INTRODUCTION:

Maintaining the necessary drug concentration at the site of action is essential for medication therapy in chronic conditions such as hypertension, and this concentration is directly correlated with the drug's plasma concentration¹. When typical oral dose forms are administered often, the drug's plasma level fluctuates, which might have harmful consequences or render the drug ineffective for therapeutic purposes².

Maintaining steady-state plasma concentrations of the drug throughout the course of therapy might help address these undesirable concerns, such as dosing frequency and unwanted effects. This is now attainable by designing in various modern drug-delivery systems³. Over the past few decades, significant efforts have been made to develop drug delivery systems with controlled release, which release the drug gradually over an extended period of time. One widely used strategy is the use of polymeric microspheres as drug carriers, which investigates the possibilities of drug localization at the target site, decreases drug toxicity and side effects, regulates drug release, maintains consistent drug bioavailability, and shields sensitive drugs (such as peptides and proteins) from enzymatic and chemical breakdown by entangling inside polymers^4-6. By creating microspheres, it is possible to investigate the use of an oral controlled release drug delivery system, reduce dosage and frequency of doses compared to traditional dosage forms, and accomplish all of these desired results⁷. When it comes to their conventional equivalents and dose, microspheres containing sustained release of amlodipine besylate are more well-tolerated.

Amlodipine besylate (benzenesulfonic acid salt of 3-O-ethyl-5-O-methyl-2-(2-aminoethoxymethyl)-4-(2-chlorophenyl)-6-methyl-1,4-dihydropyridine-3,5-dicarboxylate) is a synthetic dihydropyridine calcium channel blocker with antihypertensive and antianginal effects. Because of its slow and nearly total absorption from the digestive system, it is a good option for a formulation intended to be released in microsphere form over time. It is common practice to employ microspheres made of biodegradable and biocompatible polymers to enable prolonged, controlled medication release^3,8. The pharmaceutical industry frequently uses the single emulsion solvent evaporation method to manufacture microspheres⁹. To create the microspheres, amlodipine and Drugcoat® RLPO were utilized as the model drug and polymer, respectively. Ammonio methacrylate copolymer, Type A, also known as Drugcoat® RLPO, is a pH-independent, water-insoluble swelling polymer.

Microspheres' entrapment effectiveness and drug release properties are contingent upon both processing and formulation variables, including drug concentration, polymer types and levels, surfactant concentration, and amount of organic solvents used in preparation. In order to develop and optimize formulation variables for new goods and to improve existing products without raising costs, a number of experimental designs have gained a lot of traction^10,11. The Box-Behnken screening design, which outlines the ideal relationship between the formulation variables and responses for the development of the microspheres, is used in the design of experiments approach^12,13. For the process to yield a product with ideal qualities, the least amount of experimentation and time are needed. It has been demonstrated to be an effective method for analyzing a large number of variables and determining which ones are important¹⁴.

The purpose of this work was to create and evaluate response surface methodology-based amlodipine-loaded microspheres as a means of achieving a sustained release system for the treatment of hypertension. The effects of the independent factors, namely the drug to polymer ratio, the concentration of surfactant, and acetone, on the characteristics of amlodipine-loaded microspheres were investigated using a computer-aided optimization technique utilizing Box-Behnken design. The effectiveness of trapping, realistic drug loading, and in vitro drug release were assessed for microspheres.

MATERIALS AND METHODS:

Materials:

Amlodipine besylate was a gift sample from M/s. Hetero Drugs Limited. Drugcoat®RLPO was obtained from M/s. Vikram Thermo (India) Ltd., Ahmedabad, India. Span 80 were obtained from M/s. Loba Chemie Pvt. Ltd. Acetone, n-hexane and potassium dihydrogen phosphate were obtained from M/s. CDH Pvt. Ltd., New Delhi. Sodium hydroxide is obtained from M/s. Qualigens Fine Chemicals, Mumbai. All other materials used in this study were of analytical grade.

Box-Behnken experimental design (BBD):

To find the circumstances in which the procedure produced microspheres, several pilot tests were carried out prior to the design being implemented. This approach also defined the component levels. A three-level, three-factorial design (3³ factorial design) was used to investigate the effects of factors on the physicochemical properties of microspheres. With the aid of Design-Expert® Software (Version 11 Free Trial, Stat-Ease Inc., Minneapolis, MN), which enables evaluation by 13 experiments, the factors were investigated using the Box-Behnken design^15–17. BBD's goal was to assess each processing component's impact and determine which one was most important in determining the overall percentage of drug release and the efficiency of entrapment (dependent variables). The independent variables chosen were the drug to polymer ratio (A), the acetone volume (C, ml), and the surfactant proportion (B, %w/v). Table 1 lists the level of screening variables examined in this investigation. In order to determine whether or not an increase in factor value is advantageous, the BBD experimental design examines the input data, displays the rank ordering of the variables, and assigns a sign to the impact^18-20. Higher R² values were used to compare statistical measures and assess the design's relevance. Design-Expert® software was used to create two-dimensional (2D) contour plots and three-dimensional (3D) response plots that were obtained from the equations.

Preparation of encapsulated microspheres:

The process of water-in-oil (w/o) single emulsion solvent evaporation was used to create amlodipine-loaded microspheres. Table 1 lists the screening level of independent factors evaluated in this study. In short, according to the design, precisely weighed amounts of medication and polymer (factor A) were combined with a factor C. In order to create a w/o single emulsion, the resultant solution was injected using a syringe fitted with a 21.5 G needle into a 60 ml liquid paraffin mixture (light to heavy at a 1:1 ratio). The external phase of the combination contained different concentrations of span 80 (factor B), which is an emulsifier. Until the organic solvent evaporated from the solid microspheres, four bladed lab stirrers (Remi electrotechnik limited, Thane, India) were used to stir continuously for four hours at a speed of 1000 rpm. After being recovered through filtering, the microspheres were cleaned with petroleum ether, allowed to air dry for 48 hours, and then utilized in additional research^21,22.

Table 1. Screening variables and their levels in the Box-Behnken design

Independent variables	Code	Levels used (Coded)
Independent variables	Code	Low, (-1)	Intermediate, (0)	High, (+1)
Drug: Drugcoat RLPO	A	1:1	1:2	1:3
Surfactant (%, w/v)	B	0.05	0.075	0.1
Acetone (ml)	C	2	3	4

Characterization and evaluation of the microspheres

a) Fourier Transform Infrared (FTIR) spectroscopic study:

With FTIR spectroscopy, the chemical interactions between the medication and excipients were examined. FTIR Spectrometer (FTIR-8400S, Shimadzu, Japan) was used to record the IR spectra of amlodipine and the produced microsphere. To do that, a mixture of 1 mg of sample and 40 mg of KBr was shaped into a disk using a manual press. The scan range that was used to record the spectra was 4000 – 400 cm⁻¹ ²³.

b) Percentage recovery:

After preparation, the microspheres were recovered and dried overnight at room temperature and percentage recovery (yield) is calculated using the below equation 1 ^24,25.

Quantity of microspheres

Yield (%) = ------------------------------------ × 100 …(1)

Total Quantity of solid added

c) Drug loading and Entrapment efficiency (% EE)

The dried microspheres were powdered and 50 mg of powder sample was dispersed in 10 ml acetone. It was placed in an ultrasonic bath for 15 min and then filtered through a 0.22 mm Millipore filter. The filtrate was suitably diluted with phosphate buffer (pH 6.8) and the absorbance of the solution was taken at 238 nm using a UV-Vis spectrophotometer (Shimadzu, Japan) to determine the amount of Amlodipine present in the microspheres. Drug loading and entrapment efficiency (% EE) were determined using the following equations 2 and 3 respectively^24,25.

Quantity of drug in microspheres

Drug loading (%) = --------------------------- × 100 …(2)

Quantity of microspheres

Actual quantity of drug determined

Entrapment efficiency (%) = ---------------- × 100 …(3)

Theoretical quantity of drug

Among all the formulation, microspheres having higher encapsulation efficiency were chosen for further characterization study.

d) Scanning Electron Microscopy (SEM):

Scanning electron microscopy (FESEM-S 4800, Hitachi, Japan) was used to examine the morphology of microspheres at an 8.6–8.8 mm working distance and 1.0 kV accelerating voltage. The size, shape, and surface properties of the particles were investigated²⁶.

e) In vitro drug dissolution studies:

Using 900 ml of phosphate buffer (pH 6.8) as the dissolve medium, drug release from microspheres was tested for 12 hours at 37 ± 0.5°C and 100 rpm in a USP XXVII, type I tablet dissolving tester (Electrolab TDT–06T, Mumbai, India). Phosphate buffer pH 6.8 dissolving media was made in accordance with Indian Pharmacopoeia 2007²⁷. For the test, microspheres corresponding to 10 mg of amlodipine were utilized. To keep the sink condition, aliquots of the dissolving medium were taken out at prearranged intervals and replaced with new dissolving media. A Whatman filter paper no. 41 was used to filter the samples. After an appropriate dilution, the amlodipine concentration of each sample was measured using a 1 cm cell and UV-visible spectroscopy with a λ_max of 238 nm. To keep the total volume constant and preserve sink conditions, the removed volume was promptly supplied with the equivalent volume of new medium. Three copies of each experiment were conducted. In order to investigate a potential mechanism for drug release from the microspheres, the release data were assessed kinetically.

RESULT:

Characterization and Evaluation of Microspheres:

a) FTIR spectroscopic study:

Table 2 Box-Behnken (3³ factorial) experimental design matrix (in coded level) and experimental results

Runs	Response^a
Runs	Recovery (%)	Drug loading (%)	EE (%)	CPR
1	92.17 ± 2.43	41.26 ± 1.83	82.51 ± 1.62	79.81 ± 1.08
2	84.73 ± 3.56	26.87 ± 1.16	80.62 ± 1.63	76.59 ± 0.93
3	82.49 ± 2.68	27.24 ± 1.72	81.74 ± 2.09	81.75 ± 1.14
4	90.62 ± 3.81	37.64 ± 1.47	75.28 ± 2.16	74.52 ± 1.46
5	76.39 ± 1.59	19.36 ± 1.36	77.44 ± 2.61	61.85 ± 0.59
6	72.47 ± 3.24	17.73 ± 2.14	70.94 ± 2.26	52.47 ± 0.37
7	85.32 ± 1.75	27.09 ± 1.57	81.37 ± 2.36	84.63 ± 1.41
8	90.13 ± 4.71	40.62 ± 1.62	81.24 ± 1.83	77.94 ± 1.05
9	84.53 ± 2.48	28.21 ± 1.47	84.63 ± 1.86	86.18 ± 0.84
10	78.39 ± 3.73	19.59 ± 1.85	78.38 ± 2.51	68.48 ± 0. 97
11	79.61 ± 2.92	24.83 ± 1.16	74.58 ± 2.34	74.32 ± 1.52
12	89.12 ± 2.76	39.67 ± 2.14	79.35 ± 2.14	75.83 ± 1.36
13	77.14 ± 1.73	19.57 ± 1.27	78.28 ± 2.18	63.52 ± 0.98

Table 3. Analysis of variance for entrapment efficiency

Source	Sum of Squares	d.f.	Mean Square	F-value	p-value	Remark
Model	121.43	3	40.48	8.07	0.0064	Significant
A-DP:RLPO	22.34	1	22.34	4.45	0.0640	Significant
B-Span	81.09	1	81.09	16.16	0.0030	Significant
C-acetone	18.00	1	18.00	3.59	0.0907	Significant
Residual	45.16	9	5.02	--	--
Cor Total	166.59	12	--	--	--

d.f. = degree of freedom

a b c

d e f

Figure 4. Contour plot (a, c, e) and response surface plot (b, d, f) showing the effect of independent variables on entrapment efficiency.

Table 4. Analysis of variance for drug release

Source	Sum of Squares	d.f.*	Mean Square	F-value	p-value
Model (Quadratic)	1127.94	9	125.33	112.39	0.0012	Significant
A-DP:RLPO	477.10	1	477.10	427.86	0.0002	Significant
B-Span	212.18	1	212.18	190.28	0.0008	Significant
C-acetone	7.22	1	7.22	6.47	0.0843	Significant
AB	28.73	1	28.73	25.76	0.0148	Significant
AC	0.0484	1	0.0484	0.0434	0.8483	--
BC	0.1296	1	0.1296	0.1162	0.7556	--
A²	317.59	1	317.59	284.81	0.0005	Significant
B²	2.98	1	2.98	2.68	0.2004	--
C²	0.0720	1	0.0720	0.0646	0.8158	--
Residual	3.35	3	1.12	--	--	--
Cor Total	1131.29	12	--	--	--	--

*d.f. = degree of freedom

a b c

d e f

Figure 5. Contour plot (a, c, e) and response surface plot (b, d, f) showing the effect of independent variables on cumulative percentage release of amlodipine.

Table 5. Statistical parameters for response

Parameters	Model	Std. Dev.	Mean	C.V. %	R²	Adjusted R²	Predicted R²	Adeq. Precision
% EE	Linear	2.24	78.95	2.84	0.7289	0.6386	0.4500	7.8149
CPR	Quadratic	1.06	73.68	1.43	0.9970	0.9882	N.A.	35.720

Equation (5) represents the polynomial model that describes the relationship between the formulation process variables and cumulative percentage medication release.

CPR = + 81.75 – 7.72A + 5.15B + 0.9500C + 2.68AB – 0.1100AC – 0.1800BC – 11.79A² – 1.14B² – 0.1775C² …..(5)

Tables 4 and 5 present the model's p-value, F-value, mean square, and R² derived from the ANOVA of the cumulative percent release design.

