INTRODUCTION:

A Nanosuspension is a submicron colloidal dispersion of drug particles. A pharmaceutical nanosuspension is defined as very finely colloid, Biphasic , dispersed, solid drug particles in an aqueous vehicle, size below 1µm, without any matrix material, stabilized by surfactants and polymers, prepared by suitable methods for Drug Delivery applications, through various routes of administration like oral, topical, parenteral, ocular and pulmonary routes [1].

In addition, an increase in saturation solubility is postulated by particle size reduction due to an increased dissolution pressure. Depending on the production technique applied changes in crystalline structure of drug particles may also occur. An increasing amount of amorphous drug fraction could induce higher saturation solubility [2].

Received on 31.10.2014 Modified on 12.11.2014

Research J. Pharm. and Tech. 8(1): Jan. 2015; Page 38-43

DOI: 10.5958/0974-360X.2015.00008.6

It was hypothesized that nanosuspensions will enhance drug flux resulting from higher trans-membraneous concentration gradients. Nanosuspensions differ from nanoparticles. In nanosuspension technology, the drug is maintained in the required crystalline state with reduced particle size, leading to an increased dissolution rate and therefore improved bioavailability [3].

Felodipine is a calcium antagonist (calcium channel blocker). It is insoluble in water and is freely soluble in dichloromethane and ethanol. Felodipine is a member of the dihydropyridine class of calcium channel antagonists. The effect of Felodipine on blood pressure is principally a consequence of a dose related decrease of peripheral vascular resistance in man, with a modest reflex increase in heart rate. The bioavailability of Felodipine extended-release tablets are influenced by the presence of food. Felodipine also possesses unwanted side effects [4,5].

The main objective of the present study to prepare nanosuspensions using hydroxyl propyl methyl cellulose and hydroxyl propyl cellulose as surfectants as well as rate controlling polymer with objective to make drug release more targeting with the evident of the in vitro and ex-vivo evaluations.

MATERIALS:

Felodipine was obtained as gift sample from Ranbaxy Ltd., New Delhi. Hydroxyl propyl methyl cellulose and hydroxyl propyl cellulose were procured from S.D. Fine chemical, Kolkota. All other chemicals are of analytical grade, were procured from authorized dealer.

METHODOLOGY:

Drug polymers interaction study by DSC:

The DSC analysis of pure drug, HPMC, HPC and HPMC, HPC - drug physical mixtures (1:5) was carried out using Mettler Toledo (Model SW 810) DSC to evaluate any possible drug-polymer interaction. Samples (5.5 - 8 mg) were weighted accurately using a single pan electronic balance and heated in sealed aluminum pan at the rate of 5 °C/min in the temperature range of 25 – 45 °C under a nitrogen flow of 35 ml/min [6].

Formulation design and preparation of felodipine nanosuspension:

Precipitation method has been used to prepare nanosuspension particles of poorly soluble drug. The drug, Felodipine was dissolved in ethanol. Then this solution was mixed with a miscible anti-solvent that is water in presence of surfactants that is hydroxyl propyl methyl cellulose (HPMC K4M) and hydroxyl propyl cellulose (HPC) at concentrations of 0.25, 0.5, 0.75 and 1.0 % of respectively. Rapid addition of a drug solution to the anti-solvent leads to the super saturation of drug in the mixed solution and generation of ultra fine or amorphous drug solids. Optimized formulations of nanoemulsions were prepared by dissolving 2 % w/w of Felodipine in a 10 % w/w stabilizer. Stabilizer, lecithin is used to wet the drug particles thoroughly, prevent Ostwald’s ripening and agglomeration of nanosuspensions, providing steric or ionic barrier. Co-surfactant, bile salt is used to influence phase behavior when micro emulsions are used to formulate nanosuspensions. Ethanol is used as organic solvent. Then, 30 % w/w mixture (surfactant: co-surfactant at ratio of 1:1) was added slowly to the stabilizer, followed by the slow addition of distilled water to adjust the ﬁnal preparation to 100 % w/w. Sorbitol was used as osmogen. Sodium chloride was used as pH adjustment agent. Methyl paraben was used as preservative. All components were mixed and stirred at 3000 rpm.

Characterization of felodipine nano-suspension:

Nanosuspension droplet size analysis:

Droplet size distribution is one of the important physicochemical characteristics of a nanosuspension, was measured by a diffusion method using a light-scattering particle size analyzer Coulter LS-230. It measures the size distribution using the diffusion of laser light by particles. Polarization intensity differential scattering (PIDS) is the assembly consists of an incandescent light source and polarizing filters, a PIDS sample cell and an additional seven photodiode detectors. It is used to measure the droplets size distribution, like 0.5 ml suspension was introduced in the measure compartment (125 ml of water). The results were presented as the volume distribution [7, 8].

Viscosity determination:

The viscosity of the nanosuspension formulations was determined using a Brookﬁeld Cup and Bob Viscometer (Brookﬁeld Engi- neering Laboratories, Middleboro, MA) at 25 ± 0.3 °C and 100 rpm without diluting the nanosuspension formulations using spindle number 2 [9,10].

Refractive Index:

The refractive index, n, of a medium is defined as the ration f the speed, c, of a wave such as light or sound in a reference medium to the phase speed, Vp, of the wave in the medium [11, 12]. n=c/Vp ……………………. [1]

It was determined using an Abbes type refractrometer (Nirmal International) at 25 ± 0.5°C.

pH:

The apparent pH of the felodipine nanosuspension formulations was measured by Digital pH meter (Scientific Instruments, Mumbai) at 25 °C [13].

Tranmission Electron Microscopy (TEM):

Morphology and structure of the suspensions were studied using the transmission electron microscopy (TEM) TOPCON 002B operating at 200kV and of a 0.18nm capable point-to-point resolution. Combination of bright ﬁeld (BF) imaging at increasing magniﬁcation and of diffraction modes was used to reveal the form and size of the emulsions and to determine the amorphous or crystalline character of their components. In order to perform the TEM observations, the concentrated emulsion was ﬁrst diluted in water (1/10), a drop of the diluted suspension was then directly deposited on the holey ﬁlm grid and observed after drying. The emulsion appears dark and the surroundings are bright, a “positive” image is seen [17-19].

Drug entrapment efficiency:

About 10 ml of each nanosuspension formulation was taken and dissolved in 10 ml isotonic solution and kept overnight. About 10 mg (similar as in formulation) of drug was taken and dilution was made to 10 μg/ml. The dilutions were filtered and analyzed using UV-Visible spectrophotometer for their content uniformity. The absorbance of the nanosuspension formulations were read using one cm cell in a UV-Vis spectrophotometer. The instrument was set at 362 nm. The entrapment efficiency in each nanosuspension formulation was calculated based on the absorbance values of known standard solutions [14]. The drug entrapment efficacy of various felodipine nanosuspension was calculated by using following formula: Entrapment efficiency (%) = [(Entrapment efficiency)/ (Drug added in each formulation)] × 100.

Zeta potential:

Zeta potential is a technique which is used to measure the surface charge properties and further the long term physical stability of nanosuspensions, the instrument which is used to measure the surface charge is known as ZetaPALS .The measurements were carried out with diluted nanosuspension formulations and its values were determined from the electrophoretic mobility of the oil droplets. The minimum zeta potential of ±20 mv is desirable [15-17].

In vitro skin permeation studies:

In vitro skin permeation studies were performed using porcine abdominal skin with a Franz diffusion cell having an effective diffusion area of 0.785 cm and 4 ml receiver chamber capacity. Full-thickness porcine skin was excised from the abdominal region and hair was removed with an electric clipper. The subcutaneous tissue was removed surgically, and the dermis side was wiped with isopropyl alcohol to remove adhering fat. The leaned skin was washed with distilled water and stored in the deep freezer at 0°C until further use. The skin was brought to room temperature and mounted between the donor and receiver compartments of the Franz diffusion cell, with the stratum corneum side facing the donor compartment and the dermal side facing the receiver compartment.

The thickness of the skin was 50–70 lm as measured with a validated micrometer screw gauge. The receiver chamber was ﬁlled with phosphate-buffered saline (PBS) solution pH 6.8, stirred with a magnetic rotor at a speed of 50 rpm, and maintained at a temperature of 37 ± 1 °C. A quantity of the nanosuspension diluted with 50 % water was used for the release studies.

Since the nanosuspension had 2 % (20 mg/ml) of the drug, the ﬁnal drug concentration in the suspension was 10 mg/ml which was equivalent to the amount in the marketed formulation. One ml of the optimized nanosuspension formulation was placed in the donor compartment and sealed with parafﬁn ﬁlm to provide occlusive conditions. Samples were withdrawn at regular intervals (0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h) and sink conditions were maintained by replacement with fresh medium. Samples were ﬁltered through a 0.45- lm membrane ﬁlter and analyzed for entrapment efficiency by a validated UV-Visible spectrophotometric method at 362 nm. Each experiment was conducted in triplicate [19-21].

In vitro drug release kinetic study:

To find out the mechanism of drug release from hydrophilic matrices, the dissolution data of tablets of each batch was treated with different kinetic equations, namely zero order kinetic study, first order kinetic study, Higuchi, Hixon-Crowell, Korsemeyer and Peppas [22-25].

Histopathological examination of skin specimen:

The skin specimens subsequent to in vitro permeation studies were histopathologically examined. They were stored in 10 % formalin solution. Vertical sections were dehydrated using ethanol, embedded in parafﬁn for ﬁxing and stained with hematoxylin and eosin. These samples were then observed under a light microscope (Motic, Tokyo, Japan) and compared with the control sample. Three different sites of each were scanned and evaluated for possible abnormalities due to the formulation [26-29].

Stability studies:

During the thermodynamic stability of drug loaded nanosuspension following stress tests as reported. Nanosuspension formulations (Optimized nanosuspension formulation, F3) was subjected to six cycles between refrigerator temperature (4°C) and 40°C. Stable formulations were then subjected to centrifugation test. Nanosuspension formulations were centrifuged at 3500 rpm and those that did not show any phase separation were taken for the freeze thaw stress test. In this the formulation were subjected to three freeze thaw cycles between 21 and 25°C kept under standard laboratory conditions. These studies were performed for the period of 3 months. Three batches of formulations were kept at accelerated temperature of 30, 40, 50 and 60°C at ambient humidity. The samples were withdrawn at regular intervals of 0, 1, 2 and 3 months and were analyzed for particle size, pH, refractive index and entrapment efficiency by stability-indicating UV-Visible spectrophotometric method at maximum wave length of 362 nm [30-33].

RESULTS AND DISCUSSIONS:

The DSC thermograms of felodipine, felodipine polymers physical mixtures showed almost similar identical melting endotherm which proves the compatibility of used polymers (HPMC and HPC).

The globule size analysis of the optimized formulations was done using a light-scattering particle size analyzer Coulter LS-230. The globule size values are shown in Table 2. The droplet size was in the ranges of 61.2±0.58 (F3) to 91.4±0.79 nm (F5). The deference in the droplet size between the formulations is not statistically signiﬁcant (p > 0.05). There is only a marginal deference in the mean globule size of formulations. The minimum droplet size was obtained with nanosuspension formulation F3. The viscosity of various felodipine nanosuspension formulations was in the ranges of 10.68±0.92 to20.55±1.01 cp (Table 2). Most nanosuspension possesses a very low viscosity and, therefore, their application may be convenient. It was observed that the viscosity of all the formulations is less than 21 cP. The felodipine nanosuspension formulation F3, has the lowest viscosity (10.68±0.92 cP) which is highly signiﬁcant (p < 0.01) as compared to the other formulations. Satisfactory refractive index was obtained with all the nanosuspension formulations. The refractive index was in the ranges of 0.48±0.44 to 0.82±0.21, as given in Table 2. The maximum refractive index was obtained with nanosuspension formulation F7; where as minimum refractive index was obtained with nanosuspension formulation F5. Monitoring the pH value is important for determining the emulsions’ stability because pH changes indicate the occurrence of chemical reactions that can compromise the quality of the final product. Forearm skin testing is standard in most clinical studies of skin and has pH values in the range of 4.2 to 5.9 for both sexes (Table 3). Satisfactory pH was obtained with all the nanosuspension formulations. The pH was in the ranges of 6.42±0.34 (Nanosuspension formulation F3) to 6.91±0.35 (Nanosuspension formulation F8). The pH value of all the felodipine nanosuspension formulation was in the skin pH range, which was nearer to neutral pH range, demonstrating that all the felodipine nanosuspension will be non toxic, non irritating and non allergic. Morphology and structure of the nanosuspension were studied using Transmission electron microscopy (TOPCON 002B). The nanosuspension appears dark and the surroundings are bright, a positive image is seen using TEM. Some particles sizes are measured using TEM, as it is capable of point to point resolution. The droplet agreement with the results obtained from droplet size analysis using zeta sizer that concludes all most all the particles are having uniform size as well as all globules are nano size. The entrapment efficiency of various felodipine nanosuspension formulations was in the ranges of 68.4±1.08 to 92.3±0.98 % (Table 3). The nanosuspension formulation F8 has lowest entrapment efficiency. The felodipine nanosuspension formulation F3, has the highest entrapment efficiency (92.3±0.98 %) which is highly signiﬁcant (p < 0.01) as compared to the other formulations. Zeta potential is a technique which is used to measure the surface charge properties and further the long term physical stability of nanosuspension. The zeta potential of various felodipine nanosuspension formulations was in the ranges of 12.13±0.16 to 13.31±0.33 mV, as given in Table 3. All most all the nanosuspension formulation showed uniform Zeta potential, demonstrating that all nanosuspension formulation would be stable. All most all felodipine nanosuspension formulations were able to release drug in controlled manner over extended period of time. The in vitro drug dissolution study revealed that all nanosuspension formulations released the drug up to 9 h. The felodipine nanosuspension formulation F5 and F6 released 100 % of drug in 9 h only, where as nanosuspension formulation F1, F2, F4 and F7 released all drug in 10 h and nanosuspension formulations F3 and F8 released complete drug in 12 h (Fig 1 and 2). The more controlled and constant manner drug release was observed from felodipine controlled release nanosuspension formulation F3 (Containing HPMC K4M 80 mg and ethyl cellulose 75 mg) as it released it 100 % of drug up to 12 h with minimum fluctuation of drug (Felodipine) concentration in blood stream. From the release kinetics data Table 4, it was confirmed that, the control release formulations F1 to F9 obeyed zero order kinetic model, independent of time and concentration. All the tablet formulations obeyed Korsemeyer and Peppas kinetic model which confirms the diffusion controlled release. The diffusion co-efficient data indicates that the tablet formulations F3 to F8 released the drug by diffusion following Fickian transport mechanism, where as tablet formulations F1, F2 and F9 released the drug by diffusion following non-Fickian transport mechanism.

Untreated porcine skin (control) showed normal skin with well-deﬁned epidermal and dermal layers. Skin appendages were within normal limits both in untreated and treated with nanosuspension formulation (F3) for 24 h. These observations support the compatibility of the formulation with skin. There were no apparent signs of skin irritation (erythema and edema) observed on visual examination of the skin specimens treated with nanosuspension formulations indicating absence of any skin irritation as a consequence of nanosuspension treatment. Skin irritation test revels that no erythema was observed on visual inspection after application of felodipine nanosuspension formulations on rat skin. Thus, the developed formulation is non-sensitizing and safe for use.

Short-term stability studies of felodipine nanosuspension optimized formulation, F3 showed that the nanosuspension was physically and chemical stable in other three formulations when being stored at 4°C for 3 months, as well as in the cold and heat cycles for 15 days, as no such significant changes in nanosuspension parameters like pH, viscosity, particle size and entrapment efficiency were observed. In the long-term stability assessment, the optimized formulation, F3 was stable for at least 4 months when being stored at ambient condition in amber glass bottles.

Table 1. Formulation design of felodipine nanosuspension formulations.

Formulations/ components	F1	F2	F3	F4	F5	F6	F7	F8
Felodipine	2	2	2	2	2	2	2	2
Lecithin	10	10	10	10	10	10	10	10
Surfectant	0.5	1.0	1.5	2.0	0.5	1.0	1.5	2.0
Co-surfectant	0.1	0.1	0.1	0.1	0.1	0.1	0.1	0.1
Methyl paraben	0.02	0.02	0.02	0.02	0.02	0.02	0.02	0.02
NaCl	1	1	1	1	1	1	1	1
Sorbitol	0.5	0.5	0.5	0.5	0.5	0.5	0.5	0.5
Distilled water	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.	q.s.

q.s. – Quantity sufficient. All components are presented as percentage manner. The total volume nanosuspension is 100 ml.

