INTRODUCTION:

Sustained release dosage forms are pharmaceutical formulations designed to release the active drug ingredient at a controlled and prolonged rate over an extended period of time. They are also known as extended-release, controlled-release, or prolonged-release dosage forms. The objective of these formulations is to maintain therapeutic drug levels within the body for a longer duration, reducing the frequency of dosing and improving patient compliance. The drug is dispersed or embedded within a matrix of hydrophilic or hydrophobic polymers. The polymer matrix controls the diffusion of the drug out of the dosage form, resulting in sustained release¹.

The release rate can be modulated by the polymer composition, drug-polymer interactions, and matrix characteristics. Conventional dosage forms often require frequent dosing throughout the day, sometimes several times a day. This can be inconvenient for patients, leading to poor compliance, especially for patients with complex medication regimens or those who have difficulty remembering to take multiple doses. Immediate-release formulations rapidly release the drug into the bloodstream, resulting in peak drug levels followed by a decline as the drug is metabolized and eliminated from the body. These fluctuations in drug levels can lead to suboptimal therapeutic effects, reduced efficacy, and potential side effects². Immediate-release formulations typically provide a relatively short duration of action, requiring repeated dosing to maintain therapeutic drug levels. This can be problematic for medications that need to be maintained at a steady concentration to achieve their desired therapeutic effects³. For this kind of therapy, controlled release microsphere formulations are highly desired and preferred because they improve patient compliance, maintain consistent drug levels, lower dosage and adverse effects, and boost the safety margin for high-potency medications⁴. In order to minimize side effects and enhance the therapeutic response at the local site, microsphere delivery devices have been developed for localized drug delivery^5,6.

Metformin is an oral antidiabetic medication that belongs to the class of drugs known as biguanides. It is commonly prescribed for the management of type 2 diabetes mellitus. The biological half-life of metformin is 1.5h-1.6 h andthe majorsite of its absorption in the proximal small intestine. A sustained release microsphere formulation of an antidiabetic drug and characterization of the microsphere has been represented that a microsphere was formulated and characterized for the sustained release of metformin-loaded microsphere formulation, which was prepared by ionotropic gelation method⁷. Here the work revealed that increase in the drug entrapment up to an optimum level and a decrease in the release rate and extend the action. Metformin loaded microsphere formulation would be an acceptable pharmaceutical formulation for the treatment of diabetic patients in carrier-based drug therapy for it extend action. The present focus is to develop oral site-specific sustained release anti-diabetic drug delivery to minimize systematic side-effects.

MATERIALS AND METHODS USED METFORMIN MICROSPHERE PREPARATION AND ITS CHARACTERIZATIONS:

Chemicals and instruments used are mentioned in table no 1A and 1B.

Table 1:A) Chemicals used

Sl No	Chemicals used	Procured from
1	Metformin	Clearsynth Labs Ltd, Mumbai, India
2	Sodium alginate	Loba Chem Pvt. Ltd
3	Chitosan	Loba Chem Pvt. Ltd
4	Calcium chloride	Loba Chem Pvt. Ltd

Table 1: B) Instruments used

Sl No	Name of the instrument	Model no
1	Magnetic Stirrer	REMI 1 MLH
2	Digital electronic balance	KEROY balance– FB360H
3	Optical Microscope	Olympus, ESAW optscopes classic
4	FTIR spectroscopy	BRUKER Alpha

A) Characterization of the drug sample: The sample of metformin was characterized by various parameters like:

i) Physical observation: It is white, hygroscopic crystalline powder.

ii) Solubility: Metformin powder freely soluble in water, slightly soluble in alcohol and practically insoluble in acetone, methylene chloride.

iii) Determination of melting point (M.P): M.P of the sample was revealed to be 222-226°C which was matched with the literature assuring that the identity of the received sample.

iv) U.V spectroscopy: The sample of metformin was scanned spectroscopically in a wavelength of 200-400nm in phosphate buffer of pH 6.8.λ max was found at 234 nm wave length, which was used for quantitative analysis.

v) IR spectra: IR spectra of metformin was matched with that of the reference spectra in BP and it showed similar peak.

vi) Calibration curve of metformin: The calibration curve was prepared in phosphate buffer pH 6.8. Concentration was increased in a predetermined manner. The correlation coefficient was calculated as 0.9955. Standard curve of metformin is represented in fig no1.

Fig 1: Data for preparation of calibration curve was shown in figure no1.

