A method to prepare solid lipid nanoparticles with improved entrapment efficiency of hydrophilic drugs
ABSTRACT Introduction: Premise of the present study was to suitably select or modify the constitution of the lipid matrix to achieve significantly high entrapment of hydrophilic drugs within solid lipid nanoparticles (SLNs). Methods and Materials: Isoniazid was selected as a representative hydrophilic drug with a high solubility of 230 mg/ml and a log P of -0.402 at 25°C (determined as per OECD TG 105 and 107 respectively). Three lipids/fatty acids (Glyceryl monostearate, Compritol 888 ATO® and stearic acid) were evaluated out of which Compritol 888 ATO® and stearic acid showed favorable interactions (FTIR and DSC studies) with isoniazid. The two lipids were used alone or in combination for preparing SLNs. Formulation of SLNs by microemulsification, method involved pouring the hot microemulsion into cold water under constant stirring, which may result in expulsion of the hydrophilic drug from the lipid matrix; hence, partitioning
of isoniazid from the hot lipid melts into cold water was also determined. Results and Discussion: Results indicate that combining stearic acid with Compritol 888 ATO® in certain ratio (1:4) led to significant entrapment efficiency (EE) of 84.0±1.1%. The formulations were subjected to morphological, physiochemical and in vitro drug release studies. Developed SLNs were found to be stable for 1 year at 4 °C. Conclusion: The study demonstrates the benefit of excipient screening techniques in improving entrapment efficiency of a hydrophilic drug.
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Dataset: Encapsulation of Rifampicinn
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ABSTRACT: Rifampicin (RIF), a vital constituent of antitubercular therapy, hydrolyze at the acidic pH of the stomach. The degradation is further enhanced by its interaction with Isoniazid (INH). Extent of RIF decomposition, in the presence and absence of INH, was determined at pH 1.2 (pH of empty stomach) at 37°C for 4h (maximum stomach residence time). Both the drugs decomposed at gastric pH (26.5% and 1.43% for RIF and INH respectively). Considering that solid lipid nanoparticles (SLNs) avert drug-drug interaction and also drug degradation, we incorporated RIF into SLNs. Latter reduced its degradation to ∼9% (from 26.50% when present alone) and to ∼20% (from 48.81% when INH was also present). Subsequent to this, we also incorporated INH into SLNs and the percent degradation of RIF in this combination (RIF SLNs+INH SLNs) further reduced to 12.35%. Furthermore, the degradation of INH in combination with RIF also reduced significantly from 13.2% to 2.7% when both the drugs were encapsulated individually within SLNs. Study therefore highlights the need to develop combinations of antitubercular drugs (ATDs) with caution and also establishes the usefulness of nanoparticulate technology to avoid drug-drug interaction.International Journal of Pharmaceutics 02/2013; · 3.99 Impact Factor
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A Method to Prepare Solid Lipid Nanoparticles with Improved Entrapment
Efficiency of Hydrophilic Drugs
Current Nanoscience, 2013, 9, 000-000
1573-4137/13 $58.00+.00 © 2013 Bentham Science Publishers
Bhandari Rohit and Kaur Indu Pal*
University Institute of Pharmaceutical Sciences, Panjab University, Chandigarh-160014, India
Abstract: Introduction: Premise of the present study was to suitably select or modify the constitution of the lipid matrix to achieve sig-
nificantly high entrapment of hydrophilic drugs within solid lipid nanoparticles (SLNs). Methods and Materials: Isoniazid was selected
as a representative hydrophilic drug with a high solubility of 230 mg/ml and a log P of -0.402 at 25°C (determined as per OECD TG 105
and 107 respectively). Three lipids/fatty acids (Glyceryl monostearate, Compritol 888 ATO® and stearic acid) were evaluated out of
which Compritol 888 ATO® and stearic acid showed favorable interactions (FTIR and DSC studies) with isoniazid. The two lipids were
used alone or in combination for preparing SLNs. Formulation of SLNs by microemulsification, method involved pouring the hot micro-
emulsion into cold water under constant stirring, which may result in expulsion of the hydrophilic drug from the lipid matrix; hence, par-
titioning of isoniazid from the hot lipid melts into cold water was also determined. Results and Discussion: Results indicate that combin-
ing stearic acid with Compritol 888 ATO® in certain ratio (1:4) led to significant entrapment efficiency (EE) of 84.0±1.1%. The formula-
tions were subjected to morphological, physiochemical and in vitro drug release studies. Developed SLNs were found to be stable for 1
year at 4 °C. Conclusion: The study demonstrates the benefit of excipient screening techniques in improving entrapment efficiency of a
Keywords: Solid lipid nanoparticle; isoniazid; entrapment efficiency, lipid combination, excipient selection.
Solid lipid nanoparticles (SLNs) were first reported  in 1992
and have received considerable interest since then, due to their abil-
ity to overcome the limitations of previously reported colloidal
carriers [2-6] and offer an interesting and effective alternative to
polymeric nanoparticles . They are supposed to be identical to
oil/water emulsion for parenteral nutrition, with the liquid lipid of
the emulsion being replaced with a solid lipid .
SLNs can be prepared from fatty acids, mono, di and triglyc-
erides and phospholipids, which are normal constituents of the hu-
man body and are thus biocompatible [9-11]. SLNs can efficiently
incorporate lipophilic drugs [12-14] because the latter can be incor-
porated easily within the lipid core. However, encapsulation of
hydrophilic materials into the hydrophobic matrix of SLNs is a
challenge, as these drugs tend to partition towards the aqueous
phase during the production process . There are limited exam-
ples of hydrophilic drugs being encapsulated into SLNs [16-21].
