Drug Delivery Applications of Exopolysaccharides from Endophytic Bacteria Pseudomonas otitidis from Tribulus terrestris L.

The present study aimed to produce a novel biocompatible, stable and biodegradable exopolysaccharide (EPS) from endophytic bacteria to deliver drugs into malignant cells effectively. EPS-producing endophytic bacteria, Pseudomonas otitidis was isolated and identified from the roots of the medicinal plant Tribulus terrestris L. The ice-cold ethanol precipitation method was used to isolate EPS from an endophytic bacterial culture. The isolated EPS was partially characterized by Fourier transform infrared spectroscopy (FTIR), Nuclear magnetic resonance (NMR) spectroscopy, and the presence of reducing sugar and protein contents were also measured. The isolated EPS contained 26.665 ± 1.302% of reducing sugar and 0.912 ± 0.023% of proteins. Further, EPS-based quercetin-loaded nanoparticles (NPs) were formulated through the precipitation method to deliver quercetin into breast cancer cells. The fabricated nanoparticles size, shape, physical nature, drug release and release kinetics properties were studied and confirmed. EPS-based nanoparticles could cause the highest release of quercetin in an acidic medium at pH 5.0, and they had high biocompatibility in a physiological medium. In addition, the NPs showed significant free radicals (DPPH* and ABTS*) scavenging and ferric ion reducing potentials and concentration-dependent cytotoxic effects against breast cancer cell lines (MCF-7 cells) after 24 h treatment with IC50 value of 14.313 μg/mL. The obtained results demonstrated that the isolated EPS from endophytic bacteria can positively applied as a drug delivery vehicle and enhanced the activity of the loaded drug.


Introduction
For the last two decades biotechnological, healthcare, and pharmaceutical industries have tried to develop drug delivery systems to effectively delivering drugs to the target site without disturbing other healthy tissues/organs [1]. The identification of efficient drug delivery systems is an essential part of the design and development of newer medicines [2]. The drug delivery systems protect the loaded or attached drugs by early degradation of body fluids and keep the blood drug concentrations from fluctuating, and reduce the dosage frequency [3]. However, a thorough understanding of drug properties, biocompatibility, and route of drug administration is previously necessary in order to develop a drug delivery system effectively. Therefore, researcher and scientists are continuously looking for new strategies to deliver drugs in a safe and wellcontrolled release over a long period. Many drug delivery

Plant Sample Collection and Isolation of Endophytic Bacteria
The healthy plant, T. terrestris L. was harvested from Sundarapandiam village, Virudhunagar Dist., Tamil Nadu, India (latitude 9.59721° N, longitude 77.67405° E). The region has a large variety of plants and a high level of vegetative growth, with diverse flora. The harvested plant was brought to the lab under sterile conditions. On the same day, the plant sample was processed for the isolation of the endophytic bacteria from the root. The root part of the plant sample was incised separately into small pieces and washed with tap water. The root was then surface sterilized with deionized water for 3 min. Later, the root was soaked in 80% ethanol for 5 min and then washed in sodium hypochlorite (1%) for 2 min. Finally, the root sample was again washed with deionized water for 1 min. The externally sterilized root sample was finely crushed using a sterile mortar and pestle and serially diluted. The serial dilutions (10 −2 to 10 −6 ) were inoculated in the nutrient agar plates. The plates were maintained at 27 °C for 24 h. The control plate was also incubated to check the contamination. After the incubation period, the plates were observed for the growth of colonies. The bacterial colony which was observed to produce exudate (exopolysaccharides) was Gram's stained and observed microscopically.

