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Radioprotective effect of self-assembled low molecular weight Fucoidan–Chitosan nanoparticles
International Journal of Pharmaceutics 579 (2020) 119161
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Radioprotective effect of self-assembled low molecular weight Fucoidan–Chitosan nanoparticles
T ⁎
Szu-Yuan Wu (MD, PhD)a,b,c,d,i,j, Vijayarohini Parasuramane, Hsieh-Chih-Tsaie,f, , Vinothini Arunagirie, Srithar Gunaseelane, Hsiao-Ying Choue, Rajeshkumar Anbazhagane,f, Juin-Yih Laie,f,g, Rajendra Prasad Nh a
Department of Food Nutrition and Health Biotechnology, College of Medical and Health Science, Asia University, Taichung, Taiwan Division of Radiation Oncology, Department of Medicine, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan, Taiwan c Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan d Department of Radiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan e Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei, Taiwan f Advanced Membrane Materials Center, National Taiwan University of Science and Technology, Taipei, Taiwan g R&D Center for Membrane Technology, Chung Yuan Christian University, Zhongli District, Taoyuan City, Taiwan h Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar – 608002, Tamil Nadu, India i Big Data Center, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan, Taiwan j Department of Healthcare Administration, College of Medical and Health Science, Asia University, Taichung, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Oligofucoidan Chitosan Self-assembled Nanoparticles Radioprotection
Fucoidan, a sulphated polysaccharide, plays a vital role in reducing cellular oxidative damage by exerting potential antioxidant activity. However, because of the negative surface charges of oligofucoidan, it shows poor oral intestinal absorption. To overcome this drawback, the oligofucoidan polysaccharides self-assembled with opposite charge based polysaccharides (chitosan) to form the chitosan-fucoidan polysaccharides (C1-F3P) nanoparticles (NPs) of 190–230 nm in size. The oligofucoidan and C1-F3P NPs were studied for their radioprotective property using mice exposed to 5 Gy radiation. The C1-F3P NPs prevents radiation induced lipid peroxidation and restores intestinal enzymatic and non-enzymatic antioxidants (p < 0.05) status. In addition, hematoxylin-eosin staining revealed the radioprotective effect of oligofucoidan and C1-F3P NPs by mitigating the loss of crypt and villi in the small intestine. Thus, the present study demonstrated that C1-F3P NPs can be considered as a radioprotective agent that can be used for the prevention and treatment of Gy-radiation-induced intestine injury.
1. Introduction Fucoidan is a cheap food supplement in many Asian countries including China, Japan, and South Korea. Oligofucoidan is a naturally abundant, pharmacologically active heteropolysaccharide isolated from the extracellular matrix and cell wall of marine brown macroalgae or seaweed (phaeophyte) (Chollet et al., 2016; Yende et al., 2014; Wang et al., 2019). Oligofucoidan is a water-soluble, anionic heteropolysaccharide that is mainly composed of L-fucose and sulfate ester groups. Its backbone contains two repeating units of α-(1 → 3)-linked Lfucopyranose residues or repeating units of disaccharides containing α(1 → 3)- or α-(1 → 4)- linked L-fucopyranose residues with additional sulfate group branching from the C2 positions or C2/C4 positions of Lfucose residues (Li et al., 2008; Weelden et al., 2019; Venkadesan et al., ⁎
2016; Hamid., 2015). Depending on its structure, oligofucoidan exhibits different bioactivities, such as antithrombotic, immunomodulatory, gastric protective effects, anti-inflammatory, antiproliferative, anti-cancer, antioxidant activity, and radioprotective effect (Santos et al., 2015; Jiao et al., 2011; Ale et al., 2009). Radiation exposure creates oxidative stress in human body owing to major production of reactive oxygen species (ROS) and derived free radicals. This results in oxidative damages to lipids, proteins, and DNA. The rapidly proliferating gastrointestinal and circulatory cellular systems are highly sensitive to radiation (Akpolat et al., 2009). Oligofucoidan is an effective free radical scavenger that inhibits lipid peroxidation and prevents oxidative damage on the cellular membrane (Wu et al., 2014). However, the efficacy of the bioactivity of oligofucoidan is linked to its molecular weight, structure, position and amount of
Corresponding author at: Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei, Taiwan. E-mail addresses: [email protected] (S.-Y. Wu), [email protected] (Hsieh-Chih-Tsai).
https://doi.org/10.1016/j.ijpharm.2020.119161 Received 17 November 2019; Received in revised form 25 January 2020; Accepted 16 February 2020 Available online 17 February 2020 0378-5173/ © 2020 Published by Elsevier B.V.
International Journal of Pharmaceutics 579 (2020) 119161
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(NBT), reduced glutathione (GSH), 2-thiobarbituric acid (TBA), and trichloroacetic acid (TCA) were purchased from Sigma Chemical Company (St. Louis, MO,USA). Cellulose dialysis membrane molecular weight cut off (MWCO), 6–8 Da) was purchased from Orange Scientific. Deionized water in the experiments was obtained from a Millipore water purification system. The other chemical reagents and buffer solution components were analytical grade.
sulfates on the polymeric chain, and the route of administration (Cho et al., 2011; Tsai et al., 2017; Wanga et al., 2008; Fitton, 2011; Hosseinimehr et al., 2015; Yamamoto et al., 2017) Despite of bioactive efficacy of oligofucoidan, it suffers from poor oral availability. Owing to its hydrophilic nature and a large number of negative charges, the oral administration of oligofucoidan results in poor intestinal absorption (Chen et al., 2018; Yu et al., 2013). This adversely impacts the efficiency of its intended bioactivity, especially its antioxidant activity (Thanou et al., 2001; Zhao et al., 2016; Tsai et al., 2019). Since chitosan has some exclusive attributes such as nontoxicity, biocompatibility, biodegradation and mucoadhesion, and also it has been widely used in mucosal drug delivery, especially in oral and nasal drug delivery in the form of liposomes, micelles, nanoparticles (NPs) and so on. Chitosan is an abundant cationic polysaccharide that occurs in marine crustaceans including shrimp and crab. It is obtained by alkaline N-deacetylation of chitin, which is mainly composed of Nacetyl-D-glucosamine and D-glucosamine (Tsai et al., 2019; Huang et al., 2011). Low molecular weight positively charged chitosan and negatively charged oligofucoidan could self-assembled to form NPs. The size of the NPs plays a major role in the cellular uptake. Therefore, it is essential to characterize this property of NPs that are aimed at biomedical applications (Li et al., 2015). The smaller NPs have larger surface area, which results in increased interactions with the biological cell membrane (Jong et al., 2008). Many studies have reported that oligofucoidan NPs in the range of 100–274 nm show enhanced antitumor activity in the tumor region (Lu et al., 2017; Huang et al., 2011; Pinheiro et al.,2015; Lee et al.,2013). Notably, positive charge and mucoadhesion property of chitosan, results in opening of tight junctions of the intestinal epithelial layer. In vivo result found that the intestinal absorption of oligofucoidan could be further enhanced by self-assembled of fucoidan with chitosan (Chen et al., 2018). And these polysaccharides based NPs exhibit potential antioxidant activity for radioprotective effect against the gamma (Gy) radiation. The radioprotective property of fucoidan in intestine has not been studied. We hypothesized that oligofucoidan combination with chitosan and form the small size of chitosan-fucoidan polysaccharides (C-FP) would enhanced the intestinal absorption of oligofucoidan (Tsai et al., 2018). We assume that the enhanced uptake of oligofucoidan in the intestine would reduce the free radical formation and reduce the risk of intestine damage during the Gy radiation therapy. To keep this in mind, this study describes the preparation of selfassembled two opposite charged polysaccharide (fucoidan and chitosan) to form the (C-FP) NPs. Here, we have shown that electrostatic complexion of negatively charged fucoidan with positively charged chitosan enables enhanced intestinal absorption of fucoidan in the mice intestines. Moreover, we demonstrate that the C-FPNPs are capable of protecting the intestinal epithelium of mice from radiation induced damage. This present study demonstrated a polysaccharide NPs to reduce the risk of unavoidable damage caused by radiotherapy.
