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Fabrication of BSA-green tea polyphenols-chitosan nanoparticles and its role in radioprotection: A molecular and biochemical approach Sumit Kumar, Ramovatar Meena, and R. Paulraj J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02068 • Publication Date (Web): 07 Jul 2016 Downloaded from http://pubs.acs.org on July 7, 2016
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Journal of Agricultural and Food Chemistry
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Fabrication of BSA-green Tea Polyphenols-chitosan Nanoparticles and Its Role In
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Radioprotection: A Molecular and Biochemical Approach
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Sumit Kumar†, Ramovatar Meena‡, R. Paulraj‡,*
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†School
of Life Science, Jawaharlal Nehru University, New Delhi 110067, India.
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‡School
of Environmental Sciences, Jawaharlal Nehru University, New Delhi 110067, India.
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*: Corresponding author
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Dr. Paulraj R, School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, India-
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110 067. Telephone: 91-11-26704162 (off), Fax. 91-11-26741586, 26741502.
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E-mail.
[email protected] 1 ACS Paragon Plus Environment
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ABSTRACT:
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Normal tissue damage from ionizing radiation during radiotherapy is a major concern in cancer
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treatment. Tea polyphenols (TPs) have shown to reduce radiation-induced damage in multiple studies,
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but its pharmacological application is still limited due to poor bioavailability. Present study was aimed
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to increase TPs bioavailability by nanoformulation using BSA as matrix and chitosan as external shell.
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Encapsulated TPs nanoparticles were spherical in size and promoted TPs stability in normal and
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gastrointestinal condition without losing antioxidant activity. Oral administration of nanoparticles for 3
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days prior to irradiation exposure has shown to protect mice from haematological injuries that result in
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reduction of radiation-induced lethality. TPs reduces radiation-induced oxidative damage and apoptosis
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by restoring the redox status through Nrf2-ERK pathway and reducing Bax expression respectively.
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Regarding potency, encapsulated TPs have shown a significantly higher level of radioprotection than
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TPs suggesting that TPs nanoparticles can be explored as valuable radioprotective and
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pharmacotherapeutic agent.
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KEYWORDS: tea polyphenols; nanoparticle; antioxidants; free radical scavenging, radiation-
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protection.
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INTRODUCTION:
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Ionizing radiation (IR) exposure inflict cellular injuries as a manifestation of reactive oxygen species
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(ROS) such as superoxide anion (O2.ˉ), hydroxyl radical (HO.) and hydrogen peroxide (H2O2), etc. and
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thus causing tumour cell killing during radiotherapy.1 However despite being targeted in nature,
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radiotherapeutic radiation also cause serious injuries to normal tissues due to undefined boundary and
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uncleared location of tumour thus reducing the benefit of radiotherapy.1,2 Radioprotectors are
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compounds that reduce radiation-induced damage to normal tissues.1 In the last few decades, multiple
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investigations have been performed, but the ideal radioprotector remains to be elusive.1 Further many
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diseases like aging, diabetes, arthritis, coronary disease, cancer, etc. are also known to be mediated by
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free radicals, hence finding of a good radioprotector may also help in treating these pathological
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conditions.2
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Tea infusion, prepared from dried leaves of Camellia sinensis is the second most popular beverage
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around the world after water.3 Green tea is produced from unfermented leaves of Camellia sinensis,
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demonstrated to rich in health promoting compounds,4,5 such as catechins [epigallocatechin gallate
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(EGCG), epigallocatechin etc.].3 Previously, EGCG has shown to reduce the radiation-induced
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esophagitis in non-small-cell lung cancer patients in Phase-II clinical trial1 and enhance the tumour
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radiosensitivity in breast cancer patients.6 However, utility of tea polyphenols (TPs) in radioprotection
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or in other therapeutic application is severely limited due to rapid degradation (80% in 1 h at
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physiological pH) and poor bioabsorption (0.1–1.1%).7,8 Nanostructure-based drug delivery system is
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one of the fastest emerging areas in enhancing the bioavailability of different drugs.9-11 Coating of
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chitosan or poly lactic-co-glycolic acid, over TPs, has shown to improve the stability of TPs in addition
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to improving the transcellular delivery of TPs.7,8 However despite enhancement in stability and
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bioavailability of TPs in in-vitro,7,8 its nanoparticles (NPs) efficacy in in-vivo model system is yet to be 3 ACS Paragon Plus Environment
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tested. Further, the choice of material in nanoparticle formulation is also quite important. Previously
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many biodegradable materials such chitosan, BSA, Poly-lactic-co-glycolic acid, cyclodextrin, etc. have
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been used for the drug entrapment. However due to remarkable efficacy of BSA12 resulted in FDA
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approval of BSA for drug formulation and delivery.12,13
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Thus, present study aimed to synthesize tea polyphenols NPs using BSA as matrix and chitosan as
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covering shell and investigate the physicochemical properties and stability of TP NPs in the gastric
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environment. Further, we also tested whether the improved stability of TPs does translate into any
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enhanced radioprotective activity in the murine model system.
