Drug Delivery, 2011; 18(6): 441–450

© 2011 Informa Healthcare USA, Inc.

ISSN 1071-7544 print/ISSN 1521-0464 online
DOI: 10.3109/10717544.2011.577109

Research Article

Preparation and in vitro, in vivo evaluations of norfloxacin-

loaded solid lipid nanopartices for oral delivery
Zhao Dong, Shuyu Xie, Luyan Zhu, Yan Wang, Xiaofang Wang, and Wenzhong Zhou*

Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, 2
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Yuanmingyuan Road West, Beijing 100193, PR China

Abstract
This work aims to develop norfloxacin-solid lipid nanoparticles (NFX-SLN) as an oral delivery formulation. Hot
homogenization and ultrasonic technique was employed to prepare NFX-SLN using stearic acid as lipid matrix and
polyvinyl alcohol as surfactant. The physicochemical characteristics of SLN were investigated by optical microscope
scanning electron microscopy and photon correlation spectroscopy. Antibacterial experiments of NFX-SLN were
carried out by broth dilution technique. Pharmacokinetics was studied after oral administration in male Sprague-
Dawley rats. The results showed that NFX-SLN was spherical and the SLN of the optimized formulation had diameters
301 ± 16.64 nm, polydispersity index 0.15 ± 0.04, zeta potential -30.8 ± 0.69 mv, loading capacity 8.58 ± 0.21% and
encapsulation efficiency 92.35 ± 2.24% with good stability at 4 °C. The NFX-SLN had sustained release effect and
For personal use only.

sustained bactericidal activity. Cytotoxicity studies in cell culture demonstrated that the nanoparticles were not
toxic. NFX-SLN resulted in significantly higher plasma drug concentration than native NFX. The SLN increased the
relative bioavailability of NFX by 12 folds, prolonged the plasma drug level above the average minimum inhibition
concentration from 14 to 168 h. These studies demonstrate that NFX-SLN could be a promising oral formulation for
enhanced bioavailability and pharmacological activities.
Keywords:  Norfloxacin, solid lipid nanoparticles, antibacterial activity, sustained release, bioavailability

Introduction
Norfloxacin (NFX) is a widely used third generation qui-
nolone synthetic antibiotic which has the characteristics
of broad bacterium contradicting, small side-effects and
cross-resistance with other drugs (Han et al., 2005). NFX
is used against both gram-positive and gram-negative
bacteria as well as trimethoprim/sulfonamide resistant
microbes (Preheim et  al., 1987). It is also active against
Mycoplasma (Brown et  al., 1996). This antibiotic shows
promise as an antimicrobial agent for a variety of bac-
terial diseases (Wolfson & Hooper 1988; Carratalá
et al.,1995). NFX kills bacteria on a concentration-depen-
dent basis (Ferrero et al., 1995). The minimum inhibitory
concentration for 90% of organisms (MIC90) was below
0.12 μg/ml for E.coli, Salmonella spp, Klebsiella pneu-
monia, Klebsiellaoxytoca, Proteus vulgaris, Haemophilus
influenzae (Lavy et  al., 1995; Andersson & MacGowan
2003). NFX has been used extensively as both oral and
injectable formulations for human and many domestic
animal species (Hans & Li 1991). Oral administration is
a fundamental route for therapy and prevention such
as the standard of therapy in the prophylaxis of bacte-
rial infections in cirrhotic patients with gastrointestinal
hemorrhage; oral treatment regimen for uncomplicated
gonococcal infection or oral prophylaxis suppressing
infection caused by aerobic gram-negative bacilli during
anti-leukemic therapy (Fernández et al., 2006; Kaplowitz
et al., 1987; Verhoef & Rozenberg 1989).
However, there are drawbacks to limit the usage of
NFX. NFX is scarcely dissolved in pure water, ethanol,
methanol or aether while freely soluble in hydrochloric
acid and natrium hydroxydatum with a low bioavail-
ability mainly attributed to its low aqueous solubility and
low permeability (Guyot et al., 1995; Breda et al., 2009).
Human pharmacokinetic data for NFX show fraction
absorption indicative of a poor permeability (Hooper

Address for Correspondence: Wenzhong Zhou, Tel.: 86-10-62734702; Fax: 86-10-62734840. E-mail: zhouwz@cau.edu.cn
(Received 30 December 2010; revised 25 February 2011; accepted 17 March 2011)