B) Preparation of metformin microsphere:

Metformin microspheres were prepared by inotropic gelation method. In one beaker a particular portion of chitosan was dispersed in 100 ml of 1% acetic acidsolution. In another beaker sodium alginate was added in 100 ml of distilled water. Then the solution was heated on the hot plate to dissolve in water. The metformin was added to the sodium alginate solution and stirring was done for almost 15mins by a magnetic stirrer. Calcium chloride was taken in another beaker then calcium chloride dissolved in distilled water.Then calcium chloride was added to the chitosan solution with continuous stirring. Then the drug solution was poured into a syringe and added dropwise with 18-gauge needle to the continuous stirring chitosan solution. The chitosan and calcium chloride containing beaker was placed on a magnetic stirrer with a 4500 rpm. Then microspheres were filtered and washed with distilled water. Then dried microspheres conserved in a sealed container.

C) Characterization of metformin loaded microsphere:

1. Fourier Transform Infrared Spectroscopy (FTIR) Study:

FTIR was used to determine the chemical interactions between the drug molecule and other active ingredients used in the microsphere preparation. It was done on BRUKER Alpha Fourier transform infrared (FTIR) Spectroscopy by using KBr powder⁸.

2. Percentage yield of the microsphere:

The percentage yield of the microsphere was evaluated by using the ratio of practical yield and theoretical yield. Practical yield is the weight of the microsphere obtained. Total weight of the raw materials is theoretical yield^9,10,11,12.

Percent yield= Practical yield/ Theoretical yield 𝑋 100

3. Swelling index:

10mg of metformin microsphere was added into 100ml of phosphate buffer solution (pH 6.8) for24 hours. swelling index was measured using formula.[13]

Swelling index = (Wv −W𝑖 / W𝑖) 𝑋 100

Where, Wv represents weight of microspheres after 24 hrs

W𝑖 represents initial weight of microspheres

4. Drug entrapment efficiency:

10mg of prepared microspheres were triturated and made into powder form. Then 100 ml of phosphate buffer (pH 6.8) was added into it and put to a magnetic stirrer for 2 hours. Solution was filtered by using the Whatman filter paper. 10 ml of this stock solution diluted with phosphate buffer (pH 6.8) and evaluated for metformin content at 233 nm^{13, 14,15}.

Drug entrapment efficiency = (Experimental drug content/ theoretical drug content)𝑋 100

5. Drug release study (In- vitro):

A drug release study was performed in the dissolution test apparatus, USP -type 2(paddle type) apparatus measured at 37°C and rotated at 50RPM. The dissolution test apparatus primarily, 900ml of the phosphate buffer of pH-6.8 added, and 50mg microsphere were added. From the solution, 5ml of the sample was withdrawn every 1 hr, and it was replaced by 5ml ml phosphate buffer of pH 6.8 every hr. This process continued for 10hours. Samples were collected and filtered by using Whatman filter paper. Diluted filtrate was measured at 233nm on Shimadzu, UV 1800 UV – visible spectrophotometer^16,17.

6. Scanning electron microscopy (SEM) analysis:

Particle size, shape, and surface morphology were determined by SEM analysis. SEM was done by JEISS make (UK) model (JSM 6360 LV).

Drug release kinetics mechanisms:

Different types of kinetic model were operated to examine drug release data from the dissolution test like Zero order release kinetics (equation no 1), First order release kinetics (equation no 2), Higuchi model (equation no 3), and Korsmeyer Peppas equation (equation no 4).

C=K₀t……(1)¹⁸

Where K means zero order rate constant indicate as concentration/time and t is the time.

Log C=LogC₀–k₁t/2.303…………………………………………….(2)¹⁸

Where C₀indicates the initial concentration of the drug

K₁is the first order constant.

Q=K_ht……………………………………………….(3)¹⁸

Where K_hmeans the constant considering the the design variables of the system.

Mt/M_&=K_kp tⁿ……………………………………….(4)¹⁸

Mt/M_&means the fraction of drug release

K_kp is the release constant

t is dissolution time

n is the diffusion release exponent considering of the drug release mechanism.

RESULTS AND DISCUSSIONS ON VARIOUS PARAMETERS OF MICROSPHERE CHARECTERIZATIONS:

1. FT-IR Analysis:

The drug polymer interaction was determined using the FT-IR Spectrum shown in figure no 2. The bands points of Chitosan show in FT-IR spectrum are 1081cm^-1,1560 cm-1, 2882 cm-1, 3423cm-1, in this case the band at 1081 cm-1 shows the c-n stretching, n-h bending shows at 1560 cm-1, O-H, N-H stretching shows at 3423 cm-1that determines c-o stretching .and metformin drug shows its characterized peak at 3373 cm-1, 3147 cm-1 ,2814 cm-1, 2696 cm-1, 1634 cm-1 ,1410 cm-1 and 1063 cm-1. 3373 shows the N-H asymmetric stretching, 3147 shows symmetric stretching of N-H bond, 1634 shows C=N stretching and 1410 shows N-H bending. The blank microsphere contains all the band presence in chitosan and sodium alginate. Metformin microsphere contains allthe band present in sodium alginate, chitosan as well as metformin drug. FT-IR spectrum determined about all the band of present in metformin microsphere that shows there are no important changes of the band, there are no drug polymer interaction observed.

