Green tea catechins (GTCs) are known for their anti-oxidant activity due to presence of Epigallaocatechin-3-gallate (EGCG). Excessive degradation and poor intestinal absorption render it ineffective. Present study is focused on enhancing the cellular uptake and intestinal absorption of EGCG through nanoencapsulated GTCs (GTC-NPs) in Caco-2 cell lines, with the ultimate goal of enhancing its bioefficacy using chitosan nanocarriers. The stability, cytotoxicity and cellular uptake studies of EGCG from GTCs and GTC-NPs were studied. The net flux and net efflux were estimated to understand the overall transepithelial transport in a polarised Caco-2 monolayer. Intestinal absorption prediction studies were carried out at different temperatures as well using different absorption stimulators. H2O2-induced oxidative stress was applied to determine the protective effect of EGCG from GTCs and GTC-NPs. The results of the study showed stability of EGCG from GTCs and GTC-NPs in HBSS buffer (pH 7.4 and pH 6.5) with reducing agent (ascorbic acid) up to 95% and 97%, respectively. Cellular uptake studies showed 3-fold improvement in the uptake of EGCG from GTC-NPs. Transepithelial transport studies have confirmed 5.6-fold increase in flux and 3.9-fold decrease in the efflux of EGCG with nano-encapsulation. The cytotoxicity studies against H2O2-induced oxidative stress confirmed the increased bioefficacy of nano-encapsulated GTCs. These findings are encouraging and demonstrates that the use of EGCG in drug delivery systems with the enhanced bio-asbsorption and bio-efficacy.
Research Article
Nanoencapsulation of Green Tea Catechins : Cellular uptake, Transepithelial transport, and Bioefficacy of Epigallocatechin-3-gallate
https://doi.org/10.21203/rs.3.rs-3291923/v1
This work is licensed under a CC BY 4.0 License
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Epigallocatechin-3-gallate
Intestinal absorption
Oxidative stress
Chitosan nanoparticles
Caco-2 cell line
Green tea is known as a flavonoid-rich beverage, the most abundant flavonoids in green tea are the flavan-3-ols (catechins), viz., EGCG (~ 60%), ECG (~ 14%), EGC (~ 20%), and epicatechins (~ 6%) (Jigisha et al. 2012; Musial et al. 2020). These green tea catechins have been reported to have anti-microbial, anti-oxidant, anti-carcinogenic, anti-obesity, anti-inflammatory and heart-related properties. Among all the tea catechins, EGCG emerged as the most potentially active catechin, playing a vital role in the health benefits. The therapeutic properties of EGCG are known to regulate numerous molecular targets, which have been constantly explored and endorsed in several animal, human, and in-vitro studies, making it a promising nutraceutical for the pharmaceutical, cosmetic, and nutritional industries (Yu et al. 2014). Despite having a plethora of health benefits, EGCG is unable to deliver expected pharmacological applications as it shows compromised bio-availability due to its poor intestinal permeability and stability (Dube et al. 2010). The stunted accumulation of EGCG in the targeted tissues of the human body has been observed due to deprived pharmacokinetics, limited biodistribution, and reduced first-pass metabolism (Krupkova et al. 2016). As per the previous studies, the apparent permeability (Papp) values have been encountered in the range of 0.8 to 3.5 × 10− 7 cm/s for the EGCG in the Caco-2 cell lines (Kadowaki et al. 2008; Zhang et al. 2004) and fall into the category of compounds with attenuated permeability. The difficulty in the permeation of EGCG through intestinal cell membranes is due to its high hydrophilicity. According to Chow et al. (2005), after 1–2 hours of consumption of EGCG, the maximum plasma concentration of EGCG has been observed, followed by rapid systemic clearance. Chen et al. (1997) reported in their study that epigallocatechin, epicatechin, and EGCG are present only up to 14%, 31% and 1%, respectively, in the blood for targeted tissue delivery after oral administration of green tea to rats. Therefore, this compromised oral administration of EGCG is due to its chemical instability, excessive degradation and absorption barriers due to the lack of specific receptors in the gastrointestinal epithelia, leading to a very poor intrinsic permeability of EGCG across the intestinal epithelium (Peter et al. 2010). According to recent studies, the intestinal permeability and transport of bioactive compounds can be improved by structural amendments and micro- and nano-encapsulation modes of delivery, ultimately enhancing the overall bio-availability of the compound (Dai et al. 2020). Furthermore, micro-encapsulation of EGCG protects it from the harsh environment of the gastrointestinal tract, however, it shows poor cellular entry due to its lower surface area as compared to nano-sized materials ( Suganya and Anuradha, 2017). Therefore, nanoencapsulation within hydrophobic carriers enhances the therapeutic properties of EGCG by improving intestinal permeability and stability (Sahadevan et al. 2022). Drug delivery approaches using polymeric nanoparticles have gained significant importance as they possess properties such as bio-degradability, bio-compatibility, anti-oxidant activity, bio-adhesiveness, high-permeability and non-toxic (Baig et al. 2021). Chitosan nanoparticles obey the pH-responsive delivery system mechanism through the paracellular pathway (Sonaje et al. 2011). Chitosan is a positively charged polymer with muco-adhesive properties, which helps in opening tight junctions. Therefore, chitosan nanoparticles act as an ideal delivery system for enhancing the limited intestinal permeability and absorption of several bioactive compounds. Hence, the limited oral bioavailability of EGCG should be improved using an ideal drug delivery system by targeting permeation enhancers, metabolic enzyme inhibitors, and advanced carrier systems to counter the reduced intestinal absorption. As per our previous studies, nano-encapsulated GTCs have showed anti-oxidant activity with improved bio-accessibility (Tyagi et al. 2021)
This study aimed to analyse the cellular uptake and transport of EGCG from GTCs and GTC-NPs using human epithelium, i.e. Caco-2 cell monolayers, as an intestinal epithelial barrier model (Sambuy et al. (2005). The cytotoxicity and chemical stability of EGCG from GTCs and GTC-NPs, along with prediction studies using absorption stimulators, were also examined. Further, the study aimed to gain deep insight into the mechanism of intestinal absorption of the nano-encapsulated GTCs.
