensity was measured at 524 nm on a fluorescence spectrophotometer (Hitachi F2500, Japan) using the excitation wavelength of 494 nm. Measurement of mean diameter of liposome The mean hydrodynamic diameter of liposome free of benzyl disulfide (liposome(20:0)) and liposome incorporating benzyl disulfide (liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10)) were determined by a light scattering method [18–21]. The light scattering intensity was adjusted to 50–200 kcps by diluting the liposomal suspension with HEPES buffer (30 mM, pH 7.0), then the mean diameter was measured on a dynamic light scattering equipment (ZetaPlus 90, Brookhaven Instrument Co., USA). Transmission electron microscopy of liposome The structure of liposomes incorporating benzyl disulfide was investigated by transmission electron microscopy [18,19]. Liposome suspension obtained after column chromatography was directly used for the negative staining without dilution. Each suspension of liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) was dropped onto Parafilm of which surface was hydrophobic. PTA was dissolved in distilled water so that the concentration was 2% (w/v), then the pH value of PTA solution was adjusted to pH 6.8 using potassium hydroxide solution (1 M). PTA solution was filtered through a syringe filter (0.2 mm) and it was dropped onto each droplet of the liposome suspension on the Parafilm. After the droplets were mixed using pipette tip for a few minutes, they were left at room temperature (20–23 C) for 3 h under dust free condition. The stained liposome suspensions were transferred onto formvar/carbon-coated grids (200 mesh) by dipping carefully them into the droplets. The grids were air-dried at room temperature for 12 h under dust-free and dark condition.
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Raman spectroscopy The presence of disulfide compound (i.e., benzyl disulfide) in liposome(20:1) was confirmed using Raman spectrometer (JobinYvon/HORIBA LabRam ARAMIS Raman spectrometer (Villeneuved'Ascq, France)). 3 ml of the liposome suspension (0.67 mg/ml) in HEPES buffer (30 mM, pH 7.0) was put in a glass cuvette and the Raman spectrum was taken in the range of 300–800 cm1. In parallel, the spectrum was also taken after the liposome was solubilized using Triton X-100. The radiation wavelength was 523 nm, the grating was 1800 lines/mm, the exposure time was 60 s, and the accumulation number was 3. Observation of reduction-responsive release 0.2 ml of each suspension of liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) was put in 3.8 ml of HEPES buffer (10 mM, pH 7.4) containing various amounts of DTT, contained in a 10 ml glass vial. The final concentration of DTT was 0 mM, 5 mM, 10 mM, and 30 mM. The glass vial was tightly sealed, wrapped with an aluminum foil, and it was rolled on a rolled mixture at room temperature for 24 h. The fluorescence intensity was measured at 524 nm with the excitation wavelength of 494 nm at a given time. The release degree was calculated by the following equation [18,19]. % release = (Ff Fi)/(Ft Fi) 100 where Ff was the fluorescence intensity at a given time and Fi was the initial fluorescence intensity. Ft was the fluorescence intensity after the liposome was completely solubilized by Triton X-100. Results and discussion Determination of fluorescence quenching degree The fluorescence quenching degree of calcein enveloped in egg PC liposome incorporating benzyl disulfide decreased with increasing the benzyl disulfide content. For example, the fluorescence quenching degree of the fluorescence dye loaded in liposome (20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) was 67.2%, 66.5%, 52.5%, 50.1%, and 49.2%,
Fig. 2. TEM photos of liposome free of benzyl disulfide (liposome(20:0) (A)), and liposomes incorporating benzyl disulfide (liposome(20:1) (B), liposome(20:2) (C), liposome (20:5) (D), and liposome (20:10) (E)).
