INTRODUCTION:

The patient prefers the oral route over other administration methods, but there are drawbacks to oral drug administration as well, including hepatic first pass metabolism and enzymatic degradation in the gastrointestinal tract (GIT). In order to deliver the therapeutic dose of medication to the right location in the body and maintain the desired concentration, there has been an increase in interest in the use of transmucosal channels for drug delivery. As a result, different absorptive mucosa are taken into consideration as possible drug administration locations. For a systemic effect, transmucosal drug delivery routes—that is, the mucosal linings of the oral, nasal, rectal, vaginal, and ocular cavities—offer a number of advantages over peroral administration.¹

The mucosal membrane lining the mouth is capable of acting as a location for medication absorption, one of its less well-known uses. medications typically enter the bloodstream through the jugular vein, which richly nourishes the salivary glands and their ducts, allowing the medications to enter the systemic circulation through easy diffusion across the mucous membrane. For most medications, the roles of pinocytosis, active transport, and aqueous pore transit are negligible when it comes to drug delivery across the oral mucosa. There are two locations in the buccal cavity where drugs have been administered. When taking medication sublingually, it is frequently inserted beneath the tongue in the form of a tablet that dissolves quickly. Between the cheek and gingiva is the second anatomic place where drugs are administered; nevertheless, this second application site is referred to as buccal absorption².

Urinary incontinence is treated with darifenacin (Enablex®). The mechanism of action of darifenacin is the blockade of M3 muscarinic acetylcholine receptors, which is principally in charge of bladder muscle contractions. As a result, there is less of an urgency to urinate. Those who have urine retention shouldn't use it. It is unknown if this M3 receptor selectivity has any therapeutic benefits for managing overactive bladder syndrome symptoms. Muscarinic M3 receptors are specifically antagonistic against darifenacin. Saliva production, iris sphincter function, and smooth muscle contraction in the human bladder and gastrointestinal system are all mediated by M3 receptors³. It was chosen as the model drug for the study due to its appropriate characteristics, including its low molecular weight (426.55) and low dosage (7.5mg). Due to first pass metabolism, darifenacin has a very low bioavailability of 15%–19% following oral ingestion. To ensure that a buccal drug delivery system is retained in the mouth cavity for the intended amount of time, it should have strong bioadhesive qualities⁴.

Spray-drying is a quick and easy way to turn liquid particles into solid ones. Because it can run continuously, it is an extremely productive and efficient operation. Techniques for controlling particle shape, powder flow ability, and size distribution are also offered by spray-drying. The size distribution of particles produced by a conventional pharmaceutical spray-dryer ranges from half a micron to a few hundred microns⁵.

The dried fruits of Ablemoschus esculents, a member of the Malvaceae family, were used to make the okra mucilage. The goal of this study was to create darifenacin buccal mucoadhesive microspheres that would extend their residence time in the mouth. This would guarantee the drug released satisfactorily and unidirectionally into the mucosa, preventing drug loss.

MATERIALS AND METHODS:

Darifenacin Hbr was a gift sample from Microlab (Banglore, India). Okra fruits were purchased from local market. Ethyl alcohol, acetone and acetonitrile were purchased from Hi media lab chemicals (Mumbai, India).

Extraction of Okra Mucilage:

Through aqueous cold extraction, the mucilage was extracted. Fresh okra fruits were washed with water, sliced into tiny pieces, and sun-dried for seven days. For two hours, the dried okra was submerged in water. After that, muslin cloth was used to filter it, and methanol, ethanol, and acetone were used to precipitate the mucilage. To produce mucilage on a large scale, the solvent that produced the highest yield of mucilage was chosen. For 7-8 hours, the mucilage was separated and dried in an oven set to 55°C^6,7.

Preparation of buccal mucoadhesive microspheres:

Through the use of spray drying, microspheres were created. Water was used to dissolve darifenacin and okra mucilage in varying ratios of 1:1, 1:2, 1:3, 1:4, and 1:5 (Drug: Polymer W/W %). For 30minutes, a magnetic stirrer running at 100rpm was used to mix this mixture evenly. After that, the solution was spray dried with the following settings: feed rate of 3ml/min, aspirator speed of 45%, intake temperature of 120°C, and exit temperature of 90°C^8,9.

