Preparation, characterization and therapeutic properties of gum arabic-stabilized gallic acid nanoparticles

Gallic acid (GA) is a natural phenolic compound with therapeutic effects that are often challenged by its rapid metabolism and clearance. Therefore, GA was encapsulated using gum arabic into nanoparticles to increase its bioavailability. The formulated nanoparticles (GANPs) were characterized for physicochemical properties and size and were then evaluated for antioxidant and antihypertensive effects using various established in vitro assays, including 1,1-diphenyl-2-picrylhydrazyl (DPPH), nitric oxide scavenging (NO), β-carotene bleaching and angiotensin-converting enzyme (ACE) inhibitory assays. The GANPs were further evaluated for the in vitro cytotoxicity, cell uptake and cell migration in four types of human cancer cell lines including (MCF-7, MDA-MB231) breast adenocarcinoma, HepG2 hepatocellular cancer, HT-29 colorectal adenocarcinoma, and MCF-10A breast epithelial cell lines. The GANPs demonstrated potent antioxidant effects and have shown promising anti-cancer properties in a dose-dependent manner with a predilection toward HepG2 and MCF7 cancer cells. The uptake of GANPs was successful in the majority of cancer cells with a propensity to accumulate in the nuclear region of the cells. The HepG2 and MCF7 cancer cells also had a significantly higher percentage of apoptosis and were more sensitive to gallic acid nanoparticle treatment in the cell migration assay. This study is the first to confirm the synergistic effects of gum arabic in the encapsulation of gallic acid by increasing the selectivity towards cancer cells and enhancing the antioxidant properties. The formulated nanoparticles also had remarkably low toxicity in normal cells. Based on these findings, GANPs may have promising therapeutic applications towards the development of more effective treatments with a probable targeting precision in cancer cells.

. Powder X-ray diffraction patterns of XRD analysis of (A) the none capsulated gallic acid, (B) Gum arabic, (C) Physical mixture of GANPs, (D) the nanoparticles of gallic acid prepared using the freeze-drying technique.
Evaluation of the antioxidant properties. The free radical scavenging activity of the free and nano encapsulated gallic acid was assessed in vitro by the DPPH assay. DPPH is a stable organic free radical used to estimate the antioxidant activity of various compounds. Trolox served as a positive control due to its capacity to dissolve in the aqueous system. In this assay, the color of the DPPH changed from deep violet to a pale or colorless solution in the presence of GANPs nanoparticles. As illustrated in Fig. 6, the percentage of DPPH scavenging activity of GANPs was 35.6%, 57.9%, and 75.6% for the concentrations of 50, 100, and 200 µg/mL respectively; whereas for free gallic acid the scavinging activity was 25.4%, 46.6%, and 62.3%, respectively for the same concentrations (p < 0.05). This confirms the retention of the complete function of gallic acid after nanoencapsulation in gum arabic nanoparticles. Trolox revealed strong scavenging activity of 94.6% against DPPH at similar concentrations.
The antioxidant evaluation of free and nano encapsulated gallic acid was also determined in murine macrophage cell line (RAW 264.7). Initially, the cell viability of RAW 264.7 cells in response to treatments was evaluated using the MTT reduction assay in the presence or absence of LPS. As shown in Fig. 7, the LPS induction had shown a relative toxic effect on the RAW 264.7 cells upon treatment with GA and GANPs. Both treatment forms had negligible cytotoxicity activity against RAW 264.7 cells in comparison to the untreated LPS-stimulated cells. Treatment of RAW 264.7 cells with GA and GANPs caused a considerable inhibition of nitric oxide (NO) production as depicted in Fig. 8. Nonetheless, the nano encapsulated GA exhibited potent antioxidant activity, when compared to the free GA.  The antihypertensive activity of GANPs. The in vitro antihypertensive activity of free gallic acid and GANPs was evaluated by measuring the ACE inhibitory activity of the enzyme following the method outlined by Cushman and Cheung 18 . This assay is based on the conversion of hippuryl-histidyl-leucine to hippuric acid. The in vitro antihypertensive capacity of free GA and GANPs was investigated by measuring its absorbance      Fig. 13. According to the results, significant cytotoxicity was elicited among the HepG2, MCF7, MDA-MB231, and HT29 cell lines after treatment with IC 50 concentrations of free and nano encapsulated gallic acid as shown in Fig. 12. There was negligible cytotoxicity among the MCF-10A cells especially at concentrations equivalent to the IC 50 concentrations used in cancer cells (Fig. 11) and also in comparison with the untreated MCF-10A cells.