The potential of SLNs to incorporate hydrophilic drugs, can be
efficiently harnessed by suitably selecting or modifying the consti-
tution of the lipid matrix; a field hitherto under-explored.
A water soluble drug, isoniazid (Fig. 1), has been used in the
present investigation. It is a nucleoside reverse transcriptase inhibi-
tor and is used in the treatment of tuberculosis (TB). Isoniazid is a
Biopharmaceutical Classification System (BCS) class III drug (high
solubility and low permeability) reported to have aqueous solubility
of 140 mg/ml  and log P of -0.64 . Isoniazid finds its use in
cerebral meningitis (brain TB), however, because of its low cerebral
permeability, the dose and therapy have to be prolonged. Thus,
isoniazid is a potential candidate for delivery via lipid based
nanoparticulate system that will improve its gut permeability and
bioavailability by the avoidance of hepatic first pass metabolism
due to the lymphatic uptake of these lipid nanoparticles. These fac-
tors will reduce the therapeutic dose and the incidence of dose de-
pendent side effects (majorly hepatotoxicity and neurotoxicity)
*Address correspondence to this author at the University Institute of Phar-
maceutical Sciences, Panjab University, Chandigarh-160014. India; Tel: 91
172 2534191 (O); Fax: 91 172 2543101; E-mail: email@example.com
associated with long-term use of isoniazid. A sustained drug release
will also reduce the dosing frequency. Deol et al. made a liposomal
system for isoniazid  with similar aims. The system was how-
ever, suitable only for parenteral / inhalable route . Moreover,
liposomes show low encapsulation efficiency, rapid leakage of
water-soluble drug in the presence of blood components and poor
storage stability [26, 27]. Polymeric nanoparticles of isoniazid with
an improvement in bioavailability have been reported. However,
owing to the limitations of polymeric nanoparticles like the toxicity
of residual solvents, toxic monomer residues, toxic degradation
products and low drug payload, the authors also ventured to de-
velop SLNs of isoniazid by emulsion solvent diffusion technique
and an entrapment efficiency (EE) of 45% ± 4 was achieved by
them . They also demonstrated a significant improvement in
pharmacokinetic behavior of isoniazid upon incorporation into
SLNs. Toxicity is a multidimensional issue for drugs like isoniazid,
which need to be administered chronically for a period varying
from a minimum of 9 months upto 2 years for the control of TB. To
avoid the use of organic solvents we presently propose to use micro
emulsification method for preparing isoniazid-SLNs.
Fig. (1). Isoniazid
Main limiting factor in the entrapment of hydrophilic drugs into
SLNs is their poor loading and retention within the lipid matrix, at
the time of production. Several researchers indicate the influence of
lipid type (velocity of crystallization, hydrophilicity and self-
emulsifying properties of the lipid) on the particle size of the SLN
[11, 29-33]. In the present study, we evaluate the interaction (FTIR,
DSC) of isoniazid with different lipids and lipid/cold water parti-
tioning of isoniazid into these lipids, thereby selecting lipid(s) that
2 Current Nanoscience, 2013, Vol. 9, No. 2 Rohit and Pal
can achieve high EE for isoniazid. Three different lipids, glyceryl
monostearate (GMS) - a mixture of monostearoylglycerol (40-55
%), together with variable quantities of di- (30-45%) and
triacylglycerols (5-15%) with higher mono glyceride content;
Compritol 888 ATO®- consisting of mono- (15-30%), di-(40-60%)
and triglycerides (21-35%) with
triglycerides; and stearic acid, a fatty acid, were employed for the
study. The inclusion of these lipids was based on the fact that GMS
is having three free hydroxyl groups which could bind themselves
with the –NH2 groups of isoniazid, while Compritol 888 ATO®
being rich in triglycerides and stearic acid being a fatty acid are
known to form imperfect crystals with a sufficient space to ac-
commodate drugs of hydrophilic nature.
higher composition of
MATERIALS AND METHODS
Isoniazid was obtained as a gift sample from Panacea Biotec
Ltd. Lalru, Punjab, India. Soya lecithin (Phospholipon 90 H) was
received as a gift sample from Lipoid GmBH, Germany. Compritol
888 ATO ® was a gift sample from Colorcon Asia Pacific Pvt. Ltd,
India. Stearic acid and polysorbate 80 were purchased from Central
Drug House, Mumbai. All other chemicals and solvents used in the
study were of analytical or HPLC grade.
Solubility of Isoniazid in Different Media
Standard curves of isoniazid were prepared in triple distilled
water (TDW), chloroform: water::1:1 and n-octanol in the range of
1—100 ?g/ml. Saturation solubility of isoniazid in aqueous media
was determined at 25ºC as per Organization for Economic Co-
operation and Development (OECD) TG 105 . A preliminary
test was performed by adding increasing volume of water as de-
scribed in the guidelines, for a rough estimate of solubility, fol-
lowed by exact determination using the shake flask method. Similar
method was used for determining the solubility of isoniazid in n-
octanol at 25ºC (test conditions defined under Table 1) to be subse-
quently used for determination of partition coefficient.
