INTRODUCTION:

Nowadays the preferable choice for the medication delivery is nanoparticles¹. Improved drug loading and delivery^2,3, tailored drug delivery, giving pharmaceuticals less adverse effects than standard dosage forms ^4-6, and delivering poorly soluble drugs^7-9 are some of the advantages of nanoparticulate drug delivery systems. Aquasomes are names after the latin words "Aqua" which means water and "Some" which refers to nano structure that resembles a cells. Aquasomes are 3- layered vesicular nano carriers used to specially delivery the drugs to the site of target which has been used in various diseases such as cancer, cardiovascular diseases and infections ^10,11. Aquasomes is also called "water bodies" for its water similar properties in order to protect and preserve fragile biological molecule. In general, the aquasomes consists of ceramic core which coated with polyhydroxy compound to which the drug molecule will be loaded ¹².

Elevated glucose (or blood sugar) levels give rise to the hallmark of diabetes, a chronic metabolic disease that could have devastating impacts on the blood vessels, heart, eyes, nerves, and kidneys. Due to insulin resistance or an inadequate amount of insulin synthesis, the most common form of diabetes, type 2 diabetes, often affects adults (90-95% of all instances). In countries of all economic level in the last three decades, Type 2 diabetes has become much more prevalent ¹³.

Type 2 diabetes medications include oral medications such as Metformin (often first-line), DPP-4 inhibitors, SGLT2 inhibitors, and sulfonylureas, as well as injectable ones such as GLP-1 agonists, dual GLP-1/GIP agonists, and insulin to lower blood sugar levels via improving insulin sensitivity, decreasing glucose production by the liver, or boosting the release of insulin, according to WebMD, and sometimes combinations are chosen for better control, in the form of pills, injections, or pumps ¹⁴.

For the present study Glibenclamide which was also called as glyburide was chosen. It is an oral antidiabetic drug that is highly used in the treatment of type 2 diabetes mellitus. It is part of the sulfonylurea family of medication and it operates by encouraging the pancreas to produce more insulin. Glibenclamide is almost insoluble in water and ether. It forms water-soluble salts with alkalis hydroxides [15]. The direct determination of its pKa value in water is impossible because of its poor solubility. Also due to, the plasma levels of Glibenclamide is generally low. In order to overcome this problem with appropriate solution here in we have proposed for formulation of aquasomes filled with this drug without any complicated and expensive methods.

MATERIAL AND METHODS:

The API, Glibenclamide was procured from Orchid Health Care Pvt.Ltd, Chennai Tamil Nadu. Calcium phosphate, poly hydroxy oligomers sucrose, trehalose, cellobiose, lactose was bought from authorized local dealers. All the chemicals and reagents used were of analytical grade. The instruments used also had been well calibrated prior to use.

Pre-Formulation Studies:

Pre formulation studies Included characterisation of physical properties, determination of the melting points and solubility. It also involved determination of lambda maximum by the standard calibration curve method.

Aquasomes Formulation:

Glibenclamide aquasomes were prepared in three steps.

· Preparation of ceramic core.

· Carbohydrate coating on the ceramic core.

· Adsorption of drug on the coated ceramic.

Preparation of ceramic core:

The cores were prepared by disodium hydrogen phosphate with calcium chloride gives the colloidal precipitate without much modification. Based on the reaction stoichiometry, equivalent moles were reacted in a reaction volume of 120ml. specifically, disodium hydrogen phosphate (1 mole 8.90g) and calcium chloride (1mole = 7.35g) were taken in 60ml of water as well separately and mixed. A bath sonicator was used for sonication of the mixture for 2 hours at room temperature. After sonication, it was centrifuged with centrifuge at the room temperature and at 6000 rpm for 1 hr. After centrifugation, decanted supernatant, precipitate was washed thrice with double distilled water. The core was dried at 40degC, 24 h to get dried ceramic nano-particles. The chemical reaction involved is as follows,

2Na2HPO4 + 3CaCl2+H2O→ Са3(PO4)2 +4NaCl + 2H2 + Cl2 + (O)

Carbohydrate coating on the ceramic core:

Carbonhydrate coater on the core were done by adsorption method using sonication, the prepared cermeical core was coated with polyhydroxy oligopers like sucrose/trehahlose/ lactose /cellobiose /with 1:1, 1:1.5 or. 1:2 ratios. About 150, 225 or 300 mg of polyhydroxy oligomers were weighed and dissolved in 100ml of distilled water. In separate beaker, 150mg of ceramic core was taken and 100ml of sugar solution was added in it. (1:1, 1:1.5 or 1:2, core: sugar coat) and sonicated for 40min using sonicator. This suspension was shaken/mixed with magnetic stirrer for 30 min at room temperature and 800 rpm. Here after mixing acetone (non-solvent, 1 ml) was added to the suspension and kept aside for some time. Then, the solution was centrifuged 2000 rpm for 15 min. The supernatant was decanted off and the sugar-coated core was washed thoroughly with double distilled water and then dried for 24 h at 40^oC in a hot air drier to obtain dried carbohydrate or sugar-coated core.

Loading of drug on the coated ceramic core:

A precisely weighed quantity of sugar-coated core was placed in volumetric flasks with a 0.03% w/v glibenclamide solution (acetate buffer solution, pH 4.5). For one hour at room temperature, the flasks were stoppered and forcefully agitated at 800 rpm using a magnetic stirrer. After the medication was adsorbed on the sugar-coated ceramic core, the ceramic nanoparticles and aquasomes were decanted and dried for two hours at 700 degrees Celsius. An example of the current innovation is the Glibenclamide aquasomes, which are composed of a medication, sugar, and ceramic core in weight proportions of 30 mg, 150 mg, and 150–300 mg (the weight ratio is 1:5:5–10 by weight).

Table 1: Formulation design of Glibenclamide aquasome

Characterization of Glibenclamide aquasomes

Drug Entrapment efficiency and drug loading

The percentage of real drug entrapped on the carrier compared to the original amount of drug loaded is known as entrapment efficiency. One may determine the trapping efficiency % by

% 𝐷𝑟𝑢𝑔 𝑒𝑛𝑡𝑟𝑎𝑝𝑚𝑒𝑛𝑡 𝑒𝑓𝑓𝑖𝑐𝑒𝑛𝑐𝑦 = (𝐴𝑐𝑡𝑢𝑎𝑙 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑/ 𝑡ℎ𝑒𝑜𝑟𝑒𝑡𝑖𝑐𝑎𝑙 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑) × 100

The complete medication was encapsulated in a ceramic core covered with carbohydrates for the purpose of theoretical drug loading. A precisely weighed 30 mg of aquasomes were dissolved in 50 ml of pH 4.5 acetate buffer for practical drug loading, and the mixture was centrifuged for 30 minutes at 6000 rpm. After filtering the mixture to produce a clear supernatant, a UV spectrophotometer was used to measure the absorbance at 300nm.

%𝐷𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑖𝑛𝑔 = [(𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑢𝑔 𝑙𝑜𝑎𝑑𝑒𝑑 − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑢𝑛𝑡𝑟𝑎𝑝𝑒𝑑 𝑑𝑟𝑢𝑔)/ 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑞𝑢𝑎𝑠𝑜𝑚𝑒𝑠] × 100

Particle size and Zeta potential:

The Glibenclamide aquasomes' particle size and size distribution were determined using a nanoparticle analyzer (HORIBA SZ100 Horiba) using instrument software and water as a surfactant at 25°C. The produced aquasomal formulation's polydispersant index (PDI) and mean diameter (z average) were assessed. Zeta potential was used to assess the aquasomal compositions' stability. In order to assess the sample's zeta potential, an appropriate volume of the sample was diluted with water and injected into the electrophoretic cell of the zeta potential analyzer, where a potential of 150mV was established.

Fourier - Transform Infrared (FTIR) Spectroscopy:

Pure Glibenclamide, calcium phosphate ceramic core, poly hydroxy oligomers sucrose, trehalose, cellobiose, lactose, and formulation of carbohydrate coated Glibenclamide aquasomes were all subjected to Fourier transform infrared spectral spectroscopy. The mixture was combined with IR grade potassium bromide in a 1:100 ratio, and the pellets were created by applying 10 metric tons of pressure in a hydraulic press. The pellets were then analyzed using an FTIR spectrometer (Brucker, Germany) across a range of 4000-400 cm-1.

