BiopolymerâLipid Bilayer Interaction Modulates the Physical

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Journal of Agricultural and Food Chemistry

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Biopolymer-lipid bilayer interaction modulates the physical properties of liposomes: mechanism and structure

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Chen Tan, Yating Zhang, Shabbar Abbas, Biao Feng, Xiaoming Zhang, Wenshui Xia,

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Shuqin Xia*

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State Key Laboratory of Food Science and Technology, School of Food Science and

7

Technology, Jiangnan University, Lihu Road 1800, Wuxi, Jiangsu 214122, China

1 2

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∗Corresponding author

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E-mail: [email protected];

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Telephone: +86-510-85884496;

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Fax: +86-510-85884496

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ACS Paragon Plus Environment


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ABSTRACT

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This study was conducted to elucidate the conformational dependence of

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modulating ability of chitosan, a positively changed biopolymer, on a new type of

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liposomes composed of mixed lipids including egg yolk phosphatidylcholine

26

(EYPC) and nonionic surfactant (Tween 80). Analysis on the dynamic and

27

structure of bilayer membrane upon interaction with chitosan by fluorescence and

28

electron paramagnetic resonance techniques demonstrated that, in addition to

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providing the physical barrier for membrane surface, the adsorption of chitosan

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extended and crimped chains rigidified the lipid membrane. However, the decrease

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in relative microviscosity and order parameter suggested that the presence of

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chitosan coils disturbed the membrane organization. It was also noted that the

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increase of fluidity in lipid bilayer centre was not pronounced, indicating the

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shallow penetration of coils into the hydrophobic interior of bilayer. Microscopic

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observations revealed that chitosan adsorption not only affected the morphology of

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liposomes, but also modulated the particle aggregation and fusion. Especially, a

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number of much heterogeneous particles were visualized, which tended to confirm

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the role of chitosan coils as a “polymeric surfactant”. Accompanying by the

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particle deformation, the membrane permeability was also tuned. These findings

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may provide a new perspective to understand the physiological functionality of

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biopolymer and design the biopolymer-liposome composited structures as the

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delivery systems for bioactive components.

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Keywords: liposomes, chitosan, structure, morphology, polymeric surfactant

44 ACS Paragon Plus Environment

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INTRODUCTION

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Modifying the liposomal surface via decorating with a layer or alternate deposition

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(layer by layer) of hydrophilic polymers has emerged as a novel way to improve

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liposome delivery performance. As example, chitosan is a very promising

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glycosaminoglycan for biological applications due to its biocompatibility,

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biodegradability, and low toxicity.1 In the field of drug delivery, nanoparticles and

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formulations containing chitosan have presented promising results in the search of

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drug release systems optimizing disease treatments and reducing drug side effects.

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These advantages of chitosan contribute to the development of composite

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phospholipid-chitosan vesicles (chitosomes) as new structures for encapsulation of

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drugs and vaccines aimed as vectors and tolls for controlled antigen release.2, 3 More

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recently, food application of chitosomes has greatly increased for encapsulating

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nutraceuticals to make them water-dispersible in aqueous food formulations and

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increase their bioavailability.4, 5, 6

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As a soft matter, liposomes may be subjected to change in the morphology,

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integrity and structure upon interaction with anchored and/or adsorbed polymers.

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These changes are closely related to the liposome properties, polymer characteristics

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and preparation conditions. Through manipulating these factors, chitosan can form a

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dense mesh covering the liposomal surface, thus improving the structural properties of

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liposomes, biocompatibility, and drug delivery efficiency. Interestingly, it is also

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argued that the strong interaction of chitosan with phospholipids may disrupt the cell

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membrane7 and model liposomes,8 thereby increasing membrane permeability.9 The



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contradiction in these reports aroused our great interest to study the mechanisms of

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chitosan action. One effective way is to visualize the direct response of the membrane

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upon interaction with chitosan. It has been revealed that the presence of chitosan

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negatively influences the giant unilamelar vesicles (GUVs, 1-100 µm) due to the

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increased spontaneous tension by chitosan adsorption,10 whereas, the effect of

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spontaneous tension may not necessarily occur in the case of large unilamelar vesicles

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(LUVs, 200-1000 nm).11 However, what happens to the response of more highly

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curved vesicles (typically 2.0 mg/mL).13 Such

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conformational change of chitosan molecules suggested that they could adopt various


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interaction mechanisms with liposomal membrane, besides the flat adsorption, which

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seemed of interest to further investigate.

