INTRODUCTION:

In the past two decades, the evaluation of medical treatments has shifted from a narrow focus on clinical effectiveness and safety to include patient-centered outcomes, particularly health-related quality of life (HRQoL)^1-2.

This evolution reflects the growing recognition among healthcare providers and researchers of the importance of HRQoL measures in understanding the broader impact of illnesses and the effectiveness of treatments. Comprehensive HRQoL scales assess various aspects of physical, psychological, and social functioning, which are critical for evaluating the influence of diseases like gastroesophageal reflux disease (GERD)³^,⁴. GERD, a prevalent digestive disorder affecting approximately 7.6%-30% of the Indian population, primarily manifests through symptoms such as heartburn and acid regurgitation⁵. Due to its significant morbidity and adverse effects on quality of life, GERD represents a serious health concern, emphasizing the need for a holistic approach to treatment that prioritizes patient well-being alongside traditional clinical measures⁶.

Statistics:⁷

The worldwide GERD statistics are given in Table 1

Table 1: Statistical data of GERD

Sl No.	Continents	% of Population Affected
1	North America	18.1% - 27.8%
2	Europe	8.8% - 25.9 %
3	East Asia	2.5% - 7.8%
4	Middle East	8.7% - 33.1%
5	Australia	11.60%
6	South America	23.00%
7	Russia	18% -46%
8	India	7.6% - 30%

Figure 1: Worldwide GERD Statistics

Gastroesophageal Reflux Disease (GERD) is a common condition, affecting up to 26% of the population weekly, with its global prevalence on the rise. The primary goals of treatment for GERD are to alleviate symptoms, promote esophageal healing, and prevent recurrences. While early treatments like H2-receptor antagonists and first-generation proton pump inhibitors (PPIs) helped manage GERD symptoms, newer second-generation PPIs, such as rabeprazole sodium, have demonstrated improved efficacy in treating acid-related conditions like GERD, peptic ulcers, and Zollinger-Ellison syndrome^8-10.

Despite these advances, Rabeprazole sodium, like other PPIs, has some notable drawbacks. It has a short half-life and is unstable in acidic environments, which can lead to inconsistent therapeutic outcomes, especially in long-term treatments. Additionally, Rabeprazole sodium’s selective action on active proton pumps means that nighttime acid production can still occur, particularly when dosing is not optimized. These factors can reduce patient adherence to the treatment and result in suboptimal outcomes.

To address these challenges, the formulation of Rabeprazole sodium into liposomes has been explored. Liposomes are microscopic spherical vesicles made of lipid bilayers, which act as carriers for both hydrophilic and lipophilic drugs¹¹. By encapsulating Rabeprazole sodium in liposomes, the drug’s stability is enhanced, protecting it from degradation in acidic environments, and improving its bioavailability. Liposomes also provide controlled and sustained release, allowing the drug to maintain therapeutic levels over a longer period, reducing the need for frequent dosing^12-13.

This advanced drug delivery system not only addresses the limitations of conventional PPI formulations but also offers targeted delivery, leading to better therapeutic outcomes and improved patient adherence. Liposomal Rabeprazole sodium, therefore, represents a promising solution for overcoming the current challenges in GERD treatment and other acid-related disorders.

Design-Expert software plays a vital role in the formulation of Rabeprazole sodium-loaded liposomes by employing a central composite design (CCD) to optimize key parameters. This software facilitates systematic exploration of the formulation space, allowing researchers to identify the optimal conditions for achieving desired liposome characteristics, such as size, encapsulation efficiency, and drug release profiles. With 13 experimental runs generated, Design-Expert enables efficient testing of various formulation combinations while minimizing resource use^14-15.

By analyzing in vitro drug release and entrapment efficacy, researchers can gain valuable insights into the performance of the liposomes under controlled conditions. The software's statistical capabilities help in identifying significant factors and interactions that influence formulation outcomes. Overall, the use of Design-Expert streamlines the development process, enhances formulation efficiency, and contributes to the creation of a more effective drug delivery system, ultimately improving the therapeutic potential of rabeprazole sodium in clinical applications.