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MATERIALS AND METHODS:
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Tea Polyphenols Extraction:
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Fresh dried Darjeeling variety green tea (Camellia sinensis) leaves were purchased locally (TATA
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Tetley, Tata Global Beverages, Kolkata, India), crushed, mixed with hot distilled water [(DW) (80oC)]
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in 1:20 ratio and incubated for 20 min in water bath at 80oC. The infusion was collected by filtration
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(0.45 µm membrane filter) and the process was repeated once with residue. The infusion was cooled at
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room temperature (RT) and extracted with equal volume of chloroform to remove caffeine and pigments.
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The aqueous phase was freeze-dried in a vacuum evaporator (Labconco, MO, USA) and stored at -20oC
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as green tea polyphenols (GTP).
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Total Phenol, Flavonoid, Catechins, Caffeine, Polysaccharides Quantification in Tea Infusion:
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For total polyphenol content measurement,14, 2 ml tea infusion was mixed with equal volume of Folin–
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Ciocalteu reagent (SRL, Mumbai, India) and kept for 3 min at RT in dark. Afterwards, 6 ml of 10%
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Na2CO3 solution was added and kept for 2 h at RT. Absorbance of sample was measured
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spectrophotometrically (UV-1800, Shimadzu Inc, Tokyo, Japan) at 760 nm and amount of polyphenol 4 ACS Paragon Plus Environment
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was calculated from gallic acid standard. The result was expressed as mg/g of Gallic acid equivalents
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(GAE). The flavonoid content was measured as described previously.15 Amount of caffeine was
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quantified by lead subacetate method. Briefly, 1 ml tea infusion was treated with 0.5 ml HCl (3.8x10-4
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w/v in DW) and 0.1 ml lead acetate (0.5% in DW) and the mixture was diluted up to 10 ml by DW. The
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solution was filtered and 5 ml of filtrate was treated with 60 µl of H2SO4 (0.3% w/v in DW), and then
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filtered again. The filtrate absorbance was measured at 274 nm and caffeine amount present in the sample
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was calculated from caffeine (Sigma-Aldrich, St. Louis, MO USA) standard. The amount of catechins
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was determined by vanillin assay as described previously.16 The polysaccharide was quantified by
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anthrone–sulfuric acid method using glucose as standard as describe previously in our paper.17
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Green Tea NPs Preparation:
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Synthesis of BSA NPs (BN):
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BN were synthesized by desolvation method.13 Briefly, 25 ml BSA (Sigma-Aldrich, St. Louis, MO,
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USA) solution (4 mg/ml) was placed in a beaker under stirring condition (800 rpm), and 50 ml ethanol
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(≥99.8%, Merck Millipore, MA, USA) was added dropwise to the solution. The suspension was stirred
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for 15 min, then 1 ml glutaraldehyde was added and further kept under stirring condition for 1 h. BSA
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NPs was collected by centrifugation at 20000x g for 15 min, sediment washed thrice with DW and
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resuspended in 20 ml deionized water.
