An approach for lacidipine loaded gastroretentive formulation prepared by different methods for gastroparesis in diabetic patients

Abstract

The present work deals with various attempts to prepare a gastroretentive formulation of lacidipine for treating gastroparesis. High density sucrose beads were modified by coating with certain polymers, but unfortunately sustained release could not be achieved. Granules were prepared by wet granulation technology using different combinations of polymers and a release of the drug was observed. The method failed to release the drug as per desired specifications. Polymeric coating followed by wet granulation was thought to be a better process to sustain the dissolution rate. The release rate can be modified by the incorporation of different polymeric coatings, but the mucoadhesive potential of granules was only 4.23% which might be due to its large size and the presence of other ingredients. Further, the lacidipine loaded microparticles were prepared by different methods such as compression, ionic gelation with TPP, ionic gelation with TPP and glutaraldehyde, spray drying and coacervation techniques. The formulations were evaluated for average particle size, surface morphology, entrapment efficiency, % yield and mucoadhesive potential. The microparticles prepared by compression method using HPMC K4M and SCMC as mucoadhesive polymers and BaSO₄ as high density diluent showed poor bioadhesion (8.3%) and poor release characteristics (100% in 120 min). Ionic gelation with tripolyphosphate yielded microspheres with poor mechanical strength. In order to improve its mechanical strength, TPP ionic gelation was combined with step-wise cross-linking with glutaraldehyde. The additional solidification step to improve mechanical strength left this procedure tedious, time consuming and cytotoxic. Spray drying method gave a very low yield with 46.67% bioadhesion. The method using CaCl₂ for ionotropic gelation showed the best results with regard to physical characteristics (well formed discrete, spherical surface microcapsule), particle size (88.57 ± 0.51), in vitro bioadhesion (67.33%), yield (>85%) and loading (>70%).

1. Introduction

Gastroparesis is one of the most severe and complicated gastric motility disorders. Ordinarily, strong muscular contractions propel food through the digestive tract. But in gastroparesis, the muscles in the wall of the stomach do not contract properly which the affect gastric emptying process (Bouras and Scolapio, 2004). In some cases, the pylorus does not open spontaneously or frequently enough which results in indigestion, nausea and vomiting. Gastroparesis is complicated to treat and the treatment options are limited. There are few available medications which provide symptomatic control rather than dealing with the underlying problem, slow acting and are associated with serious side effects. For this reason, they are often ineffective. Calcium channel blockers have a dilation effect on smooth muscles and thus can enhance gastric emptying in patients suffering from gastroparesis if targeted to the pylorus (Sultana et al., 2009). Such an effect can be attained by developing a high-density mucoadhesive drug delivery system, which can be retained at the site of action or near the pylorus and provide sustained local drug delivery. The mucoadhesive polymer selected should have strong hydrogen bonding groups, strong anionic charges, high chain flexibility required for penetration, surface tension that favors spreading of their chain into mucin network with high molecular weight. These polymer characteristics coupled with their high-density approach could solve the targeting purpose to pylorus with prolonged absorption at the desired site.

Targeted formulation for treating gastroparesis can be prepared using different polymers and drug delivery system. In the present work, a wide variety of natural and synthetic biodegradable polymers such as hydroxypropyl methylcellulose (HPMC), sodium carboxy methylcellulose (SCMC), polyvinyl pyrrolidone (PVP), alginate and chitosan were investigated to obtain optimistic formulation. However, the main concern is toward chitosan and alginate owing to its non-toxicity, biodegradation and bioadhesion like properties. Chitosan obtained by the deacetylation of chitin, carries a positive charge capable of interacting with the negative charge mucin. It has been investigated for its possible role in controlling the delivery of active medicaments, which is ascribable to its unique gel forming ability. Positively charged microparticles enhance its mucoadhesive properties and make chitosan suitable for delivery of drugs via the nasal or the gastrointestinal route (He et al., 1999).

The drug delivery system enough to fulfill the demand includes beads, granules and microparticles due to the small size when compared with tablets that favor better targeting, adhesion, penetration and localized drug delivery with prolonged action (Choy et al., 2011; Mohammed et al., 2010; Dettmar et al., 2001). Different methods can be utilized to prepare beads or microparticles such as solvent evaporation (Trivedi et al., 2008), precipitation polymerization method (Flake et al., 2011), chemical denaturation (Sultana et al., 2009), spray drying (Oliveira et al., 2005), emulsion droplet coalescence (Tokumitsu et al., 1999), ionotropic gelation (Prasanth et al., 2009; Silva et al., 2006) and the thermal crosslinking method (Sinha et al., 2004). The mucoadhesive potential varies with the type of cross-linking agent and the extent of crosslinking between the polymeric molecules.

In this study, an attempt has been made to formulate the gastroretentive drug delivery system of lacidipine (calcium channel blocker) which is a potent vasodilator. A variety of mucoadhesive and high density polymers were chosen and implemented. A comparison between different formulations was made on the basis of % yield, entrapment efficiency, release profile, and in vitro mucoadhesion in order to groom optimistic formulation.