MATERIALS AND METHODS:

MATERIALS:

Rabeprazole sodium (API) from Triveni Chemicals in Gujarat. Soya lecithin was obtained from Techno lb Kraft, Mumbai. Cholesterol was obtained from S D Fine-Chem Limited, Mumbai. Additionally, ethanol and chloroform were sourced from Karnataka Fine Chem, Bengaluru.

PREPARATION OF RABEPRAZOLE SODIUM LIPOSOMES:

The preparation of Rabeprazole sodium liposomes involves several key steps, starting with the use of the rotary evaporation method. First, the required amounts of cholesterol and soya lecithin are measured and dissolved in ethanol at a predetermined ratio in a round bottom flask. Following this, a rotary evaporator is employed to evaporate the ethanol, resulting in a thin lipid film coating the walls of the flask. Next, the appropriate amount of Rabeprazole sodium is accurately weighed and dissolved in phosphate buffer, then added to the flask containing the lipid film. To facilitate liposome formation, the flask is gently shaken to hydrate the lipid film. The mixture is then filtered using a vacuum filtration system, with the filtrate reintroduced into the flask to ensure maximum recovery of the product. Finally, the liposomal formulation is collected, allowed to cool to room temperature, and stored in a sealed container for further analysis or use^16-17.

EXPERIMENTAL DESIGN:

Rabeprazole Sodium liposomes were formulated by varying the concentrations of soya lecithin and cholesterol. A Central Composite Design (CCD) was utilized, focusing on two variables—soya lecithin (phospholipid) and cholesterol (stabilizer)—at two different levels. This approach was used to assess their impact on entrapment efficiency and in vitro drug release. The experimental batches were prepared based on this design (Table 2), which evaluates combinations of the two factors and includes five axial points, resulting in a total of 13 runs. The study employed the Response Surface Design-Central Composite Design, with 2 factors at 2 levels, using Design Expert software (Version 13, Stat-Ease Inc, Minneapolis, MN). To ensure statistical rigor, the run order was randomized to avoid time-related influences and maintain the independence of observations (Table 3).

Table 2: Variables in 2²Central Composite Design

Independent variable	Levels
	Low	High
Soya Lecithin (mg)	100	150
Cholesterol (mg)	10	30
Dependent variables
Y1: Entrapment efficacy
Y2: In-Vitro Drug Release

Table 3: Matrix of Central Composite Design for Rabeprazole sodium loaded liposomes

STD	RUN	FACTOR 1	FACTOR 2
		Soya Lecithin	Cholesterol
		(mg)	(mg)
6	1	160.355	20
5	2	89.6447	20
3	3	100	30
2	4	150	10
11	5	125	20
1	6	100	10
10	7	125	20
7	8	125	5.85786
13	9	125	20
8	10	125	34.1421
12	11	125	20
4	12	150	30
9	13	125	20

Statistical analysis, including ANOVA, was conducted using Design Expert. The F-value was derived by comparing the variance between treatments to the error variance. Additionally, the multiple correlation coefficient was calculated to determine how much variation in the data was explained by the model. All assumptions required for ANOVA were validated to ensure accurate and reliable results.

EVALUATION OF LIPOSOMES:

METHODOLOGY:

Fourier-transformed infrared (FTIR) spectroscopy:

The FTIR spectra of Rabeprazole sodium was carried out by IR-Affinity-1-FTIR Spectrophotometer,

Shimadzu, Japan, FTIR Spectrophotometer in the region of 4000to 600 cm−1¹⁹.

Drug content determination:

The drug content in the liposomes was quantified by dissolving 1 ml of the formulation in 9 ml of a chloroform-methanol mixture (2:1 ratio), and then diluting the solution to 100 ml with methanol. The resulting mixture was analyzed using a UV-Visible spectrophotometer at 284 nm, with methanol serving as the blank²⁰.

Drug content = (Actual drug content in Liposomes)/(Theoretical drug content)*100

The individual values for three replicates were determined, and their mean values are reported.