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On the other hand, it is important to point out that most of the suggestions

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aforementioned were based on a simple model membrane system composed of

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synthesized

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phosphatidylcholine (EYPC) is the commonly used lipid for the preparation of

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liposomes as it is generally recognized as a safe (GRAS) food ingredient. Previously,

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we explored a new type of liposome composed of mixed lipids including egg yolk

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phosphatidylcholine (EYPC) and nonionic surfactant (Tween 80) as carriers for

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coenzyme Q10,14, 15 carotenoids,16, 17, 18, 19 and Vitamin E.20 The appropriate amount of

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Tween 80 into the formulation was demonstrated to impart high rigidity to the

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liposomal membrane, thus favoring the encapsulation efficiency and stability of

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liposomes. To the best of our knowledge, however, there is little information

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regarding the interaction of chitosan with mixed lipids-based liposomes.

phospholipids.

From

food

industry

perspective,

egg

yolk

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Taken together, further detailed knowledge of such chitosan-lipid bilayer composite

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structure is required with special attention on the role of polymer conformation.

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Herein, the effect of chitosan conformation on the physical characterization of

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liposomes is thoroughly investigated including membrane dynamics, structure,

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hydrophobicity, surface morphology and size. Several analysis techniques are

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introduced, including fluorescence anisotropy, electron paramagnetic resonance,

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transmission electron microscopy, atomic force microscopy and dynamic light



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scattering. The aggregation and fusion behaviours of decorated liposomes as well as

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membrane permeability against surfactant are also evaluated.

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MATERIALS AND METHODS

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Materials. Egg yolk phospholipid (EYPC, composed of approximately 80%

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phosphatidylcholine, 5% lysophosphatidylcholine and phosphatidic acid, and 15%

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fatty acids) was purchased from Chemical Reagent Plant of East China Normal

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University (Shanghai, China). Polyoxyethylene sorbitan monooleate (Tween 80) was

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obtained from China Medicine (Group) Shanghai Chemical Reagent Co. (Shanghai,

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China). Partially deacetylated chitosan (85%) with average molecular weight of

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200,000 was purchased from Ao Xing Biotechnology Co., Ltd (Zhejiang, China). The

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fluorescent

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phenyl]-6-phenyl-1,

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8-anilino-1-naphthalene-sulfonic acid (ANS, 98% purity), and the spin labels

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5-doxyl-stearic acid (5-DSA) and 16-doxyl-stearic acid (16-DSA) were all purchased

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from Sigma Chemical Co. (St. Louis MO, USA). All other reagents were of analytical

125

grade.

probe,

5(6)-carboxyfluorescein 3,

5-hexatriene

(CF),

(TMA-DPH,

1-[4-(trimethylammonio) 98%

purity)

and

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Preparation of Chitosan-decorated Liposomes. Bare liposomes were prepared

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according to our earlier method.14 Briefly, phospholipid (6 g) and Tween 80 (4.32 g)

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were dissolved in 12 mL warm ethanol at 55 ˚C. The ethanol solution was rapidly

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injected using a syringe as a pump into 120 mL buffer solution (0.1 M acetic

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acid/0.01 M phosphate, 75 mM NaCl, pH=4.0) at 55 ˚C in water bath under stirring at

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700 r/min using an IKA® RW 20 digital overhead stirrer (IKA® Works Guangzhou).