2.2. Preparation of chitosan–oligofucoidanpolysaccharides (C-FP) nanoparticles (NPs). C-FPNPs were prepared by previously described method (Huang et al., 2011; Huang et al., 2014; Barbosa et al., 2019). Briefly, chitosan in acetic acid and oligofucoidan in water with various weight ratio (C1:F1, C1:F3, and C3:F1 and named as C1-F1P, C1-FP3, and C3-FP1) were mixed together and probe sonicated (pulse-on 3 s and pulse-off 7 s) at 10000 rpm for 1 min maintained at room temperature. Later, the unreacted were removed from the self-assembled C-FPNPs by subjecting the obtained reaction mixture to dialysis in water for 24 h. The C-FPNPs were then centrifuged at 5000 rpm for 5 min and lyophilized for further use. 2.3. Conjugation of dyes to the chitosan and fucoidan oligomers. 2.3.1. Synthesis of FITC conjugated chitosan Briefly, FITC dissolved in DMSO and added to chitosan /DMSO in 1:1 ratio and maintained pH 9 by using 0.1 M NaOH. The reaction mixture was stirred for 12 h under dark condition. The obtained dark orange solution was dialyzed against deionized water for 48 h until no fluorescence was detected in the supernatant under dark condition. Then, the solution was centrifuged at 3000 rpm for 15 min in order to collect the supernatant for further step. Finally, the FITC dye conjugated chitosan was lyophilized and stored for further use. 2.3.2. Synthesis of Rhodamine B conjugated oligofucoidan 1:1 ratio of oligomer fucoidan and Rhodamine B was taken in round bottomed flask. Then, 6 mg of DCC and 8 mg of DMAP were both added to the reaction mixture and stirred for 4 h at room temperature. A dark pink colour solution was obtained and first dialyzed in methanol for 24 h and then dialysis in water for another 48 h in dark. Finally, the rhodamine B conjugated fucoidan oligomer was lyophilized and stored in refrigerator for further use (Fiel et al., 2014; Ding et al., 2007). 2.3.3. Preparation dye conjugated C1-F3P NPs The dye conjugated C1-F3P NPs was synthesized similar to C-FP NPs preparation described above. 2.4. Characterization of low molecular weight C-FP NPs
2. Materials and methods
The low molecular weight C-FPNPs were characterized by attenuated total reflectance (ATR) FTIR spectroscopy (JASCO, ATR-FTIR6700). Field emission scanning electron microscopy (JSM 6500F, JEOL) was used to observe the morphology. The hydrodynamic size and zeta potential of the C-FPNPs were analyzed using SZ-100 nanoparticle analyzer (Horiba). The measurements were carried out in triplicates at 25 °C and the scattering angle was fixed at 90°.All the C-FPNPs were prepared in ultra-distilled water and large aggregates were removed using a 0.45 μm syringe filter prior to measurements.
2.1. Materials. Low molecular weight chitosan was purchased from Sigma Aldrich (average molecular weight (Mw) of 50,000–190,000 Da (based on viscosity) and degree of deacetylation (DD%) was 75–85%). Oligofucoidan was derived from Laminaria japonica and prepared by Hi-Q Marine Biotech International Ltd (molecular weight around 500–1500 Da). (New Taipei City, Taiwan). All other chemicals used were of reagent grade and were purchased from Sigma Aldrich unless stated otherwise.1-Chloro-2,4-dinitrobenzene (CDNB), 2,4-dinitrophenylhydrazine,5,5′-dithiobis(2-nitrobenzoicacid) (DTNB),glutathione reductase (GR), hydrogen peroxide (H2O2), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), nitroblue tetrazolium
2.5. Stability of C-FPNPs in phosphate buffer medium To evaluate the stability of C-FP NPs in biological media, the various ratio of C-FP NPs were dissolved in phosphate saline buffer (PBS) medium. The size of the NPs was measured at 30 days-time intervals. 2
International Journal of Pharmaceutics 579 (2020) 119161
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NPs (dose: 20 mg/kg body weight) by route of oral gavage respectively. After 24 h, the irradiated group of mice was administered with the samples until end of the experiment. Gy radiation induced intestinal injury study was carried out as reported by Zhu et al., 2019. The supply of 5 Gy radiation facilities was provided by the Taipei Municipal Wanfang Hospital, Taiwan. With a single dose of 5 Gy radiation the whole body was exposed using ELEKTA Synergy. The mice were sacrificed at the end of experimental period and the tissue samples (the liver and intestine) were used for biochemical and histochemical analysis.
2.6. Cytotoxicity of low molecular weight C1-F3PNPs The biocompatibility of chitosan-fucoidan nanoparticles was evaluated using HaCaT cell line as a mode by MTT Assay l. The HaCaT cell line was seeded in 96 well plate at a density of 1.0 × 105 and incubated overnight in a DMEM (medium supplement with 10% FBS, 1%penicillin, 1% glutamine, and 1% sodium pyruvate at 37 °C and 5%CO2. After, 24 h incubation, the culturing medium was replaced with fresh medium containing NPs with various concentrations (20, 40, 60, 80, 100 µg/ml) and again incubate at 37 °C and 5%CO2 in an incubator. Subsequently, 5 mg/mL of MTT solution was prepared and replaced with old medium in the 96 well plate and incubating the plate for 2–4 h at 37 °C. Later, 50–100 μL of cell culture dimethyl sulfoxide (DMSO) was added and access the toxicity using at 570 nm and 450 nm wavelengths. The results were quantified as the percentage of viable cells after treatment compared to a control of untreated cells. Cell viability was expressed using the following formula. Cell viability (%) = (absorbance of test cells/absorbance of controlled cells) × 100
2.10. Preparation of tissue homogenate. Animals were anesthetized with an i.p injection of ketamine hydrochloride and sacrificed by cervical decapitation. The liver and intestine tissue samples were sliced immediately and rinsed with 0.9% NaCl, then homogenized using the appropriate buffer in a tissue homogenizer in cold condition at pH 7.0 to give 20% homogenates (w/ v). The supernatant was separated and used for various biochemical estimations.
2.7. In vitro cellular uptake study for dye conjugated C1-F3P NPs 2.11. Estimation of lipid peroxidation and antioxidants
HaCaT cells were grown at a density of 2.5 × 105 cells/well in confocal dish and add the DMEM supplemented with 10% FBS, 1% penicillin, 1% glutamine, and 1% sodium pyruvate at 37 °C and 5% CO2. Then, old medium was removed and the cells were incubated with fresh medium containing dye conjugated particles (Chitosan-FITC & Fucoidan-Rho B, and C1-F3P NPs) (concentration of 5 μg /mL) for 1 h at 37 °C. After incubation, test samples were aspirated. Cells were then washed with pre-warmed PBS three times before they were fixed in 4% paraformaldehyde. Finally, the fixed cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA) and the cellular internalization was visualized using a fluorescence microscope.
Lipid peroxidation was determined as described earlier by Niehaus & Samuelson 1968 method. Conjugated dienes (CD) was estimated by the methods reported by Rao & Recknagel 1968. Superoxide dismutase (SOD) activity was measured as reported by Kakkar et al., 1984. Catalase activity was performed based on the method reported by Sinha. 1972. Glutathione peroxidase activity was determined following Rotruck et al., 1973. Furthermore, totalreduced glutathione (GSH) substance was quantified as described earlier Ellman. 1959. 2.12. Histopathology study The mice intestine tissue was excised immediately and fixed in 10% formalin for 4 days. It was then dehydrated with a series of different concentrations of ethanol and embedded in paraffin wax. The biopsies (about 3–5 μm) were processed for hematoxylin and eosin (H&E) staining was measured by using light microscope (Erben et al., 2014). A minimum of three animals and at least six measurements for each specimen were observed with a light microscope (40×). All histopathology changes were examined by a pathologist.
2.8. In vivo fluorescence imaging Groups of six male Sprague Dawley mice each were used for absorption studies. A dose of 0.2 ml of 1 mg/ml of control chitosan-FITC, Fucoidan-Rhodamine-B, or dye conjugated C-F nanoparticle formulations was administered to the respective groups. The nanoparticle dispersion were dispersed in phosphate buffer and administered by oral gavage under the light influence of the anesthetic isoflurane. The mice were cannulated in the right jugular vein under isoflurane inhaled anesthesia and allowed to recover. The cannulated mice were fasted overnight (14 + 1 h) before each oral dosing and given access to food 4 h after each dose, but water was provided at all times. The mice are euthanized humanely by CO2 inhalation and the jejunum and colon parts were collected and stored at −70 C in Cryostor solution until use. Four centimeter pieces of everted mice jejunum and colon pieces were taken and the cross sections were imaged using a confocal laser scanning microscope (Zhu et al., 2019).
