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Development and optimization of solid lipid nanoparticles of amikacin by

central composite design

Article  in  Journal of Liposome Research · August 2009

DOI: 10.3109/08982100903103904 · Source: PubMed

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 Journal of Liposome Research, 2010; 20(2): 97–104

RESEARCH ARTICLE

Development and optimization of solid lipid

nanoparticles of amikacin by central
composite design
Jaleh Varshosaz1, Solmaz Ghaffari1, Mohammad Reza Khoshayand2, Fatemeh Atyabi3,
Journal of Liposome Research Downloaded from informahealthcare.com by University of Alberta on 06/03/10

Shirzad Azarmi4,5, and Farzad Kobarfard6

1
Department of Pharmaceutics, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran,
2
Department of Drug and Food Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran,
3
Department of Pharmaceutics and Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of
Medical Sciences, Tehran, Iran, 4Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton,
Alta, Canada, 5Research Center for Pharmaceutical Nanotechnology, Faculty of Pharmacy, Tabriz University of
Medical Sciences, Tabriz, Iran, and 6Department of Medical Chemistry, Faculty of Pharmacy, Shahid Beheshti
University of Medical Sciences, Tehran, Iran

Abstract
For personal use only.

Solid lipid nanoparticles (SLNs) have been studied as a drug-delivery system for the controlling of drug
release. These colloidal systems have many important advantages, such as biocompatibility, good toler-
ability, and ease of scale-up. In the preparation of SLNs, many factors are involved in the characteristics
of the particles, such as particle size, drug loading, and zeta potential. In this study, fractional factorial
design was applied to examine which variables affect the physicochemical properties of amikacin SLNs.
Study was continued by a statistical central composite design (CCD) to minimize particle size and maxi-
mize drug-loading efficiency of particles. The results showed that three quantitative factors, including the
amount of lipid phase, ratio of drug to lipid, and volume of aqueous phase, were the most important vari-
ables on studied responses. The best predicted model for particle size was the quadratic model, and for
drug-loading efficiency, was the linear model without any significant lack of fit. Optimum condition was
achieved when the ratio of drug to lipid was set at 0.5, the amount of lipid phase at 314 mg, and the volume
of aqueous phase at 229 mL. The optimized particle size was 149 ± 4 nm and the drug-loading efficiency
88 ± 5%. Polydispersity index was less than 0.3. The prepared particles had spherical shape, and the drug
release from nanoparticles continued for 144 hours (6 days) without significant burst effect.
Keywords:  Solid lipid nanoparticles; amikacin; central composite design; optimization; particle size;
drug-loading efficiency

Introduction respect to large-scale production, sterilization possibil-

Solid lipid nanoparticles (SLNs) as colloidal carrier sys-
tems combine the advantages of traditional systems but
avoid some of their major disadvantages (Muller et al.,
2000; Hu et  al., 2004). Some advantages of SLNs are
­possibility of controlling drug release and drug target-
ing, increased drug stability, high drug payload, possi-
bility of the incorporation of lipophilic and hydrophilic
drugs, lack of biotoxicity of the carrier, no problems with
ity, and good tolerability (Mehnert and Mader, 2001; Hu
et  al., 2004; Zhang et  al., 2006). However, some of the
major drawbacks of SLNs are low drug loading, unpre-
dictable drug release, and the risk of gelation due to
polymorphism of the solid lipids (Hue et al., 2006).
The main ingredients used to produce SLNs include
solid lipid(s), emulsifier, and water. The character of SLNs
is changed by involved factors, depending on the prepara-
tion methods, and some of these factors are: type of lipid,

Address for Correspondence:  Jaleh Varshosaz, Department of Pharmaceutics, Faculty of Pharmacy, Isfahan University of Medical Sciences, Hezar Jarrib
street, Isfahan, Iran 81746-73461. Fax: 0098 311 6680011 E-mail: varshosaz@pharm.mui.ac.ir
(Received 01 May 2009; revised 31 May 2009; accepted 09 June 2009)