Hygroscopic products were seen in the early experiments, and it stuck to the spray dryer's cyclone separator wall. Therefore, three different additives, such as Avicel 200, mannitol, and dextrin (0.4% each), were applied and the product was evaluated in order to prevent the product from sticking.

Preformulation studies:^10,11

Differential Scanning Calorimetry (DSC):

Using DSC, the drug-polymer interaction was examined. Using a Mettler DSC-7 Differential Scanning Calorimeter, the thermal behavior of the medication, mucilage, and microspheres was examined. 50.00μl aluminum pans were filled with approximately 2.00mg of mucilage, drug, and microsphere samples, which were then sealed. Each sample had a heat run set between 5 and 300°C with nitrogen used as the blanket gas.

FTIR Studies:

By using FT-IR spectroscopy, drug-polymer interactions were also investigated. The spectra of darifenacin Hbr, okra mucilage, and the physical combination of darifenacin and okra mucilage were recorded. KBr disks were used to prepare the samples. The 400–4000 cm-1 scanning range was used.

Evaluation of microspheres:¹²

Scanning electron microscopy:

With the use of a Japanese Hitachi S-4800 Type II scanning electron microscope, the surface morphology of the microparticles was investigated. The samples were coated with a thin layer of gold using a sputter coater Palaron E 5100 to make them electrically conductive after being secured on a brass stub with double-sided tape. Using scanning electron microscopy, the form and surface morphologies of drug-loaded microspheres were examined¹³.

X-Ray Diffraction:

To determine how the crystallinity of the medicine changed when it was mixed with polymer, powder x-ray diffraction studies of the pure drug, polymer, and formulation were conducted. More crystalline material is indicated by sharper diffraction peaks.¹⁴

Partical size analysis:

Partical size analysis was performed for microspheres from each formulation using Motic microscpe model DMWB-223 and average partical size were calculated.¹⁵

Determination of percentage yield:

The yield of microspheres was determined by the formula, % Yield = Total Weight of Microspheres/Total weight of Raw Material × 100

The percentage yield of each formulation was determined.¹⁶

Drug loading and incorporation efficiency.

After the microspheres were weighed, they were dissolved in distilled water and left overnight. Drug content was determined using spectrophotometry at a wavelength of 284nm. The following formulas were used to determine the medication loading and incorporation efficiency (%),¹⁷

Drug loading (%)

= M actual/weight quantity of powder of microspheres × 100

Incorporation efficiency (%) = M actual/M theoretical × 100

Where M actual is the actual drug content in weighed quantity powder of microspheres and M theoretical is the theoretical amount of drug in microspheres calculated from the quantity added in the spray process.

Moisture content:

Moisture content was determined by drying in a vacuum oven at 70⁰ C until constant weight.

The moisture content of the sample was calculated using the following equation.¹⁸

%W = (A - B)/B *100

Where:

%W = Percentage of moisture in the sample,

A = Weight of wet sample (grams), and

B = Weight of dry sample (grams)

In the preliminary trials the microparticles were sticking to the drying chamber and to cyclone separator to avoid this and increase the yield, 0.4% Aerosil 200 was added to every batch.

Zeta-Potential:

Nearly every particle that comes into touch with a liquid picks up an electric charge on its surface. Zeta potential is the name given to the electric potential at the shear plane. The shear plane is an imaginary surface that divides the liquid layer, which is made up of counterions, from the moving solid surface. Zeta potential is a significant and practical measure of particle surface charge that can be utilized to forecast and manage stability. Zeta potential measurement is frequently essential to comprehending the aggregation and dispersion processes that occur in applications. Using a Zeta sizer device, the electrophoretic mobility of particles in an electrical field was measured in order to calculate the zeta potential. Every sample underwent triplicate analysis.^19,20

In vitro diffusion study:

The modified Franz diffusion cell with the dialysis membrane (Mol. Wt. cut off 12,000–14,000) was used for the in vitro drug diffusion analysis of the microspheres.Glass was used to create the diffusion cell. The recipient chamber with a water jacket has a 100.00 ml total capacity. The donor chamber has an interior diameter of 1.13cm and a length of 10.00cm. The donor chamber tube was positioned so that it barely makes contact with the receptor chamber's diffusion medium. Phosphate buffer solution pH 6.8, which was kept at 37±1°C and within the pH range of the buccal cavity, was found in the receptor compartment. Prior to gently scattering the microspheres equivalent to 10.00mg of medication onto the donor side, the membrane was allowed to equilibrate. Samples were taken out of the receptor compartment on a regular basis and refilled with the same volume of new buffer solution. A spectrophotometer was used to measure the samples at 284nm.²¹

Release kinetics and mechanism:

In-vitro diffusion data from formulations were applied to different kinetic models, such as the zero order, first order, Higuchi release, and Korsemeyer-Peppas equation, in order to evaluate the release kinetics. The zero-order plot, which shows the percentage of CDR versus time, represents the release from systems in which the release rate is concentration independent. The first order figure, which shows the percentage remaining vs time, represents the release from systems where the release rate is concentration dependant. The results were fitted using the Korsmeyer-Peppas equation in order to confirm the precise mechanism of drug release from these microspheres. Diffusion-controlled drug release is indicated when n = 0.5, while swelling-controlled drug release is indicated when n = 1.0. Both phenomena can be indicated by values of n between 0.5 and 1.0 (Anomalous transport).¹⁶

Ex-vivo mucoadhesion study:

The laboratory-designed gear was utilized to ascertain the microspheres' mucoadhesive strength. The 1.6 cm² flat section of mucosal tissue, with a thickness of 1.2–2 mm, was taken from the goat buccal mucosa, which was obtained from the slaughterhouse. It was then placed on a cylindrical polyvinyl chloride plastic support, secured with an adhesive glass slide, and it was cleaned for 30 minutes using a peristaltic pump at a rate of 30mL/min with saline solution. After being hydrated with a small amount of distilled water, 20mg of microspheres were spread out across the mucosal tissue and allowed to interact with the mucosal surface for 15 minutes. The study examined the mucoadhesive strength of goat buccal mucosa under physiological settings, including 6.8 pH buffer solutions kept at 37±0.5°C. Microsphere adhesion was consistently noted. After filtering the solution collected in the buffer beaker, a UV spectrophotometer was used to measure the drug concentration at λ max 284nm.²²

Swelling property:

By letting them swell to equilibrium, the microspheres' capacity to swell in physiological media was ascertained. Using a Franz diffusion cell (12.5ml) filled with phosphate buffer (pH 6.8), an exact weight of 10 mg of microspheres were placed on a millipore filter (NY 11 0.22μm) and left for 3.5minutes.²²

The following formula was used for calculation of degree of swelling.

a = (Ws – Wo)/Ws

Where,

α = Degree of swelling

Wo = Initial weight of microspheres and

Ws = Weight of microspheres after swelling.

Stability studies:

The buccal microspheres produced the most desirable results from stability experiments. The chosen mixture was put into a glass bottle and kept for three months at 40±2ºC and 75±5% relative humidity. Physical properties of the microspheres, such as particle size and Mucoadhesion percentage, were assessed.⁸

RESULTS AND DISSCUSSION:

Okra mucilage:

To extract mucilage, a variety of solvents were tried, including ethanol, methanol, and acetone. Of these, ethanol precipitated a larger amount of mucilage than the other solvents. A maximum mucilage production of 18% was attained.

DSC studies:

Thermogramic DSC The endothermic peak near 232⁰C in Figure 1 of the pure drug powder indicates the melting temperature. The presence of okra mucilage is indicated by the enlarged endothermic peaks closer to 143⁰C. When compared to the thermogram of the pure medication, the DSC thermogram revealed little variation in the onset and peak temperatures. Therefore, there was no evidence of a drug-polymer interaction.