Zeta potential and particle size distribution:

The zeta measurement potential was carried out using a NanoZS (Malvern UK) employing a 532 nm laser at a hack scattering angle of 173°.²¹

Entrapment Efficiency (%EE):

Entrapment efficiency was measured using the centrifugation method. The liposome dispersion was centrifuged (REMI LJ 01, Mumbai, India) at 15,000 rpm for 40 minutes. The clear supernatant was collected to determine the amount of unencapsulated drug. The concentration of the free drug in the solution was analyzed using a UV spectrophotometer (Shimadzu-1800, Japan) at a wavelength of 250 nm²².

The percentage of drug encapsulation was calculated by the following equation:

Loading efficiency = (Actual drug content in Liposomes)/(Theoretical drug content)*1

In-vitro release study:

The in-vitro dissolution study was conducted using a USP Type 2 (Paddle) apparatus set at 100 rpm. Phosphate buffer (pH 7.4) was used as the dissolution medium, with the temperature maintained at 37±0.5°C. At specific intervals over a 10-hour period, 1 ml samples were taken from the dissolution apparatus and replaced with an equal volume of pre-warmed fresh buffer. The samples were then filtered using Whatman filter paper and diluted to the appropriate concentration with phosphate buffer (pH 7.4). Absorbance measurements were taken at 284 nm using a UV spectrophotometer.

Kinetic analysis of In-vitro release rates of Rabeprazole Sodium loaded Liposomes formulation:

To analyze the drug release kinetics and mechanism, the in-vitro release data for the liposomes were fitted to different kinetic models. These included zero-order (cumulative % release vs. time), first-order (log % drug remaining vs. time), Higuchi’s model (cumulative % release vs. square root of time), and the Peppas model (log cumulative % release vs. log time). The correlation coefficient (R²) and release rate constant (k) were calculated from the linear regression analysis of the curves generated by these models.

RESULTS:

The IR spectra of pure drug Rabeprazole Sodium liposomes:

Figure 2: IR spectrum of Rabeprazole sodium

The FTIR spectrum shows that there were no significant changes in the chemical integrity of drug and also indicates that the polymer and drug are compatible with each other.

EVALUATION OF RABEPRAZOLE SODIUM LOADED LIPOSOMES:

Determination of entrapment efficiency:

Table 4: percentage entrapment efficiency of formulation (F1-F13)

FORMULATION	ENTRAPMENT EFFICIENCY
F1	82.31±0.15
F2	69.76±0.19
F3	73.78±0.18
F4	82.78±0.12
F5	77.96±0.18
F6	79.36±0.17
F7	77.68±0.17
F8	80.45±0.15
F9	79.05±0.13
F10	80.88±0.16
F11	78.14±0.14
F12	85.05±0.16
F13	79.22±0.19
F14	84.97±0.16

Mean (X ± S.D) (n = 3)

Percentage in– vitro drug release of formulation (F1 – F14):

In-vitro dissolution study:(F1-F7):

Table 5: Percentage In-vitro release (F1 – F7)

Time (min)	F1	F2	F3	F4	F5	F6	F7
30	18.77±0.52	21.67±0.95	23.48±0.73	21.67±0.56	20.25±0.45	22.6±0.74	18.97±0.44
60	27.8±0.52	37.86±0.9	36.64±0.34	32.9±0.81	34.37±1.14	34.57±0.37	34.27±0.76
120	39.36±0.97	47.08±1.48	48.01±0.81	45.01±0.37	46.44±0.53	47.76±0.53	45.65±0.23
180	47.01±0.59	56.7±0.76	54.98±0.52	51.15±0.66	52.47±0.86	55.86±0.45	51.54±0.22
240	50.74±0.6	64.52±1.66	63.09±0.89	60.33±0.37	60.34±0.31	64.61±0.74	58.12±0.89
300	55.51±0.59	69.69±0.43	67.87±0.98	65.06±0.86	65.41±0.52	69.2±0.89	63.78±0.66
360	61.95±0.59	76.54±1.00	71.63±1.05	69.16±0.47	70.88±0.66	73.05±0.74	69.05±0.39
420	66.24±0.61	82.22±0.76	74.65±1.00	74.58±0.44	75.08±0.89	77.26±0.53	72.66±0.44
480	68.67±0.84	85.94±0.09	79.79±0.56	78.74±0.61	79.14±0.96	80.58±0.9	76.62±0.22
540	76.25±0.37	89.52±0.78	85.67±0.97	82.36±0.45	83.25±0.52	85.23±0.26	81.56±0.98
600	81.1±0.82	92.86±0.51	89.49±0.47	85.00±0.44	87.12±0.67	88.03±0.81	84.94±0.62

