INTRODUCTION:

Topical drug delivery involves the local delivery of drugs anywhere in the body by using vaginal, rectal, ophthalmic and skin as topical route. Skin is the largest organ of the human body for topical delivery of drugs¹. The idea of using skin for topical delivery of drugs is since ancient time. Different ancient cultures used pastes, creams and plasters in treatment of various diseases². But now a day these ancient methods of drug delivery were replaced by novel topical drug delivery systems, which offers a various advantage over conventional systems like reduction in side effects, avoidance of 1^st pass metabolism and improves patient compliance³. Various strategies are being used for the delivery of drugs by topical routes which include sonophoresis, nanoparticles, patches, microneedles, and vesicular drug delivery systems⁴.

Among these strategies vesicular drug delivery systems appear most promising. These systems are highly ordered assemblies of phospholipids and were 1^st developed by Bangham in 1965. So these systems are called as bangham bodies^5.6.7. A number of different vesicular systems have been developed like liposomes, niosomes which provides targeted and controlled and controlled release of drugs⁸. But in most of cases these vesicles becomes ineffective in drug delivery due to low entrapment efficiency, instability etc. to overcome these limitations new vesicular formulation have been introduced such as ethosomes and transferosomes. Ethosomes contain high concentration of ethanol which improves pemetration of drug across skin by increasing flexibility and deformability. But this high concentration of ethanol in ethosomes results in unfavourable results. Manca et.al., developed a new approach to enhance the vesicular properties of these systems by modifying the fluidity of lipid bilayers. These vesicular systems were named as glycerosomes⁹. These carriers do not contain any harmful substance in their composition, hence fully accepted for topical delivery¹⁰. Minoxidil is used as vasodilator and as hair tonic. We formulated this drug as topical vesicular hair tonic¹¹.

MATERIALS AND METHODS:

Characterization of model drug:

Organoleptic characteristics:

The Organoleptic characteristics i.e. color, odour and taste of the drug were determined by using descriptive terminology.

Identification of model drug:

Identification of drug sample was carried out by different analytical techniques such as UV/VIS spectroscopy, IR spectroscopy, melting point, solubility analysis.

Absorbance spectra of Minoxidil in pH 6.8 Phosphate buffer:

Stock solution of minoxidil (100μg/ml) was prepared in phosphate buffer of pH 6.8 and scanned spectrophotometrically with the help of UV double beam spectrophotometer (Shimadzu 1700) in a range of 200 nm to 400 nm¹².

FT-IR spectrum:

Fourier Transform Infrared Spectrophotometer (Bruker) was used for structural analysis of drug. A small amount of Minoxidil was taken and scanned in the range 4000 to 400cm^-1.

Melting point:

A melting point of model drug was determined by capillary melting point apparatus.

Partition Coefficient:

The partition coefficient of the model drug was determined by Shake Flask method¹³.

Concentration of drug in n – octanol

P_o/w------------------------------------------------

Concentration of drug in water

Solubility studies of drug:

Solubility determination:

The equilibrium solubility of the model drug was determined by shake flask method¹⁴

Absorbance of Test × conc.of standard × Dilution Factor

Concentration = -----------------------------------------------------------------

of Test Absorbance of Standard

Design and Development of Glycerosomes:

Fabrication of Glycerosomes:

Method of preparation of Glycerosomes:

Glycerosomes were formulated by lipid thin film hydration method¹⁵. Firstly minoxidil (50mg/ml of medium) was dissolved in ethanol in a round bottom flask. Simultaneously phosphotidylcholin (30mg/ml), stearylamine (10% of lecithin) and Cholesterol (5 mg/ml) were dissolved in chloroform. Both the solutions were mixed and solvent was evaporated until a complete dryness was achieved under reduced pressure by using rotary evaporator¹⁶. A dried thin film was formed at the bottom of the flask and hydrated with varying concentration of glycerol in phosphate buffer of pH 6.8. The resulted solution was homogenized by ultrasonication to obtained vesicles of nanometric size¹⁷.

