Tailoring of an anti-diabetic drug empagliflozin onto zinc oxide nanoparticles: characterization and in vitro evaluation of anti-hyperglycemic potential

Diabetes is a serious health issue that can be a great risk factor related to numerous physical problems. A class of drugs “Gliflozin” especially Sodium Glucose Co. Transporter 2 was inhibited by a novel drug, which is known as “empagliflozin”. While ZnO nanoparticles (NPs) had considerable promise for combating diabetes, it was employed in the treatment and management of type-2 diabetes mellitus. The new drug empagliflozin was initially incorporated into Zinc Oxide NPs in this study using the surface physio-sorption technique, and the degree of drug adsorption was assessed using the HPLC method. The tailored product was characterized by using the FTIR, EDX, Ultraviolet–Visible, XRD and SEM techniques. With an average particle size of 17 nm, SEM revealed mono-dispersion of NPs and sphere-like form. The Freundlich isotherm model best fits and explains the data for the physio-sorption investigation, which examined adsorption capabilities using adsorption isotherms. The enzymes α-amylase and α-glucosidase, which are involved in the human metabolism of carbohydrates, were used in the in-vitro anti-diabetic assays. It was discovered that the composite showed the highest levels of 81.72 and 92.77% inhibition of -α-amylase and -glucosidase at an absolute concentration of 1000 μg per ml with IC50 values of 30.6 μg per ml and 72 μg per ml.


Synthesis of ZnO NPs
One gram of zinc-sulfate heptahydrate ZnSO 4 ⋅7H 2 O was dissolved in 30 ml of bi-distilled de-ionized water, before being magnetically stirred to complete the process.Sodium carbonate solution, NaHCO 3 (2 M) was added drop by drop until pH 7 approaches followed by continuous stirring.The final product underwent additional agitation for 1 h, followed by centrifugation and washing.After that, the sample was dried at 120 °C for about 12 h.The result was then ground up such that it could pass through meshes before being put into a white crucible for calcination at 600 °C for 120 min at a particular rate of 10 °C per minute 30 .The ZnO NPs powder was obtained and used for further characterization as shown below in Fig. 1.Table 1 represents the different variables for preparation.

Adsorption of empagliflozin by ZnO NPs
The drug was absorbed by the physio-sorption batch adsorption method.Empagliflozin was dissolved in absolute alcohol to make an exact starting concentration of 1.00 mg per ml which is considered as the stock solution.The concentration of the drug which indicates the maximum elimination percentage was then determined by doing serial dilutions of empagliflozin with various concentrations (0.1, 0.2, 0.3, 0.4, and 0.5 mg per ml) by using the stock solution.The whole procedure was conducted by using the solution taken in an Erlenmeyer conical flask of 100 ml at room temperature (25 °C).Then the final solution was stirred for 5 h at a fixed stirring rate of 600 RPM using 100 mg of ZnO NPs.The solution was then filtered using a vacuum pump and 0.45 m nylon filter paper after being centrifuged at 10,000 rpm.The previously described HPLC technique was used to assess the concentration of unbound empagliflozin as illustrated in Fig. 2 31 .
To evaluate the resilience of the technique repeatability following the standards for quality control and method validation, the adsorption performance for each experiment was repeated three times (as standard without ZnO NPs and evaluated with ZnO NPs.Three experimental replicates were used to get the data, which were then  averaged.The drug removal percentage denoted by P (%), is the amount that was loaded and regarded as the "adsorption capacity" at equilibrium, denoted by the qe (mg/g), and was calculated using the following formulas.

Characterization of ZnO NPs
For the comprehensive identification of the compounds, ultraviolet (UV) spectroscopy is carried out using a UV instrument by Shimadzu UV-1800 Japan.UV and visible spectroscopy followed the principle of "Beer's-Lambert Law".To identify phases, quantify powders and thin films while providing details on crystallinity, crystalline size, and orientation.Using an X-ray diffraction spectrometer (Maker Malvern Panalytical) worked at 40 kV at normal room temperature.X-ray diffraction spectra at the angle of 2θ ranging from 10° to 80° were obtained at normal temperature and 40 mA with Cu Kα radiation (1.54 Å) 32 .Morphology, topography, and chemical composition related to phase elucidation were studied with scanning electron microscopy at length scales ranging from nanometers to millimeters.The Nova Nano SEM-450 "Field-Emission-Scanning-Electron-Microscope (FE-SEM)" was used to carry out SEM with EDX analysis.At a very low voltage (1 kV) with a great minimum resolution of 1.4 nm within the great vacuum environment through the "TLD" Detector which was only operated within a very low vacuum.The FTIR spectroscopy technique is used for the analysis of the structure of compounds.FTIR analysis is performed for the spectroscopic study of the substances by focusing on the different kinds of bonds and as well as the functional groups within the substance by placing a tablet of the KBR having the material of interest on the front of the IR radiation 33 .By using an FTIR instrument IR-Prestige 21 makes Shimadzu and a spectrum within the range of 400 cm −1 to 4000 cm −1 is recorded covering the functional and fingerprint regions in which the presence of functional group with the modes of vibrations are studied and used for the basic identification of the whole molecule 34 .