In-vitro dissolution study:(F8-F14)

Table 6: Percentage In-vitro release (F8 – F14)

F8	F14	F10	F11	F12	F13	F14
20.2±0.31	19.71±0.39	18.28±0.22	19.51±0.37	20.88±0.44	20.64±0.45	19.56±0.29
32.75±0.66	33.39±0.76	31.97±0.39	34.77±0.44	34.57±0.52	33.15±0.53	29.29±0.15
44.91±0.22	45.85±0.53	43.73±0.37	41.09±0.56	41.97±0.47	41.53±0.31	38.58±0.15
52.23±0.39	51.69±0.31	49.67±0.61	49.08±0.56	50.65±0.61	50.7±0.31	45.98±0.15
58.52±0.37	58.22±0.47	57.18±1	58.07±0.31	56.55±0.68	55.57±0.74	53.87±0.09
64.08±0.73	62.41±0.47	62.55±0.22	64.22±0.37	62.11±0.45	62.25±0.47	59.42±0.31
70.87±0.96	68.66±0.52	69.83±0.22	71.8±0.61	67.38±0.52	68.8±0.52	65.52±0.23
74.87±0.89	74.38±0.31	74.23±0.23	74.87±0.44	72.31±0.45	73.19±0.31	70.1±0.37
77.9±0.92	78.54±0.45	76.71±0.39	79.12±0.61	75.83±0.29	77.99±0.34	74.39±0.15
81.72±0.76	82.45±0.81	79.55±0.22	82.35±0.47	78.61±0.52	82.29±0.47	77.86±0.15
85.59±0.3	84.6±0.45	82.58±0.47	86.52±0.37	81.69±0.37	87.24±0.31	81.13±0.23

Determination of In-vitro drug release:

Table 7: In-vitro drug release formulation (F1-F13)

FORMULATION	*IN-VITRO* DRUG RELEASE
F1	81.09±0.82
F2	92.85±0.50
F3	89.49±0.47
F4	85.00±0.44
F5	87.11±0.66
F6	88.02±0.81
F7	84.94±0.61
F8	85.58±0.29
F9	84.60±0.44
F10	82.57±0.47
F11	86.51±0.37
F12	81.69±0.37
F13	87.24±0.30
F14	81.13±0.22

Mean (X ± S.D) (n = 3)

Observed response in Central Composite Design for Rabeprazole Sodium Liposomes:

Table 8: Observed response in Central Composite Design for Rabeprazole Sodium Liposomes

		FACTOR	FACTOR	RESPONSE	RESPONSE
		1	2	1	2
Std	RUN	A: Soya	B:	Entrapment efficiency (%)	*In-vitro* drug release (%)
Run	RUN	lecithin	Cholesterol	Entrapment efficiency (%)	*In-vitro* drug release (%)
6	1	160.355	20	82.31	81.09
5	2	89.6447	20	69.76	92.85
3	3	100	30	73.78	89.49
2	4	150	10	82.78	85.6
11	5	125	20	77.96	87.11
1	6	100	10	79.36	88.02
10	7	125	20	77.68	84.94
7	8	125	5.85786	80.45	85.58
13	9	125	20	79.05	84.6
8	10	125	34.1421	80.88	82.57
12	11	125	20	78.14	86.51
4	12	150	30	85.05	81.69
9	13	125	20	79.22	87.84

Response 1 :Entrapment efficacy:

Figure 3: percentage entrapment efficiency

Figure 4: 3D surface of percentage entrapment efficiency

Response 2: In vitro Drug release:

Figure 5: Percentage of in-vitro drug release

Figure 6: 3D surface of in-vitro drug release

OPTIMIZATION:

To obtain the optimized formulation the required limits of response values were clearly defined Entrapment efficiency of a minimum of 78.80% and maximum of 83.38%, In-vitro drug release of minimum of 81.09% maximum of 92.85%. The combinations of variables which resulted in formulation meeting the required specifications were calculated using the design expert software. The overlapping of obtained result over the predicted values confirms the practicability and validation of the model.