2. Materials and methods

2.1. Materials

Lacidipine was received as a gift sample from Ranbaxy Research laboratory (Gurgaon, India; average M.W. 455.5). Chitosan (75% medium deacetylated) was purchased from Sigma Aldrich (Bangalore, India). Medium viscosity sodium alginate was procured from Merck, India (2% w/v solution 5000 cp). Eudrajit RL 100 and HPMC K4M were received as a gift sample from Dr. Reddy’s Laboratory (Bangalore, India). Glutaraldehyde (25% w/v) and sodium carboxy methylcellulose (SCMC) was purchased from S.D Fine Chem Ltd. Calcium chloride (CaCl₂.2H₂O), Span 80, TPP (tripoly phosphate), Tween 80 was obtained from the Central Drug House, India. All other chemicals and reagents were of analytical grade.

2.2. Methods

2.2.1. Preparation of high-density sucrose beads

Drug was coated on high-density sucrose beads (1 mm) by two methods: (a) adsorption method and (b) coating method. Adsorption method involves the adsorption of SCMC (2%) and Eudragit (2%) on sucrose beads received as a gift sample from Panacea Biotech. Ltd. (Mumbai, India). Lacidipine (2 mg) was dissolved in a polymeric solution (2% SCMC and Eudragit in ethanol). The same solution (0.5 mL) was allowed to adsorb on 500 mg sucrose beads and dried at 60 °C temperature in an oven. For the coating method, a coating solution was prepared and coated on sucrose bead by a spray gun (pressure: 30 kg/cm²; nozzle size 1 mm) in a coating pan. Different compositions of polymeric solution (% w/w) as shown in Table 1 were coated on sucrose beads until the weight of the beads was increased to 5%. The temperature of the blown air was maintained at 60 °C.

Table 1.

Composition of coating polymers for the preparation of high density beads (per 500 mg sucrose beads).

S. No.	Ingredients	I	II	III	IV	V	VI	VII
1	Eudragit L100	–	–	–	60	80	–	80
2	Ehylcellulose	90	80	70	40	20	–	–
3	HPMC	10	20	30	–	–	100	–
4	PVP K30	–	–	–	–	–	–	20
5	Ethanol	q.s	–	q.s	q.s	q.s	q.s	q.s

2.2.2. Preparation of granules

Granules were prepared by wet granulation technology (Table 2). Lacidipine (0.5% w/w), dibasic calcium phosphate and magnesium stearate were passed through 80 mesh. The weighed quantities of ingredients were mixed thoroughly in a dry blender and the required quantity of granulating agent (hydroalcohlic solution of HPC, Eudrajit, and EC) as specified in Table 2 was added slowly. Ethanol was mixed in portions until a slightly cohesive product was formed. The cohesive product was passed through an extruder and the extruded material was chopped to produce slugs having a diameter of about 1 mm and a length of 2–3 mm. The slugs were then spheronised by passage through a spheroniser, and the particles thus formed were dried to constant weight at 60 °C. The dried particles were sieved to separate those having diameters between 0.7 and 1.5 mm.

Table 2.

Composition of ingredients for the preparation of high density granules (per 500 mg granules).

S. No.	Ingredients	I	II	III	IV	V	VI	VII	VIII	IX
1	Lacidipine	2	2	2	2	2	2	2	2	2
2	DCP	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s
3	HPMC K4M	20	20	20	20	20	20	–	–	–
4	Eudrajit L100	–	–	–	–	3	5	10	–	–
5	Ethylcellulose	3	5	10	–	–	–	–	5	5
6	HPC	–	–	–	–	–	–	–	10	20
7	Magnesium stearate	1	1	1	1	1	1	1	1	1
	Ethanol	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s

All ingredients were expressed in % w/w.

DCP, dibasic calcium phosphate; HPMC, hydroxypropyl methylcellulose; HPC, hydroxypropyl cellulose.

2.2.3. Preparation of coated granules

Lacidipine (0.5% w/w), hydroxymethylcellulose (10% w/w), dibasic calcium phosphate (83.5% w/w) and magnesium stearate (1% w/w) were mixed together by doubling up in a dry blender. Ethanol was added in portions until a slightly cohesive product was formed. The mixture was then extruded, chopped into suitable lengths, spheronised and dried to form the core particles of the formulation. These granules were coated with different polymers according to the formulae discussed in Table 3. The sieved core particles were rotated in a small coating pan and the coating mixture was added in portions by a spray gun (pressure 30 kg/cm²; nozzle size 1 mm) to the pan up to 5% w/w increase in the granules weight. After each addition of coating mixture, air was blown into the pan at a temperature of 60 °C to assist in solvent removal. At the end of the addition of the coating mixture, the coated core particles were dried to constant weight and sieved to produce granules having a size between 0.8 and 1.2 mm.

Table 3.

Composition of coating polymers to prepare high density coated granules (per 500 mg granules).

