INTRODUCTION:

Diabetes mellitus (DM) is an endocrinological metabolic disorder¹ resulting from a defect in insulin secretion, insulin action or both. Insulin deficiency in turn leads to chronic hyperglycemia with disturbances of carbohydrate, fat and protein metabolism. Diabetes is best controlled either by diet alone and exercise (non-pharmacological) or diet with herbal or oral hypoglycemic agents or insulin (pharmacological). Diabetes mellitus may be classified into several types such as type 1, type 2 and gestational diabetes. On the basis of aetiology type 1 and type 2 were widely used to describe insulin dependent diabetes mellitus (IDDM) and noninsulin dependent diabetes mellitus (NIDDM) respectively.

According to the American Diabetes Association (ADA) recommendation changes in 1997 the fasting glucose concentration should be used in routine screening for diabetes as well as epidemiological studies the threshold for fasting glucose was changed from 7.8mmol/L (140mg/dl) to 7.0mmol/L (126mg/dl) however the 2 hr glucose criterion remains as = 11.1mmol/L (200mg/dL). Metformin HCL is an anti-hyperglycemic agent, which improves glucose tolerance in type II diabetes. It has been reported that the absolute bioavailability of Metformin HCL when given orally is 50–60%. Biological half-life of Metformin HCL is 1.5–1.6 h and the main site of its absorption is proximal small intestines.

Drug delivery system using colloidal particulate carriers such as liposomes, niosomes or proliposomes²proved to have distinct advantage over conventional dosage with an increasingly important role in drug delivery, on account of their small size, better drug targeting³, delivery and release, with their additional potential to combine diagnosis with therapy.

MATERIALS AND METHODS:

Materials:

Metformin hydrochloride was provided as gift samples from Wockhardt Pvt.Ltd, Aurangabad.

All other chemicals used were of analytical grade.

Methods:

Preparation of Proliposomes:

Proliposomes were prepared by varying the concentration ratios of lecithin and mannitol. Accurately weighed amount of drug and excipients are taken in a clean and dry glass volumetric flask and required quantity of ethanol is added to it. All the ingredients were mixed for 10min until complete dissolving. The drug-polymer solution thus prepared was poured into a spray gun. This was sprayed against the non abrasive glass as the substrate with specific spray time (6-7 s). After each specific spray, 2-3 min was given so as to aid the particle drying, formation of the particles on the substrate and the evaporation of the solvent. The spray number was kept at 15 times after which proliposomes were collected by scrapping and further sieved through sieve number 85 and 100. (Table-1).

Compatibility Studies⁴

Fourier-transform infrared spectroscopy (FT-IR):

Physicochemical compatibility between Metformin Hydrochloride and excipients used to prepare proliposome was studied using Fourier transforminfrared (FTIR- 8300, Shimadzu Co., Kyoto, Japan) spectroscopy by the KBr pellet method and spectra were recorded in the wavelength region between 400 and 4000 cm⁻¹.

X-ray diffractometry (XRD):

X-ray scattering measurements of Metformin Hydrochloride and Metformin Hydrochloride loaded proliposome were carried out with X-ray diffractometer (Philips PW 1729 X-ray diffractometer, Holland) by exposure of samples to the radiation, over an angular range (2) of 360° at a scanning velocity of 200°/min. Scintillation detector and the DIFRAC plus software were used for signal processing.

Evaluation of proliposome formulations:

Proliposome of metformin Hydrochloride were evaluated for different parameters like micromeritics properties, numbers of vesicles forms after hydration, mean vesicle size, size distribution and zeta potential, drug content, entrapment efficiency and in vitro drug release.