In-Vitro Drug release studies:

Using USP-Type II dissolving equipment, in vitro dissolution tests were conducted for pure Glibenclamide, its chosen aquasomal forms (F-12), and a reference commercial tablet formulation (Gliford 5). This involved using 900 cc of pH 4.5 acetate buffer as the dissolving medium, keeping the temperature at 37°C ± 0.5°C, and rotating the paddle at 50 rpm. A UV-visible spectrophotometer was used to detect absorbance at 300 nm after 5 ml sample aliquots were taken out at various times and refilled with the same amount of new dissolving media to maintain sink conditions. The dissolution tests were carried out three times.

Stability studies:

Stability tests were conducted on formulation F12 from the nine batches of glibenclamide-loaded aquasomes. Three sample sets were created from Formulation F12 and stored at: 4 ± 1^oC, 25± 2^oC and 60% RH ± 5% RH, 37± 2^oC and 65% RH ± 5% RH

Following a month, the drug release of the chosen formulation (F7) was assessed using the previously stated in vitro drug release experiments, % loading efficiency, and SEM for the same formulation.

RESULTS AND DISCUSSIONS:

Pre-Formulation Studies of Glibenclamide:

Physical characterization of Glibenclamide enlisted in table 2.

Table 2: Physical properties of Glibenclamide

Parameters	Observations
Appearance	White powder
Nature	crystalline
Odor	Odorless
Molecular Weight	494.01 g/mol
Stability	Stable under ambient conditions
Melting point	173°C

Pre-formulation investigations revealed that glibenclamide was a white, crystalline powder with a melting point of 173°C, which is within the stated range of 169°C and 176°C ¹⁶.

Solubility:

Glibenclamide's solubility was investigated in a range of solvents. Table 3 presented the findings.

Table 3: Solubility profile of Glibenclamide

S. No.	Solvents/ buffers	Solubility mg/ml
1	Distilled water	0.508±0.52
2	Methanol	0.936±0.32
3	Phosphate buffer pH 6.8	0.85±0.21
4	0.1N HCl	2.851±0.33
5	NaOH	4.100±0.113
6	Dimethylformamide	4.136±0.603

Note: all values were expressed as mean ± SD n=3, SD = standard deviation

The solubility data showed that the solubility in water was less than 1 mg/ml, which is extremely low. According to the results, glibenclamide becomes less soluble as pH rises. It was discovered to be greater in alkali hydroxides, such as dimethylformamide and NaOH.

Glibenclamide λ max:

This was ascertained by utilizing a UV-visible spectrophotometer to scan the produced solution in the 200–400 nm range. 300 nm in a solution of methanol.

Figure 1: UV spectrum of glibenclamide

The methanolic solution of glibenclamide displayed UV absorption maxima at 235, 275, and 300 nm. According to the stated literature study ^{17, 18}, the absorption band at 300 nm was thought to be the most selective in order to prevent the absorbance of other substances that may be added to the absorbance of glibenclamide under inquiry.

Formulation and Characterization of Aquasomes:

Three stages are involved in the production of glibenclamide aquasomes. The main active pharmaceutical ingredient, glibenclamide, is adsorbed onto the sugar-coated inorganic core after it has been coated with poly hydroxy oligomers, sucrose, trehalose, cellobiose, and lactose.

Four kinds of polyhydroxy oligomers—sucrose, trehalose, cellobiose, and lactose—were chosen for the current study's twelve trials in the ratios of 1:1, 1:1.5, and 1:2, respectively, and are listed in table 1. Each trail's outcomes served as the foundation for the next trail's design. To achieve the intended goal, twelve similar paths were completed.

Drug entrapment efficiency, drug loading, particle size, zeta potential, FT-IR spectroscopy, and scanning electron microscopy were used to evaluate the resulting Glibenclamide aquasomes.

Drug entrapment efficiency and % drug loading:

Glibenclamide aquasomes were used to determine these. Table 4 presents a tabulation of the results.

Table 4: Drug entrapment efficiency and drug loading of Glibenclamide aquasomes.