Molecular Identification of the Endophytic Bacteria
The exopolysaccharides producing endophytic bacteria from T. terrestris L. was identified through the molecular gene sequencing [25]. The monolayer colonies of bacterial cells were grown and subjected to lysis. The lysis buffer (50 mM Tris pH 8.0, 10% glycerol, 0.1% Triton X-100, 100 µg/mL lysozyme, DNAse 3U, 1 mM PMSF (antiprotease), 2 mM MgCl final concentration) about 500 µL in a 2 mL microcentrifuge tube was mixed with the bacterial culture. The cells were finely lysed by repeated pipetting. 4 µL of RNAse and 500 µL of neutralization buffer (3/5 M potassium acetate, pH 6.0) was added to the contents. The contents were vortexed and incubated for 30 min at 65 °C in a water bath. To minimize shearing the DNA molecules, mix DNA solutions by inversion. After the incubation, the contents were centrifuged at 10,000 rpm for 10 min. Following centrifugation, the resulting viscous supernatant was transferred into a fresh 2 mL microcentrifuge tube without disturbing the pellet. Then, about 600 µL of chloroform isoamyl alcohol was added, and hand mixed vigorously. The contents were then centrifuged for 10 min at 10,000 rpm and 600 µL of the aqueous phase was transferred into a fresh 2 mL microcentrifuge tube. For binding, the 600 µL of binding buffer (6 M guanidine thiocyanate in pH 8.0) was added to the mixture by pipetting and incubated at room temperature for 5 min. Then, about 600 µL of the contents were transferred to a spin column placed in a 2 mL collection tube. The contents were centrifuged for 2 min at 10,000 rpm and discarded flow-through. The spin column was reassembled, and the remaining 600 µL of the lysate was transferred to the collection tube. The contents were centrifuged for 2 min at 10,000 rpm. For the washing process, 500 μL washing buffer I (20 mM NaCl, 2 mM Tris-HCl, 80% ethanol, in pH 7.5) was added to the spin column and centrifuged at 10,000 rpm for 2 min. The spin column was reassembled and 500 µL washing buffer II (50 mM Tris HCl, 10 mM EDTA, RNase A (100 µg/mL), in pH 8.0) was added, centrifuged at 10,000 rpm for 2 min and discard flow-through. 100 µL of elution buffer (0.5 mM EDTA, 10 mM Tris-HCl, in pH 9.0) was added in the middle of the spin column, which contains DNA and incubated at room temperature for 2 min. Single-pass sequencing was performed for bacterial identification. The ITS region of the DNA was amplified using universal 27F (AGA GTT TGA TCT GGC TCA G) forward and 1492R (TAC GGT ACC TTG TTA CGA CTT reverse primers. The fluorescent-labelled fragments were purified from the unincorporated terminators with an ethanol precipitation protocol. Using the protocols, the products were sequenced in both the forward and reverse directions using a Genetic Analyzer 3500 (Life Technologies Corporation, Applied Biosystems®, California 94404, USA).

Extraction and Purification of the Bacterial Exopolysaccharides
The endophytic bacterial exudate (exopolysaccharides) was separated by the ethanol precipitation method [26]. Ethanol precipitation is one of the frequently considered methods for the separating EPS from the bacterial culture. The bacterial broth culture was centrifuged at 5000 rpm for 10 min at 4 °C to remove the pellet containing bacterial cells. The supernatant containing the bacterial EPS fraction was mixed with an equal volume of ice-cold ethanol (96% v/v) and incubated overnight at 20 °C. After the incubation period, the mixture was centrifuged at 1000 rpm for 15 min to separate the settled EPS (pellet) in the broth. The collected EPS was lyophilized and used for further analysis.

Fourier Transform Infrared (FTIR) Spectroscopy
The structural characterization of the EPS was examined through FTIR spectroscopy to determine the functional group's presence in the EPS. Approximately 5 µg of dried EPS was finely mixed with 100 µg of KBr powder (FTIR grade) and exposed to the hydraulic press to prepare a round translucent disc (5 mmØ). The FTIR spectra was recorded in transmittance mode in a range of 4000-400 cm −1 with an accumulation of 15 scans, and a resolution was 4 cm −1 , using a Shimadzu IR Tracer-100 spectrometer [27].

NMR Spectroscopy
The purified EPS was subjected to NMR analysis. Approximately 30 mg of the purified EPS sample was solubilized in D 2 O to exchange deuterium two times by lyophilization. The sample was then put into 4 mm NMR tube with redissolved D 2 O (0.5 mL). The experiment was performed by 1 H spectra on Bruker Avance III HD 400 MHz spectrometer [28].

Total Reducing Sugar Determination
By using the phenol-sulfuric acid colorimetric method, the total reducing sugar content of the EPS was calculated by ethanol precipitation [29]. A 200 µL of EPS solution (10 mg/ mL) was mixed with 200 µL of phenol solution (5%) and 1 mL of pure H 2 SO 4 . After 30 min, the reaction mixture was cooled to room temperature. UV-Visible spectrophotometry (UV-1800 series, UV Probe 2.62 software, Shimadzu, Japan) was used to measure the absorbance of samples and standard sugar (10-100 µg/mL ribose) at 492 nm.

Protein Content Measurement
Bradford's method was used to measure the protein content present in the bacterial EPS [30]. 20 µL of the EPS (10 mg/ mL) were combined with 1 mL of Bradford's reagent (Himedia Laboratories, Mumbai, India), which was then incubated for 5 min at 37 °C. The OD of the sample as well as standard protein (bovine serum albumin (BSA) 0-50 mg/mL) were both measured at 595 nm (UV-1800 series, UV Probe 2.62 software, Shimadzu, Japan).