2.1. Materials
In this study, EGCG, verapamil, ascorbic acid, EDTA, MK-571, sodium azide, HBSS, and MTT, foetal bovine serum (FBS), high-glucose DMEM media, antibiotic solution, and sodium bicarbonate were purchased from Sigma Aldrich Pvt. Ltd, (India). Cellular transport experiments were conducted using Corning® mm Transwell inserts with 0.4 µm pore sizes of polycarbonate membrane inserts purchased from Merck life science Pvt, Ltd, (India). HPLC-grade methanol and formic acid were purchased from SD fine Chem Pvt. Ltd. (India). EGCG rich green tea catechins was procured from M/s Indovedic Pvt. Ltd.,(India)
2.2. Assessment of EGCG stability on HBSS buffer
The stability of EGCG from GTCs and GTC-NPs was studied in HBSS buffer (pH 7.4), as mentioned by Hwang et al. (2020) with minor modifications. Briefly, different concentrations of ascorbic acid (0.1 mM, 1 mM, and 10 mM) were used to understand the stability of EGCG from GTCs and GTC-NPs in HBSS buffer for a period of 24 hours. Further, the same experiment was repeated with the best-suited ascorbic acid concentration at two different pH conditions: slightly acidic (pH, 6.5) and slightly alkaline (pH, 7.4). The quantification of EGCG in HBSS buffer in all samples was estimated by HPLC as prescribed in our previous studies (Tyagi et al. (2021) and expressed in the percentage of EGCG remaining.
2.3. In-vitro cell culture experimentation
2.3.1. Maintenance of Caco-2 cell line
The Caco-2 cell line (human adenocarcinoma) was cultivated using high glucose, Dulbecco’s minimal essential media fortified with 10% FBS (heat inactivated, USA origin) and 1% 1X antibiotic solution (10000 U Penicillin, 10 mg streptomycin, and 25 µg amphotericin B) in a CO2 incubator with 95% air and 5% CO2 at 37°C. The cells were maintained until they reached 80% confluence in 25 cm2 flasks and seeded in 96-well or 6-well plates
2.3.2. Cell viability studies
. The cell viability experiment was divided into four groups: Group I: Cells were treated with only dH2O (Control); Group II: Cells were treated with EGCG; and Group III: Cells were treated with GTCs; Group IV: Cells were treated with GTC-NPs. Further, MTT assay was performed according to Katram et al. (2021) to determine the cellular viability of EGCG from GTCs and GTC-NPs. Various concentrations of EGCG, GTCs, and GTC-NPs (10 to 400 µg/mL) were studied for their effect on cell viability. After 24 hours of individual treatments, cells were subjected to MTT treatment. For that, cells were supplemented with MTT (50 µL, 0.5 mg/mL) in each well and subjected to incubation at 37ºC. The absorbance was recorded at 570 nm and cell viability was calculated by using the formulae:
2.4. EGCG cellular uptake studies from GTCs and GTC-NPs
The cellular uptake studies of EGCG from GTCs and GTC-NPs were conducted according to Redan et al. (2017) with minor changes. A washing buffer (1X PBS, pH 7.8) was used to rinse the Caco-2 cells before treatment. EGCG, GTCs, and GTC-NPs (100 µg/mL) were added individually to the Caco-2 cell line in the six-well plate. Plates were placed in a shaking incubator at 37°C and cells were extracted every hour for a period of six hours from individual wells. Extracted cells undergo lysis using Triton-X 100 (0.5%). The cells were washed 3 times with ethyl acetate and resuspended in mobile phase i.e., 0.1 formic acid in methanol and subjected to HPLC analysis.
2.5. Maintenance of epithelial monolayer
Caco-2 cell lines between passages 36 to 45 were subcultured using DMEM with high glucose media at 80% confluence after trypsinization. Cells were seeded at 2.6 x 105 cells/cm2 onto 24 mm transwell cell culture inserts with a polycarbonate membrane (0.4 µm) placed in 6 well plates (4.67 cm2). The apical and basolateral chambers were supplemented with 3 mL and 2 mL of high-glucose-DMEM, respectively. The cells were regularly maintained at every 24 hours for a period of one week; followed by every 48 hours until 21 days. Confluent monolayers of Caco-2 cells were subjected to transport and metabolism experiments.
2.6. Caco-2 monolayer integrity studies
A confluent Caco-2 cell monolayer was used to study the cellular permeability of EGCG from GTCs and GTC-NPs (Bhushani et al. (2016). Lucifer yellow (0.1 mg/mL; 0.5 mL) is a paracellular marker used to measure the cellular integrity of the monolayer. Briefly, DMEM was supplemented with and without Lucifer yellow (HBSS buffer, pH 6.5) (w/v) in apical and basolateral chambers of cell inserts, respectively and incubated for 37°C for a period of 2 hours. Basolateral media was collected at every 30 min intervals for a period of 2 hours to estimate the apparent permeability coefficient (Papp) of this paracellular marker in the monolayer using a multi-plate reader (SpectroSTAR NANO, BMG Lab Tech, Germany) at an excitation wavelength of 480 nm and an emission wavelength of 535 nm. The results were denoted in apparent permeability (cm/sec), as mentioned in the following equation.
dQ/dt = change in concentration of EGCG in the basal chamber with different intervals of time, A = area of polycarbonated membrane; and C0 is the initial concentration of EGCG from GTCs and GTC-NPs in the apical chamber. V is the volume of media in the basal chamber.
2.7. EGCG cellular transport studies
2.7.1. Transepithelial electrical resistance studies
Transepithelial/transendothelial electrical resistance (TEER) anticipates the epithelial membrane integrity indicated by a normal range of 300–500 Ω × cm2. The study was conducted using a Millicell ERS-2 epithelial volt-ohmmeter (Millipore Corp., Bedford, MA) connected with a pair of chopstick electrodes (Faralli et al. 2019). The electrode of the volt-ohm meter was activated 24 hours before the experiment by dipping it into 1X PBS (pH 7.8). The confluent monolayer of the Caco-2 cell line was observed weekly for its TEER value until the TEER value reached 450 Ω × cm2. Blank readings were taken from the well inserts without the Caco-2 monolayer, and the resistance of the monolayer was calculated as follows:
Rtissue (Ω) = Rtotal (Ω) - Rblank (Ω)
Rtissue = Specific cell resistance values,
Rblank = Resistance values of semi-permeable membrane without cells,
Rtotal = Resistance values of each cellular monolayer on the top of the semi-permeable membrane.
TEER values were denoted in Ω × cm2 and calculated by the formulae mentioned below:
TEERtissue (Ω × cm2) = Rtissue (Ω) × Amembrane (cm2)
Rtissue = Specific cell resistance values, TEERtissue = Specific cell TEER values,
Amembrane = Surface area of cell culture inserts.
2.7.2. Cellular permeability and absorption prediction
Confluent and polarized Caco-2 monolayer was used to perform the EGCG cellular permeability according to Song et al. (2014). The cells were washed two times with warm HBSS buffer (pH 7.4, 37 ºC). EGCG, GTCs, and GTC-NPs in HBSS buffer (pH 6.5) was suspended on to the apical chamber and blank HBSS buffer (3 mL, pH 7.4) was suspended on to the basolateral chamber of the cell inserts. The cell inserts were subjected to incubation at 37°C and 200 µL of basolateral media was collected at every 30 min intervals for a period of 6 hours. Quantification of EGCG from GTCs, GTC-NPs, and free form was carried out by HPLC.
The transportation of EGCG from the basolateral chamber to the apical chamber was studied as followed by Hwang et al. (2020). The HBSS buffer (pH 7.4) has aspired from cell inserts, the apical chamber was supplemented with fresh buffer (2 mL) without EGCG, GTCs, and GTC-NPs served as blank and the basolateral chamber was supplemented by HBSS buffer (3 mL) containing each sample (100 µg/mL). The aliquots were collected and analyzed as mentioned above.
2.7.3. EGCG cellular transport studies with an absorption enhancer
The absorption prediction studies of EGCG from GTCs and GTC-NPs were conducted using absorption stimulators as prescribed by Song et al. (2014). The study was conducted on the same day as cellular permeability studies to avoid cell batch variations. The absorption inhibitors viz., Sodium azide (ATP Inhibitor; 10 mM), Verapamil (Permeability glycoprotein inhibitor; 100 µM), and, MK-571 (MRP-2 inhibitor; 100 µM) or EDTA (enhancers; 10 µM) were freshly prepared at the moment of commencement of experiment and added to the apical chamber. The experiment was also performed at 4 ºC and 37 ºC without absorption stimulators to examine the effect of temperature on the transport of EGCG in Caco-2 cells. EGCG, GTCs, and GTC-NPs in HBSS buffer (100 µg/mL) was added to the apical chamber and was supplemented as mentioned above. At regular intervals, 200 µL of basolateral media has been collected for a period of 6 hours. Biotransformation of EGCG from GTCs and GTC-NPs was quantified by HPLC.
2.8. Effect of EGCG from GTCs and GTC-NPs on oxidative stress
2.8.1 Mitochondrial membrane potential
Caco-2 cells with 80% confluence were grown in 24-well plates and treatment was carried out as stated previously. Caco-2 cells were incubated with rhodamine 123 in the dark for 30 min at 37°C. Further, cells were rinsed 2 times with ice-cold 1X PBS (pH 7.8). The fluorescence intensity indicating electrochemical potential was detected at an excitation/emission wavelength (485/535 nm) using a multi-plate reader (SpectroSTAR NANO, BMG lab tech, Germany) as mentioned in Chandrasekhar et al. (2018).
Confluent Caco-2 cells (80%) were seeded in 75 cm2 cell culture flasks and treatment was carried out as stated in previously. Cell lysis was done using RIPA lysis buffer (pH; 8) in cold conditions. The cell lysate was centrifuged at refrigerated conditions at 12,000 ×g for 10 min. The ELISA kits from Cloud-clone Corp., India, were used to measure the activity of antioxidant enzymes viz., Superoxide dismutase, Catalase, Glutathione peroxidise and Glutathione reductase. The enzyme activity was expressed by U/mg or mU/mg.
2.8.2. Reactive oxygen species
The production of intracellular ROS in Caco-2 cells was examined by DCFH-DA assay as reported by Ramya et al. (2017). Caco-2 cell line at 80% confluence in 24 well plates was cultivated for the commencement of this experiment. Based on cell viability outcome, the experiment was conducted using six groups: Group I (Control): No treatment- blank culture media was added to the Cells; Group II (Negative control): Treatment of cells with H2O2 (100 µM) in culture media; Group III (Test): Treatment of cells with H2O2 (100 µM) and 100 µg/mL EGCG; Group IV (Test): Treatment of cells with H2O2 (100 µM) and 100 µg/mL GTCs; Group V (Test): Treatment of cells with H2O2 (100 µM) and 100 µg/mL Chitosan NPs; Group VI (Test): Treatment of cells treated with H2O2 (100 µM) and 100 µg/mL GTC-NPs. After exposure, the Caco-2 cells were rinsed 2 times with ice-cold 1X PBS (pH 7.4), treated with 10µM DCFH2-DA solution and kept at 37°C for incubation for 30 min in the dark. After treatment, cells were rinsed and the DCF-DA fluorescence intensity was measured at excitation/emission wavelength (485 / 535 nm) using Hidex plate chameleon TMV.
2.9. Statistical analysis
The collection, analysis and interpretation of data were conducted using Microsoft Excel software-2016. The measurements were taken in triplicates and the data was expressed in mean standard deviation. The Data was examined using one-way ANOVA and differences between the groups were compared by Tukey and Dunnet posthoc tests with a p-value of < 0.05.
3.1. Stability of EGCG from GTCs and GTC-NPs in HBSS buffer
EGCG was dissolved in HBSS buffer (pH; 7.4) with increased concentrations of ascorbic acid i.e. 0.1 mM, 1 mM and 10 mM showed significantly increased stability in a concentration-dependent manner of EGCG (p > 0.05; Fig. 1a). The addition of ascorbic acid at 0.1 mM has shown a maximum degradation of EGCG up to 50% in HBSS buffer within a period of 24 hours. However, ascorbic acid at 1 mM and 10 mM concentrations has shown significantly lesser EGCG degradation (27% and 11%, respectively). Application of GTCs and GTC-NPs significantly improved the stability of EGCG in HBSS buffer with ascorbic acid when compared to EGCG alone (p < 0.05). The degradation pattern in various concentrations of ascorbic acid with the application of GTCs and GTC-NPs is provided in Fig. 1b and Fig. 1c. Further, the study confirmed that the application of GTCs and GTC-NPs with 10 mM ascorbic acid in HBSS buffer has significantly inhibited the degradation of EGCG from 10.6–5.4%, and 2.3%, respectively.
Further experiments of the study were conducted with EGCG in HBSS buffer at 10 mM ascorbic acid concentration as it was more stable than other concentrations. As the apical media of the cell inserts used to have pH 6.5, therefore, the above experiment was repeated at pH 6.5. GTCs and GTC-NPs were not showing any significant EGCG degradation when compared to the pH 7.4 condition (p > 0.05; Fig. 2).
3.2. Cell viability studies
The cytotoxicity of EGCG from GTCs and GTC-NPs was calculated as a function of concentration as shown in Fig. 3a. Different concentrations (10–400 µg/mL) of EGCG in free form, GTCs, and GTC-NPs were used to estimate the effect of EGCG on the cell viability. The effective concentration of EGCG was found to be 100 µg/mL as it did not show any significant change in cell viability when compared to control (p < 0.05) cells. Similarly, GTCs, blank Chitosan nanoparticles and GTC-NPs were showed the same pattern for the concentrations, 100 µg/mL, 120 µg/mL, and 140 µg/mL, respectively ( p < 0.05; Fig. 3b, 3c and 3d). Further, cellular transport and toxicity studies were conducted using EGCG, GTCs, and GTC-NPs at 100 µg/mL as this concentration was determined to be a safer dose for the experiments.
3.3. Cellular uptake of EGCG from GTCs and GTC-NPs in Caco-2 cells
The cellular uptake studies of EGCG in Caco-2 cells were carried out for a period of six hours in a regular interval time and the results were presented in Fig. 4. Maximum EGCG cellular uptake was observed after two hours of incubation. Cellular uptake of EGCG from GTCs was two-fold increase when compared to the free form of EGCG. However, GTC-NPs have shown significantly improved EGCG cellular uptake as compared to GTCs and EGCG i.e. three-fold improvement when compared to EGCG free form (p > 0.05).
3.4. Cell integrity and permeability studies
A polarized and intact Caco-2 monolayer was obtained after 21 days of incubation indicated by apparent permeability coefficient values in the range of 6.8 to 8.1 X 10− 7 cm/sec using para-cellular marker, Lucifer yellow, and was maintained after the transport experiment too. The monolayer with TEER value ≤ 450 Ω × cm2 was maintained, specifying complete tight junction formation and no significant (p < 0.05) changes was observed throughout the cellular transport experiment.
3.5. EGCG transport and cell permeability studies
3.5.1. Apparent permeability coefficient (Papp) and net flux and efflux of EGCG from GTCs and GTC-NPs
The transepithelial transport mechanism of EGCG, GTCs, and GTC-NPs was examined and results were depicted in Fig. 5. During apical to basolateral flux, EGCG, GTCs and GTC-NPs showed an essentially linear nature concerning concentration for a period of 6 hours with no evident saturation levels (Fig. 5a). After 6 hours of the study, faster flux of EGCG was observed in case of GTCs upto 11-fold cumulative flux and 25-fold in GTC-NPs which is significantly much higher than the flux of EGCG across Caco-2 cells (p < 0.05). Administration of EGCG via GTCs and GTC-NPs from apical to the basolateral chamber, showed increased Papp values of GTCs (5 x 10− 6 ± 0.016 cm/sec) and GTC-NPs (1.1 X 10− 5 ± 0.03 cm/sec) as compared to free EGCG (4.88x 10− 7 ± 0.02 cm/sec) as shown in Fig. 5b. Whereas, during transport from the basolateral to the apical chamber, the Papp values of GTCs and GTC-NPs decreased significantly up to 1.6-fold and 2.2-fold, respectively, as depicted in Fig. 5c and 5d. Therefore, the net flux ratio (Papp A to B: Papp B to A; Fig. 6a) of EGCG enhanced up to 5.6-fold and the net efflux ratio (Papp B to A: Papp A to B; Fig. 6b) decreased by 3.9-fold after nanoencapsulation of EGCG using GTCs extract as compared to the free form of EGCG (p < 0.05).
3.5.2. Effect of absorbance enhancers on apparent permeability coefficient (Papp) of EGCG from GTCs and GTC-NPs
The absorption prediction studies of EGCG from GTCs and GTC-NPs were conducted on effect of temperature, absorption inhibitors and enhancers, the results were depicted in Fig. 7, and Papp values of all the stimulator groups were shown in Table 1.
| Papp ± SD (10− 6 cm/sec) | ||||||
|---|---|---|---|---|---|---|
| 37 ºC | 4 ºC | ATP inhibitor (sodium azide) | P-gp inhibitor (verapamil) | MRP2 inhibitor (MK-571) | Absorption enhancer (EDTA) | |
| EGCG | 4.88 ± 0.025 | 1.5 ± 0.05 | 1.4 ± 0.03 | 5 ± 0.015 | 5.1 ± 0.0251 | 5.3 ± 0.03 |
| GTCs | 9.13 ± 0.03 | 2.6 ± 0.025 | 2.2 ± 0.03 | 9.2 ± 0.02 | 9.3 ± 0.015 | 9.4 ± 0.02 |
| GTC-NPs | 11.15 ± 0.03 | 4.2 ± 0.015 | 2.9 ± 0.03 | 12 ± 0.03 | 13 ± 0.02 | 13.73 ± 0.025 |
| Note: Papp values of EGCG, GTCs and GTC-NPs (100 µg/mL) from the apical to the basolateral chamber with the treatment of different temperatures, ATP inhibitor (sodium azide; 10mM), P-gp inhibitor (verapamil; 10µM), MRP2 inhibitor (MK-571; 10µM), and absorption enhancer (EDTA; 10mM) | ||||||
| Papp values were reported as mean ± SD, n = 3. | ||||||
| EDTA: ethylenediaminetetraacetic acid, EGCG: epigallocatechin gallate, MRP2: multidrug resistance-associated protein 2, Papp: apparent permeability coefficient. | ||||||
The apparent permeability coefficient at 4°C was significantly lower than 37°C up to 2.65-fold ( p < 0.05). Further, the effect of absorption stimulators was studied based on apparent permeability coefficient values. ATP inhibitor, was showed significantly decreased Papp values in all groups (p < 0.05). Permeability and absorption enhancers such as P-gp inhibitor, MRP-2 inhibitor and EDTA have been observed with significantly increased Papp values when compared to control condition (1.07-fold, 1.16-fold and 1.23-fold, respectively) (p < 0.05).
3.6. Amelioration of oxidative stress by EGCG from GTCs and GTC-NPs
3.6.1.. Effect of EGCG from GTCs and GTC-NPs on mitochondrial membrane potential
Caco-2 cells treated with 100 µM H2O2 showed attenuation of mitochondrial membrane potential up to 77.7% indicating apoptosis as depicted in Fig. 8b. Post-treatment of 100 µg/ml of EGCG, GTCs, blank chitosan nanoparticles and GTC-NPs, showed significantly improved MMP when compared to the H2O2 alone treated cell line (p < 0.05). The GTC-NPs have significantly restored the mitochondrial membrane potential up to 75% as compared to EGCG (50%), GTCs (66%) and blank Chitosan nanoparticles (48%).
3.6.2 Effect of EGCG from GTCs and GTC-NPs against total ROS
Reactive oxygen species generation accelerated up to 2.29-fold after treatment with 100 µM H2O2 in Caco-2 cells concerning the control group i.e. without treatment as depicted in Fig. 8a. Post-treatment of 100 µg/ml of EGCG, GTCs, blank chitosan nanoparticles and GTC-NPs showed a significant reduction in fluorescence intensity following the control group i.e. no treatment (p < 0.05). Among all the test groups, GTC-NPs magnificently reduced the generation of ROS up to 1.69-fold in the Caco-2 cells.
3.6.3 Effect of EGCG from GTCs and GTC-NPs on antioxidant enzymes
The levels of antioxidant enzyme activity were significantly upregulated after the post-treatment of EGCG from GTCs and GTC-NPs (p < 0.05) as shown in Table 2, Post-treatment of 100 µg/ml of EGCG, GTCs, blank chitosan nanoparticles, and GTC-NPs showed a protective effect against 100 µM H2O2. The levels of SOD, GPx, GR, and Catalase were re-established concerning levels of these antioxidant enzymes in the control group. GTC-NPs showed substantially maximum amelioration when compared to free EGCG and GTCs (p < 0.05).
| Control | H2O2 | EGCG | GTCs | Chitosan NPs | GTC-NPs | ||
|---|---|---|---|---|---|---|---|
|
| 1.786 ± 0.07 | 1.033 ± 0.02 | 1.196 ± 0.04 | 1.456 ± 0.03 | 1.286 ± 0.02 | 1.573 ± 0.04 | |
| Catalase (U/mg) | 2.77 ± 0.03 | 1.24 ± 0.03 | 1.97 ± 0.02 | 2.23 ± 0.03 | 2.083 ± 0.02 | 2.43 ± 0.05 | |
| Glutathione peroxidase (U/mg) | 0.946 ± 0.02 | 0.363 ± 0.04 | 0.646 ± 0.02 | 0.836 ± 0.01 | 0.74 ± 0.03 | 0.906 ± 0.02 | |
| Glutathione reductase (mU/mg) | 23.13 ± 0.57 | 11.6 ± 0.36 | 17.82 ± 0.21 | 19.54 ± 0.32 | 18.36 ± 0.1 | 21.71 ± 0.13 | |
| Antioxidant enzymes level after exposure with 100µM H2O2. 100µg/ml of EGCG, GTCs, Blank chitosan NPs and GTC-NPs restored Antioxidant enzymes (Superoxide dismutase, Catalase, Glutathione peroxidase and Glutathione reductase) levels. Note: n=3, p<0.05. | |||||||
Green tea catechins are natural flavan-3-ol known to have anti-oxidant and anti-cancerous activities, which are attributed to the availability of six to eight hydroxyl groups in the chemical structure of tea catechins (Oh et al. 2023). Epigallocatechin-3-gallate (EGCG) is an important catechin with prompting therapeutic properties such as hypocholesterolemic, neuroprotective, anti-angiogenic, anti-diabetic, etc (Khardrawy et al. 2017; Chaundhary et al. 2023). However, the Bio-availability and intestinal stability of EGCG is very poor due to oxidativr decomposition (Dai et al. 2020). To combat the intestinal instability and poor oral bioavailability of EGCG, our previous study offered a new approach to limit the degradation and boost the intestinal absorption via nanoencapsulation technique (Tyagi et al. 2021). In this study, the GTC-NPs were developed using ion-gelation method and confirmed with an average size: 250 nm, the zeta potential: +49.8 mV, the poly-dispersion index (PDI): 0.1 and encapsulation efficiency (EE): 87%. (Tyagi et al. 2021). The present study was planned to conduct a series of experiments to confirm the beneficial properties of GTC-NPs at cellular model (i.e., Caco-2 cell line) by virtue of intestinal transport, permeability and toxicity. This cell line is a widely studied and accepted model for studying the underlying mechanism of cellular uptake and intestinal absorption of bioactive compounds having therapeutic properties like EGCG (Gonzales et al. 2015).Further, Caco-2 cell line is known for its small intestinal barrier function due to the formation of tight junctions between the individual cells ( Lopez-Escalaria and welkins, 2022). After differentiation of the cells, the apical membrane was roofed by brush-bordered microvilli indicating the morphological polarization of the confluent monolayer. Moreover, it also has multiple drug transporters such as P-glycoprotein, ATP, MRP-2, and MRP-5 and secretes membrane enzymes that participate in the metabolism and intestinal transport of numerous compounds (Hubatsch et al. 2007). Hence, transport studies of EGCG from GTCs and GTC-NPs were conducted across confluent polarized Caco-2 monolayer cultivated in cell culture inserts where the transport was studied under a proton gradient, and total flux ratio and efflux ratio was analyzed across both the chambers of the inserts. The reasons to carry out this study are poor intestinal absorption of this remarkable EGCG compound. It hardly penetrates the intestinal membrane due to its high hydrophilicity. It undergoes only passive diffusion for permeation through the membrane, which might be the reason for the extensive efflux of EGCG (Hwang et al. 2020).
Cellular transport studies were carried out at suitable pH conditions as the change in pH can affect the cellular transport of the desired compound in the Caco-2 monolayer (Hubatsch et al. 2007). As per our earlier study, the release kinetics of GTC-NPs showed maximum release of GTCs at pH 7.4, mimicking the pH conditions of the small intestine suitable for drug transport studies (Tyagi et al. 2021). In order to increase the stability of EGCG, Ascorbic acid at 10 mM (pH 7.4 and pH 6.5) in HBSS buffer was used. Ascorbic acid is reported to enhance the stability of catechins in in-vitro digestive models and alter the intestinal accumulation of catechius ( Green et al. 2007). Therefore, in the present study, the enhanced stability of EGCG is observed by the addition of a reducing agent and preventing redox reaction at neutral or slightly alkaline pH. The acidic micro-climate of the intestine was mimicked by adjusting the pH of HBSS buffer of the apical chamber to pH 6.5 and recommended pH for the basolateral chamber should be 7.4 to determine the absorption and permeability of the drug through the Caco-2 monolayer (Hwang et al. 2020). The Caco-2 cell monolayer was found to have Papp values ranging from 0.66 to 0.75 x 10− 6 cm/sec, signifying the formation of an intact cellular monolayer.
The MTT assay (Tetrazolium assay) was conducted for cell viability studies. Tetrazolium assay is a colorimetric assay indicating the metabolic function of viable cells. The tetrazolium dye was taken into mitochondria through endocytosis and reduced to water-insoluble formazan particles by the dehydrogenase enzyme present in mitochondria, indicating the normal metabolic activity of cells (Lu et al. 2012). The effective concentration of a drug is the dose at which there is no significant cell death observed. The treatment of EGCG, GTCs, blank chitosan NPs and GTC-NPs revealed effective concentrations up to 100 µg/ml, 120 µg/ml, 120 µg/ml, 140 µg/ml, respectively. Usage of these safer dosages ensures that there will not be any disruption of tight junctions in the intestinal membrane. The integrity of tight junctions was reassured by checking the intestinal permeability of Lucifer yellow, paracellular maker, after the completion of the transport experiment.
Nanoencapsulation is the best suitable technique for enhancing catechin cellular uptake. In this study, the uptake of EGCG from GTCs extract was significantly 2-fold higher than that of free EGCG. GTCs have been reported with the following phytocompounds such as gallic acid, RC, EGC, ECG and EGCG (Tyagi et al. 2021). Vaidyanathan and Walle (2003) reported enhanced permeability of EGC due to delayed saturation and effective binding of EGC to Caco-2 cells. This binding of EGC can be helpful for the absorption of EGCG as they both were absorbed faster by the Caco-2 cells as MRP transporters were involved in the transport of gallated catechins. Cellular uptake of catechin, gallic acid and EGCG employing active transport when added to the apical chamber because glucuronides or sulfates transformed tea catechins into hydrophilic form, suitable for simple diffusion (Song et al. 2014; Faralli et al. 2019). These studies help in predicting the synergistic effect of other catechins on the cellular uptake of EGCG. Further, the study confirms that nanoencapsulated GTCs have better cellular uptake when compared to free form EGCG (3-fold). The data revealed that chitosan as a carrier molecule, triggers the cellular uptake of the EGCG. Chitosan nanoparticles improve the permeability of EGCG by opening of tight junction, and endocytosis (Hu et al. 2013). Chitosan nanoparticles can easily enter the enterocytes and undergo controlled release of catechins. Due to the presence of mucoadhesive properties, chitosan persist positive charge and will be able to retained in the GI tract for a longer time (Fathima et al. 2022).Therefore, chitosan based nano-encapsulation is best suitable as nano-carriers.
In the present study, the transepithelial transport of EGCG from the apical chamber to the basolateral chamber was found to be increased in the presence of chitosan as a carrier. The net flux of EGCG from GTC-NPs enhanced up to 5.6-fold when compared to free form, which indicates improved permeability coefficient value. These enhanced permeability of EGCG was due to the endocytosis pathway of GTC-NPs which is supported by cellular uptake studies. The chitosan nanoparticles are known to have the mechanism disrupt tight junction, meanwhile EGCG may release from the carrier for paracellular means of transport (Liang et al. 2017). In our study, the net efflux of EGCG was decreased by up to 1.6-fold by incorporation of GTC-NPs. This may be because the absorption-boosting properties of chitosan nanoparticles restrict the functioning of intestinal P-glycoprotein transporter and eventually improved intestinal absorption. The interactio n between chitosan and efflux pumps establish employing ATP depletion, mucoadhesion, and formation of drug-polymer conjugates to prevent the efflux of EGCG, creating interference with sites specific for binding of ATP and jamming sites specific for binding of the drug at the intestinal membrane (Werle 2008).
The absorption prediction studies revealed that the transport of EGCG using GTC-NPs across the intestinal membrane is energy-driven, active transport. The Papp values of EGCG at 4°C and at 10 mM Sodium azide (ATP inhibitor) concentration were decreased up to 62.3% and 73.9%, respectively indicating the absorption-limiting factors. The Papp value of EGCG in combination with 100 µM Verapamil (P-gp inhibitor), 100 µM MK-571 (MRP-2 inhibitor), and absorption enhancer (10 mM EDTA) increased up to 1.07-fold, 1.16-fold and 1.23-fold, respectively (p < 0.05). The ABC transporters such as P-gp and MRPs are the main efflux transporter located at brush border microvilli of the intestinal membrane, restricting the absorption of the drug in intestinal mucosa by pumping out the drugs (Lohner et al. 2007). These inhibitors deactivate the efflux pump and improved intestinal absorption as the EGCG acts as the substrate for these membrane transporters. 10 mM EDTA works as an absorption enhancer by enabling paracellular expansion of tight junctions amid the cells. Furthermore, it acts as a penetration enhancer by extracting the membrane lipids, thus, reducing the barrier properties of the intestinal membrane and aiding in drug permeability (Rojanasakul et al. 1990).
H2O2 is a metabolite present in living cells at very low concentrations, generated by dis-mutation reactions. It is converted into highly reactive hydroxyl radicals, which reacts with any hydrogen donating compounds and causes oxidative stress (kumar et al. 2013). The GI tract is the main target of hydroxyl radicals and many studies were reported on the involvement of H2O2 in the development of colon cancer (Wijeratne et al. 2005). These reactive oxygen species cause shrinkage of cells, nuclear condensation, DNA fragmentation and ultimately apoptosis. The major therapeutic advantage of EGCG is its antioxidant activity. The results of the present study indicated the enhanced radical scavenging property, reinstatement of mitochondrial membrane potential and up-regulated antioxidant enzyme levels by GTC-NPs. Recently, many studies confirmed the mechanism of action of drug-loaded chitosan nanoparticles and well known mechanism is cellular pinocytosis (Conner and Schmid, 2003). The mechanism of cellular pinocytosis initiates with entrance of nano-encapsulated particles in the cells and intracellularly trafficked onto endosomes. Then, nanoparticles will get trapped by lysosomes, where they will be metabolized by glycosylase and methylase enzyme, later, the EGCG may release in the cells to restore the pro-oxidant: anti-oxidant ratio and then enhance mitochondrial membrane potential (Hu et al. 2013). In the present study, GTC-NPs have shown significantly decreased oxidative stress in H2O2-induced Caco-2 cell when compared to other treatment as EGCG is not metabolised and it may directly attack free-radical (p < 0.05). Moreover, the same treatment also up-regulated all the anti-oxidant enzymes such as SOD, CAT, GPx and GR. Further, the study confirms that GTC-NPs acted via cellular pinocytosis and restores MMP. Therefore, the study demonstrates that EGCG-rich GTCs loaded chitosan nanoparticles will be an ideal delivery system for EGCG by enhancing its intestinal absorption and bio-efficacy.
Epigallocatechin-3-gallate is a tea flavanol gaining substantial attention due to its magnificent biological activities in recent times. Nevertheless, its biological efficacy is reduced due to the harsh environment of the gastrointestinal tract and poor intestinal absorption. In the present study, EGCG-rich GTCs extract-loaded chitosan nanoparticle was evaluated for its cellular uptake and intestinal absorption. In comparison with free EGCG, GTCs and GTC-NPs have shown significant improvement in stability, cellular uptake and transepithelial transport in Caco-2 monolayer model. The study revealed that the intestinal absorption of EGCG from GTC-NPs was temperature-dependent and energy-dependent, which indicated an active transport mechanism. The cellular uptake studies showed endocytosis as a mode of uptake of EGCG. The inhibitor of P-gp and MRP-2 transporter proteins and penetration enhancer (EDTA) significantly improved the uptake of EGCG from GTC-NPs. The biological activity of EGCG against H2O2-induced oxidative stress was also improved after nanoencapsulation using chitosan. The outcomes of this study imply that chitosan nanocarriers are proficient for oral administration of EGCG imparting wholesome health benefits and can be used as efficient nanoceuticals in the health sector.
ACKNOWLEDGMENT
The authors acknowledge the Director, DFRL for granting them permission to conduct their study and facilitate institutional support with necessary amenities to carry out their research.
CONFLICTS OF INTEREST
The authors declare that they do not have any financial affiliation or personal relationships that could impact the integrity of the work conducted in this paper.
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No competing interests reported.