Optimization of various parameters of Glycerosomes by Full Factorial Design:

Nine formulations of minoxidil loaded glycerosomes were prepared based on 3² factorial designs¹⁸, as summarized in Table 1. Two independent variables were selected which were Sonication time (X₁), and Glycerol concentration (X₂) as given in Table 2 and with respect to these two dependent variables were selected which were % cumulative drug release after eight hours(Y₁), and Entrapment efficiency (Y₂).

Table 1: Formulations of Glycerosomes

Batch code	X₁ (sec)	X₂ (%w/v)
F1	-1(50)	-1(20)
F2	0 (60)	-1(20)
F3	+1(70)	-1(20)
F4	-1(50)	0(30)
F5	0(60)	0(30)
F6	+1(70)	0(30)
F7	-1(50)	+1(40)
F8	0(60)	+1(40)
F9	+1(70)	+1(40)

Table 2: Test Factors for Optimization of process parameters

Factor

Name

Units

Low Level (-)

Middle Level (0)

High

Level (+)

A (X₁)

Sonication Time

sec

B (X₂)

Glycerol conc.

% w/v

Characterization of Glycerosomes:

Surface and shape analysis by using Transmission Electron Microscopy:

The surface characteristics of prepared vesicles were determined via Transmission Electron Microscope¹⁹ (H-7500 Hitachi, Japan).

Zeta potential analysis:

Zeta Potential of optimized formulation (F7) was determined by using laser doppler electrophoresis (Zetasizer, Malvern).

Drug entrapment efficiency (%):

Method for drug entrapment efficiency (%):

Small quantity of glycerosomal suspension was applied drop wise in the center of the sephadex gel G-50 bed columns. Columns were centrifuged²⁰ at 2500rpm for 4 min. Elutes was take off. After this vesicles were lysed by absolute alcohol and sonicated for 10 minutes^21,22. Concentration of minoxidil in sample was determined with the help of UV Visible spectrophotometer at 288.1 nm.

The percentage drug entrapment was determined by using following equation:

Observed drug content

Entrapment Efficiency = ------------------------------------- × 100

Initial drug content

In vitro drug release studies:

In vitro drug release studies were carried out by using modified Franz diffusion cell²³. To perform the drug release study, cellophane membrane was fitted horizontally on the receptor compartment of franz diffusion cell. The receptor compartment was filled with phosphate buffer of pH 6.8. The diffusion cell was mounted on magnetic stirrer and temperature was maintained at 37± 0.5ºC²⁴. Vesicular formulation equivalent to 5% drug was applied on cellophane membrane and covered with aluminum foil. At appropriate time interval aliquots (5ml)²⁵ was withdrawn and restored by an equal volume of fresh phosphate buffer of pH 6.8 to maintain sink conditions. The sample was determined spectrophotometrically at 288.1nm.

Study of in vitro drug release kinetics:

The in vitro release kinetics of prepared formulations was evaluated model dependent methods (Higuchi, Korsmeyer-Peppas, zero order, and first order model²⁶. The obtained after in-vitro release studies was subjected to fit in above four models and evaluation was done on the basis of value of regression coefficients.^23,27

Stability studies:

A stability study of optimized formulation (F7) was performed by storing them in air tight container at several temperatures [4 ± 1ºC²⁸ and 25ºC]. Samples were studied for drug content at different time intervals. Initial drug content was taken as 100% for each formulation²⁹.

The value of degradation rate constant k, t_1/2 and t_10% of the optimized formulation was determined by plotting a graph between log % residual drug content vs. time.

RESULTS:

Organoleptic Characteristics:

Under investigation physical appearance of minoxidil was consider to be same as that of the official specifications.

Preformulation studies:

Identification of model drug:

UV Spectrum analysis:

Absorbance spectrum of minoxidil in pH 6.8 phosphate buffer:

On scanning of minoxidil in phosphate buffer of pH 6.8 over the recommended range in UV spectrophotometer, the drug exhibited three absorption maxima at 229.7, 262.1, 288.1 nm.