Exopolysaccharides as a Drug Delivery Vehicle
In the present investigation, EPS from endophytic bacteria is used as a drug delivery vehicle to deliver the loaded chemotherapeutic agents into cancer cells. Here, quercetin was loaded into EPS (vehicle) as a model drug. Quercetin is a plant-derived pigment (flavonoid), it's mainly present in the human diet [31]. It has a wide range of pharmacological actions, including maintaining free radicals, eliminating cancer cells, reducing swelling, regulating blood sugar levels, and helping to prevent cardiovascular diseases [32].

Quercetin-Loaded Nanoparticles
Quercetin-loaded EPS nanoparticles (NPs) was formulated according to our previously published work [33]. In short, 2 mg of quercetin was dissolved in 5 mL of cell culture grade methanol and added drop-wise manner into deionized water (50 mL) containing 10 mg of EPS and 0.1 mL glutaraldehyde (cross-linking agent). The contents were kept in a magnetic stirring at room temperature (~ 28 ± 2 °C). The absence of precipitate in the stirring mixture serves as a visual indicator of the self-assembled nanoparticles, and UV visible spectrophotometric examination for further confirmation of nanoparticles formation.

Characterization of the Quercetin-Loaded Nanoparticles
The functional groups and their interaction between the EPS and quercetin in the nanoparticles were characterized by FTIR spectroscopy [34]. The physical nature of the formulated quercetin-loaded EPS NPs was determined by X-ray diffractometer (BRUKER D 8 Advance ECO XRD system equipped with SSD160 1 D Detector). The XRD pattern was collected in 2θ at the range from 10 to 80° at the intensity between 2 and 12 [35]. The step counting of about 0.78 s/step was used for the analysis. The average particle size and zeta potential of the formulated quercetin-loaded EPS NPs was analyzed through Nano ZS ZEN 3600, Malvern Instrument, UK. The DLS analysis evaluates the presence of nano sized particles of the sample with respect to the stability [36]. Thus, 300 µL of sample are dispersedly placed onto the cell and exposed to laser irradiation. The morphological analysis of the formulated quercetin-loaded EPS NPs was performed in Scanning Electron Microscope (CARL ZEISS EVO 18). The dried sample was mounted on the carbon strip and dried thoroughly under mercury lamp for 5 min. In high resolution scanning electron microscope at 15 kv voltage the images of the encapsulated material were taken [37].

Drug Release Studies
In vitro release of quercetin from quercetin-loaded EPS NPs and quercetin were performed in 0.01 M acetate (pH 3.5 and pH 5.0), and 0.01 M phosphate (pH 7.2) systems at 36 ± 1 °C, according to our previously reported method [35].

Drug Release Kinetic Studies
Additionally, the above-measured drug release data from quercetin-loaded EPS NPs was computed using the DD solver 1.0 software (Excel-plugin module), and the resulting data was fitted to the zero-order (cumulative% drug release vs time), first-order (log% drug remaining vs time), Higuchi (cumula-tive% drug release vs square root of time), Hixson-Crowell (cube root of drug% remaining vs time), and Korsmeyer-Peppas (log drug release vs log time) models to verify the kinetics of drug release, based on our previously published research [38].

DPPH Radical Scavenging Assay
The 2,2-diphenyl-1-picrylhydrazyl solution (prepared by dissolving 8 mg of DPPH in 100 mL of 95% ethanol) was used to determine the DPPH* radical scavenging effect of the quercetin-loaded exopolysaccharides nanoparticles and quercetin, with minor adjustments, according to a previously reported procedure [39]. Thus, 1 mL of DPPH reagent was mixed with 3 mL of quercetin-loaded exopolysaccharides nanoparticles with varied concentrations (6.25, 12.5, 25, 50 and 100 µg/mL), quercetin (25 µg/mL) and the blank. This arrangement was permitted to incubate for 30 min at room temperature (vortexed in between), following which the absorbance was measured using a UV-visible spectrophotometer at 517 nm against a blank. Ascorbic acid was used as a comparative reference. The scavenging activity was calculated by using the Eq. (1):