Formulations	% Drug entrapment efficiency	% Drug loading
F-1	79.8±0.03	7.1±0.03
F-2	83.4± 0.04	7.3 ± 0.09
F-3	85.6± 0.06	7.4± 0.12
F-4	82.7± 0.08	7.1 ± 0.05
F-5	85.0 ± 0.01	7.3 ± 0.11
F-6	88.3 ± 0.02	7.2 ± 0.09
F-7	86.7 ±0.03	7.3 ± 0.06
F-8	89.0 ± 0.06	7.2 ± 0.03
F-9	90.2 ± 0.03	7.8 ± 0.12
F-10	89.4 ± 0.01	7.8 ± 0.05
F-11	92.5 ± 0.02	7.8 ± 0.04
F-12	93.0 ± 0.05	7.8 ± 0.03

Note: all values were expressed as mean ± SD n=3, SD = standard deviation

The drug loading capacity and entrapment efficiency percentages, which ranged from 7.1±0.03 to 7.8±0.03 and 79.8±0.03 to 93.0±0.05, respectively, were determined to be adequate for every trail.

The lactose-coated aquasomes of the F-12 trail yielded the greatest entrapment efficiency and % drug loading of all the trails (93.0±0.05 and 7.8±0.03, respectively).

Particle size and zeta potential:

For calcium phosphate ceramic core, four carbohydrates used in formulation design—sucrose, trehalose, cellobiose, and lactose coated ceramic cores—pure Glibenclamide, and the prepared aquasome formulation, particle size, size distribution, polydispersity index (PDI), and zeta potential were measured. Tables 5 through 10 present the tabulated findings.

Table 5: Particle size of sucrose coated Glibenclamide aquasomes

Formulation	Particle Size (Nm)
F-1	381.4
F-2	284.8
F-3	263.0

Table 6: Particle size of trehalose coated Glibenclamide aquasomes

Formulation	Particle Size (Nm)
F-4	426.7
F-5	386.2
F-6	298.2

Table 7: Particle size of cellobiose coated Glibenclamide aquasomes

Formulation	Particle Size (Nm)
F-7	315.5
F-8	297.0
F-9	265.5

Table 8: Particle size of lactose coated Glibenclamide aquasomes

Formulation	Particle Size (Nm)
F-10	297.9
F-11	284.8
F-12	250.4

Table 9: Zeta potential of Glibenclamide aquasomes with different carbohydrates

S.No.	Carbohydrates Used in Aquasome	Zeta Potential (Mv)
1	Sucrose	+16.7
2	Trehalose	-17.6
3	Cellobiose	-17.1
4	Lactose	-31.6

Table 10: Particle size, PDI and zeta potential of ETM

Formulation	Particle size nm	PDI	Zeta potential mV
Ceramic core	113.5	0.328	-24.7
Lactose ceramic core	200.7	0.205	+17.6
Pure Glibenclamide	6171.5	0.699	-8.6
Glibenclamide aquasome (F12)	250.4	0.266	-31.6

Each carbohydrate in the 1:2 ratio had a suitable particle size (nm). From F-1 to F-12, the carbohydrate-coated Glibenclamide aquasome formulations had particle sizes of 381.4 nm, 284.8 nm, 263.0 nm, 426.7 nm, 386.2 nm, 298.2 nm, 315.5 nm, 297.0 nm, 265.5 nm, 297.9 nm, 284.8 nm, and 250.4 nm. Aquasome formulation trails yielded good results (less than 300 nm), with trail F-12 (250.4 nm) being the most satisfactory.

Particle size, PDI, and zeta potential of ceramic core, lactose-coated ceramic core, pure Glibenclamide, and the optimized formulation are listed in Table 10. The ceramic core, lactose-coated core, pure drug, and lactose-coated Glibenclamide aquasome formulation all had particle sizes of 113.5, 200.7,6171.5 and 250.4 nm. The final formulation F-12 had a particle size of 250.5 nm, while the pure drug's particle size was determined to be 6171.5 nm. This demonstrated adequately that the technique used to create the aquasomes led to a decrease in size, which increased the surface area accessible for the polyhydroxy oligomer coating.

