Formulation and Evaluation of Mucoadhesive Microspheres of Metformin Hydrochloride for Controlled Drug Delivery system.

Over the past thirty years, oral controlled release medications have been created due to their significant therapeutic benefits like simple administration, patient adherence, and flexibility in formulations. Nonetheless, this method faces challenges such as the difficulty in controlling and directing the drug delivery system to the intended area of the gastrointestinal tract (GIT) due to the inconsistent movement and short gastric emptying time (GET) in humans, typically lasting around 2-3 hours across the primary absorption zones, mainly the stomach and upper intestine. Consequently, incomplete drug release from the delivery system occurs, reducing the effectiveness of the dose administered. The main goal of designing a controlled release system is to provide the drug at a pace that achieves and maintains a consistent blood concentration of the drug. This pace should mimic that of a continuous intravenous infusion where the drug is given to the patient at a rate equal to its elimination rate, regardless of the remaining amount in the dosage form, ensuring a constant release over time, following zero-order kinetics.  Microsphere carrier systems, crafted from naturally degradable polymers, have garnered significant interest for sustained drug delivery over the years. Recently, there has been a substantial impact on the formulation and advancement of new drug delivery systems by dosage forms capable of precisely regulating release rates and directing drugs to specific body sites. Microspheres play a vital role in these innovative drug delivery systems. Although microspheres prepared using various polymers have diverse applications, their effectiveness is limited due to their brief stay at the absorption site. To enhance drug bioavailability and extend the residence time of the dosage form in the stomach, efforts have been made to develop bioadhesive drug delivery systems that create close contact between the drug delivery system and the absorbing membranes. This can be achieved by integrating mucoadhesive properties into microspheres to form mucoadhesive microspheres. These microspheres offer benefits such as improved drug absorption and bioavailability due to their high surface-to-volume ratio, close interaction with the mucus layer, and precise drug targeting to the absorption site. Gastric mucoadhesive drug delivery presents numerous opportunities for drugs with low bioavailability due to a narrow absorption window in the upper part of the gastrointestinal tract. It retains the dosage form at the absorption site, thereby boosting bioavailability.

2. Material and methods.

Metformin HCl was received as a complimentary sample from Strides acrolabs Pvt. Ltd, Bangalore. Sodium alginate and Carbopol 934P were bought from Rolex Chemical Industries, Mumbai. HPMC K-100M and HPMC K-4M were acquired from NR Chem, Mumbai. Calcium chloride was obtained from S.D. Fine Chem Ltd, Mumbai. Hydrochloric acid was procured from Reachem Laboratory Chemicals Pvt. Ltd, Chennai. All other chemicals were of analytical quality.

Formulation of mucoadhesive microspheres:

Mucoadhesive microspheres that contain an antidiabetic drug as the core material were created using the orifice ionic gelation technique. Sodium alginate and mucoadhesive polymers such as Carbopol 934P, HPMC K100 M, and HPMC K-4M were dissolved in 40 mL of purified water to create a uniform polymer solution. The active ingredient, metformin hydrochloride (500mg), was mixed into the polymeric solution and thoroughly blended with a mixer to form a thick dispersion. This dispersion was then carefully added drop by drop into a 10% w/v calcium chloride solution (40 mL) using a syringe (no.20). The droplets were left in the calcium chloride solution for 15 minutes to finish the curing process and form spherical rigid microspheres. The microspheres were gathered by decantation, washed multiple times with water, and dried at 40 °C for 3 hours in a hot air oven. They were stored in a desiccator with fused CaCl2 for future analysis. The batches of mucoadhesive microspheres were prepared accordingly.