3. Results and discussion 3.1. Measurement of particle size and charge The size of the as prepared low molecular weight (LMW) chitosanfucoidan polysaccharides C-FP nanoparticles (NPs) were found to be in the range of 190–230 nm diameter (Z-average) as measured by dynamic light scattering (DLS) method (Table 1). The polydispersity index (PI) of LMW C-FP NPs were found to be between 0.32 and 0.38, which was suitable for various biological applications. Since it can excrete through mononuclear phagocytic system in the aforementioned size range (Jo et al., 2015). Among the prepared NPs, C1-F3P NPs exhibit smaller size and smaller PI values compared to other two compositions. We select
2.9. Animal and drug treatments Mice (18–22 g) were used in this study. The mice were housed at a constant temperature (25 ± 1 °C) and humidity (50–60%) under a 12 h: 12 h light-dark cycle with free access to standard food (American Institute of Nutrition, Rodent Lab Diets (AIN-93G diet) and water. All the experiments were reviewed and approved by the Institutional Animal Care and Use. After one week of acclimatization, the mice were randomized into three groups (n = 24): Group-I: Irradiation (IR 5 Gy) group; it served as control, Group-II: Irradiation (IR 5 Gy) with Oligofucoidan, Group-III: Irradiation (IR 5 Gy) with fucoidan and C-F NPs. Prior to irradiation with gamma (Gy) radiation, the mice in group II and III were administered with oligofucoidan and fucoidan and C-F
Table 1 DLS measurement of size (nm) and its polydispersity index of C-FPNPs of various compositions.
3
Sample
LMW C-FP NPs
Size (nm)
PI
1 2 3
C1-F3P C1-F1P C3-F1P
190.9 ± 1.23 205.6 ± 1.84 225.4 ± 2.31
0.321 0.384 0.394
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C1-FP3 NPs for further analysis due to its smaller size and PI a value which is considered to be good for biomedical application (Danaei et al., 2018). The morphology and size of C-FPNPs were consistent and the results were discussed in supporting information Fig. S1.
Table 2 DLS measurement of the C-FP NPs of different compositions after 30 days’ incubation in PBS.
3.2. Stability of the C-FP NPs A major challenge in the utilization of biomaterials is to retain their stability in the target microenvironments of the host. Therefore, it is essential to assess the stability of NPs in various pH environments not only for their effective storage but also considering their ability to withstand the conditions of cell culture or in the tissues for in vivo applications. To validate this, we evaluated the stability of C-FP NPs by suspending the prepared NPs in phosphate-buffered saline (PBS) medium and observed the size using DLS method. Notably, no significant size change was observed after incubation of the C-FP NPs in the PBS medium for 30 days, which demonstrated the excellent stability of the C-FPNPs.
Sample
LMW C-FP NPs
Size (nm) in 30 days
1 2 3
C1-F3P C1-F1P C3-F1P
190.9 ± 1.90 205.6 ± 1.32 197.6 ± 2.64
Table 3 Zeta potential measurements of the prepared C1-F3PNPs at acidic and basic pH environment. pH
C1-F3P
LMW oligofucoidan
2.5 (Stomach pH) 7.4 (Intestinal pH)
−3.2 mV −1.5 mV
−10.9 mV −12.2 mV
result, the surface charge of C1-F3P was shifted to neutral when compared with oligofucoidan. Thus, the latter was determined to be suitable for the intended oral administration (Barbosa et al., 2019).
3.3. FT-IR characterization of C1-F3P NPs The FT-IR spectrum was recorded to analyze the interactions between the polymer chains in the C-FP NPs. The electrostatic and hydrogen bonding interactions between carboxyl group of fucoidan at 1601 cm−1 and ammonium group of chitosan at 1651 and 1593 cm-1in C-FPNPs was indicated by a shift in the bands to 1597 cm−1 Fig. 1. Further, the bands of C–O–C, S=O, and C–O–S group of fucoidan shifted from 1426 cm−1, 1028 cm−1, 883 cm−1, to 1406 cm−1, 1022 cm−1, and 878 cm−1, respectively in C1-F3PNPs. This showed changes in the environment of these functional groups possibly due to electrostatic interaction and hydrogen-bonding between fucoidan and chitosan results the formation of C-FP NPs. The obtained results were consistent with the previous published literature (Huang et al., 2011; Huang et al., 2016; Barbosa et al., 2019; Dai et al.,2019) (see Tables 2 and 3).
3.5. Intestinal absorption of oligofucoidan-Rhodamine B, chitosan-FITC and dye-conjugated C1-F3P NPs in mice In order to evaluate the biocompatility assay of C1-F3P NPs, various concentrations of C1-F3P NPs incubated with HaCaT cells for 24 h. The results showed a maximum viability of more than 85%even at 100 mg/ ml demonstrating the excellent biocompatibility of as prepared C1-F3P NPs Fig S2. Therefore, we then conjugated RhodamineB and FITC to C1F3P NPs, confirmed using FT-IR and fluorescence spectroscopy Fig. S3 and S4, and finally investigated the uptake efficiency in HaCaT cells. The obtained uptake results proved that the C1-F3P NPs incubated HaCaT cells exhibited enhanced fluorescence intensity for both FITC and Rhodamine B relative to only oligofucoidan-Rhodamine B and only chitosan-FITC incubated HaCaT cells Fig. S5. The enhanced uptake of C1-F3P NPs may have related to its small size and reduced surface charge (zeta potential) which was higher in oligofucoidan and chitosan. Further, the permeability of C1-F3P NPs in HaCaT cells were studied Fig. S5. Furthermore, biocompatible C1-F3P NPs were administered orally to mice and their intestinal absorption was studied using single pass perfusion method Fig. 2. The upper portion of the intestine consists of jejunum (small intestine), which has finger-like projections so called villi that help in the absorption of nutrients, such as sugars, fatty acids and amino acids from food to the bloodstream. The lower portion of the intestine consists of colon (large intestine) which is involved in the elimination of waste products after digestion. In this study, the efficiency of oligofucoidan intestinal absorption is directly related to the fluorescent intensity of jejunum and colon. Oligofucoidan-Rhodamine B and FITC-chitosan show poor intestinal absorption due to its poor absorption ability when they were administered individually, as indicated by higher and lesser fluorescence intensity in colon and jejunum part, respectively. Interestingly, strong fluorescence intensity in jejunum part was found in case of C1-F3P NPs, which indicated enhanced intestinal absorption by the endoderm finger-like thin villi projections. However, C1-F3P NPs showed lesser intensity in colon part as most of the C1-F3P NPs was absorbed in jejunum confirming the oligofucoidan uptake efficiency enhanced by NPs formulations with chitosan (Nagamani et al., 2015; Kim et al., 2018; Liu et al., 2019; Simovic et al., 2015). The enhanced absorption of fucoidan in C1-F3P NPs may be due to its reduced zeta potential may cause increased bioavailability of C1-F3P NPs in bloods for longer time than only oligofucoidan and only chitosan.
3.4. Charge correlation of C1-F3P NPs and fucoidan for intestinal absorption Surface charge on the C1-F3PNPs and oligofucoidan were determined by zeta potential measurements. The changes in the surface charges between these particles in acidic pH, mimicking stomach and neutral pH, mimicking intestines were monitored. In LMW oligofucoidan, as pH was increased, weak acids deprotonate resulting increased net negative surface charge from −10.9 to −12.2. In case of C1-F3PNPs, as pH was increased, the net negative surface charge decreased due to strong interactions with quaternary ammonium ions of chitosan with sulfates and carboxylic groups of oligofucoidan. As a
Fig. 1. FT-IR spectra of self-assembledC1-F3 PNPs. 4
International Journal of Pharmaceutics 579 (2020) 119161
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Fig. 2. In vivo intestinal absorption of DAPI stained (a) Rhodamine-B-Fucoidan. (b) FITC-Chitosan. (c), (d), (e), and (f) are DAPI stained dye conjugated C1-F3P NPs.