Interestingly, the HepG2 and MCF7 cells were more sensitive to the treatments than the HT29 and MDA-MB231 cells with a significantly higher selectivity with IC 50 of 16.61 and 8.52 µg/mL for free GA and GANPs respectively as shown in Fig. 12. The IC 50 value of GANPs was approximately half that of free gallic acid in HepG2 cells as shown in Fig. 12. As demonstrated, the HT29 and MDA-MB231 cells were less sensitive to the treatments with an IC 50 concentration of 35.45 and 19.62 µg/mL respectively.
The GANPs demonstrated variable patterns of cellular uptake in HepG2, MDA-MB231, HT29, and MCF7 cancer cell lines. As shown in Fig. 14, the GA/C6NPs labeled with C6 green fluorescent dye were successfully uptake by the majority of the cancer cell types with condensation in the nuclear region as shown in Fig. 15A. in section B, the empty nanoparticles labeled with C6 didn't have any of the cells stained with PI indicating non of . Inhibition (%) of free GA and Gallic acid nanoparticles by β-carotene bleaching assay with statistical analysis using a one-way ANOVA test. Data shown are mean value ± SD (n = 3, *p-value ≤ 0.05, **p-value ≤ 0.01). Figure 10. ACE inhibition (%) for free gallic acid and GANPs after 30, 60, and 90 min. Data shown are mean value ± SD (n = 3, t test, *p-value ≤ 0.05, **p-value ≤ 0.01).  19 . Therefore, with more dead cells due to GA/C6NPs, it will be challenging to have the real background color of C6 nanoparticles. The cell uptake study findings were consistent with the cytotoxicity study of cancer cells using the MTT reduction assay. The GA/C6NPs showed higher cellular uptake and toxicity in HepG2 and MCF7 cells by internalizing in the nucleus and cytoplasm of cancer cells. The MDA-MB231 and HT29 cells had a lower fluorescence intensity of PI attributed to the lower toxicity of GA/C6NPs.  www.nature.com/scientificreports/ The migration assay was carried out to assess the effect of GANPS and free GA with the IC 50 value of concentrations on MCF7, MDA-MB-231, HepG2, and HT29. The pictures were captured using an inverted light microscope at 0 h and 24 h and the cell migration was calculated as percentages as illustrated in Fig. 16. The findings of this study had shown that the GANPs treatment significantly retarded the migration of HepG2 and MCF7 cells compared with MDA-MB231 and HT29 cancer cells (Fig. 16). GANPs had also demonstrated potent anti-proliferative properties in the wound zone.

Discussion
The GANPs were prepared by the freeze-drying method with slight modifications including the high-pressure of homogenization at the pressure of 1,000 bar for 8 cycles to formulate smaller particles. Deionized water was used in the preparation to improve the dispersion of nanoparticles during the sonication process.
The X-ray diffraction technique was carried out to investigate the amalgamation between gallic acid and gum arabic as shown in Fig. 1. The physical mixture pattern as shown in the figure demonstrated GA peaks with a reduction of intensity. However, the diffraction peaks of the GANPs were completely diffused due to the amorphous state and the lack of crystallinity of the nanostructure indicating the formation of a new state in the GANPs. These changes were significantly related to the interaction between GA and gum arabic. Furthermore, the lack of a clear peak in the X-ray diffraction of GA might be the cause for the appearance of non-crystalline large peaks. Hence, the absence of crystallinity of pure compounds indicates the formation of a new phase, specifically by the conversion to the amorphous state.