ABTS Radical Scavenging Assay
The quercetin-loaded EPS NPs was tested for the ability to scavenge ABTS radical [40]. The reagent was prepared by mixing 5 mL of potassium persulfate (4.9 mM) with 14 mM of 2,2′-azino-bis (ethylbenzthiazoline-6-sulfonic acid) (ABTS) reagent (v/v) about 5 mL and the contents are kept in dark place for 14-16 h. The reagent was mixed with 0.1 mL of the quercetin-loaded exopolysaccharides nanoparticles with varied concentrations (6.25, 12.5, 25, 50 and 100 µg/mL) and quercetin (25 µg/mL), the reagent was diluted by using 0.3 mL ethanol. The whole reaction setup was incubated for 6 min and the absorbance was measured at 734 nm. After the 6 min of incubation, the absorbance at 734 nm was determined and adjusted to 0.700 (0.0020) in UV-visible spectrophotometer. Distilled water was used as blank and the obtained results were compared with the control (only ABTS solution). As given in Eq. (2), the percent ABTS radical scavenging activity was calculated using a standard curve that included Ascorbic acid in 80% of the ethanol:

Ferric Reducing Antioxidant Power (FRAP) Assay
According to the standard protocol the 2,4,6-tripyridyl-striazine (TPTZ) was treated with the quercetin-loaded EPS NPs for knowing its antioxidant effect [13]. At lower pH, electron-giving antioxidants decrease Fe 3+ TPTZ (a colorless complex) into Fe 2+ TPTZ (a blue coloured complex). This mechanism was seen using variations in absorbance at 593 nm. 300 mM acetate buffer (3.1 g sodium acetate, 16 mL acetic acid) at pH 3.6, 10 mM TPTZ solution in 40 mM HCl solution and 20 mM FeCl 3 ·6H 2 O solution were used to make the FRAP reagent. The acetate buffer (25 mL) and TPTZ (2.5 mL) were mixed together before adding FeCl 3 ·6H 2 O. (2.5 mL). In a dark condition, 40 µL of quercetin-loaded exopolysaccharides nanoparticles with varied concentration (6.25, 12.5, 25, 50 and 100 µg/mL) and quercetin (25 µg/mL) was allowed to react with newly prepared FRAP reagent for 30 min before measuring the absorbance at 593 nm. The standard was linear at 200 and 1000 M FeSO 4 . M Fe(II)/g was used to calculate the findings. To compare quercetin-loaded EPS NPs, ascorbic acid was selected as a standard.

MTT Assay
The cytotoxic potential of quercetin-loaded EPS NPs was initially assessed in MCF-7 cells using the MTT method [41]. Thus, MCF-7 cells were seeded in a 96-well microtiter plate and maintained around for 24 h at 37 °C with 5% CO 2 . This promotes the exponential development of the cells. The cells were then exposed to quercetin-loaded exopolysaccharides nanoparticles at a range of doses (100, 50, 25, 12.5, 6.25, and 3.125 µg/mL) and quercetin (25 µg/mL) for 24 h. After the treatment, the cells undergo a medium change and a 4 h MTT treatment at 37 °C. The excessive MTT stain was removed with DMSO (1-100 μL). By leaving the plate undisturbed for few minutes, the air bubbles were eliminated. The ELISA plate reader measured at the absorption at 570 nm (Bio-Rad, Model 680, Hercules, USA). The control cells were also used to test the cytotoxicity. The proportion of cell viability is determined from Eq. (3). *n = 3, where n is the number of independent experiments.

Apoptotic Morphological Detection by Dual Staining Assay
The quercetin-loaded EPS NPs was used to treat the MCF-7 cells at their IC 50 concentration in a 24-well microplate. The treated cells were incubated for 30 min and observed under ×40 magnification in a fluorescent microscope. Following treatment, cells were rinsed in ice-cold 1× PBS and stained with two fluorescent DNA-binding dyes [(10 μL/mL) (Acridine orange and Ethidium bromide)] [42]. Cells that were not treated with NPs, served as the control for the study. The proportion of cell apoptosis was calculated using Eq. (4).

Apoptosis Analysis by Flow Cytometer
MCF-7 cells were cultured overnight at 37 °C in a CO 2 incubator after being seeded on a 24-well microplate. The cultured cells were treated to the quercetin-loaded EPS NPs at an IC 50 concentration for 24 h. The cells were then centrifuged for 5 min at 500×g at 4 °C after being incubated and rinsed in PBS. The cell pellets are mixed at 1 × 10 5 /mL in ice-cold 1× binding buffer after the supernatant is removed. The tubes are placed on ice before being filled with 1 μL of annexin V-fluorescein isothiocyanate reagent and 1 μL of propidium iodide (PI). The tubes were then incubated in ice-cold conditions at dark for about 15 min. As per the protocol, it was then mixed with 400 μL of ice-cold 1× binding buffer, and the sample was injected to a flow cytometer to observe the cell preparations [13].