3.6. Radio protective effects of oligofucoidan and C1-F3P NPs against γradiation induced intestinal damage in mice
ionizing radiation performed for cancers of the total body region. This liver also is the primary organ responsible for drug metabolism and mainly detoxifies damaging electrophiles generated during oxidative stress. We designed several biochemical assays to study the effect of Gyradiation when orally administrated with oligofucoidan and C1-F3P NPs. According to the results of the present study, when mice were exposed to a single dose (5.0 Gy) the total body Gy-irradiation-induced lipid peroxidation products, such as thiobarbituric acid reactive substance assay (TBARS) and conjugated diene (CD) levels significantly increased in the liver and intestine tissue samples of radiation alone treated group Fig. 4A and B. Thus, these findings confirm that exposure to Gy-radiation induces oxidative stress by increasing TBARS and CD levels of the small intestinal and liver tissues in mice (Jeong et al., 2016). As a result, overproduction of ROS formation due to oxidation of unsaturated fatty acids in the cell membranes, ROS targets lipids, and DNA (Zhu et al., 2012) On the other hand, treatment with oligofucoidan and C1-F3P NPs prior to γ-irradiation reduce the TBARS and CD levels in liver and intestinal tissues compare to radiation alone group, which indicated the strong antilipoperoxidative property in present in the intestine and liver of mice treated with oligofucoidan and C1-F3P NPs. Notably, C1-F3P NPs treated mice before and after exposure to Gy-radiation exhibited very low TBARS and CD levels confirming the strong antilipoperoxidative property of C1-F3P NPs. This kind of results
One of the major problems associated with irradiation during radiotherapy is the unavoidable damage to the connective tissues. A few studies indicated that oligofucoidan can be acted as radioprotector, which reduces the damage in human leukemia cells, diminishes hematological changes, and decreases radiation induced death in mice. Hence, the present study deals with the in vivo administration of oligofucoidan and C1-F3PNPs before and after irradiation protect mice from radiation-induced damage. Ten days after Gy-5 irradiation, animals were sacrificed, the tissue sections were collected and biochemical assay and histological studies were performed. The colon was found abnormally dark in the case of control, but the oligofucoidan and C1F3P NPs administered mice showed normal colon (see Fig. 3). 3.7. Effect of fucoidan and C1-F3P NPs and /or Gy- radiation on lipid peroxidation status The small intestine has been reported to have an amazing capacity for repair. A delay in the rate of revival after irradiation could be explained on the basis of antioxidants depletion by oxidative stress the liver of mammals has been reported as a highly radiosensitive organ. This liver one of the most frequently injured organs during exposed to
Fig. 3. Gy-radiation induced intestinal damage in mice after administration of oligofucoidan, and C1-F3PNPs. 5
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Fig. 4. Effect of oligofucoidan and C1-F3P NPs treatment on 5 Gy-radiation mediated lipid peroxidation (TBARS & CD) in the liver (A) and intestine (B) of mice. Values are given as means ± SD of six experiments in each group. Values (a, b, c) differ significantly (P < 0.05 (DMRT)).
previously observed in fucoidan obtained from Fucus vesiculosus and exhibited excellent scavenging capacities on the hydroxyl and superoxide radicals (Kruk et al., 2009). 3.8. Effect of oligofucoidan and C1-F3P NPs and /or Gy-radiation on antioxidants status Another important finding in our study antioxidant status, may contribute to mitigate the oxidative stress. Enzymatic and non-enzyme antioxidants constitute a mutually supportive team of the defense system by alleviating tissue damage. SOD and CAT work together to eliminate reactive oxygen species and small deviation in physiological concentrations may have a dramatic effect on the resistance on cellular lipids, proteins, and DNA to oxidative damage (Nita et al., 2016). When mice were exposed to a single dose (5.0 Gy) total body Gyradiation, results showed significantly lower level of enzymic antioxidants such as SOD, CAT and GPx Fig. 5A, 5B and non-enzymatic antioxidants GSH Fig. 5C in liver and intestinal tissue sample compared to both Gy-irradiated + oligofucoidan and C1-F3P NPs treated group of mice. These results proved that the γ-radiationtreated mice experienced unfavorable effects in its antioxidant systems. It has been shown that over production of free radicals interrupts the stability between oxidation and antioxidant mechanism (Kurutas, 2016). In addition, H2O2 also formed during radiolysis of water, which is easily reached to DNA and lipids. Oral administration of oligofucoidan and C1-F3P NPs prior to Gy-irradiation reversed in enzymatic antioxidants of SOD, CAT, and GPx and non-enzymatic antioxidants of GSH activities in tissues samples compared to control group, which indicate the strong ability of C1F3P NPs to scavenge free radicals and toxic carcinogenic electrophiles. Moreover, oligofucoidan and C1-F3P NPs significantly increases endogenous SOD and CAT due its strong antioxidants property, an enzyme which converts hydrogen peroxide into water and prevents the formation of hydroxyl radicals, which may be one of the contributing factors for its radioprotective activity (Wang et al., 2019).
Fig. 5. Effect of oligofucoidan and C1-F3P NPs on 5 Gy-radiation mediated enzyme antioxidants (SOD, CAT and GPx) and non-enzymatic antioxidants GSH in liver (A) and intestine (B) of Swiss albino mice. Values are given as means ± SD of six experiments in each group. Values (a, b and c) differ significantly at P < 0.05 (DMRT). * The enzyme concentration required for 50% inhibition of nitroblue tetrazolium reduction in one minute, ** μmol of hydrogen peroxide consumed per minute, and *** μg of glutathione consumed per minute.
3.9. Histopathology analysis Following a single dose (5.0 Gy) total body Gy-irradiation, the animals were euthanized and intestine biopsies were processed for hematoxylin and eosin staining. The intensity of the color is proportional to the concentration of hydroxyl groups. The obtained results showed that crypt–villus units of the intestinal mucosa were severely destroyed, as indicated by bright magenta color Fig. 6 (I) and also evidenced by the flattened villi and decreased number of surviving crypts. In contrast, oligofucoidan and C1-F3P NPs treated, Gy-irradiated mice showed reduced intensity of magenta color (Fig. 6 (II and III)). Thus, indicating that the loss of villi and crypts was mitigated by fucoidan and C-F NPs. In conclusion of this study, self-assembled chitosan-fucoidan polysaccharide (C-FP) nanoparticles (NPs) were prepared successfully for the purpose of enhancing the intestinal absorption of oligofucoidan. For
the purpose of producing stable NPs, the ratio between oligofucoidan and chitosan was tuned during NPs preparation and selected the more stable C1-F3P NPs. An improved intestinal absorption of C1-F3P NPs observed over only oligofucoidan treated mice with increased antioxidant activity when Gy-radiation was irradiated. The radioprotective effect of oligofucoidan and C1-F3P NPs was demonstrated using various biochemical assays after Gy-irradiation i.e lipid peroxidation, antioxidants system, and histopathology in the mice intestine and liver. Further, C1-F3P NPsand oligofucoidan provided protection against Gyradiation induced damage. Notably, self-assembledC1-F3P NPsexhibited enhanced antioxidant property than its counterparts proved the 6
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Fig. 6. (A and B) Effect of oligofucoidan and C1F3P NPs on 5 Gy-radiation induced intestinal damage. Twenty-four hours after 5 Gy-radiation, the animals were euthanized, and intestinal biopsies were processed for Hematoxylin-Eosin (H&E) staining. Group (I) Gy-radiation alone, (II) Gy-radiation + oligofucoidan (III) GyRadition + C1-F3P NPs treated in mice. A minimum of three animals and at least six measurements for each specimen were taken for intestine damage.
potential for mitigating radiation induced damages due to radiotherapy.