ISSN 0898-2104 print/ISSN 1532-2394 online © 2010 Informa UK Ltd

DOI: 10.3109/08982100903103904 http://www.informahealthcare.com/lpr
 98   Varshosaz et al.
type and concentration of emulsifier, and temperature.
There are many studies on SLNs, even for hydrophilic
drugs and peptide delivery (Mehnert and Mader, 2001;
Hu et al., 2004; Zhang et al., 2006; Jaspart et al., 2007).
Amikacin is a hydrophilic drug from aminoglyco-
sides, which is active against most of the gram-negative
bacteria, including gentamycin- and tobramycin-
­resistant strains, and it is used for the treatment of seri-
ous infections (e.g., cystic fibrosis or skin infections), but
its adverse effects, such as ototoxicity and nephrotoxic-
ity, are the same as other aminoglycosides (Nicoli and
Santi, 2006; Zawilla et al., 2006). Many studies have been
Journal of Liposome Research Downloaded from informahealthcare.com by University of Alberta on 06/03/10
carried out, so far, to reduce these toxicities, such as the
producing of liposomal amikacin dry powder inhaler
(Shah and Mirsa 2004) intratracheal delivery strategy of
gentamycin (Cullen et al., 1999), and high-dose nebuli-
zation of amikacin (Erhmann et al., 2008), and also, thi-
olated chitosan nanoparticles of amikacin were prepared
(Atyabi et al., 2009), but there are no published data on
the SLNs of amikacin. Previous studies reported that
encapsulated tobramycin showed considerable anti-
bacterial effect at concentrations below the minimum
inhibitory concentration (MIC) of the free antibiotic
in vitro (Schiffelers et  al., 2001). Several studies were
For personal use only.
done to show the effect of different factors on SLN(s)
(Zhang et  al., 2008; Lui et  al., 2008; Asasutjarit et  al.,
2006; Freitas and Muller, 1998).
In this study, the effect of different variables on the
particle size and drug-loading efficiency, zeta potential,
and polydispersity index (PDI) of particles were evalu-
ated. Also, the validation of the high-performance liquid
chromatography (HPLC) method for the analysis of
amikacin was done by the pre–column derivatization
method. There are some studies on HPLC with an ultra-
violet (UV) detector for the assay of amikacin (Nicoli
and Santi, 2006; Freitas and Muller, 1998; Ovalles et al.,
2005). In most studies, ready kits were used for the assay
of amikacin and other aminoglycosides because these
drugs do not have any chromophor group to be detected
by UV or fluorescence detectors directly (Lovering et al.,
1999; Ngui et al., 1984; Schwenzer and Anhalt, 1983).
To achieve the optimum condition of making ami-
kacin SLN(s), a statistically experimental design meth-
odology was employed properly. After selecting the
critical variables affecting particle size and drug-loading
efficiency by factorial screening design, response surface
methodology (RSM), followed by CCD were employed
to optimize the level of these variables.
Materials and methods
Materials
Cholesterol and stearic acid (Merck, Germany) were
used as lipid materials of SLNs. Tween 80 (Merck) and
Poloxamer 167 (Sigma-Aldrich, US) were used as sur-
factants. Amikacin sulphate (Darupakhsh, Tehran, Iran)
was used as the active ingredient. Ethanol and acetone
(Merck Chemical Company, Germany) were organic sol-
vents. Acetonitrile (Merck Chemical Company, Germany)
and 1-fluoro,2,4-dinitro benzene (FDNB) (Merck
Chemical Company, Germany) were used for HPLC
assay. Hydrochloric acid and NaOH (Merck Chemical
Company, Germany) were used for pH adjustment.
Methods
Preparation of SLNs
Different amounts of lipids were added to 6 mL of acetone
and 18 mL of ethanol, and the mixture was heated in a
water bath at 70°C. Amikacin was added to different vol-
umes of deionized water containing 1% (w/w) surfactant
and the pH adjusted to 2 by hydrochloric acid (1N) (Hu
et al., 2004). Then, the hot oily phase was added to water
at room temperature under homogenizing at 11,000 rpm,
using IKA® (Staufen, Germany) T-18 basic, Ultra-Turrax®
(Germany) for 7 minutes, and then the mixture was soni-
cated at 45–50° C for 10 minutes, using a bath-sonicator
system (Tecna 6; Tecno-Gaz, Sala Baganza, Italy). SLNs
were made when the mixture temperature was brought
to room temperature. Considering that the HPLC
method used in the determination of drug-­loading effi-
ciency of nanoparticles was time-­consuming, SLNs were
prepared in 3 days. The particle size of the nanoparticles
was measured by a Zetasizer (Nano ZS3000; Malvern
Instruments, Malvern, UK). For the determination of
drug-loading efficiency, the samples were centrifuged at
26,000 rpm for 35 minutes, at 4°C by a Sigma Laboratories
centrifuge (Osterode am Harz, Germany). The drug con-
centration in the supernatant was analyzed by using the
HPLC method, and the drug-loading efficiency was cal-
culated by using the reverse method applying Equation 1
(Kimberly and Tabrizian, 2005):
Drug – Loading Efficiency (LE %)
Drug total − Drug supernatant (1)
= ×100
Drug total 
Experimental design
Optimization of particle size and drug-loading efficiency
of amikacin SLN(s) were performed in two stages. In the
first stage, the parameters that showed significant effects
on particle size and loading efficiency have been identi-
fied. At the second stage, the optimum values of these
parameters were determined by CCD properly.
Screening study
Statistical evaluation and experimental design can eval-
uate and identify the most important parameters and
their interactions in the experiments with the minimum
 Development and optimization of solid lipid nanoparticles   99