In-vitro antidiabetic activity
Inhibition of α-amylase.For the preparation of the stock solution, a 10 ml volumetric flask was taken and 10 mg of the sample was added followed by the addition of 2 ml of alcohol to dissolve in it then make up the volume to mark with alcohol.The 1 mg/ml solution is equal to 1000 µg/ml which is considered as stock solution and then serial dilution of 750, 500, 250, 100, and 50.0 µg per ml of the solutions were prepared from the already prepared stock solution.To prepare the buffer solution having pH 6.9, 0.36 g of disodium hydrogen phosphate, 0.24 g of sodium dihydrogen phosphate, and 0.04 g of sodium chloride were poured into the volumetric flask and filled with 100 ml of distilled water and dissolved it.To prepare the 2 unit α-amylase solution 0.004 g of α-amylase was dissolved into 100 ml of buffer solution and after complete dissolution kept in the refrigerator.
To prepare the 0.5% of starch solution 0.05 g of water-soluble starch was taken in 10 ml of hot boiling water and gently shaken to dissolve.To prepare the 3, 5-Dinitrosalicyclic acid (DNS) solution 0.1 g of DNS acid was taken and 0.5 N sodium hydroxide was added to it and heated to dissolve.3 g of sodium potassium tartrate was added and dissolved by sonication and made up the volume to 10 ml with 0.5 N NaOH solution.The potential of empagliflozin loaded on ZnO NPs to inhibit the α-amylase enzyme was examined [35][36][37] .
Experimental design.50 μl of the above-mentioned sample at various concentrations (50.0, 100, 250, 500, 750, and 1000 μg/ml) were dropped to a 50 μl of an α-amylase solution containing (2 Units), and after adding the  1 mM phosphate buffer having (pH 6.9) the volume was increased to 250 μl.The sample solution was placed in an incubator at 37 °C for about 15 min.A starch solution with a concentration of (0.5%) was then added to the above mixture in a volume of 50 μl.It was again placed in an incubator for 15 min at 37 °C.Then a 500 μl DNS solution with the 0.1 M concentration was added to it.The synthesized product was then placed in a furnace at 100 °C in an oil-containing bath for 5 min before being cooled to room temperature.Then, at 540 nm, a sample mixture that had been diluted five times was found.In this study, Dapagliflozin served as a positive control.The catalyst was not used in the preparation of the control.Then proceed as mentioned in the sample solution as above in Fig. 3 38 .
Inhibition of α-glucosidase.To prepare the 2-unit α-Glucosidase solution 0.004 g of α-glucosidase was poured into 100 ml of cool distilled water and dissolved and kept in the refrigerator.To prepare the 5 mM solution of nitrophenyl-β-d-glucopyranoside 0.015 g of nitrophenyl-β-d-glucopyranoside was added in cool distilled water and dissolved and kept on the refrigerator.To prepare the 0.1 M sodium carbonate solution 0.10 g of sodiumcarbonate was taken in 10 ml of double distilled water and dissolved it.To prepare the Phosphate buffered saline solution, a described amount of Sodium chloride (NaCl), Na 2 HPO 4 , (NaH 2 PO 4 , KH 2 PO 4 and KCl were taken and added to distilled water and dissolved 39,40 . Experimental design.The ZnO NPs' ability to inhibit α-Glucosidase was assessed.For each sample, 25 µl of α-Glucosidase having (1 unit) was added to 50 µl of each sample at serial concentrations of 50, 100, 250, 500, 750, and 1000 g per ml followed by the addition of 1.0 mM phosphate buffer having (pH 6.9) and the volume was increased to 200 µl.After 15 min at 37 °C, the mixture was added to 50 µl of nitrophenyl-d-glucopyranoside (5 mM).The reaction mixture with the addition of substrate was placed in an incubator at the optimum temperature of 37 °C for about 15 min (Fig. 4).After the completion of the reaction, a 100 µl solution of Na 2 CO 3 with a concentration of 0.1 M was dropped down into it to discontinue the reaction.The control used phosphate buffer saline having pH 6.9.Dapagliflozin was considered to work as a positive control.The resultant mixture was then analyzed using a UV-Vis spectrophotometer at 405 nm.IC 50 calculation.IC 50 is the value of concentration at which 50% of the inhibitory activity has been completed.It is illustrated as the percentage of inhibition is drawn graphically with a concentration on the x-axis (Figs. 5, 6).When the Trend line was selected as an addition, a slope equation that could be used to determine the IC 50 value appeared.After finding IC 50 values for both the Standard and Sample, the concentration that prevented protein denaturation was noted (Fig. 6).The Formula used for the calculation of IC 50 is The IC 50 value for Glucosidase can be calculated by using the above formula

UV-Visible analysis
UV-Vis absorption spectra (Fig. 7) showed a maximum peak at 365 nm, which has confirmed that the material's inherent band-gap absorption is generated when a transition is made by the outermost valance band electron to the conduction band (O 2 p → Zn 3d ).The band gap was calculated by using the equation, where 'λg' is the wavelength of absorption and results to be 3.40 eV for ZnO NPs 41 .