From these peaks, peak at 288.1 nm was selected, due to the highest intensity of the peak as shown in figure 1.

Figure 1: UV spectrum of Minoxidil in Phosphate buffer of pH 6.8

FT-IR Spectral analysis:

The FT-IR spectrum of Minoxidil as recorded in Figure 2 was found in accordance with the FT-IR of standard Minoxidil.

Figure 2: IR spectrum of Minoxidil

Melting point:

Drug melting point was found to be at 245ºC, which is in the standard melting point range of the model drug.

Partition Coefficient:

Drug Partition coefficient was found to be at 1.27, which is in the standard range of partition coefficient of the drug.

Solubility studies:

The solubility profile of drug in different media was as follows.

Table 3: Solubility data of Minoxidil in different media

Media	Solubility
Phosphate buffer of pH 6.8	Soluble
Propylene glycol	Soluble
Purified Water	Insoluble
Glycerol	Soluble
Methanol	Soluble

Drug excipient compatibility study:

Compatibility study showed that drug was compatible with all the excipients.

Design and Development of Glycerosomes:

Preparation of Glycerosomes:

Glycerosomes were successfully prepared via lipid thin film hydration.

Optimization of various parameters by Full Factorial Design:

The results obtained after implementing 3²Full Factorial design are summarized in Table 4.

Calculation of Main and Interaction effects:

a). For Y₁: Cumulative Drug Release (%):

The main and interaction effects are summarized in Table 5 and 6. The effect of various coefficients is shown in Figure 3 and in Eq. 1.

Table 5: Complete matrix, including interactions, with calculated effects (Y₁)

Batch code	Main Effects		Interaction Effects			Response
Batch code	A	B	AB	AA	BB	Y₁
G1	-	-	+	+	+	58.92
G2	0	-	0	0	+	53.18
G3	+	-	-	+	+	50.12
G4	-	0	0	+	0	65.99
G5	0	0	0	0	0	61.16
G6	+	0	0	+	0	59.78
G7	-	+	-	+	+	68.17
G8	0	+	0	0	+	66.12
G9	+	+	+	+	+	61.08
Effect	-3.2	6.0	-0.29	0.04	-3.19	62.28

Table 6: Summarised results of main and interaction effects (Y₁)

Coefficient	Effect
X1	-3.2
X2	6.0
X12	-0.29
X11	0.04
X22	-3.19

The regression equation obtained after calculation of main and interaction effect is represented in Eq. 1 and the corresponding Pareto chart is shown in Figure 3.

Y₁= 62.28-3.2X₁+6.0X₂-0.29X₁X₂+0.04X₁²-3.19X₂²

Eq. 1

In Eq.1, X₁ coefficient had negative sign which indicate that the drug release from the glycerosomes were inversely related to time of sonication (X₁) and X₂coefficient had positive sign which indicate that the drug release from the glycerosomes were directly related to glycerol concentration (X₂). Magnitude of X₂ coefficient was greater than X₁coefficient; hence release is highly dependent on glycerol concentration. Simultaneous effect of both variables was negative but negligible.

Figure 3: Pareto Chart (Y₁)

b). For Y₂: Entrapment Efficiency (% w/w):

The main and interaction effects are summarized in Table 7 and 8. The effect of various coefficients is shown in Figure 4 and in Eq. 2.

Table 7: Complete matrix, including interactions, with calculated effects (Y₂)

Batch code	Main Effects		Interaction Effects			Response
Batch code	A	B	AB	AA	BB	Y₂
G1	-	-	+	+	+	80.24
G2	0	-	0	0	+	78.92
G3	+	-	-	+	+	77.12
G4	-	0	0	+	0	75.11
G5	0	0	0	0	0	73.31
G6	+	0	0	+	0	70.29
G7	-	+	-	+	+	87.91
G8	0	+	0	0	+	85.04
G9	+	+	+	+	+	82.12
Effect	-2.3	3.09	-0.6	-0.25	9.02	73.07

Table 8: Summarised results of main and interaction effects (Y₂)

Coefficient	Effect
X1	-2.3
X2	3.09
X12	-0.6
X11	-0.25
X22	9.02

The regression equation obtained after calculation of main and interaction effect is represented in Eq 2 and the corresponding Pareto chart is shown in Figure 4.