S. No.	Ingredients	I	II	III	IV	V	VI	VII	VIII	IX
1	HPC	100	85	75	50	25	25	25	75	–
2	Eudragit L100	–	–	–	–	–	–	75	25	–
3	EC	–	15	25	50	75	–	–	–	25
4	HPMC K100	–	–	–	–	–	75	–	–	75
5	Ethanol	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s	q.s

2.2.4. Preparation of microparticles

2.2.4.1. Compression method

The lacidipine microparticles were prepared using different formulae as shown in Table 4, which included the formation of slugs (10 mm) by tablet press and then passing them through a sieve (60 mesh) to obtain microparticles.

• (i)Preparation of slugs: Weighed quantity of lacidipine (0.5%) and tween 80 (0.2–0.4%) was allowed to dissolve in methanol. The mixture was adsorbed on diluents (DCP; Mannitol; BaSO₄), mixed with a polymer in different ratios and passed through a 30 mesh sieve to form granules. The granules were then mixed with a lubricant (magnesium stearate; 1%) and finally compressed to 250 mg tablets using 10 mm punches.
• (ii)Preparation of microparticles: The prepared slugs were passed through a 60-mesh sieve to form microparticles and finally filled into capsules for in vitro dissolution.

Table 4.

Different formulae for the preparation of lacidipine microparticles.

Composition	CF 1	CF 2	CF 3	CF 4
Lacidipine	0.5	0.5	0.5	0.5
Lactose	q.s	–	–	–
Mannitol	–	q.s	q.s	q.s
BaSO4	–	–	–	50
DCP	10	–	50	–
SCMC	–	20	20	20
HPMC	–	–	20	20
Magnesium stearate	1.0	1.0	1.0	1.0
Tween 80	0.2%	0.4%	0.4%	0.4%

All ingredients were expressed in % w/w.

This batch was coded as F-CF.

2.2.4.2. Ionic gelation with tripolyphosphate

The chitosan microspheres were prepared with TPP by ionic crosslinking by the method described by (method A) (Ko et al., 2002). An attempt was made to improve the rigidity of the formulation by cross-linking of the prepared microspheres with glutaraldehyde (method B) (Table 5).

Table 5.

Methods for cross linking of chitosan TPP microspheres.

Method	Formulation code	Formulation
Method A	TPP	Ionic gelation with TPP
Method B	TPG	Ionic gelation combined with step-wise cross linking with glutaraldehyde

Method A: The drug (2 mg) was dissolved in methylene chloride (2:10) due to its water insoluble behavior. This organic phase was mixed with aqueous phase (5 mL) containing 4% w/v chitosan solution in acetic acid (2% v/v) by continuous stirring for 20 min. O/W emulsion was sprayed onto 20 mL of TPP solution (10% w/v) with continuous stirring using a spray gun having a pressure of 40 kg/cm² with a nozzle size of 1 mm. After the cross linking time (60 min), the chitosan microspheres were washed with distilled water repeatedly, filtered through a Whatman filter paper (No. 542, pore size 2.7 mm) and allowed to dry in a vacuum desiccator for 24 h at room temperature. The batch was coded as F-TPP.

Method B: In this method ionic gelation with TPP is combined with stepwise crosslinking with glutaraldehyde. Microspheres obtained by method A were further dipped in glutaraldehyde (2.5% w/v, 10 mL) for 8 h. The microspheres were collected over Whatman filter paper (No. 542, pore size 2.7 mm), washed (with distilled water to remove excess of glutaraldehyde) and dried at room temperature (37 °C) for 24 h in a vacuum desiccator. This formulation was coded as F-TPG.

2.2.4.3. Spray drying

The chitosan micropsheres were prepared by the spray drying technique. The chitosan solution to be spray dried was prepared by dissolving chitosan (0.5% w/v) in deionized water containing acetic acid (2% w/v). Lacidipine (100 mg) was dissolved in methylene chloride (10 mL) and then dispersed in a polymer solution by continuous stirring on a magnetic stirrer (1000 rpm) for 15 min. Glutaraldehyde solution (7.5 mL, 1% w/v) was added as a cross-linking agent to the above solution and stirred again for 10 min. Then the solution was sprayed dried by a mini spray drier (Table top model, SM Scientech, Mumbai, India) using an inlet temperature of 120 °C, outlet temperature of 80 °C with spray flow at a rate of 1.2 ml/min and a pressure of 50 kg/cm². This batch is denoted by code F-SD.

2.2.4.4. Ionotropic gelation with CaCl₂

A sodium alginate solution (6% w/v) was prepared by dissolving a weighed amount of sodium alginate in warm water (60 °C) with continuous stirring. After thorough mixing, the solution was kept for 20–30 min in order to make the solution bubble-free. In another flask, lacidipine (2 mg) was dissolved in methylene chloride (2.5 mL) and then the drug solution was allowed to disperse in a polymer solution (5 mL) by continuous stirring for 10 min. The resulting drug polymer solution was then sprayed onto 50 mL of cross-linking solution containing calcium chloride solution (10% w/v), chitosan (1% w/v solution in 2% acetic acid) and tween 80 (0.1% w/w). The solution was atomized through a spray gun having a nozzle size of 1 mm maintained at a pressure of 40 kg/cm² by an oil free air compressor piston. After the formation of sodium-alginate microcapsules, the suspension was kept in freezer at 8 °C for 12 h for solidification. The formed microparticles were then filtered through Whatman filter paper (No. 542, pore size 2.7 mm), washed twice with distilled water and allowed to dry in a vacuum desiccator for 24 h at room temperature. The batch is referred to as F-CC.