The ceramic core has a particle size of 113.5 nm, the lactose-coated core has a particle size of 200.7 nm, and the drug-adsorbed formulation has a particle size of 250.4 nm. This suggests that the size of the particles rises when the core is subsequently coated with drug and carbohydrate.

The polydispersity index (PDI) for ceramic core, lactose coated core, pure glibenclamide, and drug-adsorbed aquasome formulation was determined to be 0.328, 0.205, 0.699, and 0.266, according to table no. 10. The PDI for the drug-adsorbed aquasome formulation, lactose-coated core, and ceramic core was determined to be less than 0.51, indicating a reasonably narrow particle dispersion and homogeneity of the sample's particles. The pure drug's PDI of 0.699, which is more than 0.5, suggests heterogeneity and a wide particle dispersion.

An important technique for examining the surface of nanoparticles and determining their stability is zeta potential. The zeta potential is a potential at the hydrodynamic shear plane that may be ascertained by particle mobility and in the presence of an electric field. The mobility of sufficiently tiny molecules and particles will depend on the electrolyte concentration and surface charge; a strong zeta potential will offer stability, preventing the particles from aggregating. When the potential is low, the particles tend to group together because attractive forces can outweigh this repulsion.

Electrostatic and steric forces, or both when using carbohydrates, stabilize drug particles dispersed in a liquid continuous media. Table 9 lists the zeta potential of the Glibenclamide aquasomal formulations including four polyhydroxy oligomers: sucrose +16.7 mV (± 20 mV to ±30 mV range), trehalose -17.6 mV (±20 mV to ±30 mV range), cellobiose -17.1 mV (± 20 mV to ±30 mV range), and lactose -31.6 mV to ±30 mV to ±40 mV range).

Nanoparticles that have a zeta potential of more than +30 mV or less than -30 mV are generally very stable. Due to interparticle interactions such as van der Waals, hydrophobic interactions, and hydrogen bonding, dispersions having zeta potential values of less than +25 mV or larger than -25 mV will ultimately agglomerate.

The lactose coated Glibenclamide (F-12) aquasome formulation was found to be more stable because the higher the zeta potential value, the more stable the aquasomes. In contrast, the sucrose (F-3), trehalose (F-6), and cellobiose (F-9) coated aquasome formulations were all well within the acceptable range of zeta potential for stability.

The zeta potential values of ceramic core, lactose-coated ceramic core, pure Glibenclamide, and aquasome formulation were determined to be -24.7mV, +17.6, -8.6 and -31.6 mV, which falls within the range of -15 mV to -30 mV for well-stabilized nanoparticles, according to table 10. Both the pure medication and its aquasome formulation had zeta potentials of -8.6 mV and -31.6 mV, respectively, indicating that the lactose-coated Glibenclamide aquasome formulation (F-12) is more stable than the pure Glibenclamide.

FT-IR Spectroscopy Studies:

FTIR analyses were conducted on drug-coated aquasomes, pure glibenclamide, poly hydroxy oligomer lactose, and a ceramic core containing calcium phosphate. The following figures (figures 2 through 9) show the results. The study's FT-IR research results reveal various bond strains at various peaks. FT-IR analysis was used to assess the drug-excipient interaction research.

Figure 2: FT-IR spectra of Pure Glibenclamide

Figure 3: FT-IR spectra of Calcium phosphate ceramic core

Figure 4: FT-IR spectra of Trehalose ceramic core

Figure 5: FT-IR spectra of Trehalose coated Glibenclamide aquasome formulation(F-6)

Figure 6: FT-IR spectra of Cellobiose ceramic core

Figure 7: FT-IR spectra of Cellobiose coated Glibenclamide aquasome formulation (F-9)

Figure 8: FT-IR spectra of A. Lactose coated ceramic core

Figure 9: Lactose coated Glibenclamide aquasome formulation (F-12)

Table 11: FT-IR interpretation of pure drug and optimized formulation (F-12)