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respectively. This indicates that the efficiency of liposome formation decreased with increasing the benzyl disulfide content because the fluorescence quenching degree is a measure of the liposome formation. Benzyl disulfide would be intercalated into the phospholipid bilayer due to its non-polar property. The amount of benzyl disulfide the phospholipid bilayer could accommodate without disintegration would be limited, and the bilayer vesicle could be more readily disintegrated into a non-bilayer structure (e.g., mixed micelle) as the content of benzyl disulfide increased. This would explain why the fluorescence quenching degree decreased with increasing the benzyl disulfide content. Measurement of mean diameter of liposome The mean diameter of liposome incorporating benzyl disulfide decreased as the benzyl disulfide content increased. For example, the mean diameter of liposome(20:0), liposome(20:1), liposome (20:2), liposome(20:5), and liposome(20:10) was 276 nm, 267 nm, 254 nm, 226 nm, and 206 nm, respectively. As described previously, benzyl disulfide is a non-polar compound and it would be solubilized in the phospholipid bilayer. Upon the intercalation of benzyl disulfide into the bilayer, the curvature of the liposomal membrane could increase, leading to a decrease in the mean diameter. Since the size measured by light scattering method was hydrodynamic diameter, the diameter reported here would be larger than the real diameter. If the sonication intensity had been stronger and the sonication duration had been longer, the diameter could have been smaller. But more intensive sonication would have chemically deteriorated egg PC and benzyl disulfide. Transmission electron microscopy of liposome Fig. 2 shows the TEM photos of liposome free of benzyl disulfide (liposome(20:0)) and liposomes incorporating benzyl disulfide (liposome(20:1), liposome(20:2), liposome(20:5), and liposome (20:10)). Multi lamellar vesicles were observed regardless of whether the liposome was incorporating benzyl disulfide. Benzyl disulfide would be incorporated in the bilayer of liposome owing to its non-polar property. Non-polar compounds were reported to be solubilized in the liposomal bilayer membrane without affecting the lamellarity [22,23]. The major factors affecting the lamellartiy of liposome are known to be the preparation method and the homogenization energy (e.g., sonication energy) [24–26]. For example, multi-lamellar vesicles are obtained by hand-shaking method and uni-lamellar vesicles are obtained by reversed-phase evaporation method and sonication method. The liposome in present study was prepared by hand-shaking and sonicating the aqueous mixture suspension of egg PC/benzyl disulfide. The sonication intensity and the sonication duration were thought not to high enough to convert multi-lamellar vesicle to uni-lamellar one. Raman spectroscopy Fig. 3(A) shows the Raman spectrum before liposome(20:1) was solubilized by Triton X-100. The disulfide signal is known to appear in the range of 425–550 cm1 [27–29]. There seemed to be a weak signal in the range but it was not apparent. Fig. 3(B) shows the Raman spectrum after liposome(20:1) was solubilized by Triton X100. An evident signal was found between 425 and 550 cm1 and it could be ascribed to the disulfide bond of benzyl disulfide [30,28]. Benzyl disulfide is a non-polar compound and it could be intercalated in the liposomal membrane [22]. Moreover, the liposome was multi-lamellar on the TEM photo (Fig. 2). In this circumstance, a small amount of laser photons could reach benzyl disulfide and a small amount of Raman scattered light photons
Fig. 3. Raman spectrum before (a) and after (b) liposome (20:1) was solubilized by Triton X-100.
could emit due to the light-shielding effect of the liposomal bilayer [31,32]. This could be one of reasons why no appreciable signal of disulfide bond was found even if benzyl disulfide was in the liposomal sample. Furthermore, laser light is scattered by liposomal particles and the scattered light intensity would be much higher than Raman scattered light intensity produced by the disulfide bond [33–35]. Thus, the signal of Raman scattered light would hardly appear due to the strong background signal. On the other hand, liposome can be disintegrated into mixed micelle in the presence of Triton X-100 (a surfactant). Due to the smaller size of micelle, the incident light-shielding effect would be less and the background signal would be weaker [31,32]. This could elucidate the reason why an evident signal of disulfide bond could be obtained after the liposome was solubilized. Based on the Raman spectrum obtained before and after liposome (20:1) was solubilized, it could be said the benzyl disulfide was loaded in the liposomal bilayer. Observation of reduction-responsive release Fig. 4 shows the release profile of calcein enveloped in liposome (20:1) at 25 C, 37 C, and 42 C when the concentration of DTT was 0 mM, 5 mM, 10 mM and 30 mM. The release degree increased rapidly in early stage and the release was almost completed in 8 h. The release degree was higher as the DTT concentration was higher. The maximum release degree at 25 C was 2%, 3.5%, 5% and 11%, respectively, when the DTT concentration was 0 mM, 5 mM, 10 mM, and 30 mM (Fig. 4(A)). DTT is a reducing agent and it can break down the disulfide bond of benzyl disulfide in the liposomal membrane, leading to the formation of benzyl thiols. Due to the reduction-caused polarity change, the fragments of benzyl disulfide (i.e., benzyl thiols) would re-orient in the liposomal membrane. Thiol groups would face water phase with benzyl groups embedded in the interior of phospholipid bilayer. During the re-orientation, the liposomal membrane could be subjected to fluctuation, leading to a promoted release. This could account for why the release increased with increasing the DTT concentration. However, the release was not promoted markedly by the reducing agent. DTT is water-soluble and it would not readily penetrate into the nonpolar interior of the liposomal membrane where benzyl disulfide exists. Accordingly, only a small amount of benzyl disulfide would undergo the reduction and the membrane fluctuation would be weak, resulting in a small increase in the release. The maximum release degree at 37 C was 2.5%, 5.1%, 7.2%, and 12.5%, respectively, when the concentration of DTT was 0 mM,
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Fig. 4. Release profile of calcein enveloped in liposome (20:1) at 25 C (A), 37 C (B), and 42 C (C) when the concentration of DTT was 0 mM (*), 5 mM (), 10 mM (!) and 30 mM (D).