FTIR analysis
Using the FT-IR Spectroscopy technique, the exact chemical environment, bonding between the molecules, and cleanliness of the composite under study were verified.Figure 8 displays the given spectrum of the FT-IR transmittance, the resultant spectrums were of ZnO NPs, empagliflozin, and empagliflozin-ZnO-NPs in the 400-4000 cm −1 region at retention time.It was observed that the tested ZnO and Empagliflozin-ZnO NPs exhibited the wurtzite crystal structure of Zn-O NP's transmittance at 430 and 445 cm −1 confirming the stretching modes of it.Some stretching modes are shown below the 500 cm −1 implying that ZnO-NPs were successfully produced 42 .The band with a core at 877-880 cm −1 is frequently discovered when the samples for FT-IR are Eg = 1240/ g,  studied without vacuum confirming the presence of an O-C-O bond which was the bending transmittance of carbonates 43 .The considerable transmittance bands at 3415-3415 cm −1 and 1625-1664 cm −1 were connected to the (O-H) stretching mode and (O-H-O) bending vibration by the water molecules that were absorbed on the surface, respectively 44 .The spectra obtained directly from the solid standard might be used to identify various empagliflozin distinguishing bands, as shown in Fig. 8.The FTIR spectra here is the research simply presenting the attachment of the drug with the nanoparticles by the physisorption mode not disturbing the actual chemistry of the concerned molecules.The primary bands detected were those connected to the stretching of axial bonds and methyl group's C-H bending, which were located in 2966-2985 cm −1 and 1367-1374 cm −1 ranges, correspondingly.Another prominent band in the spectrum includes those observed in the 1130-1225 cm −1 range, which appeared owing to absorption associated with stretching of C-O axial bonds.A very broad band between 1710 and 1722 cm −1 is due to the C=O group in the lactone 45 .

XRD analysis
The composite's crystalline phase makeup of the fabricated Zn-O composite sample was evaluated using XRD analysis.Empagliflozin loaded ZnO NPs XRD patterns are displayed in Fig. 9 and XRD data is presented in Table 2. Strong and precise diffraction peaks were visible in the sample, which confirmed the extent of the degree of crystallization of Zn-O NPs.When ZnO is crystallized, its hexagonal (wurtzite) phase, no other crystalline phases associated with impurities could be found, demonstrating the formation of wurtzite crystal structure purity of the synthesized ZnO NPs.Additionally, the empagliflozin Loaded ZnO NPs sample's maximum peak intensity was at 2θ = 35.105,It was coupled to the wurtzite ZnO phase via 101 Miller indices.
The results were calculated by using Debye-Scherer formula, so, the ZnO sample's exact average crystallite size denoted by (Dv) was measured.
FWHM value which was equal to the full width at half maximum expressed in radians (120 planes), diffraction angle denoted by the "ϴ" and λ was the wavelength of 1.5418 Å of the X-ray source.The crystallite size was found to be 12.35 nm.Dv = 0.94 (FWHM) cos θ .

SEM analysis
SEM investigation revealed that ZnO NPs were mono-dispersed and had a sphere-like form.The huge aggregation of nearly spherical forms with various sizes of a particle of the synthesized materials was visible in typical SEM micrographs of ZnO NPs and empagliflozin-ZnO samples, as illustrated in Fig. 10a.The adsorption of empagliflozin on ZnO NPs has no impact on the morphology of the particles.Additionally, Fig. 10b displays the matching diameter distribution histogram of the empagliflozin-ZnO and ZnO NPs samples, and it was discovered that the particle size was 17 nm.The SEM analysis confirmed the exact shape and size of the NPs as there was no change in the morphology of the particles.The size of the particles matters a lot as in the literature the NPs having a size less the 30 nm have better permeability as they can move into and out of the blood membranes but the NPs having greater size cannot move in or out of the membranes due to their large size (Fig. 10c).The NPs with an ideal size of 17 nm can possess the better permeability of the drug due to the large surface area with maximum adsorption and attachment sites 46 .

EDX analysis
The chemical makeup of the Zn-O NPs sample was examined using EDX spectroscopy to assess the sample's elemental and compositional stoichiometry properties (EDXS).Figure 11 depicts the EDAX spectrum of pure and mixed ZnO nanostructure samples.The produced powder samples show great purity, which was validated by the measured EDAX spectra, which showed that it was made entirely of zinc (Zn) and oxygen (O) in the absence of impurities.Langmuir Ce/qe = (1/q L K L ) + (1/q L )Ce R L = 1/(1 + K L Cmax) q L : "monolayer adsorption capacity" ZnONPs (mg/g) K L : "Langmuir energy of adsorption constant" (l/mg) R L : sensitive equilibrium parameter or separation factor Cmax: "Highest initial drug concentration" in the solution (mg/l) www.nature.com/scientificreports/outcomes showed that ZnO NP with 1000 g/ml of empagliflozin loaded on it exhibits a considerable increase in α-Glucosidase inhibitory action compared to the other samples.Significantly it was observed that the empagliflozin-loaded ZnO NPs reduce the activity that inhibits α-Glucosidase (Viladgliptin).Empagliflozin-loaded ZnO NPs were observed in 50 μg/ml samples and showed reduced enzyme inhibitory activity.Further, it was discovered that Dapagliflozin has an IC 50 of 72.44 μg/ml for 1000 μg/ml against the α-Glucosidase.Among the remaining fractions, the empagliflozin-loaded ZnO NPs displayed the least IC 50 concentration for α-Glucosidase and were significantly associated with Dapagliflozin (p < 0.05).According to the study, ZnO NPs loaded with empagliflozin inhibit α-Glucosidase as well as the α-amylase having IC 50 concentrations of 5 μg per ml and 2.1 μg/ml, respectively [48][49][50] .Aside from that, earlier studies confirmed that dapagliflozin significantly inhibits the enzymes α-amylase and α-α-Glucosidase, having IC 50 concentrations of 58.3 ± 0.8 µg per ml and 64.9 ± 1.1 µg per ml, respectively.

Conclusion
The drug was then adsorbed using a batch adsorption approach with serial dilution after the ZnO NPs were made using the precipitation process.The study revealed that in a novel drug when tailored with ZnO NPs the efficiency of the drug was increased as the surface area and attachment level of the drug increased, and maximum adsorption was observed at the 100 mg/l solution concentration.The morphology, topology, particle size, and grain size distribution, as well as the elemental and identification of the ZnO NPs, were studied by SEM, XRD, FTIR, EDX and UV-Visible analysis, confirming the fabrication of ZnO NPs.A clear absorption maximum at 365 nm was observed in UV analysis and tailoring of zinc oxide was confirmed by the FTIR analysis as the clear transmittance at wavenumber 430 to 445 cm −1 is detected which showed the stretching type of vibrations.ZnO wurtzite crystal structure was depicted in the synthesized composite confirming the generation of ZnO NPs-empagliflozin composite with an average crystallite size of 17 nm.The SEM analysis and XRD analysis confirmed the wurtzite crystal structure formation with an average particle size distribution of 12.35 nm.Adsorption isotherms were studied and observed that the Frendulich isotherm was more suitable for the adsorption isotherms model.By the statistical analysis, it was observed that at the 100 mg/l concentration, 59.76% of the drug adsorption by the ZnO NPs displayed the maximum adsorption capacity of 360 mg/g of the drug.An in-vitro anti-diabetic assay with the two metabolic enzymes was performed at different concentrations and it was concluded that the tailored drug with the ZnO NPs showed great and enhanced anti-diabetic activity as the product induced 81.72% of α-amylase inhibition and 92.77% of α-Glucosidase inhibition showing the maximum and improved anti-diabetic activity.A dose of 1000 µg/ml of the tailored empagliflozin with ZnO NPs was sufficient to control diabetes in the patients suffering from hyperglycemia.Further studies will be required and in progress as analytical testing, stability studies, and the clinical efficiency of Tailored empagliflozin-ZnO NPs in the treatment of chronic diabetes. https://doi.org/10.1038/s41598-024-52523-4
Percentage of drug in mg/l conc.= Areaof drug after loading Conc of drugs without loading × Area of drug without loading Conc of drug after loading × Percentage of drugs after loading, Drug Removed = 100 − Percentage of Drug adsorbed %, Amount of drug adsorbed qe mg/g = Initial concentrations of drug − Equilibrium concentration of drug × The volume of the drug/Mass of ZnO NPs = Amount of Drug.

Figure 2 .
Figure 2. Schematic illustration for the adsorption of empagliflozin by ZnO NPs.

Figure 10 .
Figure 10.(a) SEM Analysis of Empagliflozin Loaded ZnO NPs and (b) SEM Analysis of Empagliflozin Loaded ZnO NPs at 200 nm.(c) Histogram of SEM Analysis of Empagliflozin Loaded ZnO NPs.

Table 1 .
Different variables for preparation.