441
 442  Zhao Dong et al.
& Wolfson 1985). The absolute bioavailability of NFX in
humans and in laboratory animals is reported 40% post
oral administration (Gips & Soback 1996). Examination
of the individual serum concentration/time curves of
the volunteers indicated that there may be a “first-pass”
effect after oral administration and the site of metabo-
lism is the liver, first-pass hepatic metabolism may be in
part responsible for their lesser bioavailability (Hooper
& Wolfson 1985). Poor bioavailability may provide lower
antimicrobial activity and give rise to the development
of resistance by the microorganisms (Aleem et al., 2008).
Furthermore, repeated administrations are required to
attain a steady-state level of drug. The common usage
of NFX is twice a day for 7 to 10 days or even longer for
uncomplicated lower urinary tract infections leading to
a frequent dosage (Arredondo et al., 2004). Prophylactic
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NFX to patients with cancer who were neutropenic or
likely to develop cytotoxic therapy–induced neutropenia
or gastrointestinal hemorrhage are given orally 400 mg
twice daily lasting more than 7 days (Fernández et  al.,
2006; Carratala et al., 1998). Repeated dosage may result
in higher incidence of adverse effect. Additionally, treat-
ment of some diseases requires high dosage, such as uri-
nary tract infection required high dose NFX for months
(Boerema & Saene 1986). Long-term high dose therapy
has caused lenticular opacities, decreased spermato-
genesis and testicular atrophy in experimental animals
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nausea and headache in patients(Smith 1987; Pittman
et al., 1993).
Solid lipid nanoparticles (SLN) are valuable as an
oral delivery carrier to increase the absorption of a
poor water soluble drug and enhance the permeabil-
ity and retention effect (Li et  al., 2009; Maeda 2001;
Zhang et  al., 2008). It can improve the bioavailability
of poorly hydrophilic and lipophilic drugs when these
drugs are encapsulated in SLN (Luo et  al., 2006). The
mechanisms of bioavailability enhancement by SLN is
that they possess adhesive properties that make them
adhere to the gut wall and release the drug exactly
where it should be absorbed (Müller & Keck 2004). In
addition, the lipids are known to have properties that
promote the oral absorption of lipophilic drugs and
drugs in general (Porter &Charman 2001; Charman
2000). SLN can protect labile macromolecules from
stomach acid and from the “first-pass” metabolism in
the gastrointestinal tract (Manjunath & Venkateswarlu
2005). Better absorption and bioavailability enhance-
ment enables dosage reduction and minimizes side
effects (Munoz et al., 1994; Kalaria et al., 2008). The use
of biodegradable materials for nanoparticles prepara-
tion allows for sustained drug release within the target
site over a period of days or even weeks, lowering the
frequency of administration (Panyam & Labhasetwar
2003; Vasir et al., 2003).
Stearic acid is a saturated 18-carbon chain fatty acid
and usually synthesized from acetyl-CoA, metabolized
by β-oxidation and expected to have good biocompat-
ibility and low toxicity in body. The fatty acid is solid at
 Drug Delivery
room or body temperature, thus it is chosen for com-
monly pharmaceutical use as lipid matrix to prepare SLN
(Zhang et al., 2000). Polyvinyl alcohol (PVA) is the most
commonly used surfactant in the formulation of nano-
particles due to its excellent mechanical strength, bio-
compatibility, and non-toxicity and has been approved
by the US Food and Drug Administration for medical and
food applications (Davda & Labhasetwar 2002). In this
work, NFX-SLN was prepared using stearic acid as lipid
matrix and PVA as surfactant. The performances and the
pharmacological activities of NFX-SLN were studied in
vitro and in vivo.
Materials and methods
Materials
Stearic acid was obtained from Shanghai Sangon
Biological Engineering Technology & Services Co., Ltd.
(Shanghai, China). PVA and MTT (3-[4, 5-dimehyl-
2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide)
were purchased from Sigma (St. Louis, MO, USA). NFX
was bought from Shandong Lukang Pharmaceutical
Co., Ltd. (Jining, China). NFX reference standard and
Staphylococcus aureus (CCVCC2248) were purchased
from China Institute of Veterinary Drug Control (Beijing,
China). The solvents used for HPLC were of liquid chro-
matography (LC) grade. Methanol and acetonitrile were
purchased from Fisher Scientific (Fair Lawn, NJ, USA).
The water for HPLC was prepared with a Milli-Q system
(Millipore, Bedford, MA). The citric acid was purchased
from Sinopharm Chemical Reagent Co., Ltd. (Beijing,
China) and the ammonium acetate was obtained from
Beijing Chemical Reagents Company (Beijing, China).
Other chemicals and reagents not specified in the text
were of analytical grade or equivalent. Baby hamster kid-
ney cell line (BHK-21) was from American Type Culture
Collection (ATCC) (Manassas, VA, USA). Sprague-
Dawley (SD) rats were from Academy of Military Medical
Sciences (Beijing, China). They were acclimatized for 1
week before use with free access to food and water. All
animal procedures were conducted with the approval of
the China Agricultural University Institutional Animal
Care and Use Committee. Humane care of all animals
was stringently followed throughout all animal-involved
studies.
Preparation of norfloxacin-loaded nanoparticles
Norfloxacin-solid lipid nanoparticles were formulated
by hot homogenization and ultrasonic technique (Xie
et al., 2010). Briefly, NFX was mixed with 3 g stearic acid
in a 50 ml tube and heated in a boiling water bath. After
the lipid was melted and the drug was dissolved in the
melted lipid, 30 ml PVA solution preheated on a boiling
water bath was poured into the lipid phase under mag-
netic stirring to form an o/w emulsion. The emulsion
was sonicated for 5 min (VC X 750 Vibra-CellTM, Sonics
& Materials, Inc., Newtown, CT, USA, using the 13 mm
microprobe with amplitude 35%) to form a nanoemulsion.