Assessment of Mitochondrial Transmembrane Potential
Rhodamine-123, a lipophilic dye, was used to measure the mitochondrial transmembrane potential [36]. The technique was carried out in such a way that the MCF-7 cells were seeded in the 24-well microplate, which was then kept in a CO 2 incubator at 37 °C for 24 h. The halfmaximal inhibitory concentration of the quercetin-loaded EPS NPs (IC 50 ) value was measured, and the cells were retained for 48 h. Rhodamine-123 dye was then used to stain the cells for around 30 min. Additionally, they were fixed for 30 min with paraformaldehyde (4%) after being rinsed with PBS. The fluorescent microscope was used to look at the membrane's permeability and morphological changes.

Nuclear Integrity Measurement by DAPI Staining
For the preservation of the genetic material and cellular proliferation, nucleus stability is more essential. The MCF-7 cells were used to evaluate the nucleus integrity concerning the quercetin-loaded EPS NPs. MCF-7 cells that had been exposed to the extract were then rinsed with PBS in a 24-well flat-bottom microplate before being fixed in paraformaldehyde (4%) for 30 min. The cells were then washed twice, once in PBS and once in Triton X100 (0.4%), for approximately 20 min each. The cells were then rinsed after being stained with DAPI (nuclear counterstain) (0.5 µg/mL) for 1 min in a dark environment. The cell images were taken using a fluorescent microscope using the proper filter [13]. (4) % apoptotic cells = Total number of apoptotic cells Total number of normal cells × 100 Determination of ROS Using 2′,7′-dichlorofluorescein diacetate, the increase in intracellular ROS was observed. After being exposed to 10% FBS and the IC 50 concentration of quercetin-loaded EPS NPs, the MCF-7 cells were cultured for 24 h and 48 h. The cells were then labelled with 10 M H 2 DCF-DA on ice after being washed twice with PBS. The plates were shaken for 10 min under the dark condition. Using a fluorescence spectrophotometer, the difference in fluorescence intensity at 475 nm (λ ex ) and 525 nm (λ em ) was detected [37].

Statistical Analysis
Statistical Package for Social Science (SPSS) version 20.0 software (SPSS Inc., Chicago, IL, USA) was used to analyse the experimental data. At least three times each study was repeated. The mean and ± standard deviation was used to express the obtained quantitative data. The significance of the statistical method is investigated using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison method. A statistically significant difference was determined to exist when the p value was 0.05 or lower. The IC 50 was determined for cytotoxicity investigations using the robust curve fit non-linear regression approach from the percentage viable measurements. The paired t-test method was used to assess the ROS production data.

Endophytic Bacteria Isolation
For the isolation of endophytic bacteria, separate triplicate efforts were made. After the incubation period, the nutrient plates with three different types of endophytic bacterial colonies were observed. The bacterial colony with white slimy or mucoid morphology which represent the production of bacterial exudate (exopolysaccharides) was taken for the further work. The slimy colony was Gram's stained which shows Gram-negative short rods (Fig. 1).

Phylogenetic Analysis of Endophytic Bacteria
The phylogenetic tree was built using character-based and distance-based approaches. The identical topology was found while performing phylogenetic analysis using several methods. The endophytic bacterial sequence was used for the phylogenetic evolution by neighbor-joining technique.
The phylogenetic interpretation reveals that the endophytic bacteria was closer to the genus Pseudomonas. According to the ideal phylogenetic tree analysis, the isolated endophytic bacteria was found to be Pseudomonas otitidis which was classified as distinct branches based on their morphological population (Fig. 2). The identified endophytic bacterial sequence was submitted in the GenBank (Accession Number: OM736191).

Characterization of the Bacterial Exopolysaccharides
The FTIR spectrum (Fig. 3a) (Fig. 3b). The dried EPS from the endophytic bacterial colony was determined for the total reducing sugar content by the phenol-sulfuric acid method. The ice-cold ethanol precipitation method of exopolysaccharides recovery from endophytic bacteria is estimated to contain 26.665 ± 1.302% of the reducing sugar. Further, the endophytic bacterial EPS was analysed for the presence of protein. The total protein content was found to be 0.912 ± 0.023% in the endophytic

Quercetin-Loaded Nanoparticles Formulation
The quercetin-loaded EPS NPS was formulated by a precipitation method. Following quercetin was added to the exopolysaccharide's solution, the combination initially had a dark yellow colour with suspended particles, but after continuous stirring, the colour changed to clear with light yellow. The synthesis of quercetin-loaded EPS NPs was validated by the observed colour shift and the elimination of opalescence, which demonstrated that quercetin was completely loaded with EPS. The quercetin-loaded EPS NPS was further examined and used in biological processes. According to measurements, quercetin can be loaded into exopolysaccharides with an encapsulation efficiency of 81.4066 ± 4.758% and a drug loading capacity of 8.03 ± 0.018%, respectively.