Fig 1. In vitro drug release comparative profile of felodipine nanosuspension formulation F1 to F4.

All points (Percentage drug release) represent mean value (n = 3).

Fig 2. In vitro drug release comparative profile of felodipine nanosuspension formulation F5 to F8.

All points (Percentage drug release) represent mean value (n = 3).

Table 2. Globule size, viscosity and refractive index of the nanosuspension formulations.

Formulation	Globule size (nm) (X±S.D.)	Viscosity (cp) (X±S.D.)	Refractive Index (X±S.D.)
F1	70.9±0.78	20.55±1.01	0.79±0.22
F2	75.5±0.91	15.00±0.98	0.57±0.34
F3	61.2±0.58	^a10.68±0.92	0.62±0.25
F4	88.3±0.88	14.25±1.05	0.53±0.48
F5	91.4±0.79	16.08±0.99	0.48±0.44
F6	84.6±0.94	19.34±1.11	0.66±0.31
F7	77.8±0.91	17.32±1.04	0.82±0.21
F8	62.5±0.78	15.78±1.06	0.58±0.19

Each value are represented as mean ± standard deviation

(n = 3). ^ap < 0.01 when compared to other nanosuspension formulations. Standard error of mean (SEM) < 0.641.

Table 3. pH, entrapment efficiency and zeta potential of the felodipine nanosuspension formulations.

Formulation	pH value (X±S.D.)	Entrapment efficiency (%)(X±S.D.)	Zeta potential (mV)(X±S.D.)
F1	6.65±0.31	69.3±1.08	12.63±0.19
F2	6.44±0.26	76.5±0.97	12.81±0.28
F3	6.42±0.34	^a92.3±0.98	12.13±0.16
F4	6.84±0.54	82.4±0.88	12.41±0.34
F5	6.69±0.29	87.9±1.07	13.22±0.44
F6	6.51±0.43	85.7±1.04	12.52±0.51
F7	6.75±0.22	75.3±1.01	13.05±0.23
F8	6.91±0.35	68.4±0.87	13.31±0.33

Each value are represented as mean ± standard deviation (n = 3). Standard error of mean (SEM) < 0.335.

CONCLUSION:

The felodipine nanosuspension formulation F3 also released drug in a constant manner irrespective of time with minimum fluctuation in drug concentration in blood stream explaining exhibition of less side effects.Thus the felodipine nanosuspension formulation F3 containing 0.75% HPMC K4M, could be concluded as the best optimized formulation for safe management of hypertension. The optimized felodipine nanosuspension formulation F3 was found to be stable in various storage conditions. Histopathological study reveals that no erythema was observed on visual inspection after application of felodipine nanosuspension formulations on rat skin. Thus, the developed formulation is non-sensitizing and safe for use.

ACKNOWLEDGEMENT:

The authors are grateful to H.O.D., Department of Pharmaceutics, L.B. Rao Institute of Pharmaceutical Education and Research, Khambhat, Anand, Gujarat, Department of Pharmaceutics, Royal College of Pharmacy and Health Sciences, Andhapasara Road, Berhampur, Ganjam, Odisha and Department of Chemistry, Khallikote (Auto.) College, Ganjam, Berhampur, Odisha for providing research facilities.

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Formulation	Zero order kinetics	First order kinetics	Higuchi equation	Korsemeyer- Peppas	Release Exponent (n)
Formulation	Regression co-efficient (r²)				Release Exponent (n)
F1	0.924	0.7261	0.8349	0.972	1.28
F2	0.861	0.7967	0.7665	0.906	1.66
F3	0.909	0.786	0.8159	0.933	1.12
F4	0.927	0.750	0.856	0.937	1.015
F5	0.898	0.589	0.809	0.873	1.035
F6	0.887	0.714	0.797	0.925	1.235
F7	0.899	0.658	0.810	0.927	1.176
F8	0.913	0.740	0.816	0.942	1.164