The nanoemulsion was quickly poured into 200 ml cold
water to obtain a nanoparticle suspension. The nano-
particles were collected by centrifugation at 9000 rpm
(Centrifuge 5810 R; Eppendorf, Germany) for 30 min and
washed 3 times with distilled water. The SLN were re-sus-
pended in 100 ml distilled water and lyophilized for 72 h
(LGJ-12 Freeze Dryer; Beijing Songyuanhuaxing Science
Technology Development Co., Ltd., China). The control
SLN was prepared similarly without adding NFX.
Determination of mean diameter (MD), polydispersity
index (PDI) and zeta potential (ZP)
The mean diameter, PDI and zeta potential of the SLN
were determined by photon correlation spectroscopy
(PCS) using Zetasizer Nano ZS90 (Malvern Instruments,
UK). The samples of SLN were suspended in distilled
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water at a concentration of 2.7 mg/ml for particle size and
PDI analysis, and a concentration of 0.3 mg/ml for zeta
potential determination.
Determination of loading capacity (LC) and
encapsulation efficiency (EE)
The wavelength scan using a UV spectrophotometer
(U-1800, Hitachi Tech Co., Japan.) showed that NFX
had a specific absorption at 272.4 nm. To determine the
drug content in SLN, a weighed amount of freeze-dried
SLN was suspended in 1% NaOH solution in a 15 ml
For personal use only.
tube and heated on a boiling water bath for 5 min. After
cooling down, the solution was centrifuged at 9000 rpm
(Centrifuge 5810 R; Eppendorf, Germany) at 4 °C for
15 min. The supernatant was analyzed at 272.4 nm. The
control nanoparticles without NFX were treated similarly
as blanks for measurements. Loading capacity (LC) was
calculated directly. NFX in total SLN was calculated by LC
multiply the recovered freeze-dried SLN. Loading capac-
ity (LC) and encapsulation efficiency (EE) are defined as
follows:
Weight of norfloxacin inSLN
LC = 100%
Weight of SLN
LC weight of total SLN
EE = 100%
Weight of norfloxacin added
Microscopic analysis
One milligram of NFX-SLN prepared with 3g strearic
acid, 300mg NFX and 30ml 1% PVA was suspended
in 1 ml distilled water. Two microliters of the suspen-
sion were placed on a microscope slide and the nano-
particles were dried at room temperature for 5 min.
Photomicrographs of the SLN were taken using an
inverted optical microscope (Olympus 1X71, Olympus,
Japan).
Morphology analysis
The morphology of NFX-SLN prepared with 3g strearic
acid, 300mg NFX and 30ml 1% PVA was studied by SEM
© 2011 Informa Healthcare USA, Inc.
Norfloxacin-loaded solid lipid nanopartices  443
(SE S-3400N; Hitachi, Japan). Briefly, 1 mg samples
were suspended in 1 ml distilled water and 2 µl of the
suspension were placed on a cover glass and dried
at room temperature over 30 min. The samples were
coated with gold and examined at an accelerating volt-
age of 20 kV.
In vitro release study
Norfloxacin-solid lipid nanoparticles (3 mg) prepared
with 3g strearic acid, 300mg NFX and 30ml 1% PVA
were suspended in 2 ml 0.1 mol/ml hydrochloric acid
(HCl) to mimic the in vivo stomach decomposition
process (Luo et al., 2006) in a dialysis bag (MW: 8000–
14400) and dialyzed against 38 ml 0.1 mol/ml HCl in a
50 ml tube at 37 °C under magnetic stirring at 60 rpm.
Since NFX was freely soluble in hydrochloric acid, the
volume of receiver solution was large enough to com-
pletely dissolve the released drug. Native NFX suspen-
sion with the same drug concentration was used as
the control. To determinate the NFX amount diffused
through the dialysis bag, at fixed time intervals samples
(2 ml) were withdrawn from the receiver solution and
the same amount of 0.1 mol/ml HCl was added to main-
tain a constant volume. NFX in the release medium was
measured spectrophotometrically at 272.4 nm (U-1800,
Hitachi Tech Co., Japan). The control nanoparticles
without NFX were treated similarly and used as blanks
for measurement. To determine the drug remained
in SLN within dialysis bags after release, the SLN was
collected by centrifugation of the suspension and the
measurement was carried out by the method in deter-
mination of LC. All the experiments were carried out in
triplicate.
Stability studies
Stability studies of SLN were performed after the samples
were stored at 4 °C for 6 months. The values of MD, PDI,
ZP and LC were measured for the evaluation of the physi-
cal stability of the nanoparticles. In vitro release was also
carried out and compared with that of newly prepared
SLN.
Antibacterial activity studies
Minimum inhibitory concentration (MIC) and mini-
mum bactericidal concentration (MBC) were deter-
mined by the broth dilution technique of National
Committee for Clinical Laboratory Standards (NCCLS;
now renamed as Clinical and Laboratory Standards
Institute, CLSI, 2000). Briefly, 11 mg NFX-SLN (con-
taining 944 μg NFX) was suspended in 4 ml distilled
water and serial dilutions were made with distilled
water (NFX concentration: 1.2, 0.9, 0.6, 0.45 0.3 and
0.225 µg/ml). The dilutions (2.7 ml) were mixed with 10
µl Staphylococcus aureus (CCVCC2248) containing 0.5
McFarland (1.5 × 108 CFU/ml) to a final bacterial con-
centration of 5.5 × 105 CFU/ml. The mixture was incu-
bated at 37 °C for 12 h and 24 h in an incubator (Jiangsu
Taicang Experimental Equipment Company, China)
 444  Zhao Dong et al.
with shaking at 130 rpm. The MIC of NFX was defined
as the lowest concentration inhibiting visible growth
of bacteria. The MBC is measured by subculturing the
broths used for MIC determination onto fresh agar
plates. The MBC is the lowest concentration of the drug
that results in killing 99.9% of the bacteria being tested.
