Chapter 5

FELODIPINE FORMULATIONS AND EVALUATION

5.1. PREPARATION, CHARACTERIZATION AND EVALUATION OF

FELODIPINE LOADED EUDRAGIT® RS100 NANOPARTICLES

5.1.1. Preparation of felodipine nanoparticles

Introduction

The bioavailability enhancement of poorly water soluble drugs is one of the main

targets of drug development during the last decades. Several techniques like particle

size reduction, solubilization, salt formation and preparation of solid dispersion

systems are frequently used for improving the bioavailability of these drugs.

Nevertheless, there are several disadvantages and limitations in the use of these

techniques. An effective way to address the bioavailability issues of the poorly soluble

drug is by making the polymeric nanoparticles. The embedded drug inside the suitable

polymer can prevent the crystal growth and particle aggregation, producing the

amorphous system (Sarkari et al., 2002) that can be stable for long period of time. The

polymeric nanoparticulate systems are also the promising carriers for oral sustained

drug delivery ( Vauthier et al., 2003; Amaral et al., 2007), that will be beneficial to the

patients for the long term treatment.

Felodipine is a calcium channel blocker used to treat systemic arterial hypertension.

Being BCS class II (Biopharmaceutics Classification System) drug, it has very low

water solubility and high permeability (Kim et al., 2006). The oral bioavailability of

this drug is limited and, thus, the enhancement of its bioavailability would be a useful

achievement. The incorporation of felodipine in polymeric nanoparticles can be used

for improving the bioavailability and for reducing the dosing frequency, which can

also improve the patient compliance.

Eudragit®

RS100, the co-polymer of poly (ethylacrylate, methyl-methacrylate and

chlorotrimethyl-ammoniumethyl methacrylate) is commonly used polymer for

developing the controlled release dosage forms (Pignatello et al., 2001). It is insoluble

in physiological pH and capable of swelling, which represents the good material for

the drug dispersion (Perumal et al., 1999). This characteristic may maximize the

cellular uptake of drug-polymer complex. Eudragit®

RS100 has been previously used

for delivery of anti-inflammatory and antihypertensive drugs (Pignatello et al., 2002;

Adibkia et al., 2007; Jana et al., 2014). In the present study felodipine-Eudragit®

RS100 nanoparticles were prepared, characterized and evaluated with the aim to

achieve the prolonged and effective delivery felodipine.

Materials

Felodipine was a kind gift from Cadila Healthcare Limited (Ahmedabad, India).

Eudragit®

RS100 (Evonik Industries AG, Germany) was obtained from Sandoz Ltd.

Mumbai. Lutrol®

F-68 (Poloxamer 188) was obtained from Sigma, Mumbai.

Distilled- deionized water was prepared with Milli-Q plus System (Elix 10, Millipore

corp. India). All other chemicals used were of the highest available grade.

Method of preparation

The felodipine loaded Eudragit®

RS100 nanoparticles were prepared using the

solvent evaporation technique with slight modification (Jain, 2000). Different ratios of

felodipine and Eudragit®

RS100 were dissolved in acetone at room temperature as

described in table 5.1.1. The resultant primary solution was added with a constant

flow rate (0.5 ml/min) into separately prepared aqueous phase containing poloxamer-

188. The mixture was then homogenized at various agitation speeds in an ice bath.

The resultant oil-in-water (O/W) emulsion was kept at room temperature for 24 h

under gentle stirring to evaporate the organic solvents. The obtained nanoparticle

formulations were centrifuged at 40,000 rpm, 4oC for 20 min (Sorvall Ultracentrifuge,

USA). The un-entrapped drug was removed by washing the pellets with double

distilled water. The recovered nanoparticulate suspension was then freeze dried to get

powdered sample and kept at freeze for further use.

The impact of various formulation and process variables on entrapment efficiency

were studied to optimize the nanoparticle formulation to obtain maximum drug

entrapment in the nanoparticles.

• The agitation speed was varied from 5000, 10,000, 15,000 and 17,000 rpm

while keeping the other parameters constant ( for further experiment the agitation

speed which produced the lowest particle size with highest entrapment efficiency will

be chosen);

• The various weight ratios of drug and polymer were taken for the nanoparticle

formulation keeping the agitation speed, organic phase to the aqueous phase ratio

constant.

Table 5.1.1. Formulation of felodipine loaded Eudragit® RS100 nanoparticles

Sl.

No. Formulation

Drug:

Polymer

Ratio

Wt. of

Drug

(mg)

Wt. of

Polymer

(mg)

Vol. of

OP (ml)

Vol. of

AP (ml)

Agitation

(rpm)

1 FEN1 1:2 20 40 10 20 15,000

2 FEN2 1:3 20 60 10 30 15,000

3 FEN3 1:4 20 80 10 20 15,000

4 FEN4 1:5 20 100 10 30 15,000

5 FEN5 1:4 20 80 10 20 17,000

6 FEN6 1:4 20 80 10 30 10,000

7 FEN7 1:4 20 80 10 50 5,000

Note: FEN indicates felodipine loaded Eudragit® RS100 nanoparticle; OP: organic phase; AP:

aqueous phase.

5.1.2. Characterization of nanoparticles

Determination of particle size and Zeta potential

Particle size analysis was performed by Photon Correlation Spectroscopy (PCS) with

Zetasizer 3000 (Malvern Instruments). The freeze dried powdered samples were

suspended in Milli-Q water (1mg/ml) at room temperature (25 °C) and sonicated for

30 sec in an ice bath before measurement to prevent clumping. The mean particle

diameter and size distribution of the suspension were assessed. Analysis was carried

out for three times for each batch of sample under identical conditions and mean

values were reported. The Zeta potential was also measured using the same suspension

and same equipment.

Determination of entrapment efficiency and drug loading

The entrapment efficiency (EE) was estimated by reverse phase High Performance

Liquid Chromatography (RP-HPLC) method (Mohanty et al., 2010). The drug loaded

nanoparticle solution of 1 mg/ml was prepared in methanol and 20 µL of the sample

was injected manually to HPLC equipped with Shimadzu LC-20AD PLC pump and

SPD-M20A PDA detector. The chromatographic separation was achieved by using

Phenomenex C18 (150×4.6 mm, 5µ) analytical column. The mobile phase used

consisting of methanol and water (80:20 v/v) was passed through 0.45 µm membrane

filter and degassed by ultrasonication. The flow rate was maintained at 1.0 ml/min and

the measurements were made at 240 nm. The column was maintained in ambient

condition using thermostat. The amount of the felodipine in the sample was

determined from the peak area correlated with the standard curve. The standard curve

was prepared under the same identical condition. The drug entrapment efficiency (EE)

and drug loading (DL) were calculated using following formula:

Weight of the drug in nanoparticles

EE (% w/w) = ×100

Weight of the drug added

Weight of the drug in nanoparticle

DL (% w/w) = ×100

Weight of the polymer and drug added

Scanning electron microscopy (SEM)

The particle shape and surface morphology of felodipine nanoparticles were

examined by scanning electron microscopy (SEM) (JEOL JSM-5610LV). Moisture

free lyophilized samples were consigned on aluminium stubs using adhesive tapes and

coated with gold using sputter coater (JEOL auto fine coater, Japan) and observed for

morphology at an acceleration voltage of 20 kV at high vacuum.

Atomic force microscopy (AFM)

The surface morphology of prepared nanoparticles was carried out using

atomic force microscopy (AFM). The nanoparticle suspension was prepared with

milliQ water and dried overnight in air on a clean glass surface and observation was

performed with AFM consisting of silicon probes with pyramidal cantilever having

force constant of 0.2 N/m. To avoid damage of the sample surface, the tip to sample

distance was kept constant. The scan speed of 2 Hz and 312 kHz resonant frequency

was used for displaying amplitude, signal of the cantilever in the trace direction and to

obtained images (Trapani et al., 2009).

Transmission electron microscopy (TEM)

Morphology of the particles was also examined using transmission electron

microscope. A sample of particle suspension was diluted with 3% w/v

phosphotungstic acid adjusted to pH 7.5 with potassium hydroxide corresponding to a

1:1 ratio before examination. One drop of sample was placed for 1 minute on a copper

grid coated with a formvar carbon film. The excess of sample was wicked away with

the aid of filter paper. The sample was then ready for analysis by TEM.

Fourier transforms infrared spectroscopy (FTIR)

The possible chemical interaction between the drug, polymer and prepared

nanoparticles was determined by FTIR analysis. Samples were mixed separately with

potassium bromide (200 - 400mg) and compressed by applying pressure of 200

kg/cm2 for 2 min in hydraulic press to prepare the pellets. The pellets of the

felodipine, polymer and the drug loaded nanoparticles were scanned with resolution of

2 cm-1

in the range of 4000–400 cm-1

.

Differential scanning calorimetry (DSC)

The physical status of the drug inside the nanoparticles was ascertained by the DSC

analysis (DSC-60, Shimadzu, Japan). Approximately, weighed 2 mg of native drug,

polymer and nanoparticles were placed separately into the different sealed standard

aluminium pan and were scanned between 25 ºC to 300

ºC with heating rate of 10 ºC/

minute under nitrogen atmosphere. An empty aluminium pan served as reference.

5.1.3. Evaluation of nanoparticles

5.1.3.1. In vitro evaluation

5.1.3.1.1. Drug release study

The in vitro drug release study of felodipine nanoparticles was carried out by

using bottle method (Devarajan and Sonavane, 2007; Jain and Saraf, 2009). The

nanoparticles and pure felodipine (each containing 5 mg felodipine) were suspended

in glass bottles containing 100 ml of phosphate buffer pH 6.8 (simulated intestinal

fluid). Glass bottles were placed in beaker and kept in incubator shaker throughout the

study (37 oC, 50 rpm). The in vitro release study was also carried out for marketed

tablet formulation (each containing 5 mg of felodipine) of felodipine. At specified

time intervals (1, 2, 4, 6, 8, 12, 18, 24, 48, 72 and 96 h) 10 ml

samples were collected and centrifuged. The supernatants were collected for analysis

and the precipitate resuspended in 10 ml of fresh phosphate buffer. The analysis was

carried out by RP-HPLC at 240 nm. All the measurements were carried out in

triplicate.

Analysis of drug release data: The in vitro drug release data were analyzed by various

mathematical models to determine the kinetics and the drug release mechanism from

the developed nanoparticle formulation. The drug release data were fitted with

mathematical models including zero order kinetic [Eq. (1)], first order kinetic [Eq.

(2)], Higuchi kinetic [Eq. (3)] and Korsmeyer-Peppas model [Eq. (4)].

Qt = K0t (1)

ln Qt = ln Qt – K1t (2)

Qt = Kh t1/2

(3)

Mt / M = Kp tn (4)

The plots were made: Qt vs. t (zero order kinetic), ln (Q0 - Qt) vs. t (first order kinetic)

and Qt vs. t1/2

(Higuchi model), where Qt is the percentage of drug release at time t, Q0

is the initial amount of drug present in the formulation and K0, Kt and Kh are the

constants of the equations and calculated from the slope of the line. The first 60%

drug release was fitted in Korsmeyer-Peppas model, Where Mt / M are the fraction of

drug release at time t, Kp is the rate constant and “n” is the release exponent. The value

of “n” is calculated from the slop of the plot of log of fraction of drug released (Mt /

M ) vs. log of time to characterize the different release mechanism (Shoaib et al.,

2006; Aydin and Pulat, 2012).

According to Korsmeyer- Peppas law value of release exponent, n, is

indication of mechanism of drug release from spherical particles. When the value of

‘n’ is 0.43, it indicates that the mechanism of drug release follows fickian diffusion;

when ‘n’ > 0.43 and < 1.00, drug release follows non- fickian (anomalous) diffusion.

A value of n=1 means that the drug release is independent of time, regardless of

geometry and follows zero order.