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CRediT authorship contribution statement Szu-Yuan Wu: Project administration, Conceptualization. Vijayarohini Parasuraman: Investigation. Hsieh-Chih-Tsai: Supervision, Writing - review & editing. Vinothini Arunagiri: Formal analysis. Srithar Gunaseelan: Writing - original draft. Hsiao-Ying Chou: Methodology. Rajeshkumar Anbazhagan: Validation. Juin-Yih Lai: Funding acquisition. Rajendra Prasad N: Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to thank the Ministry of Science and Technology of the Republic of China (Taiwan) (Grant Nos. MOST 1052221-E-011-151-MY3 and 105-2221-E-011-133-MY3), National Taiwan University of Science and Technology, National Taiwan University and Taipei Medical University Joint Research Program (Grant Nos. TMUNTUST-104-10, TMU-NTUST-105-07 and TMU-NTUST-106-06), Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital (Funding Number: 10908 & 10909) and Hi-Q Marine Biotech International Ltd., New Taipei City, Taiwan for providing the financial support. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2020.119161. References Akpolat, M., Kanter, M., Uzal, M.C., 2009. Protective effects of curcumin against gamma radiation-induced ileal mucosal damage. Arch. Toxicol. 83, 609–617. Ale, M.T., Jorn, D., Mikkelsen, D., Meyer, A.S., 2009. Important determinants for fucoidan bioactivity: a critical review of structure-function relations and extraction methods for fucose-containing sulfated polysaccharides from brown seaweeds. Mar. Drugs 9,
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International Journal of Pharmaceutics journal homepage: www.elsevier.com/locate/ijpharm
Radioprotective effect of self-assembled low molecular weight Fucoidan–Chitosan nanoparticles
T ⁎
Szu-Yuan Wu (MD, PhD)a,b,c,d,i,j, Vijayarohini Parasuramane, Hsieh-Chih-Tsaie,f, , Vinothini Arunagirie, Srithar Gunaseelane, Hsiao-Ying Choue, Rajeshkumar Anbazhagane,f, Juin-Yih Laie,f,g, Rajendra Prasad Nh a
Department of Food Nutrition and Health Biotechnology, College of Medical and Health Science, Asia University, Taichung, Taiwan Division of Radiation Oncology, Department of Medicine, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan, Taiwan c Department of Internal Medicine, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan d Department of Radiology, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan e Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei, Taiwan f Advanced Membrane Materials Center, National Taiwan University of Science and Technology, Taipei, Taiwan g R&D Center for Membrane Technology, Chung Yuan Christian University, Zhongli District, Taoyuan City, Taiwan h Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar – 608002, Tamil Nadu, India i Big Data Center, Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital, Yilan, Taiwan j Department of Healthcare Administration, College of Medical and Health Science, Asia University, Taichung, Taiwan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Oligofucoidan Chitosan Self-assembled Nanoparticles Radioprotection
Fucoidan, a sulphated polysaccharide, plays a vital role in reducing cellular oxidative damage by exerting potential antioxidant activity. However, because of the negative surface charges of oligofucoidan, it shows poor oral intestinal absorption. To overcome this drawback, the oligofucoidan polysaccharides self-assembled with opposite charge based polysaccharides (chitosan) to form the chitosan-fucoidan polysaccharides (C1-F3P) nanoparticles (NPs) of 190–230 nm in size. The oligofucoidan and C1-F3P NPs were studied for their radioprotective property using mice exposed to 5 Gy radiation. The C1-F3P NPs prevents radiation induced lipid peroxidation and restores intestinal enzymatic and non-enzymatic antioxidants (p < 0.05) status. In addition, hematoxylin-eosin staining revealed the radioprotective effect of oligofucoidan and C1-F3P NPs by mitigating the loss of crypt and villi in the small intestine. Thus, the present study demonstrated that C1-F3P NPs can be considered as a radioprotective agent that can be used for the prevention and treatment of Gy-radiation-induced intestine injury.
1. Introduction Fucoidan is a cheap food supplement in many Asian countries including China, Japan, and South Korea. Oligofucoidan is a naturally abundant, pharmacologically active heteropolysaccharide isolated from the extracellular matrix and cell wall of marine brown macroalgae or seaweed (phaeophyte) (Chollet et al., 2016; Yende et al., 2014; Wang et al., 2019). Oligofucoidan is a water-soluble, anionic heteropolysaccharide that is mainly composed of L-fucose and sulfate ester groups. Its backbone contains two repeating units of α-(1 → 3)-linked Lfucopyranose residues or repeating units of disaccharides containing α(1 → 3)- or α-(1 → 4)- linked L-fucopyranose residues with additional sulfate group branching from the C2 positions or C2/C4 positions of Lfucose residues (Li et al., 2008; Weelden et al., 2019; Venkadesan et al., ⁎
2016; Hamid., 2015). Depending on its structure, oligofucoidan exhibits different bioactivities, such as antithrombotic, immunomodulatory, gastric protective effects, anti-inflammatory, antiproliferative, anti-cancer, antioxidant activity, and radioprotective effect (Santos et al., 2015; Jiao et al., 2011; Ale et al., 2009). Radiation exposure creates oxidative stress in human body owing to major production of reactive oxygen species (ROS) and derived free radicals. This results in oxidative damages to lipids, proteins, and DNA. The rapidly proliferating gastrointestinal and circulatory cellular systems are highly sensitive to radiation (Akpolat et al., 2009). Oligofucoidan is an effective free radical scavenger that inhibits lipid peroxidation and prevents oxidative damage on the cellular membrane (Wu et al., 2014). However, the efficacy of the bioactivity of oligofucoidan is linked to its molecular weight, structure, position and amount of
Corresponding author at: Graduate Institute of Applied Science and Technology, National Taiwan University of Science and Technology, Taipei, Taiwan. E-mail addresses: [email protected] (S.-Y. Wu), [email protected] (Hsieh-Chih-Tsai).
https://doi.org/10.1016/j.ijpharm.2020.119161 Received 17 November 2019; Received in revised form 25 January 2020; Accepted 16 February 2020 Available online 17 February 2020 0378-5173/ © 2020 Published by Elsevier B.V.
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(NBT), reduced glutathione (GSH), 2-thiobarbituric acid (TBA), and trichloroacetic acid (TCA) were purchased from Sigma Chemical Company (St. Louis, MO,USA). Cellulose dialysis membrane molecular weight cut off (MWCO), 6–8 Da) was purchased from Orange Scientific. Deionized water in the experiments was obtained from a Millipore water purification system. The other chemical reagents and buffer solution components were analytical grade.
sulfates on the polymeric chain, and the route of administration (Cho et al., 2011; Tsai et al., 2017; Wanga et al., 2008; Fitton, 2011; Hosseinimehr et al., 2015; Yamamoto et al., 2017) Despite of bioactive efficacy of oligofucoidan, it suffers from poor oral availability. Owing to its hydrophilic nature and a large number of negative charges, the oral administration of oligofucoidan results in poor intestinal absorption (Chen et al., 2018; Yu et al., 2013). This adversely impacts the efficiency of its intended bioactivity, especially its antioxidant activity (Thanou et al., 2001; Zhao et al., 2016; Tsai et al., 2019). Since chitosan has some exclusive attributes such as nontoxicity, biocompatibility, biodegradation and mucoadhesion, and also it has been widely used in mucosal drug delivery, especially in oral and nasal drug delivery in the form of liposomes, micelles, nanoparticles (NPs) and so on. Chitosan is an abundant cationic polysaccharide that occurs in marine crustaceans including shrimp and crab. It is obtained by alkaline N-deacetylation of chitin, which is mainly composed of Nacetyl-D-glucosamine and D-glucosamine (Tsai et al., 2019; Huang et al., 2011). Low molecular weight positively charged chitosan and negatively charged oligofucoidan could self-assembled to form NPs. The size of the NPs plays a major role in the cellular uptake. Therefore, it is essential to characterize this property of NPs that are aimed at biomedical applications (Li et al., 2015). The smaller NPs have larger surface area, which results in increased interactions with the biological cell membrane (Jong et al., 2008). Many studies have reported that oligofucoidan NPs in the range of 100–274 nm show enhanced antitumor activity in the tumor region (Lu et al., 2017; Huang et al., 2011; Pinheiro et al.,2015; Lee et al.,2013). Notably, positive charge and mucoadhesion property of chitosan, results in opening of tight junctions of the intestinal epithelial layer. In vivo result found that the intestinal absorption of oligofucoidan could be further enhanced by self-assembled of fucoidan with chitosan (Chen et al., 2018). And these polysaccharides based NPs exhibit potential antioxidant activity for radioprotective effect against the gamma (Gy) radiation. The radioprotective property of fucoidan in intestine has not been studied. We hypothesized that oligofucoidan combination with chitosan and form the small size of chitosan-fucoidan polysaccharides (C-FP) would enhanced the intestinal absorption of oligofucoidan (Tsai et al., 2018). We assume that the enhanced uptake of oligofucoidan in the intestine would reduce the free radical formation and reduce the risk of intestine damage during the Gy radiation therapy. To keep this in mind, this study describes the preparation of selfassembled two opposite charged polysaccharide (fucoidan and chitosan) to form the (C-FP) NPs. Here, we have shown that electrostatic complexion of negatively charged fucoidan with positively charged chitosan enables enhanced intestinal absorption of fucoidan in the mice intestines. Moreover, we demonstrate that the C-FPNPs are capable of protecting the intestinal epithelium of mice from radiation induced damage. This present study demonstrated a polysaccharide NPs to reduce the risk of unavoidable damage caused by radiotherapy.