Determination of Partition Coefficient
An estimated partition coefficient was determined as per OECD
TG 107  from the ratio of solubility of isoniazid in n-octanol to
that in water. The experimental partition coefficient was determined
by shake-flask method; n-octanol and water were mutually presatu-
rated for 24 h with constant stirring on a magnetic stirrer. A stock
solution of isoniazid was prepared in n-octanol (presaturated with
water) such that the maximum concentration of the test substance,
in each phase, always remains below 0.01 mol per liter during the
experiment. Partitioning of isoniazid was determined at different
ratios of water is to n-octanol (1:1, 1:2, 2:1 volume to volume ra-
tios, as defined by OECD) for determining the associa-
tion/dissociation among isoniazid molecules. The two phases were
added to a centrifuge tube and mixed by rotating the tube at 180°
along the transverse axis (100 times/5 min) at 25°C after which the
organic and the aqueous phase was separated by centrifugation at
2,000 rpm for 10 min. Both the layers were analyzed by UV spec-
trophotometric method and the partition coefficient was calculated
Partition Coefficient; Log10 P n-octanol/water =
drug ofion concentrat Saturation
(Caq) in water
(Coct) octanol-nin drug ofion concentrat Saturation
A mass balance of isoniazid in both the phases was established.
Physical mixture (PM) of isoniazid with lipid(s)/lipid blends
and heat-cooled isoniazid-lipid mixture (D/L-Melts) were evaluated
using FTIR and DSC, to determine the interaction, if any, of isoni-
azid with various lipids to be used for preparing SLNs.
mixtures (lipid: drug: 1:1) and D/L-Melts (lipid: isoniazid: 1:1)
were recorded using KBr pellet technique on an IR spectropho-
tometer (60 MHz Varian EM 360 Perkin Elmer) over a range 400-
FTIR spectra of pure isoniazid, proposed lipids, their physical
lipids and their 1:1 combinations were recorded on a Q20 Differen-
tial Scanning Calorimeter (TA Systems, USA). Samples were
weighed accurately (~5mg) in aluminum pans and heated at a pre-
defined rate of 10ºC/min over the temperature range from 20 to
300ºC in nitrogen atmosphere. Thermal data analysis of DSC ther-
mograms was conducted using TA instruments universal analysis
2000 software (version: 4.5A). The scans were recorded and plots
between heat flow (W/g) and temperature (ºC) were obtained. In-
dium was used as a standard to calibrate the calorimeter.
DSC thermograms of isoniazid, PM and D/L-Melts of proposed
A stock solution (100mcg/ml) of the drug was prepared in
TDW. Known volume of the stock was transferred to different test
tubes containing fixed amounts of melted lipids alone or lipid com-
binations in different ratios. The test tubes were shaken at tempera-
tures 10oC above the respective lipid melting points for 24 h. Sam-
ples were cooled, and centrifuged (10,000 rpm for 20 min) to sepa-
rate the aqueous phase. Both the phases were analyzed spectropho-
tometrically to establish the mass balance. Partition coefficient was
determined as described below-
Where, PCLIPID/WATER = Lipid-water partition co-efficient of
isoniazid; CLipid = the concentration of drug in lipid/lipid mixtures;
CTDW = the concentration of drug in TDW
Preparation and Characterization of Isoniazid-SLN
SLNs were prepared by microemulsification method [36-38].
Briefly the lipidic phase (lipid-8%; stearic acid-STERI-SLN; Com-
pritol 888 ATO®- COMPI-SLN or their 1:4 combination -
COMBI-SLN) and the aqueous phase (polysorbate 80, soy lecithin
and water) were heated to ~10 degree above the lipid melt tempera-
ture. The proportion of surfactant and the volumes of two phases
were adjusted so that a microemulsion was formed spontaneously
upon mixing the two phases. Hot microemulsion, thus formed was
transferred into cold water (~2oC) under constant stirring (WiseTis
HG-15 D, 10,000 rpm) to obtain SLNs. Prepared SLN dispersions
were used for subsequent studies.
Particle Size, PDI and Zeta Potential
SLN formulation(s) was characterized for particle size, PDI and
zeta potential using DelsaNano C, Beckman Coulter, Inc. TDW was
used as a dispersant medium.
Particle Shape and Surface Morphology
Microscopic analysis of the prepared SLNs was carried out to
study the morphology like sphericity and aggregation using a
Transmission Electron Microscope (Hitachi, H-7500). Samples
were stained with phosphotungstic acid (PTA, 2%, 5 minutes and
excess PTA removed), spread on a gold grid and examined.
Total Drug Content (TDC) and EE
Total amount of drug per unit volume present in the formulation
was determined by suitably disrupting 0.1 ml of the SLN dispersion
in 5 ml chloroform: methanol (1:1) volumetrically. The amount of
isoniazid was determined by HPLC. Each experiment was per-
A Method to Prepare Solid Lipid Nanoparticles Current Nanoscience, 2013, Vol. 9, No. 2 3
formed in triplicate. A similarly processed blank SLN sample was
taken as control. The total drug content was determined by using
the equation given below.
