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Natural poly- and oligosaccharides as novel delivery systems for plant protection compounds Olga Selyutina, Irina Apanasenko, Salavat Khalikov, and Nikolay E. Polyakov J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02591 • Publication Date (Web): 21 Jul 2017 Downloaded from http://pubs.acs.org on July 28, 2017

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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Journal of Agricultural and Food Chemistry

Natural poly- and oligosaccharides as novel delivery systems for plant protection compounds

O. Yu. Selyutinaa*, I. E. Apanasenkoa, S. S. Khalikovb, N. E. Polyakova a

Voevodsky Institute of Chemical Kinetics and Combustion of Siberian Branch of the Russian Academy of Sciences, Institutskaya St., 3, 630090, Novosibirsk, Russia, e-mail: [email protected] b

Nesmeyanov Institute of Organoelement Compounds of Russian Academy of Sciences, Vavilova St., 28, 119334, Moscow, Russia

*

Corresponding author (Tel: +7(383)333-29-47; Fax:+7(383) 330-73-50; E-mail:[email protected])

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Abstract

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To increase the bioavailability of plant protection products we have applied new approach

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based on non-covalent association with natural water soluble polysaccharides and

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oligosaccharides as delivery systems (DSs). The mechanochemical technique has been

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applied to prepare the solid state nano-dispersed compositions of antidote 1,8-naphthalic

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anhydride (NA) with arabinogalactan, sodium salt of carboxymethylcellulose, and

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glycyrrhizin as DSs. The effect of DSs on the solubility and the penetration of NA into the

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seeds of barley and wheat has been investigated by various physicochemical techniques. All

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DSs considerably enhance the solubility of NA and improve its penetration into the grain.

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The influence of polysaccharides and oligosaccharides on artificial lipid membrane was

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studied by NMR relaxation method. It was concluded, that the effect of polysaccharides and

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oligosaccharides on the penetration efficacy of plant protection products might be associated

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with the detected solubility enhancement and the affinity of DSs to the surface of cell

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membranes.

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Keywords

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1,8-naphthalic anhydride; antidotes; delivery systems; glycyrrhizic acid; arabinogalactan; sodium salt of

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carboxymethyl cellulose; nuclear magnetic resonance; mechanochemistry.

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Introduction

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Nowadays more than 30 thousand of compositions based on more than 600 active agents are

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involved in protection of plants. But all over the world increasingly high demands are made

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on the safety of chemicals used. Development of new forms of plant protection agents (PPA)

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may results in a more effective and safe products.

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Last decades the tremendous progress was achieved in pharmacology due to applying of

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nanosized drug delivery systems (DDS) in order to enhance the bioavailability and safety of

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the drugs1-6. The idea is based on using water soluble carriers (polymers, micelles and so on)

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to increase the solubility of low soluble drugs and their permeation ability7-19. The use of

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nanotechnology in the agricultural sector has lagged far behind, despite the growth of

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scientific papers on the subject10,

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agriculture started less than a decade ago20. The authors point out the obvious prospects for

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the introduction of nanotechnology in agriculture. Nanotechnological approach allows to

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reduce the use of agricultural inputs (pesticides, herbicides, fertilizers, etc.) by increasing the

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efficiency of its delivery to the plants22. Thus, in the field of plant protection significant

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decrease consumption rates of plant protection products and their harmful effects on the

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environment are indicated as benefits. In the present study we have applied the same

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approach to increase the effectiveness of known antidote 1,8-naphthalic anhydride (anhydride

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of naphthalene-1,8-dicarboxylic acid (NA), Fig. 1). NA helps to accelerate the detoxification

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of herbicides in cereals and increase harvest resistance to them.

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Large amount of experimental data indicates that NA is the most effective agent for

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neutralizing the phytotoxic action of sulfonylureas among the commercial antidotes23.

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Antagonistic effect of NA on plant hormones associated with the implementation of activity

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. Research on the applications of nanotechnology in

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auxin-like herbicides; it can also restore the synthesis of lipids and inhibit absorption of

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herbicides by plants under seeds treatment24.

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We focused our attention on associates of NA with various delivery systems (arabinogalactan

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(AG), glycyrrhizic acid (GA), and sodium salt of carboxymethyl cellulose (Na-CMC)) which

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demonstrated high effectiveness as the DDS7-19. We compared permeation of NA into the

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seeds in pure form and in the form of associates mentioned above. Also we have made an

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attempt to study the interaction (affinity and penetration) of these delivery systems with

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model lipid membrane.

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Glycyrrhizic acid (GA, glycyrrhizin (Figure 2a)) is a triterpene saponin extracted from the

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licorice root. Glycyrrhizin has a number of properties that make it an attractive delivery

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system. The key feature is the amphiphilicity of glycyrrhizin molecule: the hydrophilic part

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consists of glucose rings, hydrophobic one is a glycyrrhetic acid residue (Fig. 2a). Owing to

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this, glycyrrhizic acid is capable to form micelles in water solution and "host-guest"

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complexes with various hydrophobic molecules7-11.

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Different studies show that formation of GA complexes with drugs results in a significant

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increase in their solubility, enhancement the therapeutic effect and, consequently, reduce the

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therapeutic doses of drug8-12. In addition, previous studies showed increase of the cell

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membrane permeability for small molecules in the presence of GA and increase of lipid

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mobility in model cell membranes (liposomes) after GA treatment12-14.

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Arabinogalactan (AG, Fig. 2b) is a natural polysaccharide consisting of arabinose and

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galactose fragments. AG demonstrates strong binding with different hydrophobic biologically

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active molecules which results in significant increase of their solubility and oxidative

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resistance15-17. AG also increases the permeability of cell membranes for small molecules18. 5

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So, GA and AG are natural compounds which are known to form complexes with different

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drugs and to influence on cell permeability25-28. We hope that they could be used as effective

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delivery systems (DS) for plant protection agents also. For preparation of water soluble solid-

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state nanodispersed compositions, the mechanochemical technique was applied. This

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approach permits solid-state preparation of water soluble compositions of insoluble

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compounds in one technological step without use of any organic solvents27.

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Materials and methods

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Materials

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1,8-Naphthalic anhydride (Shenzhen Sunrising Industry Co., Ltd. China, purity > 96.0%) was

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used in the present study. Arabinogalactan of the Siberian larch, glycyrrhizic acid and sodium

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salt of carboxymethyl cellulose (Na-CMC) (CEKOL 700, pharmacopeia purity,

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http://cpkelco.com/products/cellulose-gum.1) were used for mechanochemical modification

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of NA.

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Liposomes were formed from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC,

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Avanti Polar Lipids, purity > 99%).

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Preparation of NA compositions

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The method of water soluble complex preparation used in the present work was a

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mechanochemical treatment of the solid mixture of NA crystals with polysaccharides or

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oligosaccharides powder. Typical mechanochemical reactions are those activated by co-

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grinding or milling of powder materials27-29. In the present study, we used electro-

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mechanochemical equipment. Co-grinding of solid materials results in penetration of NA

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molecules into the polymer without use of any organic solvents. This approach allowed us to 6

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prepare the water soluble composites in one technological step. For preparation of NA

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complexes pre-mixed powders of 1,8-naphthalic anhydride and delivery systems (AG, Na-

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CMC, GA) with mass ratios 1:9, 1:2 and 1:9, respectively, were loaded into a caprolon

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cylinder of the roller mill LE-101 with metal balls. The cylinder was filled on 65%,

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activation modulus was 1:15. Total mass of metal balls was 600 g, ball diameter was about

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12-15 mm. Then physical mixture was activated for 7 hours. The rotation rate of cylinder

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was in range 60-80 rpm. The described procedure resulted in nano-dispersed powder

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composition of NA with delivery systems. The particle size (dispersion composition) of the

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particles was determined using a laser particle size analyzer Microsizer-20la ("BA Instalt",

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St. Petersburg).

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Solubility analysis was performed using HPLC technique (Agilent 1200; column Hypersil

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5µ HyPURITY Elite C18 (150×4.6 mm); eluent acetonitrile/water 1:1, 1 mL/min, T = 30

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°C; detection at 230 and 340 nm). Blue Ribbon Quantitative Filter Paper was used for fine

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precipitates separation.

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Preparation of grain samples

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Experiments were done with wheat “Omsk 36” and spring barley “Acha”. Barley and wheat

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grains (1.5 g) were placed in tube with 0.1 mL of NA complex dispersions or with water

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dispersion of NA. Equal amounts of NA were used for all experiments. Then the tube was

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rotated for 15 minutes for homogeneous putting of agents on samples. Then grains were

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dried for 24 hours. After that 0.5 g of grains was washed with acetone to obtain solution of

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NA remained on the surface of grains. Then washed grains were milled and mixed in

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acetone for 24 hours to extract NA from the inside of grains. Grain solids were removed by

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centrifugation. 7

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Another 0.5 g of treated grains was placed in Petri dish in humid atmosphere for 3 days for

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germination. Then washing and extract of NA were prepared in the way described above.

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Washed and extracted samples in acetone were analyzed by means of NMR. 1H NMR

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spectra were recorded on Bruker Avance III 500 MHz spectrometer.

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Each NMR sample contains extract from ~50 grains. All experiments were repeated twice

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and resulted values were calculated as an arithmetic mean, and resulted error was calculated

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as mean value of least squares errors, the differences were considered statistically significant

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at p < 0.01 using a t-test.

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Preparation of liposome samples

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Powder components were pre-dissolved in chloroform. After removing the solvent, the dry

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lipid film was hydrated with D2O. The final concentration of lipid was 10 mM. The

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suspension was then sonicated (about 37 kHz, 1 hour) to obtain unilamellar liposomes. NMR

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spectra were recorded for samples of 0.6 mL of vesicle suspension supplemented with 4 mM

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PrCl3 in 5-mm NMR tubes. After addition of PrCl3, the liposome suspension was

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supplemented with different delivery systems (GA, AG, Na-CMC).

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NMR study

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H NMR spectra were recorded on Bruker Avance III 500 MHz spectrometer, spin-spin

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relaxation time T2 was measured by the Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence.

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Deuterated solvent (D2O (99.9% D), Acetone-D6 (99.8% D), Aldrich) were used as received.

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The study of lipid mobility

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Spin-spin and spin-lattice relaxation times (T1 and T2) are sensitive to molecular motions.

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The T1 relaxation parameter is sensitive to high frequency lipid motions such as trans-gauche

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methylene isomerization, whereas the T2 time is sensitive to low frequency (e. g., large

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amplitude chain wagging) lipid motions30,

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functional groups (-N+(CH3)3-group in lipid polar head and terminal CH3-groups in

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hydrophobic tails) were measured for characterization of delivery systems influence. The

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spin-spin relaxation time T2 is closely related to the mobility of the molecule and is inversely

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proportional to the rotational correlation time. Thus, using T2 data, one can form conclusions

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about changes of environment or state (free / bound) of molecules. The ability to resolve lipid

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resonances in the inner and outer headgroup region using shift-reagent and in the terminal

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methyl groups has allowed us to study the effect of delivery systems on lipid motions from

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the surface to the center of the membrane. Measurement temperature was 300 K.

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Statistical analysis

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independent experiments, and the differences were considered statistically significant at p