- Email: [email protected]
Preparation, microstructure and function of liposome with light responsive switch
Accepted Manuscript Title: Preparation, Microstructure and Function of Liposome with Light Responsive Switch Authors: Yangyang Liu, Xueqin An PII: DOI: Reference:
S0927-7765(18)30762-8 https://doi.org/10.1016/j.colsurfb.2018.10.068 COLSUB 9752
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
1-6-2018 14-9-2018 24-10-2018
Please cite this article as: Liu Y, An X, Preparation, Microstructure and Function of Liposome with Light Responsive Switch, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.10.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation, Microstructure and Function of Liposome with Light Responsive Switch
IP T
Yangyang Liu and Xueqin An*
School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China *
SC R
Corresponding author, E-mail: [email protected]
CC E
PT
ED
M
A
N
U
Graphical Abstract
Highlights
A
Liposome was prepared by combined method with supercritical carbon dioxide technique
Microstructure of liposomes was proved by fluorescence probe embedded in liposome
Liposome photo-responsiveness was studied by microstructure and the macro property
Drug release in liposomes was controlled by altering UV and vis light irradiation
1
Abstract
A novel liposome with photo-responsiveness was prepared by a combined method of thin film hydration and supercritical carbon dioxide technique. In liposomes, curcumin and 4butylazobenzene-4-hexyloxy-trimethyl-ammoniumtrifluoro-acetate (BHA) were used as model drug and photo-responsive reversible switch, respectively. Optimum incubation
IP T
temperature of drug was determined by using phase transition temperature of the liposomes. Morphology and size of liposomes were obtained by transmission electron microscope and
dynamic light scattering technology. Microstructure of liposomes was explored by fluorescent
SC R
probes, and it was found that the BHA was encapsulated in polar groups of phospholipid in the bilayer of liposomes. Light responsiveness and reversibility of BHA in liposomes were
studied from the viewpoint of the microstructure and the macro property. It was shown that the reversibility of structural isomerization of BHA can be used as photo-responsive switch to
N
U
control drug release.
M
A
Abbreviations:
ED
thin film hydration and supercritical carbon dioxide technique (TE-scCO2) 4-butylazobenzene-4-hexyloxy-trimethyl-ammoniumtrifluoro-acetate (BHA)
PT
phosphatidyl choline (PC)
BHA-liposomes (BHA-lipo)
CC E
BHA-curcumin-liposomes (BHA-cur-lipo)
A
Keywords: liposomes; light responsive switch; microstructure; drug delivery; control release; reversibility of structural isomerization
1 Introduction
Liposomes are microscopic spherical vesicles composed of lipids like phospholipids, and hydrophilic materials can be entrapped within the aqueous interior and hydrophobic materials can 2
be bound to the bilayer membrane[1]. Liposomes with high transfection efficiencies, good biological compatibility and high encapsulation efficiency[2, 3] might serve as a depot for sustained and controlled release of dermally active compounds[4]. Functional liposomes also can be used for controlled drug release, fluorescent imaging and sensing and so on[2, 5, 6]. It has been utilized in pharmaceutical industry, such as drug delivery in transdermal systems and targeted therapy for cancer[7, 8]. There are many traditional methods to prepare liposomes, such as thin film hydration and ethanol injection. However, there are some shortages for liposomes prepared by traditional
IP T
methods, such as bad monodispersion, poor stability, high residual organic solvent, side effects etc[9]. These disadvantages limit their applications in biomedicine fields. Supercritical carbon
dioxide has the characteristics of high density, high solubility, high mass transfer rate and nontoxicity, which has been used in the preparation of liposomes[10]. Liposomes prepared by using
SC R
combined method of thin film hydration and supercritical carbon dioxide technology have high encapsulation efficiency, good stability, low side effects and low residual solvent[11].
Recently, controlling drug release in the liposomes has attracted much attention. Liposomes
U
modified with corresponding sensitive response materials have been designed to release their contents in response to enzyme, pH, temperature, magnetic and light. [9]. Aptamer-enzyme
N
conjugated liposome can trigger stimuli-responsive release by destroying the phospholipid chain of
A
lecithin[12, 13]. Hemisuccinate was commonly used as a pH-sensitive complementary molecule[14]. Drug in liposomes encapsulated magnetic nanoparticles can be released by
M
controlling magnetic field intensity, upon exposure to an alternating current electromagnetic field[15, 16]. The cargo in thermal stimulus response liposomes can be released by a process of gel-
ED
to-liquid crystal transition [17]. In the stimulus response liposomes, drug release in liposomes by the light stimulation has a number of advantages over other stimulations, including no secondary pollution, low toxicity, non-invasive energy source etc. Drug release in liposomes with light-
PT
sensitive materials, such as organic polymers[18-20] and inorganic nanocomposites[21], has already been done by using light irradiation. Recently, a photosensitive materials, 4-butylazobenzene-4hexyloxy-trimethyl-ammoniumtrifluoro-acetate (BHA) was designed and synthesized[22]. BHA
CC E
contained stimuli-responsive azobenzene unit can achieve structure isomerization under different light radiation conditions, and it showed reversible transition between trans and cis form by UV and visible light irradiation.[20]. BHA has advantages of providing a broad range of adjustable parameters, such as wavelength, duration, intensity and so on. Additional, cationic amphipathic
A
compound BHA has similar structure with phosphatidyl choline (PC) and much easier inserted into the bilayers of the liposomes[23]. The aim of this study was to design stable liposomes with intelligent photo-responsive function for drug delivery system. The photo responsive BHA-liposomes were prepared by a combined method of thin film rehydration and supercritical carbon dioxide technology (TE-scCO2). BHA as photo-responsive material and curcumin as model drug were encapsulated in the bilayer of liposomes. Physicochemical characteristics such as size, morphology and zeta potential were 3
investigated. The microstructure of liposomes was studied by using pyrene and DPH as fluorescent probes. Photo-isomerization of BHA in the liposome was studied, and the reversibility of structural heterogeneity was used as photo-responsive switch. Curcumin in the liposome was released by isomerization of BHA in the bilayer of liposomes at UV and visible light irradiation alternately.
2 Experiments
IP T
2.1 Materials Egg phosphatidyl choline (PC) was from East China Normal University chemical factory
and curcumin was purchased from Macklin Inc. Diphenylhexatriene (DPH) and pyrene were purchased from Sigma-Aldrich Inc. And main material 4-butylazobenzene-4-hexyloxy-
SC R
trimethyl-ammoniumtrifluoro-acetate (BHA) is synthesized by a series of synthetic experiments. All other organic reagents were the best grade commercially available. 2.2 Liposome preparation
U
Liposomes were prepared by a combined method of thin film hydration and supercritical carbon dioxide technology (TE-scCO2). Briefly, egg phosphatidyl choline (20 mg) was
N
dissolved in mixture solution (chloroform: methanol = 3:1 total 20 mL). The organic solvent
A
was removed under a vacuum rotary evaporator in a dark environment. The thin lipid film formed on the wall of flask was hydrated with double distilled water and incubated in CO 2
M
autoclave at temperature of 40℃ and pressure of 20 MPa for an hour[11]. The liposomal solution was achieved by self-assembly and remaining CO 2 in the liposome was removed at
ED
room temperature and pressure, finally a transparent liposome solution was obtained. For preparation of BHA-lipo, EPC was replaced by mixture of EPC and BHA, and other process were the same as above. Free BHA in the BHA-lipo solution was removed by centrifugation
PT
and the absorbance of BHA solution was obtained by UV-Vis absorption spectrum. In the process of BHA-cur-lipo preparation, EPC was replaced by mixture of EPC, cur and BHA.