dispersion SLN of ml / added drug ofamount Total
dispersion SLN of ml / drug of amount alculated
by centrifuging the developed SLN dispersions at 8.02 Lac g for 2 h
at 4oC using Beckman Coulter Ultracentrifuge (L100 K and 100 Ti
rotor). The EE was calculated as follows:
EE was determined by analyzing the clear supernatant obtained
where Df = amount of drug in clear supernatant fluid
Solution stability of isoniazid was confirmed for 24 hrs using
HPLC before venturing into in vitro drug release studies for isoni-
azid-SLN. Isoniazid was analyzed on a Waters e-2695 ALLIANCE
separation module (HPLC) comprising of a solvent management
system (quaternary gradient mode), auto injector, column oven and
a 4 channel in line degasser), a sample management system (sample
heater cooler) and a 2998 PDA detector. Chromatographic separa-
tion was performed using a symmetry shield RP-C18 column. Data
acquisition was performed using Empower 2® software. The mobile
phase consisting of a mixture of pH 6.8 phosphate buffer and
methanol (85:15), was delivered at a flow rate of 0.9 ml/min and
isoniazid was detected at 254 nm. The injection volume was 20 ?L
and the analysis was performed at 30°C. The experiments were
carried out at a temperature of 37.0±0.5 °C that was maintained
within the HPLC sample heater/cooler compartment. Briefly, a
stock solution of isoniazid was prepared in methanol. The stock was
suitably diluted with pH 6.8 phosphate buffer as the latter was used
as the receptor media for the in vitro drug release studies. Samples
were prepared immediately prior to the zero time analysis. Run time
of the validated isocratic HPLC method was adjusted to accommo-
date sampling at specific time intervals from each of the vials.
Samples were collected up to 24 hrs and the % drug degraded, if
any, was calculated by calculating the % change in area under the
curve (AUC) of samples at each time point with respect to the AUC
obtained at 0 time.
Powder X-ray Diffraction (PXRD)
The crystalline/amorphous nature of formulated nanoparticles
was confirmed by X-ray diffraction measurements carried out with
an X-ray diffractometer (XPERT-PRO, PANalytical, Netherlands).
PXRD studies were performed by exposing the samples to CuK?
radiation (45 kV, 40 mA) and scanning from 5° to 50°, 2? at a step
size of 0.017° and scan step time of 25 s. Samples used for PXRD
analysis were same as those of DSC analysis. The instrument meas-
ures interlayer spacing d which is calculated from the scattering
angle ?, using Bragg’s equation n? = 2d sin ?, where ? is the wave-
length of the incident X-ray beam and n is the order of the interfer-
ence. Obtained PXRD patterns were compared with the characteris-
tic drug peak intensity obtained for the pure drug.
In vitro Drug Release Studies
In vitro release of isoniazid as a free drug and from isoniazid
loaded STERI-, COMPI- and COMBI-SLNs was determined by
dialysis bag method using dialysis membrane with a molecular
weight cut off of 12000-14000 Da. An accurate volume (1 ml) of
SLN dispersion or free drug solution, containing 2.6 mg of isoni-
azid (as per the calculated TDC) was placed inside the dialysis tub-
ing tied tightly at both the ends. The bag so formed was dipped in
80 ml of dissolution media (pH6.8 phosphate buffer maintained at
37.0±0.2 °C) and stirred continuously at 100 rpm on a magnetic
stirrer. Two milliliters aliquots were withdrawn at pre-set time in-
tervals (0.25, 0.5, 1, 2, 3, 4, 6, 8, 12, 24 h) and replaced with an
equal quantity of fresh release medium. After suitable dilution, the
samples were analyzed by HPLC.
All three SLN formulations (STERI-, COMPI- and COMBI-
SLNs) were packed in glass vials and kept at 4oC for twelve
months. Stability parameters viz. TDC, EE and particle size were
evaluated at 0, 2, and 12 months.
Solubility of Isoniazid in Different Media
Standard curves were found to be linear with correlation co-
efficient in TDW, chloroform: methanol::1:1 and n-octanol being
0.999 for each of these. The solubility of isoniazid (determined as
per OECD TG 105) in TDW and n-octanol at 25 °C is presented in
Table 1. Solubility of Isoniazid in TDW and n-Octanol at
25ºC (Determined as per OECD Guideline 105)
Solvent Solubility (mg/ml)
Preliminary test (TDW) 200.000
*Main test: (TDW) 230.000
Preliminary test (n-Octanol) 4.100
*Main test: (n-Octanol) 5.709
*Test conditions for main test: amount of drug 5 times of preliminary solubility, stir-
ring speed 100 rpm, stirring time 72 hrs: sample was withdrawn at 24 hr, 48 hr and 72
hr, kept at 25°C for 24 hrs, filtered through 0.2 micron nylon filter and then absorbance
of each was determined. The difference between 48 hr and 72 hr sample was 0.25%
Determination of Partition Coefficient
Isoniazid demonstrated a negative LogP value of -0.402, indi-
cating a hydrophilic nature of the molecule. The relationship of
partition co-efficient of isoniazid with change in volume ratios is
presented in Table 2.
FTIR spectra of isoniazid and respective PM with three se-
lected lipids/fatty acid viz GMS, Compritol 888 ATO® and stearic
acid alone and in combinations were performed. -NH and -C=O
stretch of isoniazid were observed in the PM and the respective
D/L-Melts. A shift of –NH stretch from 3301.7 cm-1 (isoniazid) to
3335.79 cm-1 for PM and 3358.2 cm-1 for D/L-Melts and -C=O
stretching from 1665.6 cm-1 (isoniazid) to 1653.4 cm-1 for PM and
to 1653.4 cm-1 for D/L-Melts was observed in isoniazid + Compri-
tol 888 ATO® samples (Fig. 2d, 2e). Similarly, for isoniazid +
stearic acid samples, a shift in –C=O stretch was observed from
1665.6 cm-1 to 1700.4 cm-1 for PM and 1699.5 cm-1 for D/L-Melts
while, no –NH stretchings were observed (Fig. 2f, 2g). The samples
containing isoniazid + Compritol 888 ATO® + stearic acid also
pointed towards an interaction. This was evident from a shift in -
C=O stretch from 1665.6 cm-1 to 1705.5 cm-1 for PM and to 1706.4
cm-1 for D/L-Melts; and shift in –NH stretch from 3301.7 cm-1
(isoniazid) to 3303.0 cm-1 for PM and to 3303.8 cm-1 for D/L-
Melts) (Fig. 2h, 2i).
4 Current Nanoscience, 2013, Vol. 9, No. 2 Rohit and Pal
Fig. (2). FTIR Spectra of isoniazid, lipids and their combinations.
A: Compritol 888 ATO®, B: Stearic acid, C: Isoniazid, D: Isoniazid
Compritol 888 ATO® PM, E: Isoniazid Compritol 888 ATO® D/L-Melts, F:
Isoniazid Stearic PM, G: Isoniazid Stearic D/L-Melts, H: Isoniazid+
Compritol 888 ATO®+stearic acid PM, I: Isoniaizd+Compritol 888
ATO®+stearic acid D/L-Melts
Isoniazid, Compritol 888 ATO ® and stearic acid showed endo-
thermic peak at 170.96 °C (247.4 J/g), 70.53 °C (108.6 J/g) and
53.95 °C (185.6 J/g) respectively corresponding to their respective
melting points. A significant decrease in enthalpy of the peak corre-
sponding to lipid was observed for isoniazid + Compritol 888
ATO® D/L-melts [PM: isoniazid + stearic acid + Compritol 888
ATO® (46.09 J/g) < isoniazid + stearic acid (73.52 J/g) < isoni-
azid + Compritol 888 ATO® (87.59 J/g); D/L-melts: isoniazid +
Compritol 888 ATO® (4.49 J/g) < isoniazid + stearic acid + Com-
pritol 888 ATO® (59.35 J/g) < isoniazid + stearic acid (92.70
J/g)]. A maximum % lowering of enthalpy, corresponding to
isoniazid peak, was also observed with isoniazid + Compritol 888
ATO® samples i.e. in PM [PM: isoniazid + Compritol 888 ATO®
(50.75 J/g) < isoniazid + stearic acid (82.25 J/g) < isoniazid +
Compritol 888 ATO® + stearic acid (88.25 J/g); D/L-melt: isoni-
azid + stearic acid (83.42 J/g) < isoniazid + Compritol 888 ATO®
+ stearic acid (86.15 J/g) < isoniazid + Compritol 888 ATO®
Fig. (3). Overlay of DSC thermograms of isoniazid, lipids, physical mix-
tures of isoniazid & lipids and their respective D/L-Melts.
(104.6 J/g)]. The reduction in enthalpy of isoniazid when isoni-
azid-lipid PMs were melted together was observed only in case of
isoniazid + Compritol 888 ATO® + stearic acid samples (Fig. 3,
Partitioning of isoniazid was greater towards Compritol 888
ATO® (PCLIPID/WATER= 0.054). The PC Lipid/Water value was almost
half in stearic acid. A significant increase in partitioning towards
the lipid phase was however, observed when Compritol 888 ATO®
was combined with different ratios of stearic acid. A 1:1 stearic
acid: Compritol 888 ATO® showed a partitioning of 0.200 (4 times
that of Compritol 888 ATO® alone). A decrease in partitioning
towards the lipid phase with further increase in stearic acid was
observed. Combinations with an increasing proportion of Compritol
times that when Compritol 888 ATO® was used alone) was ob-
served with a 1:4 combination of stearic acid: Compritol 888 ATO®
(Fig. 4). Thus, the above combination was selected for preparing
were also tried and a maximum partitioning of 0.302 (~6
CHARACTERIZATION OF SLN
Particle size, TDC and EE
SLNs were prepared using two different lipids i.e., Compritol
888 ATO® and Stearic acid (alone and in 1:4 combination) and a
combination of a surfactant i.e. polysorbate 80 and a co surfactant
(Soy lecithin; Phospholipon 90H). All the SLN formulations exhib-
ited a small particle size below 120 nm (D90) and a TDC of > 92%
w/v (the PDI for all the developed systems was below 0.3¸ indicat-
Table 2. Partition Coefficient of Isoniazid (Determined as per OECD Guideline 107) at 25ºC
Preliminary test 0.023 (Log Pest= -1.64)
n-Octanol : water Concentration in
n-octanol phase (mg/ml)
Concentration in aqueous
1:1 0.287 0.724 0.396 -0.402
1:2 0.190 0.673 0.282 -0.550
2:1 0.697 1.333 0.522 -0.282
*Test conditions for main test: pH of aqueous phase 6.15.
Log Pest = estimated log P
A Method to Prepare Solid Lipid Nanoparticles Current Nanoscience, 2013, Vol. 9, No. 2 5
ing a narrow particle size distribution). COMBI-SLN, as expected
showed a significantly (p ? 0.01) enhanced EE of 84.0±1.1% in
comparison to 77.0±1.9% and 69.0±0.6% for STERI-SLN and
COMPI-SLN, respectively (Table 4).