5.1.3.1.2. Permeation study across the intestine

The everted intestinal sac model was used to study the permeability of the

nanoparticles across the intestine following the reported procedure with slight

modifications (Schilling and Mitra, 1990; Agarwal and Khan, 2001). The isolated

intestinal segment from the albino rat was carefully everted using the glass rod and

rinsed with saline solution. Then the segment was cut and secured to the tip of a 1 ml

disposable syringe barrel. Modified Kreb’s Ringer bicarbonate (MKRB) solution of

pH 7.4 was filled within the intestinal sac as serosal fluid and placed in a bath

containing 50 ml of nanoparticle suspension (1 mg/ml) in MKRB solution on mucosal

side. The fluid was continuously bubbled with carbogen gas (mixture of 5% CO2 and

95% O2 gas). The temperature of the organ bath was maintained at 37 ± 0.5 oC. The

amount of felodipine was analyzed spectrophotometrically at 240 nm using standard

curve obtained from serial dilution of felodipine in phosphate buffer pH 7.4.

5.1.3.1.3. Stability study

Stability study ensures the safety and efficacy and possible storage condition

for the pharmaceutical formulations. Decomposition or degradation of the

pharmaceutical formulations may develop due to environmental factors like

temperature, humidity, radiation, light, air etc. and due to interaction with other

chemical constituents/excipients in formulation or due to the nature of container used

for packing. Hence, it is necessary to perform stability testing for assuring safety and

efficacy and acceptability of the pharmaceutical formulations.

The lyophilized felodipine nanoparticle formulation was kept in glass vials and

stability study was carried out in three different storage conditions [ICH Q1A (R2)]

viz. long term study (25 ± 2 °C / 60% RH ± 5% RH) for one year, intermediate study

(30 ± 2 °C / 65% RH ± 5% RH) for six months and accelerated study (40 ± 2 °C / 75%

RH ± 5% RH) for six months.

The nanoparticles were evaluated at intervals of 0, 3, 6 and 12 months for long

term study and 0, 3 and 6 months for intermediate study and accelerated study. During

stability testing samples were evaluated for physical appearance, particle size, Zeta

potential and drug content.

5.1.3.2. In vivo evaluation

Wistar albino mice, Wistar albino rats were selected for the in vivo evaluation

of prepared nanoparticle and procured from Central Animal House, RMMCH,

Annamalai University and housed in the Institutional animal house under standard

environmental conditions (22 ± 30 C, 55 ± 5% humidity and 12h/12h dark/light cycle)

and maintained with free access to standard diet and water ad libitum. All

experimentations were approved by IAEC, (Proposal No.967).

5.1.3.2.1. Toxicity study

The acute toxicity study was carried out in Wistar albino mice. The healthy female

albino mice were divided into five groups for single dose oral acute toxicity study

(OECD 423) and following treatment regimen was followed:

Acute toxicity was measured by mortality and survival time and also by clinical

picture of intoxication and behavioral reactions. Animals on study were observed for

any adverse reaction, like changes of body weight, condition of eye and nose, motor

activity and also examined for internal abnormalities viz. size and appearance of heart,

lungs, liver, spleen and kidney at necropsy (Gelperina et al., 2002). Mice were bled

via the retro orbital plexus before sacrificing.

Biochemical assay: The blood samples were collected at 14th day and centrifuged at

4000 rpm for 5 min. The serum was kept at – 20 oC until analyzed. The levels of

serum glutamate oxaloacetic transaminase (SGOT), serum glutamic pyruvic

transaminase (SGPT), serum creatinine, serum bilirubin and proteins were analyzed

with automatic analytical instrument (Hitachi, Japan) (Lam et. al., 2004; Oberdorster

and Oberdorster, 2005).

5.1.3.2.2. Bioavailability study

The plasma drug concentration-time profile of the developed nanoparticle

formulation in Wistar albino rats was constructed to calculate the bioavailability.

Adult albino rats of either sex weighing 150 to 180 gm were divided into two groups

of six animals each and fasted overnight before commencing the experiment with free

access to water. The felodipine suspension and prepared nanoparticle formulation

were administered in a dose of 20 mg/kg body weight orally with the help of cannula

after anaesthetizing for a very short period of time with diethyl ether. After

administration of dose 0.5 ml blood samples were collected from the retro-orbital

plexus into the heparinized tubes at preset time points of 0.5, 1, 2, 4, 8, 12, 24, 48, 72

and 96 h. The blood samples were centrifuged at 4000 rpm for 10 minutes and the

separated plasma samples were stored at – 20 oC until analysis.

Estimation of felodipine in plasma sample by RP-HPLC analysis: The

plasma sample of 0.3 ml and 50 L of chlorzoxazone (50 ng/ml) was added in a

centrifuge tube and volume made to 2 ml with acetonitrile to precipitate the protein.

Then the sample was centrifuged at 4000 rpm for 25 min and the supernatant was

collected. The supernatant was dried under nitrogen air. The residue was dissolved in

200 L of mobile phase and a volume of 20 L was injected into HPLC system. The

plasma samples were analyzed using Phenomenex C18 (150×4.6 mm, 5µ) analytical

column. The mobile phase used consisting of methanol and water (80:20 v/v) was

passed through 0.45 µm membrane filter and degassed by ultrasonication. The flow

rate was maintained at 1.0 ml/min and the measurements were made at 240 nm. The

amount of the felodipine in the sample was determined from the peak area ratio

correlated with the standard curve prepared under the same identical condition.

Pharmacokinetic analysis: The pharmacokinetic parameters were determined from

plasma concentration data by non-compartmental model. The parameters such as area

under the plasma concentration-time curve (AUC 0 - t), maximum plasma

concentration (Cmax) and the time taken to reach the maximum plasma concentration

(Tmax) were calculated directly from the plasma concentration time curve. The relative

bioavailability (Fr) of felodipine was calculated using the following equation:

5.1.3.2.3. Antihypertensive study

Blood pressure and heart rate measurement: Systolic blood pressure (SBP)

and heart rate (HR) were measured using tail cuff method (Oh et al., 2007). Rat tails

were occluded with size tubular tail cuff (7/16 inch, 12 mm) connected to the

photoplethysmograph (IITC Life Sci., CA, USA) and pulses were detected as the cuff

pressure become lowered. For measuring the SBP, rats were pre-warmed at 32 0C for

5-10 min in a restraining cage in a warming box. This procedure was followed at least

2 weeks before experiments to habituate the rats.

Measurement of blood pressure and heart rate in L-NAME induced

hypertensive rats: The SBP and HR were measured after oral administration of

nanoparticles in L-NAME (N-nitro-L-arginine methyl ester) induced hypertensive rats

using the tail cuff method. L-NAME (40 mg/kg) was dissolved in drinking water and

given orally to rats at an interval of 24 hrs for 4 weeks. The hypertensive rats with

SBP of more than 170 mmHg were used in this study. To evaluate the

antihypertensive effect of the nano formulation 24 healthy male rats (weighing 200-

250g) were divided into four groups (6 each group). First group was treated with

normal saline (normal control) and second group treated with L-NAME (disease

control). Third and fourth groups (pre-treated with L-NAME) received orally native

felodipine (20m/kg), and felodipine nanoparticles respectively, in a dose equivalent to

20 mg of felodipine. All the measurements were performed three times.

Statistical analysis

For statistical analysis the experimental data was tested by one-way analysis of

variance (ANOVA) and Student’s t-test. Data represented as mean values ± SD

(standard deviation). The values of p < 0.05 (*) were indicative of significant

difference, very significant difference if p < 0.01 (**) and highly significant difference

if p < 0.001 (***).

5.1.4. Results and discussion

Preparation of nanoparticles

The felodipine nanoparticles were prepared by solvent evaporation technique using

different ratios of drug and polymer, various agitation speed and different organic

phase and aqueous phase ratios (Tab. 5.1.1). This method is comparatively easy to

prepare the nanoparticles than the other technique due to high drug entrapment

efficiency for poorly water soluble drug, narrow particle size distribution and high

batch to batch reproducibility. The solution of polymer and drug in methanol-acetone

mixture forms the organic phase. This organic phase was poured into an aqueous

phase containing stabilizer (poloxamer 188). The organic solvent rapidly partitioned

into the external aqueous phase and the polymer precipitated around the drug particles.

The evaporation of entrapped solvents led to the formation of polymeric nanoparticles.

The various optimized parameters were studied in the formation of desired

nanoparticle formulation and characterized.

Particle size and Zeta potential measurement

The particle size is an important parameter as it has direct effect on the stability,

cellular uptake, drug release and biodistribution. The mean particle sizes of the

prepared nanoparticles as measured by the Photon Correlation Spectroscopy (PCS)

were in size range of 492 to 738 nm and the size distributions were monodispersed

(0.214 to 0.517) in all the formulations (Tab. 5.1.2). There were no noticeable

differences between the sizes of nanoparticles obtained with different ratio of drug and

polymer. The results were consistent with the previous report described for the

nanoparticles of anti-inflammatory drugs with Eudragit®

RS100 (Pignatello et al.,

2002). In the present study, the decrease in size of the particles has been reported.

Table 5.1.2. Particle size, Zeta potential, polydispersity index, entrapment

efficiency and drug loading of felodipine loaded Eudragit® RS100 nanoparticles

Formulation

Particle

size

(nm)*

Zeta

potential

(mV)*

Polydispersity

index*

Entrapment

efficiency

(%w/w)*

Drug

loading

(%w/w)*

FEN1 492 ± 3.12 +14.1 ± 0.47 0.483 ± 0.053 57.78 ± 0.480 16.35 ± 0.19

FEN2 517 ± 6.36 +17.6 ± 0.56 0.356 ± 0.078 69.89 ± 0.861 14.26 ± 0.23

FEN3 526 ± 3.47 +19.8 ± 0.81 0.214 ± 0.007 75.87 ± 0.242 11.54 ± 0.61

FEN4 738 ± 3.91 +21.5 ± 1.04 0.352 ± 0.068 74.65 ± 0.621 11.09 ± 0.27

FEN5 597 ± 5.23 +18.7 ± 0.91 0.517 ± 0.029 54.39 ± 0.457 16.73 ± 0.96

FEN6 583 ± 4.26 +19.1 ± 0.58 0.319 ± 0.012 69.25 ± 0.293 13.59 ± 0.04

FEN7 607 ± 4.18 +20.3 ± 1.16 0.243 ± 0.006 64.47 ± 0.531 13.23 ± 0.12

* The values are expressed as mean ± SD for n=3

The Zeta potential is one of the important characteristics of the nanoparticles, as it

determines the physical stability and in vivo distribution of nanoparticles. The Zeta

potential values were measured in water and exhibited positive values of 14.1 to 21.5

mV (Tab. 5.1.2). This positive charge can facilitate for the effective adhesion of the

nanoparticles with the negatively charged mucus of the gastro-intestinal tract,

prolonging the effective residence time of the formulations.

Drug entrapment efficiency and drug loading

The entrapment efficiency and drug loading has direct impact on the drug release

profile from the formulations. Both EE and DL depend on the characteristics of

polymer, drug, surfactant and process variables. The high entrapment efficiency

results from the high affinity of both drug and polymer to the same solvent. In present

study the EE and DL were affected by the drug and polymer ratio in the formulation.

There were no noticeable changes found in the entrapment efficiency and drug loading

with the increase of agitation speed from 5000 rpm to 17,000 rpm (Tab. 5.1.1)

keeping drug-polymer ratio constant. But keeping the agitation speed constant, the

improved entrapment efficiency was observed with the increasing proportion of

polymer in the formulation from FEN1 to FEN4. The results were consistent with the

previous findings of Dongming et al and Adibkia et al (Dongming et al., 2007;

Adibkia et al., 2011). The greater proportion of polymer with respect to the amount of

drug improved entrapment efficiency. Since the drug is hydrophobic in nature, there

was no chance of diffusion of drug away from the polymer. The percentage of

felodipine entrapment in the formulation was found to be good. The increased drug

loading enhances the drug leakage in the organic phase lead to drug loss due to the

formation of channels in the polymer structure through which drug can easily escape

to the outer phase.