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After agitation for 30 min, the liposomal system was transferred to a round-bottom

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flask attaching to a rotary evaporator at 55 ˚C and reduced pressure to remove

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ethanol. The obtained liposomal suspension was then submitted to a probing

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sonication process in an ice bath for 10 min at 240 W with a sequence of 1 s of

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sonication and 1s rest using a sonicator (Sonics & Materials, Inc., 20 kHz). The final

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samples were filled into vials (The headspace of the vials was blanketed with nitrogen)

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and kept in the refrigerator (4 ˚C in the dark). The CF-entrapped liposomes was

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prepared in the same way expect for using 20 mM CF solution (0.1 M acetic acid/0.01

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M phosphate, 75 mM NaCl, pH=4.0) instead of buffer solution. Un-encapsulated CF

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was removed from the liposomal suspension by gel filtration through a Sephadex

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G-25 column.

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The chitosan-decorated liposomes were prepared as follows: the chitosan was

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dissolved in the same buffer solution for bare liposomes and then added dropwise to

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bare liposomal suspension at equal volume at 1000 r/min for 30 min using an

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overhead stirrer at room temperature. The final concentration of chitosan was adjusted

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from 0.5 to 4 mg/mL. The phospholipid concentration was kept at 25 mg/mL. The

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final samples were filled into vials and stored at the same conditions as bare

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liposomes.

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Liposomal Membrane Fluidity. A weighted amount of ANS probe was dissolved

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in ethanol solution, and the final concentration was 6×10-3 mol/L. The TMA-DPH

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stock

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tetrahydrofuran/water (1:1, v/v) and adjusting the concentration to 10-2 mol/L. The

solution

was

prepared

by

dissolving

the

TMA-DPH


powders

in


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final TMA-DPH concentration was adjusted to 10-4 mol/L by PBS dilution before use.

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When determining the liposomal fluidity, aliquots of probe solution was first mixed

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with bare liposomes in a 10 mL tube, incubating at 37 oC for 30 min. Then, the

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chitosan of different concentration in the same buffer was added dropwise to the

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incubated sample under stirring at 1000 r/min for 30 min. Note that before mixed with

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liposomal samples, the ANS solution should be dried by nitrogen to avoid the

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destroying effect of ethanol to liposomal membrane.

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Microviscosity of liposomal membrane was measured at 25 oC using a fluorescence

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spectrometer (Hitachi F-7000, Japan) equipped with excitation and emission

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polarization filters. The excitation and emission wavelengths were respectively: for

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ANS: λex=337 nm and λem=480 nm; TMA-DPH: λex=365 nm and λem=430. The slit

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widths for both excitation and emission were 5 nm. The fluorescence intensities were

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measured according to our previous study

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by fluorescence polarization (P) according to the following equation:21

19

. The microviscosity (η) was calculated

η=2P/(0.46-P)

168 169

P= (I0,0-GI0,90)/( I0,0+GI0,90), G= I90, 0 / I90, 90.

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where I0,0 and I0,90 are the fluorescence intensities of the emitted light polarized

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parallel and vertical to the exciting light, respectively, and G is the grating correction

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coefficient.22

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Electron Paramagnetic Resonance (EPR). EPR technique was applied to monitor

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the molecular dynamics of lipids by observing the changes in the spin tropic

175

movement of an unpaired electron. EPR measurements were performed using a


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Bruker EMX spectrometer (Bruker, Rheinstetten, Germany) at the X-band (9 GHz).

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The spectra were recorded on microwave power 20 mW with sweep width 120 G,

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centre field 3511 G, and 20 s sweep time. All measurements were performed in a

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triplicate at room temperature. In the present study, 5-DSA and 16-DSA were used as

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spin markers. These compounds consist of a radical group and a hydrocarbonated

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chain (stearic acid) which acts as a radical support (Figure 1). The radical in the 5 or

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in the 16 position of the alkyl chain will determine local motional profiles in the two

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main regions of the lipid bilayer, near the polar head group (5-DSA) or at the end of

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the hydrophobic chain (16-DSA). A weighed amount of 5-DSA or 16-DSA was

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dissolved in ethanol solution, adjusting the final concentration to 1.0 and 0.9 mol/L,

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respectively. One millilitres of spin probe solution was added into a 10 mL tube

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followed by nitrogen dryness to remove the ethanol solution. An aliquot of 2.5 mL

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bare liposomes was added into the tube, incubating at 25 oC for 30 min. Then, the

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chitosan solution was added dropwise to the incubated sample under stirring for 30

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min.

