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Preparation, physicochemical characterization, and bioactivity evaluation of berberine-entrapped albumin nanoparticles

Berberine (BBR) is an isoquinoline alkaloid with several clinical therapeutic applications. Its low water solubility, absorption, and cellular bioavailability diminish BBR's therapeutic efficacy. In this study, BBR was encapsulated into bovine serum albumin nanoparticles (BSA NPs) core to reduce BBR limitations and enhance its clinical therapeutic properties. Several physicochemical characterization tools, such as Dynamic Light Scattering and Ultraviolet–Visible spectroscopic measurements, field emission transmission electron microscopy surface morphology, Fourier transforms infrared spectroscopy, thermal stability analysis, and releasing studies, were used to evaluate the BBR-BSA NPs. Compared to BBR, BBR-BSA nanoparticles demonstrated superior free radical scavenging and antioxidant capacities, anti-hemolytic and anticoagulant efficacies, and antimicrobial activities, as demonstrated by the findings of the in vitro studies. Furthermore, a stressed pancreatic rat model was induced using a high-fat, high-sucrose diet plus carbon tetrachloride injection. The in vivo results revealed that BBR-BSA NPs substantially restored peripheral glucose metabolism and insulin sensitivity. Oral administration of BBR-BSA NPs also improved pancreatic β-cells homeostasis, upregulated pancreatic antioxidant mechanisms, inhibited oxidants generation, and attenuated oxidative injury in the stressed pancreatic tissues. In conclusion, our in vitro and in vivo results confirmed that BBR-BSA NPs demonstrated more potent antioxidant properties and restored pancreatic homeostasis compared to BBR.

The FTIR characteristic spectra of the synthesized BBR-BSA NPs and BSA NPs. The characteristic FTIR spectra of standard BBR-chloride, crystalline BSA, BBR-BSA NPs, and BSA NPs are demonstrated in Fig. 2a-d. Significant crystalline BSA peaks were observed at 3415.58 cm −1 (O-H stretching vibration band), 2924.53 cm −1 (amide A band, related to N-H stretching vibration), 2856.73 cm −1 (amide B band, related to N-H stretching vibration of NH 3 + free ions), 1645.90 cm −1 (amide I band related to C=O stretching vibration of the peptide linkages, indicating the secondary structure of the BSA globular protein), 1540.55 cm −1 (amide II band considered as the standard protein peak, which referred to the coupling of the in-plane bending vibrations of N-H and the stretching vibrations of C-N and C-C), 1444.36-1395.20 cm −1 (CH 2 bending groups, indicated the C-H bending vibrations), and 1239.93 cm −1 (amide III bands, related to the combination of the in-plane N-H bending vibrations and the C-N stretching vibrations) (Fig. 2b). There was a relative change in the band intensities of the different functional characteristic peaks of crystalline BSA (Fig. 2b), compared to the FTIR spectra of BBR-BSA NPs and BSA NPs (Fig. 2c,d and Supplementary Table S2). It was induced by the chemical interactions and the conformational changes between the BSA molecules following the formulation and preparation of the NPs (Fig. 2b-d). Significant peaks of standard BBR-chloride were observed at 3407.33 cm −1 (O-H stretching vibration), 3052.67 cm −1 (hydrophilic quaternary ammonium (N + ) group, which was related to the binding of this group to four hydrophobic aromatic heterocyclic hydrocarbons groups through covalent C-N bonds), 2944.96 cm −1 (saturated C-H stretching), 2845.13 cm −1 (-OCH 3 Table S3).
