International Journal of Pharmaceutics 468 (2014) xxx–xxx
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Light- and temperature-responsive liposomes incorporating cinnamoyl Pluronic F127 MinHui Wang, Jin-Chul Kim * College of Biomedical Science and Institute of Bioscience and Biotechnology, Kangwon National University, 192-1, Hyoja 2 dong, Chunchon, Kangwon-do 200701, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 April 2014 Received in revised form 3 April 2014 Accepted 3 April 2014 Available online 05 April 2014
Light- and temperature-responsive liposomes were prepared by immobilizing cinnamoyl Pluronic F127 (CP F127) on the surface of egg phosphatidylcholine liposomes. CP F127 was prepared by a condensation reaction, and the molar ratio of cinnamoyl group to Pluronic F127 was calculated to be 1:1.4 on 1H NMR spectrum. The cinnamoyl group of CP F127 was readily dimerized under the irradiation of a UV light (254 nm, 6 W). CP F127 decreased the absolute value of the zeta potential of liposome possibly because it can shift the hydrodynamic plane away from the liposome surface. The size of liposome decorated with CP F127, measured on a dynamic light scattering machine and observed on a TEM, was larger than that of bare liposome. The liposome bearing CP F127 seemed to fuse and aggregate each other. The liposome released calcein, a fluorescence dye, in response to a UV irradiation, possibly because the photodimerization of cinnamoyl group perturbs the liposomal membrane. Moreover, the liposome released the dye in response to a temperature change, possible due to the phase transition of Pluronic F127 layer on the liposomal surface or the hydrophobic interaction of the polymer with liposomal membrane. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Liposomes Cinnamoyl Pluronic F127 Light-responsive Temperature-responsive Release
1. Introduction Light-responsive liposomes have been extensively studied recently by including photo-reactive molecules in the liposomal membranes (Alvarez-Lorenzo et al., 2009; Friesen et al., 2009; Roy et al., 2010; Nicoletta et al., 2012). Coumarin, cinnamic acid and their derivatives are representative photo-triggers to cause the release of a water-soluble molecule from the liposome under the irradiation of a UV light (Seo and Kim, 2011, 2012; Seo et al., 2013; Seo and Kim, 2013). A cyclobutane bridge is built by a UV light irradiation between two molecules of the photo reactive molecules, leading to the formation of dimers. Also, the bridge is cleaved by a shorter UV irradiation, resulting in the regeneration of the monomers. In other words, the photo-dimerization and the photocleavage reversibly take place (Gnanaguru et al., 1985; Chujo et al., 1990; Tieke 2004). When included in liposomal membranes, the photo-dimerization and de-dimerization are supposed to perturb the membrane, leading to a photo-triggered release (AlvarezLorenzo et al., 2009; Wells et al., 2010; Yavlovich et al., 2010; Roy et al., 2010). On the other hand, temperature-responsive liposomes have also been extensively studied by decorating the liposomal
Abbreviation: CP F127, Cinnamoyl Pluronic F127. * Corresponding author. Tel.: +82 33 250 6561; fax: +82 33 253 6560. E-mail address: [email protected] (J.-C. Kim). http://dx.doi.org/10.1016/j.ijpharm.2014.04.014 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
surface with temperature-sensitive polymers (Kono et al., 1996, 1999; Kim et al., 1997, 1999). Poly(N-isopropylacrylamide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) tri-block copolymers (Pluronics) are representative thermal triggers to cause the release of a water-soluble molecule from the liposomes when the temperature is raised across their lower critical solution temperatures (LCSTs) (Chandaroy et al., 2001; Ma et al., 2008). The temperatures-sensitive polymers become hydrophobic upon increasing the solution temperature across LCSTs and they interact hydrophobically with the liposomal membranes, triggering release from the liposomes. Recently, the copolymers of hydroxylethyl acrylate and non-polar co-monomers (e.g. octadecyl acrylate, ethylhexyl acrylate, cinnamoyl acrylate) were proposed to be used as thermal triggers for temperature-responsive liposomes (Mok and Kim, 2014). In this study, light- and temperature-responsive liposomes were prepared by modifying the surface of egg phosphatidylcholine liposomes with cinnamoyl Pluronic F127 (CP F127). CP F127 is surface-active because cinnamoyl group is hydrophobic and the PEO-PPO-PEO chain of Pluronic F127 is hydrophilic. So it can be immobilized on the liposomal surface with cinnamoyl group anchored into the liposomal membranes. And the cinnamoyl groups of CP F127 are photo-dimerized under the irradiation of a UV light. Thus, they can perturb the liposomal membrane by the photo-dimerization and trigger the release of a water-soluble payload from the liposomes in response to the irradiation of a UV
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light. In addition, CP F127 undergoes micellization and gelation at a certain temperature when the concentrated aqueous solution is heated up (Dai and Kim, 2014). So, it can perturb the liposomal membrane due to the phase transitions, and cause a temperatureresponsive release from the liposomes. 2. Materials and methods 2.1. Materials Pluronic F127 (MW 12,600), cinnamoyl chloride (MW 166.6), cinnamic acid (MW 148.2), triethylamine (MW 101.19), calcein (MW 622.55), and 1,2-diacyl-sn-glycero-3-phosphocholine from egg yolk (EPC) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Diethyl ether (MW 74.12) and dichloromethane (MW 84.93) were provided by Dae Jung Co. (Siheung, Republic of Korea). Water was doubly distilled in a Milli-Q water purification system (Millipore Corp.) until the resistivity was 18 MV/cm. All other reagents were in analytical grade. 