5 mM, 10 mM, and 30 mM (Fig. 4(B)). At all the DTT concentrations tested, the release degree at 37 C (Fig. 4(B)) was higher than the release degree at 25 C (Fig. 4(A)). The maximum release degree at 42 C was 2.7%, 6.7%, 8.5%, and 13.3%, respectively, when the DTT concentration was 0 mM, 5 mM, 10 mM, and 30 mM (Fig. 4(C)). At all the DTT concentrations tested, the release degree at 42 C (Fig. 4(C)) was higher than the release degree observed at the lower temperatures (Fig. 4(A) and (B)). The fluidity of liposomal membrane is higher at a higher temperature [36,37], thus calcein could release out of liposome more readily at a higher temperature. The diffusivity of a solute (calcein) itself would also increase with increasing the temperature. According to Stoke–Einstein’s equation, the diffusion coefficient is proportional to the root square of absolute temperature [38,39]. In addition, DDT would also penetrate into the liposomal membrane and reduce benzyl disulfide more easily at a higher temperature. Thus, the reduction of benzyl disulfide and the fluctuation of the liposomal membrane would be more favorable at a higher temperature. These could account for why the release was more extensive as the temperature was higher. Fig. 5 shows the release degree in 24 h of calcein enveloped in liposome(20:0), liposome(20:1), liposome(20:2), liposome(20:5), and liposome(20:10) at 25 C, 37 C, and 42 C with the DTT concentration. At all the DTT concentrations tested, the release degree of dye enveloped in liposome(20:0) increased a little as the temperature of release medium increased (Fig. 5(A)). For example, the release degree at the DTT concentration of 30 mM was 5%, 6.3%, and 6.5%, respectively, when the temperature of release medium was 25 C, 37 C, and 42 C. On the other hand, at all the temperatures tested, the DTT concentration had no
significant effect on the release degree. For example, the release degree increased only 2–3% when the DTT concentration increased from 0 to 30 mM. The slight increase in the release degree was possibly because the osmotic pressure difference between the aqueous core of liposome and the release medium could be developed by DTT existing in the release medium. Like the release degree of calcein loaded in liposome(20:0), the release degree of calcein loaded in liposome(20:1) somewhat increased with increasing the temperature at all the DTT concentrations tested (Fig. 5(B)). For example, the release degree at the DTT concentration of 30 mM was 11%, 12.5%, and 13.3%, respectively, when the temperature of release medium was 25 C, 37 C, and 42 C. Unlike the release degree of calcein loaded in liposome(20:0), the release degree of calcein loaded in liposome(20:1) seemed to be affected by the DTT concentration at all the temperatures tested. For example, the release degree at 25 C increased from 2% to 11% when the DTT concentration increased from 0 to 30 mM. Benzyl disulfide was contained in liposome(20:1) and it could be reduced to benzyl thiols by the reducing agent (DTT). Since the polarity of the fragment of benzyl disulfide (i.e., benzyl thiol) is different from that of the disulfide compound, the fragment would re-orient in the membrane. It was thought that thiol groups would orient toward water phase with benzyl groups in the interior of liposomal bilayer. The liposomal membrane would be stressed and fluctuated upon the re-orientation and it could promote the release. The release degree of calcein enveloped in liposome(20:2) was also proportional to the temperature at all the DTT concentrations tested (Fig. 5(C)). The DTT concentration seemed to have an effect on the release degree of calcein enveloped in liposome(20:2) at all
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
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Fig. 5. Release degree in 24 h of calcein enveloped in liposome (20:0) (A), liposome (20:1) (B), liposome (20:2) (C), liposome (20:5) (D), and liposome (20:10) (E) at 25 C (*), 37 C (), and 42 C (!) with DTT concentration.