DISCUSSION:

The microspheres' recovered total weight varied from 72.47±3.24% to 92.17±4.71%. The yield and percentage loading of the formulations declined as the Drugcoat® RLPO concentration increased (Table 2). Amlodipine's FTIR spectrum revealed principal peaks at 3296 cm⁻¹, secondary N-H stretching, C–C stretching of the aromatic ring signals at 1494 cm⁻¹, C=O stretching of the carbonyl group signals at 1697 cm⁻¹, C–H stretching of the aromatic ring signals at 2983 cm⁻¹, and aliphatic C–H stretching vibrations at 2926 cm⁻¹. The FTIR spectra of optimized amlodipine-loaded microspheres showed values that were quite close to each other (Figure 1b), indicating that there was no evidence of a distinct kind of amlodipine interaction with polymers. It was discovered that microspheres ranged in size from 10 μm to 100μm. The microspheres were distinct, roughly spherical, and had less porosity, according to SEM pictures.

Water-insoluble swelling polymer Drugcoat® RLPO is capable of regulating medication release from microspheres. The cumulative percentage of drug release from the pure drug and the microspheres loaded with amlodipine is displayed in Figure 2 as a function of the dissolving time. Among the prepared microspheres Run 7 and Run 9 and the optimized formulation (Run 14) showed maximum drug release of 84.63% w/w, 86.18% w/w and 86.76% w/w respectively. The possibility of changes in the drug release mechanism was evaluated by fitting the data to various kinetic model equations of zero order, first order, Hixson–Crowell, Higuchi, and Korsmeyer–Peppas models. It was found that the data were best fitted into Korsmeyer–Peppas model equation with good linearity (R² = 0.99) and a value of the slope (n) < 0.45. This value of release exponent, n, corresponds that the Fickian diffusion is the dominant mechanism of drug release with these formulations.

The polynomial model and the ANOVA for entrapment efficiency and cumulative percent drug release, it can be found that the Drugcoat® RLPO had a significant negative effect and the span 80 had a positive effect on the % EE and CPR. It means that higher entrapment efficiency or CPR can be obtained at a lower level of factors A and a higher level of factor B. From 3D response surface plots and 2D contour plots, it was observed that the %EE was increased with a decreased level of Drugcoat® RLPO-Drug ratio (Figure 4: a & b; c & d) and an increased level of Span 80 (Figure 4: a & b; e & f). Surface response plots and contour plots represent the CPR is greater with a medium level amount of Drugcoat® RLPO-Drug ratio (Figure 5: a & b; c & d) and with a higher level of span 80 (Figure 5: a & b; e & f).

The ANOVA for drug release indicating that the p values of 0.0289 and 0.0012 respectively for both linear and quadratic models were found to be less than 0.05 indicating that both models are significant.

Optimization and Validation:

Utilizing the Design Expert® software's point prediction function based on a desirability factor's proximity to 1, an optimized microsphere formulation was created. Its components included a drug/polymer ratio of 1:1.53 (A); 0.1% w/v Span 80 in the external oil phase (B); and 3.99 ml of acetone (C). The dependent variables' projected values (EE: 84.63% and CPR: 86.18%) for the composition mentioned above fell within the intended range. Using the anticipated independent factors, a checkpoint experimental batch was created and its dependent variables were described. The response variables' anticipated and observed values (EE: 85.12% and CPR: 86.76%) showed a strong connection, indicating the validity of the optimized formulation. The predicted values, observed values, and percentage prediction error for the optimized formulation is shown in Table 6.

CONCLUSION:

The amlodipine loaded microspheres were successfully formulated using the single emulsion-solvent evaporation technique. Box-Behnken (3³ factorial) design was used to study and optimize the effect of three independent factors on the % entrapment efficiency and percentage drug release. The optimized formulation (Run 14) exhibited entrapment efficiency 85.12% and cumulative percentage drug release 86.76% at 12 h. The morphology of optimized microparticles was roughly spherical in shape. Such experimental design approach may be applied in the development of microparticulate systems to produce microspheres with high entrapment efficiency and provide sustained release of amlodipine besylate.

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Received on 13.06.2024 Revised on 02.12.2024

Accepted on 12.02.2025 Published on 02.08.2025

Available online from August 08, 2025

Research J. Pharmacy and Technology. 2025;18(8):3655-3661.

DOI: 10.52711/0974-360X.2025.00526

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Run	Factors				Response
	A	B (% w/v)	C (ml)		EE (%)	CPR
14	1:1.53	0.1	3.99	Observed value^a	85.12±1.94	86.76±1.32
				Predicted value	84.41	86.18
				% Prediction error	0.83	0.66