2.2. Preparation of cinnamoyl Plurionic F127 Cinnamoyl Pluronic F127 (CP F127) was prepared by a method described in a previous report (Dai and Kim, 2014). 0.42 ml (3.0 mmol) of triethylamine was mixed with 100 ml of dichloromethane, and then 10 g (0.8 mmol) of Pluronic F127 was dissolved in the solution. While the mixture being stirred in an ice bath, 1 g (6 mmol) of cinnamoyl chloride was added to the mixture, and it was stirred for 12 h. The reaction mixture was further stirred for 24 h at room temperature. In order to precipitate out CP F127, the reaction mixture was poured into 2 L of diethyl ether, and it was stirred for 1 h. The precipitate was filtered through a filter paper (Whatman Ltd., Maidstone, Kent, UK), and it was dried in an oven thermostat at 40 C for 48 h. 2.3. Spectrometry CP F127 was dissolved in DMSO-d6, and the 1H NMR spectrum was taken on a Bruker Avance 400 spectrometer (Karlsruhe, Germany) in the Central Laboratory of Kangwon National University. CP F127 was pelleted with KBr, and the FTIR spectrum was taken on a FTIR spectrophotometer (FT-3000, MX, Excalibur, BIO-RAD, Cambridge, USA, located at the Central Laboratory of Kangwon National University). 2.4. Photo-dimerization Cinnamic acid and CP F127 were dissolved in distilled water so that the concentration of each solution was 1% (w/w). The solutions were subjected to the irradiation of a UV light (254 nm, 6 W) for 80 min. The dimerization degree was calculated using the following equation (Eq. (1)) (Jackson et al., 1998; Jin et al., 2010; Seo and Kim, 2013): At 100% (1) Dimerizationð%Þ ¼ 1 A0 where, A0 and At are the absorbance at 276 nm before and after being exposed to the irradiation of the UV light, respectively. 2.5. Preparation of liposomes incorporating CP F127 Liposomes were prepared by a hand-shaking and sonication method (Kirby and Gregoriadis, 1984; Jo et al., 2008a, b; Jo and Kim, 2009; Hong et al., 2011). EPC dry thin film, 20 mg, on the wall of a 50 ml-round bottom flask was dispersed into 2 ml of HEPES buffer (30 mM, pH 7.4) by whirling the flask with a hand-shake. In order
to entrap calcein, a fluorescence dye, in the liposomes, calcein solution (50 mM) in the buffer solution was used as a dispersing medium. In order to modify the liposomal surfaces with CP F127, the functional polymer was dissolved in the dispersing medium so that the EPC to polymer ratio was 100:0, 100:2 and 100:20 (w/w), and the polymer-containing solution was used as a dispersing medium. After the liposomal dispersion was hand-shaken, it was homogenized in a bath-type sonicator (Sonics & Materials, USA) for 30 min and then annealed for 6 h at room temperature. The liposome was separated from free calcein using a Sephadex G-100 column (1.6 cm ca. 38 cm). Liposome prepared using the EPC to CP F127 ratio is 100:0, 100:2 and 100:20 (w/w) will be termed liposome (100:0), liposome (100:2), and liposome (100:20), respectively. 2.6. Characterization of liposomes The fluorescence of calcein is quenched when it is entrapped into liposomes in a high concentration. Thus, the fluorescence quenching degree can be used as a measure of liposome formation efficiency. The fluorescence quenching degree of calcein entrapped in liposomes was calculated using a well-known equation (Eq. (2)) (Jo and Kim, 2009): F (2) Quenching degreeð%Þ ¼ 1 i 100 Ft where Fi is the fluorescence intensity of a liposomal dispersion, and Ft is the fluorescence intensity after the liposome was completely disintegrated in deoxycholate solution. The fluorescence intensity was measured at 520 nm using the excitation wavelength at 492 nm. Liposome (100:0), liposome (100:2), and liposome (100:20) were investigated in terms of the mean diameter and the zeta potential on a particle size analyzer (ZetaPlus 90, Brookhaven Instrument Co., USA). And the shapes of the liposomes were observed on a transmission electron microscope (LEO 912AB OMEGA, Germany) installed at Korea Basic Science Institute (KBSI, located in Chuncheon, Republic of Korea using a negative staining technique (Jackson et al., 1998; Harris et al., 1999; Wang and Kim, 2013, 2014). 2.7. Light- and temperature-responsive release from liposomes incorporating CP F127 The light-responsive release was observed using the method described in a previous report (Jo and Kim, 2009; Seo and Kim, 2012). A UV (l = 254 nm, 6 W) light was irradiated to the suspension (0.056 mg lipid/ml) of liposome (100:0), liposome (100:2) and liposome (100:20) for 180 min at room temperature. The fluorescence intensity of calcein was determined at 520 nm at a given time using the excitation wave length of 492 nm. The release degree was calculated by a well-known equation (Eq. (3)): %release ¼
ðF F i Þ 100 ðF t F i Þ
(3)
where F is the fluorescence intensity after UV was irradiated for a certain period, Fi is the initial fluorescence intensity before the UV irradiation, and Ft is the fluorescence intensity after liposome is disintegrated in deoxycholate solution. And the temperatureresponsive release was observed using the method described in a previous report (Kim and Kim, 2002). The suspension (2.9 mg EPC/ ml) kept at 6 C of liposome (100:0), liposome (100:2), and liposome (100:20), 0.04 ml of each, was injected into 2 ml of HEPES buffer (30 mM, pH 7.4) contained in a cuvette, which was preadjusted to the temperature of 10 C, 20 C, 30 C, and 37 C. The release degree was determined as described above except that F is the fluorescence intensity at a given time and at a certain temperature, and Fi is the initial fluorescence intensity, measure at 6 C.
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Fig. 1. 1HNMR spectrum of CP F127.
3. Results and discussion
around 1.4 molecule of cinnamoyl group was attached to 1 molecule of Pluronic F127 which has two hydroxyl groups. Thus, there will be some free hydroxyl groups in CP F127.
