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Der Pharmacia Lettre, 2013, 5 (4):43-50
ISSN 0975-5071

Development and enhancement of entrapment efficiency of isoniazid loaded

poly--caprolactone nanoparticle
Atanu Kumar Behera*1, S. Shah1 and B. B. Barik2

Monad University, Hapur, Uttar Pradesh, India

Jazan University, Saudi Arabia

The present study was aimed at enhancing the entrapment efficiency of highly water soluble Isoniazid (INH)
nanoparticles using the biodegradable polymers, Poly--caprolactone (PCL) by varying the different formulation
parameters such as polymer ratio, amount of drug loading (w/w), solvent selection, electrolyte addition and pH in
the formulation. The PCL loaded INH were prepared by water-in-oil-in-water double emulsion technique. The
prepared nanoparticles were characterized by atomic forced microscopy (AFM), differential scanning calorimetry
(DSC) and for in vitro drug release study. Fourier transform infrared spectroscopy (FTIR) study, differential
scanning calorimetry analysis and in vitro release kinetics study. The percentage entrapment can be enhanced up to
63 by changing different formulation parameters.
Key words: Isoniazid, Polycaprolactone, AFM, DSC, Nanoparticle
Isoniazid is the one of the first line drug used for the treatment of tuberculosis. It is a bactericidal agent active
against organisms of the genus Mycobacterium, specifically M. Tuberculosis, M. bovis and M. kansasii. Isoniazid is
bactericidal to rapidly-dividing mycobacteria, but is bacteristatic if the Mycobacterium is slow growing. Isoniazid is
a prodrug activated by catalase-peroxidase hemoprotein, KatG. Isoniazid inhibits InhA, a nicotinamide adenine
dinucleotide (NADH) -specific enoyl-acyl carrier protein (ACP) reductase involved in fatty acid synthesis [1].
Isoniazid is readily absorbed when administered either orally or parentally. The plasma half-life of isoniazid ranges
from 1-4 h, those who are fast acetylators because of genetic variations, having short half-lives. The most important
drawback of the current therapy used for the treatment of tuberculosis is the frequency and amount of the drug used.
The limitation is because of the instability in the biological environment and premature loss through rapid
clearance and metabolism [1]. Moreover, high concentration of these agents may be toxic to healthy tissues. Thus, to
enhance the therapeutic efficacy, modern drug delivery plays an important role in controlled delivery of these agents
to the target site of the body at a therapeutically optimal rate and concentration. These controlled release systems are
proficient to maintain optimum therapeutic drug concentration in the blood with minimum fluctuation giving
predictable and reproducible release rates for a longer period of time, enhancing the duration of action of drugs with
a short half-life, eliminating the side effects of frequent dosing and limiting wastage of drugs, and providing an
optimized therapy and improved patient compliance [2]. All of the features are strictly related to the nature of
materials that constitute the continuous matrix of the delivery system.