Micromeritic properties of proliposome powders⁵

The micromeritic properties of proliposome powder like flow property was checked by angle of repose, Carr's compressibility index, and Hausner's ratio. The angle of repose was determined by using the fixed funnel method

Number of vesicles per cubic mm^6,7

One of the vital parameter to evaluate the proliposome powder is the number of vesicles formed after hydration. The proliposome powder was hydrated with phosphate buffer (pH 7.4), and the formed liposomes were counted by using a hemocytometer in an optical microscope. The liposomes distributed in 80 small squares were counted and calculated by using the following formula:

Total no. of proliposomes per cubic mm=

Total Liposome counted X dilution factor X 4000

-----------------------------------------------------------------

Total no of Squares counted

Determination of vesicle size and zeta potential:

Mean vesicle size, size distribution and zeta potential of loaded liposomal dispersions was determined by photon correlation spectroscopy using the Zetasizer Nano ZS 90 (Malvern Instruments, Malvern, UK). Proliposomal powder was hydrated with distilled water and sonicated for 5 min. The resulting liposome dispersion was used for the determination of size of the vesicles, polydispersity index (PDI) of liposomes and zeta potential

Determination of drug content and entrapment efficiency⁸

Proliposome was determined by dissolving 10mg of proliposome powder in 10ml of methanol. The resultant solution was diluted properly and analyzed for drug content by UV method. For determination of entrapment efficiency within Metformin loaded proliposome, drug encapsulated in liposome was determined after hydration of proliposome powder (10mg) with water (10ml) by sonicating the dispersion for 10 min. Free Metformin was separated from the liposome entrapped Metformin Hydrochloride by centrifugation method. Metformin in supernatants was estimated as free Metformin by UV method Entrapment efficiency was calculated using the following formula.

Amount of Free Drug

EE% =--------------------------------------------- X 100

Total Amount of Drug

Table 2: Formulation design of proliposomes

Ingredients	MPFI	MPF2	MPF3	MPF4	MPF5	MPF6	MPF7	MPF8	MPF9
Metformin (mg)	100	100	100	100	100	100	100	100	100
Lecithin (mg)	100	150	200	100	150	200	100	150	200
Mannitol (mg)	500	500	500	250	250	205	125	125	125
Cholesterol (mg)	10	10	10	10	10	10	10	10	10
Ethanol (ml)	10	10	10	10	10	10	10	10	10

In vitro dissolution study⁹

In vitro dissolution study of proliposomal powders and pure Metformin was performed using USP type II (paddle) apparatus (Electrolab, Mumbai, India) in phosphate buffer (pH 6.8). Pure Metformin and proliposome powder equivalent to 100mg of metformin were transferred to 900ml of dissolution medium maintained at a temperature of 37 ± 0.5°C with paddle speed set at 50rpm throughout the experiment. An aliquot of 5ml was collected at predetermined time intervals up to 3 h and replaced with fresh dissolution medium to maintain constant volume. Samples were filtered by passing through a 0.45-μm membrane filter (Millipore, Billerica, Massachusetts, USA) and analyzed by UV Spectrometer

Scanning electron microscopy (SEM):

Scanning electron micrographs of the pure Metformin Hydrochloride powder, Metformin Hydrochloride loaded proliposome were taken using analytical scanning electron microscopes (Leo 440i, LEO Electron Microscopy Ltd., Cambridge, UK). The samples were fixed on an aluminum stub using double-side adhesive tape and coated with platinum to a thickness of about 10 Å under an argon atmosphere using a gold sputter module in a high-vacuum evaporator.

Stability studies¹⁰

The optimized proliposome powder was filled into glass vial and stored for 6 months. The stability was assessed at 40 ± 2°C/75 ± 5% RH, 30 ±2°C/70 ± 5% RH and 25 ±2°C/65±5% RH as per ICH guideline. After period of 1, 3 and 6month, the samples were withdrawn and evaluated for vesicle size, any sign of drug crystallization after hydration under an optical microscope and drug content for checking physical and chemical stability.