The thermogravimetric analysis of the BBR-BSA NPs and BSA NPs. The thermal gravimetric analysis (TGA) thermograms revealed the variations in the mass degradation rates of standard BBR-chloride, crystalline BSA, and BBR-BSA NPs related to the increased temperature ( Fig. 3 and Supplementary Fig. S1a). Compared to the thermogram of standard BBR-chloride at 170 °C, the mass degradation rate of the TGA thermogram of BBR-BSA NPs (Fig. 3b) revealed a significant decrease in the mass degradation rate before 210 °C (Fig. 3a). After formulation, the thermal stability of the entrapped BBR was increased. The Differential thermal analysis (DTA) and differential scanning calorimetry (DSC) thermograms of standard BBR-chloride demonstrated two points for reaction, the first at 35.7 °C (DTA); 35.8 °C (DSC), and the second at 517.2 °C (DTA); 518.5 °C (DSC) (Fig. 4a,b). In the temperature range of 535.7-634.2 °C (DTA); 520.7-638.5 °C (DSC), a large, sharp, and slender melting endothermic peak was observed. It indicated the crystalline nature of BBR-chloride (Fig. 4a,b). The DTA/DSC thermograms of BSA revealed two points for reaction, the first at 41.5 °C (TGA); 41.5°C (DSC), and the second at 549.2 °C (DTA); 549.8 °C (DSC) (Supplementary Fig. S1b,c). A large sharp, slightly narrow melting endothermic peak was demonstrated at the 535.7-634.2 °C (DTA); 520.7-638.5 °C (DSC) temperature range ( Supplementary Fig. S1b,c). The DTA/DSC thermograms of BBR-BSA NPs revealed two different points for reaction, the first at 41.1 °C (DTA); 41.4 °C (DSC), and the second at 259.5 °C (DTA); 528.0 °C (DSC) (Fig. 4c,d). These points varied from the points of reaction of standard BBR-chloride and crystalline BSA. The DTA/DSC thermograms of BBR-BSA NPs have also demonstrated a decrease in heat change values from -1454.36 and 2578.15 µVs/mg to -993.23 and 2507.50 µV/mg (Fig. 4c) and a reduction in the values of the enthalpy from -8888.39 and 7749.44 J/g to − 2477.03 and 7391.38 J/g (Fig. 4d), compared to the DTA/ DSC thermograms of standard BBR-chloride (Fig. 4a,b).
In vitro studies. Evaluation of the BBR releasing rates (%). Figure 5 shows a significant decrease in the releasing and local dissolution rates of BBR-BSA NPs compared to BBR in different dialysis media. Simulated intestinal fluid (SIF, pH 7.4) and simulated gastric fluid (SGF, pH 2) was used. The SGF medium reduced the releasing and local dissolution rates of BBR and BBR-BSA NPs compared to their rates in the SIF medium. After 48 h, the releasing rates of the entrapped BBR (BBR-BSA NPs) were only about 40 and 35% in SIF and SGF, compared to about 65 and 50% release of BBR, respectively.   50 values and lower antioxidant capacities (FRP and phosphomolybdenum complex) compared to BBR and BBR-BSA NPs, as illustrated in Table 1 and  Supplementary Table S4. Interestingly, the antioxidant potential of BSA NPs was significantly (P < 0.05) increased   Fig. S11 and Table S4). Supplementary Table S4 illustrates that BBR-BSA NPs had the highest anticoagulant activity and activated partial thromboplastin time (APTT) of 92.7 ± 0.8 s compared to the coagulation time of BBR (79.4 ± 0.9 s), ascorbic acid (82.9 ± 1.1 s) at the same concentration of 1.8 mg/mL. In contrast, BSA and BSA NPs had lower APTT values. After formulation, BSA NPs significantly (P < 0.05) enhanced the anti-platelet coagulation activity and elevated the APPT value (67.7 ± 1.2 s) compared to BSA (58.8 ± 0.6 s) at 40 mg/mL (Supplementary Table S4).    Table S5). The encapsulated BBR (0.01-1.8 BBR mg/mL) in the BSA NPs core substantially improved its antimicrobial activities against these pathogens compared to BBR (Supplementary Table S6). As illustrated in Table 2, BBR-BSA NPs demonstrated a significant (P < 0.05) increase in their inhibition zones (mm), with significantly decreased inhibitory concentrations (MICs) towards this microbial growth compared to BBR. In contrast, E. coli illustrated an inhibition zone of 32 In vivo studies. The effect of BBR and BBR-BSA NPs on serum glycemic markers and pancreatic oxidative stress biomarkers. In the current study, the control (C)-BBR-BSA NPs group demonstrated a significant (P < 0.05) improvement in glucose metabolism and insulin sensitivity compared to the control rats, C-BBR, and C-BSA NPs groups. The stressed pancreatic rat model demonstrated a significant (P < 0.05) increase in the levels of serum glucose, insulin, and HOMA-IR index associated with a significant (P < 0.05) decrease in the level of HOMA-β index compared to the control group. The BBR-BSA NPs-treated rats (T-BBR-BSA NPs) significantly (P < 0.05) reduced the hyperglycemia and the peripheral insulin resistance features and elevated the insulin sensitivity and β-cell function (HOMA-β) compared to the stressed rats, BBR-, BSA NPs-, and Ator-treated rats ( Fig. 6a,b). In this study, the C-BBR-BSA NPs group demonstrated significantly decreased (P < 0.05) levels of pancreatic TBARS and NO and substantially increased (P < 0.05) the level of reduced pancreatic GSH as well as GSHPx, GST, and SOD activities compared to the control, C-BBR and C-BSA NPs groups. The administration of HFHSD plus carbon tetrachloride (CCl 4 ) injections significantly (P < 0.05) increased the levels of pancreatic lipotoxicity and free radicals' generation (elevated TBARS and NO levels) and significantly (P < 0.05) decreased the antioxidant modulators (reduced GSH level as well as GSHPx, GST, and SOD activities). On the contrary, the BBR-BSA Table 1. The inhibition or the effective concentrations (IC 50 or EC 50 ) of free radicals scavenging activities, total antioxidant capacities, and anti-hemolytic properties of the tested compounds. NO · , nitric oxide; H 2 O 2 , hydrogen peroxide; O 2 − , superoxide anion; OH − , hydroxyl radical; DPPH, 1, 1-diphenyl-2-picrylhydrazyl; TBARS, thiobarbituric acid reactive substances; TAC, Total antioxidant capacities; FRP, ferric reducing power; RBCs, red blood cells. Data values are expressed as means ± SD (n = 5). Different letters in the same row indicated statistically significant differences (P < 0.05) that evaluated using the analysis of one-way ANOVA with post hoc LSD test. The same letters in the same row indicated non-significant differences (p > 0.05). www.nature.com/scientificreports/ NPs-treated rats demonstrated a significant (P < 0.05) retardation in pancreatic toxicity and improved the pancreatic antioxidant biomarkers compared to the stressed, T-BBR, T-BSA NPs, and T-Ator groups (Fig. 6c,d).
The effect of BBR and BBR-BSA NPs on pancreatic histopathological evaluations. The histopathological investigations revealed that control, C-BBR, C-BBR-BSA NPs, and C-BSA NPs groups had normal pancreatic endocrine parts (islets of Langerhans) and healthy exocrine acinar cells (ACs) (Fig. 7a-d). While the pancreatic tissue of stress-rats model (HFHSD administration plus CCl 4 injection) revealed marked histological alterations in both endocrine and exocrine parts ( Fig. 7e-g). The Islets of Langerhans appeared smaller in size and lower in number compared to the control islets ( Fig. 7e) and showed vacuolar degeneration and necrosis (Fig. 7f). Moreover, the necrotic cells appeared either with pyknotic nuclei or homogenous eosinophilic structureless without nuclear structure. The pancreatic duct also showed mild to a moderate proliferation of its epithelial linings (Fig. 7e,f). Figure 7g demonstrates that the stressed pancreatic rat model demonstrated severe swelling of the acinar cells with marked vacuolar degeneration and loss of zymogen granules. Regarding the treated groups, BBR-the treated group (T-BBR) showed a moderate curative effect on the pancreatic tissue with some degenerated and scattered necrotic islets cells and mild pancreatic congested vessels (Fig. 7h). BBR-BSA NPs treatment was described as a good restorative formulation that significantly improved the pancreatic exocrine and endocrine sections and showed near to normal appearance of both islets cells as well as the acinar cells (Fig. 7i). The BSA NPs-treated rats also demonstrated normal pancreatic tissue with mild cytoplasmic vacuolation of few islets' cells (Fig. 7j). In addition, the Ator-treated group demonstrated a mild degree of islet cells degeneration, scattered necrosis, and few pyknotic cells (Fig. 7k). Interestingly, treatment with BBR-BSA NPs had a more restorative effect on pancreatic tissue than the treatment with BBR followed by the treatment with reference drug (Ator).

Discussion
Bovine serum albumin, a nano-carrier, can encapsulate several bioactive components and deliver different drugs and natural products with high selectivity, high bioavailability, and potent therapeutic properties 23,26,27 . In the current study, BBR-BSA NPs were synthesized by the desolvation method, using ethanol as a desolvating agent and glutaraldehyde as the crosslinking agent. The PDI value indicates the homogeneity of the prepared NPs. The lower PDI value revealed a better uniform distribution of the prepared NPs, demonstrating good stability 28 . In the present study, the synthesized alkaline BBR-BSA NPs have better properties than the neutral ones as the particle size diameter, and PDI values decreased, and their zeta potential increased. Previous studies reported that shifting away from the isoelectric point of BSA by increasing the pH of the NPs preparation media reduced the coagulation interactions and induced the electrostatic repulsive forces between the BSA molecules 27,29,30 . . Data values are expressed as means ± SD (n = 5). Different letters indicated statistically significant differences (P < 0.05) between the experimental groups within the same parameter, which were evaluated using the analysis of one-way ANOVA with the post hoc LSD test. www.nature.com/scientificreports/ Moreover, the surface charge of the formulation is considered an indicator of the stability of the NPs distribution, which indicates the degree of electrostatic repulsion between the particles 28 . The highly charged particles have a high zeta potential value, considered stable due to the strong electric repulsion forces between the molecules 28 . NPs with a particle size < 200 nm can significantly avoid renal clearance and permeate into different tissues 28 . In this study, the alkaline BBR-BSA NPs size diameter was < 200 nm. Interestingly, the hydrodynamic size diameter of the alkaline BBR-BSA NPs (155 ± 0.6 nm, hydrated form) was relatively higher than their diameter from FE-TEM measurements (125 ± 23.60 nm, dried form). During DLS measurements, the presence of water around BBR-BSA NPs increased their size diameter compared to the TEM measurements 14,15 .
In agreement with Stoyanova et al. report., our results confirmed that the entrapment of BBR in the BSA NPs core increased its thermal stability by decreasing the values of the mass-loss rate, heat change, and enthalpy of BBR related to the increased temperature 31 . In addition, the retardation in the mass degradation rates and the intensities of the melting peaks of the individual target compounds related to the increased temperature indicated the transformation from their physical crystalline states to the NPs' amorphous form 31 . Therefore, the nano-particulate drug delivery system is introduced as a vital tool to increase the thermal stability of the entrapped bioactive compounds 31 .
BSA NPs could increase the half-life time, LC%, and the antioxidant properties of the entrapped bioactive ingredients 24 . The solubility of BBR-BSA NPs formed in distilled water, 0.1 M HCl, and PBS (pH 6.8) was significantly increased compared to BBR 24 . After formulation, the synthesized BSA NPs entrapped BBR in an amorphous state, reducing the agglomeration and aggregation between the hydrophobic BBR particles 24 . Our www.nature.com/scientificreports/ study targeted the encapsulation of standard BBR-chloride in the core of BSA NPs that delayed and minimized the BBR releasing rates in different dialysis media (SIF or SGF), which agrees with Patra et al. 32 and Saleh et al. 33 . Wu et al. also reported that chitosan/fucoidan-taurine conjugate NPs released BBR in SIF (pH 7.4) faster than its releasing rate in SGF (pH 2.0) 15 . Furthermore, the current in vitro results revealed that BBR-BSA NPs greatly enhanced the antioxidant properties of its entrapped BBR against NO · , H 2 O 2 , O 2 − , OH − , DPPH, TBARS, and potassium ferricyanide/ferric chloride, and ammonium molybdate radicals compared to BBR. BBR-loaded polymeric nanostructures have been used to improve the physicochemical characteristics (solubility, dissolution rate, hydrophilicity, and releasing profile), pharmacodynamics and pharmacokinetics properties, selectivity, and the therapeutic potentials of BBR 28,34 . BBR was reported as a potential anti-radical alkaloid that scavenges several free radicals such as DPPH, NO · , H 2 O 2 , O 2 − , lipid peroxidation, and ferric ions 35,36 and BBR NPs formulations were found to increase the radical scavenging activity 31 . In the current study, BSA NPs as a nano-carrier markedly elevated the anti-hemolytic potent activity and reduced the cytotoxicity of the entrapped BBR. Previous work confirmed that BSA NPs could improve the therapeutic properties and attenuate the toxicity of several bioactive compounds 22 . By augmenting the anticoagulant activity of BBR, our findings suggested that BSA nanoparticles could significantly stimulate and improve blood flow .
BBR has potent antimicrobial properties against bacteria, viruses, protozoa, fungi, and yeasts 37 , which supports its antibiotic action toward harmful pathogenic bacteria 38,39 . Previous studies aimed to develop a natural antimicrobial nano-particulate drug delivery formulation that could enhance the sensitivity of the antimicrobial drugs 40,41 . Our study demonstrated that BBR-BSA NPs demonstrated the highest antimicrobial activities and the lowest MIC values compared to BBR against the studied pathogenic microbes, which confirmed the results of the Sahibzada et al. work 42 . Moreover, BBR-conjugated chitosan or alginate nanostructures revealed more potent antibacterial activities compared to BBR hydrochloride 34 .
Excessive consumption of high fat/ high sugar diets mainly develops hepatic steatosis, oxidant generation, chronic liver injury, necro-inflammation, and mitochondrial dysfunction [43][44][45] . CCl 4 , a strong hepatotoxin, is metabolized to produce a highly reactive substance known as trichloromethyl radical (CCl 3 * ). This radical can bind with cellular molecules to generate oxidants and cellular damage and disturb mitochondrial homeostasis 46,47 . CCl 3 * can react with oxygen to form highly reactive species that initiate the chain reaction of lipid peroxidation 46,47 . Furthermore, HFHSD feeding plus streptozotocin (STZ) injection also developed a diabetic mice model that described hyperglycemia and hyperinsulinemia and a severe injury in the islets of Langerhans (β-cells) and pancreatic ACs 48 . Furthermore, previous studies reported that exposure to HFD and/ or CCl 4 developed severe degeneration and necrosis in the pancreatic tissues and inhibited pancreatic β-cells survival [49][50][51] . These findings agree with the current results where HFHSD/ CCl 4 stress model induced pancreatic oxidative stress, hyperglycemia, and hyperinsulinemia and demonstrated significant histological alterations in the pancreatic endocrine parts (islets of Langerhans) and the exocrine acinar cells. These findings indicated a reduction in the function and sensitivity of β-cells islet upon HFHSD/CCl 4 stress 52,53 .
The current in vivo studies evaluated the enhanced protective effect of BBR-BSA NPs on HFHSD/ CCl 4 stressed pancreatic tissue. Natural products have an essential role as insulin sensitizers. BBR, as an alkaloid compound, is a potent hypoglycemic modulator, significantly improved the function of pancreatic β-cells, restored glucose homeostasis, and enhanced the peripheral insulin sensitivity 54 of HF fed 55 , HFHSD fed 53 , and STZinduced diabetic rats 2 . In the current study, BBR administration was found to improve the level of the glycemic markers (serum glucose, insulin, as well as HOMA-IR and HOMA-β index) and the level of the pancreatic oxidative stress parameters (TBARS, NO, GSH level, and GSHPx, GST and SOD activities). BBR-BSA NPs administration demonstrated powerful therapeutic antioxidant properties, which substantially improved the pancreatic injury features and restored pancreatic homeostasis compared to the other treatments under control and stress conditions. In addition, BBR-BSA NPs administration has therapeutic efficacy against the induced pancreatic injury and exhibited a significant curative effect on the histopathological alterations of pancreatic areas and the function of pancreatic ACs and β-cells. These findings are compatible with previous studies conducted on BBR 2,53,54,56 .

