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Comparative Fluorescence Resonance Energy Transfer Study in Pluronic Tri Block Co-Polymeric Micelle and Niosome Composed of Biological Component Cholesterol : An Investigation of Effect of Cholesterol and Sucrose on the FRET parameters Arpita Roy, Niloy Kundu, Debasis Banik, and Nilmoni Sarkar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b09761 • Publication Date (Web): 16 Dec 2015 Downloaded from http://pubs.acs.org on December 29, 2015
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Comparative Fluorescence Resonance Energy Transfer Study in Pluronic Triblock Copolymer Micelle and Niosome Composed of Biological Component Cholesterol : An Investigation of Effect of Cholesterol and Sucrose on the FRET parameters
Arpita Roy, Niloy Kundu, Debasis Banik, and Nilmoni Sarkar* Department of Chemistry, Indian Institute of Technology, Kharagpur 721302, WB, India E-mail:
[email protected] Fax: 91-3222-255303 Abstract In this article, the formation of pluronic triblock copolymer (F127)-cholesterol based niosome and its interaction with sugar (sucrose) molecules have been investigated. The morphology of F127-cholesterol based niosome in presence of sucrose has been successfully demonstrated using dynamic light scattering (DLS) and transmission electron microscopic (TEM) techniques. The DLS profiles and TEM images clearly suggest that the size of the niosome aggregates increases significantly in presence of sucrose. In addition to structural characterization, a detailed comparative fluorescence resonance energy transfer (FRET) study has been carried out in these F127 containing aggregates, involving coumarin 153 (C153) as donor (D) and rhodamine 6G (R6G) as an acceptor (A) to monitor the dynamic heterogeneity of the systems. Besides, time resolved anisotropy and fluorescence correlation spectroscopy (FCS) measurements have been carried out to monitor the rotational and lateral diffusion motion in these F127-cholesterol based aggregates using C153 and R6G respectively. During the course of FRET study, we have observed multiple time constants of FRET inside the F127-cholesterol based niosomes in contrast to the F127 micelle. This corresponds to presence of more than one preferential donoracceptor (D–A) distances in niosomes than in F127 micelle. FRET has also been successfully used to probe the effect of sucrose on the morphology of F127-cholesterol based niosome. In presence of sucrose, the time constant of FRET further increases as the D-A distances increases in sucrose decorated niosome. Finally, the excitation wavelength dependent FRET studies have indicated that as the excitation of donor molecules varies from 408 nm to 440 nm, the contribution of the faster rise component of the acceptor enhances considerably which clearly establishes the dynamics heterogeneity of both systems. Our findings also indicate that FRET is completely intra-vesicular in nature in these block copolymer-cholesterol based aggregates. 1 ACS Paragon Plus Environment
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Keywords: Triblock copolymer (F127), Cholesterol, Fluorescence Resonance Energy Transfer (FRET), Niosome, Sugar.
1. Introduction: The spherical aggregates formed in aqueous solution composed of amphiphiles (lipids, surfactants or polymers) having one or more concentric bilayer called vesicles. The inner water pool of vesicles is surrounded by the concentric bilayer.1–9 Therefore, on the basis of microstructural characteristic, the vesicles are mainly classified as multilamellar and unilamellar ones.2 The unique amphipathic structural features of vesicle allow it to solubilize substances in hydrophilic aqueous compartment, hydrophobic bilayer or even solubilize in both compartments.10,11 The artificially prepared vesicles using phospholipids are called liposomes1,2 which are extensively used as drug and gene delivery vehicle over the past few years.11–16 But the degradation possibility of phospholipids, low stability and difficulties in preparation techniques hinder its applicability.17 Therefore, in recent times, different amphiphilic molecules (surfactants, surface active ionic liquids or polymers) are used as suitable substitutes to prepare non-phospholipids vesicular assemblies. In aqueous medium, the self assemblies of the amphiphilic molecules in presence of oppositely charged molecules or other external additives can form vesicle and exhibits colloidal stability.18–25 These vesicular aggregates are widely used as medium for chemical synthesis, template for nanomaterial preparation or pharmaceutical applications.26–29 In recent years, nanoscopic vesicles are prepared using hydrated mixture of various single tail amphiphilic molecules and cholesterol.23,30–36The nonionic surfactant containing vesicles are termed as niosomes. These niosomes are of much more advantageous over other vesicles due to their non toxicity and biodegradability. Various nonionic surfactants are used to formulate niosomes using different additives.3,4,30–32,37-40Previously, we have investigated various photophysical studies in ionic surfactant forming vesicles.35,36,41However, this is the first report where the effect of cholesterol has been investigated into the biocompatible triblock copolymer micellar assembly using any spectroscopic tool. To probe the microstructural transitions of the pluronic triblock copolymer F127 micelle into niosome in presence of cholesterol, we have employed dynamic light scattering (DLS) and transmission electron microscopy (TEM), timeresolved anisotropy and fluorescence correlation spectroscopy (FCS) measurements. Now-a2 ACS Paragon Plus Environment