Figure 1: DSC thermo gram of A) pure Darifenacin drug and B) drug, polymer combination

FTIR Studies:

FTIR spectral analysis was used to assess physical mixes of drugs and polymers for any changes to the drug's properties, both chemical and physical. Given their chemical compatibility, Figure 2 indicates that there was no interaction in the principal peaks of the okra seed mucilage and darifenacin.

Figure 2: FT-IR spectra of A) Okra polymer B) Darifenacin, and C) physical mixture of drug and polymer

Scanning Electron Microscopy:

Using scanning electron microscopy, the surface morphology of microspheres for the optimal formulation (F5) was examined. Figure 3 displays the external morphology and surface texture of the microspheres. The exterior morphology demonstrated the production of spherical particles, and the microspheres' surface texture was adequately porous.

Figure 3: SEM images of microspheres formulation

XRD:

The drug-loaded microsphere's lack of crystalline darifenacin peaks The drug's molecular dispersion in the polymer and its transformation from its crystalline form to its amorphous form were both verified by Figure 4A. Figure 4B shows that the typical crystalline form of the medication was evident at diffraction angles 9, 12, 17, 18, 19, 20, and 22 on the 2θ scale, where the characteristic sharp peaks were observed. These outcomes also line up with the DSC analysis's findings. The overlay in the XRD spectrum of the chosen formulation, the polymer, and the pure medication is shown in Figure 4.

Figure 4: Comparison of diffractograms of A) Formulation, B) Drug, C) Polymer.

Particle size analysis:

The microspheres exhibited spherical, discrete, and homogeneous characteristics; nevertheless, their particle size range expanded and changed in proportion to the concentration of mucilage. Table 1 illustrates the microspheres particle size range, which was 8.8 µm to 16.6µm.

Percentage yield:

The experiments were carried out and the results of % yield of mucoadhesive microspheres were 6% to maximum of 10.8%. The maximum yield was obtained with formulation F5.

Drug loading and Incorporation efficiency:

Incorporation efficiency was high since it always exceeded 90%. An increasing the ratio of drug to polymer, the drug loading of microspheres was increased (showed Table 1)

Moisture content:

The moisture content of the prepared microspheres was analyzed. The moisture content of the F5 was found to be 0.5%. Aerosil 200 added, avoided stickiness of particles to cyclone separator and collector of the spray dryer.

Ex-vivo Mucoadhesion Study:

Every formulation was the subject of an ex vivo mucoadhesion research. Mucoadhesion increased along with the increase in mucilage content. Mucilage's maximal level of mucoadhesion was found to be between 87.5% and 94.4. The outcomes are displayed in Table 2.

Table 2: % mucoadhesion and swelling properties of all formulation of microspheres (F1-F5).

Fmulation Code	*Percentage Mucoadhesion (% ± SD)**	*Percentage of Swelling(% ±SD)**
F1	87.5 ± 1.55	0.5133 ± 1.25
F2	88.2 ± 2.50	0.6153 ± 1.53
F3	90.3 ± 1.10	0.6987 ± 0.85
F4	91.7 ± 1.50	0.7572 ± 1.32
F5	94.4 ± 2.25	0.8722 ± 1.52

* Values expressed as Mean ± SD, n=3

Swelling property :

Okra mucilage was the only ingredient in the spray-dried system with the ability to swell, hence the amount of okra mucilage in the preparation largely dictated the microspheres' ability to swell. For formulation F5, the maximum degree of edema was recorded at 0.8722 (refer to Table 2). The ability to swell was shown to be lower in microspheres with lower okra mucilage content (F1) compared to those with higher mucilage concentration (F5). This demonstrated that the ability of microspheres to expand is greatly enhanced by an increase in the mucilage ratio.

Zeta Potential:

It is in charge of the formulation's coating and stability. Zeta potential values for okra mucilage and plane medication were -9.21 and -18.3, respectively. Zeta potential for formulation was -7.86. It suggests that the charge on the mucilage was not changed by the spray drying procedure. Mucilage's anionic properties considerably aid mucoadhesion. Additionally, the negative zeta potential value showed that, in colloidal conditions, a repulsive attraction exists between the mucoadhesive agent particles in the aqueous solution, aiding in the rapid settling of the mucilage formation process.