The formulation FEN1 showed high drug content and small particle size than other

formulations. But this formulation was not selected for further studies for its low drug

entrapment (57.78%). The nanoparticle formulation (FEN3) with drug-polymer ratio

of 1:4 with the agitation speed of 15,000 rpm shows the good entrapment efficiency

(75.87%), smaller particle size of 526 nm and Zeta potential value of +19.8 (Tab.

5.1.2, Fig. 5.1.1 & 5.1.2). Based on the particle size and entrapment efficiency the

formulation FEN3 was selected and validated for the further studies.

Figure 5.1.1. Particle size distribution of felodipine loaded nanoparticle

formulation (FEN3) prepared with drug-polymer ratio (1:4).

Figure 5.1.2. Zeta potential of felodipine loaded nanoparticle formulation (FEN3)

prepared with drug-polymer ratio (1:4).

Validation of nanoparticle formulation FEN3

Three batches of FEN3 prepared using the drug to polymer ratio (1:4),

agitation speed (15,000 rpm), organic phase to aqueous phase ratio (1:2) and stabilizer

concentration (1% w/v) confirmed the reproducibility of the formulations. The particle

size, polydispersity index and entrapment efficiency (Tab. 5.1.3) of three batches of

formulations show no noticeable differences among the batches.

Table 5.1.3. Particle size, polydispersity index and drug entrapment efficiency of

reproducible batches of FEN3

Note: The values are expressed as mean ± SD for n=3

Surface morphological properties of felodipine nanoparticles (FEN3)

The surface morphology of felodipine loaded nanoparticles was measured using

scanning electron microscopy. The SEM image of nanoparticles showed the spherical

shape with smooth surface (Fig. 5.1.3).

The AFM investigations revealed the disc like shape of the particles surrounded by

soft layer (Fig. 5.1.4). The particle sizes obtained by SEM were relatively smaller

compared with the particle sizes obtained by Zetasizer. The Zetasizer measures the

particles surrounded by hydrodynamic layer whereas the scanning electron

microscope measures only size of the particle.

Figure 5.1.3. SEM image of the felodipine nanoparticle formulation (FEN3)

Formulation Particle size

(nm)

Polydispersity

index

Entrapment

efficiency (%w/w)

FEN3a 531 ± 2.09 0.230 ± 0.048 76.81 ± 0.480

FEN3b 529 ± 2.16 0.216 ± 0.082 75.69 ± 0.752

FEN3c 532 ± 3.11 0.272 ± 0.091 76.15 ± 0.541

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Fourier transform infrared spectroscopy (FTIR)

The FTIR spectral analysis is useful in determining the compatibility of drug

and excipients. The preformulation study sometimes showed no interaction among the

drug and polymer. But there may be possibility of changes in drug even after

manufacturing process. Hence, to confirm further, whether there is any change in the

drug molecule in the nanoparticle, FTIR spectroscopy done. FTIR study of pure

felodipine, polymer and felodipine loaded Eudragit®

RS100 nanoparticles was carried

out for the interaction study between the drug and polymer (Fig. 5.1.6).

Figure 5.1.6. FTIR spectra of (a) native felodipine, (b) Eudragit® RS100 and (c)

felodipine - Eudragit® RS100 nanoparticles (FEN3)

The spectroscopy showed no distinctive changes in the peak confirming no

interaction among the components. This is in consistent with the compatibility results

showed in chapter 3. Thus FTIR spectroscopy further validated the compatibility

between felodipine and Eudragit® RS100 in nanoparticles.

Differential scanning calorimetry (DSC)

Differential scanning calorimetry was used to analyze the physiochemical

interaction of the drug encapsulated and the polymer. The analysis was performed for

the pure felodipine, polymer and felodipine-Eudragit®

RS100 nanoparticles (Fig.

5.1.7). The prominent and sharp endothermic peak at 147 ºC in the thermogram of

native felodipine represents its melting point. This sharp endothermic peak indicated

that the pure felodipine was in crystalline anhydrous state (Mura et al., 2001). No

distinctive peak was observed in the DSC profile of the felodipine loaded nanoparticle

confirming the decreased crystallinity of felodipine.

Figure 5.1.7. DSC thermogram of (a) Eudragit® RS100, (b) native felodipine and

(c) felodipine - Eudragit® RS100 nanoparticles (FEN3)

In vitro drug release study

The goal of in vitro drug release study (dissolution testing) is to provide a

possible prediction of or correlation with the product’s in vivo bioavailability. The in

vitro drug release rate was influenced by the drug-polymer composition. However, a

complex phenomenon may occur between the drug and polymer, including entrapment

of the drug in the polymer and the adsorption of drug on the surface of the polymer

matrix as a result of electrostatic adhesion (Douglas et al., 1987).

Drug release profile from drug powder, prepared nanoparticles and marketed

formulation are shown in (Tab. 5.1.4 & Fig. 5.1.8). The felodipine loaded Eudragit®

RS100 nanoparticles show slower drug release in comparison with drug powder and

marketed formulation. The drug release from the marketed formulation was observed

~91 % within 10 h. Within the first hour a release of about 41 % was observed for

felodipine powder. The initial burst release was reduced to 18 % and a slow release

was observed for felodipine loaded Eudragit®

RS100 nanoparticles at least 96 h due to

the formation of wall around the drug by the polymer. It signifies that they possess

sustained release properties. The initial burst release from the nanoparticles is

expected as a result of surface adsorbed drug (Kim and Martin, 2006).

Table 5.1.4. In vitro drug release profile of native felodipine, felodipine loaded

Eudragit® RS100 nanoparticles (FEN3) and marketed formulation in phosphate

buffer pH 6.8

Time

(h)

Native

felodipine FEN3

Marketed

formulation

0 0 0 0

1 39.73 ± 0.69 17.8 ± 0.56 45.97 ± 0.83

2 48.58 ± 0.97 20.3 ± 0.93 59.49 ± 0.63

4 56.75 ± 0.63 24.8 ± 0.49 73.91 ± 0.82

6 63.24 ± 0.48 27.1 ± 1.02 87.14 ± 1.26

8 66.17 ± 1.02 42.4 ± 0.68 89.54 ± 0.98

10 69.97 ± 0.92 47.6 ± 1.13 90.72 ± 0.84

12 74.06 ± 1.28 54.6 ± 1.17 -

18 79.54 ± 0.81 58.3 ± 0.93 -

24 86.84 ± 0.73 64.6 ± 2.31 -

48 87.54 ± 1.05 68.1 ± 1.07 -

72 87.98 ± 0.82 71.4 ± 2.05 -

96 89.61 ± 0.65 74.6 ± 0.73 -

T50 3.0 ± 0.49 11.4 ± 0.49 1.5 ± 0.71

The values are represented as Mean ± SD; n=3

After 6 h the rapid drug release was identified and this could be as a result of

polymer erosion in the surface of nanoparticles and consequent release of drug which

has been loaded near the surface of nanoparticles (Adibkia et al., 2011). It suggests

that the combination of dissolution, diffusion and erosion are the possible mechanism

for drug release from the nanoparticles.

Figure 5.1.8. In vitro drug release profile of marketed formulation, native

felodipine and felodipine loaded Eudragit® RS100 nanoparticles (FEN3) in

phosphate buffer pH 6.8

Drug release kinetics: The drug release data are plotted in various kinetic models, viz.

zero order, first order, Higuchi and Korsmeyer-Peppas. Derived data and

corresponding plots with respect to various models are furnished in tables and figures

as follows:

Zero order : Tab. 5.1.5 and Fig. 5.1.9.

First order : Tab. 5.1.6 and Fig. 5.1.10.

Higuchi : Tab. 5.1.5 and Fig. 5.1.11.

Korsmeyer-Peppas : Tab. 5.1.7 and Fig. 5.1.12.

Table 5.1.5. Data for Zero order plot (Cumulative % drug release vs. Time) and

Higuchi plot (Cumulative % drug release vs. Sq. root of time) obtained from in

vitro drug release profile of felodipine nanoparticles (FEN3)

Table 5.1.6. Data for first order plot (Log cumulative % drug remaining vs.

Time) obtained from in vitro drug release profile of felodipine loaded Eudragit®

RS100 nanoparticles (FEN3)

Time (h) Sq. root of time (h) Cumulative % drug release

0 0.000 0

1 1.000 17.8

2 1.414 20.3

4 2.000 24.8

6 2.449 27.1

8 2.828 42.4

10 3.162 47.6

12 3.464 54.6

18 4.243 58.3

24 4.899 64.6

48 6.928 68.1

72 8.485 71.4

96 9.798 74.6

Time (h)

Log (% drug remaining)

0 -

1 1.9191

2 1.9031

4 1.8808

6 1.8633

8 1.7634

10 1.7243

12 1.6628

18 1.6232

24 1.5563

48 1.5051

72 1.4624

96 1.4150

Table 5.1.7. Data for Korsmeyer-Peppas model (Log cumulative % drug

remaining vs. Log time) for felodipine loaded Eudragit® RS100 nanoparticles

(FEN3)

Model fitting of the drug release data into zero and first order model indicate

that the drug release followed first order kinetics during 1 to 18 h and zero order

during 18 to 96 h. The results were consistent with the previous study report by Jain

and Saraf (Jain and saraf, 2009).

Figure 5.1.9. Felodipine loaded Eudragit® RS100 nanoparticles (FEN3) – Zero

order release in phosphate buffer pH 6.8 between 18 to 96 h.

Time (h) Log time (h) Log cumulative

% drug released

0 0 0

1 0.000 1.23

2 0.301 1.30

4 0.602 1.38

6 0.778 1.43

8 0.903 1.62

10 1.000 1.67

12 1.079 1.73

18 1.255 1.76

24 1.380 1.81

48 1.681 1.83

72 1.857 1.85

96 1.982 1.87

Figure 5.1.10. Felodipine loaded Eudragit® RS100 nanoparticles (FEN3) - First

order release in phosphate buffer pH 6.8between 1 to 18 h.

Figure 5.1.11. Drug release from felodipine loaded Eudragit® RS100

nanoparticles (FEN3) in phosphate buffer pH 6.8 – Higuchi kinetic.

Figure 5.1.12. Drug release from felodipine loaded Eudragit® RS100

nanoparticles (FEN3) in phosphate buffer pH 6.8 – Peppas model.

Drug release from the nanoparticles also obeyed Higuchi as well as Peppas

models, indicating that the drug release was by diffusion mechanism. The release

exponent ‘n’ value (0.359 ) was calculated from the slope of the Peppas model and is

less than 0.5, indicating the release mechanism from the nanoparticles was diffusion

controlled (Tab. 5.1.8). As the r2 values are closed it is difficult to find the suitable

release mechanism of the felodipine from the nanoparticles. Although the values are

close proximity the possible drug release from the nanoparticles well suited with

Higuchi kinetic followed by diffusion and erosion mechanism.

Table 5.1.8. Régression coefficient (r2) values plots of drug release data obtained

from the felodipine Eudragit® RS100 nanoparticles (FEN3)

Formulation Zero order 1st order Higuchi

Korsmeyer-

Peppas Peppas ‘n’

Period (18- 96h) (1-18h) (1-96h) (1-96h) -

FEN3 0.894 0.936 0.956 0.903 0.359

In vitro permeation across the intestine

The biological effect of the drug is influenced by its absorption in the blood

from the gastro intestinal tract. To assess the permeability of the formulation across

the intestine, everted intestinal sac model was used. The result showed that about

61.48 ± 0.39 % of felodipine reached across the intestine to the serosal fluid from the

nanoparticles after 8 h study (Fig. 5.1.13.). On the other hand, the pure felodipine was

found to be only 29.56 ± 0.71 % in the serosal fluid. The increased paracellular uptake

of felodipine may be mucoadhesive nature of nanoparticles (Pignatello et al., 2002).

Figure 5.1.13. In vitro intestinal permeation study of felodipine loaded

nanoparticles (FEN3) in everted intestinal sac model.