The native NFX and the released NFX were also made
serial dilutions to the final concentrations as above and
the MIC and MBC were measured by the same way.
Sustained antibacterial studies were conducted by
broth dilution technique. Ten microliters bacterial cul-
ture containing 0.5 McFarland (1.5 × 108 CFU/ml) of
organisms were added to 2 ml Mueller-Hinton broth
medium in a 4 ml tube containing NFX or NFX-SLN with
a drug concentration of 0.34 µg/ml. Control nanopar-
ticles (CNP) and Mueller-Hinton broth (MHB) were used
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as controls. The mixtures were incubated at 37 °C in an
incubator with shaking (130 rpm). At fixed time points (6,
12, 24, 36, 48, 72 h), and ten serial dilutions of the mix-
tures were cultured on Luria broth agar plates at 37 °C.
The colonies were counted when they could be observed
by naked eyes. Growth graphs were plotted by calculating
the number of colonies in 1 ml of the mixture versus the
incubating time (hour). All experiments were carried out
in triplicates.
Cytotoxicity evaluation
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Cytotoxicity evaluation of NFX-SLN was conducted by
MTT assay in BHK-21 cell line. The cells were seeded into
a 96-well microplate at a density of 1 × 104 cells per well
in 0.1 ml DMEM supplemented with 10% fetal bovine
serum (FBS) and antibiotics and cultured in a humidi-
fied 5% CO2 incubator at 37 °C. After 24 h, the medium
was replaced by 100 µl DMEM complete medium con-
taining 58.3 µg NFX-SLN (drug concentration: 50 µg/ml).
Native NFX or control nanoparticles (CNP) at same con-
centrations were also used. After 24 h co-incubation, cell
viability was assessed by MTT assay. Typically, 5 mg/ml
of MTT in PBS were added to each well reaching a final
MTT concentration of 0.5 mg/ml and incubated for 4 h.
Then the supernatants were removed and the formazan
crystals were dissolved in 150 µl DMSO. Aliquots were
drawn from each well and the absorbance at 490 nm was
determined by a microplate reader (Bio-Rad 680). Cells
without adding SLN or NFX were taken as control and set
to 100% viability. The cell viability (%) compared to con-
trol cells were calculated by (OD490sample/OD490control) × 100.
Pharmacokinetics
A group of 20 male SD rats weighing on an average of
360 ± 10 g were used for the pharmacokinetic studies.
They were randomly divided into 4 groups with 5 animals
in each group. NFX was administrated to them orally
(20 mg/ kg of body weight) in the form of either NFX or
NFX-SLN suspended in 2 ml sterilized double-distilled
water. Each treatment contained 2 groups. At different
times (0, 0.17, 0.5, 1, 1.5, 2, 3, 4, 8, 14, 24, 36, 48, 72, 96, 120,
144, 168 h) post dosage, blood samples were collected
 Drug Delivery
from tail vein and at one time point only one group of
each treatment were collected. The blood samples were
collected by turns from 2 groups over the observation
period. The experimental protocols concerning the han-
dling of rats were in accordance with the requirements
of the Institutional Animal Care and Use Committee at
China Agricultural University.
HPLC assay
Sample extracts were prepared by mixing 150 µl plasma
with 300 µl mixture of methyl alcohol and acetonitrile
(1/1 v/v) and vortexed for 3 min, and then centrifuged at
14000 rpm (Sigma 1-14; Sartorius, Germany) for 25 min.
Two hundred microliters supernatant were taken for high
performance liquid chromatography (HPLC) analysis
using VP-ODS: 250 mm × 4.6 mm (Shimadiu Coperation,
Kyoto, Japan). The HPLC condition was: mobile phase:
(0.05 mol/l citric acid and 0.01 mol/l ammonium acetate,
pH of the aqueous phase was adjusted by triethylamine
to 4.5)/methyl alcohol (65/35, v/v); flow rate: 1 ml/min;
UV detector wavelength: 278 nm; the injection Volume:
50 µl. The plasma concentration of NFX was found to be
linear over the range 0.125–10 µg/ml. The correlation
coefficient was 0.9991. The limit of quantification (LOQ)
of UV detection was 0.1µg/ml. The relative standard
deviations (R.S.D.) of accuracy and precision for three
different plasma concentrations of NFX (0.1, 1 and 4 µg/
ml) were 12.33%, 5.57% and 0.78% for intra-day analysis,
and 7.10%, 6.58% and 6.03% for inter-day analysis.
Statistical analysis
The values of maximum serum concentration (Cmax) and
time to peak serum concentration (Tmax) were obtained
directly from the concentration–time plotting. The area
under the concentration–time curve (AUC0-inf ), mean
residence time (MRT) and elimination half-life (T½) was
analyzed based on noncompartmental pharmacokinet-
ics statistical moment. The relative bioavailability at
infinity was calculated as: Fr = AUCNFX-SLN, 0-inf /AUCNFX, 0-inf.
Results were expressed as mean ± SD. Statistical analysis
was performed using the one-way ANOVA. The differ-
ences were considered significant at the level of P < 0.05.
Microsoft Excel (2003) (Microsoft Inc., USA) was used for
all statistical analysis.
Results and discussion
Characteristics of NFX-loaded nanoparticles
The characteristics of SLN were listed in table 1. The val-
ues of MD, PDI, ZP, LC and EE were significantly affected
by the amount of NFX added in the preparation. With the
increase of NFX from 150 to 600 mg, the values of MD,
PDI, ZP and LC increased significantly. Although SLN
with 600 mg NFX had higher LC value, its EE value was
lower than those with both 150 and 300mg NFX, because
of more drugs diffusing into the continuous phase. SLN
with 300 mg NFX had higher drug loading capacity than
SLN with 150 mg NFX and lower values of MD and PDI