3. Evaluations.

1. Percentage yield calculation was done for microspheres by comparing the weight of the final dried product to the initial combined weight of the drug and polymer. The formula used for this calculation was:

Percentage Yield = Actual mass (microspheres) / Theoretical mass × 100

2. For Drug content and Drug Entrapment Efficiency (DEE) assessment, 50 mg of the drug in microspheres was examined. Drug entrapment was determined by crushing the microspheres and extracting them with successive aliquots of 0.1N HCl. The resulting extract was then transferred to a 50 ml volumetric flask, diluted with 0.1N HCl, filtered, and its absorbance measured at 233 nm using a UV 1800 spectrophotometer from Shimadzu, Japan. The formula used to calculate the Drug Entrapment Efficiency was:

% DEE = (Actual drug amount / Expected drug load) × 100

3. Particle Size analysis for the microspheres was conducted through optical microscopy. A calibrated optical microscope was used to count around 10 microspheres for determining their size.

Techniques in laboratory settings

Assessing bonding strength

The calculation of the adhesive forces between polymeric microspheres and the mucosal tissue is a valuable parameter in determining the adhesive properties of microspheres. Laboratory methods have been employed to examine the polymeric microspheres against various types of artificial and biological tissue samples, including synthetic and organic mucus, frozen and freshly removed tissue, etc.

Different laboratory techniques utilized include:

Approach based on measuring tensile strength

The Wilhelmy plate method is a traditional approach for evaluating dynamic contact angles and requires a microtensiometer or a microbalance. The CAHN dynamic contact angle analyzer (model DCA 322, CAHN instruments, Cerritos, California, USA) has been adjusted to conduct measurements of adhesive microforces. The DCA 322 setup comprises an IBM compatible computer and a microbalance system. The microbalance component includes fixed sample and tare loops along with a motor-driven translation stage. The device gauges the mucoadhesive strength between mucosal tissue and a single microsphere affixed to a thin metal wire hanging from the sample loop in the microtensiometer. The tissue, typically rat jejunum, is positioned inside the tissue chamber containing Dulbecco’s phosphate.

4.Method of Preparation

The technique of microencapsulation allows the integration of solids, liquids, or gases into one or more polymer coatings. The methods for preparing various microspheres are chosen based on factors such as particle size, administration route, and drug release duration, along with parameters like rpm, cross-linking method, cross-linking agent, evaporation time, and co-precipitation. The diverse methods of preparation include:

Phase separation coacervation technique

This method relies on reducing the polymer's solubility in the organic phase to induce the creation of a polymer-rich phase known as coacervates. In this approach, drug particles are dispersed in a polymer solution, and an incompatible polymer is introduced to prompt the initial polymer to separate into phases and encapsulate the drug particles. The introduction of a nonsolvent leads to the polymer solidifying. It is crucial to carefully manage process variables as the speed of coacervate formation impacts the distribution of the polymer film, particle size, and agglomeration of particles. To prevent agglomeration, it is necessary to stir the suspension at an appropriate speed, as the polymer globules formed during microsphere creation tend to adhere and form agglomerates. Hence, controlling process variables is vital as they influence the kinetics of particle formation due to the absence of a defined equilibrium state.

Evaporation of Solvent

The procedures are conducted in a liquid production medium.The coating of microcapsules is scattered in a volatile liquid that doesn't mix with the liquid phase of the production medium. The material at the center that will be encapsulated into microcapsules is dissolved or dispersed in the solution of the coating polymer. By stirring, the mixture of the central material is dispersed in the liquid phase of the production medium to achieve the correct size of microcapsules. The blend is then heated if needed to remove the liquid for the polymer of the central material to scatter in the polymer solution, causing the polymer to contract around the core. If the central material is dissolved in the solution of the coating polymer, microcapsules of the matrix type are produced. The central materials can be materials that are soluble or insoluble in water. The process of solvent evaporation includes creating an emulsion between the polymer solution and a phase that does not mix.

4. Evaluation Parameters

Particle size

measurement will be conducted using optical microscopy [8].