3.1. Spectrometry 3.2. Photo-dimerization In 1H NMR spectrum of CP F127 shown in Fig. 1, the signal in the range of 6.8–7.92 ppm is due to the cinnamoyl protons, and the signal of 1.02 ppm is due to the methyl protons of poly(propylene oxide) block. The molar ratio of Pluronic F127 to cinnamoyl group was calculated to be about 1:1.4 using the area of the cinnamoyl protons signal (6.75) and that of the methyl protons signal (142.77). The molar ratio of cinnamoyl chloride to Pluronic F127 was 7.5:1 in the reaction mixture so the amount of cinnamoyl chloride was stoichiometrically excessive. Nevertheless, only 1.4 of 2 hydroxyl groups of Pluronic F127 participated in the reaction with cinnamoyl chloride. Once one molecule has been covalently attached to the one end of Pluronic F127, CP F127 could be assembled into reversed micelles in the reaction organic solvent. So the hydroxyl group on the other end of the polymer chain will be sequestered and it would hardly take a part in the reaction (Dai and Kim, 2014). The molar ratio of Pluronic F127 to cinnamoyl group in the present study (1:1.4) was somewhat higher than that of molar ratio obtained in a previous report (1:1) (Dai and Kim, 2014). This might because the input molar ratio of cinnamoyl chloride to Pluronic F127 here (7.5:1) was somewhat higher than that of previous report (4:1) (Dai and Kim, 2014). Fig. 2 shows the FT-IR spectra of cinnamic acid, Pluronic F127 and CP F127. The characteristic peak of cinnamic acid was observed around 1686 cm1, corresponding to the carboxylic carbonyl group (Spectrum A). However, it disappeared when conjugated to Pluronic F127. Moreover, a strong signal appeared at 1745 cm1, attributed to the carbonyl group of the ester bond formed by the esterification of cinnamic acid and Pluronic F127 (Spectrum C). Accordingly, it was confirmed that cinnamoyl group had been attached to Pluronic F127. On the other hand, a peak at 3500 cm1 is ascribed to the hydroxyl group of Pluronic F127 (Spectrum B), after conjugated with cinnamic acid, the signal of the hydroxyl group was still shown at 3437 cm1 (Spectrum C). According to the 1H NMR spectrum of CP F127, only
Fig. 3 shows the photo dimerization degree of cinnamic acid and CP F127. The dimerization degree of cinnamic acid increased with the irradiation time, but the increasing rate decreased with the time lapse and it approached 0 at the irradiation time of 60 min. The UV irradiation can not only dimerize the cinnamoyl group but also de-dimerize the dimer (Gnanaguru et al., 1985; Chujo et al., 1990; Tieke, 2004). The dimerization will takes place
Fig. 2. FT-IR spectra of cinnamic acid (Spectrum A), Pluronic F127 (Spectrum B) and CP F127 (Spectrum C).
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Fig. 3. Photo dimerization degree of cinnamic acid (*) and CP F127 ().
dominantly in the early stage of the UV irradiation where monomer cinnamic acid is abundant, and it will become equilibrated with the de-dimerization as the UV irradiation time increases. The dimerization degree of CP F127 increased with the irradiation time in a similar manner to how the dimerization degree of cinnamic acid increased with the time. However, the dimerization degree of CP F127 was almost two times higher than that of cinnamic acid. For example, the maximum dimerization degree of CP F127 was 56.7% and that of cinnamic acid was 29.7%. CP F127 would be an amphiphile because the cinnamoyl group is hydrophobic and the polymer chain is hydrophilic. Thus, the molecules could be assembled into micelles in an aqueous phase. In this circumstance, cinnamoyl groups will be close to one another in the core of the micelles, and the photo dimerization will be more favorable than that of free cinnamic acid. 3.3. Characterization of liposomes Table 1 shows the fluorescence quenching degree calculated by Eq. (2), the zeta potential, and the mean diameter of liposome (100:0), liposome (100:2), and liposome (100:20). The fluorescence quenching degree of calcein entrapped in liposome (100:0), liposome (100:2), and liposome (100:20) was 74.5%, 74.4%, and 68.6%, respectively. The quenching degree of calcein entrapped in liposome (100:20) was somewhat less than those of calcein entrapped in the other liposomes, indicating that the former liposome was formed less in number than the latter liposomes. As described previously, CP F127 is a surface-active polymer so it can form a mixed micelle with EPC. This may account for why the liposome containing a higher amount of CP F127 (liposome (100:20)) exhibited a lower fluorescence quenching. It was
Table 1 Fluorescence quenching degree, the zeta potential, and the mean diameter of liposome (100:0), liposome (100:2), and liposome (100:20). Weight ratio of EPC:CP F127
100:0
50:1
5:1
Quenching degree (%) Zeta potentials (mV) Mean diameter (nm)
74.5 20.3 1.8 185.8
74.4 13.4 4.3 120.5
68.6 8.7 2.4 1113.7
reported that the inclusion of a surface-active polymer in the liposome preparation led to a lower quenching degree (Seo and Kim, 2011, 2012; Seo et al., 2013). The zeta potential of liposome (100:0), liposome (100:2), and liposome (100:20) was 20.3 mV, 13.4 mV, and 8.7 mV, respectively. The liposomes containing CP F127 exhibited less negative potentials than the liposome free of the polymer. This is possibly because CP F127 immobilized on the liposomal surface can shift the hydrodynamic plane away from the surface. It was reported that the zeta potential of liposome of which the surface is decorated with a non-ionic hydrophilic polymer showed a less zeta potential than bare liposome owing to the steric effect of the polymer (Liu et al., 2003). The mean diameter of liposome (100:0), liposome (100:2), and liposome (100:20) was 185 nm, 120 nm, and 1113 nm, respectively. The liposome containing a higher amount of CP F127 (liposome (100:20)) was much bigger in the mean diameter than the other liposomes. The layer of CP F127 immobilized on the liposome would contribute to an increase in the mean diameter. One of the major reasons would be the agglomeration of the liposomal particles. Since the absolute value of the zeta potential of liposome (100:20) was relatively small (e.g. 8.7 mV), the agglomeration of liposomal particles can readily take place due to the lack of an inter-particular repulsive force. And, the amount of CP F127 immobilized on the surface of liposome (100:20) is thought to be high enough for the polymer chains to inter-particularly interact each other through the hydrophobic interaction of PPO blocks. Another reason would be the fusion of liposomes. PEO is known to be fusogenic for a biological membrane (Aldwinckle et al., 1982; Barauskas et al., 2010). Thus, Pluronic F127, which contains PEO blocks, could help the liposomes to fuse each other. Fig. 4 shows the TEM photos of liposome (100:0), liposome (100:2), and liposome (100:20). Multi-lamellar vesicles were observed with all three kinds of liposome. Liposome (100:0) and liposome (100:2) were a few hundreds of nanometers in diameter. Some of liposome (100:20) seems to be fused and they were obviously bigger than the other liposomes. As described previously, Pluronic F127 may help the liposomal particles to fuse each other. 