In the TEM, the prepared nanoparticles had spherical elliptical shapes with the size distribution of individual particles ranging from 35 to 65 nm as shown in Fig. 4. According to the zeta sizer, the nanoparticles size measurement was in a higher range between 35 to 250 nm in aqueous solution probably due to the dry phase of TEM analysis. As demonstrated, the produced droplets had a small size to increase the surface area and the bioavailability of GA incorporated into gum arabic nanoparticles.
The use of GA is limited by its fast metabolism, low bioavailability, and poor absorption. These pharmacokinetic challenges reduce the concentration of drug reached (Cmax) and therefore lead to its rapid elimination 20 . Therefore, the encapsulation of GA into gum arabic nanoparticles was justified to effectively enhance its bioavailability and improve the therapeutic effects in cancer tissue by enhancing the uptake via transcellular or paracellular mechanisms 21 . Increasing the dispersion of the small nanoparticles aimed to increase the reactive surface area of gallic acid in the formulated nanoparticles 22 .
The release profile of gallic acid is depicted in Fig. 5 where the GANPs released a burst of gallic acid with a remarkably higher percentage of 41.39% at pH 4.8 compared to 25.66% at pH 7.4 after 2,000 min. This release pattern may be attributed to the weak bonds between gallic acid and gum arabic that are prone to break faster in acidic pH. This feature might offer a therapeutic privilege in cancer tissue by enhancing the release of gallic acid among cancer cells. These cells are continuously exposed to rapid shifts in the acid-base balance due to the limited blood supply exposing them more to acidic pH 23 . There was also a slow release of GA from GANPs within 3,500 min at pH 4.8 and pH 7.4. The release of GA was relatively slow at this phase compared with the initial release pattern. This pattern of release is probably due to the diminution of GA content in gum arabic www.nature.com/scientificreports/ nanoparticles. Hence, there was a plateau stage that lasted till 4,000 min at pH 4.8 and pH 7.4, in which about of 95.96% and 74.56% of GA was released from GANPs at pH level of 4.8 and pH 7.4, respectively. The release behavior of GA from GANPs was dependent on the negative charge of phosphate anion which gave a higher affinity for ion exchange with GA as an encapsulated agent. At pH 7.4, solubility decreased with an increase in particle binding properties due to the ionization properties of gum arabic. These findings are consistent with other studies in which the gum arabic polymer tended to disturb NPs at pH close to and higher than 5.5 22 .
Antioxidants play a crucial role in the protection against the attack of free radicals thus helping to prevent cardiovascular and cancer diseases which represent the most common causes of mortality. Oxidative stress significantly contributes to the etiology of these diseases through multiple pathways. Therefore, it is vital to keep the physiological balance between antioxidants and free radicals through the administration of potent antioxidants. Therefore, several in vitro assays were used in this study to confirm the antioxidant properties of GANPs.