Characterization Studies of the Nanoparticles
The FTIR spectrum of quercetin-loaded EPS NPs (Fig. 4a) shows a broad peak at 3250 cm −1 signifying the presence of strong O-H bonding of carboxyl groups, and a stretching peak at 2978 cm −1 , indicating the presence of -CH 2 group. Peak at 1650 cm −1 signifies aromatic conjugation (-C=C-) between quercetin and exopolysaccharides. The X-ray diffractogram of the quercetin-loaded EPS NPS was presented in Fig. 4b. The broad diffraction peak observed at 2θ = 22° suggested that the formulated nanoparticles are highly amorphous. Figure 4c and d presented the average particle size, and zeta potential of the quercetin-loaded EPS NPs, respectively. The average particle size distribution and zetapotential of the formulated NPs were 780 nm and − 20 mV respectively. Further, the formulated quercetin-loaded EPS NPs were found to be non-aggregated, distributed uniformly and spherical shape with individual particle size were measured from 200 to 800 nm in diameter when observed under SEM (Fig. 5a-d).

Release of Quercetin from Exopolysaccharides Nanoparticles
The therapeutic effectiveness of the loaded drug is primarily regulated by the rate of drug release from EPS NPs. The dialysis bag was used to assess the in vitro release mechanism of quercetin from quercetin-loaded EPS NPs at 37 °C in a variety of buffer solutions, including acetate buffer (pH 3.5 and 5.0) and PBS (pH 7.2). The cumulative percentage of quercetin release was calculated after the release data was collected at pre-determined time intervals. Figure 6 shows the outcome of the proportion of quercetin released from EPS NPs and quercetin. In the beginning, ~ 100% of quercetin released from control in acetate buffer at pH 3.  of loaded quercetin (82.523 ± 0.854%) was released from EPS NPs in 25 h at pH 5.0. The loaded quercetin release from EPS NPs is slightly altered by pH variations. Additionally, the experimental results showed that at pH 5.0 compared to the other examined pH ranges, quercetin release from EPS NPs was higher and more effective.

Kinetics of Drug Release
The results of drug release data were fitted into multiple kinetic models (Zero-order, first-order, Higuchi, Korsmeyer-Peppas and Hixson-Crowell) to further examine the mechanism of quercetin release from quercetin-loaded EPS NPs, as shown in Table 1. The regression coefficient (r 2 ) value and release rate constant were predicted for each kinetic model. In general, the value (r 2 ) closer to 1, the better the fit or relationship between the two factors. From the analysis of kinetic models, it was observed that, the zeroand first-order kinetics regression coefficient (r 2 ) values are very closer to 1, hence these kinetic models were considered to be a good fit. Additionally, based on the Korsmeyer-Peppas model, it was found that the r 2 value was higher than 0.96 in all of the studies that were examined. Fickian diffusion was the desired result of these quercetin release kinetics. The quercetin release from quercetin-loaded EPS NPs, which was primarily regulated by the diffusion mechanism, may exhibit Fickian behaviour. This kinetics model indicated that the best-fitting model for the mechanism of quercetin release from exopolysaccharides nanoparticles was firstorder kinetics. As a result, the kinetic model mechanism also illustrates the nanoparticles uniform disintegration and controlled release.

ABTS Radical Scavenging Assay
The standard drug has been used to compare the relative antioxidant effects to scavenge the ABTS radical. Using potassium persulphate, the stable form of the ABTS* radical cation was generated. After producing stable absorbance, the reaction medium is supplemented with an antioxidant quercetin-loaded exopolysaccharides nanoparticles, and the antioxidant power is measured by observing decolorization. The varied concentrations of 6.25, 12.5, 25, 50, and 100 µg/mL of quercetin-loaded EPS NPS scavenged ABTS radical in a concentration-dependent mode as shown in Fig. 7b. At a maximum concentration (100 µg/mL) of ethyl acetate of quercetin-loaded EPS NPs exhibited highest (75.806 ± 1.452%) decolourization of ABTS* radicals.

FRAP Assay
When quercetin-loaded EPS NPs react with a ferric tripyridyltriazine (Fe 3+ -TPTZ) complex to produce a coloured ferrous tripyridyltriazine (Fe 2+ -TPTZ), the FRAP assay determines the reducing potential of the ferric ions. The free radical chain breaking takes place by donating an electron. Varied concentrations of quercetin-loaded EPS NPs were screened by FRAP assay and standard ascorbic acid. In the results obtained, the better-reducing potential was exhibited 164.06 ± 6.159 μg/mol (Fe(II))/g at a concentration of 100 μg/mL of quercetin-loaded EPS NPs. In the observed results, quercetin-loaded EPS NPs showed higher activity than reference standard ascorbic acid as shown in Fig. 7c.

Cytotoxicity Assay
The cytotoxic efficacy of quercetin-loaded EPS NPs was studied against MCF-7 cells through MTT assay. From the results, it was found that 72.453 ± 0.878% of the viability of cells was observed for the 6.125 μg/mL concentration of the quercetin-loaded EPS NPs. The control drug, Doxorubicin 0.25 μM showed 42.83 ± 2.424% viability of the cells. The IC 50 value of the quercetin-loaded EPS NPs was calculated as 14.313 μg/mL. Figure 8a illustrates the % viability of cells at various concentrations (μg/mL). And Fig. 8) and c shows the control cells and changes in the morphology of the MCF-7 cells after treatment with the quercetin-loaded exopolysaccharides nanoparticles, respectively. The IC 50 concentration (14.313 μg/mL) was then selected in order to further examination of anticancer activities of the quercetinloaded EPS NPs.

AO/EtBr Staining for Analysis of Apoptosis
The abnormalities in the cells were observed when the cells were treated with the quercetin-loaded exopolysaccharides nanoparticles (IC 50 concentration: 14.313 μg/mL) and then stained with AO/EtBr. By using fluorescent microscopy, the morphology of MCF-7 cells during apoptosis, living cells, and necrotic cells was noted. The cells treated with quercetin-loaded EPS NPs showed early apoptotic cells in orange and late apoptotic cells with abnormal shapes, chromatin in the nucleus, and distorted membranes in red (Fig. 9b). The control cells have not undergone any notable alteration (Fig. 9a).

Apoptosis Analysis by Flow Cytometry
The results of flow cytometry analysis of apoptosis induction by quercetin-loaded EPS NPs were represented in Fig. 10. In comparison to control cells (early apoptosis 1.63%, dead cells 0.020%), MCF-7 cells treated with 14.313 µg/mL (IC 50 conc.) of quercetin-loaded exopolysaccharides nanoparticles induced early apoptosis at a rate of 0.23% and dead cells were 6.87%. When compared to control cells (Late apoptosis: 0.44%), the quercetin-loaded EPS NPs was found to have significantly increased the proportion of late apoptosis (10.8%). The percentage proportion of living, early-stage, late-stage, and dead cells is shown in Table 2.

Analysis of Mitochondrial Membrane Potential (∆Ψ m )
The dye Rhodamine-123 was used to detect changes in mitochondrial membrane in the MCF-7 cells after 24 h of exposure with quercetin-loaded exopolysaccharides nanoparticles (IC 50 conc.). The mitochondrial membrane The disruption and depolarization of the mitochondrial transmembrane was observed, which was indicative of the earliest intercellular event of apoptosis (Fig. 11). The penetration of the quercetin-loaded EPS NPs into mitochondrial membrane results in loss of the membrane and cell death (MCF-7). The event releases cytochrome C from the mitochondria, activating the mitochondrial permeability transition pore and triggering the apoptosis [43].

DAPI
DAPI fluorescent labelling, made it possible to determine the type of the nucleus and the overall morphological alterations that quercetin-loaded exopolysaccharides nanoparticles caused in MCF-7 cells. The results are shown in Fig. 12. The nuclear DNA's adenine and thymine sections attach to the DAPI dye. The induction of apoptosis in the MCF-7 cells after 48 h of treatment with quercetin-loaded exopolysaccharides nanoparticles is evident. The treated cells saw cell shrinkage, increased chromatin condensation, and damage to the nuclear DNA. The blue fluorescence was also less intense in the quercetin-loaded EPS NPs-treated MCF-7 cells than in the control group. All these processes increase the number of cells that undergo apoptosis.

ROS
The imbalance between the amounts of reactive oxygen species and antioxidants has a significant role in the initiation and progression of cancer. The outcomes of analysing the generation of ROS by quercetin-loaded exopolysaccharides nanoparticles in MCF-7 cells using the H 2 DCF-DA staining technique. In comparison to the control, cells treated with 14.313 µg/mL of quercetin-loaded exopolysaccharides nanoparticles showed an increased green fluorescence (Fig. 13a,  b), confirming the initiation of apoptotic nature in MCF-7 cells. ROS generation measured as relative fluorescence intensity using fluorescence microscope. Results expressed as mean ± standard deviation of triplicate measurements (p < 0.05) (Fig. 13c).

Discussion
Endophytic bacterial EPS was isolated and characterized, further it was converted into nanoparticles encapsulated with quercetin. The drug loading and drug release kinetics were studied followed by the characterization of nanoparticles. The endophytic bacteria were isolated from T. terrestris L. and was identified as Pseudomonas otitidis. Endophytic bacteria are associated with plants and are highly beneficial for the plant growth. The metabolites of endophytic  bacteria exhibited for various applications including therapy.
Here, the exopolysaccharides of P. otitidis is used to deliver quercetin to control proliferation of cancer cells. The crude exopolysaccharides and nano-formulations with quercetin was characterized by FTIR and NMR. Structural analysis of exopolysaccharides and its derivatives was identified by   [49]. Indeed, in an another reports the IC 50 value of quercetin was reported as 37 µM against MCF-7 cells but not for MDA-MB-231 cells [50]. Delivery of quercetin with exopolysaccharide nanoparticles provide better results than previous reports since it is 14.313 μg/mL in 24 h. The induction of apoptosis by quercetin loaded nanoparticles was analyzed with live/dead cell assay. Apoptotic cells were observed in quercetin-nanoparticles treated cells due to the DNA damage. The appearance of yellowish orange indicates DNA damage which is hall mark of dual staining method. Whereas the control cells are observed in green color. Differentiating the apoptotic-and normal cells by live/dead cell assay was validated by previous reports [50]. The cells treated with quercetin-loaded exopolysaccharides nanoparticles and control cells were analyzed with flow-cytometry with annexin-V to confirm the formation apoptotic bodies. Compared to control, cells treated with quercetin revealed the presence of early apoptotic cells (14%), and cells treated with quercetin exopolysaccharide nanoparticles exhibited late apoptotic bodies (38.9%). The anti-proliferative effect of Graviola fruit extract was analyzed against MCF-7 cells and its apoptosis mechanism was proved with flow-cytometry.
The annexin-V/FITC assay proved the formation of apoptotic bodies in Graviola fruit extract treated cells after 72 h [51]. Our results corroborate with the results of Gao et al., [52], where increase in annexin V positive cells were observed concentration dependent manner in quercetin treated group compared to control [52]. Further, the mitochondrial membrane potential was studies with the Rhodamine-123 dye with the incubation period of 24 h. In comparison to control, the cells treated with quercetin-loaded exopolysaccharides nanoparticles displayed significant reduction in mitochondrial membrane potential. As a part of apoptosis, disrupted and depolarized mitochondrial transmembrane was observed. The mitochondrial membrane potential of polymer micelle-encapsulated quercetin was evaluated on ovarian cancer cells. Following quercetin treatment, a decrease in Rh123 accumulation in ovarian cancer cells (A2780S) was observed, which indicated the collapse of mitochondrial membrane potential followed by the treatment of quercetin [52]. DAPI staining was performed followed by mitochondrial potential analysis. The DAPI staining results unveiled the increased chromatin condensation, and damage to the nuclear DNA. Further, the effect of quercetin in influencing the balance between ROS and antioxidants was studied. Generation of ROS in quercetin treated cells was observed with H 2 DCF-DA staining technique. Our results corroborated with the work reported by Jeon et al. [53]. The antiproliferative effect of quercetin was experimented in the Hepatocellular Carcinoma Cell Line HepG2. Quercetin is reported for the upregulation of intracellular ROS levels, activation of p53 a tumor suppressor protein, and for inducing the secretion of antioxidants such as glutathione. Increase in intracellular ROS levels were proved in HepG2 cells by H2DCFDA assay [53].

Conclusion
In conclusion, this study revealed that endophytic bacterial EPS was successfully delivered loaded quercetin into malignant cells. Initially, the EPS-producing endophytic bacteria was screened from the roots of Tribulus terrestris L. and it was confirmed as Pseudomonas otitidis. The EPS from the bacterial culture was isolated, and their structure and chemical properties were characterized and confirmed. Further, quercetin-loaded EPS NPs was fabricated through the precipitation method. The fabricated quercetin-loaded NPs characterized by FTIR, XRD, DLS, and SEM for their functional integrity, physical properties, zeta potential, size distribution and morphological characteristics and confirmed. Further, the drug release studies of the fabricated quercetin-loaded EPS NPs were performed via varied physiological buffer medium (acetate buffer (pH 3.5 and 5.0) and