3.4. Light- and temperature-responsive release from liposomes incorporating CP F127 Fig. 5A shows the calcein release profiles from liposome (100:0), liposome (100:2) and liposome (100:20) without UV irradiation. There was no significant release for 180 min with all three kinds of liposome. Fig. 5B shows the calcein release profiles from liposome (100:0), liposome (100:2), and liposome (100:20) with UV irradiation (254 nm, 6 W). Liposome (100:0) exhibited no significant release for 180 min under the UV irradiation. However, liposome (100:2) released their content in response to the UV irradiation. The cinnamoyl group of CP F127 was readily dimerized under the irradiation of UV light (Fig. 3). The dimerization of cinnamoyl group is believed to perturb the liposomal membrane and trigger the release, since the cinnamoyl group will be incorporated in the liposomal membrane due to a hydrophobic interaction. It was reported that the photo dimerization of photoreactive compounds (e.g. coumarin and cinnamic acid) and the photo de-dimerization of the dimers caused the release of water soluble dyes from liposomes (Seo and Kim, 2011, 2012; Seo et al., 2013). The release increased in a saturation manner with the irradiation time and no significant release took place after 90 minUV irradiation. This is possibly because the dimerization degree of cinnamoyl group increased in a saturation manner (Fig. 3). The release profile of liposome (100:20) resembled that of liposome (100:2). However, the release degree of the former liposome was almost two times higher than that of the latter liposome. If a higher amount of CP F127 is included in liposome, it is likely to perturb the
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Fig. 4. TEM photos of liposome (100:0) (A), liposome (100:2) (B), and liposome (100:20) (C). The scale bars are 200 nm.
liposomal membranes more efficiently because a higher amount of photo dimerizable group is in the liposome. Fig. 6A shows the release profiles of liposome (100:0) at 10 C, 20 C, 30 C, and 37 C. No marked release was observed for 180 s at all the temperatures tested. Liposome is known to release their content markedly at the membrane phase transition temperature (Grüll and Langereis, 2012; Wang and Kim, 2013, 2014). Liposomal
membrane, phospholipid bilayer, undergoes a solid gel to liquid crystal transition at a certain temperature. Since the phase transition temperature of EPC liposomal membrane is in the range of 5.8 C–1.5 C (Marsh, 1992; Koynova and Caffrey, 1998), the liposome will not undergoes the phase transition in the full range of temperature tested. Fig. 6B shows the release profiles of liposome (100:20) at 10 C, 20 C, 30 C, and 37 C. When the
Fig. 5A. Calcein release profiles from liposome (100:0) (*), liposome (100:2) () and liposome (100:20) (!) without UV irradiation.
Fig. 5B. Calcein release profiles from liposome (100:0) (*), liposome (100:2) (), and liposome (100:20) (!) with UV irradiation (254 nm, 6 W).
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micellization and the gellation of Pluronic F127 can take place on the surface of liposome, and the phase transitions may impose a mechanical stress on the liposomal membrane, triggering the release. Another reason for the triggered release is possibly due to the hydrophobic interaction between Pluronic F127 chains and the liposomal membrane. The dehydrated and hydrophobicized PPO blocks at a higher temperature could hydrophobically interact with liposomal membranes, giving a rise to the triggered release. 4. Conclusion
Fig. 6A. Release profiles of liposome (100:0) at 10 C (solid line), 20 C (dotted line), 30 C (dash-dotted line), and 37 C (dash line).
temperature of release medium was 10 C, there was no significant release for 180 min. When the temperature was 20 C, a marked release took place and it was completed in a few seconds, and the maximum release degree was around 20%. The maximum release degree was higher at a higher temperature. For example, when the release temperature was 30 C and 37 C, the maximum release degree was about 27% and 45%, respectively. As the temperature increases, the PPO blocks of Plurionic F127 in an aqueous phase become dehydrated and the hydrophobic interaction among the blocks will increase. As a result, the polymer chains are assembled into micelles at a certain temperature and the solution becomes a gel at a higher temperature. The micellization temperature of Pluronic F127 in an aqueous solution ranges from 8 C to 24 C, and the gellation temperature is from 20 C to 29 C depending on the concentration (Chandaroy et al., 2002; Pisal et al., 2004; Jiang et al., 2008). The
Liposome which releases its water-soluble payload in response to a UV irradiation and a temperature change were prepared by decorating the surface of egg phosphatidylcholine liposomes with CP F127. The cinnamoyl group to Pluronic F127 molar ratio of CP F127 was calculated to be about 1:1.4 on 1H NMR spectrum. The immobilization of CP F127 on the liposomal membrane resulted in a decreased absolute value of the zeta potential of liposome possibly due to its steric effect. And, it led to an increased size possibly due to the fusion caused by the fusogenic effect of CP F127 and due to the aggregation caused by the low zeta potential and the inter-particular interaction of CP F127 chains. The liposome was UV light-sensitive in terms of release. The photo-dimerization of cinnamoyl group of CP F127 is believed to perturb the liposomal membrane and trigger the release. And the liposome was also temperature-sensitive. The phase transition of Pluronic F127 layer on the liposomal surface or the hydrophobic interaction of the polymer chains with liposomal membrane would account for the temperature-sensitive release. Light- and temperature-responsive liposomes developed in the present study could be used as a drug carrier which releases its content in a controlled manner in response to UV irradiation and temperature. Conflict of interest We confirm that there are no conflicts of interest associated with this publication. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2007898). References
Fig. 6B. Release profiles of liposome (100:20) at 10 C (solid line), 20 C (dotted line), 30 C (dash-dotted line), and 37 C (dash line).