The DPPH test was based on the reduction of the purple-colored stable free radical DPPH to the yellow diphenylpicrylhydrazine in the presence of free gallic acid and GANPs. Due to the existence of hydrogen-donating www.nature.com/scientificreports/ properties of GANPs, the phenolic compound has been shown to quench oxygen derived from free radicals by donating an electron or hydrogen atom to the free radicals in various systems from in vitro studies 24,25 . Trolox was used as a positive control to calculate the percentage of DPPH inhibition. Interestingly, GANPs nanoparticles and gallic acid exhibited an in vitro scavenging activity in a dose-dependent manner at the concentrations gradient of 50-200 µg/mL in DPPH as depicted in Fig. 6. This property of nanostructured gallic acid was reported in the literature 26 in which the underlying mechanism of DPPH radical scavenging activity was attributed to the Single Electron Transfert (SET) and Hydrogen Atom Transfer (HAT) 26 . SET refers to a rearrangement of structure with a loss of a proton. Usually, this mechanism produces quinone derivatives, resulting in the formation of a double bond with a change of proton signals. The gallic acid is stabilized after the reaction as a radical. In contrast, HAT is attributed to the loss of proton with the stabilization action of nearby groups on the charge of the molecule.  www.nature.com/scientificreports/ The quick proton exchange is considered as a contraction of the proton signals and it doesn't indicate any change in the chemical structure of the molecules 27 . Although the shown antioxidant effect of GA was lower than that of Trolox, the formulated nanoparticles form had significantly enhanced these effects compared with free gallic acid due to the DPPH scavenging properties of gallic acid combined with the hydroxyl units of gum arabic. From the data in Fig. 6, it is apparent that the DPPH test has examined the impact of free radical on test compounds. Therefore, the strong absorption at 517 nm in visible spectroscopy was influenced by the odd electron of DPPH. The odd electron was paired in the presence of hydrogen of a free radical scavenger, indicating the capacity of gallic acid nanoparticles to scavenge free radicals without prior enzymatic activity 28 . The antioxidant properties of gum arabic may be attributed to its hydroxyl groups, polypeptide, and its highly branched structure which may improve the stability and antioxidant properties of gallic acid within the nanoparticles 17 . Gum arabic is, therefore, a suitable encapsulant capable of forming stable nanoparticles for safe delivery and can be used as a potent antioxidant in future formulations. The in vitro cytotoxicity effect of the free GA and GANPs in RAW 264.7 cells was evaluated using MTT assay as demonstrated in Fig. 7. The MTT results which reflect the number of viable cells confirmed that both GANPs and GA did not have a significant cytotoxic effect on the RAW 264.7 cells. In these cells, another antioxidant test was performed known as the nitric oxide inhibition assay which is important for evaluating the antioxidant and anti-inflammatory properties of potential agents. The assay is based on the production of nitrite metabolite from Nitric oxide and its quantification using Griess reagent 29 . Nitric oxide is an important molecule in the regulation of numerous physiological mechanisms such as blood pressure and pathological conditions such as inflammation, shock and neurotoxicity 30 . Due to the scavenging capacities of antioxidants, these agents are used to treat those deleterious disorders. The cells were challenged with LPS which induces the expression of nitric oxide synthase protein. This enzyme in turn will enhance the production of nitric oxide from sodium nitroprusside and oxygen that are also induced by the oxidative challenge of LPS. The production of peroxynitrite anion, a strong oxidant is based on the interactions between superoxide radical and NO 29,31 .
The results of the nitric oxide inhibition assay confirmed significant antioxidant effects in RAW 264.7 cells. The free and nano encapsulated gallic acid at various concentrations accordingly inhibited the production of nitrite radical in the culture medium in a dose-dependent manner. As depicted in Fig. 8, the hydroxyl radical scavenging activity of GANPs was 2 times higher than that of GA at concentrations ranging between 31.5 and 500 µg/mL with IC 50 values of 56.03 µg/mL and 105.53 µg/mL, respectively (P < 0.05). This result also confirmed that gallic acid maintained its antioxidant properties that were consistent with other reports 32 . Gallic acid was reported to have cyclooxygenase 2 (COX-2) and nitric oxide synthase (iNOS) inhibitory properties in LPSinduced RAW 264.7 cells modulating the pro-inflammatory cytokines such as IL-17, IFN-γ, and TNF-α 33 . The GANPs nanoparticles exhibited strong inhibition on NO production at concentrations ranging from 62.5 to 500 µg/mL. Gum arabic was also reported to enhance the free radical scavenging properties of therapeutic agents by neutralizing and absorbing free radicals 34 . Nitric oxide produced as a proinflammatory mediator during the immunopathological phenomenon may cause chronic inflammation that is circumvented by macrophages. These cells were enhanced by gum arabic to exhibit antioxidant and hepatoprotective properties 35 .
The β-carotene bleaching assay is a quick and simple test based on the competition between GANPs and β-carotene to neutralize the hydroperoxide-derived free radicals produced by the oxidation of linoleic acid. As shown in Fig. 9, a concentration-dependent inhibition of β-carotene bleaching was recorded due to the presence of phenolic groups in gallic acid. The highest reducing power was recorded at the concentration of 500 µg/mL, which exhibited approximately 68.9% of antioxidant activity (P < 0.05). Although both GA and GANPs showed potent antioxidant activity; however, the nano encapsulated gallic acid appeared to be significantly more effective than free gallic acid. These findings may be attributed to the phenolic groups in gallic acid 36 and the functional groups of gum arabic used as the coating material 37 .
Oxidative stress is largely implicated in the pathogenesis of hypertension disease; where it is responsible for the activation of several mechanisms leading to vasoconstriction 38 . Therefore, the in vitro antihypertensive activity of the free and nano encapsulated gallic acid was assessed by measuring the ACE-inhibitory capacity using a UV-visible spectrophotometer at 228 nm. The inhibition of ACE function is also one of the widely used therapeutic options in the hypertension disease as this enzyme is involved in the renin-angiotensin axis of blood pressure regulation which is largely involved in the pathogenesis of hypertension 39 . The ACE that is renowned now being the target of the recent coronavirus infection is the catalyst converting angiotensin I to angiotensin II in lung and kidneys 40 . This study is the first evidence confirming the ACE inhibitory activity of GANPs in addition to its antioxidant activity providing a promising treatment for hypertension disease. As shown in Fig. 10, the ACE-inhibitory capacities of gallic acid and GANPs were time-dependent in which the ACE inhibitory activity of GANPs and Gallic acid at 90 min was 69.14% and 54.12%, respectively using 100 µL of 5 µg/mL treatment solution. The use of gum arabic polymer as nanocarrier improved the antihypertensive capacity of GANPs that explains the high percentage of ACE inhibition compared to that of free gallic acid. Such antihypertensive effects were reported in animal models of hypertension disease using free gallic acid 41 and gum arabic 42 treatments. Several phenolic natural products including gallic acid are known to have ACE inhibitory activity but with a poorly understood mechanism 43 . The enhanced antihypertensive property GANPs could be imputed to the high amount of gallic acid released from the nanoparticles.
The evaluation of cell viability is a common assay to determine the in vitro cytotoxicity of various biomaterials. The MTT assay is largely known as a rapid quantitative and colorimetric assay to evaluate cell viability for different purposes. The assay depends on the reduction of MTT salt by mitochondrial dehydrogenases inside the viable cells. Upon reduction, the yellow tetrazolium salt changes to the corresponding purple formazan. In this study hepatocellular, colorectal, and breast adenocarcinoma cell lines were used in addition to normal epithelial www.nature.com/scientificreports/ cells. Figure 11 demonstrates the cytotoxic effect of GA, GANPs in these cell lines after 72 h of exposure. It has been observed that both GA and GANPs exerted potent anti-proliferative effects in cancer cells with minimal toxicity in normal epithelial cells as shown in section A of Fig. 12. As depicted, the IC 50 values were significantly low in all the cancer cells compared with epithelial cells. These results were consistent with previous reports confirming the antineoplastic effects of gallic acid where it significantly increased apoptosis and apoptotic markers in esophageal and melanoma cancer cells by interfering with Akt/mTOR pathway with no apparent toxicity in normal cells 44,45 . Interestingly as shown in Fig. 11, hepatocellular cancer (HepG2) and breast cancer (MCF-7) cells appeared to be more sensitive to gallic acid nanoparticles than the MDA-MB231 breast, and HT29 colorectal cancer cells. GANPs at the concentration of 15.63 µg/mL caused a reduction to 80.16% of viable HepG2 cells n compared to untreated cells (p < 0.05). The nanoparticles appeared to be more potent in HepG2 cells where a lower percentage of cell viability (20.11%) was recorded at (100 µg/mL) concentration compared to 25.6% of free gallic acid. These findings could be attributed to the effect of gum arabic. In one study, gum arabic was found to have a targeting specificity towards hepatic cells by its galactose groups that attach with asialoglycoprotein receptors (ASGRP) of liver cells in which the nanocarriers stimulated receptor-mediated endocytosis of the therapeutic agents 46 . Thus, the chain in gum arabic reacted as natural targeting ligands for asialoglycoprotein receptors (ASGRP) on hepatocytes, which have improved the cytotoxicity effect of gallic acid nanoparticles. Consequently, the high cytotoxicity effect of GANPs against HepG2 cells might be attributed to the galactose units of gum arabic polymer. GANPs had also demonstrated enhanced cytotoxicity in MCF7 breast cancer cells compared to MDA-MB231 breast cancer cells. This significant potency in MCF7 cancer cells may be attributed to the modulation of estrogen receptor function as reported in several studies [47][48][49] . These receptors are present in MCF7 cells and are potent targets approached in the treatment of breast cancer; however, the MDA-MB231cells lack these receptors. Therefore GANPs have a better selective index toward hepatic cancer cells and MCF7 breast cancer cells probably due to the chemical structure of gum arabic polymer and the selectivity toward hepatic and breast cancer cell receptors as demonstrated in Fig. 12.