Conclusion. According to physicochemical characteristics and in vitro and in vivo studies of BBR-BSA NPs,
BBR-BSA NPs delayed the mass degradation rate and increased thermal stability of the entrapped BBR compared to BBR-chloride. Furthermore, BBR-BSA NPs successfully reduced in vitro BBR releasing rates in different dialysis media (SIF and SGF), which decreased its local dissolution rates compared to BBR-chloride. BBR-BSA NPs demonstrated potent free radical scavenging activities, total antioxidant capacities, anti-hemolytic and anticoagulant properties, and antimicrobial potentials compared to BBR-chloride. In addition, BBR-BSA NPs significantly reduced the levels of pancreatic injury and oxidants generation and improved the glucose homeostasis, and insulin sensitivity of the HFHSD/CCl 4 -induced stressed pancreatic rat model compared to BBR-chloride ( Supplementary Fig. S12a-d). www.nature.com/scientificreports/ Co., Ltd. (Egypt). Other chemicals and reagents were an analytical grade. In this study, all solutions and buffers were prepared in ultrapure deionized water. Afterward, the prepared BBR-BSA NPs were crosslinked with a specific volume of 8% glutaraldehyde. The suspended NPs were stirred until all ethanol and glutaraldehyde were evaporated. The resulting NPs pellet was purified using differential centrifugation at 14,000 rpm through 6 cycles (each cycle 20 min) at 4 °C until the supernatant became transparent. The NPs pellet was washed with deionized water to remove the free drug, BSA, and glutaraldehyde molecules from the surface of the NPs. The NPs pellet was also suspended and redispersed to reach the original volume (5 mL) with deionized water as a vehicle using a stick-type ultrasonicator 15 . Specific formulations were stalled as suspended forms, and others were lyophilized at − 48 °C for 24 h, weighed, and stored in the sealed vials at 4 °C. The pH was adjusted with 0.1 M NaOH solution through the NPs preparation step (Supplementary Table S1). Determination of the mean particle diameter, particle size distribution (polydispersity indices), zeta potential, and surface morphology of the synthesized NPs. The hydrodynamic particle size diameter, polydispersity indices (PDI), and zeta potential of the synthesized NPs were measured by Dynamic Light Scattering (DLS)/photon correlation spectroscopy using a particle seizer at a fixed angle of 90° at 25 °C [Zetasizer Ver. 6.20 (Serial Number: MAL1054905), Malvern Instruments Ltd., UK]. Furthermore, field emission-transmission electron microscopy (FE-TEM) photomicrographs were used to evaluate the surface morphological characterization and the size of BBR-BSA NPs and BSA NPs. For analysis, the TEM samples were measured by JSM 1400 PLUS-JEOL. To perform the FE-TEM observations, the suspended NPs were first diluted with deionized water (40 µL of suspended NPs was added to 15 mL of deionized water). Then, a drop of the diluted suspended NPs was placed onto a copper grid coated with carbon film. The excess was drawn off with a dry filter paper. The copper grid was allowed to dry at room temperature before its scanning at various magnifications.

Preparation and physicochemical characterization of BBR-loaded albumin nanoparticles. Preparation of BBR-BSA
Ultraviolet-Visible spectroscopic measurements. The unloaded BBR and BSA contents (mg) were determined by measuring the free BBR and BSA concentrations in the supernatant using an Ultraviolet-Visible (UV-Vis) spectrophotometer. Moreover, the BBR encapsulation efficiency (EE%), its loading capacity (LC%), and the NPs synthesis yield (SY%) were calculated by the following equations: Fourier transforms infrared spectroscopic analysis. Fourier transforms infrared (FTIR) spectroscopy is an absorption spectrophotometer that can identify the different chemical compounds depending on their chemical structures and functional groups 57 . The FTIR samples were prepared by grinding 98.99% KBr with 1% of the samples that were compressed to form a tablet 26 . Pure BBR-chloride, pure BSA, BBR-BSA NPs, and BSA NPs were characterized using an FTIR spectrometer (Bruker Tensor 37 FTIR spectroscopy with ATR method, German).