2.2. Preparation of chitosan–oligofucoidanpolysaccharides (C-FP) nanoparticles (NPs). C-FPNPs were prepared by previously described method (Huang et al., 2011; Huang et al., 2014; Barbosa et al., 2019). Briefly, chitosan in acetic acid and oligofucoidan in water with various weight ratio (C1:F1, C1:F3, and C3:F1 and named as C1-F1P, C1-FP3, and C3-FP1) were mixed together and probe sonicated (pulse-on 3 s and pulse-off 7 s) at 10000 rpm for 1 min maintained at room temperature. Later, the unreacted were removed from the self-assembled C-FPNPs by subjecting the obtained reaction mixture to dialysis in water for 24 h. The C-FPNPs were then centrifuged at 5000 rpm for 5 min and lyophilized for further use. 2.3. Conjugation of dyes to the chitosan and fucoidan oligomers. 2.3.1. Synthesis of FITC conjugated chitosan Briefly, FITC dissolved in DMSO and added to chitosan /DMSO in 1:1 ratio and maintained pH 9 by using 0.1 M NaOH. The reaction mixture was stirred for 12 h under dark condition. The obtained dark orange solution was dialyzed against deionized water for 48 h until no fluorescence was detected in the supernatant under dark condition. Then, the solution was centrifuged at 3000 rpm for 15 min in order to collect the supernatant for further step. Finally, the FITC dye conjugated chitosan was lyophilized and stored for further use. 2.3.2. Synthesis of Rhodamine B conjugated oligofucoidan 1:1 ratio of oligomer fucoidan and Rhodamine B was taken in round bottomed flask. Then, 6 mg of DCC and 8 mg of DMAP were both added to the reaction mixture and stirred for 4 h at room temperature. A dark pink colour solution was obtained and first dialyzed in methanol for 24 h and then dialysis in water for another 48 h in dark. Finally, the rhodamine B conjugated fucoidan oligomer was lyophilized and stored in refrigerator for further use (Fiel et al., 2014; Ding et al., 2007). 2.3.3. Preparation dye conjugated C1-F3P NPs The dye conjugated C1-F3P NPs was synthesized similar to C-FP NPs preparation described above. 2.4. Characterization of low molecular weight C-FP NPs
2. Materials and methods
The low molecular weight C-FPNPs were characterized by attenuated total reflectance (ATR) FTIR spectroscopy (JASCO, ATR-FTIR6700). Field emission scanning electron microscopy (JSM 6500F, JEOL) was used to observe the morphology. The hydrodynamic size and zeta potential of the C-FPNPs were analyzed using SZ-100 nanoparticle analyzer (Horiba). The measurements were carried out in triplicates at 25 °C and the scattering angle was fixed at 90°.All the C-FPNPs were prepared in ultra-distilled water and large aggregates were removed using a 0.45 μm syringe filter prior to measurements.
2.1. Materials. Low molecular weight chitosan was purchased from Sigma Aldrich (average molecular weight (Mw) of 50,000–190,000 Da (based on viscosity) and degree of deacetylation (DD%) was 75–85%). Oligofucoidan was derived from Laminaria japonica and prepared by Hi-Q Marine Biotech International Ltd (molecular weight around 500–1500 Da). (New Taipei City, Taiwan). All other chemicals used were of reagent grade and were purchased from Sigma Aldrich unless stated otherwise.1-Chloro-2,4-dinitrobenzene (CDNB), 2,4-dinitrophenylhydrazine,5,5′-dithiobis(2-nitrobenzoicacid) (DTNB),glutathione reductase (GR), hydrogen peroxide (H2O2), reduced nicotinamide adenine dinucleotide (NADH), reduced nicotinamide adenine dinucleotide phosphate (NADPH), nitroblue tetrazolium
2.5. Stability of C-FPNPs in phosphate buffer medium To evaluate the stability of C-FP NPs in biological media, the various ratio of C-FP NPs were dissolved in phosphate saline buffer (PBS) medium. The size of the NPs was measured at 30 days-time intervals. 2
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NPs (dose: 20 mg/kg body weight) by route of oral gavage respectively. After 24 h, the irradiated group of mice was administered with the samples until end of the experiment. Gy radiation induced intestinal injury study was carried out as reported by Zhu et al., 2019. The supply of 5 Gy radiation facilities was provided by the Taipei Municipal Wanfang Hospital, Taiwan. With a single dose of 5 Gy radiation the whole body was exposed using ELEKTA Synergy. The mice were sacrificed at the end of experimental period and the tissue samples (the liver and intestine) were used for biochemical and histochemical analysis.
2.6. Cytotoxicity of low molecular weight C1-F3PNPs The biocompatibility of chitosan-fucoidan nanoparticles was evaluated using HaCaT cell line as a mode by MTT Assay l. The HaCaT cell line was seeded in 96 well plate at a density of 1.0 × 105 and incubated overnight in a DMEM (medium supplement with 10% FBS, 1%penicillin, 1% glutamine, and 1% sodium pyruvate at 37 °C and 5%CO2. After, 24 h incubation, the culturing medium was replaced with fresh medium containing NPs with various concentrations (20, 40, 60, 80, 100 µg/ml) and again incubate at 37 °C and 5%CO2 in an incubator. Subsequently, 5 mg/mL of MTT solution was prepared and replaced with old medium in the 96 well plate and incubating the plate for 2–4 h at 37 °C. Later, 50–100 μL of cell culture dimethyl sulfoxide (DMSO) was added and access the toxicity using at 570 nm and 450 nm wavelengths. The results were quantified as the percentage of viable cells after treatment compared to a control of untreated cells. Cell viability was expressed using the following formula. Cell viability (%) = (absorbance of test cells/absorbance of controlled cells) × 100
2.10. Preparation of tissue homogenate. Animals were anesthetized with an i.p injection of ketamine hydrochloride and sacrificed by cervical decapitation. The liver and intestine tissue samples were sliced immediately and rinsed with 0.9% NaCl, then homogenized using the appropriate buffer in a tissue homogenizer in cold condition at pH 7.0 to give 20% homogenates (w/ v). The supernatant was separated and used for various biochemical estimations.
2.7. In vitro cellular uptake study for dye conjugated C1-F3P NPs 2.11. Estimation of lipid peroxidation and antioxidants
HaCaT cells were grown at a density of 2.5 × 105 cells/well in confocal dish and add the DMEM supplemented with 10% FBS, 1% penicillin, 1% glutamine, and 1% sodium pyruvate at 37 °C and 5% CO2. Then, old medium was removed and the cells were incubated with fresh medium containing dye conjugated particles (Chitosan-FITC & Fucoidan-Rho B, and C1-F3P NPs) (concentration of 5 μg /mL) for 1 h at 37 °C. After incubation, test samples were aspirated. Cells were then washed with pre-warmed PBS three times before they were fixed in 4% paraformaldehyde. Finally, the fixed cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, St Louis, MO, USA) and the cellular internalization was visualized using a fluorescence microscope.
Lipid peroxidation was determined as described earlier by Niehaus & Samuelson 1968 method. Conjugated dienes (CD) was estimated by the methods reported by Rao & Recknagel 1968. Superoxide dismutase (SOD) activity was measured as reported by Kakkar et al., 1984. Catalase activity was performed based on the method reported by Sinha. 1972. Glutathione peroxidase activity was determined following Rotruck et al., 1973. Furthermore, totalreduced glutathione (GSH) substance was quantified as described earlier Ellman. 1959. 2.12. Histopathology study The mice intestine tissue was excised immediately and fixed in 10% formalin for 4 days. It was then dehydrated with a series of different concentrations of ethanol and embedded in paraffin wax. The biopsies (about 3–5 μm) were processed for hematoxylin and eosin (H&E) staining was measured by using light microscope (Erben et al., 2014). A minimum of three animals and at least six measurements for each specimen were observed with a light microscope (40×). All histopathology changes were examined by a pathologist.
2.8. In vivo fluorescence imaging Groups of six male Sprague Dawley mice each were used for absorption studies. A dose of 0.2 ml of 1 mg/ml of control chitosan-FITC, Fucoidan-Rhodamine-B, or dye conjugated C-F nanoparticle formulations was administered to the respective groups. The nanoparticle dispersion were dispersed in phosphate buffer and administered by oral gavage under the light influence of the anesthetic isoflurane. The mice were cannulated in the right jugular vein under isoflurane inhaled anesthesia and allowed to recover. The cannulated mice were fasted overnight (14 + 1 h) before each oral dosing and given access to food 4 h after each dose, but water was provided at all times. The mice are euthanized humanely by CO2 inhalation and the jejunum and colon parts were collected and stored at −70 C in Cryostor solution until use. Four centimeter pieces of everted mice jejunum and colon pieces were taken and the cross sections were imaged using a confocal laser scanning microscope (Zhu et al., 2019).