Y₂= 73.07-2.3X₁+3.09X₂-0.6X₁X₂-0.25X₁²+9.02X₂²

Eq. 2

In Eq. 2, X₁ coefficient had negative sign which indicate that the entrapment efficiency of the Glycerosomes were inversely related to time of sonication (X₁) and X₂coefficient had positive sign which indicate that the entrapment efficiency of the Glycerosomes were directly related to glycerol concentration (X₂). Simultaneous effect of both variables was negative but negligible.

Figure 4: Pareto Chart (Y₂)

Characterization of Glycerosomes:

Surface analysis and shape by using Transmission Electron Microscopy:

A surface characteristic of the optimized formulation (G-7) was determined by TEM as shown in Fig. 5. Vesicles were observed smooth, spherical and vesicular in nature, and morphologically same without clusters.

Fig. 5: TEM photograph of optimized formulation of (G-7) Minoxidil loaded Glycerosomes.

Zeta potential analysis:

Zeta potential optimized formulation (G-7) was analyzed by zetasizer (Malvern) and average zeta potential was found as 19.2 (mV).

Entrapment Efficiency:

Entrapment efficiency of nine formulations (G1-G9) is summarized in Table 9.

Table 9: Entrapment efficiency of Glycerosomes

Batch Code	Entrapment Efficiency (%) ± S.D.
G1	80.24±0.13
G2	78.92±0.24
G3	77.12±1.01
G4	75.11±0.12
G5	73.31±0.87
G6	70.29±0.75
G7	87.91±0.23
G8	85.04±1.03
G9	82.12±0.12

In vitro drug release study:

Drug dissolution study of nine formulations (G1-G9) is summarized in Table 10 and in Figure 6.

Table 10: In vitro drug release data of Glycerosomes

TIME (hrs)	G1	G2	G3	G4	G5	G6	G7	G8	G9
0.5	13.01	11.1	9.95	14.07	12.89	10.21	16.88	15.11	12.76
1	22.1	22.03	21.07	25.79	24.15	22.86	27.73	25.94	23.57
2	33.07	30.89	32.08	32.98	30.91	29.66	35.71	34.12	29.62
4	43.82	42.09	39.15	44.93	44.19	42.95	50.13	47.08	45.83
6	55.93	53.04	46.17	58.65	56.07	55.03	61.98	60.09	56.41
8	58.92	53.18	50.12	65.99	61.16	59.78	68.17	66.12	61.08
10	65.57	64.81	62.09	70.09	67.19	66.02	72.81	69.86	67.15
12	70.65	68.21	66.06	74.18	72.41	67.14	78.16	75.28	72.36
14	74.85	72.91	73.07	79.62	76.18	75.67	84.13	83.29	81.44
16	76.91	73.56	72.45	81.21	78.31	75.95	87.14	84.03	82.67
18	79.12	74.65	73.12	82.14	81.23	79.24	89.34	82.67	85.78

Figure 6: In vitro drug release profiles of Glycerosomes

Drug Release Kinetics of drug release:

In order to study the release mechanism of present drug delivery system, the data collected from in vitro release of final optimized formulation (G7) were fitted into equations for the zero-order, first-order, Higuchi release model and Peppas equation. Table 11 enlists the values of regression coefficient obtained from various kinetics models.