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Loading of BN with Green Tea Polyphenols:
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Twenty ml of GTP extract (2 mg/ml in deionized water) was added dropwise into 10 ml of BN under
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stirring (800 rpm) condition at RT and left for 30 min under stirring condition.
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Coating of Chitosan over BSA-GTP NPs (BGN):
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Chitosan solution (4 ml, 1 mg/ml) was added dropwise in stirring BGN (20 ml) suspension and kept for
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30 min under stirring condition at RT. After NPs were collected by centrifugation (20000x g), then
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washed with DW, freeze-dried and stored at 4oC in anhydrous condition as BSA green tea chitosan
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nanoparticles (BGCN). The drug-free NPs were prepared by following similar steps except GTP solution
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was replaced with DW.
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Physicochemical Characterisation of BGN and BGCN:
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The BN, BGN, and BGCN were characterized by transmission electron microscopy (TEM), scanning
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electron microscope (SEM), zeta potential analyser and dynamic light scattering (DLS), which have been
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broadly described in our previous paper.18 Briefly, a drop of homogeneous NPs suspension (50 µg/ml)
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was placed over copper grid with a laser carbon film and air dried. NPs were observed with a JEOLJEM-
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2100F TEM (MA, USA) operating at 200 kV and image was captured. NPs size were calculated from
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diameters of randomly selected particles. For examining the NPs surface morphology, a small drop of
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NPs was placed over a cut glass and air dried. Gold particles were deposited on its surface using spray
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gun under vacuum and micrographs were taken by SEM (Zeiss EVO40, Oberkochen, Germany). NPs
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size distribution pattern and zeta potential were measured by DLS (NanoZS, Malvern, UK) and zeta
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potential analyser (ZC-2000, Microtec, Chiba, Japan) as describe previously.18
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Drug Loading and Entrapment Efficiency:
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To calculate the NPs drug loading and entrapment efficiency8,19 dried NPs (20 mg) was sonicated for 30
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min in 2 ml of 0.4 N NaCl solution containing 20 mM ascorbic acid and 13 mM Tris[2-
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carboxyethyl]phosphine hydrochloride as reducing agent. Supernatant was collected by centrifugation
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and absorbance was recorded at 274 nm in spectroscope (UV-1800, Shimadzu Inc, Tokyo, Japan) against
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sample containing GTP-free NPs. Tea extract showed peak absorbance at 274 nm owing to the presence
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of EGCG.19 Amount of tea polyphenols was calculated from polyphenols standard (r2: 0.971) and used 6 ACS Paragon Plus Environment
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for calculation of entrapment efficiency (Quantity of GTP present in NPs/Quantity of GTP used x 100)
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and drug-loading efficiency (Quantity of GTP present in NPs/dry weight of NPs x 100).
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In-vitro Release Assay:
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BGN (10 mg) and BGCN (9.84 mg) equivalent to 2.57 mg of tea extract and same amount of GTP-free
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NPs was dispersed in 1.5 ml of phosphate buffer (pH 7.4) and incubated at 37oC under stirring condition
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(200 rpm). At constant time interval (2 h), supernatant was collected by centrifugation (20000x g for 10
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min), pellet resuspended in 1.5 ml phosphate buffer and process was repeated till 24 h. Release of tea
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extract (%) in supernatant was quantified by measuring sample absorbance at 274 nm, Folin–Ciocalteu’s
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method and vanillin reagent as describe above. The result was subtracted from sample containing GTP-
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free NPs and expressed as % value of reference sample containing 2.57 mg of tea extract under similar
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condition.