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Line widths 2Amax, 2Amin, and ∆H, in Gauss (G) and heights of the high- and

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mid-field lines, h-1 and h0, respectively, were obtained from the first derivative of each

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absorption spectrum. Spectral parameters used to study the location of spin labels in

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liposomal membrane were shown in Figure 1.

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The order parameter (S) reflects the segmental order parameter of the segment to

196

which the nitroxide fragment is attached.23 It can be obtained from the conventional

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EPR spectra and calculated as S=0.5407×(Amax-Amin)/a0, where a0 =(Amax+2Amin)/3.



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The order parameter S is equal to zero for the membrane in total disorder. When it

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equals 1, the membrane has a crystal structure.24

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The EPR spectrum of 16-DSA incorporated into the lipid bilayer reflects an

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isotropic motion of the acyl-chain. In this case, rotational correlation time (τ, ns),

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assuming isotropic rotational diffusion of 16-DSA can be used to measure the motion

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of the phospholipid acyl-chains near the hydrophobic end.25 An increase in τ indicates

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a decrease of membrane fluidity. It can be calculated from the following equation:

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ℎ0 τ = 6.5 × 10−10 × ∆‫( × ܪ‬ඨ − 1) ℎ−1

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Transmission Electron Microscopy (TEM). TEM micrographs were performed

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on a HITACHI-7650 at an accelerating voltage of 200 kV. The samples (bare

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liposomes and chitosan-decorated liposomes) were prepared as described above. An

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aqueous suspension of the samples was deposited upon a carbon-coated copper grid,

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followed by air-drying for 1 min. Then, the sample was instantly stained with 2 wt%

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phosphotungstic acid for 15 s.

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Atomic Force Microscopy (AFM). AFM imaging was carried out using a

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commercial AFM (Dimension icon, Bruker Co., USA). The ScanAsyst mode was

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applied using silicon tip (TESP, Bruker, nom. frep. 320 kHz, nom. Spring constant of

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42 N/m) with scan resolution of 512 samples per line. Mica sheets were freshly

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cleaved to obtain a flat and uniform surface and were used as the substrate for AFM

217

observations. Just before the analysis, the liposomal samples were diluted in water to

218

obtain a less sticky fluid. An aliquot of 1 µL liposome suspension were deposited on

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the mica surface and incubated for 30 min at room temperature. Afterward, samples


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were carefully washed with ultrapure water to eliminate salts and non-adsorbed

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vesicles, and dried for 24 h at room temperature. Height mode images are acquired

222

simultaneously at a fixed scan rate (0.997 Hz) with a resolution of 512 × 512 pixels in

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the dimension of 5 µm × 5 µm. Processing of images and analysis was carried out

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using NanoScopeTM software (Digital Instruments, version V614r1). All height

225

images are presented after the zero-order two-dimensional flattening.

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Particle Size Distribution. Size measurements were performed at (25±0.1)˚C

227

using a commercial zeta-sizer (Nano-ZS90, Malvern Instruments Ltd, United

228

Kingdom) with a He / Ne laser (λ = 633 nm) and scattering angle 90°. To evaluate the

229

aggregation and fusion of particles, the liposomal sample was heated at 37 ˚C for 5 h.

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After heat treatment, 2 mL liposomal dispersion was transferred into the polystyrene

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cuvette for size determination and then the particle size distribution were recorded by

232

dynamic light scattering (DLS). The measurements were repeated three times.

