Biopolymer–Lipid Bilayer Interaction Modulates the Physical

Jul 14, 2015 - positively charged biopolymer, on a new type of liposome composed of mixed lipids ..... liposomes maintained a spherical and well-defin...
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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

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

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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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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

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

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structure of bilayer membrane upon interaction with chitosan by fluorescence and

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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

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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

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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

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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

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. The microviscosity (η) was calculated

η=2P/(0.46-P)

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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

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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

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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

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

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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

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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

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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

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

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

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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

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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

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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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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immediately monitored every second by spectrofluorimeter.

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

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

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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

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

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

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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

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

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

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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,

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

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

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Considering the appearance of hydrophobic interaction accompanied by crimped

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

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

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supported by the earlier research which concluded that the involvement of

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hydrophobic interaction could promote the physical adsorption and deposition of

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

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

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

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

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chitosan coils might be correlated with the enhancement of hydrophobic interaction

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

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

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

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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.

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Figure captions

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

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corresponding EPR spectrum.

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

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

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

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

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

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

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mean value ± standard deviation (n=3).

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

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

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mean value ± standard deviation (n=3).

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

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

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

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

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

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

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size 5 µm×5µm.

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

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after 5 h incubation at 37 oC.

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

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

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

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

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

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

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

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CF fluorescence was immediately monitored every second by spectrofluorimeter.

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Figures

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Figure 1. 5-DSA

16-DSA

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Figure 2. 5 ANS T MA-DP H

Relative microviscosity

4 3 2 1 0 0

633

1 2 3 Chitosan concentration (mg/mL)

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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

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1 2 3 Chitosan concentration (mg/mL)

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Rotational correlation time (ns)

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Figure 4. 15.0 5-DSA 16-DSA

a0 (G)

14.5

14.0

13.5 0

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1 2 3 Chitosan concentration (mg/mL)

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Figure 5.

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Figure 6.

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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)

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Figure 8.

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Figure 9. 40

8

CF release (%)

4

CF release (%)

0.0 0.5 1.0 1.5 2.0 3.0 4.0

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0.0 0.5 1.0 1.5 2.0 3.0 4.0

30

20

10

2 A

0 0

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B

0

10 20 Incubation time (min)

30

0

20

40 60 80 100 Incubation time (sec)

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Graphic for table of contents

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