Table 11: Regression coefficient (R²) found from various kinetics models

Batch code	Zero Order Kinetics	Higuchi Kinetics	Korsmeyer Peppas Kinetics	First Order Kinetics
G1	0.883	0.984	0.986	0.759
G2	0.888	0.981	0.978	0.745
G3	0.909	0.982	0.960	0.746
G4	0.936	0.984	0.980	0.629
G5	0.946	0.988	0.982	0.638
G6	0.944	0.985	0.984	0.659
G7	0.884	0.99	0.981	0.784
G8	0.890	0.99	0.978	0.772
G9	0.910	0.993	0.971	0.781

The interpretation of obtained data was carried out on the basis of values of the regression coefficients. The in vitro drug release shown that the regression coefficient values of optimized formulation (G7) for Zero order (R²= 0.887) shown in Figure 7, Higuchi’s model (R²= 0.99) as shown in Figure 8, Peppas model (R²= 0.988) Figure 9 First order (R² = 0.784) as shown in Figure 10. The value of regression coefficient (R²) for Higuchi model is highest. Hence, formulations follow Higuchi release kinetics and Fickian diffusion.

Figure 7: Zero order plot of the optimized formulation (G-7).

Figure 8: Higuchi plot of the optimized formulation (G-7).

Figure 9: Korsmeyer-peppas plot of the optimized formulation (G-7).

Figure 10: First order plot of the optimized formulation (G-7).

Stability studies:

Short term accelerated stability studies were performed for optimized formulation as per the procedure. The samples were analyzed for physical change, cumulative % drug release and drug content.

Effect of aging on residual drug content:

The stability studies indicate no physical change in appearance and color, indicating that the optimized formulation G7 was physically stable at the accelerated conditions. On chemical evaluation, it was observed that the % residual drug content of the optimized batch at the end of 3 months was found to be 97.45 ± 0.14% at 4 ± 1ºC and 86.04 ± 0.13% at room temperature as shown in Table 12, 13 and Figure 11, 12.

Table 12: Effect of aging on residual drug content at 4 ± 1˚C

Sr. No.	Days	Physical Change	Mean % Residual Drug Content (at 4 ± 1˚C)
1	0	No Change	100
2	15	No Change	99.69 ± 0.18
3	30	No Change	99.12 ± 0.06
4	45	No Change	98.58 ± 0.27
5	60	No Change	97.82 ± 0.09
6	90	No Change	97.45 ± 0.14

Table 13: Effect of aging on residual drug content at room temperature

Sr. No.	Days	Physical Change	Mean % Residual Drug Content (at room temp.)
1	0	No Change	100
2	15	No Change	98.54 ± 0.05
3	15	No Change	96.92 ± 0.12
4	45	No Change	93.84 ± 0.15
5	60	Color changes to slight yellow	89.92 ± 0.12
6	90	Color changes to slight yellow	86.04 ± 0.13

Figure 11: Effect of aging on percent residual drug content at 4 ± 1˚C and at room temperature.

Figure 12: Effect of aging on log percent residual drug content at 4 ± 1˚C and at room temperature.

Table 14: Effect of aging on cumulative % drug release before storage, storage at 4 ± 1˚C and room temperature.

Time (hrs)	Cumulative % drug release before storage	Cumulative % drug release (at 4 ± 1˚C)	Cumulative % drug release (at room temp.)
0	0	0	0
0.5	16.88±0.21	15.95 ± 0.45	12.75 ± 0.49
1	27.73±0.42	26.39 ± 0.35	23.19 ± 0.28
2	35.71±0.67	33.15 ± 1.12	28.97 ± 0.34
4	50.13±0.23	48.84 ± 0.76	39.54 ± 0.19
6	61.98±0.56	60.19 ± 0.24	44.76 ± 0.75
8	68.17±0.23	65.59 ± 0.39	49.69 ± 0.13
10	72.81±0.12	71.49 ± 0.23	54.06 ± 1.26
12	78.16±0.34	76.39 ± 0.98	58.96 ± 0.59
14	84.13±0.12	83.03 ± 1.45	65.83 ± 0.27
16	87.14±0.32	85.26 ± 0.62	69.27 ± 0.46
18	89.34±0.53	88.25±0.15	74.24±0.34

Effect of aging on cumulative percent drug release:

The stability studies indicate that the cumulative percent drug release of the optimized batch at the end of 3 months was found to be 85.26±0.62% at 4 ± 1˚C and 69.27±0.46% at room temperature as shown in Table 14 and Figure 13.