3. Results and discussion 3.1. Measurement of particle size and charge The size of the as prepared low molecular weight (LMW) chitosanfucoidan polysaccharides C-FP nanoparticles (NPs) were found to be in the range of 190–230 nm diameter (Z-average) as measured by dynamic light scattering (DLS) method (Table 1). The polydispersity index (PI) of LMW C-FP NPs were found to be between 0.32 and 0.38, which was suitable for various biological applications. Since it can excrete through mononuclear phagocytic system in the aforementioned size range (Jo et al., 2015). Among the prepared NPs, C1-F3P NPs exhibit smaller size and smaller PI values compared to other two compositions. We select
2.9. Animal and drug treatments Mice (18–22 g) were used in this study. The mice were housed at a constant temperature (25 ± 1 °C) and humidity (50–60%) under a 12 h: 12 h light-dark cycle with free access to standard food (American Institute of Nutrition, Rodent Lab Diets (AIN-93G diet) and water. All the experiments were reviewed and approved by the Institutional Animal Care and Use. After one week of acclimatization, the mice were randomized into three groups (n = 24): Group-I: Irradiation (IR 5 Gy) group; it served as control, Group-II: Irradiation (IR 5 Gy) with Oligofucoidan, Group-III: Irradiation (IR 5 Gy) with fucoidan and C-F NPs. Prior to irradiation with gamma (Gy) radiation, the mice in group II and III were administered with oligofucoidan and fucoidan and C-F
Table 1 DLS measurement of size (nm) and its polydispersity index of C-FPNPs of various compositions.
3
Sample
LMW C-FP NPs
Size (nm)
PI
1 2 3
C1-F3P C1-F1P C3-F1P
190.9 ± 1.23 205.6 ± 1.84 225.4 ± 2.31
0.321 0.384 0.394
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C1-FP3 NPs for further analysis due to its smaller size and PI a value which is considered to be good for biomedical application (Danaei et al., 2018). The morphology and size of C-FPNPs were consistent and the results were discussed in supporting information Fig. S1.
Table 2 DLS measurement of the C-FP NPs of different compositions after 30 days’ incubation in PBS.
3.2. Stability of the C-FP NPs A major challenge in the utilization of biomaterials is to retain their stability in the target microenvironments of the host. Therefore, it is essential to assess the stability of NPs in various pH environments not only for their effective storage but also considering their ability to withstand the conditions of cell culture or in the tissues for in vivo applications. To validate this, we evaluated the stability of C-FP NPs by suspending the prepared NPs in phosphate-buffered saline (PBS) medium and observed the size using DLS method. Notably, no significant size change was observed after incubation of the C-FP NPs in the PBS medium for 30 days, which demonstrated the excellent stability of the C-FPNPs.
Sample
LMW C-FP NPs
Size (nm) in 30 days
1 2 3
C1-F3P C1-F1P C3-F1P
190.9 ± 1.90 205.6 ± 1.32 197.6 ± 2.64
Table 3 Zeta potential measurements of the prepared C1-F3PNPs at acidic and basic pH environment. pH
C1-F3P
LMW oligofucoidan
2.5 (Stomach pH) 7.4 (Intestinal pH)
−3.2 mV −1.5 mV
−10.9 mV −12.2 mV
result, the surface charge of C1-F3P was shifted to neutral when compared with oligofucoidan. Thus, the latter was determined to be suitable for the intended oral administration (Barbosa et al., 2019).
3.3. FT-IR characterization of C1-F3P NPs The FT-IR spectrum was recorded to analyze the interactions between the polymer chains in the C-FP NPs. The electrostatic and hydrogen bonding interactions between carboxyl group of fucoidan at 1601 cm−1 and ammonium group of chitosan at 1651 and 1593 cm-1in C-FPNPs was indicated by a shift in the bands to 1597 cm−1 Fig. 1. Further, the bands of C–O–C, S=O, and C–O–S group of fucoidan shifted from 1426 cm−1, 1028 cm−1, 883 cm−1, to 1406 cm−1, 1022 cm−1, and 878 cm−1, respectively in C1-F3PNPs. This showed changes in the environment of these functional groups possibly due to electrostatic interaction and hydrogen-bonding between fucoidan and chitosan results the formation of C-FP NPs. The obtained results were consistent with the previous published literature (Huang et al., 2011; Huang et al., 2016; Barbosa et al., 2019; Dai et al.,2019) (see Tables 2 and 3).
3.5. Intestinal absorption of oligofucoidan-Rhodamine B, chitosan-FITC and dye-conjugated C1-F3P NPs in mice In order to evaluate the biocompatility assay of C1-F3P NPs, various concentrations of C1-F3P NPs incubated with HaCaT cells for 24 h. The results showed a maximum viability of more than 85%even at 100 mg/ ml demonstrating the excellent biocompatibility of as prepared C1-F3P NPs Fig S2. Therefore, we then conjugated RhodamineB and FITC to C1F3P NPs, confirmed using FT-IR and fluorescence spectroscopy Fig. S3 and S4, and finally investigated the uptake efficiency in HaCaT cells. The obtained uptake results proved that the C1-F3P NPs incubated HaCaT cells exhibited enhanced fluorescence intensity for both FITC and Rhodamine B relative to only oligofucoidan-Rhodamine B and only chitosan-FITC incubated HaCaT cells Fig. S5. The enhanced uptake of C1-F3P NPs may have related to its small size and reduced surface charge (zeta potential) which was higher in oligofucoidan and chitosan. Further, the permeability of C1-F3P NPs in HaCaT cells were studied Fig. S5. Furthermore, biocompatible C1-F3P NPs were administered orally to mice and their intestinal absorption was studied using single pass perfusion method Fig. 2. The upper portion of the intestine consists of jejunum (small intestine), which has finger-like projections so called villi that help in the absorption of nutrients, such as sugars, fatty acids and amino acids from food to the bloodstream. The lower portion of the intestine consists of colon (large intestine) which is involved in the elimination of waste products after digestion. In this study, the efficiency of oligofucoidan intestinal absorption is directly related to the fluorescent intensity of jejunum and colon. Oligofucoidan-Rhodamine B and FITC-chitosan show poor intestinal absorption due to its poor absorption ability when they were administered individually, as indicated by higher and lesser fluorescence intensity in colon and jejunum part, respectively. Interestingly, strong fluorescence intensity in jejunum part was found in case of C1-F3P NPs, which indicated enhanced intestinal absorption by the endoderm finger-like thin villi projections. However, C1-F3P NPs showed lesser intensity in colon part as most of the C1-F3P NPs was absorbed in jejunum confirming the oligofucoidan uptake efficiency enhanced by NPs formulations with chitosan (Nagamani et al., 2015; Kim et al., 2018; Liu et al., 2019; Simovic et al., 2015). The enhanced absorption of fucoidan in C1-F3P NPs may be due to its reduced zeta potential may cause increased bioavailability of C1-F3P NPs in bloods for longer time than only oligofucoidan and only chitosan.
3.4. Charge correlation of C1-F3P NPs and fucoidan for intestinal absorption Surface charge on the C1-F3PNPs and oligofucoidan were determined by zeta potential measurements. The changes in the surface charges between these particles in acidic pH, mimicking stomach and neutral pH, mimicking intestines were monitored. In LMW oligofucoidan, as pH was increased, weak acids deprotonate resulting increased net negative surface charge from −10.9 to −12.2. In case of C1-F3PNPs, as pH was increased, the net negative surface charge decreased due to strong interactions with quaternary ammonium ions of chitosan with sulfates and carboxylic groups of oligofucoidan. As a
Fig. 1. FT-IR spectra of self-assembledC1-F3 PNPs. 4
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Fig. 2. In vivo intestinal absorption of DAPI stained (a) Rhodamine-B-Fucoidan. (b) FITC-Chitosan. (c), (d), (e), and (f) are DAPI stained dye conjugated C1-F3P NPs.