Table No 2: Various parameters of microsphere characterizations:

Batch Code	Metformin (mg)	chitosan (mg)	Sodium alginate (mg)	Calcium chloride	% Yield	Swelling index	Particle size (µm)	%Drug entrapment efficiency	% of drug release	Sphericity
B1 (3:1)	10 mg	3	1	2	62.34 ± 0.30	18.66 ± 2.39	102± 0.4	77.83. ± 0.66	81± 0.97%	Spherical
B2 (2:1)	10mg	2	1	4	63.63 ± 0.37	44.66 ± 5.56	537± 0.5	77.10 ± 0.38	98.46±0.2%	Spherical
B3 (1:1)	10mg	1	1	3	46.52 ± 0.31	77.33 ± 5.80	306± 0.5	39.63 ± 0.28	99.46±0.6%	Irregular Spherical

Various parameters on microsphere characterizations shown in table no 2.

2. Swelling index:

Data of swelling index shown in table no 2. The swelling index data shows that when the percentage of crosslinker (calcium chloride) in the matrix increases, the equilibrium water uptake rises significantly. This is as a result of risinga higher quantity of calcium chloride results in a more significant crosslinking with sodium alginate.

3. %Drug entrapment efficiency:

Data of % drug entrapment efficiency shown in table no 2. Entrapment efficiency determination was used to quantify the amount of metformin contained in the different microsphere formulations. Drug entrapment efficiency revealed a dependence on chitosan concentration. The entrapment efficiency increased together with the concentration of chitosan in the formulation. As the ratio of drug to polymer increases, there is a noticeable rise in drug entrapment.It took place as a result of the droplets being stabilized by the increasing aqueous phase viscosity caused by a rise in polymer concentration. The stability of these droplets stops metformin from leaving the microspheres further¹⁹.

4. In-vitro drug release studies:

In- vitro drug release study of B1, B2 and B3 batches are represented in table no 2and 3 and it was shown that drug release rate was between 81%-99%. Data suggests that the rate of drug release is slowed down by higher chitosan concentrations. B1 batch is the optimized formulation which showing 81% drug release rate in 10 hours.

Table 3: In-Vitro drug release studies of 3 batches of microspheres

TIME(min)	BATCH 1	BATCH 2	BATCH 3
0	0	0	0
15	10.7±0.10	13.14±0.21	15.84±.19
30	17.24±0.08	26.84±0.19	38.35±.23
45	22.96±0.12	44.68±0.04	50.64±.45
60	27.47±0.15	58.17±0.06	63.89±.19
120	39.38±0.21	65.92±0.17	72.1±.01
180	45.51±0.19	67.41±0.23	93.45±.04
240	53.66±0.07	81.17±0.14	97.65±.23
300	58.81±0.15	84.33±0.07	99.46±.17
360	68.05±0.03	86.83±0.21	--------
480	79.53±0.12	97.53±0.18	--------
600	81.10±0.23	98.46±0.03	--------

5. Mechanisms of drug release kinetics:

Drug release kinetics of 3 batches of prepared microspheres are shown in figure 3 and table no 4.

6. Selection of optimized batch:

Selection of 3 formulation of metformin microspheres was estimated on size and shape of the microspheres, % entrapment efficiency, and cumulative % drug release data. From the all parameters it can be mentioned that B1 is the optimized formulation which was further used for SEM study.

7. SEM Study of metformin microsphere:

The morphology of B1 formulation microsphere was examined by SEM using JEOL JSM -6360 (JSM 6360 LV) to examine metformin microsphere with a focused electron beam to images with details regarding shape and topography of the sample. Drug containing microsphere was planted on conducting stubs and vacuum coated gold palladium film using a Gold Sputter Coater (Model Edwards – S 150 B Mfg. BOC Edwards UK). Photograph were obtained using 17 kv electron intensity in SEM to explore the surface morphology. SEM study of B1 formulation determines spherical structure and rough surface of microsphere. Photographs of SEM analysis of optimized batch is shown in figure no 4.

Fig 4: SEM study of B1 batch of microsphere

CONCLUSION:

Ionotropic gelation method was used for the preparation of 3 batches (B1-B3) of microspheres. Various parameters like particle size, Percentage yield, swelling index, Drug entrapment efficiency, Drug release study (In- vitro) and drug release kinetics studies were assessed. From the results it was found that increased concentration of calcium chloride increases the swelling index whereas increased chitosan concentration causes decrease in drug release. From the various results it can be concluded that B1 batch is the optimized batch and were utilized for further studies.

REFERENCES:

1. Kumar AR. Aeila AS. Sustained release matrix type drug delivery system: An overview. World J Pharma Pharm Sci. 2019; Oct 24; 8(12): 470-80. DOI: 10.20959/wjpps20201-15241.