Thermal stability of the synthesized nanoparticles. The thermodynamic behavior, decomposition rates with increasing temperature, thermal stability, and the variations in the loss rates of the amorphous matter (BBR-BSA NPs and BSA NPs) and its crystalline form (BBR and BSA) were carried out using LINSEIS STA PT 1000 (TG-DTA/DSC). The temperature of this program began from 0 to 700 ℃, with a heating rate of 10 ℃/min. Furthermore, the thermal gravimetric analysis (TGA) profile showed the temperature stability and the variations in the mass change rates of specific compounds (crystalline forms or amorphous states) related to the increased temperature 58 . Differential thermal analysis (DTA) and differential scanning calorimetry (DSC) profiles were also used to investigate the existence of the drug in the NPs form 16,59 . In vitro studies. BBR releasing profile. In this study, the releasing profile of BBR from BBR-BSA NPs (pH and time-dependent stability) was estimated using a dialysis tubing (Nadir®-Dialysierschläuche, 38  www.nature.com/scientificreports/ In 100 mL beaker, the dialysis bag which contained 5 mg of BBR or BBR-BSA NPs was immersed in 50 mL of different dialysis media (SIF or SGF). Aliquots of the dialysis media that contained released BBR were withdrawn at time intervals (0, 0. 5, 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 28, 32, 36, 40, 48 h.). In order to maintain the releasing media, the media was completed to 50 mL after each withdrawn step. The concentration of the released BBR was evaluated by determining the absorbance of BBR at 344 nm using a UV-Vis spectrophotometer. The cumulative BBR releasing rate (%) in the dialysis medium was calculated according to Wu,et al. 15 as follows: Determination of the nitric oxide (NO · ), hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 − ), and hydroxyl radicals (OH − ) scavenging activities. NO · scavenging activities (%) of standard BBR-chloride (0.01-1.8 mg/mL), BBR-BSA NPs (0.01-1.8 mg BBR/mL), BSA (0.5-40 mg BSA/mL) and BSA NPs (0.5-40 mg BSA NPs/mL) in addition to ascorbic acid (0.01-1.8 mg/mL, reference standard) and sodium nitrite (NaNO 2 , 5-232 µM, as positive controls), were determined using the Griess reaction method with minor modifications 58 . Furthermore, hydrogen peroxide (H 2 O 2 ), superoxide anion (O 2 − ), and hydroxyl radicals (OH−) scavenging activities of tested compounds were determined according to standard methods with slight modifications 36,61,62 . The modified procedures used to determine the free radical scavenging activities of these compounds are explained in detail in our supplementary file. The percentage of the scavenged free radicals was estimated as follows: Ac, the absorbance of the negative control against its blank; At, the absorbance of the different diluted samples and the reference standard (positive control) against their blank. The value of the inhibitory concentration 50 (IC 50 ) represented the sample concentration (mg/mL) that inhibited 50% of the oxidant agent (radical) activity.
Determination of the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity. The dark purple DPPH gradually turns to a clear yellow color when it is reduced by an antioxidant agent. These samples' DPPH radical scavenging activities were evaluated using the standard method with minor modifications (Supplementary file) 1,58 . The percentage of the scavenged DPPH was determined as the following: Determination of the thiobarbituric acid reactive substances (TBARS) scavenging activity. TBARS scavenging activities (%) of BBR, BBR-BSA NPs, BSA, BSA NPs, and ascorbic acid were determined according to the standard method 62,63 with some modifications explained in detail in the supplementary file. The TBARS scavenging activities (%) were evaluated as the following: Ac, the absorbance of the negative control; At, the absorbance of the different diluted samples and the reference standard (positive control) against their blank.
Determination of the total antioxidant capacities (TAC). The total antioxidant capacities (TAC) of tested compounds and corresponded NPs besides ascorbic acid (reference drug) were determined using the potassium ferricyanide/ferric chloride reducing power (FRP) and green phosphomolybdenum complex methods with slight modifications (Supplementary file) 58,62 . The effective concentration value 50 (EC50) represented the compound concentrations (mg/mL) that had 50% antioxidant reducing power.
Determination of the anti-hemolytic powerful and the anticoagulant activity. The effect of BBR, BBR-BSA NPs, BSA, BSA NPs, and ascorbic acid on the red blood cells lysis rate (%)was determined with minor modifications mentioned in the supplementary file 24,64 . After the incubation period with the prepared compounds, the released hemoglobin was quantified to measure the red blood cells lysis rate (%), which normalized to the 100% released hemoglobin of the positive control (Triton X-100). The hemolysis (%) was estimated as follows: where A sample was the absorbance of the different diluted tested samples; A C+ was the absorbance of the positive control (100% hemolysis); A C-was the absorbance of the Mcllvaine's buffer (blank) or the diluted erythrocytes suspended form (4% hematocrit) in the Mcllvaine's buffer (negative control). The value of the inhibition concentration (IC 50 ) was introduced as the compound concentrations (mg/mL) that had 50% anti-hemolytic power.