the temperatures tested. The release sensitivity of liposome(20:2) with respect to the DTT concentration was not markedly different from the sensitivity of liposome(20:1). The release degree of calcein enveloped in liposome(20:5) was also dependent on the temperature at all the DTT concentrations tested (Fig. 5(D)). DTT had a significant effect on the release degree of calcein enveloped in liposome(20:5) at all the temperatures tested. For example, the release degree at 25 C increased from 2.3% to 15.5% when the DTT concentration increased from 0 to 30 mM. The release sensitivity of liposome(20:5) with respect to the DTT concentration was significantly higher than the sensitivity of liposome(20:1) and that of liposome(20:2). The benzyl disulfide content of liposome (20:5) was much higher than that of liposome(20:1) and that of liposome(20:2). Accordingly, upon the reduction of the disulfide compound, the membrane of former liposome would be fluctuated more than the membrane of latter one. This could explain why liposome(20:5) was more sensitive to the DTT concentration. Like the other liposomes described above, liposome(20:10) released its content more at a higher temperature (Fig. 5(E)). Liposome(20:10) also released its content in a DTT concentration-dependent manner at all the temperatures tested, and its release sensitivity to the DTT concentration was the highest among the liposomes tested. For example, the release degree at 25 C increased from 3.1% to 17.9% when the DTT concentration increased from 0 to 30 mM. The highest content of disulfide compound would be responsible for the highest sensitivity with respect to the DTT concentration. Conclusions Reduction-responsive liposome bearing benzyl disulfide was prepared by hydrating the dry mixed film of egg PC and the disulfide compound and sonicating the suspension. The fluorescence quenching degree decreased from 62.7% to 49.2% as the egg
PC to benzyl disulfide weight ratio increased from 20:0 to 20:10, possibly due to the disintegration of the liposome by the intercalation of benzyl disulfide into the bilayer. Increase in the egg PC to benzyl disulfide weight ratio (from 20:0 to 20:10) led to decrease in the mean diameter of the liposome (from 276 nm to 206 nm), probably because the curvature of the liposomal membrane could increase upon the intercalation of benzyl disulfide into the liposomal bilayer. Multi-lamellar vesicles were found on the TEM photos of the liposomes bearing benzyl disulfide. Benzyl disulfide was confirmed to be loaded in the liposomal bilayer by Raman spectroscopy. At all the DTT concentrations tested (0 mM, 5 mM, 10 mM, 30 mM), the release degree in 24 h was more extensive as the temperature was higher, possibly because the membrane fluidity and the dye diffusivity could be greater at a higher temperature. At all the temperatures tested (25 C, 37 C, and 45 C), the release degree became more extensive as the DTT concentration increased. It was thought that benzyl thiol, produced by the reduction of benzyl disulfide, would re-orient in the liposomal membrane due to its polarity change and it could cause the liposomal membrane fluctuation and the promoted release. At all the temperatures and all the DTT concentrations tested, the release sensitivity with respect to the DTT concentration was higher as the benzyl disulfide content in liposome was higher. This means that the promoted release was due to the reduction of the disulfide compound in the liposomal membrane. The liposome bearing benzyl disulfide developed in this study could be simply prepared by hydrating the dry mixed film of egg PC and the disulfide compound at room temperature. It could release its content more extensively at a higher DTT concentration. The liposome bearing benzyl disulfide is thought to deliver a pharmaceutical drug to intracellular space in a selective manner because the intracellular concentration of a reducing agent (i.e., glutathione) is much higher than the extracellular one.
Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018
G Model JIEC 3053 No. of Pages 7
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Please cite this article in press as: H. Guo, J.-C. Kim, Reduction-responsive release property of egg phosphatidylcholine liposomes incorporating benzyl disulfide, J. Ind. Eng. Chem. (2016), http://dx.doi.org/10.1016/j.jiec.2016.08.018