Stability study

Stability plays an important role in the drug development process. Stability

studies are conducted during product development stage to access the storage

condition and shelf-life of products. Lack of drug substance or drug product stability

may affect the purity, potency, and safety of the drug product. Changes in drug

stability could risk patient safety, since the dosage amount to patient may be lower

than expected. Instability may also lead to formation of toxic degradants. Stability

testing therefore allows the establishment of recommended storage conditions, retest

periods, and ultimately product shelf-life and expiry date.

Table 5.1.9. Stability study data of selected felodipine nanoparticles (FEN3)

subjected to three different storage conditions (25 °C/60% RH, 30 °C/65% RH and

40 °C/75% RH).

Test parameters Long term storage condition (25 °C/60% RH)

Periods (months) 0 3 6 12

Physical change No change No change No change No change

Particle size (nm) 532 ± 2.09 535 ± 1.94 536 ± 3.62 538 ± 2.41

Zeta potential (mV) +19.5 ± 0.93 +20.1 ± 1.02 +19.4 ± 0.86 +19.6 ± 1.12

Drug content (%) 100 98.18± 0.87 97.73± 0.63 96.15 ± 0.94

Intermediate storage condition (30 °C/65% RH)

Physical change No change No change No change -

Particle size (nm) 531 ± 1.16 529 ± 1.84 529 ± 2.31 -

Zeta potential (mV) +19.6 ± 0.92 +18.4 ± 0.71 +17.9 ± 0.59 -

Drug content (%) 100 98.29± 0.78 97.24± 0.63 -

Accelerated storage condition (40 °C/75% RH)

Physical change No change No change Clumping -

Particle size (nm) 528 ± 3.14 539 ± 2.91 553 ± 2.63* -

Zeta potential (mV) +19.2 ± 0.95 +20.9 ± 1.03 +22.3 ± 0.91 -

Drug content (%) 100 98.87 ± 1.03 96.04 ± 0.87 -

The values are represented as Mean ± SD; n=3

(*) p< 0.05 against time t=0

The stability study was carried out to ensure the suitability of the

nanoparticulate system and its storage condition. The results of stability studies for

nanoparticulate system (FEN3) are given in (Tab. 5.1.9). No visible physical changes

(colour and cake formation) were observed for the nanoparticles stored in 25 °C/60%

RH and 30 °C/65% RH. But some clumping was observed for the nanoparticles that

are stored in 40 °C/75% RH after 6 months. This may be due to the absorption of

moisture on the nanoparticle surface at high humidity.

The drug degradation is not significant as only less than 4% drug decomposed at long

term storage condition. Even in accelerated condition drug decomposition was less

than 4% after 6 months of storage. No appreciable changes has been observed in size

of particles during storage at 25 °C/60% RH and 30 °C/65% RH over 6-12 months,

but some appreciable change was observed when stored at accelerated condition.

Toxicity evaluation

Preliminary toxicity (safety) evaluation of drug/drug products are essential before

proceeding further in developmental process. The in vivo acute toxicity study of the

felodipine nanoparticle formulation (FEN3) was performed using female albino mice.

The smaller particles exhibit interaction with local tissues and provoke the dysfunction

of the organs. Hence, the toxicity study was carried to observe the dysfunction of the

organs.

The acute toxicity studies performed in female albino mice showed no mortality or

behavioral changes up to dose level of 240 mg/kg body weight. However, complete

mortality was observed at dose level of 480 mg/kg body weight. Serum biochemical

parameters (Tab. 5.1.10 and Fig.5.1.14) also showed no noticeable changes between

the test group (60, 120 and 240 mg/kg body weight) and the control group. It confirms

that the product has no influence on liver function parameters. The study also

confirms that the nanoparticle formulation does not influence the serum sodium,

chloride and potassium.

Table 5.1.10. Serum biochemical reports of control and treated mice.

Parameter

Referenc

e

value

Control

(Normal

saline)

60mg/kg 120mg/kg 240mg/kg

Serum creatinine

(mg/dL) 0.2 - 0.9 0.2 ± 0.08 0.17±0.07 0.22±0.01 0.26±0.02

Total bilirubin

(mg/dL) 0 - 0.9 0.6 ± 0.02 0.79±0.04 0.69±0.08 0.64±0.04

Total proteins

(g/dL) 3.5 - 7.2 4.8 ± 1.2 5.5±1.4 5.1±1.2 4.9±1.0

ALT (SGPT) U/L 17 - 77 24.1 ± 2.4 24.6±2.6 24.8±2.6 24.1±3.0

AST (SGOT)

U/L 54 - 298 61.2 ± 2.8 61.1±3.2 61.7±2.6 62.2±2.2

Sodium (mEq/L) 140 - 160 148.2 ± 4.8 147.3±5.2 146.1±4.6 144.2±3.2

Chloride (mEq/L) 88 - 110 102.4 ± 2.8 102.2±3.4 102.1±3.8 102.6±2.2

Potassium

(mEq/L) 5 - 7.5 4.81 ± 1.2 4.89±1.4 4.78±1.6 4.82±1.8

The values are represented as Mean ± SD; n=3

Ser

um cre

atinine

Total b

iliru

bin

Total p

roteins

ALT (SGPT)

AST (SGOT)

Sodium

Chloride

Potass

ium

0

50

100

150

200Control

60mg/kg

120mg/kg

240mg/kg

Biochemical parameters

Figure 5.1.14. Effect of felodipine-Eudragit® RS100 nanoparticles (FEN3) on

serum biochemical parameters in control and treated mice of acute toxicity

studies.

Bioavailability study

The availability of the drug to the biologic system is integral to the goals of dosage

form design and paramount to the effectiveness of the medication. To achieve the

antihypertensive effect it is necessary to reach the drug in the blood from the dosage

form.

Figure 5.1.15. Plasma concentration-time profiles of felodipine in rats after oral

administration of free felodipine suspension and felodipine loaded Eudragit®

RS100 nanoparticles (20 mg/kg). Each data was given as mean ± S.D. (n=4).

The plasma drug concentration- time profile of rat after single dose (20 mg/kg) oral

administration of pure felodipine suspension and felodipine loaded nanoparticles is

illustrated in (Fig. 5.1.15) and the pharmacokinetic parameters are summarized in

(Tab. 5.1.11). The experimental results showed a significant difference between the

pharmacokinetic profiles of felodipine loaded nanoparticles and free felodipine

suspension (Fig. 5.1.15). At each time points, the plasma concentration of felodipine

from the nanoparticles was higher than that of free felodipine suspension.

The peak concentration (Cmax) of felodipine in rats treated with nanoparticles was

1098 ± 41 ng/ml, which was significantly improved (*p< 0.05) compared with that of

free felodipine suspension (821 ± 28 ng/ml). It was also observed that AUC0-72 value

of felodipine in rats treated with FEN3 was enhanced than that of pure drug

suspension. This enhanced AUC of felodipine nanoparticles may be due to the uptake

of FEN3 in the intestine and the sustained release of the felodipine from the

nanoparticle formulation. The improved permeability of the FEN3 may also be the

reason for improved AUC.

Table 5.1.11. Pharmacokinetic parameters of felodipine after oral administration

of free drug and nanoparticle formulation (FEN3) (20 mg/kg) in rats ( n=4).

The values are represented as Mean ± SD (n = 4). *p< 0.05

The Tmax value was observed 2 h to achieve the Cmax after oral administration of

felodipine suspension, but shortened to 1.5 h after administration of nanoparticle

formulation (FEN3). The rapid absorption could attribute to the nanometric size of the

felodipine nanoparticles.

Antihypertensive activity study

The spontaneous or experimentally induced hypertensive rats are widely used for

screenings of antihypertensive compounds. The indirect tail cuff method is used to

determine the systolic blood pressure which is analogous to sphygmomanometry in

human and can also be applied in dogs and small primates.

Parameters Pure drug

suspension

Nanoparticles

(FEN3)

Tmax(h) 2 ± 0.12 1.5 ± 0.18

Cmax(ng/ml) 821 ± 28 1098 ± 41*

AUC 0-72(ng.h/ml) 16,033 ± 189 19,493± 240*

Relative bioavailability (%) - 121.58

Tab

le 5

.1.1

2. E

ffec

t of

felo

dip

ine

nan

op

art

icle

s (F

EN

3)

on

SB

P, D

BP

an

d H

R i

n L

-NA

ME

in

du

ced

hyp

erte

nsi

ve

rats

.

Para

met

ers

Gro

up

s P

re-t

reatm

ent

Po

st-a

dm

inis

tra

tio

n (

ho

urs

)

6

24

48

72

SB

P

(mm

/Hg

)

Neg

ativ

e co

ntr

ol

(No

rmal

sal

ine)

1

25 ±

5.2

1

24 ±

4.3

12

5 ±

6.1

1

26 ±

4.5

1

23

± 7

.1

Po

siti

ve

con

tro

l (

L-N

AM

E 4

0 m

g/k

g /

day

) 1

85 ±

4.3

18

8 ±

5.1

a**

*

19

2 ±

3.4

a**

*

19

4 ±

4.2

a**

*

193 ±

4.5

a**

*

L-N

AM

E 4

0 m

g/k

g /

day

&

FE

N3 (

20 m

g/k

g)

18

3 ±

2.5

15

1 ±

4.6

b**

*

15

4 ±

2.3

b**

*

161 ±

3.1

b*

**

165 ±

2.9

b**

*

L-N

AM

E 4

0 m

g/k

g /

day

& P

ure

Dru

g (

20 m

g/k

g)

18

6 ±

2.7

160 ±

4.3

c***

17

1 ±

3.6

c**

17

8 ±

2.9

182 ±

3.3

DB

P

(mm

/Hg

)

Neg

ativ

e co

ntr

ol

(No

rmal

sal

ine)

84 ±

4.4

86 ±

2.1

84 ±

2.5

83 ±

2.4

85 ±

3.1

Po

siti

ve

con

tro

l (L

-NA

ME

40

mg/k

g /

day

) 1

10 ±

2.3

10

6 ±

3.4

a**

*

11

1 ±

2.2

a**

*

11

5 ±

3.5

a**

*

113 ±

2.3

a**

*

L-N

AM

E 4

0 m

g/k

g /

day

&

FE

N3 (

20 m

g/k

g)

11

6 ±

2.1

86 ±

3.5

b*

**

87 ±

2.1

b***

89 ±

2.7

b***d

***

93 ±

3.4

b***d

***

L-N

AM

E 4

0 m

g/k

g /

day

& P

ure

Dru

g (

20 m

g/k

g)

11

0 ±

2.8

81 ±

1.6

c**

*

93 ±

2.4

c**

*

10

4 ±

3.3

c**

108 ±

1.9

HR

(Bea

ts/m

in)

Neg

ativ

e co

ntr

ol

(No

rmal

sal

ine)

3

56

± 1

0.4

3

54 ±

8.1

35

2 ±

8.3

3

58 ±

6.2

3

56

± 7

.1

Po

siti

ve

con

tro

l (

L-N

AM

E 4

0 m

g/k

g /

day

) 3

65 ±

5.3

3

67 ±

3.1

36

9 ±

4.5

3

70 ±

5.3

3

68

± 4

.4

L-N

AM

E 4

0 m

g/k

g /

day

&

FE

N3 (

20 m

g/k

g)

35

1 ±

3.8

32

2 ±

3.1

b**

*

32

4 ±

2.9

b**

*

33

3 ±

3.6

b**

*d

**

337 ±

2.8

b**

*d

**

L-N

AM

E 4

0 m

g/k

g /

day

& P

ure

Dru

g (

20 m

g/k

g)

36

8 ±

3.7

317 ±

2.4

c***

339 ±

3.2

c***

35

5 ±

4.1

361 ±

3.1

Val

ues

rep

rese

nte

d a

s m

ean ±

S.E

.M (

n=

6)

On

e w

ay A

NO

VA

bet

wee

n n

egat

ive

and

posi

tiv

e co

ntr

ol

gro

up a

nd

the

trea

ted

gro

ups

foll

ow

ed b

y D

un

net

t’s

test

.