2.2.5. Particle size analysis

The average particle size was measured using an optical microscope with a calibrated ocular micrometer. 100 particles of each formulation were measured for analysis.

2.2.6. Morphological characterization

The shape and surface characteristics of the microparticles were followed by a scanning electron microscope (JSM 5610 LV SEM, JEOL, Datum Ltd., Japan) chamber in which samples of the microcapsules were dusted onto a double-sided tape on an aluminum stub. Afterwards, the coating was performed with gold using a cool sputter coater (Polaran E 5100) to a thickness of 400 A⁰. Photomicrographs were taken at the accelerated voltage of 20 kV and a chamber pressure of 0.6 mmHg.

2.2.7. Drug content of microparticles

Drug entrapment for microparticles was determined by the method previously reported with a slight modification (Sultana et al., 2009). Accurately weighed quantity of microparticles (50 mg) were crushed in a glass mortar-pestle and suspended in 10 mL of methanol. The suspension was sonicated (bath sonicator) for 30 min and filtered through a 0.22 μm Millipore filter. The filtrate was analyzed for drug content at 241.5 nm spectrophotometrically.

2.2.8. In-vitro release studies

The optimized formulations prepared by different methods were evaluated for in vitro release by USP dissolution apparatus 2 (paddle apparatus), using 900 mL of the dissolution medium (0.1 N HCl, pH: 1.2) and containing 1% PEG 400 as solubilizing agent. The rotating speed was adjusted to 50 rpm. At predetermined time intervals 10 mL sample was removed and the same sample was replaced with fresh medium. The samples were filtered using Millipore filter (0.22 μm) assembly and the drug content was estimated spectrophotometrically at 241.5 nm.

2.2.9. In-vitro bioadhesion

The mucoadhesive property of the prepared microparticles was evaluated by an in vitro adhesion testing method known as wash-off method as reported previously by Sultana et al. (2009). A constant number of microspheres (50) were spread onto rat stomach mucosa tied onto the glass slide using a thread. The prepared slide was hung onto one of the groves of a USP tablet disintegrating test apparatus and was given up and down movements for 1 h in the beaker of the disintegration test apparatus containing the gastric fluid (pH 1.2). At the end of the experiment, the number of microparticles still adhering to the tissue was counted. % bioadhesion was determined by the following formula:

$$\text{Percent}\text{bioathesion}=\frac{\text{No}\text{of}\text{athered}\text{microparticles}}{\text{No}\text{of}\text{applied}\text{microparticles}}\times 100$$

2.2.10. Drug release pattern from microspheres

Different kinetic equations like zero order (% release vs t), first order (log% release vs t) and Higuchi model (% release vs square root of time) were applied and R² values were calculated for the linear curves obtained by regression analysis of the above plots. In order to define a better release mechanism, the experimental data were further analyzed by Peppas equation, Qt/Qx = Ktn, where Qt/Qx was the fraction of drug release at time t, K was the kinetic constant and n (diffusional exponent) values could be obtained from the slope of a plot of log Qt/Qx vs log time.

2.2.11. High density potential test

A constant number of particles (50) were added in a beaker containing 20 mL of glycerol (100 mL, 1.261 g/cm³) as a medium placed on a magnetic stirrer. The speed of stirring was adjusted to 50 rpm. The time of settling of microparticles and the total number of particles settled in glycerol were noted by visual inspection.

3. Results and discussion

3.1. Polymer coated sucrose beads

The high density sucrose beads were used to target lacidipine directly to the pylorus. As these beads showed immediate drug release (solubilizes within few seconds), it required polymeric coating which was able to retard the drug release. Different polymers/combinations (Table 1) were tested to fulfill the desired target. SEM photomicrograph of the sucrose bead showed a spherical shape with approx 1 mm size. The drug and polymer particles adsorbed on its surface can be clearly visible in the images (Fig. 1a and b). Fig. 1c revealed a smooth surface of the coated bead free from any cracks or imperfections. As shown in Fig. 2, release by this method could not extend more than 40 min which was attributed to high solubility of sucrose in the release medium (0.1 N HCl). Thus the method was dropped meanwhile and not further continued.

Figure 1.

Lacidipine loaded sucrose beads prepared by (a) adsorption method in which 2% SCMC and Eudragit ethanolic solution was adsorbed on the beads; (1a) individual beads containing the adsorbed drug; (2a) zooming of the bead surface (b) coating method in which polymeric solution was coated over the bead in a coating pan; (1b) individual bead containing the adsorbed drug; (2b) zooming of the bead surface.