Particle Shape and Surface Morphology
The TEM (COMBI-SLN) image shows spherical particles with
a small size (67-98 nm) (Fig. 5).
Isoniazid was found to be stable for 24 hrs, at 37±0.5 °C in pH
6.8 phosphate buffer and water with not more than 0.6 and 0.3%
change in AUC, respectively, being observed in either case.
Powder X-Ray Diffraction (PXRD)
Overlaid PXRD patterns of isoniazid, COMBI-SLN, STERI-
SLN and COMPI-SLN are shown in (Fig. 6). PXRD pattern of
isoniazid exhibited sharp peaks at 2? scattered angles 15.4, 16.7,
25.1, 25.9, 26.1, 26.9 and 27.1 indicating a crystalline nature. How-
ever, amorphous halo was obtained in all the SLNs with degree of
amorphicity decreasing in the order COMBI-SLN>STERI-
SLN>COMPI-SLN as evidenced by the presence of peaks at 2?
scattered angles of 21.4 (insignificant), 19.4 and 19.3/21.4 for
COMBI-SLNs, STERI-SLNs and COMPI-SLNs respectively.
In Vitro Drug Release Studies
The drug release study was carried out for all the three devel-
oped SLNs (COMPI-SLNs, STERI-SLNs and COMBI-SLNs).
Release showed triphasic (more appropriately four phasic, includ-
ing a lag phase) behaviour comprising an initial fast release of free
drug (the free drug was not removed from the prepared SLN disper-
sions which were loaded into the dialysis tubing), followed by a
hump and finally a delayed release phase starting approximately at
5 h. The release kinetics followed peppas model (r2 = 0.997, 0.999
and 0.999 for COMPI-SLNs, STERI-SLNs and COMBI-SLNs,
respectively) and 65.09 %, 79.98% and 86.76% of the drug was
released in a period of 24 h for COMPI-SLNs, STERI-SLNs and
COMBI-SLNs respectively (Fig. 7).
All the three SLN formulations were stable with no significant
change in TDC (n.m.t 1%) and EE (n.m.t 6%) upon storage at 4 °C
for 1 year.
Table 3. Differential scanning calorimetry peaks and corresponding enthalpy values of isoniazid, its PM and D/L-melts with
stearic acid and compritol 888 ATO®
Description Melting (°C) Enthalpy (J/g)
Isoniazid 170.96 247.4
Stearic acid 53.95 185.6
Compritol 888 ATO® 70.53 108.6
Melting (°C) Enthalpy (J/g) Melting (°C) Enthalpy (J/g)
Lipid Isoniazid Lipid Isoniazid Lipid Isoniazid Lipid Isoniazid
Isoniazid + stearic acid 54.62 166.10 82.25 73.52 52.91 166.57 83.42 92.70
Isoniazid + Compritol 888 ATO® 69.84 169.36 50.75 87.59 71.90 164.69 104.6 4.49
Isoniazid + stearic acid + Compritol
50.87 166.03 88.25 46.09 49.33 165.62 86.15 59.35
Fig. (4). Lipid-water partitioning profile of isoniazid using different ratios of stearic acid and Compritol 888 ATO®
All values are significantly different from one another (P =<0.05)
6 Current Nanoscience, 2013, Vol. 9, No. 2 Rohit and Pal
Fig. (5). TEM micrograph of isoniazid-SLNs using combination of Stearic acid and Compritol 888 ATO® (COMBI-SLNs).
Table 4. Particle Size, Total Drug Content and Entrapment Efficiency of Developed SLNs
S.No. SLN Composition *D (90%) (nm) PDI TDC (%) **EE (%) Zeta potential (mV)
1 COMPI-SLN 120±0.70 0.281 94±0.8 69±0.6 - 0.101
2 STERI-SLN 116±4.04 0.113 92±2.1 77±1.9 - 0.609
3 COMBI-SLN 113±4.00 0.207 92±1.4 84±1.1 - 0.069
*Particle size D (90%) represents volume distribution pattern; No significant difference between values (P ?0.05) was observed for various SLN compositions.
**All values are significantly different from one another (P ? 0.001)
Fig. (6). Overlaid PXRD patterns of isoniazid (a), COMPI-SLN (b), STERI-SLN (c) and COMBI-SLN (d).
A Method to Prepare Solid Lipid Nanoparticles Current Nanoscience, 2013, Vol. 9, No. 2 7
Fig. (7). In vitro drug release of prepared SLNs in pH 6.8 phosphate buffer (n=6).
All values are significantly different from one another (P =<0.05) except those marked with*.
Fig. (8). Four phasic drug release: phase A (free drug, triangle), Phase B (drug release from outer phospholipid membrane), phase C (release of surface ad-
sorbed drug and small solid lipid nanoparticle), phase D (drug release from matrix).
A measure of the aqueous solubility of a drug molecule is of
utmost importance because it will have a direct influence on its
entrapment in the SLNs. Different values ranging from 125 to
140mg/ml (at 25°C) [22, 39, 40] are reported for the aqueous solu-
bility of isoniazid. Varying values prompted us to experimentally
determine the solubility of isoniazid by standard methods. Isoni-
azid, showed a solubility of 230mg/ml, at 25°C in water, falling in
the category of very soluble drug as per I.P. 2007 . Studies were
performed at two different times and in strict adherence to OECD
guidelines [34, 35].
The difference between log P values at three different ratios of
n-octanol and water were within the limits i.e. ± 0.3  defined for
non-association/dissociation in n-octanol or water (Table 2). A
negative log P (-0.402) confirms the hydrophilic nature of isoniazid.