2. Rada SK. Kumari A. Fast dissolving tablets: waterless patient compliance dosage forms. Journal of Drug Delivery and Therapeutics. 2019; Jan15; 9(1): 303-17. DOI: https://doi.org/10.22270/jddt.v9i1.2292 .

3. Saxena AK. Sharma A. Verma N. Microspheres as therapeutically effective multiparticulate drug delivery system: A systemic review. Research Journal of Pharmacy and Technology. 2021; 14(6): 3461-70.

4. Vinod R. Kumar PA. Yadav AS. Rao BS. Kulkarni SV. Formulation and Evaluation of Controlled Release Microspheres of Zidovudine. Research Journal of Pharmaceutical Dosage Forms and Technology. 2010; 2(1): 96-9.

5. Kumbhar DM. Mali KK. Dias RJ. Havaldar VD. Ghorpade VS. Salunkhe NH. Formulation and development of ethyl cellulose coated pectin based capecitabine loaded microspheres for colorectal cancer. Research Journal of Pharmaceutical Dosage Forms and Technology. 2016; 8(4): 261-8.

6. Chandarsekaran N. Balamurugan M. Formulation, Characterization and In-vitro Evaluation of Abacavir Sulphate Loaded Microspheres. Research Journal of Pharmacy and Technology. 2013; 6(7): 731-5.

7. Patel NR. Patel DA. Bharadia PDet al. Microsphere as a novel drug delivery. International Journal of Pharmacy and Life Sciences. 2011; Aug 1; 2(8): 992-997.

8. Patil UK. Sahu R. Yadav SK. Formulation and Evaluation of Controlled Release Microspheres Containing Metformin Hydrochloride. Research Journal of Pharmacy and Technology. 2009; 2(1): 176-9.

9. Ratnaparkhi MP, Gupta jyoti P. Sustained release oral drug delivery systeman overview. International Journal of Pharma Research and Review. 2013; 2(3): 11-21.

10. Bose S. Kaur A. Sharma SK. A review on advances of sustained release drug delivery system. Int. Res. J. Pharm. 2013; 4: 1-5.

11. Gavini V. Murthy MS. Kumar PK. Radhika DL. Formulation and in-vitro evaluation of mucoadhesive microspheres loaded with stavudine using hydrophilic macromolecular polymers. Research Journal of Pharmaceutical Dosage Forms and Technology. 2014; 6(2): 99-104.

12. Talokar SS. Vakhariya RR. Formulation and evaluation of lomefloxacin microspheres. Research Journal of Pharmaceutical Dosage Forms and Technology. 2017; 9(1): 1-5.

13. Khan R. Ashraf MS. Afzal M. Kazmi I. Jahangir MA. Singh R. Chandra R. Anwar F. Formulation and evaluation of sustained release matrix tablet of rabeprazole using wet granulation technique. Journal of Pharmacy and Bioallied Sciences. 2014; Jul; 6(3): 180. DOI: 10.4103/0975-7406.130961

14. Das MK. Ahmed AB. Saha D. Microsphere a drug delivery system: A review. Int J Curr Pharm Res. 2019; 11(4): 34-41.

15. Hajare AA. Shetty YT. Formulation, characterization and in-vitro evaluation of floating microspheres of diltiazem hydrochloride by ionotropic gelation technique. Research Journal of Pharmacy and Technology. 2008; 1(1): 52-6.

16. Saraswathi B. Satyanarayana T. Manasa G. Mounika C. Chandh PG. Krishnaveni K. Formulation and evaluation of Candesartan microspheres. Research Journal of Pharmaceutical Dosage Forms and Technology. 2019; 11(2): 81-6.

17. Nighute AB. Bhise SB. Preparation and evaluation of rifabutin loaded polymeric microspheres. Research Journal of Pharmacy and Technology. 2009; 2(2): 371-4.

18. Ahmed TA. Aljaeid BM. Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery. Drug Design, Development and Therapy. 2016; Jan 28: 483-507.

19. Denkbaş EB. Seyyal M. Pişkin E. Implantable 5-fluorouracil loaded chitosan scaffolds prepared by wet spinning. Journal of Membrane Science. 2000; Jul 1; 172(1-2): 33-8. https://doi.org/10.1016/S0376-7388(00)00314-8 .

Received on 27.12.2023 Modified on 08.03.2024

Research J. Pharm. and Tech 2024; 17(10):5079-5084.

DOI: 10.52711/0974-360X.2024.00781

R² Value Kinetic	Batch 1	Batch 2	Batch 3
Zero order kinetics	0.9946	0.9437	0.9667
First order kinetics	0.937	0.875	0.899
Korsmeyer Peppas kinetics	0.8482	0.9188	0.8994
Kinetics model followed	Zero order kinetics	Zero order kinetics	Zero order kinetics