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Atanu Kumar Behera et al

Der Pharmacia Lettre, 2013, 5 (4):43-50
PCL is an important member of the aliphatic polyester family can be used in alone or combined with several
polymers to formulate different formulations. It has been shown that the entrapment of hydrophilic drugs inside
hydrophobic polymeric nanoparticles is a difficult task [3-5]. This is because the entrapment efficiency of the
hydrophilic drug inside the hydrophobic nanoparticle is low due to the low affinity of hydrophilic drugs for
hydrophobic polymers. Furthermore, the interaction between the polymer and the entrapped drug is weak and the
drug has a tendency to move from the organic phase to the outer aqueous phase during nanoparticle formation [6].
The objective of this study is to encapsulate RIF inside PCL nanoparticles and to increase the encapsulation
efficiency by optimizing different parameters, like amount of polymer amount of drug loaded, solvent selection, pH
value of inner aqueous phase and the addition of salt to outer aqueous phase. The present study included
preparation and characterization of INH loaded PCL nanoparticles.
2.1 Materials
Isoniazid was a gift sample from Macleod Pharmaceuticals, Mumbai; Poly- -caprolactone (PCL MW 65000 g/Mol)
was purchased from Sigma Aldrich. Polyvinyl alcohol (PVA MW 88000 g/Mol), DCM, Phosphate buffer, all are of
analytical grade was purchased from S.D. Fine Chem Ltd., Mumbai.
2.2 Method of preparation of PCL Nanoparticles
Isoniazid (INH) loaded nanoparticles were prepared with water in oil in water double emulsion solvent evaporation
method [7]. In this method INH equivalent to 5-14 % w/v was dissolved in 500 l of phosphate buffered saline (0.01
M, pH 7.4) to form INH aqueous solution. The INH aqueous solution was emulsified in an organic phase consisting
of 100-500 mg of the PCL polymer in 5 ml of organic solvent (DCM) to form primary water in oil emulsion by
probe sonicator using a micro tip probe sonicator (VC 505, Vibracell Sonic, Newton, MA, USA) set as 55 W of
energy output for 3 minutes over an ice bath. The emulsion was further emulsified in an aqueous PVA solution
(20ml 2.5% w/v) to form water in oil-in-water emulsion. The emulsification was carried out using a micro tip
probe sonicator (VC 505, Vibracell Sonic, Newton, MA, USA) set as 55 W of energy output for 5 minutes over an
ice bath by adding the primary emulsion drop wise to the 20 ml of phosphate buffer (0.01 M, pH 7.4). The emulsion
was stirred for 2 hours on a magnetic stir plate at room temperature to allow the evaporation of organic solvent.
Further one hour vacuum drying was also performed to remove any residual organic solvent present. Any excess
amount of PVA was removed by ultracentrifugation at 16000 rpm at 40C for 20 minutes ( Remi, India) followed by
washing with double distilled water. The supernatant was collected and kept for an estimation of the amount of the
drug which was not encapsulated. The recovered nanoparticulate suspension was lyophilized for two days (-800 C
and < 10 mm mercury pressure, LYPHILOCK 12, Labconco, Kansas City, MO, USA) to provide the lyophilized
powder for further use.
2.3 Physical characterization
2.3.1 Measurement of particle size, polydispersity index and zeta potential
Particle size distribution of isoniazid loaded PCL nanoparticles was determined by a laser scanning technique using
Malvern instrument after appropriate dilution with distilled water. Approximately 5 mg of dried particles was resuspended in 1.0 ml of distilled water and the resulting solution was briefly vortexes and treated in a bath sonication
for 3 min. This suspension was analyzed at an obscuration of 10-20% on a Malvern Mastersizer. The mean particle
size and polydispersity index zeta potential were calculated for each formulation maintained at 250C. Polydispersity
index will measure the size distribution of nanoparticles population.
2.3.2 Differential scanning calorimetry (DSC)
DSC analysis was performed in order to investigate the melting and crystallization behavior of crystalline materials
like PCL nanoparticles. The samples were sealed in aluminum pans and measurements were recorded using DSC
instrument. The samples were heated from 25 to 2000C at a heating rate of 100C /min under nitrogen atmosphere.
2.3.3 Atomic Force Microscopic studies (AFM)
The shape of Drug loaded NPs was further characterized by AFM (JPK nanowizard II, JPK instrument,
Bouchestrasse, Berlin, Germany) consisting of pyramidal cantilevers with silicon probes having force constants of
0.2 N/m. Samples for AFM imaging were prepared by placing a drop of the NPs suspension (1 mg/ml) on a freshly
cleaved mica sheet. After 5 minutes of incubation, the surface was gently rinsed with deionized water to remove
unbound NPs. The sample was air dried at room temperature and mounted on the microscope scanner. The shape