3.6. Radio protective effects of oligofucoidan and C1-F3P NPs against γradiation induced intestinal damage in mice
ionizing radiation performed for cancers of the total body region. This liver also is the primary organ responsible for drug metabolism and mainly detoxifies damaging electrophiles generated during oxidative stress. We designed several biochemical assays to study the effect of Gyradiation when orally administrated with oligofucoidan and C1-F3P NPs. According to the results of the present study, when mice were exposed to a single dose (5.0 Gy) the total body Gy-irradiation-induced lipid peroxidation products, such as thiobarbituric acid reactive substance assay (TBARS) and conjugated diene (CD) levels significantly increased in the liver and intestine tissue samples of radiation alone treated group Fig. 4A and B. Thus, these findings confirm that exposure to Gy-radiation induces oxidative stress by increasing TBARS and CD levels of the small intestinal and liver tissues in mice (Jeong et al., 2016). As a result, overproduction of ROS formation due to oxidation of unsaturated fatty acids in the cell membranes, ROS targets lipids, and DNA (Zhu et al., 2012) On the other hand, treatment with oligofucoidan and C1-F3P NPs prior to γ-irradiation reduce the TBARS and CD levels in liver and intestinal tissues compare to radiation alone group, which indicated the strong antilipoperoxidative property in present in the intestine and liver of mice treated with oligofucoidan and C1-F3P NPs. Notably, C1-F3P NPs treated mice before and after exposure to Gy-radiation exhibited very low TBARS and CD levels confirming the strong antilipoperoxidative property of C1-F3P NPs. This kind of results
One of the major problems associated with irradiation during radiotherapy is the unavoidable damage to the connective tissues. A few studies indicated that oligofucoidan can be acted as radioprotector, which reduces the damage in human leukemia cells, diminishes hematological changes, and decreases radiation induced death in mice. Hence, the present study deals with the in vivo administration of oligofucoidan and C1-F3PNPs before and after irradiation protect mice from radiation-induced damage. Ten days after Gy-5 irradiation, animals were sacrificed, the tissue sections were collected and biochemical assay and histological studies were performed. The colon was found abnormally dark in the case of control, but the oligofucoidan and C1F3P NPs administered mice showed normal colon (see Fig. 3). 3.7. Effect of fucoidan and C1-F3P NPs and /or Gy- radiation on lipid peroxidation status The small intestine has been reported to have an amazing capacity for repair. A delay in the rate of revival after irradiation could be explained on the basis of antioxidants depletion by oxidative stress the liver of mammals has been reported as a highly radiosensitive organ. This liver one of the most frequently injured organs during exposed to
Fig. 3. Gy-radiation induced intestinal damage in mice after administration of oligofucoidan, and C1-F3PNPs. 5
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Fig. 4. Effect of oligofucoidan and C1-F3P NPs treatment on 5 Gy-radiation mediated lipid peroxidation (TBARS & CD) in the liver (A) and intestine (B) of mice. Values are given as means ± SD of six experiments in each group. Values (a, b, c) differ significantly (P < 0.05 (DMRT)).
previously observed in fucoidan obtained from Fucus vesiculosus and exhibited excellent scavenging capacities on the hydroxyl and superoxide radicals (Kruk et al., 2009). 3.8. Effect of oligofucoidan and C1-F3P NPs and /or Gy-radiation on antioxidants status Another important finding in our study antioxidant status, may contribute to mitigate the oxidative stress. Enzymatic and non-enzyme antioxidants constitute a mutually supportive team of the defense system by alleviating tissue damage. SOD and CAT work together to eliminate reactive oxygen species and small deviation in physiological concentrations may have a dramatic effect on the resistance on cellular lipids, proteins, and DNA to oxidative damage (Nita et al., 2016). When mice were exposed to a single dose (5.0 Gy) total body Gyradiation, results showed significantly lower level of enzymic antioxidants such as SOD, CAT and GPx Fig. 5A, 5B and non-enzymatic antioxidants GSH Fig. 5C in liver and intestinal tissue sample compared to both Gy-irradiated + oligofucoidan and C1-F3P NPs treated group of mice. These results proved that the γ-radiationtreated mice experienced unfavorable effects in its antioxidant systems. It has been shown that over production of free radicals interrupts the stability between oxidation and antioxidant mechanism (Kurutas, 2016). In addition, H2O2 also formed during radiolysis of water, which is easily reached to DNA and lipids. Oral administration of oligofucoidan and C1-F3P NPs prior to Gy-irradiation reversed in enzymatic antioxidants of SOD, CAT, and GPx and non-enzymatic antioxidants of GSH activities in tissues samples compared to control group, which indicate the strong ability of C1F3P NPs to scavenge free radicals and toxic carcinogenic electrophiles. Moreover, oligofucoidan and C1-F3P NPs significantly increases endogenous SOD and CAT due its strong antioxidants property, an enzyme which converts hydrogen peroxide into water and prevents the formation of hydroxyl radicals, which may be one of the contributing factors for its radioprotective activity (Wang et al., 2019).
Fig. 5. Effect of oligofucoidan and C1-F3P NPs on 5 Gy-radiation mediated enzyme antioxidants (SOD, CAT and GPx) and non-enzymatic antioxidants GSH in liver (A) and intestine (B) of Swiss albino mice. Values are given as means ± SD of six experiments in each group. Values (a, b and c) differ significantly at P < 0.05 (DMRT). * The enzyme concentration required for 50% inhibition of nitroblue tetrazolium reduction in one minute, ** μmol of hydrogen peroxide consumed per minute, and *** μg of glutathione consumed per minute.
3.9. Histopathology analysis Following a single dose (5.0 Gy) total body Gy-irradiation, the animals were euthanized and intestine biopsies were processed for hematoxylin and eosin staining. The intensity of the color is proportional to the concentration of hydroxyl groups. The obtained results showed that crypt–villus units of the intestinal mucosa were severely destroyed, as indicated by bright magenta color Fig. 6 (I) and also evidenced by the flattened villi and decreased number of surviving crypts. In contrast, oligofucoidan and C1-F3P NPs treated, Gy-irradiated mice showed reduced intensity of magenta color (Fig. 6 (II and III)). Thus, indicating that the loss of villi and crypts was mitigated by fucoidan and C-F NPs. In conclusion of this study, self-assembled chitosan-fucoidan polysaccharide (C-FP) nanoparticles (NPs) were prepared successfully for the purpose of enhancing the intestinal absorption of oligofucoidan. For
the purpose of producing stable NPs, the ratio between oligofucoidan and chitosan was tuned during NPs preparation and selected the more stable C1-F3P NPs. An improved intestinal absorption of C1-F3P NPs observed over only oligofucoidan treated mice with increased antioxidant activity when Gy-radiation was irradiated. The radioprotective effect of oligofucoidan and C1-F3P NPs was demonstrated using various biochemical assays after Gy-irradiation i.e lipid peroxidation, antioxidants system, and histopathology in the mice intestine and liver. Further, C1-F3P NPsand oligofucoidan provided protection against Gyradiation induced damage. Notably, self-assembledC1-F3P NPsexhibited enhanced antioxidant property than its counterparts proved the 6
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Fig. 6. (A and B) Effect of oligofucoidan and C1F3P NPs on 5 Gy-radiation induced intestinal damage. Twenty-four hours after 5 Gy-radiation, the animals were euthanized, and intestinal biopsies were processed for Hematoxylin-Eosin (H&E) staining. Group (I) Gy-radiation alone, (II) Gy-radiation + oligofucoidan (III) GyRadition + C1-F3P NPs treated in mice. A minimum of three animals and at least six measurements for each specimen were taken for intestine damage.
potential for mitigating radiation induced damages due to radiotherapy.
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CRediT authorship contribution statement Szu-Yuan Wu: Project administration, Conceptualization. Vijayarohini Parasuraman: Investigation. Hsieh-Chih-Tsai: Supervision, Writing - review & editing. Vinothini Arunagiri: Formal analysis. Srithar Gunaseelan: Writing - original draft. Hsiao-Ying Chou: Methodology. Rajeshkumar Anbazhagan: Validation. Juin-Yih Lai: Funding acquisition. Rajendra Prasad N: Writing - review & editing. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements The authors would like to thank the Ministry of Science and Technology of the Republic of China (Taiwan) (Grant Nos. MOST 1052221-E-011-151-MY3 and 105-2221-E-011-133-MY3), National Taiwan University of Science and Technology, National Taiwan University and Taipei Medical University Joint Research Program (Grant Nos. TMUNTUST-104-10, TMU-NTUST-105-07 and TMU-NTUST-106-06), Lo-Hsu Medical Foundation, Lotung Poh-Ai Hospital (Funding Number: 10908 & 10909) and Hi-Q Marine Biotech International Ltd., New Taipei City, Taiwan for providing the financial support. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ijpharm.2020.119161. References Akpolat, M., Kanter, M., Uzal, M.C., 2009. Protective effects of curcumin against gamma radiation-induced ileal mucosal damage. Arch. Toxicol. 83, 609–617. Ale, M.T., Jorn, D., Mikkelsen, D., Meyer, A.S., 2009. Important determinants for fucoidan bioactivity: a critical review of structure-function relations and extraction methods for fucose-containing sulfated polysaccharides from brown seaweeds. Mar. Drugs 9,
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