CC E
2.3 Characterizations
The size distribution and zeta potential of these liposomes was determined by a dynamic
light scattering instrument equipped with a computer controlled image analysis system. The
A
morphology of liposomes was observed using transmission electron microscope (TEM). 2.4 Photo-isomerization of BHA-lipo solution The BHA exhibited a trans to cis shift at UV and visible light irradiation, and the
isomerization process of BHA-lipo was recorded by using a UV-vis spectrophotometer. The UV absorption of BHA in liposome solution was observed by the spectra, and the changes of wavelength and absorption were achieved by the altering of UV and vis light. 2.5 Liposome microstructure 4
Diphenylhexatriene (DPH) was used to explore the membrane fluidities of the BHA-lipo. The fluorescent anisotropy value of DPH in the bilayer membranes was measured by fluorescent spectrophotometer. Fluorescent probe pyrene was used to prove that BHA and curcumin were encapsulated in the bilayers of liposome. In this method, incorporation of BHA to membranes containing pyrene may lead to polarity changing of environment around pyrene, which could be indicated by the structure of the pyrene monomer emission spectrum. According to the peaks at 373 nm (I) and 384 nm (III), it was found that the ratio II/IIII
2.6 Light-responsive release from liposomes incorporating BHA
IP T
determined the micropolarity of the particles.
As a kind of drug carrier, liposome can not only loaded hydrophilic drugs, but hydrophobic
SC R
drugs. The controlled release behavior for the designed BHA-lipo composite vesicles could
be triggered by photo irradiation directly. Free BHA was removed by centrifugation and the amount of encapsulation drug was determined by the UV-vis absorption spectrum and calculated by absorbance of curcumin, according to the following eq1：
U
Encapsulation efficiency (%) = ( Wencapsulated)/(Wtotal)*100
(1)
The drug release behavior in vitro was studied at room temperature. The designed
N
composite vesicles loaded with the curcumin were loaded into dialysis bag (MOCW 14KDa)
A
and immersed into 5 mL, 0.1 % sodium dodecyl sulphate (SDS) solution. The amount of drug was determined by fluorescent spectrum. Release rate was calculated according to the
M
following eq2：
Release (%) = (W r e l e a s e /W e n c a p s u l a t e d )*100
(2)
release, respectively.
ED
Wtotal Wencapsulated and Wrelease were the amount of total drug, encapsulated drug and drug
PT
3 Results and discussion
CC E
3.1 Preparation of liposomes BHA-curcumin-liposomes (BHA-cur-lipo) were prepared by a combined method of thin
film hydration and supercritical carbon dioxide technique (TE-scCO2)[11]. The major influencing factors for preparation of BHA-cur-lipo are temperature, pressure and molar ratio of BHA to phosphatidyl choline (PC). Encapsulation efficiency (EE) of curcumin in
A
liposomes was used to evaluate the parameters of preparation condition. The EE of curcumin in liposomes changed with preparation temperature at pressure of 20 MPa, as shown in Fig1 (a). The EE of curcumin in liposomes increased with temperature when preparation temperature was below 40 ℃, and it decreased with temperature when temperature was above 40 ℃. Therefore, 40 ℃ should be considered as optimum temperature. It is well known that there existed a phase transition temperature (Tc) in transition process from gel to liquid crystalline for liposomes[24]. PCs in the bilayer of liposomes were closely aligned 5
when the temperature was below Tc, but PCs were loosely aligned when the temperature was above Tc. In order to further theoretically prove why the optimum incubation temperature is 40 ℃. The temperature of Tc for BHA-cur-lipo was obtained by micro-DSC measurements as shown in Fig1 (b), it was about 42 ℃ which close to the optimal incubation temperature[19]. When the temperature was below Tc, the close arrangement of PC resulted in poor fluidity and permeability of liposome bilayer, which made it difficult for curcumin to enter liposome through bilayer. However, the distance enlargement between adjacent PC
IP T
molecules increased when temperature was close to Tc, which resulted in increase of fluidity and permeability of liposome membranes, and the curcumin was easy to get into the bilayer
of liposomes. This result may explain the optimum incubation temperature of 40 ℃. Effect of scCO2 pressure on EE of curcumin in liposomes was probed at incubation temperature 40
SC R
℃, and relationship of the EE with pressure was demonstrated in Fig1(c). The EE of
curcumin increased with the scCO 2 pressure when the pressure was below 20 MPa, and it was almost constant when the pressure was above 20 MPa. It indicated that curcumin was encapsulated into the layer of liposomes when the pressure was below 20 MPa, and the
U
curcumin was almost saturated in bilayer of liposome at the pressure above 20 MPa.
N
Therefore, the pressure of 20 MPa was determined as optimal value for preparation of
CC E
PT
ED
M
A
liposomes.
Figure1. Selection of optimum parameters in the preparation method (a) Curcumin encapsulation changes
A
with temperature at the fixed scCO2 pressure (20 MPa); (b) Micro-DSC heating curve for BHA-cur-lipo at a scanning rate of 1 ℃/min; (c) Curcumin encapsulation changes with the scCO2 pressure at the fixed
temperature (40 ℃); (d) Relation between absorbance of BHA in the BHA-lipo and molar ratio of BHA to PC
In order to select the optimal composition of liposomes, BHA-lipo with various molar ratios of BHA to PC were prepared. Absorbance of the BHA-lipo solution with various molar ratios was obtained (Fig1 (d)). The absorbance of BHA-lipo solution increased with the 6
increasing of the concentration of the BHA. When the molar ratio was above 0.02, it was nearly constant. It was suggested that best molar ratio of BHA to PC should be 0.02, and inserted BHA could be saturated in the bilayer of the liposomes. 3.2 Morphology and Size distribution The morphology and size of BHA-cur-lipos by different methods were determined by transmission electron microscope (TEM) as shown in Fig. 2a and 2b. The BHA-cur-lipo is spherical nanoparticles with a typical vesicle structure, and there existed some multichamber
IP T
near the edge of liposomes, which suggested the existence of multi-lamellar structures. The size of liposomes prepared by different methods was determined by dynamic light scattering
technology (DLS). It was found that the BHA-cur-lipo prepared by TE-scCO2 have good
SC R
monodispersion, and the average size was about 140±15 nm. Whereas, the BHA-lipo prepared by traditional film hydration showed polydispersity, which has two main peaks, their average sizes were 100±20 nm and 830±23 nm, respectively. The reason may be that lipid molecules in the liposomes are rearranged by the self-assembling process in scCO2
U
micro-environment, and which is easy to form stable liposomes with monodispersion. This result indicated that the TE-scCO2 method is better than thin film hydration for preparation
A
CC E
PT
ED
M
A
N
of BHA-lipo.
Figure2. The TEM of BHA-cur-lipo (prepared by thin film hydration (a) and TE-scCO2 (b)); (c) the size intensity distribution of BHA-cur-lipo
3.3 Stability of liposomes Surface charge of liposomes is an important indication for the stability. The zeta potential of liposomes was obtained by a Malvern Zetasizer Nano ZS (UK) at temperature of 25 ℃. 7
It was about -23 mv and +28 mv for control-lipo and BHA-lipo, respectively. The zeta potential of control liposomes is negative (about -23 mv) because the liposome comprised of negatively charged PC. The zeta potential of BHA-lipo is positive (28 mv) because positive BHA with positive charge has been inserted the bilayer of the liposomes[16]. It prove that BHA has been successfully insert into the bilayer of liposome, and majority of BHA was encapsulated in the bilayer of liposomes and the portion of positive polar groups of BHA may be exposed to the surface of liposomes. In order to probe relationship of BHA
IP T
to the zeta potential, the zeta potentials for BHA-lipo with various BHA concentrations were determined as shown in Fig 3(a). The zeta potential was almost unchanged when the concentration was below 0.8 mM, but it increased with the concentration of BHA from 24
to 28 mv when the concentration was above 0.8 mM. It indicated that cationic surfactant
SC R
BHA was inserted into bilayer of liposomes and banded with the head groups of PC. The results indicated that BHA was banded to liposomes in two different ways in the binding process, one was interpolation (inserted into bilayer of the liposomes) and other was adsorption on the surface of the liposomes. For the retaining of BHA in the liposomes, the
U
former was better than the latter, and the retaining of BHA in the liposomes was decreased
PT
ED
M
A
N
with reducing of BHA concentration in the solution in the adsorption.
Figure3.(a) Zeta potential of BHA-lipo changes with the concentration; (b) Transmittivity of BHA-lipo
CC E
changes with time.