a P

osi

tiv

e co

ntr

ol

gro

up v

s. N

egat

ive

con

tro

l g

rou

p,

b F

orm

ula

tio

n t

reat

ed g

rou

p v

s. P

osi

tive

contr

ol

gro

up,

c P

ure

dru

g t

reat

ed g

rou

p v

s. P

osi

tive

con

tro

l g

rou

p,

d P

ure

dru

g t

reat

ed g

rou

p v

s. F

orm

ula

tio

n t

reat

ed g

rou

p.

p v

alu

es *

*<

0.0

1,

**

*<

0.0

01

The indirect tail cuff method is used to evaluate the influence of antihypertensive

drugs in experimentally induced hypertensive rats. The antihypertensive activity of

felodipine loaded Eudragit®

RS100 nanoparticles was evaluated by measuring SBP,

DBP and HR in L-NAME induced hypertensive rats. The results are tabulated in

(Tab. 5.1.12). The antihypertensive effect of FEN3 and pure drug with positive

control rats are depicted in Fig. 5.1.16, Fig. 5.1.17 and Fig. 5.1.18.

hth

6hth

24 hth

48 hnd

72

0

50

100

150

200

250Negative control

Positive control

Formulation treated

Pure drug treated

Time (h)

SB

P (

mm

Hg)

Figure 5.1.16. Effect of felodipine loaded nanoparticles (FEN3) and native drug

suspension on SBP in hypertensive rats.

hth

6hth

24 hth

48 hnd

72

0

50

100

150Negative control

Positive control

Formulation treated

Pure drug treated

Time (h)

DB

P (

mm

Hg)

Figure 5.1.17. Effect of felodipine loaded nanoparticles (FEN3) and native drug

suspension on DBP in hypertensive rats.

hth

6 hth

24 hth

48h

nd

72

0

100

200

300

400Negative control

Positive control

Formulation treated

Pure drug treated

Time (h)

Heart

rate

(b

eats

/min

)

Figure 5.1.18. Effect of felodipine loaded nanoparticles (FEN3) and native drug

suspension on HR in hypertensive rats.

The SBP was notably reduced after oral administration of FEN3 (20 mg/kg) as

compared with positive control group. While treating with pure felodipine suspension,

the SBP, DBP and HR were significantly reduced (**p< 0.01) within 6h as compared

with positive control group and again increased within 24h, but after treating with

felodipine nanoparticle formulation (FEN3) the SBP, DBP and HR were significantly

decreased (***p< 0.001) as compared with pure felodipine treated group and the

activity remained up to 48h. The DBP at 48h and 72h of the felodipine nanoparticles

formulation (FEN3) treated group was significantly decreased (***p< 0.001) as

compared to the pure felodipine suspension treated groups. Similarly, the heart rate of

the felodipine nanoparticle formulation (FEN3) treated group was decreased (***p<

0.001) as compared to the pure felodipine suspension treated groups. Hence, the

prepared nanoparticle formulation showed improved antihypertensive activity as

compared with pure felodipine.

5.2. PREPARATION, CHARACTERIZATION AND IN VITRO EVALUATION

OF FELODIPINE LOADED POLY (D, L-LACTIC-CO-GLYCOLIC ACID) OR

(PLGA) NANOPARTICLES

5.2.1. Preparation of felodipine nanoparticle

Introduction

Felodipine, a calcium channel blocker is widely used for the treatment of high blood

pressure and other cardiovascular complications. Being poor soluble in nature the oral

administration of felodipine exhibits irregular absorption in the gastrointestinal tract

and poor bioavailability. The desired bioavailability and constant plasma drug

concentration can be achieved by multiple daily dosing, which leads to poor patient

compliance.

Out of many ways to increase the oral bioavailability, the decrease in the particle size

of the native drug has received much interest. An effective way to address the issues

of the drug is by making the polymeric nanoparticles. By encapsulating the native

drug in the polymeric material the crystallinity can be prevented. This polymeric

nanoparticulate system have been considered as promising carriers (Vauthier et al.,

2003; Amaral et al., 2007) for oral sustained drug delivery, which will be beneficial to

the patients for the long term treatment. The intracellular uptake of drug and stability

can be improved by nanoparticulate formulation (Ourique et al., 2008).

Poly (D, L-lactic-co-glycolic acid) or PLGA is one of the most popular biodegradable

and non toxic polymers used for preparing microparticles and nanoparticles (Zimmer

and Kreuter, 1995; Bala et al., 2004). PLGA is randomly hydrolyzed into their

biocompatible metabolite, lactic acid and glycolic acid and eliminated from the body

as carbon dioxide and water (Crotts and Park, 1998; Fulzelv, 2003). PLGA is widely

used for the manufacture of implants, internal sutures and also used for controlled and

targeted drug delivery systems (Uhrich et al., 1999; Cheng et al., 2007). The changes

in tissue distribution and pharmacokinetic profile were observed, when the drug was

incorporated into PLGA nanoparticulate system (Mainardes and Evangelista, 2005;

Dillen et al., 2006). As the particle sizes of the nanoparticles are small enough they are

expected to reach angiogenetic area and circulate through the capillaries (Jin et al.,

2008). These may lead to improve therapeutic efficacy and patient compliance.

In the present study felodipine nanoparticles were developed using biodegradable

carrier PLGA with the aim to get more prolonged and effective delivery of felodipine.

So, felodipine loaded nanoparticles were prepared, characterized and evaluated for in

vitro drug release study.

Materials

Felodipine was a kind gift from Cadila Healthcare Limited (Ahmedabad, India). Poly

(D, L-lactic-co-glycolic acid) or (PLGA 50:50) (Mw = 18,000) and Lutrol®

F-68

(Poloxamer 188) was obtained from Sigma, Mumbai. Distilled- deionized water was

prepared with Milli-Q plus System (Elix 10, Millipore corp. India). All other

chemicals used were of the highest available grade.

Method of preparation

The felodipine loaded nanoparticles were prepared with the different ratios of

drug and PLGA polymer using solvent evaporation technique (Jain, 2000) as

described in table 5.2.1. The polymeric solution of PLGA 50:50 was prepared in

acetone at room temperature and felodipine was dissolved in it. The resultant solution

was then added into water containing poloxamer-188 (aqueous phase) with a constant

rate (0.5 ml/min). The mixture was then homogenized using a probe homogenizer

(VIRTIS, Cyclone IQ, USA), at various agitation speeds in an ice bath. The formed

oil-in-water (O/W) emulsion was kept at room temperature for 24 h under gentle

stirring to evaporate the organic solvent.

The prepared nanosuspensions were centrifuged at 40,000 rpm at 4oC for 20

min (Sorvall Ultracentrifuge, USA). The pellets were collected and washed with

double distilled water to remove un-entrapped drugs. The recovered nanosuspension

was freeze dried kept at freeze for further use. The impact of formulation and process

variables on particle sizes and entrapment efficiency was studied in an attempt to

optimize formulation with less particle size and maximum entrapment of the drug in

the nanoparticles as described in section 5.1.1, Chapter 5.

Table 5.2.1. Formulation of felodipine loaded PLGA nanoparticles

S.

No Formulation

Drug:

Polymer

Ratio

Wt. of

Drug

(mg)

Wt. of

Polymer

(mg)

Vol. of

OP

(ml)

Vol. of

AP (ml)

Agitation

(rpm)

1 FP1 1:1 20 20 10 20 5000

2 FP2 1:2 20 40 10 20 10,000

3 FP3 1:3 20 60 10 50 15,000

4 FP4 1:2 20 40 10 40 15,000

5 FP5 1:2 20 40 10 30 17,000

6 FP6 1:4 20 80 10 20 15,000

7 FP7 1:3 20 60 10 20 15,000

Note: FP indicates felodipine loaded PLGA nanoparticle; OP: organic phase; AP: aqueous

phase.

5.2.2. Characterization of nanoparticles

Determination of particle size and Zeta potential

Particle size analysis was performed by Photon Correlation Spectroscopy

(PCS) with Zetasizer 3000 (Malvern Instruments, Malvern, UK) as described in

section 4.1.2, Chapter 4. The freeze dried powdered samples were suspended in Milli-

Q water (1mg/ml) at 25 °C and sonicated for 30 sec in an ice bath to prevent the

clumping. The mean particle diameter and size distribution of the suspension were

assessed with a fixed angle of 90o. The Zeta potential also measured using the same

sample. Analysis was carried out for three times for each batch of sample under

identical conditions.

Determination of entrapment efficiency and drug loading

The entrapment efficiency (EE) was estimated by reverse phase High

Performance Liquid Chromatography (RP-HPLC) method (Mohanty et al., 2010). The

drug loaded nanoparticle solution of 1 mg/ml was prepared in methanol and 20 µL of

the sample was injected manually to HPLC equipped with Shimadzu LC-20AD PLC

pump and SPD-M20A PDA detector. The chromatographic separation was achieved

by using Phenomenex C18 (150×4.6 mm, 5µ) analytical column. The mobile phase

used consisting of methanol and water (80:20 v/v) was passed through 0.45 µm

membrane filter and degassed by ultrasonication. The flow rate was maintained at 1.0

ml/min and the measurements were made at 240 nm. The amount of the felodipine in

the sample was determined from the peak area correlated with the standard curve. The

drug entrapment efficiency (EE) and drug loading (DL) were calculated using

following formula:

Weight of the drug in nanoparticles

EE (% w/w) = ×100

Weight of the drug added

Weight of the drug in nanoparticle

DL (% w/w) = ×100

Weight of the polymer and drug added

Scanning electron microscopy (SEM)

The particle shape and surface morphology of felodipine nanoparticles were

examined by scanning electron microscopy (SEM) (JEOL JSM-5610LV). Lyophilized

and completely moisture free samples were consigned on aluminium stubs using

adhesive tapes and coated with gold using sputter coater (JEOL auto fine coater,

Japan) and observed for morphology at an acceleration voltage of 20 kV at high

vacuum.

Atomic force microscopy (AFM)

Atomic force microscopy (AFM) studies were carried out to characterize the

surface morphology of prepared drug loaded nanoparticles. The nanoparticle

suspension was prepared with milliQ water and dried overnight in air on a clean glass

surface and observation was performed with AFM as describe in section 4.1.2.6,

Chapter 4. The scan speed of 2 Hz and 312 kHz resonant frequency was used for

displaying amplitude, signal of the cantilever in the trace direction and to obtained

images (Trapani et al., 2009).

Transmission electron microscopy (TEM)

Transmission electron microscopy was used to examine the morphology of the

particles. A sample of particle suspension was diluted with 3% w/v phosphotungstic

acid adjusted to pH 7.5 with potassium hydroxide corresponding to a 1:1 ratio before

examination. One drop of sample was placed for 1 minute on a copper grid coated

with a formvar carbon film. The excess of sample was wicked away with the aid of

filter paper. The sample was then ready for analysis by TEM.

Fourier transform infrared spectroscopy (FTIR)

The FTIR analysis was performed to know the chemical integrity and possible

chemical interaction between the drug and polymer inside the prepared nanoparticles

as described in section 4.1.2, Chapter 4. FTIR analysis was carried out using FT-IR

Spectrometer. Samples were made into pellets separately for drug, polymer and

nanoparticles with potassium bromide and scanned with resolution of 2 cm-1

in the

range of 4000–400 cm-1

.

Differential scanning calorimetry (DSC) analysis

The physical status of the native drug inside the nanoparticles was ascertained

by the DSC analysis (DSC-60, Shimadzu, Japan). Approximately, weighed 2 mg of

native drug, polymer and nanoparticles were placed separately into the different sealed

standard aluminium pan and were scanned between 25 ºC to 300

ºC with heating rate

of 10 ºC/ minute under nitrogen atmosphere. An empty aluminium pan served as

reference.