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Contents lists available at ScienceDirect
International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Pharmaceutical nanotechnology
Light- and temperature-responsive liposomes incorporating cinnamoyl Pluronic F127 MinHui Wang, Jin-Chul Kim * College of Biomedical Science and Institute of Bioscience and Biotechnology, Kangwon National University, 192-1, Hyoja 2 dong, Chunchon, Kangwon-do 200701, Republic of Korea
A R T I C L E I N F O
A B S T R A C T
Article history: Received 3 April 2014 Received in revised form 3 April 2014 Accepted 3 April 2014 Available online 05 April 2014
Light- and temperature-responsive liposomes were prepared by immobilizing cinnamoyl Pluronic F127 (CP F127) on the surface of egg phosphatidylcholine liposomes. CP F127 was prepared by a condensation reaction, and the molar ratio of cinnamoyl group to Pluronic F127 was calculated to be 1:1.4 on 1H NMR spectrum. The cinnamoyl group of CP F127 was readily dimerized under the irradiation of a UV light (254 nm, 6 W). CP F127 decreased the absolute value of the zeta potential of liposome possibly because it can shift the hydrodynamic plane away from the liposome surface. The size of liposome decorated with CP F127, measured on a dynamic light scattering machine and observed on a TEM, was larger than that of bare liposome. The liposome bearing CP F127 seemed to fuse and aggregate each other. The liposome released calcein, a fluorescence dye, in response to a UV irradiation, possibly because the photodimerization of cinnamoyl group perturbs the liposomal membrane. Moreover, the liposome released the dye in response to a temperature change, possible due to the phase transition of Pluronic F127 layer on the liposomal surface or the hydrophobic interaction of the polymer with liposomal membrane. ã 2014 Elsevier B.V. All rights reserved.
Keywords: Liposomes Cinnamoyl Pluronic F127 Light-responsive Temperature-responsive Release
1. Introduction Light-responsive liposomes have been extensively studied recently by including photo-reactive molecules in the liposomal membranes (Alvarez-Lorenzo et al., 2009; Friesen et al., 2009; Roy et al., 2010; Nicoletta et al., 2012). Coumarin, cinnamic acid and their derivatives are representative photo-triggers to cause the release of a water-soluble molecule from the liposome under the irradiation of a UV light (Seo and Kim, 2011, 2012; Seo et al., 2013; Seo and Kim, 2013). A cyclobutane bridge is built by a UV light irradiation between two molecules of the photo reactive molecules, leading to the formation of dimers. Also, the bridge is cleaved by a shorter UV irradiation, resulting in the regeneration of the monomers. In other words, the photo-dimerization and the photocleavage reversibly take place (Gnanaguru et al., 1985; Chujo et al., 1990; Tieke 2004). When included in liposomal membranes, the photo-dimerization and de-dimerization are supposed to perturb the membrane, leading to a photo-triggered release (AlvarezLorenzo et al., 2009; Wells et al., 2010; Yavlovich et al., 2010; Roy et al., 2010). On the other hand, temperature-responsive liposomes have also been extensively studied by decorating the liposomal
Abbreviation: CP F127, Cinnamoyl Pluronic F127. * Corresponding author. Tel.: +82 33 250 6561; fax: +82 33 253 6560. E-mail address: [email protected] (J.-C. Kim). http://dx.doi.org/10.1016/j.ijpharm.2014.04.014 0378-5173/ ã 2014 Elsevier B.V. All rights reserved.
surface with temperature-sensitive polymers (Kono et al., 1996, 1999; Kim et al., 1997, 1999). Poly(N-isopropylacrylamide) and poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) tri-block copolymers (Pluronics) are representative thermal triggers to cause the release of a water-soluble molecule from the liposomes when the temperature is raised across their lower critical solution temperatures (LCSTs) (Chandaroy et al., 2001; Ma et al., 2008). The temperatures-sensitive polymers become hydrophobic upon increasing the solution temperature across LCSTs and they interact hydrophobically with the liposomal membranes, triggering release from the liposomes. Recently, the copolymers of hydroxylethyl acrylate and non-polar co-monomers (e.g. octadecyl acrylate, ethylhexyl acrylate, cinnamoyl acrylate) were proposed to be used as thermal triggers for temperature-responsive liposomes (Mok and Kim, 2014). In this study, light- and temperature-responsive liposomes were prepared by modifying the surface of egg phosphatidylcholine liposomes with cinnamoyl Pluronic F127 (CP F127). CP F127 is surface-active because cinnamoyl group is hydrophobic and the PEO-PPO-PEO chain of Pluronic F127 is hydrophilic. So it can be immobilized on the liposomal surface with cinnamoyl group anchored into the liposomal membranes. And the cinnamoyl groups of CP F127 are photo-dimerized under the irradiation of a UV light. Thus, they can perturb the liposomal membrane by the photo-dimerization and trigger the release of a water-soluble payload from the liposomes in response to the irradiation of a UV