The aforementioned toxicity findings were consistent with the cellular uptake study. In this assay, coumarin-6 (C6) was selected for the observation of GANPs penetration to the cells with propidium iodide (PI) counterstain that is characterized by its penetration into nonliving cells. As shown in Fig. 14, there was a significant increase in the uptake of PI among HepG2 and MCF7 cells compared with other cancer cells that may be attributed to the higher uptake of GANPs leading to more apoptosis. The high fluorescence of PI as shown in Fig. 15 masked the green color of C6 due to the Förster resonance energy transfer phenomena. As mentioned earlier these cells were more sensitive to GANPs due to the chemical structure of gum arabic as confirmed in another report where the use of gum arabic assisted the delivery of curcumin and C6 into hepatocellular carcinoma cells 50 . The enhanced uptake of PI was consistent with several reports of the proapoptotic and cell cycle arrest properties of gallic acid in cancer cells 44,45,51,52 . In these studies, the mechanism of action was revealed in the form of enhancement of caspase, endonuclease dependant pathways, extrinsic apoptotic pathways, and disruption of p27Kip1/Skp2 Complexes that are crucial in regulating cell cycle procession in addition to modulating the level of IL-8. These findings confirm the therapeutic properties and the efficient cell internalization of gallic acid nanoparticles. Thus the use of gum arabic as a carrier permit facile passage of gallic acid through the cell membrane as well as increase its solubility.
GANPs treatment was further evaluated for its effect on the migration of HepG2, HT29, MCF7, and MDA-MB231 cancer cells as a measure of invasiveness and metastasis 53 . The treatment of cells using GANPs has revealed a significant reduction in the percentage of migration of HepG2 and MCF cells. These findings are consistent with the reported antimetastatic properties of gallic acid in gastric, cervical, melanoma, and oral cancer cells that are attributed to the interference of gallic acid with NF-kappaB activity, matrix metalloproteinases, RAS-ERK and the PI3K/AKT signaling pathways [54][55][56][57] .

Conclusion
This study is the first to report the augmenting property of gum arabic in encapsulating gallic acid improving the therapeutic outcomes of several in vitro assays. The potent antioxidant properties of gallic acid were significantly enhanced by gum arabic coating attributed to the chemical properties of both compounds; This study also revealed several antineoplastic properties offered by the nanoformulation. The GANPs enhanced the selective uptake in hepatic cells enhanced the cytotoxicity in breast cancer (+ estrogen receptor) and confirmed its proapoptotic effects. Therefore GANPs formulation is a potential candidate for the prevention and treatment of hepatic and breast cancers. Extrapolation of the in vitro cytotoxicity effects of GANPs to in vivo cytotoxicity effects demands further investigation in light of its application as a cancer chemotherapeutic agent.