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CF Release Study. It is generally assumed that the fluorescence emission of CF is

234

quenched to the high extent when the molecules were encapsulated in the interior

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aqueous compartments of liposomes at high concentration.26 Upon release of CF from

236

liposomes to the bulk aqueous phase, the fluorescence intensity would increase

237

greatly. Therefore, the release of CF was taken as a measure of the membrane

238

permeability after incubating liposomes with chitosan. Briefly, an aliquot of chitosan

239

solution (1 mL) at different concentrations was added into the 1 mL of liposomal

240

suspension. At proper time intervals, 10 µL of sample were withdrawn and diluted

241

with 2 mL of tris buffer (50 mM, pH 7.5). The fluorescence intensity of the samples



242

was then measured at 25 oC with a spectrofluorimeter (Hitachi F-7000, Japan) with

243

excitation at 488 nm and emission at 520 nm. The percentage of CF release from

244

liposomes was calculated from the equation: % Release=100×(Ft-F0)/( F∞-F0), where

245

F0 and Ft are the fluorescence intensities of the liposome samples before and after

246

incubation with chitosan for the time period t, F∞ is the total flurescence intensity

247

after liposome lysis with 10% Triton X-100. To evaluate the stability of

248

decorated-liposomes against surfactant Triton X-100, 100 µL of 10% Triton X-100

249

solution was added to the liposomal sample (1 mL). The CF fluorescence was

250

immediately monitored every second by spectrofluorimeter.

251

Statistical Analysis. Data are presented as a mean value with its standard deviation

252

indicated (mean ± SD). All experiments were carried out in triplicate using freshly

253

prepared samples.

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RESULTS AND DISCUSSION

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Fluidity of Liposomal Membrane. Fluidity of liposomal membrane quantitatively

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characterizes the mobility and molecular rotation rate of lipid.27 Lower membrane

257

fluidity represents a higher structural order. It is well known that ANS and TMA-DPH

258

serve as markers for the molecular movement on the exterior membrane surface28 and

259

superficial region including the surface and glycerol side chain region,29 respectively.

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The relative microviscosity η/η0 as a function of chitosan shows that the influence of

261

chitosan binding was more pronounced on the fluidity in membrane surface than

262

glycerol side chain region (Figure 2). As the chitosan concentration increased from

263

0.25 to 1.5 mg/mL, microviscosity in membrane surface region was considerably


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increased, but remained virtually unchanged in the superficial region. Apparently,

265

chitosan chains mainly adsorbed to membrane surface and restricted the motion of

266

polar headgroup. Notably, restriction was stronger in the case of crimped chains.

267

Considering the appearance of hydrophobic interaction accompanied by crimped

268

chains, we speculated that besides electrostatic attraction, a weak hydrophobic force

269

also contributed to the stabilizing effect on liposomal membrane. This hypothesis was

270

supported by the earlier research which concluded that the involvement of

271

hydrophobic interaction could promote the physical adsorption and deposition of

272

chitosan on membrane.11 However, when concentration was > 1.5 mg/mL, η/η0 values

273

were decreased greatly surrounding ANS, while slightly surrounding TMA-DPH. It

274

indicated that the presence of coils not only increased the fluidity of membrane

275

surface but also somewhat disturbed the superficial region. The destabilization of

276

chitosan coils might be correlated with the enhancement of hydrophobic interaction

277

and consequently, different adsorption patterns are expected instead flat adsorption.

278

Motion of Alkyl Chains. EPR is a useful technique for determining the fluidity

279

and structural changes of the lipid bilayers of liposomes. Figure 3 reveals that

280

decoration of chitosan chains ( 248-286, a Dramatic Enhancer of HIV Infectivity, Promotes Lipid Aggregation and Fusion. Biophys. J. 2009, 97, 2474-2483. 43. Heurtault B; Saulnier P; Pech B; Proust J-E; Benoit J-P, Physico-chemical stability of colloidal lipid particles. Biomaterials 2003, 24, 4283-4300.