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light. In addition, CP F127 undergoes micellization and gelation at a certain temperature when the concentrated aqueous solution is heated up (Dai and Kim, 2014). So, it can perturb the liposomal membrane due to the phase transitions, and cause a temperatureresponsive release from the liposomes. 2. Materials and methods 2.1. Materials Pluronic F127 (MW 12,600), cinnamoyl chloride (MW 166.6), cinnamic acid (MW 148.2), triethylamine (MW 101.19), calcein (MW 622.55), and 1,2-diacyl-sn-glycero-3-phosphocholine from egg yolk (EPC) were purchased from Sigma–Aldrich Co. (St. Louis, MO, USA). Diethyl ether (MW 74.12) and dichloromethane (MW 84.93) were provided by Dae Jung Co. (Siheung, Republic of Korea). Water was doubly distilled in a Milli-Q water purification system (Millipore Corp.) until the resistivity was 18 MV/cm. All other reagents were in analytical grade. 2.2. Preparation of cinnamoyl Plurionic F127 Cinnamoyl Pluronic F127 (CP F127) was prepared by a method described in a previous report (Dai and Kim, 2014). 0.42 ml (3.0 mmol) of triethylamine was mixed with 100 ml of dichloromethane, and then 10 g (0.8 mmol) of Pluronic F127 was dissolved in the solution. While the mixture being stirred in an ice bath, 1 g (6 mmol) of cinnamoyl chloride was added to the mixture, and it was stirred for 12 h. The reaction mixture was further stirred for 24 h at room temperature. In order to precipitate out CP F127, the reaction mixture was poured into 2 L of diethyl ether, and it was stirred for 1 h. The precipitate was filtered through a filter paper (Whatman Ltd., Maidstone, Kent, UK), and it was dried in an oven thermostat at 40 C for 48 h. 2.3. Spectrometry CP F127 was dissolved in DMSO-d6, and the 1H NMR spectrum was taken on a Bruker Avance 400 spectrometer (Karlsruhe, Germany) in the Central Laboratory of Kangwon National University. CP F127 was pelleted with KBr, and the FTIR spectrum was taken on a FTIR spectrophotometer (FT-3000, MX, Excalibur, BIO-RAD, Cambridge, USA, located at the Central Laboratory of Kangwon National University). 2.4. Photo-dimerization Cinnamic acid and CP F127 were dissolved in distilled water so that the concentration of each solution was 1% (w/w). The solutions were subjected to the irradiation of a UV light (254 nm, 6 W) for 80 min. The dimerization degree was calculated using the following equation (Eq. (1)) (Jackson et al., 1998; Jin et al., 2010; Seo and Kim, 2013): At 100% (1) Dimerizationð%Þ ¼ 1 A0 where, A0 and At are the absorbance at 276 nm before and after being exposed to the irradiation of the UV light, respectively. 2.5. Preparation of liposomes incorporating CP F127 Liposomes were prepared by a hand-shaking and sonication method (Kirby and Gregoriadis, 1984; Jo et al., 2008a, b; Jo and Kim, 2009; Hong et al., 2011). EPC dry thin film, 20 mg, on the wall of a 50 ml-round bottom flask was dispersed into 2 ml of HEPES buffer (30 mM, pH 7.4) by whirling the flask with a hand-shake. In order
to entrap calcein, a fluorescence dye, in the liposomes, calcein solution (50 mM) in the buffer solution was used as a dispersing medium. In order to modify the liposomal surfaces with CP F127, the functional polymer was dissolved in the dispersing medium so that the EPC to polymer ratio was 100:0, 100:2 and 100:20 (w/w), and the polymer-containing solution was used as a dispersing medium. After the liposomal dispersion was hand-shaken, it was homogenized in a bath-type sonicator (Sonics & Materials, USA) for 30 min and then annealed for 6 h at room temperature. The liposome was separated from free calcein using a Sephadex G-100 column (1.6 cm ca. 38 cm). Liposome prepared using the EPC to CP F127 ratio is 100:0, 100:2 and 100:20 (w/w) will be termed liposome (100:0), liposome (100:2), and liposome (100:20), respectively. 2.6. Characterization of liposomes The fluorescence of calcein is quenched when it is entrapped into liposomes in a high concentration. Thus, the fluorescence quenching degree can be used as a measure of liposome formation efficiency. The fluorescence quenching degree of calcein entrapped in liposomes was calculated using a well-known equation (Eq. (2)) (Jo and Kim, 2009): F (2) Quenching degreeð%Þ ¼ 1 i 100 Ft where Fi is the fluorescence intensity of a liposomal dispersion, and Ft is the fluorescence intensity after the liposome was completely disintegrated in deoxycholate solution. The fluorescence intensity was measured at 520 nm using the excitation wavelength at 492 nm. Liposome (100:0), liposome (100:2), and liposome (100:20) were investigated in terms of the mean diameter and the zeta potential on a particle size analyzer (ZetaPlus 90, Brookhaven Instrument Co., USA). And the shapes of the liposomes were observed on a transmission electron microscope (LEO 912AB OMEGA, Germany) installed at Korea Basic Science Institute (KBSI, located in Chuncheon, Republic of Korea using a negative staining technique (Jackson et al., 1998; Harris et al., 1999; Wang and Kim, 2013, 2014). 2.7. Light- and temperature-responsive release from liposomes incorporating CP F127 The light-responsive release was observed using the method described in a previous report (Jo and Kim, 2009; Seo and Kim, 2012). A UV (l = 254 nm, 6 W) light was irradiated to the suspension (0.056 mg lipid/ml) of liposome (100:0), liposome (100:2) and liposome (100:20) for 180 min at room temperature. The fluorescence intensity of calcein was determined at 520 nm at a given time using the excitation wave length of 492 nm. The release degree was calculated by a well-known equation (Eq. (3)): %release ¼
ðF F i Þ 100 ðF t F i Þ
(3)
where F is the fluorescence intensity after UV was irradiated for a certain period, Fi is the initial fluorescence intensity before the UV irradiation, and Ft is the fluorescence intensity after liposome is disintegrated in deoxycholate solution. And the temperatureresponsive release was observed using the method described in a previous report (Kim and Kim, 2002). The suspension (2.9 mg EPC/ ml) kept at 6 C of liposome (100:0), liposome (100:2), and liposome (100:20), 0.04 ml of each, was injected into 2 ml of HEPES buffer (30 mM, pH 7.4) contained in a cuvette, which was preadjusted to the temperature of 10 C, 20 C, 30 C, and 37 C. The release degree was determined as described above except that F is the fluorescence intensity at a given time and at a certain temperature, and Fi is the initial fluorescence intensity, measure at 6 C.
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Fig. 1. 1HNMR spectrum of CP F127.
3. Results and discussion
around 1.4 molecule of cinnamoyl group was attached to 1 molecule of Pluronic F127 which has two hydroxyl groups. Thus, there will be some free hydroxyl groups in CP F127.