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Gastrointestinal (GI) Stability of NPs:
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TP NPs stability was measured in an in-vitro simulated GI digestive environment (salivary, gastric and
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duodenal digestion).20 For salivary digestion, BGN (20 mg), BGCN (19.6 mg) equivalent to 5.14 mg of
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tea extract and same amount of GTP free NPs were suspended in 10 ml PBS. Suspension was treated
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with 23 ml of simulated saliva [KCl (89.6 mg/ml), KSCN (20 mg/ml), NaH2PO4 (88.8 mg/ml), Na2SO4
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(57.0 mg/ml), NaCl (175.3 mg/ml), NaHCO3 (84.7 mg/ml), urea (25.0 mg/ml), α-amylase (145 mg), pH
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6.8] and kept in rocking condition at 37oC for 3 min. For subsequent gastric stage, pepsin (0.1 g, SRL,
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Mumbai, India) was added, pH adjusted to 2 with HCl (5 mol/L) and incubated at 37oC for 30 min in an
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orbital shaker (100 rpm). For pancreatic stage, pH was adjusted to 6.5 by Na2CO3 (0.5 mol/L) and then
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5 ml each of pancreatin (0.2 mg/mL, SRL, Mumbai, India) and bile salts (10 mg/mL, SRL, Mumbai,
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India) solutions was added. Samples were incubated at 37oC for 30 min in an orbital shaker (100 rpm).
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At each stage, 3 ml solution was withdrawn, centrifuged (20000x g for 5 min at 4oC), and supernatant 7 ACS Paragon Plus Environment
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use for polyphenol quantification. The polyphenols present at the end of each step was quantified by
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absorbance (274 nm) measurement, Folin–Ciocalteu’s method and vanillin reagent as describe above.
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Parallel sample containing GTP-free NPs served as blank.
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Free Radical Scavenging Assay:
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For free radical scavenging activity21 measurement, 1 ml DW containing 5–200 µg/mL tea extract, NPs
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possessing equivalent amount of tea extract and GTP-free NPs were mixed with 3 ml DPPH (0.1 mM in
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methanol, Sigma-Aldrich, St. Louis, MO, USA) and incubated for 30 min in dark at RT under shaking
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condition (400 rpm) at RT. Sample absorbance (Abs) and blank absorbance (Abb) was measured at 517
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nm against methanol:water (3:1) mixture. Free radical scavenging activity (%) was calculated =[1-
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(Abs/Abb)]x100. Free radical scavenging activity of GTP-free NPs was deducted from BGN and BGCN
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free radical scavenging activity.
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In-vivo Experiments:
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Animals and Experimental Protocol:
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Inbred 6-7 weeks old male Swiss albino mice (30±3 gram) were used in present study. Animals were
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housed at University animal house (22±30C, relative humidity: 60±5%, 12-h light/dark cycle) in
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polypropylene cages and provided with food pellet (Hindustan Animal Feeds, GJ, India) and water ad
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libitum. The experimental protocol was approved by Institutional Animal Ethical Committee (IAEC-
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JNU) and guidelines of Committee for the Purpose of Control and Supervision of Experiments on
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Animals, GoI was followed. For different experiments, animals were randomly divided into eight groups
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(n=32) as detailed in Fig 1. Animals were treated orally using oral gavage with 100 µL PBS containing
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50, 194 and 191 mg/kg body wt of GTP, BGN and BGCN respectively once a day for 3 days. Dose of
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BGN and BGCN was calculated on the basis of drug loading. The animals were exposed to whole body 8 ACS Paragon Plus Environment
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radiation (WBI) after 3 h of last dose. For irradiation mice were kept inside the ventilated perspex
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container and exposed to WBI using
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(Bhabhatron 4000A, DAE, Mumbai, India). Radiation dose was 4-8.5 Gy depending on need of
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experiments.
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Survival Assay:
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For survival study,17 10 mice in each group were allocated as detailed in Fig. 1 and treated with GTP,
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BGN, BGCN for 3 days. Mice were exposed to WBI (8.5 Gy) and kept in animal house for 30 days.
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Animal were monitored daily up to 30 days and radiation-induced sickness, mortality were recorded.