number of runs (Myers and Montgomery, 2002). The
one-factor-at-a-time studies are not able to gather such
useful information.
In screening studies, the estimation of higher order
interactions is not useful. Therefore, in this study, a resolu-
tion IV 2(4-1) fractional factorial design was employed. This
means that k = 4 factors (the first number in parentheses)
have been accompanied in the study and p = 1 of these fac-
tors (the second number in parentheses) was generated
from the interactions of a full factorial design. Resolution
IV designs are characterized by the D = ABC genera-
tor and, therefore, can be a good choice for a screening
Journal of Liposome Research Downloaded from informahealthcare.com by University of Alberta on 06/03/10
response surface methodology (i.e., CCD) was used to
determine the optimum levels of these variables (Neter
et al., 1996). A CCD has three groups of design points,
including two-level factorial design points, axial or star
points, and center points. Therefore, selected variables
[ratio of drug to lipid (A), amount of lipid phase (C),
and volume of aqueous phase (D)] were studied at five
different concentrations coded as –, -1, 0, 1, and +.
The value for alpha (–1.682) is calculated to fulfill both
rotatability and orthogonality in the design. The coded
and actual values of the variables are given in Table 2.
According to the central composite design matrix gen-

design, since the main effects will be clear of two-factor
interactions. In the preliminary laboratory study, it was
found different factors (i.e., rate of homogenization, dura-
tion of homogenization, temperature condition during
the homogenization and sonication, type of lipid phase,
volume of aqueous phase and ratio of drug to lipid, and
surfactant type) may be effective on the properties of the
SLNs. Factors including ratio of drug to lipid (A), surfactant
type (B), amount of lipid phase (C), and volume of aque-
ous phase (D) were implemented in this study to find the
most important variables. The design matrix was built by
the statistical software package, Design-Expert (version
For personal use only.
erated by Design-Expert software, a total number of 20
experiments, including 8 factorial points, 6 axial points,
and 6 replicates at the center point for estimation of
pure error sum of squares, were performed (Table  3).
Using this design, we were able to choose the best
model among the linear, two-factor interaction model
and quadratic model due to the analysis of variance
(ANOVA) F-value. Predicting the response through the
full second-order polynomial equation is as shown in
Equation 2:
Y = b0 + b1 X 1 + b2 X 2 + b3 X 3 + b11 X 12 + b22 X 22 + b33 X 32
(2)