The experimental solubility and partition coefficient values substan-
tiate the suitable candidature of isoniazid for the presently proposed
study on hydrophilic drugs.
GMS, Compritol 888 ATO® and stearic acid were selected as
discussed under the introduction section. It is expected that a posi-
tive drug-lipid interaction could help in improving the EE as well as
prevent the drug expulsion when the hot microemulsion is poured
into cold water for the formation of SLNs. In view of this, the inter-
action between PM and the respective D/L-Melts (as the method of
preparation for SLNs involves melting of the lipids during micro-
emulsification followed by abrupt cooling) was determined by
FTIR and DSC. The FTIR spectra of isoniazid showed an –NH
8 Current Nanoscience, 2013, Vol. 9, No. 2 Rohit and Pal
stretch at 3301.7 cm-1 and –C=O stretch at 1665 cm-1. A shift in the
–NH and –C=O stretch indicated the participation of these groups
during the interaction between the drug and the lipid molecules.
The extent of interaction is proportional to the magnitude of the
shifts in the stretching frequencies. Thus, a higher shift in –C=O
stretching with stearic acid and significant –NH stretching observed
with Compritol 888 ATO® samples directed our interest towards
stearic acid and Compritol 888 ATO®. No significant (-C=O and –
NH) stretching was observed between isoniazid-GMS PM and
D/L-Melts and thus GMS was abandoned from further studies
(results not shown). Compritol 888 ATO® showed an interaction at
both the -C=O and –NH groups while stearic acid samples showed
only –C=O stretching. An elaborate –C=O stretching observed in
isoniazid + Compritol 888 ATO® + stearic acid (PM and D/L-melt)
could account for a significantly better EE of isoniazid within the
melts with stearic acid or Compritol 888 ATO® point towards inter-
actions (Fig. 3). It may be noted that both solubility of a drug in
lipid and the presence of lipid imperfections (indicating spaces for
drug incorporation into lipidic crystal lattice) are important consid-
erations monitoring drug loading. In the present study, the results
were analyzed for a decrease in enthalpy change that correlates with
the generation of imperfections (loss of crystallinity) or spaces to
better accommodate the drug molecule within the lipid crystals.
This decrease in crystallinity should however be maintained during
SLN preparation, in order for the drug molecule to be retained in-
side the solid lipid carcass, and hence the study was extended to
analyzing D/L-melts also.
A lowering of enthalpy (Table 3) corresponding to lipid from
87.59 J/g (PM) to 4.478 (D/L-melt) was observed for Compritol
888 ATO® which could be because of the interactions of isoniazid
and Compritol 888 ATO® at multiple groups (-C=O and –NH, refer
FTIR results), and, the possible distortion of crystal lattice of Com-
pritol 888 ATO®. Latter can exert a direct influence on EE and an
improvement in drug loading. On the other hand, a reduction in
enthalpy of isoniazid upon melting was observed in case of combi-
nation (isoniazid + stearic acid + Compritol 888 ATO®). This could
be because of a more significant stretching of the –C=O group of
isoniazid by both Compritol 888 ATO® and stearic acid synergisti-
cally, which further distorted the crystalline structure of isoniazid.
The reduction in enthalpy of isoniazid, specifically in case of D/L-
melts, could be correlated to a possible increase in solubility of
isoniazid, which is again an essential parameter for improving the
EE, drug loading as well as preventing the expulsion of isoniazid
from the SLNs, during preparation and storage.
We also observed an increase in enthalpy of the lipids in case of
stearic acid alone and in combination lipids, but the increase was
more significant for stearic acid D/L-melts than the combination
lipids. This would indirectly mean a creation of fewer imperfections
on the melting of stearic acid (D/L-melts), or a greater drug loading
in a combination. It may be concluded, in other words, that the drug
+ stearic acid + Compritol 888 ATO® combination shows a reduc-
tion in crystallanity, of both the lipid as well as the drug such that a
higher EE is achieved for COMBI-SLNs.
Shifts in the melting endotherms of isoniazid in PMs and D/L-
Since isoniazid is hydrophilic in nature, it becomes important to
identify or use lipids in which it has a favourable solubility and
partitioning. This will result in a better loading and entrapment of
the drug. FTIR spectra pointed towards an interaction of isoniazid
with two of the selected lipid / fatty acids i.e. Compritol 888 ATO®
and stearic acid.
Both the DSC and FTIR studies pointed towards a suitability of
combining stearic acid with Compritol 888 ATO® to prepare SLNs
with a better loading of the drug and a higher EE (Table 4). Simi-
lar suggestions have been made by other workers . When these
two lipids were combined in different ratios, it was found that
isoniazid showed a maximum partitioning towards the lipid phase
comprising a 1:4 proportion of stearic acid: Compritol 888 ATO®.
CHARACTERIZATION OF SLN
Particle Size, TDC and EE
SLNs were prepared using microemulsification method as it
results in small particles. Small particle size of below 200 nm
(120nm; volume distribution data) was also desired as the particles
smaller than 200 nm usually remain invisible to the reticulo-
endothelial system (RES) and thus show a prolonged circulation.
A high recovery is a prerequisite for development of any formu-
lation as it indicates smaller losses during the production process.