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Atanu Kumar Behera et al

Der Pharmacia Lettre, 2013, 5 (4):43-50
was observed and imaged in contact mode set at a frequency of 13 kHz and scanned at a speed of 1 Hz. The images
were analyzed using JPK data processing software.
2.3.4 Entrapment Efficiency
The prepared PCL nanoparticles dispersion was centrifuged at 15000 rpm for 30 min at 00C using REMI cooling
centrifuge. Then the supernatant is analyzed for the free drug content. The concentration of RIF in the supernatant
was determined by UVVisible spectrophotometry at 262 nm. Entrapment efficiency was using the below Equation.
Entrapment efficiency (%) = (RFMinitial RFM supernatant) / RFMinitial 100
2.3.5 Drug Loading
The INH content of nanoparticle was determined for all the formulations by dissolving 10mg of nanoparticle in
200L of DCM has taken in 2mL Eppendorf tubes and vortexed for 10 min. The ethanol (1800L) was added and
vortexed for another 5 min followed by centrifugation at 3000 rpm for 10 min to settle down the PCL precipitated.
The supernatant containing INH was suitably diluted using ethanol and the absorbance was measured at 262nm by
UVVis spectrophotometry and equivalent concentration was determined using the calibration curve prepared using
the same proportion of solvents. The percentage (%) drug loading (DL) of the nanoparticle was calculated using the
following formula.
DL (%) = WDL/WNP 100
Where WDL is the weight of the drug in INH nanoparticles and WNP is the weight of INH nanoparticles.
2.3.6 In Vitro Drug Release
In vitro release studies from NPs was determined in PBS buffer (0.01 M, pH 7.4), containing 1% Na CMC at 37oC
0.5oC. 50 mg of drug loaded NPs was dispersed in 15 ml of above buffer. The NP suspension was equally divided in
three tubes containing 5ml each. These tubes were kept in a shaker at 37 C and 150 RPM (Wadegati Labequip,
India). At particular time intervals, these tubes were taken out from shaker and centrifuged at 13,800 RPM, 4 C for
10 minutes (REMI 1-16K, Mumbai). The supernatants were taken out to estimate the amount of drug release, at that
particular time by using a spectrophotometer (UV-1700, Schimadzu). To the residue same amount of fresh PBS
(0.01 M, pH 7.4, containing 1% w/v Na CMC was added and kept in shaker for further study [8].
In vitro drug-release data were fitted to kinetic models such as zero order, first order, Higuchi equation and
KorsmeyerPeppas equation. The regression analysis of Q vs. t (zero order), log Q vs. t (first order), Q vs. square
root of t (Higuchi), log%Q vs. log%t (KorsmeyerPeppas), where Q is the amount of drug released at time t, was
performed [9].
2.3.7 Statistical analysis
The results are expressed as mean standard deviation (SD, n = 3). The data were statistically analyzed by one-way
analysis of variance using Graph Pad Instat, version 3.05 (USA), and a significant difference was set at p < 0.05.
The nanoparticle formation from double emulsion/solvent evaporation system involves preparation of oil/water
(o/w) emulsions with subsequent removal of the oil phase (i.e., typically a volatile organic solvent) through
evaporation. The emulsions are usually prepared by emulsifying the organic phase containing the drug, polymer and
organic solvent in an aqueous solution containing emulsifier. The organic solvent diffuses out of the polymer phase
and into the aqueous phase, and is then evaporated, forming drug-loaded polymeric nanoparticles.
3.1 Optimization of Solvent
The polarity of the organic solvent used in the emulsion formation during the nanoparticle formulation might affect
the entrapment efficiency. Therefore, nanoparticles with two different organic solvents, acetonitrile (ACN) and
dichloromethane (DCM) were formulated under identical conditions. The water solubility of ACN was greater than
DCM and therefore the rate of precipitation was higher than the rate with DCM. Due to the high water solubility of
ACN, it had a higher diffusion rate before hardening. This was the major reason for the low entrapment efficiency in