3.1. Spectrometry 3.2. Photo-dimerization In 1H NMR spectrum of CP F127 shown in Fig. 1, the signal in the range of 6.8–7.92 ppm is due to the cinnamoyl protons, and the signal of 1.02 ppm is due to the methyl protons of poly(propylene oxide) block. The molar ratio of Pluronic F127 to cinnamoyl group was calculated to be about 1:1.4 using the area of the cinnamoyl protons signal (6.75) and that of the methyl protons signal (142.77). The molar ratio of cinnamoyl chloride to Pluronic F127 was 7.5:1 in the reaction mixture so the amount of cinnamoyl chloride was stoichiometrically excessive. Nevertheless, only 1.4 of 2 hydroxyl groups of Pluronic F127 participated in the reaction with cinnamoyl chloride. Once one molecule has been covalently attached to the one end of Pluronic F127, CP F127 could be assembled into reversed micelles in the reaction organic solvent. So the hydroxyl group on the other end of the polymer chain will be sequestered and it would hardly take a part in the reaction (Dai and Kim, 2014). The molar ratio of Pluronic F127 to cinnamoyl group in the present study (1:1.4) was somewhat higher than that of molar ratio obtained in a previous report (1:1) (Dai and Kim, 2014). This might because the input molar ratio of cinnamoyl chloride to Pluronic F127 here (7.5:1) was somewhat higher than that of previous report (4:1) (Dai and Kim, 2014). Fig. 2 shows the FT-IR spectra of cinnamic acid, Pluronic F127 and CP F127. The characteristic peak of cinnamic acid was observed around 1686 cm1, corresponding to the carboxylic carbonyl group (Spectrum A). However, it disappeared when conjugated to Pluronic F127. Moreover, a strong signal appeared at 1745 cm1, attributed to the carbonyl group of the ester bond formed by the esterification of cinnamic acid and Pluronic F127 (Spectrum C). Accordingly, it was confirmed that cinnamoyl group had been attached to Pluronic F127. On the other hand, a peak at 3500 cm1 is ascribed to the hydroxyl group of Pluronic F127 (Spectrum B), after conjugated with cinnamic acid, the signal of the hydroxyl group was still shown at 3437 cm1 (Spectrum C). According to the 1H NMR spectrum of CP F127, only
Fig. 3 shows the photo dimerization degree of cinnamic acid and CP F127. The dimerization degree of cinnamic acid increased with the irradiation time, but the increasing rate decreased with the time lapse and it approached 0 at the irradiation time of 60 min. The UV irradiation can not only dimerize the cinnamoyl group but also de-dimerize the dimer (Gnanaguru et al., 1985; Chujo et al., 1990; Tieke, 2004). The dimerization will takes place
Fig. 2. FT-IR spectra of cinnamic acid (Spectrum A), Pluronic F127 (Spectrum B) and CP F127 (Spectrum C).
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Fig. 3. Photo dimerization degree of cinnamic acid (*) and CP F127 ().
dominantly in the early stage of the UV irradiation where monomer cinnamic acid is abundant, and it will become equilibrated with the de-dimerization as the UV irradiation time increases. The dimerization degree of CP F127 increased with the irradiation time in a similar manner to how the dimerization degree of cinnamic acid increased with the time. However, the dimerization degree of CP F127 was almost two times higher than that of cinnamic acid. For example, the maximum dimerization degree of CP F127 was 56.7% and that of cinnamic acid was 29.7%. CP F127 would be an amphiphile because the cinnamoyl group is hydrophobic and the polymer chain is hydrophilic. Thus, the molecules could be assembled into micelles in an aqueous phase. In this circumstance, cinnamoyl groups will be close to one another in the core of the micelles, and the photo dimerization will be more favorable than that of free cinnamic acid. 3.3. Characterization of liposomes Table 1 shows the fluorescence quenching degree calculated by Eq. (2), the zeta potential, and the mean diameter of liposome (100:0), liposome (100:2), and liposome (100:20). The fluorescence quenching degree of calcein entrapped in liposome (100:0), liposome (100:2), and liposome (100:20) was 74.5%, 74.4%, and 68.6%, respectively. The quenching degree of calcein entrapped in liposome (100:20) was somewhat less than those of calcein entrapped in the other liposomes, indicating that the former liposome was formed less in number than the latter liposomes. As described previously, CP F127 is a surface-active polymer so it can form a mixed micelle with EPC. This may account for why the liposome containing a higher amount of CP F127 (liposome (100:20)) exhibited a lower fluorescence quenching. It was
Table 1 Fluorescence quenching degree, the zeta potential, and the mean diameter of liposome (100:0), liposome (100:2), and liposome (100:20). Weight ratio of EPC:CP F127
100:0
50:1
5:1
Quenching degree (%) Zeta potentials (mV) Mean diameter (nm)
74.5 20.3 1.8 185.8
74.4 13.4 4.3 120.5
68.6 8.7 2.4 1113.7
reported that the inclusion of a surface-active polymer in the liposome preparation led to a lower quenching degree (Seo and Kim, 2011, 2012; Seo et al., 2013). The zeta potential of liposome (100:0), liposome (100:2), and liposome (100:20) was 20.3 mV, 13.4 mV, and 8.7 mV, respectively. The liposomes containing CP F127 exhibited less negative potentials than the liposome free of the polymer. This is possibly because CP F127 immobilized on the liposomal surface can shift the hydrodynamic plane away from the surface. It was reported that the zeta potential of liposome of which the surface is decorated with a non-ionic hydrophilic polymer showed a less zeta potential than bare liposome owing to the steric effect of the polymer (Liu et al., 2003). The mean diameter of liposome (100:0), liposome (100:2), and liposome (100:20) was 185 nm, 120 nm, and 1113 nm, respectively. The liposome containing a higher amount of CP F127 (liposome (100:20)) was much bigger in the mean diameter than the other liposomes. The layer of CP F127 immobilized on the liposome would contribute to an increase in the mean diameter. One of the major reasons would be the agglomeration of the liposomal particles. Since the absolute value of the zeta potential of liposome (100:20) was relatively small (e.g. 8.7 mV), the agglomeration of liposomal particles can readily take place due to the lack of an inter-particular repulsive force. And, the amount of CP F127 immobilized on the surface of liposome (100:20) is thought to be high enough for the polymer chains to inter-particularly interact each other through the hydrophobic interaction of PPO blocks. Another reason would be the fusion of liposomes. PEO is known to be fusogenic for a biological membrane (Aldwinckle et al., 1982; Barauskas et al., 2010). Thus, Pluronic F127, which contains PEO blocks, could help the liposomes to fuse each other. Fig. 4 shows the TEM photos of liposome (100:0), liposome (100:2), and liposome (100:20). Multi-lamellar vesicles were observed with all three kinds of liposome. Liposome (100:0) and liposome (100:2) were a few hundreds of nanometers in diameter. Some of liposome (100:20) seems to be fused and they were obviously bigger than the other liposomes. As described previously, Pluronic F127 may help the liposomal particles to fuse each other. 3.4. Light- and temperature-responsive release from liposomes incorporating CP F127 Fig. 5A shows the calcein release profiles from liposome (100:0), liposome (100:2) and liposome (100:20) without UV irradiation. There was no significant release for 180 min with all three kinds of liposome. Fig. 5B shows the calcein release profiles from liposome (100:0), liposome (100:2), and liposome (100:20) with UV irradiation (254 nm, 6 W). Liposome (100:0) exhibited no significant release for 180 min under the UV irradiation. However, liposome (100:2) released their content in response to the UV irradiation. The cinnamoyl group of CP F127 was readily dimerized under the irradiation of UV light (Fig. 3). The dimerization of cinnamoyl group is believed to perturb the liposomal membrane and trigger the release, since the cinnamoyl group will be incorporated in the liposomal membrane due to a hydrophobic interaction. It was reported that the photo dimerization of photoreactive compounds (e.g. coumarin and cinnamic acid) and the photo de-dimerization of the dimers caused the release of water soluble dyes from liposomes (Seo and Kim, 2011, 2012; Seo et al., 2013). The release increased in a saturation manner with the irradiation time and no significant release took place after 90 minUV irradiation. This is possibly because the dimerization degree of cinnamoyl group increased in a saturation manner (Fig. 3). The release profile of liposome (100:20) resembled that of liposome (100:2). However, the release degree of the former liposome was almost two times higher than that of the latter liposome. If a higher amount of CP F127 is included in liposome, it is likely to perturb the
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Fig. 4. TEM photos of liposome (100:0) (A), liposome (100:2) (B), and liposome (100:20) (C). The scale bars are 200 nm.
liposomal membranes more efficiently because a higher amount of photo dimerizable group is in the liposome. Fig. 6A shows the release profiles of liposome (100:0) at 10 C, 20 C, 30 C, and 37 C. No marked release was observed for 180 s at all the temperatures tested. Liposome is known to release their content markedly at the membrane phase transition temperature (Grüll and Langereis, 2012; Wang and Kim, 2013, 2014). Liposomal
membrane, phospholipid bilayer, undergoes a solid gel to liquid crystal transition at a certain temperature. Since the phase transition temperature of EPC liposomal membrane is in the range of 5.8 C–1.5 C (Marsh, 1992; Koynova and Caffrey, 1998), the liposome will not undergoes the phase transition in the full range of temperature tested. Fig. 6B shows the release profiles of liposome (100:20) at 10 C, 20 C, 30 C, and 37 C. When the
Fig. 5A. Calcein release profiles from liposome (100:0) (*), liposome (100:2) () and liposome (100:20) (!) without UV irradiation.
Fig. 5B. Calcein release profiles from liposome (100:0) (*), liposome (100:2) (), and liposome (100:20) (!) with UV irradiation (254 nm, 6 W).
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micellization and the gellation of Pluronic F127 can take place on the surface of liposome, and the phase transitions may impose a mechanical stress on the liposomal membrane, triggering the release. Another reason for the triggered release is possibly due to the hydrophobic interaction between Pluronic F127 chains and the liposomal membrane. The dehydrated and hydrophobicized PPO blocks at a higher temperature could hydrophobically interact with liposomal membranes, giving a rise to the triggered release. 4. Conclusion
Fig. 6A. Release profiles of liposome (100:0) at 10 C (solid line), 20 C (dotted line), 30 C (dash-dotted line), and 37 C (dash line).
temperature of release medium was 10 C, there was no significant release for 180 min. When the temperature was 20 C, a marked release took place and it was completed in a few seconds, and the maximum release degree was around 20%. The maximum release degree was higher at a higher temperature. For example, when the release temperature was 30 C and 37 C, the maximum release degree was about 27% and 45%, respectively. As the temperature increases, the PPO blocks of Plurionic F127 in an aqueous phase become dehydrated and the hydrophobic interaction among the blocks will increase. As a result, the polymer chains are assembled into micelles at a certain temperature and the solution becomes a gel at a higher temperature. The micellization temperature of Pluronic F127 in an aqueous solution ranges from 8 C to 24 C, and the gellation temperature is from 20 C to 29 C depending on the concentration (Chandaroy et al., 2002; Pisal et al., 2004; Jiang et al., 2008). The
Liposome which releases its water-soluble payload in response to a UV irradiation and a temperature change were prepared by decorating the surface of egg phosphatidylcholine liposomes with CP F127. The cinnamoyl group to Pluronic F127 molar ratio of CP F127 was calculated to be about 1:1.4 on 1H NMR spectrum. The immobilization of CP F127 on the liposomal membrane resulted in a decreased absolute value of the zeta potential of liposome possibly due to its steric effect. And, it led to an increased size possibly due to the fusion caused by the fusogenic effect of CP F127 and due to the aggregation caused by the low zeta potential and the inter-particular interaction of CP F127 chains. The liposome was UV light-sensitive in terms of release. The photo-dimerization of cinnamoyl group of CP F127 is believed to perturb the liposomal membrane and trigger the release. And the liposome was also temperature-sensitive. The phase transition of Pluronic F127 layer on the liposomal surface or the hydrophobic interaction of the polymer chains with liposomal membrane would account for the temperature-sensitive release. Light- and temperature-responsive liposomes developed in the present study could be used as a drug carrier which releases its content in a controlled manner in response to UV irradiation and temperature. Conflict of interest We confirm that there are no conflicts of interest associated with this publication. Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2007898). References
Fig. 6B. Release profiles of liposome (100:20) at 10 C (solid line), 20 C (dotted line), 30 C (dash-dotted line), and 37 C (dash line).
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