RESULTS AND DISCUSSION:

FTIR Study:

To check compatibility of the Metformin hydrochloride with selected excipient, FT-IR was carried out. The FT-IR spectra of pure Metformin hydrochloride had major peaks (Figure 1) at 3030–3060 cm−¹ (-CH aromatic), 2962 cm−¹ (aliphatic-CH stretching), 1760 cm−¹ (C=O), 1510–1600 cm−1 (C-C aromatic) and 1350 cm−¹ (C-N). The characteristic absorption peaks of Metformin hydrochloride were retained in proliposome formulation (Figure 2) without any additional peaks and therefore no interaction between drug and other component was concluded.

Figure 1: FTIR study of Metformin Hydrochloride

Figure 2: FTIR study of optimized proliposome formulation

XRD study:

X ray diffraction pattern of Metformin hydrochloride shows sharp peaks at 12.35°, 14.709°, 15.432°, 16.348°, 18.170°, 18.828°, 19.696°, 21.551°, 22.654°, 24.233°, 26.277° and 63.480° indicating crystalline nature of drug (Figure 3a). Proliposome powder shows sharp peaks at 13.783°, 14.759°, 16.90°, 17.398°, 18.862°, 19.939°, 20.527°, 21.228°, 23.517°, 25.254°, 27.101°, 28.240°, 29.620°, 33.272°, 34.063°, 36.166°, 64.037° (Figure 3b). The absence of typical Metformin hydrochloride peaks and decreased intensity of peaks in proliposome formulation confirms the amorphization of the drug.

Figure 3: XRD study of a) Metformin Hydrochloride, b) optimized formulation

Micromeritic properties of proliposome powders:

All the proliposomes batches were evaluated like angle of repose, Carr’s index and Hausner’s ratio. It is given in Table 3.

Table 3: Micromeritic properties of proliposome powders

Formulation	Angle of Repose (θ)	Carr’s Index	Hausner’s Ratio
MPF1	29.6 ± 0.08	12.7 ± 0.12	1.12 ± 0.06
MPF2	21.2 ± 0.20	10.6 ± 0.06	1.03 ± 0.01
MPF3	27.3 ± 0.17	11.8 ± 0.25	1.11 ± 0.09
MPF4	28.9 ± 0.38	9.8 ± 0.53	1.19 ± 0.06
MPF5	25.4 ± 0.29	13.3 ± 0.32	1.16 ± 0.04
MPF6	26.8 ± 0.43	10.1 ± 0.19	1.09 ± 0.03
MPF7	29.3 ± 0.21	13.3 ± 0.32	1.15 ± 0.06
MPF8	28.4 ± 0.39	13.1 ± 0.18	1.18 ± 0.03
MPF9	25.3 ± 0.21	12.1 ± 0.12	1.16 ± 0.03

Characterization of various proliposome powder formulations:

All the other parameters like particle size, polydispersity index (PDI), zeta potential, entrapment efficiency, number of vesicle sizes and drug content were evaluated. All the formulations were in accepted limits. It is shown in Table 4.

In-vitro dissolution Study:

The in vitro drug release characteristics were studied and the cumulative percentage drug release for MPF1, MPF2, MPF3, MPF4, MPF5, MPF6, MPF7, MPF8 and MPF9 were 86.5, 84.6, 90.4, 92.2, 79.4, 85.4, 88.6, 92.3 and 97.4 at the end of 12 h respectively.

Table 4: Characterization of proliposome formulations

Formulation	Particle size (µm)	PDI	ZP(-mV)	EE%	No of vesicle per mm³× 10³	Drug Content (%)
MPF1	16.17± 0.11	0.226	17.8 ± 3.6	89.6 ± 2.5	2.91	96.02± 0.06
MPF2	35.86± 0.21	0.109	32.4 ± 4.3	96.8 ± 1.9	3.79	95.01± 0.03
MPF3	19.76± 0.33	0.386	20.9 ± 4.6	90.7 ± 1.6	3.02	92.11± 0.16
MPF4	12.35± 0.41	0.297	23.8 ± 6.0	90.3 ± 1.2	2.86	98.03± 0.06
MPF5	26.44± 0.32	0.218	22.3 ± 2.3	94.6 ± 1.8	2.76	93.01± 0.07
MPF6	38.77± 0.28	0.268	29.6 ± 4.2	91.8 ± 1.5	2.98	93.01± 0.06
MPF7	39.12± 0.27	0.221	31.33 ± 4.2	82.13± 1.5	2.81	96.01± 0.06
MPF8	49.78± 0.32	0.253	29.11±3.6	79.33± 1.8	2.73	95.01± 0.16
MPF9	50.33± 0.46	0.108	31.33 ± 4.2	97.22± 1.7	2.99	98.01± 0.26