Antimicrobial activity assay. The effect of BBR (0.01-1.8 mg/mL), BBR-BSA NPs (0.01-1.8 mg BBR/mL), BSA (0.5-40 mg BSA/mL) and BSA NPs (0.5-40 mg BSA NPs/mL) on the inhibition growth rate of different pathogenic microbes was detected using Luria-Bertani (LB) agar diffusion well-variant method 67 . Where, Gram-   The experimental work was designed to evaluate the harmful effects of the high-fat high sucrose diet (HFHSD) feeding an addition to carbon tetrachloride (CCl 4 ) injection on the cellular homeostasis and the oxidative stress response of the pancreas. Furthermore, the standard control diet and HFHSD were prepared with minor modifications and fed along the experimental period of 15 weeks 48,68 . Moreover, CCl 4 , a cytotoxin, was also subcutaneously injected with slight modifications as a 50% CCl 4 dosage with olive oil (3 ml/kg, twice weekly during the last three experimental weeks) 36,50,69 .
Animal design. After two weeks of acclimatization, the rats were categorized into nine groups (8 per each): the control (C) group received a standard control diet plus saline via intra-gastric gavage during the last four experimental weeks; the C-BBR, C-BBR-BSA NPs, and C-BSA NPs groups were orally received standard control diet plus BBR (150 mg BBR/kg/day, 6 days per week) 55 , BBR-BSA NPs (15 mg BBR/kg/day, 6 days per week), and BSA NPs (150 mg BSA NPs/kg/day, 6 days per week), respectively via intra-gastric gavage during the last four experimental weeks. The induced group received HFHSD feeding plus CCl 4 injection. The T-BBR, T-BBR-BSA NPs, T-BSA NPs, and T-Ator groups were received HFHSD feeding plus CCl 4 injection combined with the oral administration of BBR (150 mg BBR/kg/day, 6 days per week), BBR-BSA NPs (15 mg BBR/kg/day, 6 days per week), BSA NPs (150 mg BSA NPs/kg/day, 6 days per week), and Atorvastatin (10 mg Ator/kg/day, 6 days per week) 70 , respectively via intra-gastric gavage during the last four experimental weeks. Atorvastatin (Ator), a cholesterol-lowering agent, was used to upregulate the sensitivity and function of pancreatic β-cells of hyperglycemic and dyslipidemic animal models 71 .
At the end of the experiment, the rats fasted overnight and were sacrificed by decapitation under anesthesia (isofuran). Blood samples were collected by heart puncture and allowed for clotting at room temperature for 30 min. The serum samples were centrifuged at 4,000 rpm for 10 min at 4 °C and stored at −20 °C until the biochemical assays were done. The pancreas tissue was immediately removed and washed in ice-cold saline. For biochemical analysis, one gram of pancreas tissue was homogenized (10%, w/v, 1:9) in ice-cold 10 mM sodium-potassium phosphate buffer (pH 7.4) containing 1.15% potassium chloride (KCl). The tissue homogenate was centrifuged at 10,000 rpm for 20 min at 4 °C. The resulted supernatant was stored at -80 °C to evaluate the oxidative stress biomarkers and antioxidant modulators' levels and activities. The pancreas tissue was fixed overnight in 10% neutral buffered formalin for histopathological investigations.

Routine biochemical measurements.
The level of fasting serum glucose was determined by the colorimetric method using a VITRO SCIENT kit, Egypt, according to the kit instructions. Serum insulin concentration was measured by the quantitative sandwich enzyme immunoassay technique using an insulin quantitative immunoassay ELISA kit, USA, according to the manufacturer's instructions. The homeostatic model assessment of insulin resistance (HOMA-IR) index was calculated as the following: The homeostatic model assessment of the β-cell function (HOMA-β) index was evaluated as the following 52 : The levels of pancreatic NO (μmol/mg protein), TBARS (nmol/mg protein), and reduced GSH (μmol/mg protein) were evaluated using methods that were determined previously by certain studies [72][73][74] . While the activities of pancreatic GSHPx (EC 1.11.1.9), GST (EC 2.5.1.18), and SOD (EC 1.15.1.1,) enzymes in IU/mg protein were estimated using methods that reported previously [75][76][77] .
Histopathological examinations. Pancreatic specimens were fixed in 10% neutral buffered formalin for histopathological analysis. After fixation, these tissues were processed and embedded in paraffin blocks. The solid sections were cut into a 5-6 µm thick, stained with hematoxylin and eosin (H&E) stain for histological evaluations. The pancreatic sections were examined under light microscopy (Olympus, Tokyo, Japan), and their photomicrographs were taken (X200 magnifications).