Figure 13: Effect of aging on cumulative % drug release before storage, storage at 4 ± 1˚C and at room temperature.

The percent residual drug content and log percent residual drug content was plotted against time (t) (Figure 11 and 12), which reflected an almost linear relationship. Degradation rate constant (k) was calculated from which the time required for 10% drug leaching was calculated.

Over three month investigation on stability of prepared formulation at 4±1˚C and room temperature, formulation stored at room temperature shows faster degradation and low shelf life i.e. 169 days. The formulations stored at 4 ± 1˚C have shelf life i.e. 356 days, which indicates that the ideal storage of formulation is cold storage

DISCUSSION:

Minoxidil, a pyrimidine N-oxide, is a potent K_ATP channel opener, and has been shown to act as a vasodilating agent, and the drug is used externally for treatment of androgenic alopecia with a plasma half-life of approximately 4.2 hours. Vesicular drug delivery system in the form of glycerosomes was formulated to a satisfactory level in terms of drug release and entrapment efficiency and to be used as an alternative to conventional dosage forms. Nine formulations of minoxidil loaded glycerosomes (G1-G9) were prepared based on 3²full factorial designs for optimization of glycerosomes. Two independent variables were selected which were sonication time (X₁), and glycerol concentration (X₂) and with respect to these two dependent variables were selected which were % cumulative drug release after eight hours (Y₁), and Entrapment efficiency (Y₂). On the basis of entrapment efficiency, and % cumulative drug release, G7 formulation was chosen as best formulation. Size and surface morphology of the prepared glycerosomes were evaluated by zetasizer and TEM. It was found that particles were fairly spherical in shape. Entrapment Efficiency of optimized formulation was found to be 87.91± 0.23%; this high level of encapsulation efficiency could be attributed to optimized parameters. The in vitro drug release profile of optimized formulation was performed in phosphate buffer of pH 6.8. Cumulative percent drug released was found to be 89.34±0.23% at 18^th hour. This could be accounted for by the slower release of drug from glycerosomes.

CONCLUSION:

In this study, a new vesicular carrier containing different amount of glycerol, has been developed and characterized, which exhibit many features for dermal application of cosmetic and pharmaceutical products, such as, the controlled release and targeting of drugs, occlusion associated with penetration enhancement, increase of skin hydration and excellent tolerability. Glycerosomes with anti-alopecia drug was successfully developed by lipid thin film hydration method. Morphological investigations showed that all vesicles exhibit a spherical shape with absence of aggregates and a smooth surface independent of their composition. Selection of the appropriate experimental conditions result in the production of minoxidil loaded glycerosomes having high entrapment efficiency (87.91 ± 0.23%) and high cumulative percent drug release (89.34 ± 0.23%) at 18^th hour.

Over three month of investigation on stability at 4±1˚C and room temperature, formulation shows the faster degradation at higher temperature. The results indicate that the ideal storage temperature is a cold place.

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Received on 09.05.2020 Modified on 17.06.2020

Research J. Pharm. and Tech. 2021; 14(5):2367-2374.

DOI: 10.52711/0974-360X.2021.00418

Batch Code	A: Sonication time (sec)	B: Glycerol conc. (%w/v)	Y1: Cumulative Drug Release After 8 hrs (%)	Y2: Entrapment Efficiency (% w/w)
G1	-1(50)	-1(20)	58.92	80.24
G2	0(60)	-1(20)	53.18	78.92
G3	+1(70)	-1(20)	50.12	77.12
G4	-1(50)	0(30)	65.99	75.11
G5	0(60)	0(30)	61.16	73.31
G6	+1(70)	0(30)	59.78	70.29
G7	-1(50)	+1(40)	68.17	87.91
G8	0(60)	+1(40)	66.12	85.04
G9	+1(70)	+1(40)	61.08	82.12