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Atanu Kumar Behera et al

Der Pharmacia Lettre, 2013, 5 (4):43-50
case of ACN [10]. Thus, DCM is the more appropriate solvent to use when encapsulating hydrophilic drugs in a
hydrophobic polymer [11-12].
3.2 Optimization of Polymer and Drug Ratio
For enhanced drug entrapment, varying concentrations of INH were incorporated into the aqueous medium from 25
to 70 mg in 500l of water. At the same time the amount of polycaprolactone was also varied from 300 mg to 500
mg in 5 ml of organic solvent i.e., DCM. The percentage of drug loading varied from 25 to 70 mg corresponding to
the amount of polymer 300-500mg. Different possible combination were taken and the formulation was prepared
with 2.5% w/v PVA as a surfactant. The entrapment efficiency of INH in the nanoparticles was found to be highest
with 45 mg in 500 l of water and 500 mg of PCL was found to be 48% which was the highest one but at the same
time the particle size was around 349 12.84 so the formulation NP 5 was chosen as the though the entrapment
efficiency is 42 % but the size was 229 6.37nm which was detailed in table no.1.
This may be due to the saturation level of INH inside the nanoparticles after 45 mg drug loading. As the amount of
drug loaded increases, a more porous polymeric matrix structure may be formed with a large number of channels
and hollow spaces, through which the drug could easily escape to the outer phase thereby decreasing the content of
drug inside the polymeric matrix [13]. Furthermore, owing to the increased concentration of the drug inside the
polymer, a difference in osmotic pressure between the outer and inner aqueous phase results, this may cause the
drug to escape from the inner aqueous phase [14].
3.3 Optimization of pH of the inner aqueous phase (in which the drug is dissolved)
In the preparation of INH nanoparticle, by changing the pH of the inner aqueous phase from 7.4 to 4 and by adding
4% w/v NaCl to the outer aqueous phase the encapsulation efficiency increased from 42 to 53% table no. 2. The pH
of the water phase affects the ionization of the drug substance and, hence, the solubility. An ionic drug substance is
likely to stay in the water phase, while the molecular form is more likely to be attached to the hydrophobic polymer
phase, and, in this case, the drug substance is more efficiently encapsulated [15]. Based on this finding, by simply
adjusting and controlling the pH value, the entrapment efficiency of INH inside nanoparticles can be increased. By
changing the pH of the inner aqueous phase from 7.4 to 4 the drug is dissolved in this acidic aqueous solution and
does not diffuse to the outer aqueous phase. As a result the drug may be more easily entrapped in the polymeric
matrix leading to a higher encapsulation efficiency of nanoparticles [16].
3.4 Optimization of addition of an electrolyte to the outer aqueous phase
The addition of an electrolyte affects the osmotic gradient between the inner and outer aqueous phases; this may
have an impact on drug entrapment. With the addition of salt, the concentration of the outer aqueous phase (PVA
solution) increases and becomes hypertonic; therefore, the drug does not diffuse into the outer aqueous phase and
remains in the polymeric matrix [17-18]. That result an increment of entrapment of efficiency up to 63% detailed in
table no.3.
3.5 DSC Studies
DSC study shows the nature of the drug encapsulated in the NPs. This analysis was performed on native PCL, native
Isoniazid and Isoniazid loaded NPs. Different compounds show their characteristic peaks in DSC (figure 1). The
endothermic peaks of PCL and was found approximately at 65 C due to glass transition temperature (Tg) of PCL.
The peak of PCL was slightly shifted in drug-loaded NPs as compared to that of native PCL. The endothermic peak
of native Isoniazid was found approximately at 173.12 C.
3.6 FTIR of INH nanoparticle
The FTIR analysis was used to study the chemical modifications or changes that occurred in the polymer in the form
of a band stretching or bending due to the addition of the drug during the synthesis of NPs. Fig. 2, shows the FTIR
spectra of native isoniazid, void NPs .Loaded NPs. FTIR results confirmed the chemical stability of rifampicin in the
nanoparticles. Native Isoniazid showed characteristic bands due to the presence of different functional groups. A
stretching vibrations, while those observed at 2971.63, 2865.79cm-1 are due to the C-H stretching vibrations,
1756.59 cm-1 due to C=O stretching, 1657.20 cm-1 due to N-H bend (due to substituted amide group), 1539.39 due
to C=C stretching (due to pyridine ring), 1323.47 cm-1 due to C-N stretching (due to aromatic ring), 1002.82 cm-1,
1055.17 cm-1 is due to C-N stretching (due to aliphatic) and 648 cm-1 due to =CH. The bands occurring in void NPs
are almost similar to the bands in isoniazid loaded NPs in addition with some extra bands due to the presence of
isoniazid. Thus, in this study the bands appearing at 3300, 3691.91, 3697.7, 3724.7, 3982.21, cm-1 for native

