Accepted Manuscript

Biodegradable chitosan-glycolipid hybrid nanogels: A novel approach to encapsulate

fucoxanthin for improved stability and bioavailability

Ravi Hindupur, Vallikannan Baskaran

PII: S0268-005X(14)00279-3
DOI: 10.1016/j.foodhyd.2014.08.004
Reference: FOOHYD 2687

To appear in: Food Hydrocolloids

Received Date: 8 March 2014

Accepted Date: 1 August 2014

Please cite this article as: Hindupur, R., Baskaran, V., Biodegradable chitosan-glycolipid hybrid
nanogels: A novel approach to encapsulate fucoxanthin for improved stability and bioavailability, Food
Hydrocolloids (2014), doi: 10.1016/j.foodhyd.2014.08.004.

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1 Biodegradable chitosan-glycolipid hybrid nanogels: A novel approach to
2 encapsulate fucoxanthin for improved stability and bioavailability

3 Ravi Hindupur and Vallikannan Baskaran*

5 Department of Molecular Nutrition,

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6 CSIR-Central Food Technological Research Institute,

Mysore-570020,

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8 Karnataka,

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9 India.

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13 Corresponding author. Tel: +91-8212514876; fax: +91-8212517233.

14 E-mail: baskaranv@cftri.res.in (V. Baskaran).

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1 Abstract

2 This study aimed to improve the stability and biological availability of

3 carotenoid fucoxanthin (FUCO) encapsulated in chitosan (CS) - sodium
4 tripolyphosphate (TPP) - glycolipid (GL) nanogels, prepared by ionic gelation
5 method. Scanning Electron Microscopic analysis and Dynamic Light Scattering
6 examination revealed smooth and spherical nanogels with size range of 200-550

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7 nm in weight ratios of CS: TPP (2.5:1), CS: GL (1:0.5). The zeta potential
8 values (30 to +50 mV) indicate that CS-NGs with FUCO+GL were more stable
9 than that of CS-NGs+ FUCO with no GL (+15 mV). The Fourier Transform

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10 Infrared Spectroscopy (FTIR) showed an extensive hydrogen bonding
11 interaction between the FUCO and CS. X-ray Diffraction revealed FUCO is

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12 distributed in a disordered (amorphous) state in CS-NGs. Encapsulation
13 efficiency, loading capacity and the yield of CS-NGs with FUCO+GL were
14 90%, 47% and 70%, respectively which is, significantly higher, than that of
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CS-NGs+FUCO without GL. Stability studies illustrated that glycolipid offers
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16 enhanced FUCO stability (t1/2, 45 h) with CS-NGs compared to that of with no
17 GL (t1/2, 15 h) and standard FUCO (t1/2, <5 h). The bioavailability of FUCO in
18 vitro from CS-NGs with GL was higher (68%) compared to that of CS-
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19 NGs+FUCO without GL (51%), FUCO with GL (35.5%) and control (21.5%).

20 In conclusion, the stability and bioavailability of FUCO was improved by
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21 nanoencapsulation of FUCO with CS+GL.

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23 Keywords:
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24 In vitro Bioavailability; Stability; Fucoxanthin; Chitosan; Nanogels; Glycolipid

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1 1. Introduction

2 Fucoxanthin (FUCO), a non-provitamin A, allenic and epoxy marine

3 xanthophyll carotenoid, exerts beneficial effects including hypolipidemic (Woo
4 et al., 2010), anti-obesity (Maeda, 2009), anti-diabetic (Maeda, Hosokawa,
5 Sashima, & Miyashita, 2007) and anti-carcinogenic effects (Miyashita,
6 Nishikawa, Beppu, Tsukui, Abe & Hosokawa, 2011). However, FUCO has

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7 limited bioavailability due to its lipophilic nature, and its biological availability
8 can be enhanced by incorporating lipids as carriers and polysaccharides as
9 encapsulating/coating matrices with the fact that lipids improve carotenoid

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10 bioavailability (Gorusupudi & Vallikannan, 2012). β-Carotene bioavailability
11 improved with the dietary fat incorporation (Ribaya-Mercado, 2002). The

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12 absorption of dietary lutein, and its accumulation in adult rats is influenced by
13 olive oil (Lakshminarayana, Raju, Krishnakantha and Baskaran, 2007). Lutein
14 absorption and activity of antioxidant enzymes in rats is influenced by
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phospholipid, oleic acid micelles and dietary olive oil (Lakshminarayana, Raju,
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16 Prakash, & Baskaran, 2009; Nidhi, Mamatha, & Baskaran, 2013). Gorusupudi
17 & Vallikannan (2012) studied the effect of wheat germ oil and its glycolipid
18 fraction in mice and reported an improved lutein absorption and tissue
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19 accumulation. Similarly, entrapment of carotenoids with the polymers reported

20 improving the stability and bioavailability of carotenoids (Arunkumar, Harish
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21 Prashanth, & Baskaran, 2013). These studies reveal that specific lipids and
encapsulation helps in improving biological potential of carotenoids.
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23 Encapsulation in the form of liposomes, micelles or nanogels, offers

24 targeted delivery of nutrients/drugs to the body. Nanogels (NGs) is known to
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25 transport and deliver drugs, which are unstable in the biological systems and
26 cannot readily diffuse across the intestinal mucosal barrier (Prego, Torres, &
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27 Alonso, 2005). Oral NGs are promising nutrient/drug delivery systems due to
28 improved bioavailability, targetability, bioadhesion which execute the
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29 controlled release of them in the gastrointestinal tract (Mozafari, Khosravi-

30 Darani, Borazan, Cui, Pardakhty, & Yurdugul, 2008; Chaudhry et al., 2008).
31 They can be directly adhered to the mucosa, which is a pre-requisite step before
32 the translocation process of nanogels to occur. Further, encapsulation of
33 carotenoids enhances the stability of encapsulated bioactives by protecting them
34 from light, heat, low pH (gastric) and ionic variation (during food processing)
35 when interact with other molecules (Yurdugul & Mozafari, 2004). In addition,
36 the advantages of encapsulation are to (1) reduce the reactivity of the core

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1 material with the environment (light, heat and water), (2) reduce the loss or
2 degradation of the core compound, (3) promote easier handling/processing, (4)
3 control the release of active compound and (5) mask the core taste (Wilson &
4 Shah, 2007).