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days, these pluronic micellar aggregates as well as niosomes are extensively used as drug delivery vehicle and in other pharmaceutical applications.15,19,37,39 Therefore, a detailed study with this type of pluronic micelle and its further interaction with cholesterol are needed to unravel their heterogeneity and overall structural diversity. The aim of this work is also to use fluorescence resonance energy transfer (FRET) for the measurement of donor–acceptor (D–A) distances
during the
spontaneous
micelle
to
niosome transition
and
explore the
microheterogeneity of these different selfassemblies. Over the last decades, FRET has been extensively used to characterize different heterogeneous assemblies including micelles, vesicles, proteins, lipids, DNA, nucleic acid, microemulsions etc.42–51 In recent times, FRET52,53 has been broadly used as a powerful optical technique to determine the distances between the donor and acceptor located at the two particular regions of a organized assembly. Thus FRET can provide a better insight regarding the dynamics and structural aspects of various self assembled systems.54,55 In FRET process, the nonradiative excitation energy is transferred between the electronically excited “donor” chromophore and the nearby “acceptor” molecules in ground state. The efficiency of FRET processes depends on spectral overlap between the fluorescence spectra of donor molecules and absorption spectra of acceptor molecules, distance between FRET pair, relative orientation of transition dipoles of acceptor and donor, quantum yield, absorption coefficient of donor and acceptor respectively.42Förster theory reveals that the efficiency and rate of FRET strongly depends upon the distance by which the donor-acceptor is separated and the particular direction in which the transition dipole moment is oriented. Later Fleming et al. observed that in the systems of having closer distance between donor and acceptor, the standard form of Förster theory is not practicable.56–59 They developed a new methodology utilizing generalized Förster theory to calculate the rate of energy transfer in these type of systems. Such kind of framework is also applied to light harvesting supramolecular assemblies in artificial photosynthesis processes where more than one FRET processes are in action. Very recently, FRET has been used as a tool for self-assembled light-harvesting system from chromophores in lipid vesicles.60The standard form of Förster theory provides excellent estimation of donor-acceptor distances in surfactant containing aggregates. In recent times, upon progress in the field of quantum dot based FRET, the significant development have occurred to understand long-range conformational motions in
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complex systems61 and nanometal surface energy transfer (NSET).62,63In these systems, it is observed that energy transfer rate deviated from R−6 distance dependency. While reasonable efforts have been paid to the investigation of FRET in different supramolecular aggregates including micelles, reverse micelles, microemulsions and bile salt aggregate,44,45,48,49, 51,78,79,
till now, there is no report of FRET in a block copolymer forming niosomal system,
despite of extensive applications of niosomes in the pharmaceutical field. In the present study, we have used nontoxic triblock copolymer (F127) and cholesterol to formulate niosomes and investigated the difference in FRET behavior between hydrophobic coumarin 153 as donor molecule and hydrophilic rhodamine 6G (R6G) as acceptor molecule in F127 micelle and F127cholesterol niosomes at different cholesterol concentration. Due to the difference of hydrophobicity between donor and acceptor molecules, the location of C153 differs significantly with respect to acceptor during micelle to niosome transformation. Because of strong dependence of FRET on donor–acceptor distance, variation in the location of donor during polymer micelle to niosome formation can provide some significant information regarding morphological transformation of these self-assembled systems. Therefore, FRET study can provide some significant information about the dynamics of selfassembled systems with changes in the efficiency of FRET in these multiple fluorophore systems. The present study not only capable of monitoring the gross microstructural transition from micelle to niosome but also the changes in the heterogeneity inside a particular self-assembly by using excitation wavelength dependent FRET. Using FRET technique we have been able to reveal the multiple time constant of FRET which are operative in the niosome system but not in the F127 micellar aggregate. Also, due to this multiple time constant of FRET, we have obtained multiple D-A distances in the F127-cholesterol based niosomes. Therefore, using a hydrophobic dye C153 as a donor molecule and a hydrophilic probe R6G as an acceptor, we can easily investigate the different micro heterogeneous region of the self assembled systems which DLS and TEM measurements fail to serve. Further, we have also investigated the effect of sucrose on the cholesterol-block copolymer forming niosomes. Very little is known about the interactions between lipids and the sugars in the fully hydrated state, while most of the studies on the interactions between phospholipid membranes and sugars emphasize either on the phase transition temperature (Tm) or on the preservation against solute leakage and fusion of liposomes or whole cells. In solution, 4 ACS Paragon Plus Environment