In vitro diffusion study:

Figure 5, which illustrate the % cumulative drug release as a function of time for various formulations, display the drug release data obtained for those formulations. All five microsphere formulations exhibited an initial burst effect during their in vitro release. For formulations F1 through F5, the drug release was 29.6%, 32.1%, 29.5%, 34.4%, and 44.27% in the first two hours, respectively.

Figure 5: Comparative in vitro diffusion of okra mucilage based mucoadhesive microspheres by using 6.8 pH buffers.

Drug release mechanism:

The regression coefficient (R2) and the release constant were computed from the slope of the relevant plots. Higuchi was found to have the best explanation for the in-vitro drug release of formulation F5, as the curve displayed the maximum linearity (R2 = 0.963). Drug release rate and concentration are related with formulations F2 and F3, which have the maximum linearity for first order (0.996, 0.994). Formulations F1, F4, and F5 adhered to the Higuchi kinetics, indicating that diffusion governs drug release. To verify the precise mechanism of drug release from these microspheres, the data was fitted using Korsemayer-Peppas equation simulation. It was noted that the n value for each formulation fell between 0.442 and 0.552, indicating that the fickian diffusion came next.

Stability of the microspheres:

Formulations exhibiting the ideal particle size and polymer mucoadhesion were chosen for stability investigations. Selected formulations (F5) were kept for three months at 400C and 75% relative humidity (RH) in accordance with ICH recommendations. The evaluation parameters are all within acceptable bounds and do not exhibit any significant differences.

CONCLUTION:

The results showed that formulation of buccal mucoadhesive microspheres of darifenacin Hbr based on okra mucilage is one of the alternative administration of darifenacin to avoid first pass effect and provide prolonged release. From the results, it was concluded that the in vitro drug release. Mucoadhesion strength of the optimized formulation is suitable for buccal delivery. The release pattern followed fickian diffusion with swelling controll release, which follows Higuchi kinetics. FTIR and DSC studies concluded that there was no interaction between drug and excipients.

CONFLICT OF INTEREST:

None.

ACKNOWLEDGMENT:

The authors are thankful to Microlab Pvt.Ltd, Banglore for providing gift samples. Authors are also thankful to the Principal of R.C. Patel Institute of Pharmaceutical Education and Research, Shirpur for permitting to carry out research work.

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Received on 29.02.2024 Modified on 18.05.2024

Research J. Pharm. and Tech 2024; 17(9):4465-4471.

DOI: 10.52711/0974-360X.2024.00690

Formulation code	Percentage Yield (%)	Drug loading (% ± SD)	*Drug Incorporation efficiency (%)**	Particle size(µm)
F1	33.5± 2.56	16.36 ± 1.25	90.08 ± 1.25	16.4 ± 2.25
F2	38.2± 2.69	19.20 ± 2.50	92.13 ± 1.75	15.6 ± 1.59
F3	40.2± 1.86	23.94 ± 1.74	93.76 ± 1.35	8.8 ± 1.11
F4	43.7± 1.83	31.04 ± 0.75	94.02 ± 0.75	14.2 ± 1.26
F5	47.5± 1.59	46.04 ± 1.52	96.18 ± 1.15	16.6 ± 1.83

Formulation code	Zero order (r²)	First order (r²)	Higuchi (r²)	Korsemayer-Peppas K value n Value		Best fitted model	Mechanism of release
F1	0.952	0.991	0.993	0.429	0.486	Higuchi	Fickian Diffusion
F2	0.955	0.996	0.991	0.412	0.458	First order	Fickian Diffusion
F3	0.964	0.994	0.991	0.479	0.552	First order	Fickian Diffusion
F4	0.983	0.929	0.968	0.446	0.442	Zero order	Fickian Diffusion
F5	0.963	0.912	0.991	0.464	0.471	Higuchi	Fickian Diffusion