Scientific Reports
| (2020) 10:17808 | https://doi.org/10.1038/s41598-020-71175-8 www.nature.com/scientificreports/ Preparation of gallic aid nanoparticles. The GANPs were synthesized using the freeze-drying technique with few modifications. The aqueous solution containing gallic acid (GA) and gum arabic in 1:1 molar ratio was obtained by dissolving 0.05 g of GA in 50 mL deionized water containing 0.8 g/mL of gum arabic. The mixture was kept under mild agitation at room temperature for 72 h. The final suspension was subjected to a high-pressure homogenizer at a pressure of 1,000 bar for 8 cycles and was then frozen at − 80 °C. The final product was freeze-dried for 24 h at − 55 °C. In the confocal microscope study of cell uptake and apoptosis, The GANPs labeled with C6 was prepared using a modified protocol of the same freeze-drying technique as described in some reports 58 . An aqueous solution containing gallic acid (GA) and gum arabic in 1:1 molar ratio was obtained by dissolving 0.01 g of GA and 0.1 mg of coumarin-6 in 1 mL of ethanol then mixed with 0.1 g of gum arabic dissolved in 10 mL of DMSO. The mixture was kept under mild agitation at room temperature for 72 h. The final product was then freeze-dried for 24 h at − 55 °C.
Preparation of the physical mixture. The physical mixture of gallic acid (GA) and gum arabic was performed in a 1:1 ratio as lyophilized complex. GA and gum arabic were admixed into homogeneous powder using pestle and mortar.
Size and zeta potential. The Zeta potential and size of GANPs were characterized using a zeta sizer (Malvern Nano-ZS-ZS, Zeeman) with dynamic size. www.nature.com/scientificreports/ deionized water were added to the flask with vigorous shaking for 15 min. Blank emulsion, devoid of β-carotene was prepared. Aliquots (200 µL) of β-carotene emulsion were transferred into the 96-well plate containing 50 µL of GA and GANPs at various concentrations and incubated in the dark at 50 °C. Different concentrations of α-tocopherol were used as a positive control reflecting nearly complete inhibition of β-carotene bleaching. The absorbance measurements were recorded immediately at 470 nm at 20 min intervals for 100 min. The antioxidant activity (AA) was assessed using the following equation:

Transmission electron microscopy (TEM). This technique was
where A 0, A t , and A 0c , A tc are the absorbances of sample at t = 0, t = 100 min and absorbance of control at t = 0, t = 100 min.
The anti-hypertensive assay using angiotensin-converting enzyme (ACE) inhibition assay. The in vitro ACE inhibition activity of gallic acid GA in gum arabic nanoparticles was assessed in vitro based on the conversion of hippuryl-histidyl-leucine to hippuric acid in the presence of ACE enzyme 62 . One hundred microliters of gallic acid GA and GANPs were mixed with ACE (25 µL, pH 8.3) and incubated at 37 °C for 5 min. Next, 3.5 Mm of hippuryl-histidyl-leucine (10 µL) was added to the mixture and incubated for 30, 60, and 90 min. The enzymatic reaction was halted after the addition of 50 µL of 3 mol/L HCI. Hence, 1 mL of ethyl acetate was added to remove the resultant hippuric acid. The solvent was evaporated at 120 °C and re-dissolved in 3 mL of 1 N of NaCl. The concentration of hippuric acid was evaluated by measuring the absorbance at 228 nm. A blank devoid of GA and GANPs was prepared at background subtraction.