Table 5: In-vitro dissolution study of formulated batches

Time(h)	MPF1	MPF2	MPF3	MPF4	MPF5	MPF6	MPF7	MPF8	MPF9
1	4.5	5.2	6.4	8.5	10.3	9.8	12.5	7.5	13.2
2	9.5	8.6	10.3	14.2	15.6	14.2	17.4	11.5	16.4
3	15.4	13.4	19.5	25.7	19.5	18.9	23.9	14.5	22.5
4	22.1	19.6	25.4	32.6	25.4	29.4	28.5	25.6	28.7
5	29.4	25.9	38.2	37.2	30.7	34.5	35.4	36.5	35.4
6	34.8	32.2	44.5	44.8	39.9	42.7	40.7	45.8	46.5
7	43.5	39.4	48.2	52.3	43.9	48.6	46.2	52.6	54.6
8	55.6	46.8	55.8	58.4	55.9	52.8	50.4	56.4	62.4
9	62.3	56.4	66.4	64.5	60.8	60.7	62.8	64.5	70.5
10	70.9	62.5	74.8	75.6	69.8	68.3	72.3	75.4	82.6
11	82.4	73.4	83.2	84.8	75.5	75.4	80.5	82.8	89.7
12	86.5	84.6	90.4	92.2	79.4	85.4	88.6	92.3	97.4


Figure 1a: SEM of pure metformin hydrochloride		Figure 1b: SEM of optimized proliposome formulation MPF9

Table 6: Result of stability study conducted at various temperature

Parameter	Particle Size (µm)		EE%		Drug Content%
*Days*	*At 25*± 2⁰ C	*At 4*± 2⁰ C	*At 25*± 2⁰ C	*At 4*± 2⁰ C	*At 25*± 2⁰ C	*At 4*± 2⁰ C
0	50.33± 0.46	50.33± 0.46	97.22± 1.7	97.22± 1.3	98.01± 0.26	98.01± 0.26
30	50.31± 0.15	50.32± 0.26	97.19± 1.6	97.12± 1.2	98.01± 0.23	98.01± 0.22
60	50.23± 0.26	50.22± 0.36	97.02± 1.4	97.02± 1.1	97.99± 0.22	97.21± 0.24
90	51.33± 0.43	49.93± 0.46	96.99± 1.73	96.92± 1.3	97.91± 0.24	96.99± 0.23

SEM study:

The SEM images were recorded for pure Metformin hydrochloride and Metformin hydrochloride loaded proliposome powder to examine the physical state and surface characteristics (Figure 1a). From the figure it is evident that Metformin hydrochloride exists in a crystalline form. The disappearance of crystalline form of LPV in proliposome confirms transformation to amorphous form.

Stability studies:

The stability studies suggest that the proliposome formulation was comparatively more stable when stored at refrigerated conditions, compared with room temperature. There were no significant changes in particle size, EE% and drug content in storage upto 3 months.

CONCLUSION:

The proliposome drug carriers could be successfully developed for the oral delivery of metformin hydrochloride. The proliposome powders did obviate the physicochemical problems of the liposomes. The formulation MPF9 seems to be more stable with desired physicochemical and intestinal permeability characteristics.

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Received on 07.01.2020 Modified on 10.03.2020

Research J. Pharm. and Tech. 2021; 14(2):747-751.

DOI: 10.5958/0974-360X.2021.00130.X