The TDC values of above 92% indicate the scale up applicability of
The high entrapment efficiencies obtained (greater than 69%)
have never been reported before for isoniazid. An even higher EE
obtained with combination lipids is probably due the additive or
synergistic interaction of isoniazid with both the lipids, as elabo-
rated under FTIR and DSC studies. FTIR showed a stretching of
both –C=O and –NH- bonds of isoniazid when it combined with
Compritol 888 ATO®. Similarly, DSC studies indicated that
isoniazid probably dissolves in stearic acid melt while Compritol
888 ATO® melt itself undergoes significant reduction in enthalpy
to incorporate more of the isoniazid. Hence, combining the two
lipids may result in an enhanced incorporation of isoniazid into
the lipid mix melt by two different mechanism of solubility and
incorporation into empty spaces of the imperfect lipid matrix.
Lipid-water partitioning studies indicate that mixing these two
lipids in a specific proportion (1:4: stearic acid: Compritol 888
ATO®) has a significantly beneficial influence (~84.0±1.1% EE).
Particle Shape and Surface Morphology
The permeability of a particle / nanoparticle largely depends on
its size . Particles greater than 200 nm are usually detected and
filtered out by the reticular meshwork. The shape and surface mor-
phology of prepared SLNs were studied by TEM. The particle size
observed with TEM micrographs corresponded well with the num-
ber distribution pattern (99% particles of size not more than 25 nm)
as determined by DLS. The TEM images confirmed that the parti-
cles are spherical in shape.
Solution stability of isoniazid indicated absence of degradation
for upto 24 hrs. This was done to establish stability of isoniazid in
solution under period of test extending to 24 hrs during the drug
Powder X-ray Diffraction (PXRD)
The presence of sharp peaks in the PXRD pattern of isoniazid
indicated crystalline nature. However, the SLNs showed absence of
all the major characteristic peaks of isoniazid indicating its amor-
phousness upon incorporation into lipid matrix. The effect was
observed most profoundly with COMBI-SLNs.
In vitro Drug Release
All the three types of SLNs showed an initial phase of very fast
release (upto 1 hr) which could be attributed to the presence of
unentrapped isoniazid (free drug; phase A, Fig. 8). Isoniazid is a
highly water soluble drug and hence its incorporation (in part) into
the outer phospholipid layer of the lipidic nanoparticles and its
consequent release into the dialysis bag which acts as second bar-
rier before the drug passes on to the dialysis media, may have re-
sulted in a lag phase (between 1 to 2 hr; phase B). However, this lag
phase is short lived and a second phase of burst release (phase C)
A Method to Prepare Solid Lipid Nanoparticles Current Nanoscience, 2013, Vol. 9, No. 2 9
commenced and further continued until the start of the sustained
release phase (post 5 hr). The burst effect could be attributed to the
release of isoniazid adsorbed on the surface of SLNs or precipitated
from the superficial lipid matrix [44-46]. Furthermore, we feel that
burst release may at least partially be accounted for, by the free
passage of very small SLNs across the dialysis membrane. A sus-
tained release (phase D) was observed in all the formulations and
suggests release of drug by dissolution and/or diffusion from the
lipid core, wherein the drug is homogeneously distributed . A
slow release of isoniazid from COMPI-SLN in contrast to STERI-
SLN was observed. This is probably caused by a high lipophilicity
of Compritol 888 ATO® (C69H134O6) compared with stearic acid
(C18H36O2) i.e. the higher carbon chain length contributing to the
more sustained release effect . The release pattern between the
three formulations follows the order COMBI-SLN>STERI-
SLN>COMPI-SLN. All the formulations exhibited an amorphous
nature (Fig. 6) and the extent of amorphicity followed the order:
COMBI-SLN>STERI-SLN>COMPI-SLN. It may be noted that in
general solubility increases as the amorphicity increases and hence
highest release was obtained with COMBI-SLN and a compara-
tively sustained release was obtained with COMPI-SLN. Higher
amorphicity could similarly be responsible for the relatively faster
release of isoniazid from COMBI-SLN during the initial phase also.
This initial phase of fast drug release (COMPI-SLN: ~32%, STERI
SLN: ~44%, COMBI-SLN: ~45%) can however, be exploited bene-
ficially to provide a loading dose of isoniazid within 1 h of its ad-
ministration while the sustained release of remaining drug may help
to maintain the plasma concentration for >24 h.
Our studies clearly demonstrate the advantage of using a sys-
tematic approach of lipid selection based on FTIR and DSC studies,
which may help to identify suitable lipid candidates for preparing
SLNs with high EE. Use of microemulsification method and a 1:4
combination of stearic acid with Compritol 888 ATO® as the lipid
component ensured a high EE of 84.0%. Such a high EE is hitherto
unreported for hydrophilic drug molecules. Pandey et al. have re-
ported an EE of 45% for isoniazid . Elaborate evaluation of
FTIR and DSC data could explain the reasons for high EE and the
release pattern for SLNs with different lipid compositions. The
developed SLN formulation was also found to be stable for 1 year
with an insignificant change in the stability parameters.
CONFLICT OF INTEREST
The authors confirm that this article content has no conflicts of
The work was funded by a grant from the Department of Bio-
technology, Government of India, New Delhi, India. We
acknowledge the use of Sophisticated Analytical Instrumentation
Facility (SAIF), Panjab University, Chandigarh and Mr Dinesh
Sharma for the TEM studies.
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Received: ?????? 28, 2011
Revised: ?????? 22, 2012 Accepted: ????? 6, 2012