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carbohydrates interact directly with phospholipid bilayer as indicated by the earlier studies.64It may also affect the liposome surface to prevent protein adsorption, liposome aggregation and encapsulated dye leakage.65–67 Disaccharides have been shown to associate with the lipid membrane68 and forms hydrogen bond to the phosphate groups,69 and consequently replace water molecules at the membrane surface.64,70,71 Previously, Andersen et al have investigated the effect of sugars on the lipid bilayer through SANS and thermodynamic measurements.72 Therefore, our present study may reveal some interesting aspects towards the overall effect of sucrose on the nonionic surfactant forming niosome as well as the effect of cholesterol on the triblock copolymer forming micelle F127 through different techniques. 2. Experimental Section: 2.1. Materials and Method: The triblock copolymer F127 was purchased from Sigma-Aldrich. Cholesterol was purchased from Sisco Research Laboratories Pvt. Ltd. (SRL), India. Coumarin 153 (C153) and rhodamine 6G perchlorate (R6G ClO4) were purchased from Exciton. All these materials were used as received. The micellar solution of F127 was prepared by dissolving required amount of F127 in triply distilled Milli-Q water. The concentration of F127 was 11 mM. All the experiments were executed at 298 K. For the overall study of FRET the concentration of donor (C153) was 7 µM and that of the acceptor (R6G) 24 µM. Hence, the ratio between them will be, [Acceptor]/[Donor] = 3.42. The detailed proportion of the membrane components (F127 and cholesterol) and dyes (donor and acceptor) has been provided in the Table S1, Supporting Information. The structures of F127, cholesterol, C 153 and R6G and sucrose are shown in Scheme 1. 2.1.1. Preparation of F127 and Cholesterol Containing Niosome Aggregates. The niosome solution has been prepared by thin film hydration method.73,74 The composition of cholesterol to F127 is expressed by R value (R = [Cholesterol]/[F127]). To prepare niosome, initially, triblock copolymer F127 and cholesterol at two different R values (R =0.5 and 1.0) were dissolved in CHCl3 in a round-bottomed flask. The mixtures were then mixed well. After the evaporation of the solvents, the dried film was hydrated by double distilled Milli-Q water. After that the aqueous solution mixtures were probe sonicated using an ultrasonicator (processor SONOPROS PR-250 MP, Oscar Ultrasonics Pvt. Ltd.). Required amount of C153 and R6G dyes were added in the resulting solution mixtures for spectroscopic measurements. After gentle shaking of the 5 ACS Paragon Plus Environment
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resulting solution mixtures, it becomes homogenous as the concentration of C153 and R6G were very low (micro molar range) in comparison to niosome components. It can be assumed that the amount of free dye is negligible because the concentration of the dyes used in the experiments is very low. Besides, the size of the dyes is many times smaller than the niosomes ( ̴ 500 nm) which ensures almost complete dye encapsulation. Previously, researchers have used the same procedure of dye encapsulation that we have mentioned here.39,42,43 2.2. Instrumentation: 2.2.1. Spectroscopic Techniques: UV-vis absorption and emission spectra of C153 and R6G were monitored using Shimadzu (model number UV-2450) spectrophotometer and a Hitachi (model number F-7000) spectrofluorimeter, respectively. We have used time correlated single photon counting (TCSPC) picosecond spectrometer to record the time resolved decay of fluorophore in solution. The detailed of TCSPC set-up was depicted in our earlier publication.63 Generally, picosecond diode lasers (IBH, UK, Nanoled) were utilized as excitation source and the emission decays were detected in magic angle (54.70) polarization by Hamamatsu microchannel plate photomultiplier tube (MCP PMT) (3809U). The picosecond diode laser of 408 nm and 440 nm were used as excitation source. The instrument response function of TCSPC set up is ~90 ps. During the analysis of time resolved decays, we have used IBH DAS-6 decay analysis software. Similarly, the anisotropy decays were measured using the same TCSPC instrument. During anisotropy measurements, the motorized polarizer in the emission side was utilized to collect the emission decays at parallel, ∥ (t) and perpendicular, (t) polarizations alternatively using vertically polarized excitation source until a certain peak difference between ∥ (t) and (t) decays were reached. The anisotropy decay function, was defined as:75
.
= ∥ .
∥
1
Here, G is the correction factor and for our set up is 0.6. 2.2.2. Calculation of Quantum Yield: The quantum yield of rhodamine 6G dissolved in ethanol (Φ = 0.95) taken as reference and the following equation was used to calculate the quantum yield:42
= × ×
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(2)
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Here, the subscript ‘S’ and ‘R’ indicate sample and reference, respectively. The quantum yield of fluorophore, refractive index solvent denote by ‘Φ’ and ‘n’, respectively. ‘Abs’ and ‘A’ signify absorbance and area under the fluorescence curve of fluorophore molecule. 2.2.3. Calculation of FRET Parameters: The rate of fluorescence resonance energy transfer (kFRET) calculated according to the Förster theory applying the following equation:75 =
! ' "#$%&
=
! ) "(
) *
(3)
('
Where +,-. denotes the rise time of the acceptor decay in the presence of donor (D) and
+/0 represents lifetime of donor (D) in the absence of acceptor (A). The distance within the molecular centers of A and D denoted by RDA. R0 is called the Förster distance and it represents the distance at which the energy transfer efficiency is assumed to be 50%. R0 is calculated using following equation: 10 = 0.211[ 5 6 7/ 89]
!; *
(4)
Where QD, the quantum yield of D in the absence of A and 5 is the refractive index of the medium. is the orientation factor, and 89 represents the spectral overlap among the fluorescence spectrum of D and absorption spectrum of A. 89 is related to the normalized emission intensity of D in the absence of A [0 0