Figure 2.

Release profile of lacidipine loaded sucrose beads prepared by coating of different polymeric compositions in a coating pan by using a spray gun.

3.2. Granules

The granules were prepared by wet granulation technique according to the formulae given in Table 2. Granulation is a process in which particle components are held together by the chemical bonds of infinite strength (Radhika et al., 2009). This granulation process was carried out for two purposes, firstly to densify the mass containing drug and to increase the content uniformity as the dose is very small. Sustained effect can be achieved by the addition of certain polymers as a binder. The results of dissolution studies shown in Fig. 3 indicated that the formulation F-I, containing 20% HPC released 97.98% of the drug in about half an hour (Fig. 3a). Ethylcellulose has been used as a release retardant polymer in controlled release dosage form (Roni et al., 2009). Because of its hydrophobic nature, it reduces the penetration of the solvent molecules into the system. Thus the release rate can be governed by the permeability of the matrix system by solvent molecules (Zarzycki et al., 2010). Ethyl cellulose addition in the above formulation favored sustained effect with 81.29% (F-II), 55.98% (F-III) and 55.98% (F-IV) release at the end of 45 min (Fig. 3a).

Figure 3.

Release profile of lacidipine loaded granules containing different proportions of polymeric solution as binders.

Eudragit is a copolymer of acrylic and methacrylic acid esters, which is usually supplied as an aqueous dispersion containing approximately 30% solids. It is employed for several oral dosage forms allowing controlled release throughout the whole GIT when embedded in a matrix system. A formulation containing 10% Eudragit (F-VII) released 81.33% drug in 1.5 h when compared with F-V and F-VI containing 3% and 5% Eudrajit, respectively (Fig. 3b). Incorporation of a high concentration of Eudragit produces a more controlled effect, which is attributed to decreased penetration of the solvent in a matrix system containing the hydrophobic polymer, which consequently decreases the diffusion of the drug molecule. The formulations F-VIII and IX containing 10% and 20% HPC, release 100% drug within 60 min (Fig. 3b).

3.3. Coated granules

The sustained effect could not be achieved simply by the granulation process. After drug administration, the contents of the core of each granule are thought to dissolve quickly. This makes coating of polymeric solutions (preferably hydrophobic in nature) onto the granules mandatory to design the controlled release system. It is thought that polymeric coating around the core limits the subsequent release of the components of the core and, hence, produces the controlled sustained-release shown by the formulation.

As shown in Fig. 4a, when the granules were coated employing the mucoadhesive polymer alone (F-I), the release could not be sustained for more than 4 h. Whereas when ethylcellulose was incorporated along with the mucoadhesive polymer, the granules were found to be intact over a period of 6 h due to water insoluble nature of ethyl cellulose and the lacidipine release was spread over a longer period of time. When compared with ethyl cellulose concentration, the release was relatively faster with F-II (99.28% in 240 min) containing 15% ethyl cellulose than F-IV (99.82% in 6 h) possessing 75% ethyl cellulose. Formulation (F-VI) containing 25% HPC and 75% HPMC released 100% of the drug within 4 h. This bursting effect may be due to fast erosion of the two polymers in a dissolution medium that creates pores through which rapid diffusion of drug molecules is quite possible. Furthermore, to potentiate the mucoadhesive and sustained effect of granules, combinations of Eudragit and hydroxypropylcellulose were tried. Fig. 4b shows that F-VII (75% Eudrajit) and F-VIII (25% Eudrajit) release 81.25% and 91.2% drug, respectively, in 5 h. Thus the combination of polymers and their concentration must be optimized in order to provide sustain release. The best sustained effect was attained with formulation F-VII and, hence, selected for further studies. This batch of formulation was further coded as F-GC.

Figure 4.

Release profile of lacidipine loaded granules coated with different proportions of polymeric solution.

3.4. Microparticles

The microparticles prepared by compression method (slugging process), glutaraldehyde crosslinking, ionic crosslinking with TPP, spray drying, and ionotropic gelation with calcium chloride were compared for particle size, surface morphology, % yield, entrapment efficiency, in vitro drug release and% bioadhesion. Fig. 5 showed schematic representation for the preparation of different types of microparticles. The results of evaluation for different formulations are shown in Table 6.

Figure 5.

Schematic representation of different methods for the preparation of lacidipine loaded microparticles.

Table 6.

Physicochemical characteristics and entrapment efficiency of microspheres prepared by different methods.

Formulation code	Particle size (μm ± SD)⁎	Entrapment efficiency (% ± SD)	Bioadhesion (% ± SD)
F-GC	1420 ± 0.76 mm	100	4.23 ± 0.58
F-CF4	51.76 ± 0.12	100	8.33 ± 0.32
F-TPP	46.22 ± 0.86	87.68 ± 0.78	52.67 ± 1.15
F-TPG	39.37 ± 0.63	87.68 ± 0.78	46.33 ± 1.53
F-SD	63.33 ± 0.45	11.65 ± 0.92	46.67 ± 3.05
F-CC	88.57 ± 0.51	78.58 ± 0.85	67.33 ± 1.53

GC, wet granulation with coating; CF4, indicates compression method; TPP, tripolyphosphate; TPG, tripolyphosphate combines with glutaraldehyde; SD, spray drying and CC, ionotropic gelation.

^⁎Standard deviation (n = 3).

3.4.1. In vitro release study

Microparticles prepared by compression method were optimized on the basis of dissolution rate. Comparative drug release profiles of different formulations were given in Fig. 5. The percent drug release from Formulation CF-1 was found to be only 41.26% after four hours due to the poor solubility of lacidipine in 0.1 N HCl. In order to improve the dissolution rate of lacidipine, formulation CF-2 was prepared by replacing lactose with mannitol, which was used as a diluent. The SCMC was included in this formulation as a mucoadhesive polymer. This time there was an increase in the dissolution rate of lacidipine and release in 1 h was found to be 81.53%, which was desired. It has been reported that the combination of HPMC and SCMC was found to affect bioadhesion strength and drug release (Rathi et al., 2012). Based on these observations, further formulation (CF-3) was modified by incorporation of HPMC K4 100 as a mucoadhesive polymer. Although, the release was 91.33% at this time, the microparticles were floated in the dissolution medium. To make them sink to the bottom of the flask, dicalcium phosphate was replaced with barium sulfate, a well known high density diluent in formulation CF-4. This time the microparticles remained at the bottom of the flask, which was desirable.

The technique enhanced the dissolution of lacidipine, which was poorly soluble (0.47 μg/ml) with an aim of targeting directly to pylorus with the controlled release of active medicament. A 100% yield without any loss of drug was obtained. Although the dissolution rate was enhanced from 44.6% (F-CM: marketed formulation) to 84.33% (CF-4: microparticles), poor release characteristics (84.33% in 2 h) were a main drawback associated with this method (see Fig. 6).

Figure 6.

Comparative release profile of marketed formulation (CM) and microparticles prepared by the compression method (CF).

TPP is nontoxic and consists of multivalent anions which can form gel by ionic interaction with positively charged amino group of chitosan (SzymaSka and Winnicka, 2012). Ko et al. reported an increased probability of interaction of TPP with a higher concentration of chitosan (Ko et al., 2002). As a result strong walled microparticles having better entrapment efficiency and slow release characteristics could be obtained. Microspheres prepared by this method have a very poor mechanical strength that can be explained by their preparation method. The OH^- ions compete with tripolyphosphosphoric ions (${\text{P}}_3{\text{O}}_{10}^{5-}$) to react with protonated amino group of chitosan present on the surface of chitosan particles. After the formation of an outer layer, because of small size, OH⁻ ions possess a higher ability to permeate through the gelled layer into the inside matrix than ${\text{P}}_3{\text{O}}_{10}^{5-}$ ions. As a result, the cross-linking process was greatest on the chitosan droplet surface, and it decreased toward the center, resulting in poor mechanical strength (Park et al., 2002; Bodmeier and Paeratakul, 1989). As a result microparticles were more easily deformed in washing and drying steps. This drawback can be overcome by utilizing a stepwise cross-linking method with glutaraldehyde (GTA). It is noteworthy that the release profile was greatly improved when TPP ionic gelation was adopted before GTA crosslinking (Wang et al., 2006). The release of the lacidipine from the chitosan matrix involved initial swelling followed by diffusion of the drug molecules (kumar et al., 2012). Fig. 7 showed that a significant decrease in drug release was observed after stepwise cross-linking. Formulation TPP released 81.4% drug while TPG released only 54.85% in 6 h. Obviously both faster and high burst rates were observed when microparticles were prepared without GTA. As shown the release was greatly improved with reduced burst effect when the same particles were further cross-linked with GTA. This fact was due to the large amounts of amino groups (−NH₂) on chitosan that had been consumed by TPP and glutaraldehyde that allowed lacidipine to diffuse steadily. Furthermore, the structure of lacidipine-loaded microspheres was tightly packed, that retarded the diffusion of drug. These results are further supported by Sun et al. where release was observed for >48% after modifying GTA concentration (Sun et al., 2010).

Figure 7.

Comparative release profile of microparticles prepared by different methods: TPP ionotropic gelation of chitosan with TPP; TPG stepwise crosslinking with gluatarldehyde; SD spray drying; CC ionotropic gelation with calcium chloride and chitosan.

Particles obtained by a spray drying process followed by a chemical treatment with glutaraldeyde showed 98.64% drug release at the end of the 5th hour (Fig. 7). These results were similar to those in the study of He et al. who also reported fast release, accompanied by a burst effect (He et al., 1999). On the other side Raval and coworkers reported controlled release for 10 h when 1:2 ratio of drug: polymer was chemically cross-linked (Raval et al., 2010).

Alginate gel microspheres have been conventionally prepared by ionic interaction of alginate and CaCl₂. This approach has been associated with a certain drawback, namely poor entrapment efficiency which was due to the high porosity of alginate gel that resulted in loss of the entrapped molecule during the hardening and washing process; low stability of beads such as Ca²⁺ that could be removed by the chelating agents or high concentration of ions such as Na⁺, Mg²⁺. As Ca²⁺ was removed from gels, cross-linking diminishes and facilitated gel erosion which ultimately led to the loss of the entrapped materials (Onishi et al., 2010). These disadvantages can be overcome by the incorporation of polycations such as chitosan in the encapsulation medium strengthening the alginate beads and thus reducing drug loss (Sultana et al., 2012). The same system will reduce porosity and improve the stability of the capsule (Batubara1 et al., 2012). The interaction of chitosan and alginate results in the formation of such a dense matrix system that is expected to settle down near the pylorus. In vitro release studies showed that the drug was released from the optimized formulation in a sustained release manner for more than 7 h. Similar sustained effect up to 12 h through the optimization of chitosan and alginate concentration was observed by previous researchers (Arora and Budhiraja, 2012).

3.4.2. Particle size analysis

Granulation technology yielded lacidipine loaded granules of average size of (F-GC) of 1.42 ± 0.76 mm. Average size of microparticles prepared by the compression method (F-CM) was 51.76 ± 0.12 μm (Table 6). The average particles size of formulation F-TPP was 46.22 ± 0.86 μm. The particle size was decreased from 46.22 ± 0.86 (method A) to 39.37 ± 0.63 μm (method B) when the same microparticles (F-TPP) were cross-linked with glutaraldehyde which was due to an increase in shrinking during the cross-linking process. The particles prepared by spray drying and coacervation processes were small with an average size of 63.33 ± 0.45 and 88.57 ± 0.51 μm, respectively.

3.4.3. Drug content

Entrapment efficiency was 100% in the case of particles prepared by granulation technology and compression method as no loss occurred at any stage of processing. The particles prepared by ionotropic gelation process with TPP showed excellent entrapment efficiency (87.68% ± 0.78). Previous researchers reported 78.5% entrapment efficiency when 5-fluorouracil loaded micropsheres were prepared by a two step solidification process (24). However, 11.65 ± 0.92% entrapment efficiency was observed in particles prepared by spray drying technique which was attributed to the low yield of the particles (12.23%). These results were in agreement with the previous data reported by Desai and Park, where 39.3–45.8% yield of microparticles was observed with the spray drying process (Desai and Park, 2005). Entrapment efficiency was found to be 78.58% for the particles prepared by the coacervation process using CaCl₂ and chitosan as a crosslinker. Similarly, high entrapment efficiency, i.e., 84% was reported by previous researchers when microparticles were formulated using 2% w/v alginate, 0.75% w/v chitosan (pH 5.0), and 1.0% w/v calcium chloride concentration (Arora and Budhiraja, 2012).

3.4.4. Morphological characterization

SEM photomicrographs of particles prepared by compression method are shown in Fig. 8. The particles are irregular in shape with varying size distribution. Micropheres prepared by ionotropic gelation with TPP were rounded with irregular surfaces (Fig. 91a). In fact some images showed a clumping of particles (Fig. 92a). However well defined change in surface morphology was observed when the same particles were further cross-linked with glutaraldehyde. In this instance, microparticles were well rigidized with free flowing characteristics (Fig. 9b). SEM photomicrographs showed that spherical microspheres with smooth surfaces were obtained by the spray drying technique (Fig. 9c). The particles prepared by ionotropic gelation with CaCl₂/coacervation mechanism were spherical, discrete with smooth surfaces (Fig. 9d).

Figure 8.

Scanning electron microscope photomicrograph of microparticles prepared by compression method (a) zoomed (b) microparticles showing clumping and irregular shaped surfaces.

Figure 9.

Scanning electron microscope photomicrograph of microparticles prepared by different methods: (a) TPP: ionotropic gelation of chitosan with TPP; (b) TPG: stepwise crosslinking with gluatarldehyde; (c) SD: spray drying; (d) CC: ionotropic gelation with calcium chloride and chitosan.

3.4.5. In-vitro bioadhesion

As shown in Table 5, least mucoadhesions i.e., 4.23 ± 0.58% and 8.3 ± 0.32% were observed by the particles prepared by the granulation method (GM) and the compression method (CM). This is due to the presence of large amounts of excepients (like diluents, barium sulfate, and magnesium stearate) other than mucoadhesive polymers. The formulation F-TPP showed 52.67% mucoadhesion. Chitosan possess protonated amino groups which can interact with several negatively charged surfaces of mucin. It was found that bioadhesion was decreased from 52.67% (Method A; F-TPP) to 46.33% (Method B; F-TPG) depending upon the method of cross-linking which was due to the consumption of amino groups of chitosan by glutaraldehyde when stepwise cross-linking was employed. This decreased the availability of the free amino group to cross link with mucin. TPP ionic gelation combined with step-wise cross-linking with GTA resulted in microspheres having good mechanical strength with decreased bioadhesion. 46.67 ± 3.05% bioadhesion was observed when the chitosan microparticles were prepared by the spray drying method. In vitro wash off method showed 67.33% mucoadhesion for the formulation prepared by coacervation (F-CC) which was attributed to the fact that alginates had hydrogen-bonding groups (−OH and −COOH) that favored chemical bond formation at the mucin surface. The bioadhesion property of these microparticles can result in prolonged retention in the stomach mucosa and selectively release the drug at its target site.

3.4.6. Drug release pattern from microparticles

The drug release data of different microparticles were extrapolated by zero order, first order, Higuchi and Korsmeyer–Peppas equations to predict the mechanism of drug release and the results are shown in Table 7. The in vitro release profile of formulations could be best expressed by the Higuchi’s equation, as the plots showed highest linearity (R²: 0.98–0.99). The mechanism involved the absorption of water into the hydrophilic polymer matrix and the diffusion of drug molecules into the medium. To explore the diffusion mechanism, the data were further fitted into the Korsmeyer–Peppas equation. The formulations showed good linearity (R²: >0.99) with slope (n) values ranging from 0.29 to 0.46 indicating Fickian diffusion as a mechanism for different formulations from the compression method and ionotropic gelation with TPP. Fickian diffusional release occurs by the molecular diffusion of the drug molecule driven by a chemical potential gradient, while formulations SD and CC showed high linearity with slope values >0.6, which indicated non-fickian diffusion as a predominant release mechanism. The same system favored polymer disentanglement with erosion due to swelling of the hydrophilic glassy polymers (Maggi et al., 2002).

Table 7.

R² and n values for selected formulations.

Formulation code	Zero order	First order	Higuchi	Peppas	n	Mechanism of drug release
F-GC	0.9607	0.9451	0.9848	0.9925	0.2941	Fickian release
F-CF4	0.9855	0.9623	0.9965	0.9974	0.4552	Fickian release
F-TPP	0.9694	0.87	0.9954	0.9972	0.4179	Fickian release
F-TPG	0.9585	0.8932	0.9924	0.9965	0.3466	Fickian release
F-SD	0.9491	0.8415	0.9899	0.9911	0.6248	Non-Fickian release
F-CC	0.9563	0.8339	0.9932	0.9947	0.7249	Non-Fickian release

F-GC, wet granulation with coating; F-CF4, indicates compression method; F-TPP, tripolyphosphate; F-TPG, tripolyphosphate combines with glutaraldehyde; F-SD, spray drying and F-CC, ionotropic gelation.

3.4.7. High density potential

Due to the high density of glycerol, it was selected as the medium for analyzing the high density potential of the formulation. It was also expected that the density of the chyme in the stomach could not extend to that of glycerol. The result of the high density study is shown in Table 8. All formulations were settled in the glycerol within 4–11 s. The particle settlements ranged from 70.0% to 100%, respectively, depending upon the preparation method.

Table 8.

High density test for selected formulations.

S. No.	Formulation code	Particles settled (% ± S.D⁎)	Settling time (s)⁎⁎
1	F-GC	100 ± 0.00	5
2	F-CF4	96.67 ± 1.154	6
3	F-TPP	92.0 ± 2.00	7
4	F-TPG	95.33 ± 1.154	4
5	F-SD	70.0 ± 4.00	11
6	F-CC	96.67 ± 2.31	9

F-GC, wet granulation with coating; F-CF4, indicates compression method; F-TPP, tripolyphosphate; F-TPG, tripolyphosphate combines with glutaraldehyde; F-SD, spray drying and F-CC, ionotropic gelation.

^⁎Standard deviation (n = 3).

^⁎⁎Time of settling of particles in second.

4. Conclusion

Different formulations of lacidipine were prepared keeping in mind the density, high entrapment and high mucoadhesive potential. The high density sucrose bead was not able to maintain its release profile for more than 40 min. Granulation technology in fact was unable to provide a once daily lacidipine dosage regimen for gastroparesis. Therefore, the granules were coated with different polymers. The coating polymers granules can be manipulated in order to formulate an optimistic formulation. But the formulation F-GC lacks mucoadhesive potential which might be due to its large size and the presence of other ingredients (diluents). The formulation prepared by ionic gelation with TPP (F-TPP) has poor mechanical strength, relatively fast release and low mucoadhesive potential when compared with others (F-CC). The formulation can be cross-linked with glutaraldehyde to overcome these drawbacks. This additional solidification step to improve mechanical strength left this procedure tedious and time consuming. Moreover, rigidization with GLA, further reduces the mucoadhesive potential and increases the cytotoxicity of the formulation. The formulation prepared by spray drying produced small and spherical particles, but relatively suffered from low yield. Moreover, glutaraldehyde was added as a cross-linker, make it unsuitable for in vivo utility. The formulation F-CC (coacervation process) was found to be best owing to its surface properties, sustained effect and its comparatively high mucoadhesive potential.

Conflict of interest

The authors declare no conflict of interest.