than SLN with 600 mg NFX, thus, 300 mg of NFX was
selected for the preparation of SLN for further studies.
NFX was usually orally given a single dose of 400 mg for
patients (Wella M et al., 1998; Eandi M et al., 1983), which
equaled to 4.662 g of the SLN prepared with 300 mg NFX.
Considering the improvement of the drug bioavailability
and the enhanced therapeutic efficacy, the dose could be
reduced (Pandey R & Khuller GK 2005; Pandey R et al.,
2005).
PVA concentration had significant effects on the values
of MD, PDI and EE. When PVA concentration increased
from 0.5% to 1%, EE increased significantly and MD and
PDI decreased. But further increase of PVA concentration
from 1% to 2% resulted in no significant changes of MD,
PDI and EE. Therefore, 1% PVA was chosen for prepara-
tion of SLN.
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Norfloxacin-loaded solid lipid nanopartices  445
A high ZP (>30 mV) could provide an electric repulsion to
avoid the aggregation of particles (Levy et al., 1994). The
nanoparticle system with a high LC and EE could reduce
the quantity of carrier required for the administration of
a sufficient amount of pharmacologically active agent to
the target site.
In vitro release of NFX from SLN
This 0.1 mol/ml HCl was used to mimic stomach acidity
(Li et  al., 2000). The in vitro release behavior of NFX-
SLN and native NFX is summarized as the cumulative
percentage release of NFX-SLN, as shown in Figure 2.
The release of native NFX was almost complete by 2h.
For the NFX-SLN, an initial fast release was observed
during the first 4 h, the release reached 47 ± 2.86% by 2h
and 59.09 ± 2.02% by 4h. The fast release phase could be

Optical and SEM micrographs showed that NFX-SLN
were well dispersed with good particle size distributions
(Figure 1a), and were spherical with smooth surfaces
(Figure 1b). NFX-SLN had higher values of MD, PDI and
ZP than that of control (Table 1), indicating that the drug
played an important role in these characteristics. Lipid
matrix encapsulated drug leading to a larger volume. NFX
with negative charge enhanced the total surface charge.
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attributed to the large surface area of nanoparticles with
drug enrichment or precipitation in outer shell of the
particles, leading to a relatively short distance of diffusion
and hence fast release of the drug (Mühlen et al., 1998; Hu
et al., 2002; Üner 2006). From a therapeutic standpoint, a
fast release profile could be considered as an advantage
as a sufficient amount of a drug and improvement of
the penetration of the drug (Jenninga et al., 2000). After
the fast release, it was a sustained release and the total
release reached 77.21 ± 3.63% by 24 h. Then a prolonged,
relatively steady and slow release was observed. The

release reached 88.36 ± 1.27% by 72 h. After 72 h releas-

ing, the remaining drug in SLN within dialysis bags was
8.62 ± 1.39%. This release trend is similar to our previous
studies on other drug-loaded SLN (Xie et al., 2008; Han
et al., 2009; Xie et al., 2009).

Stability studies
After 6 months of storage at 4°C, ZP and LC displayed
Figure 1.  Micrographs of NFX-SLN: (a) optical microscope (400×); no significant changes, but the values of MD and PDI
(b) scanning electron microscope (15000×) increased significantly (Table 2). In vitro release studies

Table 1.  Characteristics of SLN (mean ± SD, n = 3).

Formulation
PVA (%) NFX (mg) MD (nm) PDI ZP (-mv) EE (%) LC (%)
1 150 298.67 ± 17.24d 0.05 ± 0.23ad 29.43 ± 1.25d 83.41 ± 1.23ad 4.20 ± 0.16ad
1 300 301.13 ± 16.64d 0.15 ± 0.04d 30.80 ± 0.69d 92.35 ± 2.24d 8.58 ± 0.75d
1 600 379.33 ± 10.50 abd
0.53 ± 0.10abd
31.97 ± 1.43d
68.86 ± 0.54abd
16.21 ± 0.64abd
0.5 300 373.33 ± 44.08 ad
0.72 ± 0.14ad
29.67 ± 1.38d
85.31 ± 0.76 ad
8.05 ± 0.38d
2 300 304.67 ± 2.52cd
0.15 ± 0.05cd
31.23 ± 0.78d
93.04 ± 0.34cd
8.35 ± 0.44d
1 0 160.31 ± 5.29 0.09 ± 0.01 19.77 ± 0.99 0 0
The SLNs were prepared with 3g strearic acid, 0, 150, 300 or 600 mg NFX and 30ml 0.5%, 1%, or 2% PVA.
MD: mean diameter; PDI: polydispersity index; ZP: zeta potential; EE: encapsulation efficiency; LC: loading capacity.
Statistical significances are p< 0.05 compared with: aPVA 1%/NFX 300 mg; bPVA 1%/NFX 150 mg; cPVA 0.5%/NFX 300 mg; dPVA 1%/NFX
0 mg.

Table 2.  Comparison of characteristics of stored and newly prepared SLN (mean ± SD, n = 3).
SLN MD (nm) PDI ZP (-mv) LC (%)
Newly prepared 301.13 ± 16.64 0.15 ± 0.04 30.80 ± 0.69 8.58 ± 0.75
Stored 344.71 ± 64.20* 0.27 ± 0.05* 29.20 ± 2.36 8.37 ± 0.20
MD: mean diameter; PDI: polydispersity index; ZP: zeta potential; LC: loading capacity.
* Statistical significances between newly prepared SLN and Stored SLN are p < 0.05

© 2011 Informa Healthcare USA, Inc.

 446  Zhao Dong et al.
showed that stored SLN had the same release ability as
that of newly prepared SLN. At different time points
(Figure3), the cumulative release values of the stored
SLN were nearly the same as those of the newly pre-
pared SLN, indicating the SLN maintained their physi-
cal characteristics as a controlled release formulation.
These results demonstrate that the SLN had good sta-
bility at 4°C.
Antibacterial activity
In vitro antibacterial activity studies showed that the
released NFX had the same MIC and MBC as that of
the native NFX (Table 3), indicating that the bioactiv-
ity of NFX was not changed during the preparation
and release procedures. This preparation method
avoided the using of organic solvent, at the same time,
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maintained drug stability since the temperature in the
preparation did not exceed 100 °C while the melting
point of NFX is 228 °C (Gao 1995). Moreover, the MIC
and MBC of NFX-SLN were the same as that of native
NFX at 12 h, and lower than that of native NFX at 24 h,
suggesting that SLN increased the antibacterial activity
of NFX. These results are similar to those obtained by
Ranjita Misra et al and Esmaeili et al (Misra et al., 2009;
Esmaeili et  al., 2007). Fusion of the lipid composition
with the bacterial membranes might account for the
enhanced antibacterial effect of NFX-SLN (Sachetelli
For personal use only.
et al., 2000). Due to membrane merging, the antibiotic
could easily penetrate inside the bacteria allowing the
increased bactericidal efficacy observed with the drug,
Table 3.  MIC and MBC of NFX (µg/ml, n = 3).
12 h
MIC Native NFX 0.3
Released NFX 0.3
NFX-SLN 0.3
MBC Native NFX 0.6
Released NFX 0.6
NFX-SLN 0.6
MIC: minimum inhibitory concentration; MBC: minimum
bactericidal concentration
Figure 2.  In vitro release studies (mean ± SD, n = 3). NFX-SLN:
norfloxacin-nanoparticles; NFX: native norfloxacin
 Drug Delivery
thus circumventing the normal pathway of penetration
(Beaulac et al., 1996; Beaulac et al., 1998).
Sustained antibacterial activity studies were carried
out based on the growth kinetics of Staphylococcus aureus
and a viable colony count method was conducted. At 6 h
and 12 h NFX-SLN was less effective (Figure 4a), but at
all other time points, NFX-SLN was much more effec-
tive than native NFX (Figure 4b). Lower concentration of
NFX might count for the less effectiveness of NFX-SLN
at the early stages because of an incomplete release of
NFX from the nanoparticles. After 12h, NFX-SLN exhib-
ited its advantage over native NFX. Colony number of
native NFX sharply increased between 36 h and 48 h and
reached almost the same level as that of the controls by
72 h, indicating the exhaustion of the antibiotic. In case
of NFX-SLN, the increase of bacterial colonies was much
slower (Figure 4b). NFX-SLN remained effective for a
longer period of time owing to the sustained release of
the drug. The two curves representing control nanopar-
ticles and Muller-Hinton broth were in the same trend,
indicating that control nanoparticles had no antibacte-
rial effect.
Cytotoxicity
As demonstrated in Figure 5, cytotoxicity study on
BHK-21 cell line showed no significant differences among
the NFX-SLN, native NFX and the control (P > 0.05) on
average cell viability, indicating the nanoparticles were
not toxic to the cells. Stearic acid and PVA are commonly
used pharmaceutical lipid matrix and surfactant with
good biocompatibility and low toxicity.
24 h
Pharmacokinetics
0.6
Figure 6 shows the Plasma NFX concentration vs. time
0.6
plotting after oral administration of NFX-SLN or NFX,
0.3
table 4 shows their pharmacokinetic parameters. At
0.9 all time points, the plasma concentrations were sig-
0.9 nificantly higher with NFX-SLN than with native NFX.
0.6 NFX-SLN reached the peak plasma concentration
(Cmax) of 2.7 ± 0.6 μg/ml at 2 h. The drug concentration
decreased between 2 h and 4 h, then sustained above
Figure 3.  Comparison of In vitro releases of stored and newly
prepared NFX-SLN (mean ± SD, n = 3).