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Der Pharmacia Lettre, 2013, 5 (4):43-50
isoniazid are also appearing in isoniazid loaded NPs (with minor shifting) indicating the chemical stability of
isoniazid inside the NPs.
3.7 AFM of Rifampicin
The AFM technique was used to study the detailed morphology of NPs, as the prepared NPs are too small to be
closely investigated by SEM due to its limited magnification. Hydrodynamic radii measured by dynamic light
scattering were confirmed by AFM micrographs. AFM results confirmed the smooth and spherical surface of the
formulated NPs along with the absence of aggregation or adhesion among NPs.
3.8 Zeta Potential.
Zeta Potential of the prepared formulation found to be average of -26.94 mv which gives stability to the formulation
on the storage (Fig 4).
3.9 In vitro dissolution test
Figure 5 shows the in vitro release profile of Isoniazid, loaded NPs. A slow sustained release of Isoniazid due to the
entrapment of drugs inside the core of nanoparticles as the surface modification increased. In the formulations
highest regression co-efficient was observed in zero order equations starting from day one to tenth day (Table 6).
The slower and sustained release of the drug from the beginning can be attributed to the erosion of polymeric matrix
which releases the encapsulated drug [19].
Table. 1 Optimization of Polymer and Drug Ratio
NP 1
NP 2
NP 3
NP 4
NP 5
NP 6
NP 7
NP 8
NP 9

Drug Loading in 500 l of aq.

Solvent (mg)

Polymer Amount in 5 ml
DCM (mg)

pH of inner aqueous



Table. 2 Optimization of pH of the inner aqueous phase

NP 10
NP 11
NP 12
NP 13
NP 14
NP 15

Polymer Amount in 5 ml

Drug Loading in500 l of aq.


pH of inner aqueous


Table. 3 Optimization of addition of an electrolyte to the outer aqueous phase

NP 16
NP 17
NP 18
NP 19
NP 20
NP 21
NP 22

Polymer Amount in 5
ml DCM

Drug Loading in500 l of

aq. Solvent

pH of inner
aqueous phase

% Salt addition in outer

aqueous medium


Table 6. R2 value for Isoniazid Dissolution Profile

R2 Value

Zero Order

First Order

Hixon Crowell

Korsmeyer Pappas

Higuchi Plot

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Der Pharmacia Lettre, 2013, 5 (4):43-50
Figure. 1

Figure. 2

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Der Pharmacia Lettre, 2013, 5 (4):43-50
Figure. 3

Figure. 4

Figure 5

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Der Pharmacia Lettre, 2013, 5 (4):43-50
Overall, formulation NP19 which has a suitable particle size, drug entrapment efficiency and controlled release
profile was the most satisfactory among all the NPs. The entrapment efficiency of the formulation can be enhanced
up to 63 % having a drug loading of 11.93% by optimizing the formulation parameters and process parameters.
The authors are thankful to Macleod Pharmaceuticals, Mumbai, India, for the gift sample. The authors are also
grateful to the Head of Department, University Department of Pharmaceutical Sciences, Utkal University,
Bhubaneswar, Odisha, India for making available the research facilities used.
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