5.2.3. Evaluation of nanoparticles

5.2.3.1. In vitro drug release study

5.2.3.1.1. Drug release study

The in vitro drug release study of felodipine nanoparticles was carried out by

using bottle method (Devarajan and Sonavane, 2007; Jain and Saraf, 2009) in

phosphate buffer pH 6.8. The prepared nanoparticles, pure felodipine and marketed

tablet formulation (each containing 5 mg felodipine) were suspended in glass bottles

containing 100 ml of phosphate buffer pH 6.8. Glass bottles were placed in beaker and

kept in incubator shaker throughout the study (37 oC, 50 rpm). At specified time

intervals (as described in Chapter 4) 10 ml samples were collected and centrifuged at

13,800 rpm for 30 min. The supernatants were collected for analysis and the

precipitate resuspended in 10 ml of fresh phosphate buffer. The analysis was carried

out by RP-HPLC at 240 nm. All the measurements were carried out in triplicate.

Analysis of drug release data: The in vitro drug release data were analyzed by various

mathematical models to determine the kinetics and the drug release mechanism from

the developed nanoparticle formulation. The drug release data were fitted with

mathematical models including zero order kinetic, first order kinetic, Higuchi kinetic

and Korsmeyer-Peppas model as described in section 5.1.3.1.1, Chapter 5. The plots

were made from the in vitro drug release data: time vs. cumulative % drug release

(zero order kinetic), time vs. log cumulative % drug remaining (first order kinetic),

square root of time vs. cumulative % drug release (Higuchi model) and log time vs.

log cumulative % drug release (Peppas model) respectively. The first 60% drug

release was fitted in Korsmeyer-Peppas model and release exponent “n” was

calculated from the slop of the plot to characterize the different release mechanism

(Aydin and Pulat, 2012; Bagre et al., 2013).

5.2.3.1.2. In vitro permeation study across the intestine

The everted intestinal sac model was used to assess the permeability of the

nanoparticles across the intestine (Schilling and Mitra, 1990; Agarwal and Khan,

2001) with some modification as described in section 5.2.3.1.3, Chapter 5. The

isolated intestinal segment from the albino rat was carefully everted using the glass

rod and rinsed with saline solution. Modified Kreb’s Ringer bicarbonate (MKRB)

solution of pH 7.4 was filled within the intestinal sac as serosal fluid and placed in a

bath containing 50 ml of nanoparticle suspension (1 mg/ml) in MKRB solution on

mucosal side. The fluid was continuously bubbled with carbogen gas (mixture of 5%

CO2 and 95% O2 gas). The temperature of the organ bath was maintained at 37 ± 0.5

oC. The amount of felodipine was analyzed spectrophotometrically at 240 nm.

5.2.3.1.3. Stability study

Stability study was carried out according the procedure described in section

5.1.3.1.3, Chapter 5. The lyophilized felodipine PLGA nanoparticle was kept in glass

vials and stability study was carried out in three different storage conditions viz. long

term study (25 ± 2 °C / 60% RH ± 5% RH) for one year, intermediate study (30 ± 2 °C

/ 65% RH ± 5% RH) for six months and accelerated study (40 ± 2 °C / 75% RH ± 5%

RH) for six months. The nanoparticles were evaluated at intervals of 0, 3, 6 and 12

months for long term study and 0, 3 and 6 months for intermediate study and

accelerated study. Physical appearance, particle size, Zeta potential and drug content

were evaluated at predetermined time intervals.

5.2.3.2. In vivo evaluation

Wistar albino mice, Wistar albino rats were selected for the in vivo evaluation

of prepared nanoparticle and procured from Central Animal House, RMMCH,

Annamalai University and housed in the Institutional animal house under standard

environmental conditions (22 ± 30 C, 55 ± 5% humidity and 12h/12h dark/light cycle)

and maintained with free access to standard diet and water ad libitum. All

experimentations were approved by IAEC, (Proposal No.967).

5.2.3.2.1. Toxicity study

The acute toxicity study was carried in Wistar albino mice according the

protocol discussed in section 5.1.3.2.1, Chapter 5. The healthy female mice were

divided into five groups for the study. The toxicity was measured by mortality,

survival time, clinical picture of intoxication and behavioral reactions. Animals on

study were observed for any adverse reaction, like changes of body weight, condition

of eye and nose and motor activity. The internal abnormalities viz. size and

appearance of heart, lungs, liver, spleen and kidney were also examined (Gelperina et

al., 2002). Mice were bled via the retro orbital plexus before sacrificing.

Biochemical assay: The blood samples were collected at 14th day and centrifuged at

4000 rpm for 5 min. The serum was kept at – 20 oC until analyzed. The levels of

serum glutamate oxaloacetic transaminase (SGOT), serum glutamic pyruvic

transaminase (SGPT), serum creatinine, serum bilirubin and proteins were analyzed

with automatic analytical instrument (Hitachi, Japan) (Lam et. al., 2004; Oberdorster

and Oberdorster, 2005).

5.2.3.2.2. Bioavailability study

Bioavailability study was conducted in adult albino rats of either sex weighing

150 to 180 gm. The animals were divided into two groups of six animals each and

fasted overnight before commencing the experiment with free access to water. The

felodipine suspension and prepared nanoparticle formulation were administered orally

in a dose of 20 mg/kg body weight with the help of cannula after anaesthetizing for a

very short period of time with diethyl ether. After administration 0.5 ml blood samples

were collected from the retro-orbital plexus into the heparinized tubes at preset time

points of 0.5, 1, 2, 4, 8, 12, 24, 48, 72 and 96 h. The blood samples were centrifuged at

4000 rpm for 10 minutes and the separated plasma samples were stored at – 20 oC

until analysis.

Estimation of felodipine in plasma sample by RP-HPLC analysis: To 0.3 ml

of plasma 50 L of internal standard chlorzoxazone (50 ng/ml) was added in a micro

centrifuge tube and volume made to 2 ml with acetonitrile to precipitate the protein.

Then the sample was centrifuged at 4000 rpm for 25 min and the supernatant was

collected and transferred into an eppendorf tube. The supernatant was dried under

nitrogen air. The residue was dissolved in 200 L of mobile phase and analysis was

carried out by RP-HPLC method as discussed in section 5.1.3.2.2, Chapter 5. The flow

rate was maintained at 1.0 ml/min and the measurements were made at 240 nm. The

amount of the felodipine in the sample was determined from the peak area ratio

correlated with the standard curve prepared under the same identical condition.

Pharmacokinetic analysis: The pharmacokinetic parameters were determined from

plasma concentration data by non-compartmental model. The parameters such as area

under the plasma concentration-time curve (AUC0-t), maximum plasma concentration

(Cmax) and the time taken to reach the maximum plasma concentration (Tmax) were

calculated directly from the plasma concentration time curve. The relative

bioavailability (Fr) of felodipine was calculated using the following equation:

5.2.3.2.3. Antihypertensive study

Blood pressure and heart rate measurement: Systolic blood pressure (SBP)

and heart rate (HR) were measured using tail cuff method as describe in the section

5.1.3.2.3, Chapter 5.(Oh et al., 2007). For measuring the SBP, rats were pre-warmed at

32 0C for 5-10 min in a restraining cage in a warming box. This procedure was

followed at least 2 weeks before experiments to habituate the rats.

Measurement of blood pressure and heart rate in L-NAME induced

hypertensive rats: The Systolic blood pressure (SBP) and heart rate (HR) were

measured after oral administration of nanoparticles in L-NAME (N-nitro-L-arginine

methyl ester) induced hypertensive rats using the tail cuff method. L-NAME (40

mg/kg) was dissolved in drinking water and given orally to rats at an interval of 24 hrs

for 4 weeks. The hypertensive rats with SBP of more than 170 mmHg were used in

this study. To evaluate the BP 24 healthy male rats (weighing 200-250g) were divided

into four groups (6 each group). First group was treated with normal saline (normal

control) and second group treated with L-NAME (disease control). Third and fourth

groups (pre-treated with L-NAME) received orally native felodipine (20m/kg), and

felodipine-PLGA nanoparticles in a dose equivalent to 20 mg of felodipine

respectively. All the measurements were performed three times.

Statistical analysis

Experimental results were tested by one-way analysis of variance (ANOVA)

and Student’s t-test. Data represented as mean values ± SD (standard deviation). The

values of p < 0.05 (*) were indicative of significant difference, very significant

difference if p < 0.01 (**) and highly significant difference if p < 0.001 (***).

5.2.4. Results and discussion

Preparation of nanoparticles

Felodipine loaded PLGA nanoparticles were prepared with solvent evaporation

technique. The various parameters like drug polymer ratios, speed of agitation and

organic phase to aqueous phase ratio were studied to get desired particle size with

good entrapment efficiency. The polymer PLGA was selected because of its good

sustained release property and long circulation time in plasma.

Particle size and Zeta potential measurement

The mean particle sizes of the prepared nanoparticles as measured by the

Photon Correlation Spectroscopy (PCS) were in size range of 210 to 312 nm and the

size distributions were monodispersed (0.216 to 0.442) in all the formulations (Tab.

5.2.2). The particle size was slightly increased with the increase of polymer amount.

In the present study, the effect of agitation speed and organic phase to the aqueous

phase ratio on the particle size was studied and no noticeable changes were observed.

The Zeta potential value measured in water, exhibited negative values of 13.8 to 19.8

mV (Tab. 5.2.2). This negative charge of the nanoparticles is due to the end

carboxylic acid group present in the PLGA polymer which effectively allows the

particles to cross the lipophilic membrane of the intestine and improve the circulation

time in the plasma (Jin et al., 2008). The higher zeta potential values can also

provide more stability of the nano particles by preventing Ostwald ripening.

Drug entrapment efficiency and drug loading

The entrapment efficiency is influenced by the characteristics of the polymer,

drug, surfactant etc. The higher entrapment efficiency was observed with increasing

proportion of polymer in the formulation, which is in consistent with the previous

study by Dongming (Dongming et al., 2007). The nanoparticles formulation FP7

prepared with drug-polymer ratio of 1:3 with stabilizer concentration 1% (w/v) and

agitation speed of 15,000 rpm shows the entrapment efficiency of 85.28%, drug

loading of 9.93% and particle size of 226 nm with Zeta potential value -19.8 mV

(Tab. 5.2.2, Fig. 5.2.1 & Fig. 5.2.2).

Smaller particle size also observed with formulation FP2, but showed low

encapsulation efficiency as compared with FP7. Hence the formulation FP2 was not

selected for further study. The drug loading of formulation FP7 was less than that of

formulation FP1 and FP2. The higher drug loading forms the porous structure and

hollow channels in the polymer structure through which drug can escape to the outer

phase. Based on the particle size and entrapment efficiency the formulation FP7 was

selected, and validated for further studies.

Table 5.2.2. Particle size, Zeta potential, polydispersity index, entrapment

efficiency and drug loading of felodipine loaded PLGA nanoparticles

Formulation Particle size

(nm)*

Zeta

potential

(mV)*

Polydispersity

index*

Entrapment

efficiency

(%w/w)*

Drug loading

(%w/w)*

FP1 295 ± 2.23 -15.6 ± 0.57 0.315 ± 0.07 73.78 ± 0.38 14.55 ± 0.91

FP2 210 ± 3.51 -17.6 ± 0.72 0.293 ± 0.05 78.89 ± 0.32 13.35 ± 0.69

FP3 312 ± 5.24 -18.5 ± 0.75 0.371 ± 0.08 75.87 ± 0.87 13.04 ± 0.71

FP4 307 ± 4.51 -13.8 ± 0.38 0.442 ± 0.89 82.45 ± 0.86 10.43 ± 0.89

FP5 286 ± 5.19 -15.4 ± 1.04 0.216 ± 0.14 76.21 ± 0.47 12.41 ± 1.02

FP6 273 ± 6.28 -18.7 ± 0.54 0.254 ± 0.04 77.23 ± 0.36 12.08 ± 0.72

FP7 226 ± 5.39 -19.8 ± 0.62 0.216 ± 0.09 85.28 ± 0.32 9.93 ± 0.76

* The values are expressed as mean ± SD for n=3

Figure 5.2.1. Particle size distribution of felodipine loaded PLGA nanoparticle

formulation (FP7) prepared with drug-polymer ratio (1:3)

Figure 5.2.2. Zeta potential of felodipine loaded PLGA nanoparticle formulation

(FP7) prepared with drug-polymer ratio (1:3).

Validation of nanoparticle formulation FP7

Three batches of FP7 prepared using the drug to polymer ratio (1:3), agitation

speed (15,000 rpm), organic phase to aqueous phase ratio (1:2) and stabilizer

concentration (1% w/v) showed the reproducibility of batches. The particle size,

polydispersity index and entrapment efficiency of reproducible formulations are given

in (Tab. 5.2.3), which show there is no noticeable differences among the batches.

Table 5.2.3. Particle size, polydispersity index and drug entrapment efficiency of

reproducible batches of FP7

* The values are expressed as mean ± SD for n=3

Surface morphological properties of felodipine nanoparticles (FP7)

The scanning electron microscopy was used to examine the surface

morphology of the nanoparticles. The SEM image of FP7 formulation showed

spherical shape with smooth surface particles (Fig. 5.2.3).

Figure 5.2.3. SEM image of the felodipine nanoparticle formulation (FP7)

Formulation Particle size

(nm)

Polydispersity

index*

Entrapment

efficiency (%w/w)*

FP7a 225 ± 2.03 0.227 ± 0.094 86.14 ± 0.771

FP7b 229 ± 3.63 0.219 ± 0.038 84.69 ± 0.412

FP7c 227 ± 2.91 0.215 ± 0.079 85.74 ± 0.834

Th

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by

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particle surrounded by hydrodynamic layer. The TEM image also revealed the core

drug entrapment inside the polymer and spherical particles with smooth surface (Fig.

5.2.5).

Fourier transform infrared spectroscopy (FTIR)

The FTIR study of pure felodipine, polymer and felodipine loaded PLGA

50:50 nanoparticles was carried out for the interaction study between the drug and

polymer (Fig. 5.2.6). There were no distinctive changes in the FTIR spectrum of

felodipine and drug loaded nanoparticles indicating on intermolecular interaction.

However, the intensity of the peak at 1261 cm-1

, 1434 cm-1

and 2928 cm-1

were

slightly decreased due to the intermolecular hydrogen bonding indicating the chemical

stability of the nanoparticles.

Figure 5.2.6. FTIR spectra of (a) native felodipine, (b) PLGA 50 :50 and (c)

felodipine loaded PLGA nanoparticle formulation (FP7)

Differential scanning calorimetry (DSC)

Differential scanning calorimetry was performed for the pure felodipine,

PLGA 50:50 and felodipine nanoparticles to analyze the physiochemical interaction

between the drug and polymer inside the nanoparticles (Fig. 5.2.7). The sharp

endothermic peak at 192 ºC in the thermogram represents melting point of felodipine

indicating crystalline nature of the drug (Mura et al., 2001). No distinctive peak was

observed in the thermogram of the felodipine owing to the decreased crystallinity in

the formulation. This is because of drug solvation in the amorphous carrier as well as

solid state interaction induced by heating.

Figure 5.2.7. DSC thermogram of (a) PLGA 50 :50, (b) native felodipine and (c)

felodipine - PLGA nanoparticles (FP7)

In vitro drug release study

Drug release from the nanoparticles is usually a biphasic phenomenon

although on some occasion a triphasic profile is also seen. There is an initial rapid

removal of the drug from the nanoparticles possibly related to loss of drug associated

loosely on the surface of the nanoparticles. This initial release is rapid and

uncontrolled and is termed as burst release. Most of the particles reported in the

literature show this initial release. After the surface adsorbed drug release is complete,

slower release is observed from the nanoparticles (Kreuter, 1994; Juan and Wang,

2002). Drug release profile of drug powder, prepared nanoparticles (FP7) and

marketed formulation in phosphate buffer pH 6.8 are shown in Tab. 5.2.4 & Fig.

5.2.8. The felodipine nanoparticles show slower drug release in comparison with pure

drug powder and marketed formulation.

Table 5.2.4. In vitro drug release profile of marketed formulation, native

felodipine and felodipine loaded PLGA nanoparticles (FP7) in phosphate buffer

pH 6.8

Time (h) Native felodipine FP7 Marketed formulation

0 0 0 0

1 39.73 ± 0.69 16.1 ± 0.63 45.97 ± 0.83

2 48.58 ± 0.97 18.7 ± 0.83 59.49 ± 0.63

4 56.75 ± 0.63 22.3 ± 0.89 73.91 ± 0.82

6 63.24 ± 0.48 24.1 ± 1.16 87.14 ± 1.26

8 66.17 ± 1.02 25.5 ± 0.92 89.54 ± 0.98

10 69.97 ± 0.92 27.8 ± 1.04 90.72 ± 0.84

12 74.06 ± 1.28 37.9 ± 1.10 -

18 79.54 ± 0.81 43.7 ± 0.94 -

24 86.84 ± 0.73 51.2 ± 1.36 -

48 87.54 ± 1.05 58.3 ± 1.17 -

72 87.98 ± 0.82 61.9 ± 1.05 -

96 89.61 ± 0.65 68.3 ± 0.57 -

T50 5.0 ± 0.67 21.6 ± 0.81 1.5 ± 0.71

The values are represented as Mean ± SD; n=3

Figure 5.2.8. In vitro drug release profile of marketed formulation, native

felodipine and felodipine loaded PLGA nanoparticles (FP7) in phosphate buffer

pH 6.8

The drug release from the PLGA nanoparticles in phosphate buffer pH 6.8 was

observed ~68 % after 96 h study and the initial burst release was reduced to 16 % as

compared with pure drug powder. It signifies that the drug was uniformly coated with

the PLGA polymer which results prolongation of drug release. After 10 h the rapid

drug release was identified and this could be as a result of polymer erosion in the

surface of nanoparticles and consequent release of drug which has been loaded near

the surface of nanoparticles (Adibkia et al., 2011). It suggests combination of

diffusion and erosion are the possible mechanism for drug release from the

nanoparticles.

Drug release kinetics: The drug release data are plotted in various kinetic models, viz.

zero order, first order, Higuchi and Korsmeyer-Peppas. Derived data and

corresponding plots with respect to various models are furnished in tables and figures

as follows:

Zero order : Tab. 5.2.5 and Fig. 5.2.9

First order : Tab. 5.2.6 and Fig. 5.2.10

Higuchi : Tab. 5.2.5 and Fig. 5.2.11

Korsmeyer-Peppas : Tab. 5.2.7 and Fig. 5.2.12

Table 5.2.5. Data for Zero order plot (Cumulative % drug release vs. Time) and

Higuchi plot (Cumulative % drug release vs. Sq. root of time) obtained from in

vitro drug release profile of felodipine loaded PLGA (50:50) nanoparticles (FP7).

Time (h) Sq. root of time (h) Cumulative % drug release

0 0 0

1 1.000 16.1

2 1.414 18.7

4 2.000 22.3

6 2.449 24.1

8 2.828 25.5

10 3.162 27.8

12 3.464 37.9

18 4.243 43.7

24 4.899 51.2

48 6.928 58.3

72 8.485 61.9

96 9.798 68.3

Table 5.2.6. Data for first order plot (Log cumulative % drug remaining vs.

Time) obtained from in vitro drug release profile of felodipine loaded PLGA

nanoparticles (FP7).

Table 5.2.7. Data for Korsmeyer-Peppas model (Log cumulative % drug

remaining vs. Log time) for felodipine loaded PLGA nanoparticles (FP7)

Time (h) Log (% drug remaining)

0 -

1 1.924

2 1.914

4 1.892

6 1.881

8 1.875

10 1.857

12 1.792

18 1.748

24 1.690

48 1.623

72 1.580

96 1.505

Time (h) Log time (h) Log cumulative

% drug released

0 0 0

1 0.000 1.20

2 0.301 1.26

4 0.602 1.34

6 0.778 1.38

8 0.903 1.40

10 1.000 1.45

12 1.079 1.58

18 1.255 1.64

24 1.380 1.71

48 1.681 1.76

72 1.857 1.79

96 1.982 1.83

Figure 5.2.9. Felodipine loaded PLGA nanoparticles (FP7) - Zero order release in

phosphate buffer pH 6.8 between 18 to 96 h.

Figure 5.2.10. Felodipine loaded PLGA nanoparticles (FP7) - First order release

in phosphate buffer pH 6.8 between 1 to 18 h.

Figure 5.2.11. Drug release from felodipine loaded PLGA nanoparticles (FP7) in

phosphate buffer pH 6.8 – Higuchi kinetic.

Figure 5.2.12. Drug release from felodipine nanoparticles (FP7) in phosphate

buffer pH 6.8 - Peppas model

Table 5.2.8. Régression coefficient (r2) values plots of drug release data obtained

from the felodipine PLGA nanoparticles (FP7)

Formulation 1st order Zero order Higuchi

Korsmeyer-

Peppas Peppas ‘n’

Period (1- 18h) (18-96h) (1-96h) (1-96h) -

FP7 0.938 0.944 0.940 0.954 0.352

Model fitting of the drug release data showed that the r2 values (Tab. 5.2.8) are

almost identical and difficult to distinguish. The drug release followed first order

kinetics during 1 to 18 h and zero order during 18 to 96 h. Drug release from the

nanoparticles also obeyed Higuchi as well as Peppas models, indicating that the drug

release was by diffusion mechanism. The release exponent ‘n’ value (0.352) was

calculated from the slope of the Peppas model and is less than 0.5, indicating the

release mechanism from the nanoparticles was diffusion controlled. The felodipine

release from the nanoparticles follows the mixed order kinetics.

In vitro permeation across the intestine

To assess the permeability of the formulation across the intestine, everted

intestinal sac model was used. The result showed that about 72.47 ± 1.03 % of

felodipine reached across the intestine to the serosal fluid from the nanoparticles after

8 h study (Fig. 5.2.13.).

Figure 5.2.13. In vitro intestinal permeation study of felodipine loaded

nanoparticles (FP7) in everted intestinal sac model.

The UV-spectroscopy analysis of the serosal fluid confirmed that the felodipine-

PLGA nanoparticles were absorbed as such across the intestine. On the other hand, the

pure felodipine was found to be only 29.56 ± 0.71 % in the serosal fluid. The

increased paracellular uptake of felodipine nanoparticles contributes increased

permeability.

Stability study

The stability study was carried out to establish the suitable storage condition

and the suitability of the formulation. The results of stability studies for the developed

nanoparticles (FP7) are furnished in Tab. 5.2.9. No visible physical changes (colour

and cake formation) were observed for the nanoparticles stored in 25 °C/60% RH and

30 °C/65% RH. But particle aggregation was observed among the nanoparticles stored

in 40 °C/75% RH at 6 months. This may be due to the absorption of moisture on the

nanoparticle surface. No significant drug decomposition was found for particles stored

in long term and accelerated conditions. But some appreciable increase in particle size

was observed when stored in accelerated storage condition. Hence, after exposure in

various environmental conditions the nanoparticulate systems shows no remarkable

changes indicating the stability of the systems.

Table 5.2.9. Stability study data of selected felodipine nanoparticles (FP7)

subjected to three different storage conditions (25 °C/60% RH, 30 °C/65% RH and

40 °C/75% RH)

Test parameters Long term storage condition (25 °C/60% RH)

Periods (months) 0 3 6 12

Physical change No change No change No change No change

Particle size (nm) 227 ± 1.73 228 ± 2.05 228 ± 2.16 231 ± 3.03

Zeta potential (mV) -20.1 ± 1.26 -19.7 ± 1.03 -22.3 ± 0.91 -20.8 ± 1.05

Drug content (%) 100 98.17 ± 0.69 98.02 ± 1.01 97.33 ± 0.94

Intermediate storage condition (30 °C/65% RH)

Physical change No change No change No change -

Particle size (nm) 229 ± 2.26 231 ± 2.08 236 ± 3.11 -

Zeta potential (mV) -19.9 ± 0.82 -20.3 ± 1.07 -20.1 ± 0.76 -

Drug content (%) 100 98.73 ± 0.82 97.02 ± 1.06 -

Accelerated storage condition (40 °C/75% RH)

Physical change No change No change Aggregation -

Particle size (nm) 227 ± 3.14 249 ± 2.92* 264 ± 2.05* -

Zeta potential (mV) -19.8 ± 0.83 -20.6 ± 0.91 -18.5 ± 0.75 -

Drug content (%) 100 97.39 ± 0.96 95.81 ± 1.12 -

The values are represented as Mean ± SD; n=3

(*) p< 0.05 against time t=0

Toxicity evaluation

The in vivo toxicity study of the felodipine loaded PLGA nanoparticulate

systems (FP7) was performed using female albino mice. The toxicity study was

carried out to observe the mortality and dysfunction of the organs. No mortality or

behavioral changes were observed after single dose oral administration of the

felodipine nanoparticle formulation (FP7) up to the dose level of 240 mg/kg body

weight. But complete mortality was observed at dose level of 480 mg/kg body weight.