Stability of BHA-cur-lipo systems was evaluated by monitoring turbidity of liposomes
with time. It was found that the transmissivity of BHA-cur-lipo was nearly invariable even after six month at the environmental temperature of 4℃,as shown in Fig 3(b). The stability
A
of liposomes is beneficial to the storage and application of liposomes in drug carrier. 3.4 Microstructure of BHA-lipo Microstructure of liposomes will provide important information for their stability, EE, drug release and so on[25] [26, 27]. Fluorescence probes were used to explore the membrance fluidities and micropolarity of liposomes, respectively. Diphenylhexatriene (DPH) as a fluorescence probe was located in the lipid bilayers for both systems of control liposome 8
and BHA-lipo. The fluorescence anisotropy values of DPH in control liposome and BHA liposomes were 0.13 ± 0.01 and 0.16 ± 0.01(n=4), respectively, as shown in Fig4 (a). Anisotropy value of 0.13 for the control liposome was lower than that of 0.16 for BHA-lipo. This means that the membrance fluidity of the control liposome is higher than that of the BHA liposome, because the original structure in the bilayer was destroyed due to insertion of BHA. A similar phenomenon has been reported when hydrophobic nanoparticles was inserted in bilayer of liposomes[17]. The above results also evidence that BHA was encased
N
U
SC R
IP T
in the bilayer of the liposome.
A
Figure4. (a) The anisotropy value of control-lipo and BHA-lipo; (b) The value of II/IIII of BHA-lipo with
M
different molar ratio of BHA to PC
Pyrene, a fluorescent probe, was embedded in the bilayer of liposomes to explore the
ED
membrance micropolarity. The pyrene monomer fluorescence spectrum exhibits significant fine structure in the form of five predominant peaks. Peak I (II, at 374 nm) shows significant
PT
intensity enhancement when the environment is more polar, whereas peak III (IIII, at 384 nm) shows minimal intensity variation with polarity. Thus, the intensity ratio of II/IIII is an empiric measure of polarity[11]. The II/IIII value of control-lipo and BHA-lipo was 1.12±
CC E
0.03 and 0.79 ± 0.03, respectively. It indicates that the micropolarity of BHA-lipo was much smaller than control liposomes. This may be caused by introducing BHA in liposomes and changing the microstructure of bilayer in the BHA-lipo. Moreover, from Fig4 (b), the value of II/IIII decreased with the molar ratio of BHA to PC when the ratio was below 0.02. It means
A
that with the increasing of BHA the lipid layer is tighter and more nonpolar [28]. It decreases slightly with the ratio of BHA from 0.02 to 0.03. BHA has been saturated in the lipid layer and it would bring little effects on the lipid layer[26]. The capacity of phospholipid layer was limited and there is an optimum loading capacity with BHA to PC ratio is 0.02. 3.5 Photo-isomerization of BHA-liposomes Azobenzene group of BHA can undergo reversible trans-cis isomerization (Fig 5 (a)) under ultraviolet (365 nm) and visible light irradiation[22], and the trans- and cis9
isomerization in the BHA-liposomes are shown in Fig. 5(b). Photo-isomerization of BHAlipo was studied and the UV-Vis spectrum is presented in Fig5 (c). The absorption peak of BHA-lipo was 348 nm (black line in the Fig 5 (c)) under irradiation of visible light, which may resulted from the π → π* transition[12]. After irradiation of UV light, the intensity of the band at 348 nm decreased, whereas two absorption peaks were found at wavelengths of 315 and 440 nm (the pink line in the Fig 5 (c)). The two peaks appear due to the n → π* transition of cis-isomer. The above reversible processes could be occurred by alternating
IP T
irradiation of the visible and ultraviolet light, and the process can be repeated for many times as shown in Fig5 (d). The phenomenon indicates that the reversible trans-cis isomerization
ED
M
A
N
U
SC R
of BHA by different light irradiation could be utilized as photo switch to control drug release.
Figure 5. (a) Chemical structure and photo-isomerization of BHA; (b) The microstructure schematic of the
PT
trans-BHA-liposomes and cis-BHA-liposomes; (c) UV-Vis spectra of BHA-lipo solution at different states (initial state black line for trans-BHA and pink line for cis-BHA); (d) Changes of absorbance at 348 nm
CC E
after altering UV and visible light for cycles in BHA-lipo; (e) The II/IIII value of BHA-lipo with time after irradiating with UV and visible light alternatively
To further explore relationship of BHA-lipo microstructure to BHA isomerization in
liposome, II/IIII value of pyrene encapsulated in the liposome was obtained under altering
A
irradiation of UV and visible light. The II/IIII values were 0.81±0.05 and 1.21±0.04 under irradiation of visible and UV light, respectively. The change of II/IIII value was attributed to the isomerization of BHA in liposomes, and that resulted in alteration of the bilayer microstructure in liposomes[25]. The values also can be repeated for many times under UV and visible light irradiation alternately as shown in Fig 5(e). The results from liposome microstructure (fluidities and micro polarity achieved by fluorescence probe DPH and pyrene) and spectrum can prove the photo-isomerization of BHA in the liposome, which could be used as photo-sensitive switch to drug release in the liposomes. 10
3.6 Encapsulation efficiency Curcumin as hydrophobic model drug was encapsulated in the BHA-lipo, and free curcumin was removed by centrifugation. The concentration of curcumin can be obtained by absorbance measurement. Encapsulation efficiency (EE) of curcumin was calculated by eq. 1 (in experience section) and which was up to 88%. The encapsulation efficiency of hydrophobic drug was similar to those reported previously[29].
IP T
3.7 Drug release of liposomes To explore the role of BHA as a photo nanoswitch for drug release, the curcumin
encapsulated in liposomes was released at room temperature by UV irradiation. Curcumin
SC R
release from BHA-cur-lipo either in darkness or upon UV light irradiation for 10 min was
studied. The result was shown in Fig 6 (a). Curcumin in the BHA-cur-lipo was difficult to spontaneous release in dark (black line in Fig 6(a)). But the curcumin from BHA-cur-lipo was quickly released under UV irradiation and nearly 90% curcumin was released f in six
U
hours (the red line in Fig 6(a)). The results demonstrated that BHA should be potential
PT
ED
M
A
N
nanoswitch with photo stimulation to control release drug from the liposomes[30].
CC E
Figure6. (a) The curcumin was released from the liposomes in the dark (black line) and under UV
A
irradiation (red line); (b) The accumulated release of curcumin from BHA-cur-lipo under irradiation alternately by the UV and vis light.
As further proof of BHA as a photo-sensitive switch in liposomes, commutative release
of curcumin encapsulated in lipid was undertaken by repetitious irradiation with UV light (10 min) and visible light (40 min) as shown in Fig6 (b). However, after UV irradiation for 10 min, the curcumin release amount increased obviously. With UV stimulus, the BHA was compelled from trans to cis form and open the switch. Then visible light irradiated for 40min, optical energy was absorbed by BHA and changed configuration and phospholipid arrangement. It revealed that the amount and rate of the released curcumin could be 11
controlled by altering the UV and vis light irradiation[6]. The trans-cis isomerization of the BHA-cur-lipo can induce defects in bilayers that lead to the release of the entrapped payload. The results also reveal that BHA in liposomes as a “on-off” switch for controlling drug release[16]. This means that the drug release from liposomes can be controlled by UV and visible light irradiation alternatively.
IP T
4 Conclusions BHA, as a kind of photo-responsive compounds was synthetized by series of reactions. BHA, PC and curcumin were self-assembly into liposomes by a combined method of thin film hydration and supercritical carbon dioxide technique (TE-scCO2). Liposome is spherical nanoparticles with a
SC R
typical vesicle structure. The difference microstructures of various liposomes were proved by the
values of anisotropy obtained from the fluorescence polarization technique. The results reveal that BHA was inserted between PC polar groups. BHA in the phospholipid layer can be isomerized under light irradiation, and the isomerization of BHA can be repeated many times. The isomerization of
N
U
BHA in the bilayer of the liposomes could be used as a switch to control drug release.
M
A
Acknowledgements
This research was supported by the National Natural Science Foundation of China
PT
References
ED
(21473055 and 21773064).
CC E
[1] A. Puri, K. Loomis, B. Smith, J.-H. Lee, A. Yavlovich, E. Heldman, R. Blumenthal, Lipid-based
nanoparticles as pharmaceutical drug carriers: from concepts to clinic, Crit. Rev. Ther. Drug Carrier Syst., 26 (2009) 523-580.
[2] A. Samad, Y. Sultana, M. Aqil, Liposomal drug delivery systems: an update review, Curr. Drug
A
Delivery, 4 (2007) 297-305. [3] P. Tanner, P. Baumann, R. Enea, O. Onaca, C. Palivan, W. Meier, Polymeric Vesicles: From Drug
Carriers to Nanoreactors and Artificial Organelles, Acc. Chem. Res., 44 (2011) 1039-1049. [4] B.S. Pattni, V.V. Chupin, V.P. Torchilin, New Developments in Liposomal Drug Delivery, Chem. Rev. (Washington, DC, U. S.), 115 (2015) 10938-10966. [5] C. Flors, M.J. Fryer, J. Waring, B. Reeder, U. Bechtold, P.M. Mullineaux, S. Nonell, M.T. Wilson, N.R. Baker, Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green, J. Exp. Bot., 57 (2006) 1725-1734. [6] X. An, F. Zhang, Y. Zhu, W. Shen, Photoinduced drug release from thermosensitive AuNPs12
liposome using a AuNPs-switch, Chemical communications, 46 (2010) 7202-7204. [7] J. Khandare, M. Calderon, N.M. Dagia, R. Haag, Multifunctional dendritic polymers in nanomedicine: opportunities and challenges, Chem. Soc. Rev., 41 (2012) 2824-2848. [8] Y.I. Yoon, Y.S. Kwon, H.S. Cho, S.H. Heo, K.S. Park, S.G. Park, S.H. Lee, S.I. Hwang, Y.I. Kim, H.J. Jae, G.J. Ahn, Y.S. Cho, H. Lee, H.J. Lee, T.J. Yoon, Ultrasound-mediated gene and drug delivery using a microbubble-liposome particle system, Theranostics, 4 (2014) 1133-1144. [9] D.R. Khan, E.M. Rezler, J. Lauer-Fields, G.B. Fields, Effects of drug hydrophobicity on liposomal stability, Chemical biology & drug design, 71 (2008) 3-7. [10] M. Kamihira, M. Taniguchi, T. Kobayashi, Sterilization of Microorganisms with Supercritical
IP T
Carbon Dioxide, Journal of the Agricultural Chemical Society of Japan, 51 (2016) 407-412.
[11] L. Li, X. An, A novel combined method of thin-film evaporation and a supercritical carbon dioxide technique to prepare a fluorescent siRNA-liposome, RSC Advances, 6 (2016) 92115-92119.
[12] Z.K. Cui, T. Phoeung, P.A. Rousseau, G. Rydzek, Q. Zhang, C.G. Bazuin, M. Lafleur,
SC R
Nonphospholipid fluid liposomes with switchable photocontrolled release, Langmuir : the ACS journal of surfaces and colloids, 30 (2014) 10818-10825.
[13] P. Walde, S. Ichikawa, Enzymes inside lipid vesicles: preparation, reactivity and applications, Biomol. Eng., 18 (2001) 143-177.
U
[14] J. Li, Z. Yu, H. Jiang, G. Zou, Q. Zhang, Photo and pH dual-responsive polydiacetylene smart nanocontainer, Materials Chemistry and Physics, 136 (2012) 219-224.
N
[15] K. Elersic, J.I. Pavlic, A. Iglic, A. Vesel, M. Mozetic, Electric-field controlled liposome formation with embedded superparamagnetic iron oxide nanoparticles, Chem Phys Lipids, 165 (2012) 120-124.
A
[16] D. Qiu, X. An, Controllable release from magnetoliposomes by magnetic stimulation and thermal
M
stimulation, Colloids and Surfaces B: Biointerfaces, 104 (2013) 326-329. [17] X. Yan, X. An, Thermal and photic stimuli-responsive polydiacetylene liposomes with reversible fluorescence, Nanoscale, 5 (2013) 6280-6283.
ED
[18] A. Diguet, M. Yanagisawa, Y.J. Liu, E. Brun, S. Abadie, S. Rudiuk, D. Baigl, UV-induced bursting of cell-sized multicomponent lipid vesicles in a photosensitive surfactant solution, Journal of the American Chemical Society, 134 (2012) 4898-4904.
PT
[19] M. Karimi, A. Ghasemi, P. Sahandi Zangabad, R. Rahighi, S.M. Moosavi Basri, H. Mirshekari, M. Amiri, Z. Shafaei Pishabad, A. Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A. Vaseghi, A.R. Aref, L. Haghani, S. Bahrami, M.R. Hamblin, Smart micro/nanoparticles in stimulus-responsive drug/gene delivery
CC E
systems, Chemical Society reviews, 45 (2016) 1457-1501. [20] F. Ercole, T.P. Davis, R.A. Evans, Photo-responsive systems and biomaterials: photochromic
polymers, light-triggered self-assembly, surface modification, fluorescence modulation and beyond, Polym. Chem., 1 (2010) 37-54. [21] J. Li, X. An, Z. Pan, l. Sun, Photoinduced drug release from complexes of liposome and fluorescent
A
silver nanoparticles, RSC Adv., 4 (2014) 9476-9479. [22] S. Shi, T. Yin, X. Tao, W. Shen, Light induced micelle to vesicle transition in an aqueous solution
of a surface active ionic liquid, RSC Advances, 5 (2015) 75806-75809. [23] S. Krasnici, A. Werner, M.E. Eichhorn, M. Schmitt-Sody, S.A. Pahernik, B. Sauer, B. Schulze, M. Teifel, U. Michaelis, K. Naujoks, M. Dellian, Effect of the surface charge of liposomes on their uptake by angiogenic tumor vessels, Int. J. Cancer, 105 (2003) 561-567. [24] X. An, F. Zhan, Y. Zhu, Smart photothermal-triggered bilayer phase transition in AuNPs-liposomes to release drug, Langmuir : the ACS journal of surfaces and colloids, 29 (2013) 1061-1068. 13
[25] D. Ke, X. Wang, Q. Yang, Y. Niu, S. Chai, Z. Chen, X. An, W. Shen, Spectrometric study on the interaction of dodecyltrimethylammonium bromide with curcumin, Langmuir : the ACS journal of surfaces and colloids, 27 (2011) 14112-14117. [26] A.B. Dhanikula, R. Panchagnula, Fluorescence anisotropy, FT-IR spectroscopy and 31-P NMR studies on the interaction of paclitaxel with lipid bilayers, Lipids, 43 (2008) 569-579. [27] K. Sugikawa, T. Kadota, K. Yasuhara, A. Ikeda, Anisotropic Self-Assembly of Citrate-Coated Gold Nanoparticles on Fluidic Liposomes, Angewandte Chemie, 55 (2016) 4059-4063. [28] G.E. Savchenko, E.A. Klyuchareva, I.I. Denev, A.P. Stupak, Fluorescence of pyrene in galactolipid liposomes and native membranes of etioplasts, Journal of Applied Spectroscopy, 67 (2000) 473-481.
IP T
[29] N. Saengkrit, S. Saesoo, W. Srinuanchai, S. Phunpee, U.R. Ruktanonchai, Influence of curcuminloaded cationic liposome on anticancer activity for cervical cancer therapy, Colloids and Surfaces B: Biointerfaces, 114 (2014) 349-356.
[30] S. Geng, Y. Wang, L. Wang, T. Kouyama, T. Gotoh, S. Wada, J.Y. Wang, A Light-Responsive Self-
SC R
Assembly Formed by a Cationic Azobenzene Derivative and SDS as a Drug Delivery System, Scientific
A
CC E
PT
ED
M
A
N
U
reports, 7 (2017) 39202.
14
S0927-7765(18)30762-8 https://doi.org/10.1016/j.colsurfb.2018.10.068 COLSUB 9752
To appear in:
Colloids and Surfaces B: Biointerfaces
Received date: Revised date: Accepted date:
1-6-2018 14-9-2018 24-10-2018
Please cite this article as: Liu Y, An X, Preparation, Microstructure and Function of Liposome with Light Responsive Switch, Colloids and Surfaces B: Biointerfaces (2018), https://doi.org/10.1016/j.colsurfb.2018.10.068 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation, Microstructure and Function of Liposome with Light Responsive Switch
IP T
Yangyang Liu and Xueqin An*
School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, China *
SC R
Corresponding author, E-mail: [email protected]
CC E
PT
ED
M
A
N
U
Graphical Abstract
Highlights
A
Liposome was prepared by combined method with supercritical carbon dioxide technique
Microstructure of liposomes was proved by fluorescence probe embedded in liposome
Liposome photo-responsiveness was studied by microstructure and the macro property
Drug release in liposomes was controlled by altering UV and vis light irradiation
1
Abstract
A novel liposome with photo-responsiveness was prepared by a combined method of thin film hydration and supercritical carbon dioxide technique. In liposomes, curcumin and 4butylazobenzene-4-hexyloxy-trimethyl-ammoniumtrifluoro-acetate (BHA) were used as model drug and photo-responsive reversible switch, respectively. Optimum incubation
IP T
temperature of drug was determined by using phase transition temperature of the liposomes. Morphology and size of liposomes were obtained by transmission electron microscope and
dynamic light scattering technology. Microstructure of liposomes was explored by fluorescent
SC R
probes, and it was found that the BHA was encapsulated in polar groups of phospholipid in the bilayer of liposomes. Light responsiveness and reversibility of BHA in liposomes were
studied from the viewpoint of the microstructure and the macro property. It was shown that the reversibility of structural isomerization of BHA can be used as photo-responsive switch to
N
U
control drug release.
M
A
Abbreviations:
ED
thin film hydration and supercritical carbon dioxide technique (TE-scCO2) 4-butylazobenzene-4-hexyloxy-trimethyl-ammoniumtrifluoro-acetate (BHA)
PT
phosphatidyl choline (PC)
BHA-liposomes (BHA-lipo)
CC E
BHA-curcumin-liposomes (BHA-cur-lipo)
A
Keywords: liposomes; light responsive switch; microstructure; drug delivery; control release; reversibility of structural isomerization
1 Introduction
Liposomes are microscopic spherical vesicles composed of lipids like phospholipids, and hydrophilic materials can be entrapped within the aqueous interior and hydrophobic materials can 2
be bound to the bilayer membrane[1]. Liposomes with high transfection efficiencies, good biological compatibility and high encapsulation efficiency[2, 3] might serve as a depot for sustained and controlled release of dermally active compounds[4]. Functional liposomes also can be used for controlled drug release, fluorescent imaging and sensing and so on[2, 5, 6]. It has been utilized in pharmaceutical industry, such as drug delivery in transdermal systems and targeted therapy for cancer[7, 8]. There are many traditional methods to prepare liposomes, such as thin film hydration and ethanol injection. However, there are some shortages for liposomes prepared by traditional
IP T
methods, such as bad monodispersion, poor stability, high residual organic solvent, side effects etc[9]. These disadvantages limit their applications in biomedicine fields. Supercritical carbon
dioxide has the characteristics of high density, high solubility, high mass transfer rate and nontoxicity, which has been used in the preparation of liposomes[10]. Liposomes prepared by using
SC R
combined method of thin film hydration and supercritical carbon dioxide technology have high encapsulation efficiency, good stability, low side effects and low residual solvent[11].
Recently, controlling drug release in the liposomes has attracted much attention. Liposomes
U
modified with corresponding sensitive response materials have been designed to release their contents in response to enzyme, pH, temperature, magnetic and light. [9]. Aptamer-enzyme
N
conjugated liposome can trigger stimuli-responsive release by destroying the phospholipid chain of
A
lecithin[12, 13]. Hemisuccinate was commonly used as a pH-sensitive complementary molecule[14]. Drug in liposomes encapsulated magnetic nanoparticles can be released by
M
controlling magnetic field intensity, upon exposure to an alternating current electromagnetic field[15, 16]. The cargo in thermal stimulus response liposomes can be released by a process of gel-
ED
to-liquid crystal transition [17]. In the stimulus response liposomes, drug release in liposomes by the light stimulation has a number of advantages over other stimulations, including no secondary pollution, low toxicity, non-invasive energy source etc. Drug release in liposomes with light-
PT
sensitive materials, such as organic polymers[18-20] and inorganic nanocomposites[21], has already been done by using light irradiation. Recently, a photosensitive materials, 4-butylazobenzene-4hexyloxy-trimethyl-ammoniumtrifluoro-acetate (BHA) was designed and synthesized[22]. BHA
CC E
contained stimuli-responsive azobenzene unit can achieve structure isomerization under different light radiation conditions, and it showed reversible transition between trans and cis form by UV and visible light irradiation.[20]. BHA has advantages of providing a broad range of adjustable parameters, such as wavelength, duration, intensity and so on. Additional, cationic amphipathic
A
compound BHA has similar structure with phosphatidyl choline (PC) and much easier inserted into the bilayers of the liposomes[23]. The aim of this study was to design stable liposomes with intelligent photo-responsive function for drug delivery system. The photo responsive BHA-liposomes were prepared by a combined method of thin film rehydration and supercritical carbon dioxide technology (TE-scCO2). BHA as photo-responsive material and curcumin as model drug were encapsulated in the bilayer of liposomes. Physicochemical characteristics such as size, morphology and zeta potential were 3
investigated. The microstructure of liposomes was studied by using pyrene and DPH as fluorescent probes. Photo-isomerization of BHA in the liposome was studied, and the reversibility of structural heterogeneity was used as photo-responsive switch. Curcumin in the liposome was released by isomerization of BHA in the bilayer of liposomes at UV and visible light irradiation alternately.
2 Experiments
IP T
2.1 Materials Egg phosphatidyl choline (PC) was from East China Normal University chemical factory
and curcumin was purchased from Macklin Inc. Diphenylhexatriene (DPH) and pyrene were purchased from Sigma-Aldrich Inc. And main material 4-butylazobenzene-4-hexyloxy-
SC R
trimethyl-ammoniumtrifluoro-acetate (BHA) is synthesized by a series of synthetic experiments. All other organic reagents were the best grade commercially available. 2.2 Liposome preparation
U
Liposomes were prepared by a combined method of thin film hydration and supercritical carbon dioxide technology (TE-scCO2). Briefly, egg phosphatidyl choline (20 mg) was
N
dissolved in mixture solution (chloroform: methanol = 3:1 total 20 mL). The organic solvent
A
was removed under a vacuum rotary evaporator in a dark environment. The thin lipid film formed on the wall of flask was hydrated with double distilled water and incubated in CO 2
M
autoclave at temperature of 40℃ and pressure of 20 MPa for an hour[11]. The liposomal solution was achieved by self-assembly and remaining CO 2 in the liposome was removed at
ED
room temperature and pressure, finally a transparent liposome solution was obtained. For preparation of BHA-lipo, EPC was replaced by mixture of EPC and BHA, and other process were the same as above. Free BHA in the BHA-lipo solution was removed by centrifugation
PT
and the absorbance of BHA solution was obtained by UV-Vis absorption spectrum. In the process of BHA-cur-lipo preparation, EPC was replaced by mixture of EPC, cur and BHA.
CC E
2.3 Characterizations
The size distribution and zeta potential of these liposomes was determined by a dynamic
light scattering instrument equipped with a computer controlled image analysis system. The
A
morphology of liposomes was observed using transmission electron microscope (TEM). 2.4 Photo-isomerization of BHA-lipo solution The BHA exhibited a trans to cis shift at UV and visible light irradiation, and the
isomerization process of BHA-lipo was recorded by using a UV-vis spectrophotometer. The UV absorption of BHA in liposome solution was observed by the spectra, and the changes of wavelength and absorption were achieved by the altering of UV and vis light. 2.5 Liposome microstructure 4
Diphenylhexatriene (DPH) was used to explore the membrane fluidities of the BHA-lipo. The fluorescent anisotropy value of DPH in the bilayer membranes was measured by fluorescent spectrophotometer. Fluorescent probe pyrene was used to prove that BHA and curcumin were encapsulated in the bilayers of liposome. In this method, incorporation of BHA to membranes containing pyrene may lead to polarity changing of environment around pyrene, which could be indicated by the structure of the pyrene monomer emission spectrum. According to the peaks at 373 nm (I) and 384 nm (III), it was found that the ratio II/IIII
2.6 Light-responsive release from liposomes incorporating BHA
IP T
determined the micropolarity of the particles.
As a kind of drug carrier, liposome can not only loaded hydrophilic drugs, but hydrophobic
SC R
drugs. The controlled release behavior for the designed BHA-lipo composite vesicles could
be triggered by photo irradiation directly. Free BHA was removed by centrifugation and the amount of encapsulation drug was determined by the UV-vis absorption spectrum and calculated by absorbance of curcumin, according to the following eq1：
U
Encapsulation efficiency (%) = ( Wencapsulated)/(Wtotal)*100
(1)
The drug release behavior in vitro was studied at room temperature. The designed
N
composite vesicles loaded with the curcumin were loaded into dialysis bag (MOCW 14KDa)
A
and immersed into 5 mL, 0.1 % sodium dodecyl sulphate (SDS) solution. The amount of drug was determined by fluorescent spectrum. Release rate was calculated according to the
M
following eq2：
Release (%) = (W r e l e a s e /W e n c a p s u l a t e d )*100
(2)
release, respectively.
ED
Wtotal Wencapsulated and Wrelease were the amount of total drug, encapsulated drug and drug
PT
3 Results and discussion
CC E
3.1 Preparation of liposomes BHA-curcumin-liposomes (BHA-cur-lipo) were prepared by a combined method of thin
film hydration and supercritical carbon dioxide technique (TE-scCO2)[11]. The major influencing factors for preparation of BHA-cur-lipo are temperature, pressure and molar ratio of BHA to phosphatidyl choline (PC). Encapsulation efficiency (EE) of curcumin in
A
liposomes was used to evaluate the parameters of preparation condition. The EE of curcumin in liposomes changed with preparation temperature at pressure of 20 MPa, as shown in Fig1 (a). The EE of curcumin in liposomes increased with temperature when preparation temperature was below 40 ℃, and it decreased with temperature when temperature was above 40 ℃. Therefore, 40 ℃ should be considered as optimum temperature. It is well known that there existed a phase transition temperature (Tc) in transition process from gel to liquid crystalline for liposomes[24]. PCs in the bilayer of liposomes were closely aligned 5
when the temperature was below Tc, but PCs were loosely aligned when the temperature was above Tc. In order to further theoretically prove why the optimum incubation temperature is 40 ℃. The temperature of Tc for BHA-cur-lipo was obtained by micro-DSC measurements as shown in Fig1 (b), it was about 42 ℃ which close to the optimal incubation temperature[19]. When the temperature was below Tc, the close arrangement of PC resulted in poor fluidity and permeability of liposome bilayer, which made it difficult for curcumin to enter liposome through bilayer. However, the distance enlargement between adjacent PC
IP T
molecules increased when temperature was close to Tc, which resulted in increase of fluidity and permeability of liposome membranes, and the curcumin was easy to get into the bilayer
of liposomes. This result may explain the optimum incubation temperature of 40 ℃. Effect of scCO2 pressure on EE of curcumin in liposomes was probed at incubation temperature 40
SC R
℃, and relationship of the EE with pressure was demonstrated in Fig1(c). The EE of
curcumin increased with the scCO 2 pressure when the pressure was below 20 MPa, and it was almost constant when the pressure was above 20 MPa. It indicated that curcumin was encapsulated into the layer of liposomes when the pressure was below 20 MPa, and the
U
curcumin was almost saturated in bilayer of liposome at the pressure above 20 MPa.
N
Therefore, the pressure of 20 MPa was determined as optimal value for preparation of
CC E
PT
ED
M
A
liposomes.
Figure1. Selection of optimum parameters in the preparation method (a) Curcumin encapsulation changes
A
with temperature at the fixed scCO2 pressure (20 MPa); (b) Micro-DSC heating curve for BHA-cur-lipo at a scanning rate of 1 ℃/min; (c) Curcumin encapsulation changes with the scCO2 pressure at the fixed
temperature (40 ℃); (d) Relation between absorbance of BHA in the BHA-lipo and molar ratio of BHA to PC
In order to select the optimal composition of liposomes, BHA-lipo with various molar ratios of BHA to PC were prepared. Absorbance of the BHA-lipo solution with various molar ratios was obtained (Fig1 (d)). The absorbance of BHA-lipo solution increased with the 6
increasing of the concentration of the BHA. When the molar ratio was above 0.02, it was nearly constant. It was suggested that best molar ratio of BHA to PC should be 0.02, and inserted BHA could be saturated in the bilayer of the liposomes. 3.2 Morphology and Size distribution The morphology and size of BHA-cur-lipos by different methods were determined by transmission electron microscope (TEM) as shown in Fig. 2a and 2b. The BHA-cur-lipo is spherical nanoparticles with a typical vesicle structure, and there existed some multichamber
IP T
near the edge of liposomes, which suggested the existence of multi-lamellar structures. The size of liposomes prepared by different methods was determined by dynamic light scattering
technology (DLS). It was found that the BHA-cur-lipo prepared by TE-scCO2 have good
SC R
monodispersion, and the average size was about 140±15 nm. Whereas, the BHA-lipo prepared by traditional film hydration showed polydispersity, which has two main peaks, their average sizes were 100±20 nm and 830±23 nm, respectively. The reason may be that lipid molecules in the liposomes are rearranged by the self-assembling process in scCO2
U
micro-environment, and which is easy to form stable liposomes with monodispersion. This result indicated that the TE-scCO2 method is better than thin film hydration for preparation
A
CC E
PT
ED
M
A
N
of BHA-lipo.
Figure2. The TEM of BHA-cur-lipo (prepared by thin film hydration (a) and TE-scCO2 (b)); (c) the size intensity distribution of BHA-cur-lipo
3.3 Stability of liposomes Surface charge of liposomes is an important indication for the stability. The zeta potential of liposomes was obtained by a Malvern Zetasizer Nano ZS (UK) at temperature of 25 ℃. 7
It was about -23 mv and +28 mv for control-lipo and BHA-lipo, respectively. The zeta potential of control liposomes is negative (about -23 mv) because the liposome comprised of negatively charged PC. The zeta potential of BHA-lipo is positive (28 mv) because positive BHA with positive charge has been inserted the bilayer of the liposomes[16]. It prove that BHA has been successfully insert into the bilayer of liposome, and majority of BHA was encapsulated in the bilayer of liposomes and the portion of positive polar groups of BHA may be exposed to the surface of liposomes. In order to probe relationship of BHA
IP T
to the zeta potential, the zeta potentials for BHA-lipo with various BHA concentrations were determined as shown in Fig 3(a). The zeta potential was almost unchanged when the concentration was below 0.8 mM, but it increased with the concentration of BHA from 24
to 28 mv when the concentration was above 0.8 mM. It indicated that cationic surfactant
SC R
BHA was inserted into bilayer of liposomes and banded with the head groups of PC. The results indicated that BHA was banded to liposomes in two different ways in the binding process, one was interpolation (inserted into bilayer of the liposomes) and other was adsorption on the surface of the liposomes. For the retaining of BHA in the liposomes, the
U
former was better than the latter, and the retaining of BHA in the liposomes was decreased
PT
ED
M
A
N
with reducing of BHA concentration in the solution in the adsorption.
Figure3.(a) Zeta potential of BHA-lipo changes with the concentration; (b) Transmittivity of BHA-lipo
CC E
changes with time.