The analysis of shape and surface characteristics will be performed through scanning electron microscopy (SEM), a method that provides information on surface morphology. Information on surface morphology of microspheres and any changes due to polymer degradation can be obtained from SEM, scanning tunneling microscopy, and electron microscopy. The alterations in surface morphology resulting from polymer degradation can be examined by placing the microspheres in phosphate buffer saline for varying time intervals. Research has shown that microspheres with a rougher surface enhance adhesion through more robust mechanical interactions, while microspheres with a smooth surface exhibit weaker mucoadhesive properties [18,19]. The efficiency of drug entrapment or capture within the microspheres can be determined by immersing the microspheres in a buffer solution to allow for lysing. After lysing, the resultant lysate is filtered or centrifuged and then analyzed to determine the active components according to the requirements outlined in the monograph.

Mucoadhesion evaluation

The adhesive characteristics of the microspheres are assessed through an in-vitro wash-off experiment. A rat stomach mucosa piece measuring 1 cm by 1 cm is attached to a glass slide (3 inches by 1 inch) with thread. The wet tissue is then coated with microspheres, and the slide is placed in one of the grooves of a USP tablet disintegrating test apparatus. The apparatus moves the tissue up and down in a beaker containing simulated gastric fluid USP (pH 1.2). The number of microspheres sticking to the tissue is counted after 30 minutes, 1 hour, and hourly up to 10 hours.

Compatibility analysis

Differential scanning calorimetry (DSC)

DSC is a thermal analysis method that measures the heat needed to raise the temperature of a sample and a reference as the temperature changes. DSC thermograms of the microspheres will be recorded by placing drug samples in pans, with an empty aluminum pan as reference. The system will be purged with nitrogen gas, and heating will occur at a constant rate.

Fourier transform infrared (FTIR) spectroscopy

FTIR will be used to record IR spectra of the microspheres by creating pellets of the samples with KBr and comparing the resulting spectra with a standard reference to detect any deviations.

Drug release investigations

Typically, a standard IP/BP/USP dissolution apparatus is employed to examine the in-vitro release pattern in a dissolution medium resembling the fluid at the absorption site. The formulation is placed in baskets, immersed in a beaker with dissolution media (900 ml), and set at a specific rpm. Samples are taken at various intervals over a 12-hour period for drug release analysis.

RESULTS AND DISCUSSION

Microspheres of Metformin HCl with a coating made of different levels of sodium alginate and mucoadhesive polymers in various ratios could be created using the orifice ionic gelation method. These microspheres were identified as individual, nearly spherical, freely flowing, and resembling a monolithic matrix. They were entirely enveloped by a polymer coating.

1.Yield Percentage

The  yield percentage of microspheres from all formulations is detailed in Table 2. The yield percentage ranged from 60.22 ± 2.06% to 96.50 ± 1.01% across all formulations. With an increase in sodium alginate concentration, the yield percentage also increased. Consequently, batches F4, F8, and F12 demonstrated favorable yield percentages of 82.65±1.24%, 86.75±1.09%, and 96.50± 1.01%.

2. Drug Content and Entrapment Efficiency (%DEE)

The drug content in the dried microspheres of all formulations varied between 25.24 ± 1.06 mg to 42.07 ± 0.79 mg, while the drug entrapment efficiency ranged from 50.48 ± 1.2 to 84.15 ± 0.01. These results suggest that an increase in sodium alginate concentration led to higher drug content and entrapment efficiency. Formulations containing Carbopol 934P as a mucoadhesive polymer exhibited the highest drug content and entrapment efficiency. Therefore, the drug content and entrapment efficiency ranking for formulations based on mucoadhesive polymers are as follows: Carbopol 934P > HPMC K-100M > HPMC K-4M.

3. Particle Size Analysis

The particle size of the dried microspheres across all formulations ranged from 94 ± 0.30 to 144 ± 0.5 μm. This aligned with the observation that using Carbopol 934P as the mucoadhesive polymer resulted in smaller particles, while HPMC K-100M led to larger particles. Intermediate particle sizes were seen when HPMC K-4M was used in preparing the mucoadhesive microspheres.