5 Astaxanthin was microencapsulated with the chitosan matrix (Higuera-

6 Ciapara, Felix-Valenzuela, Goycoolea, & Argüelles-Monal, 2004) for stability

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7 enhancement (Kittikaiwan, Powthongsook, Pavasant, & Shotipruk, 2007).
8 Chitosan-dextran sulphate nanoparticles have been used for controlled delivery
9 of bioactive molecules and cells in bone regeneration (Valente, Gaspar,

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10 Antunes, Countinho & Correia, 2013). Low molecular weight chitosan has been
11 used to improve the bioavailability of lutein (Arunkumar et al., 2013). The

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12 available literature vividly demonstrates the potential application of the polymer
13 chitosan for the protection of carotenoids.

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14 Chitosan (CS), a cationic polysaccharide, is derived from deacetylation of
naturally occurring biopolymer chitin, which is composed of glucosamine
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16 namely, 2-amino-2-deoxy-(1,4)-β-D-glucopyran. It is the most widely
17 distributed biopolymer, and it is non-toxic, exhibit biocompatibility,
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18 biodegradability, non-immunogenic, non-carcinogenic and antibacterial

19 properties (Sun & Wan, 2007). The phenomenon that CS dissolves in low acidic
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20 solution at pH less than 6.5 may perhaps be exploited in formulating CS-NPs by

21 cross-linking with anionic compounds such as tripolyphosphate (TPP),
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22 polyaspartic acid sodium salt (Hu et al., 2008). In brown seaweed

23 (Pheaphycaeae), FUCO, is in association with the complex sulphated
24 polysaccharide fucoidan, which protects FUCO from various environmental
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25 factors. So, similar approach is applied in this study using a biodegradable

26 polymeric CS that complexes with FUCO thereby rendering its protection from
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27 light, heat, etc. Hence, CS would be the ideal polysaccharide for encapsulation
28 of lipophilic carotenoid FUCO. It is expected that the CS with FUCO
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29 encapsules are nano in size and may favour the mucosal and epithelial cells
30 uptake and intracellular trafficking of FUCO.

31 The present study aims at the preparation of nanoencapsulated FUCO

32 with CS and glycolipid as a surfactant and carrier. Earlier, we have reported that
33 wheat germ oil and its constituent glycolipid fraction enhance the bioavailability
34 of lutein in mice model (Gorusupudi & Baskaran, 2013). Hence, in this study,
35 wheat germ oil, being the rich source of glycolipids, has been exploited for
36 dispersion of FUCO before being encapsulated to augment its stability and
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1 bioavailability (simulated digestion model). Further, this is the first study
2 developed on hybrid nanogels for encapsulation of FUCO with chitosan-
3 glycolipid as carriers for improved stability and bioavailability. The findings
4 will have an impact in nutritional and pharmacological applications, where
5 improved FUCO absorption is essential.

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7 2. Materials and methods

8 2.1. Materials

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9 Chitosan (CS) (deacetylation degree ≥ 75%) derived from crab shell was

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10 purchased from Himedia laboratories, India. Methanol, acetonitrile (HPLC
11 grade), sodium sulphate, ammonium acetate, acetone, methanol, diethyl ether,
12 ethyl acetate, acetic acid, chloroform and silica (60-120 mesh) were purchased

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13 from Sisco Research Laboratory, Mumbai, India. Hexane was purchased from
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14 Rankem Laboratories, Mumbai, India. Penta-sodium triphosphate (TPP), pepsin
15 (porcine), bile extract (porcine) and pancreatin (porcine) were purchased from
16 Sigma–Aldrich, St. Louis, USA. Groundnut oil (GNO), food grade, was
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17 obtained from the super market (Mysore, India). Standard FUCO from brown
18 seaweed wakame was donated by Dr. T. Sugawara, Kyoto University, Japan.
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19 Indian brown seaweed for the FUCO extraction was collected from Mandapam
20 coastal region, Tamilnadu (9.28° N, 79.12° E), India. Millipore water was used
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21 for HPLC analysis. Other chemicals were of analytical grade unless otherwise
22 mentioned.
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24 2.2. FUCO extraction from Indian brown seaweed Padina tetrastomatica
25 Seaweed was collected off the coastal region of Mandapam (9.28° N,
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26 79.12° E, Tamilnadu, India), washed in de-ionised water, dried on blotting

paper followed by air drying and then ground to a fine powder. FUCO was
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28 extracted from the dried seaweed powder (100 g) with the method developed in
29 this study, with ice cold acetone: methanol (7:3) by swirling at 100 rpm in
30 shaking water bath (Scigenics Orbitek, India) three times at 40C for 2 h
31 (modified procedure of Haugan, Aakermann, & Liaaen-Jensen, 1992). The
32 pooled extract was evaporated to dryness at 300C in a flash evaporator (Hahn-
33 Shin, HS-2005V-N, Korea) and re-dissolved in methanol (200 mL) followed by
34 the addition of 20 mL of water and 200 mL hexane in a separatory funnel and
35 swirled 4 times. To the lower methanol-water phase, water (300 mL) and

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1 diethyl ether (400 mL) were added, swirled and the upper ether phase with
2 FUCO and other carotenoids was collected, flash evaporated at 300C, and the
3 crude extract obtained was dissolved in 5 mL of hexane (a few drops of acetone
4 were added if a residue is formed and isopropanol was added to remove water
5 moiety, if any).