Serum biochemical parameters also confirmed that there were no noticeable changes

between the test group and control group (Tab. 5.2.10 and Fig.5.2.14). The study

confirms that the nanoparticle formulation does not influence the liver function

parameters and the serum sodium, chloride and potassium.

Table 5.2.10. Serum biochemical reports of control and treated mice

Parameter Reference

value

Control

(Normal

saline)

60mg/kg 120mg/kg 240mg/kg

Serum creatinine

(mg/dL) 0.2 - 0.9 0.20 ± 0.08 0.18 ± 0.04 0.21 ± 0.04 0.22 ± 0.06

Total bilirubin

(mg/dL) 0 - 0.9 0.62 ± 0.06 0.64 ± 0.04 0.68 ± 0.02 0.62 ± 0.04

Total proteins

(g/dL) 3.5 - 7.2 5.2 ± 1.2 5.1 ± 1.4 4.9 ± 1.8 5.2 ± 1.3

ALT (SGPT) U/L 17 - 77 23.8 ± 2.2 22.6 ± 3.4 23.2 ± 2.2 23.4 ± 2.8

AST (SGOT) U/L 54 - 298 65.4 ± 2.2 64.9 ± 2.6 66.4 ± 3.2 66.2 ± 2.4

Sodium (mEq/L) 140 - 160 144.2 ± 3.6 145.3 ± 4.2 144.6 ± 3.6 143.2 ± 4.8

Chloride (mEq/L) 88 - 110 100.6 ± 2.4 99.2 ± 2.0 101.1 ± 3.2 102.1 ± 1.6

Potassium (mEq/L) 5 - 7.5 4.71 ± 1.8 4.70 ± 1.6 4.82 ± 1.2 4.76 ± 1.4

The values are represented as Mean ± SD; n=3

Serum

crea

tinine

Total b

iliru

bin

Total p

rotien

s

ALT (SGPT)

AST (SGOT)

Sodium

Chloride

Potass

ium

0

50

100

150

200Control

60mg/kg

120mg/kg

240mg/kg

Biochemical parameters

Figure 5.2.14. Effect of felodipine-PLGA nanoparticles (FP7) on serum

biochemical parameters in control and treated mice of acute toxicity studies.

Bioavailability study

The availability of the drug to the biologic system and its therapeutic

effectiveness is the goal of dosage form design. The plasma drug concentration-time

profile of felodipine was constructed following the oral administration of pure

felodipine suspension and felodipine PLGA nanoparticles at a dose of 20 mg/kg to

rats. The plasma concentration-time profile is illustrated in Fig. 5.2.15 and the

pharmacokinetic parameters are listed in Tab. 5.2.11.

Figure 5.2.15. Plasma concentration-time profiles of felodipine in rats after oral

administration of free felodipine suspension and felodipine loaded PLGA 50 :50

nanoparticles (20 mg/kg). Each data was given as mean ± S.D. (n=4).

The results showed a significant difference between the pharmacokinetic profiles of

felodipine loaded PLGA nanoparticles and free felodipine suspension (Fig. 5.2.15).

The plasma concentration of felodipine from the nanoparticle formulation (FP7) at

each time points was higher than that of free felodipine suspension. The peak plasma

concentration (Cmax) of felodipine in rats treated with nanoparticles was 1208 ± 62

ng/ml, which was significantly improved (*p< 0.05) compared with that of free

felodipine suspension (821 ± 28 ng/ml). The value of AUC0-72 for rats treated with

felodipine nanoparticles (FP7) was enhanced compared with pure drug suspension.

The improved AUC of felodipine nanoparticles is due to more uptake of FP7 in the

intestine and the sustained release of the felodipine from the nanoparticle formulation.

Table 5.2.11. Pharmacokinetic parameters of felodipine after oral administration

of free drug and nanoparticle formulation (FP7) (20 mg/kg) in rats (n=4).

Parameters Pure drug

suspension

Nanoparticles

(FP7)

Tmax(h) 2 ± 0.12 1.5 ± 0.22

Cmax(ng/ml) 821 ± 28 1208 ± 62*

AUC 0-72(ng.h/ml) 16,033 ± 189 21,446 ± 294*

Relative bioavailability (%) - 133.76 The values are represented as Mean ± SD (n = 4). *p< 0.05

The Tmax value for pure drug was observed 2 h, while the FP7 formulation exhibited

shorter Tmax value of 1.5 h. The reduced Tmax value is the result of rapid absorption of

the nanoparticles formulation.

Antihypertensive activity study

The antihypertensive activity of felodipine nanoparticles (FP7) against L-

NAME induced hypertensive rats were assessed by measuring the systolic blood

pressure (SBP), diastolic blood pressure (DBP) and heart rate (HR). The results are

tabulated in Tab. 5.2.12. The antihypertensive effect of FP7 and pure drug with

positive control rats are depicted in Fig. 5.2.16, Fig. 5.2.17 and Fig. 5.2.18.

The oral administration of FP7 (20 mg/kg) showed the noticeable change of

SBP in L-NAME induced hypertensive rats as compared with pure drug. The

significant reduction of DBP and HR (***p< 0.001) were also observed after the

treatment with nanoparticle formulation. The activity remained up to 24h for the pure

Tab

le

5.2

.12.

Eff

ect

of

felo

dip

ine

nan

op

art

icle

s (F

P7)

on

S

BP

, D

BP

a

nd

H

R

in

L-N

AM

E

ind

uce

d

hy

per

ten

siv

e ra

ts.

Para

met

ers

Gro

up

s P

re-t

reatm

ent

Po

st-a

dm

inis

tra

tio

n (

ho

urs

)

6

24

48

72

SB

P

(mm

/Hg

)

Neg

ativ

e co

ntr

ol

(No

rmal

sal

ine)

1

25 ±

5.2

1

24 ±

4.3

12

5 ±

6.1

1

26 ±

4.5

1

23

± 7

.1

Po

siti

ve

con

tro

l (

L-N

AM

E 4

0 m

g/k

g /

day

) 1

85 ±

4.3

18

8 ±

5.1

a**

*

19

2 ±

3.4

a**

*

19

4 ±

4.2

a**

*

193 ±

4.5

a**

*

L-N

AM

E 4

0 m

g/k

g /

day

&

FP

7 (

20 m

g/k

g)

18

6 ±

4.1

15

9 ±

3.4

b**

*

16

2 ±

4.4

b**

*

16

7 ±

6.1

b**

*

17

4 ±

4.8

b**

L-N

AM

E 4

0 m

g/k

g /

day

& P

ure

Dru

g (

20 m

g/k

g)

18

8 ±

3.7

150 ±

3.9

c***

171 ±

4.2

c***

17

9 ±

3.2

183 ±

2.8

DB

P

(mm

/Hg

)

Neg

ativ

e co

ntr

ol

(No

rmal

sal

ine)

84 ±

4.4

86 ±

2.1

84 ±

2.5

83 ±

2.4

85 ±

3.1

Po

siti

ve

con

tro

l (L

-NA

ME

40

mg/k

g /

day

) 1

10 ±

2.3

10

6 ±

3.4

a**

*

11

1 ±

2.2

a**

*

11

5 ±

3.5

a**

*

113 ±

2.3

a**

*

L-N

AM

E 4

0 m

g/k

g /

day

&

FP

7 (

20 m

g/k

g)

11

2 ±

2.2

88 ±

3.6

b*

**

89 ±

2.4

b***

91 ±

3.9

b*

**

93 ±

3.1

b*

**

L-N

AM

E 4

0 m

g/k

g /

day

& P

ure

Dru

g (

20 m

g/k

g)

11

0 ±

3.7

84 ±

2.9

c**

*

96 ±

4.1

c**

*

10

2 ±

3.5

104 ±

2.2

HR

(Bea

ts/m

in)

Neg

ativ

e co

ntr

ol

(No

rmal

sal

ine)

3

56

± 1

0.4

3

54 ±

8.1

35

2 ±

8.3

3

58 ±

6.2

3

56

± 7

.1

Po

siti

ve

con

tro

l (

L-N

AM

E 4

0 m

g/k

g /

day

) 3

65 ±

5.3

3

67 ±

3.1

36

9 ±

4.5

370 ±

5.3

a*

**

36

8 ±

4.4

a*

L-N

AM

E 4

0 m

g/k

g /

day

&

FP

7 (

20 m

g/k

g)

35

5 ±

2.1

32

0 ±

2.6

b**

*

32

5 ±

3.4

b**

*

33

4 ±

2.2

b**

*d

**

340 ±

3.1

b**

*d

**

L-N

AM

E 4

0 m

g/k

g /

day

& P

ure

Dru

g (

20 m

g/k

g)

36

3 ±

3.1

316 ±

2.9

c***

334 ±

3.7

c***

35

4 ±

3.4

359 ±

2.6

Val

ues

rep

rese

nte

d a

s m

ean ±

S.E

.M (

n=

6)

On

e w

ay A

NO

VA

bet

wee

n n

egat

ive

and

posi

tiv

e co

ntr

ol

gro

up a

nd

the

trea

ted

gro

ups

foll

ow

ed b

y D

un

net

t’s

test

.

a P

osi

tiv

e co

ntr

ol

gro

up v

s. N

egat

ive

con

tro

l g

rou

p,

b F

orm

ula

tio

n t

reat

ed g

rou

p v

s. P

osi

tive

contr

ol

gro

up,

c P

ure

dru

g t

reat

ed g

rou

p v

s. P

osi

tive

con

tro

l g

roup,

d P

ure

dru

g t

reat

ed g

rou

p v

s. F

orm

ula

tio

n t

reat

ed g

rou

p.

p v

alu

es *

*<

0.0

1,

**

*<

0.0

01

hour

th

SBP 6

hour

th

SBP 2

4 h

our

th

SBP 4

8 h

our

nd

SBP 7

2

0

50

100

150

200

250Negative control

Positive control

Formulation treated

Pure drug treated

Time (h)

SB

P (

mm

Hg

)

Figure 5.2.16. Effect of felodipine loaded nanoparticles (FP7) and native drug suspension

on SBP in L- NAME induced hypertensive rats

hour

th

DBP 6

hour

th

DBP 2

4 h

our

th

DBP

48hour

nd

DBP 7

2

0

50

100

150Negative control

Positive control

Formulation treated

Pure drug treated

Time (h)

DB

P (

mm

Hg

)

Figure 5.2.17. Effect of felodipine loaded nanoparticles (FP7) and native drug suspension

on DBP in L- NAME induced hypertensive rats.

hour

th

HR

6 h

our

th

HR 2

4hou

r

th

HR 4

8 h

our

nd

HR 7

2

0

100

200

300

400Negative control

Positive control

Formulation treated

pure drug treated

Time (h)

Heart

rate

(beats

/min

)

Figure 5.2.18. Effect of felodipine loaded nanoparticles (FP7) and native drug suspension

on HR in L- NAME induced hypertensive rats.

-drug treated group, while FP7 formulation showed its activity up to 48h. The SBP of the FP7

formulation treated group and the pure felodipine suspension treated group at different time

interval of post administration was found to be nearly equal to each other. Similarly, the DBP

and the heart rate of the both the group of animal was nearly equal to each other.