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Level of significance was calculated using log-rank/Mantel–Cox test.
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Endogenous Spleen Colony Assay and Spleen Index:
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For spleen colony assay,17 10 mice in each group were allocated as mentioned in Fig. 1 and treated with
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GTP, BGN and BGCN for 3 days. Mice were exposed to WBI (7 Gy) and sacrificed at 11th-day post-
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irradiation. Spleen was removed, washed in saline, blotted and weighed to calculate the spleen index
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(Weight of spleen/Total body weight x 100). Spleen was fixed in Bouins fixative (saturated picric acid
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30.0 ml + formaldehyde 10.0 ml + glacial acetic acid 2.0 ml) for 15 min and spleen colonies were counted
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using hand held magnifying glass.
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Hematological Parameter Analysis:
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For haematological analysis, 150 µl blood was withdrawn from mice retro-orbital sinus using glass
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capillary at 7th-day post-irradiation, immediately transferred into heparinized tube and stored at 4oC for
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blood cell parameter analysis. Subsequently, mice were sacrificed humanely by cervical dislocation and
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femur was dissected out. Bone marrow cells were removed from femur and cell viability was measured
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by trypan blue dye (0.04% in PBS) exclusion test. Leukocyte, erythrocyte, hemoglobin, platelets,
60Cobalt
at a dose rate of 2.14 Gy/min in a gamma chamber
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monocytes, lymphocytes and neutrophils count were analysed by Automated Haematology Counter (Kx-
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21, Sysmex, Mumbai, India).
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Isolation and Culture of Splenocytes:
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Spleen was removed aseptically at 6 h postirradiation, minced in PBS (pH: 7.4) using blunt forceps and
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RBC was busted out by hypotonic shock. Cells (splenocytes) were washed twice in PBS, counted and
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cultured in RPMI 1640 (Himedia, Mumbai, India) with 10% Fetal bovine serum (Himedia, Mumbai,
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India) and antibiotic-antimycotic solution (Himedia, Mumbai, India) at a density of 2x106 ml-1.
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ROS Measurements:
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Splenocytes were seeded at density of 4x104 cells/200 μl in 96 well plates and incubated for 18 h. DC-
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FDA (5 µmol, Sigma-Aldrich, St. Louis, MO, USA) was added 30 min prior to the end of incubation,
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and fluorescent emission was recorded at 520 nm (excitation: 490 nm) using Microplate reader
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(SpectraMax M2, Molecular Devices, CA, USA). Result was expressed relative to untreated control.
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The cells were also visualized under fluorescent microscope.
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Splenocytes Proliferation Assay:
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Splenocytes (20,000 cells/200 μl) were cultured for 14 h in 96 well culture plate, afterword 20 µl MTT
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(Sigma-Aldrich, St. Louis, MO, USA) solution (5 mg/ml in culture medium) was added into each well
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and again cultured for 4 h. The culture medium was aspirated, 200 µl DMSO added into each well and
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absorbance was recorded at 570 nm in ELISA reader.
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DNA Strand Break Assay:
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DNA damage was measured by comet assay as described previously.2 Briefly splenocyte were harvested
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after 16 h and ~10,000 cells were immobilized using low melting agarose (0.5% in PBS, 75 µL) over a 10 ACS Paragon Plus Environment
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glass slide (2”x1”). Cell were lysed (2.5 M NaCl, 100 mM EDTA, 100 mM Tris base, 1% Triton X-100,
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and 10% DMSO, pH 10) for 2 h at 4oC, and then electrophoresis was performed at 0.74 V/cm for 30 min
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in electrophoresis buffer (1.2% NaOH, 0.037% EDTA). DNA was stained with EtBr (5 μg/mL),
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visualised under fluorescent microscope, and comet parameter was calculated by comet score-15
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software (TriTek Corp., VA, USA).
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Apoptosis:
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Splenocytes were harvested after 18 h, washed once with PBS, and stained with EtBr/AO mixture (1:1;
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200 μg AO/mL in PBS; 200 μg EtBr/mL in PBS) for 2 min at 4oC.2 Cell were visualised under
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fluorescent microscope and live, apoptotic, and necrotic cells were distinguished on the basis of colour
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(live: green, apoptotic: yellow, necrotic cells: red).
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Mitochondrial Membrane Potential (MMP) Measurement:
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Splenocytes were cultured for 18 h. MMP was measured using JC-1 dye kit as per the manufacturer's
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instructions (Cayman Chemical, MI, USA). For microscopic analysis, mitotracker dye (Invitrogen,
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Mumbai, India) was added in culture plate at 30 min prior to the end of incubation. Follwing, cells were
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washed once with PBS, fixed in 10% formalin for 5 min at 25oC and again washed with PBS. Cells were
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observed under fluorescence microscope (Nikon, New Delhi, India).
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Estimation of Dead and Necrotic Cells by LDH:
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LDH level was quantified in culture medium as a marker of damaged, dead, and necrotic cells. Cell
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culture medium including floating cells was collected after 18 h of culture. Sample was sonicated (Q500
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sonicator, Qsonica, CT, USA) for 5 cycle at 4oC, each at peak to peak amplitude of 18 microns for 5 s
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at interval of 25 s. Following supernatant was collected by centrifugation (5000x g for 5 min at 4oC) and
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10 µl supernatant was mixed with 140 µl Tris-HCl buffer (0.2 M, pH 7.3) in 96 well Elisa plate. 11 ACS Paragon Plus Environment
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Afterword 5 µl of each NADH (6.6 mM) and sodium pyruvate (30 mM) solution was added, and change
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in absorbance/min was recorded at 340 nm spectroscopically (Shimadzu UV-2700). The change in
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absorbance/min was normalised with control and expressed as % change in LDH activity.
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Biochemical Assay:
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The liver was homogenised in 0.1 M phosphate buffer (pH 7.0) using Potter Elvehjem homogenizer. The
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homogenate was divided into two parts. One part was used for GSH estimation as described previously,22
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while another portion was used for the measurement of catalase (Cat), superoxide dismutase (SOD),
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glutathione peroxidase (GPx) and lipid peroxidation as described previously.22
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Real-time Quantitative Polymerase Chain Reaction:
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Splenocytes were prepared from mice spleen at 6 h postirradiation. Total RNA was extracted from spleen
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with TRI reagent (Sigma-Aldrich, St. Louis, MO, USA) by following manufacturer recommendations.
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RNA integrity was checked and 2 µg RNA used to synthesize cDNA by cDNA synthesis kit (Fermentas,
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Waltham, MA, USA). Twenty-five microliter reaction mixtures consisted of 12.5 μl of 2x SYBR green
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PCR reaction mix (Fermentas, Waltham, MA, USA), 1 μl (0.5 µM) of each primer (Table 1) and 2 μl (2
238
μg) of template. The amplification condition was as follow: Step 1: denaturation at 95°C for 5 min; step
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2: denaturation at 95°C for 15s; step 3: annealing at 57-60°C for 15s depending on primer set; Step 4:
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extension at 72°C for 20 s; step 5: melting curve analysis. Step 2-4 was repeated for 40 cycles. The
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threshold cycle value of amplification was used to calculate the expression of HO-1, nrf-2, NQO-1, Bax,
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ERK and β-actin gene. Result was normalised against β-actin (housekeeping gene) and expressed as fold
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change with respect to control.
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Statistical Analysis:
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Data were expressed as mean±SD, and statistical analysis was done by performing student's t-test, one-
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way ANOVA and post analysis was done by Tukey test in Graph PadPrism 5.03. p values GTP. The reduction in radiation-induced
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apoptosis was further confirmed by LDH level measurement, indicating that TPs reduce the necrotic
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events and membrane damage in splenocytes (Fig. 6c).34 At molecular level, tea polyphenols
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administration significantly (p