Functional groups	Bands range (cm^-1)	Pure Glibenclamide (cm^-1)	Aquasome formulationF-12 (cm^-1)
N-H Stretch	3500-3300	3342.62	3479.64
C=0 Stretch	1870-1540	1638.76	1648.01
C=N Stretch	1630-1690	1672.8	1647.98
C=C Stretch	1450-1600	1502.35	1495.05
C-N Vibration	1000-1400	1010.89	1055.67
C=S Stretch	1050-1200	1170.89	1127.03
C-CL Stretch	850-550	784.18	724.71
C=H Stretch	700-850	802.33	784.18

Table 11 provided an interpretation of the pure glibenclamide and its improved aquasome formulation (F-12). N-H stretch at 3342.62, C=O stretch at 1638.76, and C=N stretch at 1672.8 cm^-1 for pure glibenclamide. N-H bending ranges from 1613.15 to 1502.35 cm^-1. C=C bending between 802.33-842.51 cm^-1, C=S stretch between 1220.75-1032.97 cm^-1, and C-Cl stretch at 724.71.

Lactose's primary absorption bands were the O-H stretch at 3538.73, 3481.60 cm^-1, and 3154.49 cm^-1. C-H stretch at 3275.28 and bend at 1648.38 cm^-1, C-O stretch at 1216.46, 1127.49, and 1056.41 cm^-1, C=C stretch between 984.59 cm^-1, C=C bending between 784-663.15 cm^-1, C-N stretch at 1672.86 cm^-1, C-C stretch at 1648.01 cm^-1, 1495.05, 984.59, and 872.02 cm^-1, and C-S stretch at 1214.52 cm^-1, 1220.75, 1170.89, and 1127.03 cm^-1.

The FT-IR spectra of glibenclamide, lactose, and its aquasome formulation (F-12) were found to be within the interpretation range, suggesting that there had been no change in the chemical integrity of the medication or the excipients in the aquasome formulation.

In-Vitro Drug Release Studies:

All of the aquasome formulations underwent in vitro drug release investigations. At various time periods, cumulative drug release was computed. When compared to the original medicine, all of the formulations considerably increased the drug's release. The release profile is shown in Figure 10 and is listed in Table 12 below. The release data was fitted in a zero-order kinetic model to determine the release mechanism. Using statistical software, it was determined that all of the formulations adhered to the zero-order kinetic model based on the linear regression analysis correlating to a high value of regression coefficient. This suggests that the medicine may be delivered in a controlled release way using established formulations.

Table 12: In- vitro dissolution of Glibenclamide aquasome formulations and pure Glibenclamide with marketed formulations

Time (minutes)	% of Drug release
Time (minutes)	PD	F12	Gliford 5
0	0	0	0
5	3.86±0.12	15.03±0.11	14.78±0.11
10	5.83±0.09	29.98±0.09	28.45±0.09
15	7.45±0.11	43.67±0.1	39.32±0.1
20	7.98±0.08	52.96±0.08	51.07±0.12
30	9.45±0.7	66.84±0.13	59.43±0.13
45	10.54±0.11	74.32±0.02	72.23±0.08
60	12.09±0.09	80.46±0.03	76.59±0.07
90	13.54±0.07	91.02±0.07	88.03±0.05
120	17.25±0.06	98.46±0.05	95.99±0.03

All values were expressed as mean ± SD n=3, SD = standard deviation

Figure 10: Dissolution profiles of pure drug, its aquasome formulation and marketed formulation

The aforementioned results suggest that the created aquasome formulations had good drug release that was almost identical to that of commercial formulations.

Stability Study:

Studies on stability For one month, the formulation F-12 was stored at 4 ± 1^oC, 25± 2^oC and 60% RH ± 5% RH, 37± 2^oC and 65% RH ± 5% RH in order to conduct stability experiments of the manufactured Glibenclamide loaded aquasome.

Two metrics were used: in-vitro release tests and % drug entrapment efficiency. The results of % drug entrapment efficiency and in vitro drug release after one month of storage is shown in Table 13, under.

Parameters	4 ± 1^oC	25± 2^oC and 60% ± 5% RH	37± 2^oC and 65% ± 5% RH
Percent Entrapment Efficiency*	73.98±2.3	73.56±1.6	73.02±1.7
Cumulative % Drug Release*	88.72±2.7	88.78±2.5	88.93±1.6