Cell culture and cytotoxicity assay. A list of cancer cell lines selected to demonstrate the antineoplastic properties using the MTT assay which is based on the enzymatic reduction of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) by active living cells. The cancer cell lines were all of an epithelial type representing a list of common cancer types including colorectal adenocarcinoma (HT29), hepatocellular cancer (HepG2), breast adenocarcinoma (MDA-MB231 and MCF7), and MCF-10A breast epithelial cell lines. All the cell lines were free of mycoplasma infection and the number of passages used in the experiments ranged between (8)(9)(10). The cells were maintained in DMEM supplemented with 1% of penicillin-streptomycin and 10% of fetal bovine serum (FBS). The cells were harvested using Trypsin-EDTA solution and examined using an inverted microscope (OLYMPUS CK40). A hemocytometer was used to determine cell density. Standard solutions of GA and GANPs were prepared and dissolved to concentrations ranging between 1.56-100 µg/mL. This assay was performed as described in our previous reports to assess cytotoxicity activity of treatments in the selected cell lines 63 . Two hundred microliters of cell suspension with a density of 1 × 10 5 cells/ mL were placed into each well of 96-well plate and later incubated at 5% CO 2 and 37 °C for 24 h. After incubation time, the medium was replenished and the cells were treated with GA and GANPs at concentrations ranging between 1.56 and 100 µg/mL for cancer cells and 15.63-1,000 µg/mL for normal cell lines to determine the IC 50 values. Chemotherapeutic controls were also used with different concentration gradient from 0.156 to 10 µg/mL (5-fluorouracil for HT29, tamoxifen for HepG2 cells, and doxorubicin for MCF7, MCF-10A, and MDA-MB231. The last row of the 96-well plate was left for the control experiment under similar conditions. After 72 h of postincubation, 20 µL of 5 mg/mL of MTT solution was added to each well, in which the plate was covered with aluminum foil and incubated for 4 h. The medium was removed and replaced with 100 µL DMSO to dissolve the remaining purple formazan precipitate. The absorbance was recorded using an ELISA reader at 570 nm. This colorimetric assay focused on the reduction of MTT to purple formazan by the mitochondrial enzymes of the metabolically active cancer cells 64 . The IC 50 was calculated from the concentration of drug that inhibits 50% of cell growth. The experiment also included the MCF-10A normal breast cell lines in the MTT assay to calculate the selective index for each of the cancer cell lines. All experiments were carried out in triplicate and the outcomes are presented in standard deviation and mean values. Cellular uptake of GANPs. The study also included a qualitative assessment of cell uptake in HepG2, MCF7, MDA-MB231, HT29 cancer cell lines based on the use of coumarin 6 (C6) and propidium iodide (PI) fluorescent dyes. The use of both dyes in this experiment was adapted from Win et al. 65 . C6 is widely used for tracking of nanoencapsulation emitting green light at 500 nm 66 . While PI fluorescence dye was used as a counterstain selective to nonviable cells and cells with compromised cell membranes where it binds with base pairs of double-stranded DNA giving the unique red fluorescence emission while sparing viable cells with intact cell membranes 67 . The experiment started by culturing the cancer cells on glass coverslips with a cell suspension of 10 4 cells/ cm 2 in complete media inside the CO 2 incubator at 37 °C for 6 h allowing cell adherence to the coverslip. The cells were then subjected to the IC 50 values of GA/C6NPs in serum-free medium for 24 h. consequently, the cells were incubated with PBS buffer containing PI (10 µg/mL) for 10 min at room temperature after washing with PBS buffer; then washed again with PBS buffer followed by fixation with 70% ethanol in the freezer (− 20 °C) for 10 min. The coverslips were then examined using the confocal microscope with excitation wavelength 434 nm under multichannel mode.
Scratch migration assay. The in vitro cell migration assay was performed by scratch assay to assess cell mobility following the protocol adapted from Kovarikova et al. 68 . The HepG2, MCF7, MDA-MB231, and HT29 cells were seeded in a 6-well plate with the cell density of (2 × 10 5 ). Upon reaching 80% of confluence level, the scratch was carried out using a yellow pipette tip with an average diameter of 1 mm. The cells were then washed www.nature.com/scientificreports/ 2 times with PBS and treated with GANPs at concentrations of 1.56-100 µg/mL and were further incubated for 24 h. Based on the Dino Eye application connected to the inverted microscope, a marker line was used to select the position of the picture captured. Various pictures were captured at 0 h and 24 h after the treatment at a magnification of 40 ×. The experiment was performed in triplicate. Hence, the percentage of migration of the aforementioned cell lines was calculated using the following formula: Statistical analysis. T tests and One-way ANOVA test followed by the Holm-Sidak multiple comparisons were performed using GraphPad Prism version 8.0.1 (San Diego, California USA) to evaluate the differences among the treatment groups. A difference at p < 0.05 was considered significant.