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7 2.3. FUCO Purification by Open Column Chromatography

FUCO was purified from the crude extract by Open Column

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9 Chromatography (OCC) by the use of specific solvent systems with silica gel
10 (particle size 60-120 mesh) (Sangeetha, Bhaskar, & Baskaran, 2009). In brief,

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11 an aliquot of crude extract (5 mL) was applied to open column chromatography
12 (20 cm x 1.5 cm), the chlorophylls were eluted first with the hexane (200 mL)

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13 (modified procedure of Maeda et al, 2005) followed by hexane: acetone (9:1)
14 and hexane: acetone (8:2), respectively to elute β-carotene. Finally, hexane:
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15 acetone (7:3) was used to elute FUCO as the brownish yellow band. The FUCO
16 elute was concentrated by flash evaporator and made to 5 mL with the same
solvent system and analyzed by HPLC (procedure given). The respective
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18 spectra and absorption maxima (λmax) of FUCO were recorded and used for the
19 confirmation of purity by photodiode array (PDA) detector, and the same has
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20 been compared with the standard FUCO based on the peak area.
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22 2.4. HPLC analysis of FUCO

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23 An aliquot of crude extract, and the purified FUCO by Open Column

24 Chromatography and TLC was evaporated under a stream of nitrogen and
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25 redissolved (100 μL) in acetonitrile: methanol: water 60:35:5 (v/v/v) containing

26 0.1% ammonium acetate (mobile phase) and the extract (20 μL) was injected to
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27 HPLC system (LC-10Avp; Shimadzu, Kyoto, Japan) equipped with photodiode

28 array (PDA) detector (SPD-M20A, Shimadzu). FUCO was separated on a
29 Princeton C30 (ODS) column (250 mm × 4.6 mm; 5µm) isocratically eluting
30 with 1 ml/min of mobile phase. FUCO was monitored at 446 nm using
31 Shimadzu Class-VP version 6.14SP1 software. The peak identity of FUCO was
32 confirmed by their UV-Vis spectra recorded with the PDA detector.

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1 2.5. Extraction and purification of glycolipid from wheat germ

2 Germinated wheat was used to extract the total lipid as per Folch, Lees, &
3 Stanley (1957). Wheat germ (100 g) was dried in an oven at 370C overnight.
4 Acetone: methanol (200mL, 7:3) solvent system was used to extract total lipid,
5 after swirling at 40C for 2 h. The extract was filtered, pooled, flash evaporated,
6 and the residue was dissolved in 100 mL of ethyl acetate: water (1:1, v/v). The

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7 ethyl acetate fraction was concentrated to obtain wheat germ oil (WGO) crude
8 and the residue was dissolved in hexane: ethyl acetate (10 mL, 9:1, v/v).

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9 WGO was subjected to further purification by Open Column
10 Chromatography (OCC) to fractionate glycolipids (Sugawara & Miyazawa,

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11 1999). Pre-activated silica gel of 60-120 mesh size was used to make a slurry
12 with chloroform and packed onto the column (100 cm length x 5 cm diameter).
13 The column was equilibrated with chloroform (1 mL/min) and WGO was

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14 poured onto the column. Neutral lipids were first eluted with chloroform
fraction, which was then, followed by glycolipids and phospholipids fractions
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16 with acetone and methanol, respectively. This fraction of glycolipid was further
17 used in encapsulation studies as a carrier and surfactant to improve the stability
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18 and bioavailability of FUCO.

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20 2.6. Preparation of CS-glycolipid-FUCO hybrid nanogels

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21 CS-NGs were prepared as per Calvo, Remuñán-López, Vila-Jato, &

22 Alonso (1997) with slight modification. The preparation of CS-NGs was based
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23 on an ionic interaction between positively charged CS and negatively charged

24 sodium tripolyphosphate (TPP) solution (ionic gelation method). On
standardising the concentrations of CS and glycolipid, CS (0.1%) was dissolved
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26 in 1% acetic acid solution, and the glycolipid (0.5 mg) (chitosan-glycolipid ratio
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27 = 1:0.5) was added to the CS solution and stirred well until it was completely
28 dispersed with CS solution. TPP (0.03%) was dissolved in double distilled
29 water, and its pH was adjusted to 5.5. FUCO (1 mg) was directly dissolved in
30 TPP solution before formulation of CS-NGs. TPP solution (2 mL) containing
31 FUCO was added drop-wise in 5 mL of CS solution (weight ratio of CS: TPP
32 was 2.5:1) under magnetic stirring (600 rpm) at room temperature (250 C)
33 resulting in the formation of CS-NGs instantaneously, collected by
34 centrifugation at 10,000 rpm at 40 C for 45 min and freeze-dried for further
35 analysis.
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1 2.7. Morphology of CS-TPP nanogels

2 Scanning Electron Microscopy (SEM) was used to find out the structure
3 and morphology of CS-NGs with FUCO+GL. The lyophilized dry CS-NGs with
4 FUCO+GL were spread on a double-sided conducting adhesive tape, pasted on
5 a metallic stub, coated (100 mm) with gold in a sputter coating unit for 2 min
6 and observed in an LEO- 435-VP (LEO Electron Microscopy Ltd., Cambridge,

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7 UK) electron microscope using an accelerated voltage of 20 keV.

8 2.8. Particle size and Zeta potential measurement

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9 Average particle size and zeta potential of CS-NGs loaded with and
10 without FUCO (with and without glycolipid) were measured by Dynamic Light

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11 Scattering (DLS) using Nano-ZS (Malvern Instruments, UK) to measure the
12 surface charge and the zeta potential of the CS-NGs.
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To understand the possible interaction between the CS, FUCO and GL,
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16 the FTIR analysis was performed. CS-NGs dispersed in glycolipid (i) with
17 FUCO and (ii) without FUCO (control), was centrifuged at 10,000 rpm for 45
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18 min, the supernatant was discarded, and the pellet was centrifuged and
19 lyophilised for 24 h. The FTIR spectra of the samples were measured using an
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20 FTIR (Nicolet 5700-IR, Thermo Scientific, USA). Briefly, a small quantity of

21 CS-NGs with FUCO (2-5 mg) was mixed with KBr and compressed to form
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22 pellets. These pellets were scanned in transmission mode and in the spectral
23 region of 400-4000cm-1 using a resolution of 4 cm-1.
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25 2.10. XRD analysis

26 The physical state of FUCO in the CS-NGs matrix was assessed by X-ray
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27 diffraction (XRD) technique. XRD spectra of standard FUCO, standard CS, CS-
28 NGs dispersed in glycolipid (i) with FUCO and (ii) without FUCO (as a
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29 control) were obtained at room temperature (250 C) using X-ray diffractometer

30 (Rigaku Miniflex II desktop X-ray diffractometer, Japan) with Cu as a target at
31 a voltage of 30 kV with 15 mA current. The samples were analysed in 2θ angle
32 range of 6-600 at a scanning speed of 50/min with scan axis of 2θ/θ.
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34 2.11. FUCO entrapment efficiency, loading capacity and the yield
35 CS-NGs loaded with (a) FUCO (1 mg)+GL and (b) FUCO (1 mg)
36 without GL (control), was separated by centrifuging at 10,000 rpm at 40C for 45

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1 min and the FUCO entrapment efficiency (EE) and the loading capacity (LC) of
2 CS-NGs were evaluated by measuring the absorption of the supernatant with
3 UV-Vis spectrophotometer (UV-1800, Shimazdu, Japan) at 446 nm.
4 FUCO from the supernatant was extracted by the addition of hexane (1.8
5 mL) and methanol (2 mL), vortexed, centrifuged at 2000 rpm for 3 min. The
6 upper hexane layer was discarded and hexane: methanol: water (10:10:1) was
7 added to the lower methanol phase, vortexed and centrifuged. The upper hexane

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8 phase was discarded, and the extraction procedure was repeated twice. The
9 lower methanol: water phase (1 mL) was collected and evaporated to dryness

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10 and re-dissolved in acetonitrile: methanol: water (60:35:5, v/v/v) with 0.1%
11 ammonium acetate (mobile phase) and analysed by HPLC.

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13 EE was calculated using an equation:
14 EE (%) = TF-FF/TF x 100

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15 Where TF is the total concentration of FUCO and FF, is the free FUCO in
the supernatant (which is not entrapped).
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17 Loading capacity (LC) was calculated as:
18 LC (%) = TF-FF/weight of NPs retrieved x 100
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19 Yield (w/w) was calculated from the weight of dried NGs recovered (W1)
20 and the sum of the initial dry weight of the starting material (W2) as:
Yield (%) = W1/ W2 x 100
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23 2.12. Swelling studies

24 To determine the swelling activity of CS-NGs with FUCO (1mg) +GL,
25 they were kept at 370 C in the media of pH 1.2 (HCl: potassium chloride buffer),
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26 4 (acetate buffer) and 6.8 (phosphate buffer) for 24 h. The pre-weighed NGs (5
27 mg) were placed in each media, and the water activity was measured at 0, 4, 8,
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28 12 and 24 h intervals. Samples were centrifuged at 10,000 rpm for 45 min in

29 pre-weighed centrifuge tubes, and the wet weights were determined after
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30 decanting the supernatant. The percentage swelling of NGs was then calculated
31 gravimetrically using the following equation:
32 Esw (%) = Ww – W0/W0 x 100
33 Where, Esw is the percent swelling of NGs, W0 is the initial weight of NG
34 and Ww is the wet weight.
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1 2.13. Stability studies
2 To evaluate the stability of NGs, CS-NGs loaded with FUCO (i) with GL,
3 (ii) without GL and the standard FUCO (0.5 mg) was incubated in 30 ml
4 stoppered tubes, in a water bath, at 370C. At time intervals of 0, 2, 4, 6, 8, 12,
5 24, 48, 72 and 144 h, the samples were drawn, centrifuged at 10,000 rpm at 40C
6 and the supernatant was extracted for FUCO (as per the procedure given
7 elsewhere) and analysed by UV-Vis spectrophotometer at 446 nm and by

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8 HPLC, as well.
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10 2.14. In vitro bioavailability
11 The aim of this study was to investigate the effect of nanoencapsulation
of FUCO with glycolipid (GL) on the micellization of FUCO in vitro (simulated

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13 gastric and intestinal digestion) that is accessible for intestinal uptake, in
14 comparison with CS-NGs with FUCO (-GL), FUCO+GL alone (with no CS-

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15 TPP matrix) and FUCO dispersed in groundnut oil (GNO) as a control (with no
CS-TPP matrix). To determine the in vitro FUCO micellization in CS-NGs with
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17 GL and with no GL, the purified FUCO (200 nM) was solubilised in GL (25
18 mg) and GNO (25 mg). In brief, in a 30 mL screw capped vials, FUCO (200
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19 nM) was added to CS-NGs with glycolipid (25 mg). The samples were
20 subjected to in vitro digestion simulating the gastric and intestinal phase of
digestion according to the method proposed (Garrett, Failla, & Sarama, 1999;
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22 Nidhi & Baskaran, 2011) with slight modification. Groundnut oil (GNO) (25
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23 mg) containing 200 nM FUCO was taken as control. In brief, 3 mL of 0.5%

24 pepsin (porcine gastric mucosa 88–2,500 units/mg protein) in phosphate buffer
25 (3.6 mmol/L CaCl2, 1.4 mmol/L MgCl2.6H2O, 49 mmol/L NaCl, 12 mmol/L
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26 KCl, 6.4 mmol/L KH2PO4) was added to the samples. The pH of the digesta was
27 adjusted to 2.02 with 2 mol/L HCl. The tubes were screw capped under a stream
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28 of nitrogen and incubated for 1 h at 370C in a shaking water bath (Scigenics

29 Orbitek, India) at 120 strokes/min (gastric phase). On cooling, the pH was
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30 raised to 5.0 with 1 mol/L NaHCO3 followed by the addition of 6 mL 0.1 mol/L
31 NaHCO3 containing 16 g/L pancreatin (porcine pancreas 89 U.S.P.
32 specifications) and 25.38 g/L bile extract (porcine). Then the pH of the digesta
33 was further adjusted to 7.5 by 1 N NaOH. The test tubes were blanketed with a
34 stream of nitrogen and subjected to incubation at 370C with shaking at 120
35 strokes/ min for 2 h (intestinal phase). After incubation, an aliquot of digesta (1
36 mL) was withdrawn from each sample, centrifuged (Z 360 K, BHG Hermle,
37 Gosheim) at 12,000 x g at 40C for 10 min to separate the fraction that contain

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1 micelles and this fraction was used for quantification of micellized FUCO by
2 HPLC. The procedure for the extraction of FUCO from digesta is given
3 elsewhere.
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5 2.15. Statistical analysis
6 All the experiments were performed in triplicate. The data were
7 compared with one-way analysis of variance (ANOVA) using Graphpad prism

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8 software version 5.0 and significant difference (p< 0.05) among the groups was
9 evaluated by Turkey’s test.

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11 3. Results and discussion

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13 3.1. FUCO loaded CS-NGs+GL
14 The concentration of FUCO extracted from seaweed was 18 ± 2 mg/100g

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15 (dry weight basis). The purity of FUCO separated by OCC further confirmed by
HPLC and is 92% pure. The total lipid content of wheat germ was 10 ± 2% (dry
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17 weight basis) in which glycolipid was 70% and its purity was 95%. The cationic
18 CS (0.1%) was completely dispersed in glycolipid (CS: glycolipid- 1:0.5) and
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19 the anionic TPP (0.03%) with FUCO (Fig. 1a, b & c) in weight ratios of 2.5:1
20 (CS: TPP) added to CS solution drop-wise resulted in the formation of NGs
with the size range of 200-550 nm (Fig. 2a & b). The reason for choosing
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22 glycolipid in this study is to improve the solubility and the absorption of FUCO
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23 (Gorusupudi & Baskaran, 2013) and they reported that the glycolipid fraction of
24 the wheat germ significantly improved the bioavailability of lutein in mice. The
25 charge density of CS and TPP solutions predominantly determined by the pH of
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26 the solution and sufficient charge density are crucial for the anions to gelate and
27 cross-link the cationic CS, eventually leading to the CS-NGs formation. Charge
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28 density of the nanogels was reduced by a decrease in the pH of the TPP (Shu &
29 Zhu, 2002). The present results suggest that 200-550 nm nanogels can be
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30 produced with the critical concentration of 0.1% CS and 0.03% TPP at 2.5:1
31 weight ratios. CS in acidic media interacts with negatively charged TPP (pH
32 5.5) and forms intra- and intermolecular linkages resulting in ionically cross-
33 linked nanoparticles (De Campos, Sánchez, & Alonso, 2001). There may be
34 dissociation of the nanoparticles, in low and high pH conditions, due to the
35 weak interaction between CS and TPP (Zhang & Kosaraju, 2007).

36 Hydroxide (OH−) and tripolyphosphoric (P3O105− and HP3O104−) ions were

37 formed when the anionic TPP was dissolved in water. Both these OH− and
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1 P3O105− ions would competitively react ionically with the protonated amino
2 group (NH3+) of CS solution at pH 3 by deprotonation or an ionic cross linking
3 at basic pH (8-9). At the pH below 6, only the P3O105− ion can specifically cross
4 link with -NH3+ to form CS-NGs. Similar results were observed when TPP
5 dissociated in water when the pH of TPP solution was decreased to 4.5 and 5.5
6 for the formulation of bioadhesive CS-NGs loaded with catechins (Dudhani &
7 Kosaraju, 2010). CS-NGs were prepared loaded with drugs 5-fluorouracil and

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8 leucovorin blends by changing the pH of TPP to 5.5 to obtain the relatively
9 higher zeta potential value so as to cross link the CS (Li, Wang, Peng, She, &

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10 Kong, 2011). Hence, in our study, pH of the TPP solution was reduced to 5.5
11 for the successful preparation of CS-NGs.

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13 3.2. Zeta potential of FUCO loaded CS-NGs
14 Zeta potential and particle size are the two key characteristics of

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15 nanogels. In this study, the adopted ionic gelation method led to the formation
of CS-NGs involving the ionic interaction between the protonated amino group
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17 (-NH3+) of CS solution and the phosphate groups (-PO43-) of the anionic TPP.
18 The degree of neutralisation of the protonated amino groups by the anionic
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19 groups of TPP determines the surface charge. The more the concentration of the
20 chitosan, the more will be the -NH3+groups required to be neutralised by -PO43-
groups of TPP at any particular degree of deacetylation. Thus, the zeta potential
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22 value was directly proportional to the chitosan concentration (Fig. 3a) and the
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23 same is in agreement with the previous study (Hu et al., 2008). They reported
24 the surface charge of CS-TPP-NGs is determined by the degree of neutralisation
25 of -NH3+ groups by the polyanionic phosphate groups.
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26 However, zeta potential of CS-NGs was reduced by increasing CS

27 concentration, which may be due to variations in the molecular masses and the
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28 degree of deacetylation (Gan, Wang, Cochrane, & McCarron, 2005). The zeta
29 potential values were in the range of +30 to 50 mV and +15 mV (Fig. 3b & c)
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30 for CS-NGs loaded with FUCO with and without glycolipid respectively
31 illustrating that FUCO in the CS-NGs is completely dispersed with glycolipid
32 and is more stable under conditions adopted at room temperature (250 C). The
33 apparent increase in the zeta potential values of the CS-NGs of FUCO+GL may
34 be due to the presence of hydrophobic moieties especially fatty acid groups
35 attached to monogalactosyldiacylglycerol (MGDG) and
36 digalactosyldiacylglycerol (DGDG) that are rich in glycolipids. (Wang et al.,

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1 2010) also reported that cholesterol succinyl chitosan anchored liposomes has
2 higher zeta potential values compared to that of plain liposomes.
3
4 3.3. FTIR analysis
5 Fig. 4a show the standard CS peaks at 1031.9 cm-1and 1070.6 cm-1,
6 which indicate C-N stretch in amino groups, 1662.9 cm-1 (C=O stretch in
7 amide-I), 3532.3cm-1 (O-H stretch), 2915.7 cm-1 (C-H stretch), whereas peaks

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8 at 1586.5 cm-1 (N-H bend in amide-II), 1377.7 cm-1 (-CH3 symmetric
9 deformation) and 3440.3 cm-1 showed N-H stretch. Hu et al., (2008) reported

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10 that region between 3300 and 3450 cm-1 correspond to the stretching vibration
11 of amino groups. Fig. 4b shows the glycolipid peaks at 1462 cm-1 and 2925 cm-1
that indicates -CH2 stretch in alkanes. The peaks at 1400 cm-1 and 1710 cm-1

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13 show the C-O and C=O stretch in carboxylic acids (in fatty acids) and it shows
14 the similar spectrum as that of CS-NGs with FUCO indicating that there is no

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15 specific interaction between GL and FUCO.
In the case of CS-NGs after addition of TPP (Fig. 4c), the peak of amide-
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17 I was shifted to 1659.1 cm-1 from 1662.9 cm-1 (amide-I) and N-H bend in
18 amines was shifted to 1552.6 cm-1 from 1586.5 cm-1 (amide-II) representing the
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19 electrostatic interactions between the –NH3+ groups in CS with the phosphoric

20 groups in TPP.
In the case of spectra of the CS-NGs loaded with FUCO (Fig. 4d), the
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22 specific peaks at 1246 cm-1, 1736 cm-1 and 1926.8 cm-1 corresponded to C-O
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23 (carbonyl) stretching (carboxylic acid/acetate), C-O vibration stretch (acetate)

24 and C=C=C (allene bond), respectively and confirmed the presence of FUCO
25 functional groups (that is characteristic to CS-NGs with FUCO) in CS matrix
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26 (Fig 4b). Peaks at 1587.9 cm-1 and 1605.1 cm-1 illustrate the -NH2 bend (in the
27 plane) in amines and –NH bend (out of the plane) in amides reveals –OH and C-
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28 C (O)-O (acetate) groups in FUCO interact with – NH3+groups in CS. Whereas,

29 peaks at 1070 cm-1 and 1031.9 cm-1 (C-N stretch in amines) remained the same
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30 in CS as well as CS-NGs with FUCO.

31 The characteristic peaks at 3440.3 cm-1 and 3532.3 cm-1 that are in CS
32 disappeared in FUCO loaded CS-NGs and these peaks are shifted to the lower
33 region at 3433.3 cm-1 and 3507.7 cm-1 showing the interaction between
34 hydroxyl groups in the terminal rings and acetate groups of FUCO with the
35 amino groups of CS (Fig. 5) which leads to the formation of weak hydrogen
36 bonding between the FUCO and CS demonstrating that there would not be any
37 problem in releasing the FUCO from the CS-NGs.

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1 3.4. XRD analysis
2 X-ray diffractometry is a vital tool to examine the physical state of the
3 FUCO in the polymeric CS matrix, which can reveal the release kinetics of
4 FUCO from CS. Some drugs/nutrients are encapsulated in an amorphous form
5 or in solid solution while some others have a tendency to be in the crystalline
6 state, which in turn depend upon drug’s/nutrient’s properties and their
7 interaction with the polymer matrix.

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8 In this study, our findings with the XRD analysis revealed that FUCO is
9 distributed in solid state or in an amorphous (disordered) state in CS-TPP-NGs

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10 as shown by diffraction patterns in X-ray diffractometer (Fig. 6).
11 Fig. 6a shows two intense diffraction peaks at 2θ = 9.90 and 19.860 for CS
and two intense narrow peaks at 2θ = 33.280 and 19.320 for TPP (Fig. 6b)

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13 exhibiting a high degree of crystallinity. Whereas, CS-NGs loaded with
14 FUCO+glycolipid shows a broad peak at 2θ = 21.80 (Fig. 6c). However, one

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15 broad peak was observed at 2θ = 30.260 (Fig. 6d) for CS-NGs+ glycolipid
(without FUCO) indicating, the crystalline nature of CS was lost due to its cross
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17 linking with TPP. The width of XRD peak is related to the size of the crystallite
18 while the broadened peak usually implies the imperfect crystal (Jingou, Shilei,
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19 Weiqi, Danjun, Tengfei & Yi, 2011). In this study, the broadened peak of CS-
20 NGs may be due to cross linking of CS with TPP, which may eventually lead to
destruction of CS lattice properties (Rokhade, Agnihotri, Patil, Mallikarjuna,
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22 Kulkarni, & Aminabhavi, 2006). The diffraction spectrum of the standard
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23 FUCO (Fig. 6e) at 2θ = 23.420 reflects the amorphous nature of FUCO. Results
24 demonstrate that even though CS-NGs with FUCO+GL are slightly amorphous
25 compared to standard FUCO, the added advantage of CS-NGs with FUCO+GL
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26 is that it enhanced the half-life of FUCO (by 9-fold) thereby increasing stability
27 and higher percent micellization (68%) in comparison with control (21%).
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28 These diffraction patterns revealed the matrix between CS-TPP and their cross-
29 linking determines the native state of FUCO loaded in CS-NGs.
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30 Broad diffraction peak was observed in the combination of CS-NGs loaded

31 with FUCO in comparison with only two intense peaks with standard CS
32 reflecting the unique preparation process of CS-NGs involving the ionic cross
33 linking between two oppositely charged ions. Two types of ionic interactions
34 exist in this reaction namely (i) an intense electrostatic interaction between the
35 positively charged amine groups of CS and negatively charged TPP and (ii) the
36 electrostatic interactions between the positively charged amine groups of CS
37 and –OH and acetate groups of FUCO. These ionic interactions predominantly

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1 led to the cross linking, and subsequent formation of FUCO loaded CS-NGs,
2 which restrains the molecular chain movement of chitosan and FUCO. Thus,
3 FUCO was observed in an amorphous state in CS-NGs. A similar kind of XRD
4 patterns was shown when pramipexole was loaded into CS-NGs encapsules
5 (Papadimitriou, Bikiaris, Avgoustakis, Karavas, & Georgarakis, 2008) and 5-
6 flourouracil and leucovorin blends were nanoencapsulated with chitosan (Li,
7 Wang, Peng, She, & Kong, 2011).

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8
9 3.5. Encapsulation efficiency, loading capacity and yield of nanogels

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10 The encapsulation efficiency, loading capacity and the yield of FUCO
11 (1 mg) in CS-NGs +GL were found to be 90%, 47% and 70% which were
significantly higher than those of CS-NGs with no GL (control) (Fig. 7). This

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13 clearly illustrates GL, as a carrier that in association with FUCO improves its
14 entrapment efficiency, loading capacity and the yield in the CS-NGs matrix.

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15 The low yield of the FUCO loaded CS-NGs may be attributed to the
competitive effect between the hydroxyl groups (OH−) of FUCO and P3O105−
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17 groups of TPP (pH 5.5) for the protonated amino groups (-NH3+) of CS.
18 Previous study reported that the loading capacity of ammonium glycyrrhizinate
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19 was found to be 7.9% when the CS and TPP concentrations were 1.44 and 0.5
20 mg/ml (Wu, Yang, Wang, Hu, & Fu, 2005). This result clearly emphasises the
pH of the solution and TPP concentration plays a prominent role in nanoparticle
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22 formation. Xu & Du, (2003) reported that increasing degree of deacetylation of
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23 CS from 75.5 to 92% promoted the encapsulation efficiency and decelerated the
24 release rate. Encapsulation efficiency was highly decreased by an increase of
25 initial BSA and chitosan concentration. The above studies adopted similar a
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26 methodology for the estimation of encapsulation efficiency of CS-NGs.

27
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28 3.6. Swelling studies of CS-NGs

29 Water absorbing capacity of the CS-NGs loaded with FUCO (1 mg) was
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30 studied and demonstrated as 1700%, 400% and 200% (Fig. 8) swelling over 24
31 h at pH 1.2, 4 and 6.8 respectively. Studies demonstrated that CS swells more in
32 acidic pH and exhibits reduced swelling in alkaline pH in the intestine.
33 Tendency of CS polymer to uncoil to a fully extended structure exhibiting
34 higher molecular weight could be investigated from higher swelling properties
35 of CS (Agarwal & Mishra, 1999). The electrostatic interactions between the
36 anions and CS can be controlled by pH and CS/TPP film exhibited pH-
37 dependent swelling (Shu & Zhu, 2002).

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1 3.7. Stability studies
2 Half-life period (T1/2) of FUCO in CS-NGs with glycolipid was about
3 45 h at 370C (Fig. 9), which follows zero order rate kinetics (as the half-life
4 decreases with a decrease in FUCO concentration) when compared to the T1/2 of
5 FUCO in CS-NGs without glycolipid (15 h) illustrating that the glycolipid
6 offers more stability to FUCO (3 fold) by improving its solubility. In the case of
7 standard FUCO, T1/2 was < 5 h, which is 9 fold less stable compared to CS-NGs

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8 loaded with FUCO (without glycolipid) indicating the polymeric CS offers
9 protection to the core material FUCO by encapsulating it. By incorporating

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10 glycolipid to the CS-NGs, the stability was further enhanced thereby rendering
11 FUCO less sensitive to heat etc. Selim, Tsimidou & Biliaderis (2000), reported
that the kinetics of saffron carotenoids encapsulated in different amorphous

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13 polymeric matrices follow first-order kinetics. Storage stability and carotenoid
14 retention of spray-dried encapsulated paprika oleoresin followed both zero and

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15 first order rate kinetics using gum arabic and soy protein isolate as wall
materials (Rascón, Beristain, García & Salgado, 2011). Thus, degradation
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17 kinetics of the core material depends upon the encapsulation matrix.
18
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19 3.8. In vitro bioavailability studies

20 In vitro bioavailability studies illustrated that the percent micellization of
FUCO (Fig. 10) in the CS-NGs with GL (68%), which was significantly higher
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22 than the CS-NGs+FUCO with no GL (51%), FUCO+GL (35.5%) and the
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23 control (21.5%). This clearly demonstrates the FUCO entrapped in CS-TPP-

24 NGs, which is dispersed in GL is more bioavailable (which is more accessible
25 for uptake by the intestinal mucosa) than the CS-NGs+FUCO with no GL,
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26 FUCO+GL (that is non- encapsulated) and the control indicating the role of GL
27 and the bioavailability depends on the size of micelles. Oral bioavailability of
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28 curcumin improved 9-fold in nanoencapsulated form when compared to

29 curcumin with piperine as a absorption enhancer (Shaikh, Ankola, Beniwal,
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30 Singh & Kumar, 2009). Liposomal nanoencapsulation improved oral

31 bioavailability and tissue distribution of anti-cancer drug Flammulina velutipes
32 sterols (Yi, Fu, Cao,Tong, Zheng, Firempong, & Yu, 2013). Lutein
33 bioavailability and tissue accumulation in mice were improved by
34 nanoencapsulation of low molecular weight chitosan (Arunkumar et al., 2013).
35
36
37

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1 4. Conclusion
2 In this work, CS-NGs loaded with FUCO dispersed in glycolipid (GL)
3 were successfully fabricated by ionic gelation method. The particle size and its
4 structure were revealed by Scanning Electron Microscope (SEM) and zeta
5 potential of FUCO+glycolipid loaded CS-NGs by Dynamic Light Scattering
6 (DLS) studies. FTIR studies elucidated the extensive hydrogen bonding
7 interactions between the FUCO and CS and there is no specific interaction

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8 between FUCO and GL. Efficient FUCO encapsulation, loading capacity and
9 yield were significantly higher than that of CS-NGs without GL which were

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10 efficiently achieved by choosing suitable polymers. FUCO was found to be in
11 an amorphous (disordered) state by nature and it is in further more disordered
state when it was incorporated into CS-NGs, which was further authenticated by

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13 XRD results. The stability of FUCO was enhanced by incorporating glycolipid
14 in CS-NGs, which followed zero order rate kinetics. In vitro bioavailability

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15 studies demonstrate that the percent micellization is highest in CS-NGs with
FUCO+GL when compared to CS-NGs+FUCO with no GL, FUCO+GL alone
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17 and the control (GNO).
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20
Acknowledgements
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22 The authors thank the Director, CFTRI, for the encouragement. First
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23 author acknowledges the award of Junior Research Fellowship by University

24 Grants Commission (UGC) and Department of Biotechnology (DBT)
25 Government of India, New Delhi, for the financial support. We thank
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26 Dr. Mahendrakar, Ex-chief Editor, Journal of Food Science and Technology,

27 India (JFSTI) for helping in English language. We would like to thank Mr.
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28 Nandaprakash, Department of Physics, University of Mysore and Dr. Naveen,

29 DFRL, Mysore, for extending their help in XRD and zeta potential studies.
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27 Xu, Y., & Du, Y. (2003). Effect of molecular structure of chitosan on protein
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28 delivery properties of chitosan nanoparticles. International Journal of

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1 Yurdugul, S., & Mozafari, M. R. (2004). Recent advances in micro-and
2 nanoencapsulation of food ingredients. Cellular and Molecular Biology
3 Letters, 9(Suppl 2), 64–65.
4 Zhang, L., & Kosaraju, S. L. (2007). Biopolymeric delivery system for
5 controlled release of polyphenolic antioxidants. European Polymer
6 Journal, 43(7), 2956–2966.
7 Zhou, S., Deng, X., & Li, X. (2001). Investigation on a novel core-coated

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8 microspheres protein delivery system. Journal of Controlled Release,
9 75(1–2), 27–36.

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Scanning Electron Microscope revealed spherical nanogels with 200-550
nm in size.

X-ray diffraction reveals FUCO is distributed in a disordered state in CS-
NGs+GL.

Glycolipid offers enhanced FUCO stability and encapsulation efficiency
in CS-NGs.

In vitro bioavailability of CS-NGs with FUCO+GL is higher compared to

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control.

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