Stability of BHA-cur-lipo systems was evaluated by monitoring turbidity of liposomes
with time. It was found that the transmissivity of BHA-cur-lipo was nearly invariable even after six month at the environmental temperature of 4℃,as shown in Fig 3(b). The stability
A
of liposomes is beneficial to the storage and application of liposomes in drug carrier. 3.4 Microstructure of BHA-lipo Microstructure of liposomes will provide important information for their stability, EE, drug release and so on[25] [26, 27]. Fluorescence probes were used to explore the membrance fluidities and micropolarity of liposomes, respectively. Diphenylhexatriene (DPH) as a fluorescence probe was located in the lipid bilayers for both systems of control liposome 8
and BHA-lipo. The fluorescence anisotropy values of DPH in control liposome and BHA liposomes were 0.13 ± 0.01 and 0.16 ± 0.01(n=4), respectively, as shown in Fig4 (a). Anisotropy value of 0.13 for the control liposome was lower than that of 0.16 for BHA-lipo. This means that the membrance fluidity of the control liposome is higher than that of the BHA liposome, because the original structure in the bilayer was destroyed due to insertion of BHA. A similar phenomenon has been reported when hydrophobic nanoparticles was inserted in bilayer of liposomes[17]. The above results also evidence that BHA was encased
N
U
SC R
IP T
in the bilayer of the liposome.
A
Figure4. (a) The anisotropy value of control-lipo and BHA-lipo; (b) The value of II/IIII of BHA-lipo with
M
different molar ratio of BHA to PC
Pyrene, a fluorescent probe, was embedded in the bilayer of liposomes to explore the
ED
membrance micropolarity. The pyrene monomer fluorescence spectrum exhibits significant fine structure in the form of five predominant peaks. Peak I (II, at 374 nm) shows significant
PT
intensity enhancement when the environment is more polar, whereas peak III (IIII, at 384 nm) shows minimal intensity variation with polarity. Thus, the intensity ratio of II/IIII is an empiric measure of polarity[11]. The II/IIII value of control-lipo and BHA-lipo was 1.12±
CC E
0.03 and 0.79 ± 0.03, respectively. It indicates that the micropolarity of BHA-lipo was much smaller than control liposomes. This may be caused by introducing BHA in liposomes and changing the microstructure of bilayer in the BHA-lipo. Moreover, from Fig4 (b), the value of II/IIII decreased with the molar ratio of BHA to PC when the ratio was below 0.02. It means
A
that with the increasing of BHA the lipid layer is tighter and more nonpolar [28]. It decreases slightly with the ratio of BHA from 0.02 to 0.03. BHA has been saturated in the lipid layer and it would bring little effects on the lipid layer[26]. The capacity of phospholipid layer was limited and there is an optimum loading capacity with BHA to PC ratio is 0.02. 3.5 Photo-isomerization of BHA-liposomes Azobenzene group of BHA can undergo reversible trans-cis isomerization (Fig 5 (a)) under ultraviolet (365 nm) and visible light irradiation[22], and the trans- and cis9
isomerization in the BHA-liposomes are shown in Fig. 5(b). Photo-isomerization of BHAlipo was studied and the UV-Vis spectrum is presented in Fig5 (c). The absorption peak of BHA-lipo was 348 nm (black line in the Fig 5 (c)) under irradiation of visible light, which may resulted from the π → π* transition[12]. After irradiation of UV light, the intensity of the band at 348 nm decreased, whereas two absorption peaks were found at wavelengths of 315 and 440 nm (the pink line in the Fig 5 (c)). The two peaks appear due to the n → π* transition of cis-isomer. The above reversible processes could be occurred by alternating
IP T
irradiation of the visible and ultraviolet light, and the process can be repeated for many times as shown in Fig5 (d). The phenomenon indicates that the reversible trans-cis isomerization
ED
M
A
N
U
SC R
of BHA by different light irradiation could be utilized as photo switch to control drug release.
Figure 5. (a) Chemical structure and photo-isomerization of BHA; (b) The microstructure schematic of the
PT
trans-BHA-liposomes and cis-BHA-liposomes; (c) UV-Vis spectra of BHA-lipo solution at different states (initial state black line for trans-BHA and pink line for cis-BHA); (d) Changes of absorbance at 348 nm
CC E
after altering UV and visible light for cycles in BHA-lipo; (e) The II/IIII value of BHA-lipo with time after irradiating with UV and visible light alternatively
To further explore relationship of BHA-lipo microstructure to BHA isomerization in
liposome, II/IIII value of pyrene encapsulated in the liposome was obtained under altering
A
irradiation of UV and visible light. The II/IIII values were 0.81±0.05 and 1.21±0.04 under irradiation of visible and UV light, respectively. The change of II/IIII value was attributed to the isomerization of BHA in liposomes, and that resulted in alteration of the bilayer microstructure in liposomes[25]. The values also can be repeated for many times under UV and visible light irradiation alternately as shown in Fig 5(e). The results from liposome microstructure (fluidities and micro polarity achieved by fluorescence probe DPH and pyrene) and spectrum can prove the photo-isomerization of BHA in the liposome, which could be used as photo-sensitive switch to drug release in the liposomes. 10
3.6 Encapsulation efficiency Curcumin as hydrophobic model drug was encapsulated in the BHA-lipo, and free curcumin was removed by centrifugation. The concentration of curcumin can be obtained by absorbance measurement. Encapsulation efficiency (EE) of curcumin was calculated by eq. 1 (in experience section) and which was up to 88%. The encapsulation efficiency of hydrophobic drug was similar to those reported previously[29].
IP T
3.7 Drug release of liposomes To explore the role of BHA as a photo nanoswitch for drug release, the curcumin
encapsulated in liposomes was released at room temperature by UV irradiation. Curcumin
SC R
release from BHA-cur-lipo either in darkness or upon UV light irradiation for 10 min was
studied. The result was shown in Fig 6 (a). Curcumin in the BHA-cur-lipo was difficult to spontaneous release in dark (black line in Fig 6(a)). But the curcumin from BHA-cur-lipo was quickly released under UV irradiation and nearly 90% curcumin was released f in six
U
hours (the red line in Fig 6(a)). The results demonstrated that BHA should be potential
PT
ED
M
A
N
nanoswitch with photo stimulation to control release drug from the liposomes[30].
CC E
Figure6. (a) The curcumin was released from the liposomes in the dark (black line) and under UV
A
irradiation (red line); (b) The accumulated release of curcumin from BHA-cur-lipo under irradiation alternately by the UV and vis light.
As further proof of BHA as a photo-sensitive switch in liposomes, commutative release
of curcumin encapsulated in lipid was undertaken by repetitious irradiation with UV light (10 min) and visible light (40 min) as shown in Fig6 (b). However, after UV irradiation for 10 min, the curcumin release amount increased obviously. With UV stimulus, the BHA was compelled from trans to cis form and open the switch. Then visible light irradiated for 40min, optical energy was absorbed by BHA and changed configuration and phospholipid arrangement. It revealed that the amount and rate of the released curcumin could be 11
controlled by altering the UV and vis light irradiation[6]. The trans-cis isomerization of the BHA-cur-lipo can induce defects in bilayers that lead to the release of the entrapped payload. The results also reveal that BHA in liposomes as a “on-off” switch for controlling drug release[16]. This means that the drug release from liposomes can be controlled by UV and visible light irradiation alternatively.
IP T
4 Conclusions BHA, as a kind of photo-responsive compounds was synthetized by series of reactions. BHA, PC and curcumin were self-assembly into liposomes by a combined method of thin film hydration and supercritical carbon dioxide technique (TE-scCO2). Liposome is spherical nanoparticles with a
SC R
typical vesicle structure. The difference microstructures of various liposomes were proved by the
values of anisotropy obtained from the fluorescence polarization technique. The results reveal that BHA was inserted between PC polar groups. BHA in the phospholipid layer can be isomerized under light irradiation, and the isomerization of BHA can be repeated many times. The isomerization of
N
U
BHA in the bilayer of the liposomes could be used as a switch to control drug release.
M
A
Acknowledgements
This research was supported by the National Natural Science Foundation of China
PT
References
ED
(21473055 and 21773064).