592 593 594 595



596

Figure captions

597

Figure 1. Molecular formula of the spin probe 5-DSA and 16-DSA, and the

598

corresponding EPR spectrum.

599

Figure 2. Relative microviscosity η/η0 in liposomes for ANS and TMA-DPH as a

600

function of decorated chitosan concentration. η0 refers to the calculated microviscosity

601

in bare liposomes, η refers to the microviscosity in chitosan-decorated liposomes.

602

Each point represents the mean value ± standard deviation (n=3).

603

Figure 3. Order parameter S of spin probes and rotational correlation time of 16-DSA

604

measured as a function of decorated chitosan concentration. Each point represents the

605

mean value ± standard deviation (n=3).

606

Figure 4. Isotropic hyperfine coupling constant a0 of 5-DSA and 16-DSA in

607

chitosan-decorated liposomes as a function of concentration. Each point represents the

608

mean value ± standard deviation (n=3).

609

Figure 5. Transmission electron micrographs of bare liposomes (A) and liposomes

610

decorated by chitosan at 1.0 mg/mL (B), 1.5 mg/mL (C), 2.0 mg/mL (D), 3.0 mg/mL

611

(E) and 4.0 mg/mL (F).

612

Figure 6. Scan Asyst mode AFM images of bare liposomes and liposomes decorated

613

by chitosan at the concentration of 1.0 mg/mL, 1.5 mg/mL, 2.0 mg/mL, 3.0 mg/mL

614

and 4.0 mg/mL. The study was performed immediately after the preparation. Scan

615

size 5 µm×5µm.

616

Figure 7. Diameter distribution of bare liposomes and chitosan-decorated liposome

617

after 5 h incubation at 37 oC.


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Figure 8. Scan Asyst mode AFM images of bare liposomes (A) and liposomes

619

decorated by chitosan at the concentration of 1.0 mg/mL (B), 1.5 mg/mL (C), 2.0

620

mg/mL (D), 3.0 mg/mL (E) and 4.0 mg/mL (F). The study was performed after 5 h

621

incubation at 37 oC. Scan size 5 µm×5µm.

622

Figure 9. Time course of CF release kinetic from bare liposomes after exposure to

623

chitosan of different concentrations (A), or from chitosan decorated-liposomes after

624

exposure to Triton X-100 (B). When Triton X-100 was added to the suspension, the

625

CF fluorescence was immediately monitored every second by spectrofluorimeter.

626 627



628

Figures

629

Figure 1. 5-DSA

16-DSA

630 631


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632

Figure 2. 5 ANS T MA-DP H

Relative microviscosity

4 3 2 1 0 0

633

1 2 3 Chitosan concentration (mg/mL)

634 635


4


Figure 3. 1.3

0.56 S/5-DSA S/16-DSA τ

Order parameter S

0.54 1.2 0.52 0.13 1.1 0.12

0.11

1.0 0

637 638



4

Rotational correlation time (ns)

636

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639

Figure 4. 15.0 5-DSA 16-DSA

a0 (G)

14.5

14.0

13.5 0

640


641


4


642 643

Figure 5.

644 645


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646

Figure 6.

647 648



649

Figure 7. 15 0.0 1.0 1.5 2.0 3.0 4.0

Volumn (%)

10

5

0 10

650 651

100

1000 Diameter (nm)


10000

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652

Figure 8.

653 654 655



656

Figure 9. 40

8

CF release (%)

4

CF release (%)

0.0 0.5 1.0 1.5 2.0 3.0 4.0

6

0.0 0.5 1.0 1.5 2.0 3.0 4.0

30

20

10

2 A

0 0

657

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B

0

10 20 Incubation time (min)

30

0

20

40 60 80 100 Incubation time (sec)

658


120

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659

Graphic for table of contents

660


BiopolymerâLipid Bilayer Interaction Modulates the Physical

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BiopolymerâLipid Bilayer Interaction Modulates the Physical