Figure 4.  Sustained-antibacterial activity: (a) 6 and 12 h; (b) from 6 h to 72 h (mean ± SD, n = 3) NFX-SLN: norfloxacin-nanoparticles; NFX:
native norfloxacin; CNP: control nanoparticles; MHB: Muller-Hinton broth Statistical significances are p< 0.05 compared with: *NFX.
Drug Delivery Downloaded from informahealthcare.com by Universite De Sherbrooke on 11/22/12
Figure 5.  Cytotoxicity studies on BHK-21 cells (mean ± SD, n = 5)
For personal use only.
NFX-SLN: norfloxacin-nanoparticles; NFX: native norfloxacin;
CNP: control nanoparticles
the MIC90 (0.12 μg/ml) in a steady trend. After four-
teen hours, the plasma NFX concentration of NFX-
SLN was 0.32 ± 0.03 μg/ml and then maintained over
0.139 ± 0.012 μg/ml for a period of 168 h. In contrast,
the plasma drug concentration of native NFX increased
rapidly to the peak level of 0.54 ± 0.16 μg/ml at 0.5 h,
then declined quickly and was undetectable after
14 h. This result is similar to the in vitro release trend
between NFX and NFX-SLN. The elimination half-life
(t1/2) and the mean residence time (MRT) of NFX-SLN
were significantly longer than those of native NFX
(101.84 ± 7.52 h vs 9.23 ± 0.33 h, and 155.98 ± 5.8 h vs
14.74 ± 0.64 h), which also indicated that the NFX-SLN
had a sustained release effect. The perceived advantage
of SLN was the encapsulation of the drug in the solid
matrix for protection and controlled release purposes,
the carrier itself may exhibit certain absorption pro-
moting effect (Müller et  al., 2000; Garcia et  al., 2002).
The lipid protects the drug from chemicals as well as
enzymatic degradation, thereby delaying the in vivo
metabolism and prolonging the systemic circulation
time and controls the release of drug in blood (Luo et al.,
2006; Bala et al., 2004; Bhardwaj et al., 2005). Sustained
drug release effect over a period of days or even weeks
could provide a long-circulation effect in vivo (Panyam
& Labhasetwar 2003; Vasir et al., 2003). This NFX-SLN
should be a promising formulation to improve the ther-
apeutic efficacy and avoid frequent administrations
of NFX for treatments needed multiple and frequent
© 2011 Informa Healthcare USA, Inc.
Norfloxacin-loaded solid lipid nanopartices  447
dosages, such as cancer induced neutropenia and gas-
trointestinal hemorrhage, and consequently reduce
the adverse effects. The Cmax of NFX-SLN indicates that
there was a fast release of SLN which was consistent
with the in vitro initial fast release in this study. This
kind of curves is desirable for sustained-release prod-
ucts where the initial release rate should be sufficiently
rapid to ensure the therapeutic drug serum levels.
The AUC0-inf of NFX-SLN and native NFX were
57.81 ± 6.89 and 4.8 ± 0.14 respectively. SLN enhanced
the oral bioavailability of NFX as reflected by AUC0-inf
by 12 folds. The mechanisms of oral uptake include
diffusion of particles through mucus and accessibility
to enterocyte surface, epithelial interaction, cellular
trafficking, exocytosis and systemic dissemination (Hou
et al., 2009). Nanoparticles are preferentially absorbed
via “M-cells” in the Peyer’’s patches by the process of
endocytosis (lymphoid uptake), thereby delivering the
drug loaded particles directly into systemic circulation
through the lymphatics and circumventing the first-
pass metabolism (Jani et  al., 1992; Desai et  al., 1996).
Lymphatic uptake is influenced by lipid nature, chain
length, and hydrophobicity of nanoparticles (Hussain
et al., 2001; Nordskog et al., 2001). The stearic acid has
a long chain fatty acid, which increased in lymphatic
uptake of NFX. The size of SLN in the range of 20–500 nm
allows the efficient uptake in intestine, particularly in
the lymphoid sections of this tissue (Yuan et al., 2007).
Due to their small particle size, SLN might exhibit bio-
adhesion to the gastrointestinal tract wall or enter the
intervillar spaces thus increasing their residence time
in the gastrointestinal tract (Vasir et  al., 2003). This
increase in adhesion will result in enhanced bioavail-
ability (Luo et  al., 2006). Bioavailability enhancement
helps to lower dosage levels (Munoz et  al., 1994) and
thus decreases toxicity or side effects. The enhanced
bioavailability of NFX suggests that the NFX-SLN could
be a useful formulation for treatment of diseases spe-
cifically depended on high dosage such as treatment of
urinary tract infections.
Conclusions
Norfloxacin streaic acid-SLN prepared by hot homog-
enization and ultrasonic technique had good sustained
 448  Zhao Dong et al.

Figure 6.  Plasma NFX concentration vs. time plotting after oral administration of NFX-SLN or NFX: (a) within 24 h; (b) from 24 h to 168 h
(mean ± SD, n = 5). NFX-SLN: norfloxacin-nanoparticles; NFX: native norfloxacin.
Drug Delivery Downloaded from informahealthcare.com by Universite De Sherbrooke on 11/22/12

Table 4.  Pharmacokinetic parameters after oral administration to SD rats (mean ± SD, n = 5).
Formulation AUC0-inf MRT (h) Cmax (μg/ml) Tmax (h) T1/2 (h) Fr
NFX-SLN 57.81 ± 6.89* 155.98 ± 5.80* 2.70 ± 0.60* 2* 101.84 ± 7.52* 12*
NFX 4.8 ± 0.14 14.74 ± 0.64 0.54 ± 0.11 0.5 9.23 ± 0.33 1
AUC0-inf: the area under the concentration–time curve; MRT: mean residence time; Cmax: the values of maximum serum concentration;
Tmax: time to peak serum concentration; T1/2: elimination half-life; Fr: the relative bioavailability at infinity
*
Statistical significances between NFX-SLN and native NFX are p < 0.05

release effect and stability. The SLN effectively enhanced nanoparticles for oral delivery. J Biomed Nanotechnol. 1,
the bioavailability and antibacterial activity of NFX. It 235–258.
Boerema JB, Saene HKV. (1986). Norfloxacin treatment in complicated
could be a promising system for oral delivery of NFX.
For personal use only.

urinary tract infection, Scand. J Infect Dis. Suppl. 48, 20–26.

Declaration of interest
The authors report no conflict of interest. The authors
alone are responsible for the content and writing of the
paper.
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