4. Swelling Index (SI)

As time progressed, swelling increased due to the gradual absorption of water by the polymers' hydrophilic nature. The polymers would hydrate and swell, forming a gel barrier on the outer surface. The swelling index rose with higher polymer concentrations. Carbopol 934P exhibited less swelling than HPMC, which swelled rapidly initially due to its high viscosity. The swelling properties of different formulations depended on the viscosity of the polymer used, with the swelling index ranking as follows: Carbopol 934P > HPMC K4M > HPMC K 100M. The swelling properties of all formulations after 10 hours are depicted.

5. In-vitro Wash-off Test for Microspheres

To evaluate the mucoadhesive properties of the microspheres, an in-vitro wash-off test was conducted for all formulations. The adhesion of the polymer to the mucous membrane is facilitated by hydration, particularly in the case of hydrophilic polymers. Higher concentrations of sodium alginate led to increased mucoadhesion. Formulation F4, containing two parts sodium alginate and one part Carbopol 934P, displayed the highest mucoadhesion. The percentage mucoadhesion order after 10 hours for all formulations was as follows: F4 > F8 > F12 > F1 > F5 > F7 > F6 > F9 > F2 > F3 > F10 > F11.

CONCLUSION

An effective endeavor has been accomplished to create mucoadhesive tiny spheres containing metformin hydrochloride by utilizing sodium alginate and a sticky polymer (Carbopol 934P, HPMC K100M, HPMC K4M) that could be produced using the Orifice Ionic gelation method. These spheres demonstrate strong adhesion to mucous membranes in a lab-based wash-off test. The discharge of metformin hydrochloride from these adhesive microspheres was gradual over an extended period and relied on the makeup of the coating. The medication release from the most efficient formulation occurred through a combination of diffusion and mixed order kinetics. Consequently, these mucoadhesive microspheres are appropriate for the controlled release of metformin hydrochloride orally.

REFERENCES

1. Aulton ME, Taylor KMG. Aulton’s Pharmaceutics: The Design and Manufacture of Medicines. 5th ed. Edinburgh: Elsevier; 2018.
2. Sinko PJ. Martin’s Physical Pharmacy and Pharmaceutical Sciences. 6th ed. Philadelphia: Lippincott Williams & Wilkins; 2011.
3. Lieberman HA, Lachman L, Schwartz JB. Pharmaceutical Dosage Forms: Tablets. 2nd ed. New York: Marcel Dekker; 1989.
4. Higuchi T. Mechanism of sustained?action medication. Theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J Pharm Sci. 1963;52(12):1145-1149.
5. Korsmeyer RW, Gurny R, Doelker E, Buri P, Peppas NA. Mechanisms of solute release from porous hydrophilic polymers. Int J Pharm. 1983;15(1):25-35.
6. Peppas NA. Analysis of Fickian and non-Fickian drug release from polymers. Pharm Acta Helv. 1985;60(4):110-111.
7. Hixson AW, Crowell JH. Dependence of reaction velocity upon surface and agitation. Ind Eng Chem. 1931;23(8):923-931.
8. Costa P, Sousa Lobo JM. Modeling and comparison of dissolution profiles. Eur J Pharm Sci. 2001;13(2):123-133.
9. United States Pharmacopeia (USP 43–NF 38). Rockville, MD: United States Pharmacopeial Convention; 2020.
10. Desai S, Bolton S. A floating controlled-release drug delivery system: in vitro–in vivo evaluation. Pharm Res. 1993;10(9):1321-1325.
11. Chowdary KPR, Rao YS. Mucoadhesive microspheres for controlled drug delivery. Biol Pharm Bull. 2004;27(11):1717-1724.
12. Andrews GP, Laverty TP, Jones DS. Mucoadhesive polymeric platforms for controlled drug delivery. Eur J Pharm Biopharm. 2009;71(3):505-518.