7.0.0; Stat-Ease, Inc., Minneapolis, Minnesota, USA), and
Table 1 shows the factors and corresponding response.
In this study, all of the experiments were performed
in triplicate and the averages were considered as the
response(s).
Optimization study
After selecting the most important factors influenc-
ing amikacin SLNs particle size, the most popular
+ b12 X 1 X 2 + b13 X 1 X 3 + b23 X 2 X 3 
where Y is predicted response(s), 0 is intercept, 1, 2,
and 3 are linear coefficients, 11, 22, and 33 are squared
coefficients, 12, 13, and 23 are interaction coefficients,
and X1, X2, and X3 are independent variables. By using
this equation, it is possible to evaluate the linear, quad-
ratic, and interactive effects of the independent variables
on the responses appropriately.

Table 1.  Screening design to evaluate effect of different factors on particle size.
A B C D
Volume of aqueous Response particle
Trials Drug-to-lipid ratio Surfactant type (mg) Lipid phase type (mg) phase (mL) size (nm)
1 0.25 (−) Poloxamer (−) Cholesterol (−) 80 (−) 1,100
2 1.00 (+) Poloxamer (−) Cholesterol (−) 240 (+) 708
3 0.25 (−) Tween (+) Cholesterol (−) 80 (−) 156
4 1.00 (+) Tween (+) Cholesterol (−) 240 (+) 609
5 0.25 (−) Poloxamer (−) Stearic acid (+) 240 (+) 374
6 1.00 (+) Poloxamer (−) Stearic acid (+) 80 (−) 1,200
7 0.25 (−) Tween (+) Stearic acid (+) 80 (−) 1,640
8 1.00 (+) Tween (+) Stearic acid (+) 240 (+) 992

Table 2.  The coded and actual values of the variables used in central composite design.
Levels
Independent variables Symbol – –1 0 1 +
Ratio of drug to lipid A 0.36 0.5 0.7 0.9 1.04
Amount of cholesterol C 105.5 160 240 320 374.5
Volume of aqueous phase D 25.5 80 160 240 294.5
Dependent variables Units Constraints
Y1 = particle size nm Minimize
Y2 = loading efficacy % Maximize
 100   Varshosaz et al.

Table 3.  Central composite design in various runs and particle size and loading efficacy as the responses.
Independent variables Dependent variables
Y1 Y2
Run no. Block A B C Particle size (nm) Loading efficacy (%) PDI
1 Day 1 −1 −1 −1 1430.00 ± 100 93.00 ± 7.10 0.701
2 Day 1 1 1 −1 1010.00 ± 10 90.00 ± 6.30 0.572
3 Day 1 1 −1 1 180.00 ± 15 97.00 ± 8.00 0.263
4 Day 1 −1 1 1 175.00 ± 4 98.60 ± 4.50 0.192
5 Day 1 0 0 0 268.00 ± 9 87.30 ± 5.20 0.208
6 Day 1 0 0 0 248.00 ± 10 93.30 ± 2.57 0.166
7 Day 2 1 −1 −1 261.00 ± 15 41.00 ± 2.03 0.267
8 Day 2 −1 1 −1 361.00 ± 10 80.00 ± 7.05 0.229
9 Day 2 −1 −1 1 166.00 ± 5 59.80 ± 4.02 0.287
Journal of Liposome Research Downloaded from informahealthcare.com by University of Alberta on 06/03/10

10 Day 2 1 1 1 171.00 ± 1 39.90 ± 2.09 0.163

11 Day 2 0 0 0 193.00 ± 5 49.40 ± 5.08 0.155
12 Day 2 0 0 0 213.00 ± 7 52.00 ± 6.02 0.264
13 Day 3 −1.682 0 0 262.00 ± 9 43.70 ± 3.07 0.356
14 Day 3 1.682 0 0 218.00 ± 3 25.00 ± 1.50 0.275
15 Day 3 0 −1.682 0 227.00 ± 6 33.90 ± 3.00 0.378
16 Day 3 0 1.682 0 218.00 ± 3 41.45 ± 5.20 0.169
17 Day 3 0 0 1.682 806.00 ± 20 83.40 ± 5.01 0.791
18 Day 3 0 0 1.682 157.00 ± 3 25.00 ± 2.01 0.235
19 Day 3 0 0 0 237.00 ± 8 44.30 ± 2.03 0.214
20 Day 3 0 0 0 215.00 ± 8 46.30 ± 6.01 0.227
PDI, polydispersity index.
For personal use only.

Design-Expert software was employed for statistical
analysis and graph plotting. The effect of independent
variables on the responses was calculated by ANOVA
through Fisher’s test. The P-value less than 0.05 was
considered to be statistically significant.
To evaluate the fitness of the second-order polyno-
mial equation, multiple correlation coefficient (R2) and
adjusted R2 were employed as quality indicators. Contour
and three-dimensional surface plots were used to dem-
onstrate the relationship and interaction between the
coded variables and the responses. The optimal points
were determined by solving the equation derived from
the final quadratic model and grid search in RSM plots
regarding the constraints in which the particle size is in
its minimum and loading efficiency (%) at maximum
levels. In addition, five optimized checkpoint formula-
tions were prepared and the experimental responses
were compared by the predicted values obtained by the
equation to evaluate the precision of the model.
HPLC method
Amikacin was analyzed by some modifications on Nicoli
and Santi’s HPLC method (2006). Analysis was done on
a C18 Knauer A5 micron, 25 × 0.46 cm column (Knauer,
Berlin, Germany) at 45°C. The mobile phase was a mix-
ture of water/acetonitril/acetic acid (60/40/0.1; v/v/v)
at a flow rate of 1.6 mL/min and the UV detector, the
K-2600 (Knauer), was set at 365 nm; the HPLC system
was equipped by a K1001 Knauer pump. Precolumn
derivatization was done for each sample, using FDNB,
at 95°C, in a vacuum oven (Nicoli and Santi, 2006;
Isoherranen and Soback, 2000). The derivatization
and HPLC analysis were validated in concentrations of
2.5–50 ppm.
Drug-release study
A release study was performed by using the dialysis sack
method by DO405 Dialysis tubing 23 × 15 mm (Sigma,
Frankfort, Germany). First, 5 mL of optimum formula-
tion was placed in a dialyzing membrane (10–12 KD)
immersed in 50 mL of phosphate buffer (pH 7.4). Next,
1-mL samples were withdrawn in a predetermined time
interval, and drug concentration was analyzed by using
precolumn derivatization by the HPLC method.
Morphology study
Morphology of the nanoparticles was characterized by
scanning electron microscopy (SEM). The nanoparticles
were mounted on aluminum stubs, sputter-coated with
a thin layer of Au/Pd, and examined by using an SEM
(Philips XL30, Almelo, The Netherlands) instrument.
Results and discussion
Screening of effective factors on particle size of SLNs
Fractional factorial screening design was employed to
assess all main effective and two-factor interactions to
 Development and optimization of solid lipid nanoparticles   101

determine which independent variables and interac-
tions have influence on the particle size of amikacin
SLNs. Therefore, to screen the effective factors, a normal
plot of parameter was employed. As can be seen from
Figure 1, among the four independent variables tested,
the type of lipid phase (C) and volume of aqueous phase
(D), which deviate from the straight line considered to
have a significant effect on amikacin SLNs particle size.
Considering that the organic solvents were water misci-
ble, increasing the volume of aqueous phase volume or
decreasing the organic/aqueous phase ratio caused bet-
ter distribution of solvents in water and, consequently,
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phase (C), and volume of aqueous phase (D), were used
to minimize the particle size and maximize the loading
efficiency of amikacin nanoparticles, using central com-
posite response surface methodology (Table 3). By using
externally studentized residual (outlier t-value), trials 1,
2, and 17 (Table 3) were detected as outliers (data not
shown). Therefore, they were ignored in ­further statisti-
cal analysis of the data. According to analysis of variance
(ANOVA) results calculated by Design-Expert (version
7.0.0, Stat-Easc, Inc., Minneapolis, MN, USA) software
for choosing the best model to fit the data, a quadratic
second-order polynomial model and two-factor inter-

a reduction of SLNs particle size. In addition, this plot
shows the significant interaction between the ratio
of drug to lipid (A) and volume of aqueous phase (D).
Further analysis, using least significant difference (LSD)
calculations performed at the 95% confidence level,
showed a statistically significant difference between the
means of the particle size of amikacin SLNs with cho-
lesterol (643.3 nm) and stearic acid (1051.5 nm) as two
levels of the lipid phase. Therefore, cholesterol has been
selected for the next step in the optimization process.
Although the drug-to-lipid ratio (A) itself is not an active
factor to influence the response in screening study, for
For personal use only.
action models were found to be appropriate when the
particle size (Y1) and loading efficiency (Y2) were con-
sidered as the responses, respectively. The results of fit-
ting the polynomial equation to the data, when particle
size is the response, are shown in Table 4. As can be seen
from Table 4, the model is highly statistically significant
(P < 0.05) with insignificant lack of fit (F = 0.59; P > 0.05).
In addition, two linear [the ratio of drug to lipid (A) and
volume of aqueous phase (D)] one quadratic (aqueous
phase), and two interactions [the ratio of drug-to-lipid
aqueous phase (AD)], and the ratio of drug to lipid to
the amount of cholesterol (AC) terms were significant

correcting the hierarchical of the model as the interac- (P < 0.05). Other factors had P-values of more than 0.05
tion of A and D is significant, it was kept as a variable in and were considered to be not significant. Therefore,
optimization stage. the mathematical model describing the relationship
between variables (A, C, and D) and response (Y1) could
Central composite design response surface be reduced to that shown in Equation 3:
methodology Y1 = 231.65 − 18.27 A − 0.048C − 93.46 D + 21.46 AC
The three significant variables selected, based on the (3)
+ 22.44 AD + 31.42D2 
results of fractional factorial design, including the ratio
of drug to lipid (A), the amount of cholesterol as the lipid where Y1 is the particle size and A, C, and D are ratio
of drug to lipid, amount of cholesterol, and volume of
aqueous phase, respectively. Although the amount of
cholesterol had no significant effect on particle size, it
99 was included in the model to ensure that a hierarchical
95 AD model as the interaction with ratio of drug to lipid was
90 significant.
Normal % Probability

80
70 C
A Table 4.  ANOVA results for particle size as the response (Y1).
50
AC Sum of Mean
30 B Source squares df squares F-value P-value
20
Block 309.9517 2 154.9758 49.6932
10 D
5 Model 38,708.98 5 7,741.80 23.5177 < 0.0001
A 3,663.87 1 3,663.87 114.346 0.0009
1
D 17,814.18 1 17,814.18 6.38393 < 0.0001
AB 994.5642 1 994.5642 21.6524 0.0324
AD 3,373.26 1 3,373.26 20.6525 0.0012
–579.75 –303.50 –27.25 249.00 525.25
D2 3,217.49 1 3,217.49 0.0014
Standardized Effect
Residual 1,402.13 9 155.7919
Lack of fit 760.1271 6 126.6879 0.592 0.7325
Figure 1.  Normal probability plot for screening the most important
variables influencing the particle size. Ratio of drug to lipid (A), sur- Pure error 642 3 214
factant type (B), amount of lipid phase (C), and volume of aqueous Cor total 40,421.06 16
phase (D). ANOVA, analysis of variance.
 102   Varshosaz et al.

The negative coefficients of A and D indicate that
amikacin particle size decreases with increasing levels
of drug-to-lipid ratio and volume of aqueous phase. The
coefficient of determination (R2) and adjusted R2 were
calculated to be 0.97 and 0.95, respectively, indicating
that this model can explain 97% variability in the response
and only 3% of the variability is due to noise. Moreover,
the similarity between R2 and adjusted R2 values shows
the adequacy of the model to predict the response. The
value of the coefficient of variation (CV% = 5.63), which is
an estimation of the standard deviation associated with
experiment around the mean, also indicates the preci-
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the interaction between the ratio of drug to lipid (A) and
amount of cholesterol (C), when particle size was con-
sidered to be the response, is illustrated in Figure 2A. As
can be seen, particle size decreases as ratio of drug to
lipid (A) and amount of cholesterol (C) are increased;
it seems that this relationship is linear both in two axes
of the independent variables. In addition, the shape of
the contour plot indicates that the interaction between
ratio of drug to lipid (A) and amount of cholesterol (C)
is significant.
In Figure 2B, the response surface plot shows that
the ratio of drug to lipid (A) and level of aqueous phase

sion and reliability of the model. Finally, the predicted volume (D) influence the particle size, although the cur-
R2 of 0.82 is an indicator that shows the good prediction vature along the aqueous phase volume (D) axes reveals
of the response (i.e., particle size) by the model. the statistical significance of quadratic coefficient of the
Using loading efficiency as the response, the two- ratio of drug to lipid (A) in the model.
interaction equation is the best model, which has been
fitted to the data according to the ANOVA F-value cal-
A
culated by Design-Expert software. The results of the fit
model summaries are shown in Table 5. 272
As can be seen, the ANOVA F-value of the model
indicated that the model P-value is less than 0.05 and 251.75
considered to be significant while the lack fit of the
Particle size (nm)

model is not significant (F = 6.45; P > 0.05). Hence, it can 231.5

For personal use only.

be concluded that the following two-interaction equa-

tion can be fitted to the data appropriately, as shown in 211.25
Equation 4:
191
Y2 = 62.74 − 5.88 A + 3.37C − 4.6 D − 8.73CD  (4) 320
280 g)
0.50 m
where Y2 is loading efficiency and A, C, and D are the 240 (
0.60 r ol
ratio of drug to lipid, amount of cholesterol, and volume A: D 0.70 200 te
rug l es
of aqueous phase, respectively. to lip 0.80
160 Cho
id ra
In order to study the interaction patterns between var- tio 0.90 C:
iables, three-dimensional response surface curves were
B
plotted by calculating the amikacin particle size (Y1) and
loading efficiency (Y2) as the response at different lev- 400
els of any two variables while keeping the third variable
at its middle level. These plots are shown in Figures 2A 340
and 2B and 3, respectively. The response surface from
Particle size (nm)

280
Table 5.  ANOVA results for loading efficacy as the response (Y2).
Sum of Mean 220
Source squares df squares F-value P-value
Block 8,323.69 2 4,161.85 160
Model 1,304.30 4 326.075 8.68935 0.0027 0.50
A 349.3513 1 349.351 9.30961 0.0122 0.60 tio
80.00 ra
C 117.2295 1 117.23 3.12396 0.1076 0.70 d
120.00 lipi
D 156.9671 1 156.967 4.1829 0.0681 D: A 160.00 0.80 to
queo
CD 394.5886 1 394.589 10.5151 0.0088 us p
hase 200.00
0.90 r ug
volum 240.00 D
Residual 375.2588 10 37.5259 e (m A:
l)
Lack of fit 351.8788 7 50.2684 6.45018 0.0768
Pure error 23.38 3 7.79333 Figure 2.  (a) The effect of drug-to-lipid ratio (A) and amount of cho-
Cor total 10,003.25 16 lesterol (C) on particle size. (b) The effect of drug-to-lipid ratio (A)
ANOVA, analysis of variance. and aqueous-phase volume (D) on particle size.
 Development and optimization of solid lipid nanoparticles   103

In the same way, when loading efficiency is indicated
as the response, only the interaction of amount of cho-
lesterol (C) and aqueous phase volume (D) is statisti-
cally significant. Therefore, as can be seen in Figure 3,
by increasing the amount of cholesterol and decreasing
the volume of aqueous phase, the loading efficiency
increases in the domain of variables that were selected
in this study.
Optimization
By solving Equations 3 and 4, analyzing response sur-
Journal of Liposome Research Downloaded from informahealthcare.com by University of Alberta on 06/03/10
profile was observed, and amikacin was released for
144 hours, and after that time, 92.9% of loaded drug
was detected in samples and from 144 to 240 hours this
percentage received to 95.5%;and in the first hours,
no significant burst effect was shown. Figure 4 shows
the optimum formulation of the amikacin SLN release
profile.
Morphology study
Morphologic study for optimum formulation was done
by taking SEM pictures of prepared SLNs. Figure 5
shows the spherical shape of prepared SLNs. Studies

face plots and constraints, the optimum levels for the
ratio of drug to lipid, amount of cholesterol, and volume
of aqueous phase were determined to be 0.5 and 314 mg
and 229 mL, respectively. At these levels of independent
variables, predicted amikacin particle size and load-
ing efficiency were calculated to be 153 nm and 86%,
respectively.
Experimental validation
In order to validate the experimental model, five verifi-
cation experiments were performed by using the statisti-
For personal use only.
showed that the predicted particle size and measured
size with a nanosizer was comparable with size of par-
ticles that were observed by SEM.
Conclusions
Preparation and optimization of amikacin SLNs were
the aims of this study. The ratio of drug to lipid, amount
of cholesterol as the lipid phase, and volume of aque-
ous phase were detected as effective variables on par-
ticle size and drug-loading efficiency. The optimum

cally optimized conditions. The practical response was conditions for the preparation of amikacin SLNs was
149 ± 4 nm, loading efficiency of 88 ± 5%, and PDI < 3.
The close agreement between the observed and pre- 120
dicted values confirmed the validity and precision of the 100
Release (%)

model. 80
60
Drug release 40
20
The release profile of optimized formulation was
0
studied and as predicted for SLNs, a sustained release 0 50 100 150 200 250 300
Time (h)

Figure 4.  Release profile of optimum amikacin solid lipid

nanoparticles.
80

73
Loading efficacy (%)

66

59

52
320.00
280.00 g)
240.00 (m
240.00 ol
D: A 200.00 160.00 er
queo 200.00 est
us p 120.00 ol
hase Ch
volu 80.00 160.00 C:
me (
ml)

Figure 3.  The effect of aqueous-phase volume (D) and amount of Figure 5.  Scanning electron microscopy picture of optimized ami-
cholesterol (C) on loading efficacy. kacin solid lipid nanoparticles.
 104   Varshosaz et al.
the ratio of drug to lipid of 0.5, the volume of aqueous
phase of 229 mL, and amount of cholesterol 314 mg;
in this condition, size of particles, drug-loading effi-
ciency, and ­polydispersity index were 153 nm, 86%,
and < 0.3, respectively. These SLNs are promising for
their application in the pulmonary delivery of sus-
tained release amikacin, which is the main goal of our
future study.
Acknowledgements
Journal of Liposome Research Downloaded from informahealthcare.com by University of Alberta on 06/03/10
The authors would like to acknowledge the research vice
chancellor of Isfahan University of Medical Sciences for
financial support of this project. The technical assist-
ance of Miss Norouzi and Mrs. Saabani is appreciated.
Also, the authors thank Mr. Rezae for SEM pictures,
Miss Shafie for her help in validation methods of HPLC,
and Mr. H. Hashemi Nasl for the English editing of the
­manuscript for this article.
Declaration of interest:  The authors report no financial
conflicts of interest. The authors alone are responsible
for the content and writing of this paper.
For personal use only.
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