CC E
[1] A. Puri, K. Loomis, B. Smith, J.-H. Lee, A. Yavlovich, E. Heldman, R. Blumenthal, Lipid-based
nanoparticles as pharmaceutical drug carriers: from concepts to clinic, Crit. Rev. Ther. Drug Carrier Syst., 26 (2009) 523-580.
[2] A. Samad, Y. Sultana, M. Aqil, Liposomal drug delivery systems: an update review, Curr. Drug
A
Delivery, 4 (2007) 297-305. [3] P. Tanner, P. Baumann, R. Enea, O. Onaca, C. Palivan, W. Meier, Polymeric Vesicles: From Drug
Carriers to Nanoreactors and Artificial Organelles, Acc. Chem. Res., 44 (2011) 1039-1049. [4] B.S. Pattni, V.V. Chupin, V.P. Torchilin, New Developments in Liposomal Drug Delivery, Chem. Rev. (Washington, DC, U. S.), 115 (2015) 10938-10966. [5] C. Flors, M.J. Fryer, J. Waring, B. Reeder, U. Bechtold, P.M. Mullineaux, S. Nonell, M.T. Wilson, N.R. Baker, Imaging the production of singlet oxygen in vivo using a new fluorescent sensor, Singlet Oxygen Sensor Green, J. Exp. Bot., 57 (2006) 1725-1734. [6] X. An, F. Zhang, Y. Zhu, W. Shen, Photoinduced drug release from thermosensitive AuNPs12
liposome using a AuNPs-switch, Chemical communications, 46 (2010) 7202-7204. [7] J. Khandare, M. Calderon, N.M. Dagia, R. Haag, Multifunctional dendritic polymers in nanomedicine: opportunities and challenges, Chem. Soc. Rev., 41 (2012) 2824-2848. [8] Y.I. Yoon, Y.S. Kwon, H.S. Cho, S.H. Heo, K.S. Park, S.G. Park, S.H. Lee, S.I. Hwang, Y.I. Kim, H.J. Jae, G.J. Ahn, Y.S. Cho, H. Lee, H.J. Lee, T.J. Yoon, Ultrasound-mediated gene and drug delivery using a microbubble-liposome particle system, Theranostics, 4 (2014) 1133-1144. [9] D.R. Khan, E.M. Rezler, J. Lauer-Fields, G.B. Fields, Effects of drug hydrophobicity on liposomal stability, Chemical biology & drug design, 71 (2008) 3-7. [10] M. Kamihira, M. Taniguchi, T. Kobayashi, Sterilization of Microorganisms with Supercritical
IP T
Carbon Dioxide, Journal of the Agricultural Chemical Society of Japan, 51 (2016) 407-412.
[11] L. Li, X. An, A novel combined method of thin-film evaporation and a supercritical carbon dioxide technique to prepare a fluorescent siRNA-liposome, RSC Advances, 6 (2016) 92115-92119.
[12] Z.K. Cui, T. Phoeung, P.A. Rousseau, G. Rydzek, Q. Zhang, C.G. Bazuin, M. Lafleur,
SC R
Nonphospholipid fluid liposomes with switchable photocontrolled release, Langmuir : the ACS journal of surfaces and colloids, 30 (2014) 10818-10825.
[13] P. Walde, S. Ichikawa, Enzymes inside lipid vesicles: preparation, reactivity and applications, Biomol. Eng., 18 (2001) 143-177.
U
[14] J. Li, Z. Yu, H. Jiang, G. Zou, Q. Zhang, Photo and pH dual-responsive polydiacetylene smart nanocontainer, Materials Chemistry and Physics, 136 (2012) 219-224.
N
[15] K. Elersic, J.I. Pavlic, A. Iglic, A. Vesel, M. Mozetic, Electric-field controlled liposome formation with embedded superparamagnetic iron oxide nanoparticles, Chem Phys Lipids, 165 (2012) 120-124.
A
[16] D. Qiu, X. An, Controllable release from magnetoliposomes by magnetic stimulation and thermal
M
stimulation, Colloids and Surfaces B: Biointerfaces, 104 (2013) 326-329. [17] X. Yan, X. An, Thermal and photic stimuli-responsive polydiacetylene liposomes with reversible fluorescence, Nanoscale, 5 (2013) 6280-6283.
ED
[18] A. Diguet, M. Yanagisawa, Y.J. Liu, E. Brun, S. Abadie, S. Rudiuk, D. Baigl, UV-induced bursting of cell-sized multicomponent lipid vesicles in a photosensitive surfactant solution, Journal of the American Chemical Society, 134 (2012) 4898-4904.
PT
[19] M. Karimi, A. Ghasemi, P. Sahandi Zangabad, R. Rahighi, S.M. Moosavi Basri, H. Mirshekari, M. Amiri, Z. Shafaei Pishabad, A. Aslani, M. Bozorgomid, D. Ghosh, A. Beyzavi, A. Vaseghi, A.R. Aref, L. Haghani, S. Bahrami, M.R. Hamblin, Smart micro/nanoparticles in stimulus-responsive drug/gene delivery
CC E
systems, Chemical Society reviews, 45 (2016) 1457-1501. [20] F. Ercole, T.P. Davis, R.A. Evans, Photo-responsive systems and biomaterials: photochromic
polymers, light-triggered self-assembly, surface modification, fluorescence modulation and beyond, Polym. Chem., 1 (2010) 37-54. [21] J. Li, X. An, Z. Pan, l. Sun, Photoinduced drug release from complexes of liposome and fluorescent
A
silver nanoparticles, RSC Adv., 4 (2014) 9476-9479. [22] S. Shi, T. Yin, X. Tao, W. Shen, Light induced micelle to vesicle transition in an aqueous solution
of a surface active ionic liquid, RSC Advances, 5 (2015) 75806-75809. [23] S. Krasnici, A. Werner, M.E. Eichhorn, M. Schmitt-Sody, S.A. Pahernik, B. Sauer, B. Schulze, M. Teifel, U. Michaelis, K. Naujoks, M. Dellian, Effect of the surface charge of liposomes on their uptake by angiogenic tumor vessels, Int. J. Cancer, 105 (2003) 561-567. [24] X. An, F. Zhan, Y. Zhu, Smart photothermal-triggered bilayer phase transition in AuNPs-liposomes to release drug, Langmuir : the ACS journal of surfaces and colloids, 29 (2013) 1061-1068. 13
[25] D. Ke, X. Wang, Q. Yang, Y. Niu, S. Chai, Z. Chen, X. An, W. Shen, Spectrometric study on the interaction of dodecyltrimethylammonium bromide with curcumin, Langmuir : the ACS journal of surfaces and colloids, 27 (2011) 14112-14117. [26] A.B. Dhanikula, R. Panchagnula, Fluorescence anisotropy, FT-IR spectroscopy and 31-P NMR studies on the interaction of paclitaxel with lipid bilayers, Lipids, 43 (2008) 569-579. [27] K. Sugikawa, T. Kadota, K. Yasuhara, A. Ikeda, Anisotropic Self-Assembly of Citrate-Coated Gold Nanoparticles on Fluidic Liposomes, Angewandte Chemie, 55 (2016) 4059-4063. [28] G.E. Savchenko, E.A. Klyuchareva, I.I. Denev, A.P. Stupak, Fluorescence of pyrene in galactolipid liposomes and native membranes of etioplasts, Journal of Applied Spectroscopy, 67 (2000) 473-481.
IP T
[29] N. Saengkrit, S. Saesoo, W. Srinuanchai, S. Phunpee, U.R. Ruktanonchai, Influence of curcuminloaded cationic liposome on anticancer activity for cervical cancer therapy, Colloids and Surfaces B: Biointerfaces, 114 (2014) 349-356.
[30] S. Geng, Y. Wang, L. Wang, T. Kouyama, T. Gotoh, S. Wada, J.Y. Wang, A Light-Responsive Self-
SC R
Assembly Formed by a Cationic Azobenzene Derivative and SDS as a Drug Delivery System, Scientific
A
CC E
PT
ED
M
A
N
U
reports, 7 (2017) 39202.
14