Table 9: Optimization of Rabeprazole Sodium Liposome

Name	Goal	Lower Limit	Upper Limit	Lower Weight	Upper Weight	Import-ance
A:Soya lecithin	Is in range	100	150	1	1	3
B: Cholestrol	is in range	10	30	1	1	3
Entrapment efficacy	Maximize	69.76	85.05	1	1	3
In vitro Drug release	Minimize	81.09	92.85	1	1	3

Constraints:

Figure 7: Graphical Optimization of Rabeprazole Sodium Liposomes.

Figure 8: Percentage In- vitro drug release of formulation (F1- F14)

KINETICS STUDIES OF RABEPRAZOLE SODIUM LIPOSOMES:

The release kinetics of all five formulations were analyzed by fitting the dissolution data to various models, including zero-order, first-order, and Higuchi models (Table 11). The higher R² values indicated that drug release from all formulations followed first-order kinetics and aligned with the Higuchi model. According to the Higuchi model, the release mechanism was determined to be governed by swelling and diffusion. To further examine the release mechanism, the Peppas model was applied to differentiate between Fickian diffusion, non-Fickian diffusion, and zero-order release. The release exponent (n) from the Korsmeyer-Peppas model was found to be less than 0.50 for all formulations, indicating that drug release followed a Fickian diffusion mechanism.

In- vitro drug release for Rabeprazole sodium loaded liposomes:

KINETIC EVALUATION OF RABEPRAZOLE SODIUM LOADED LIPOSOMES:

Table 10: Kinetic studies of formulation (F1-F13)

Formulation	Zeroorder(r²)	FirstOrder(r²)	Higuchi(r²)		Korsmeyer-Peppa’s
				Mechanism	r²	n	Mechanism
F1	0.912	0.9778	0.9951	Sustain release	0.9708	0.6571	Nonfickian Diffusion
F2	0.884	0.9896	0.9916		0.9621	0.6832
F3	0.8691	0.9766	0.9871		0.9531	0.6691
F4	0.8817	0.9888	0.9922		0.9618	0.6685
F5	0.8801	0.9876	0.9907		0.9634	0.6729
F6	0.8617	0.9866	0.9862		0.9585	0.674
F7	0.8778	0.9843	0.9889		0.9642	0.6701
F8	0.8832	0.9886	0.9922		0.9654	0.6714
F9	0.8838	0.9872	0.9909		0.9645	0.6703
F10	0.882	0.9854	0.9905		0.9696	0.6716
F11	0.8989	0.9913	0.9933		0.9674	0.673
F12	0.8775	0.9838	0.9901		0.959	0.661
F13	0.9036	0.986	0.9965		0.9658	0.6683
F14	0.9113	0.9921	0.9982		0.9711	0.663

CONCLUSION:

The formulation of Rabeprazole sodium-loaded liposomes using the 2² Central Composite Design in Design Expert software successfully identified an optimized formulation, F14. This formulation, containing 150 mg of soya lecithin and 30 mg of cholesterol, demonstrated high drug entrapment efficiency and controlled drug release, making it a promising candidate for further development. The results indicate that the optimized liposomal formulation can effectively balance maximum drug encapsulation with minimal release, enhancing its potential for improved therapeutic performance

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Zeta Potential	-9.87(mV)
Polarity	Negative
Temperature	25.0 ^OC

SI NO	Entrapment efficiency (%)	*In-vitro* drug release (%)	Drug content
1.	84.97±0.08	81.13±0.23	82.46±0.16

Figure 5: Percentage of in-vitro drug release

OPTIMIZATION:

Constraints:

Figure 8: Percentage In- vitro drug release of formulation (F1- F14)

Zeta potential of Optimized Rabeprazole Sodium Liposomes: