Comparison of Ciprofloxacin Hydrochloride-Loaded Protein, Lipid,

and Chitosan Nanoparticles for Drug Delivery

Dharmendra Jain, R. Banerjee
School of Biosciences and Bioengineering, Indian Institute of Technology, Bombay-76, India

Received 5 October 2006; revised 12 May 2007; accepted 29 August 2007

Published online 20 December 2007 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.30994

Abstract: The aim of the present study was to develop single dose delivery systems based on
nanotechnology for prolonged antibiotic release in a controlled manner. Five different drug–
carrier ratios of ciprofloxacin hydrochloride-loaded nanoparticles of albumin, gelatin,
chitosan (CS), and lipid [solid lipid nanoparticles (SLNs)] were prepared and characterized.
Average particle size was found to be in the range of 73 6 2 to 98 6 44 nm for SLNs, 140 6 7
to 175 6 24 nm for albumin nanoparticles, 143 6 18 to 184 6 27 nm for gelatin nanoparticles,
and 247 6 48 to 322 6 52 nm for CS nanoparticles. A drug-to-carrier ratio of 0.5:1 was
preferred for CS nanoparticles having zeta potential of >20 mV and drug encapsulation of
35.01% 6 2.66%. Similarly, 0.6:1 ratio was preferred for albumin nanoparticles with zeta
potential >16 mV and drug encapsulation 48.20% 6 3.01%. Zeta potentials of gelatin
nanoparticles loaded with ciprofloxacin suggested that they were unstable and prone to
flocculation. SLN with 0.25:1 drug carrier ratio showed 38.71% 6 2.38% drug entrapment
and 228 6 1 mV surface charge. All the nanoparticles showed sustained drug release avoiding
‘‘burst effect’’ of the free drugs for up to 120 h for albumin nanoparticles, 96 h for CS and
gelatin nanoparticles, and 80 h for SLNs. The drug release profiles followed Higuchi model.
Results suggest that CS nanoparticles and SLNs can act as promising carriers for sustained
ciprofloxacin release in infective conditions. ' 2007 Wiley Periodicals, Inc. J Biomed Mater Res Part B:
Appl Biomater 86B: 105–112, 2008

Keywords: nanoparticles; protein; chitosan; solid lipid nanoparticles; ciprofloxacin hydro-

chloride

INTRODUCTION keratitis caused by S. aureus.2 It has been approved as a

Nanoparticulate delivery systems offer numerous advan-
tages compared with the conventional dosage forms such
as sustained drug release, improved efficacy, lesser fre-
quency of dose administration, reduced toxicity, and pro-
tection of the associated drug against enzymatic and
chemical degradation in vivo.1 Because of their higher sur-
face to volume ratio, drug entrapment efficiency of nano-
particles is greater as compared with other dosage forms.
Nanoparticles can reach lesser accessible sites because of
their smaller size; thus, the bioavailability of the drug in
the deeper tissues can be increased.
Ciprofloxacin is a broad spectrum antibiotic having effi-
cacy against gram positive and gram negative bacteria, and
the frequency of spontaneous resistance to ciprofloxacin is
very low.2 Ciprofloxacin is used as a single agent for treat-
ment of topical ocular infections like conjunctivitis and
Correspondence to: R. Banerjee (e-mail: rinti@iitb.ac.in)
Contract grant sponsor: Life Sciences Research Board, India
' 2007 Wiley Periodicals, Inc.
topical preparation for the treatment of corneal ulcer. Flour-
oquinolones are effective in the treatment of community
acquired, complicated skin and skin structure infections
like pyodermas, wound infections secondary to trauma.3 In
above-stated diseased conditions ciprofloxacin-loaded nano-
particles can be useful as local delivery systems for pro-
longed durations. This will avoid unnecessary systemic
distribution of the drug and will minimize side effects. Sys-
temic therapy with ciprofloxacin is required in several dis-
eases like typhoid, urinary tract infections, bone and joint
infections, lower respiratory tract infections, and acute si-
nusitis. Ciprofloxacin-loaded nanoparticles can be used for
pulmonary drug delivery for treatment of lower respiratory
tract infections.4,5 In systemic delivery, nanoparticles can
prove advantageous due to the sustained release of drug
from them necessitating less frequent doses. So far various
research groups have prepared ciprofloxacin-loaded nano-
particles for sustained release of the antibiotic, for example
polyisobutyl cyanoacrylate nanoparticles,6 PLGA nanopar-
ticles,2 polyethylbutylcyanoacrylate nanoparticles,7 Eudra-
git1 RS100 or RL100/PLGA nanoparticles.8 All these
carriers are based on synthetic biodegradable polymers.
105
106 JAIN AND BANERJEE
However, there has been some concern regarding the toxic-
ity of cyanoacrylate-based polymers.9–11
Colloidal carriers based on proteins (e.g., albumin, gela-
tin) and polysaccharides [e.g., chitosan (CS)] are promising,
because they are biocompatible, biodegradable, nonanti-
genic, and relatively easy to prepare.12–14 Further, protein
nanoparticles can bind to a large number of drugs in a rela-
tively nonspecific manner. Because of their surface charge,
drugs can physically adsorb onto the protein surface or can
covalently bind to the matrix. CS—a polysaccharide—is
biocompatible, biodegradable, and mucoadhesive and hence
promises as a drug carrier.15,16 Solid lipid nanoparticles
(SLNs) have the potential to carry both lipophilic and
hydrophilic drugs. They are biodegradable and nontoxic;
stable against coalescence, drug leakage, hydrolysis, and
particle growth, which are often observed in lipid emul-
sions and liposomes.17,18
The aim of the present work was to investigate the dif-
ferent approaches to obtain an optimized nanoparticulate
formulation of ciprofloxacin based on biological materials
in the form of lipids, proteins, and biopolymers. Such nano-
particulate carriers would be promising for sustained
release of ciprofloxacin in the form of regional delivery
systems for treatment of corneal ulcers and skin infections
like pyrodermas.
MATERIALS AND METHODS
Materials
Bovine serum albumin (96–98% purity) was purchased
from Sisco Research Lab, Bombay; 25% glutaraldehyde,
stearic acid (98% purity) and sodium taurocholate were
purchased from Loba Chemie, Bombay; 99% absolute alco-
hol was purchased from Changshu Yangyuan Chemical,
China; mannitol was purchased from S. K. Lab, Ahmeda-
bad; phosphate buffer saline (PBS) was prepared from di
sodium hydrogen phosphate, sodium chloride and potas-
sium dihydrogen phosphate, all purchased from Qualigen
fine chemicals, Mumbai; CS (85% deacetylated) and gelatin
(type A) were purchased from Sigma–Aldrich, St. Louis;
pentasodium tripolyphosphate (TPP) was purchased from
CDH Laboratories, New Delhi; phosphatidylcholine (leci-
thin soya) and Dialysis membrane-50 (weight cut off
12,000–14,000; pore size 2.4 nm) were purchased from
Himedia Laboratories, Nasik; Ciprofloxacin hydrochloride
I. P. was provided as a free gift by Cadila Pharmaceuticals,
Ahmedabad, India. All reagents were of analytical grade
and were used as received.
Preparation of Nanoparticles
Albumin Nanoparticles. Albumin nanoparticles with
various drug:protein ratios (w/w, from 0.25:1 to 1:1) were
prepared by desolvation based coacervation method of
microencapsulation, followed by cross-linking with glutar-
aldehyde in accordance with the procedure of Merodio
et al.19 Ciprofloxacin hydrochloride was incubated with
required amount of protein solution (2% w/v) for 1 h at
room temperature. The pH of the solution was adjusted to
5.0 by 1M hydrochloric acid using digital pH meter (Orion
meter model 720). Then, ethanol was added to the solution
in a ratio of 2:1 (v/v) under magnetic stirring. The rate of
the ethanol addition was carefully controlled at 1 mL/min
in accordance with the procedure of Langer et al.20 The
coacervates so formed were hardened with 25% glutaralde-
hyde (1.56 lg/mg of protein) for 2 h to allow cross-linking
of protein. Organic solvents were then removed under
reduced pressure by rotary vacuum evaporation and the
resulting nanoparticles were purified by centrifugation
(Sigma 3K30 laboratory centrifuge) at 48C. Three batches
of nanoparticles were centrifuged at 25,000g for 30 min.
Pellets of nanoparticles were suspended in phosphate buffer
(pH 7.4; 0.1M) and each sample was lyophilized with man-
nitol (2% w/v) at 2488C and 28 3 1023 Mbar pressure for
24 h (Ilshin freeze dryer). Albumin nanoparticle controls
were also prepared by the same procedure without drug
loading.
Gelatin Nanoparticles. Gelatin nanoparticles were pre-
pared by a desolvation technique described by Vandervoort
et al.21 An aqueous gelatin type A solution (0.5% w/v, 100
mL) was prepared, containing different amounts of cipro-
floxacin hydrochloride. The pH was adjusted to 4.0 by the
addition of 0.1N HCl solutions. The solution was stirred at
1000 rpm and the temperature was maintained at 508C. To
induce the desolvation process, ethanol was added until a
permanent faint turbidity was obtained, after which 5.0 mL
of distilled water was added. Glutaraldehyde aqueous solu-
tion (25% v/v, 2 mL) was added to harden the particles.
The preparation was then stirred for 2 h at 1200 rpm. The
cross-linking process was stopped by the addition of aque-
ous sodium metabisulfite solution (0.6 g in 150 mL). Etha-
nol and part of the water were evaporated using a rotavap.
Three batches of nanoparticles were centrifuged at 25,000g
for 30 min. Pellets of nanoparticles were suspended in
phosphate buffer (pH 7.4; 0.1M) and each sample was ly-
ophilized with mannitol (2% w/v) at 2488C and 28 3
1023 Mbar pressure for 24 h (Ilshin freeze dryer). Gelatin
nanoparticle controls were also prepared by the same pro-
cedure without drug loading.
CS Nanoparticles. CS nanoparticles were prepared
according to the procedure reported by Ma et al.22 Nano-
particles were prepared by addition of TPP aqueous solu-
tion (0.1% w/v) to CS aqueous solution (0.2–0.4% w/v in
0.25–10% v/v acetic acid) under magnetic stirring at room
temperature, until faint turbidity. TPP is nontoxic and has
multivalent anions. It can form a gel by ionic interaction
between positively charged amino groups of CS and nega-
tively charged counterions of TPP. This interaction can be
controlled by charge density of TPP and CS, which is de-
pendent on the pH of the solution.23 The formation of the
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 NANOPARTICLES FOR DRUG DELIVERY
particles was a result of the interaction between the nega-
tive groups of the TPP and the positively charged amino
groups of CS (ionic gelation). Different amounts of cipro-
floxacin hydrochloride were incorporated into the CS solu-
tion, prior to the formation of the nanoparticles. Formation
of nanoparticles was monitored by turbidity of the solution.
Nanoparticles were purified by centrifugation at 9000g for
30 min. Supernatants were discarded and pellets of nano-
particles were resuspended in distilled deionized water.
SLNs. Ciprofloxacin hydrochloride loaded-SLNs were
prepared by a warm o/w microemulsion method similar to
that used by Cavalli et al.24 with slight modifications.
Briefly, the microemulsion consisted of stearic acid (inter-
nal phase, 0.7 mmol), phosphatidylcholine (lecithin soya)
(surfactant, 0.14 mmol), sodium taurocholate (cosurfactant,
0.69 mmol), water (111.1 mmol), and ciprofloxacin hydro-
chloride (in different concentrations) and was prepared
using a high-speed homogenizer (ART-Miccra D-1 Homog-
enizer). SLNs were obtained by dispersing the warm micro-
emulsion in filtered cold water (2–38C) at a 1:10
microemulsion:water (v/v) ratio under mechanical stirring.
The SLN aqueous dispersion was washed three times with
distilled deionized water, and freeze dried for quantitative
determination of the incorporated drug.
Physicochemical Characterization
Size Determination and Morphological Characteristics
of Nanoparticles. Nanoparticle sizes and polydispersity
were measured by photon correlation spectroscopy (PCS)
based on the principle of dynamic light scattering (BI
MAS, Multiangle sizing option on Zetaplus, Brookhaven
Instruments, USA) at a scattering angle of 908 and a tem-
perature of 258C. The particle size of nanoparticles was
determined before freeze drying. The particle size was
determined in triplicates and the average values of three
replicas were calculated. Additionally, the surface morphol-
ogy of the nanoparticles was observed by transmission
electron microscopy (TEM).
Analysis of the Surface Charge of Nanoparticles (Zeta
Potential). Zeta potential is one of the important parame-
ters of the colloidal system that indicates the stability of
colloidal particles. The surface charge of the ciprofloxacin
hydrochloride-loaded nanoparticles was determined by
Zetaplus, Brookhaven Instruments, USA. The measure-
ments were carried out using a suspension of the nanopar-
ticles in deionized and distilled water at pH 7.0, at 258C
The zeta potential was determined in triplicates and the av-
erage values were calculated.
Drug Entrapment Efficiency. The drug content in the
ciprofloxacin hydrochloride-loaded nanoparticles was deter-
mined by calculating the difference between the total and
the free drug concentrations in the nanoparticle suspension
Journal of Biomedical Materials Research Part B: Applied Biomaterials
107
and the supernatant, respectively. Supernatant was sepa-
rated by centrifugation and the amount of free drug present
in the supernatant (w) was assayed by UV-spectrophotome-
try at 275 nm. A standard calibration curve of concentra-
tion versus absorbance was plotted for this purpose. The
amount of drug in supernatant was then subtracted from
the total amount of drug added (W). In effect, (W2w) will
give the amount of drug entrapped in the pellet. The per-
centage drug entrapment was calculated using the formula
ðW
wÞ3100
Percentage drug entrapment ¼
W
In Vitro Release Kinetics. In vitro release kinetics study
across a cellophane dialysis membrane (weight cutoff:
12,000–14,000) was performed in triplicate at 378C 6 28C,
under constant slow magnetic stirring at 100 rpm. Prior to
the experiment the membrane was washed with warm Milli
Q water (708C) for 1 h and then rinsed thrice with Milli Q
water to remove the glycerin. Twenty milligrams of drug-
loaded nanoparticles, suspended in 4.0 mL PBS (pH 7.4),
was placed in the cellophane membrane tied at both ends
and immersed in the receiver chamber filled with PBS (pH
7.4; 50 mL). To determine the concentration of drug in the
receiving compartment, samples (1 mL) were withdrawn
from the receiver solution at prefixed time intervals and
replaced by the same volume of PBS solution. The absorb-
ance was measured by UV spectrophotometry at 275 nm.
Finally, the concentration corresponding to the absorbance
was determined from the concentration versus absorbance
calibration curve.
Analysis of Release Profile. There are several models
which can be used for the description of the release profiles
from controlled release devices. Higuchi square root of
time model25,26 was chosen to describe the release patterns
on the basis of the known physical geometry and nature of
the nanoparticles studied. The parameters derived from fit-
ting Higuchi square root model as given in Eq. (1), to ex-
perimental data were used to characterize the release
properties.
F ¼ kt1=2 ð1Þ
where F is the fraction of drug released at any time t, and
k is release rate constant.
RESULTS
Particle Size
The formation of CS nanoparticles occurs spontaneously
upon addition of counter ion sodium tripolyphosphate. The
average particle sizes of drug-loaded CS nanoparticles
ranged between 247 6 48 nm and 322 6 52 nm (Figure
1). Also the correlation between drug:carrier ratio and
108 JAIN AND BANERJEE

The average particle sizes of the nanoparticles formula-

tions were found to be increased slightly with increasing
the amount of the drug to be entrapped. TEM confirmed
sizes of all nanoparticle formulations except in CS nano-
particles where the size on TEM was larger than obtained
by PCS. CS nanoparticle size was dependent on drug:car-
rier ratios, with lowest particle size for 0.25:1 drug-to-
carrier ratio. Particle size for albumin and gelatin nanopar-
ticles were observed to be almost independent of all drug:
carrier ratios and showed good correlation with regression
coefficient near to 1. SLNs showed smallest particle sizes
among all the carriers with smaller sizes across all the drug
carriers.
Polydispersity indicates the degree of nonuniformity of
the particle size. A low polydispersity indicates uniformity
in size distribution of nanoparticles. Range of average poly-
Figure 1. Average particle size of different nanoparticulate formula- dispersity was calculated to be 0.104 6 0.164 to 0.242 6
tions. 0.09 for CS nanoparticles, 0.129 6 0.023 to 0.273 6 0.082
for albumin nanoparticles, 0.103 6 0.092 to 0.190 6 0.034
for gelatin nanoparticles and 0.187 6 0.14 to 0.311 6
particle diameter was established. Equation for regressed
0.004 for SLNs.
straight line of this correlation for CS nanoparticles formu-
lations is y 5 0.9461x 1 235.18 with R2 5 0.8757, where
x is the amount of drug per 100 mg of carrier and y is the Surface Charge (Zeta Potential)
particle diameter.
The average zeta potential of CS nanoparticles was calcu-
It is clear from the TEM photograph of CS nanoparticles
lated between 17 6 2 and 25 6 1 mV. The higher zeta
(Figure 2) that the particle size of the drug-loaded nanopar-
potential of CS nanoparticles shows the substantial electro-
ticles is less than 100 nm. It is noteworthy that the hydro-
kinetic stability of the formulations. Only the formulation
dynamic diameter of the CS nanoparticles measured by
with 25 mg ciprofloxacin hydrochloride/100 mg of carrier
PCS is higher than the size estimated from TEM particu-
had zeta potential lesser than 120 mV, whereas all other
larly because of the bioadhesive nature and swelling char-
formulations exhibited zeta potential higher than 120 mV.
acter of the CS.
The average zeta potential of albumin nanoparticles ranged
Albumin nanoparticles displayed a particle size in the
from 11 6 1 to 17 6 3 mV, showing that the nanoparticles
range of 140 6 7 to 175 6 24 nm (Figure 1). TEM of
are positively charged but have a tendency toward aggrega-
the albumin nanoparticles also confirmed their spherical
tion to some extent. The average zeta potential of gelatin
shape and sizes comparable with those obtained from PCS.
nanoparticle formulations was ranging from 3 to 7 mV.
Equations for regressed straight line of drug:carrier ratio
The lesser values of zeta potential suggest that ciprofloxa-
and particle size correlation for albumin nanoparticles for-
cin-loaded gelatin nanoparticles are electro-kinetically
mulations is y 5 0.462x 1 127.64 with R2 5 0.9951,
unstable. The average zeta potential values for SLNs were
where x is the amount of drug per 100 mg of carrier and y
ranging from 228 6 1 to 237 6 1 mV, showing the elec-
is the particle diameter.
tro-kinetic stability of the formulations. The average zeta
The average particle size of gelatin nanoparticle formu-
potential of all the nanoparticulate formulations is repre-
lations was calculated in range of 143 6 18 to 184 6 27
nm (Figure 1). Equations for regressed straight line of
drug:carrier ratio and particle size correlation for gelatin
nanoparticles formulations is y 5 0.5729x 1 127.56 with
R2 5 0.9921, where x is the amount of drug per 100 mg of
carrier and y is the particle diameter.
SLNs were prepared by spontaneous solidification of the
nanoemulsified droplets of the stearic acid under high-
speed homogenization. SLN formulations displayed a parti-
cle size lesser than 100 nm (73 6 2 to 98 6 44 nm)
(Figure 1). Equations for regressed straight line of drug:car-
rier ratio and particle size correlation for SLN formulations
is y 5 0.2712x 1 66.249 with R2 5 0.5408, where x is the
amount of drug per 100 mg of carrier and y is the particle
diameter. Figure 2. TEM image of CS nanoparticles.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

 NANOPARTICLES FOR DRUG DELIVERY 109

appeared to be the optimal CS formulation due to a moder-

ate drug entrapment (35.01% 6 2.66%) and zeta potential
(22.95 6 2.69 mV). In case of SLN, maximum entrapment
of 38.71% 6 2.38% was observed with 0.25:1 ratio.

In Vitro Release Studies

Formulations with 50 mg drug incorporated were selected
for drug release studies. The drug release profile of differ-
ent nanoparticulate formulations after predetermined time
intervals are expressed as percentage of the drug loading.
The average cumulative percent release profile of all the
nanoparticulate formulations at prefixed time intervals is
represented in Figure 5 as a function of time. Under the
conditions used in the in vitro release studies a biphasic
Figure 3. Average zeta potential of different nanoparticulate formu- pattern of drug release was observed. The release kinetics
lations. had two distinct phases: initially a faster release profile and
thereafter sustained release for hours. Release kinetics of
albumin nanoparticles showed drug release over a period of
sented in Figure 3. Gelatin nanoparticles are not suitable
120 h. CS and gelatin nanoparticles were found to be capa-
for ciprofloxacin loading because of their low zeta poten-
ble of releasing the drug for as long as 96 h, whereas drug
tial, which suggests a tendency of aggregation of the par-
release through SLN was observed for up to 80 h. On the
ticles and thus, instability. However, this instability may be
other hand, free ciprofloxacin hydrochloride showed a burst
confirmed by particle size estimations with time. SLNs in
release with almost 50% free drug release in 30 min and
all ratios and CS nanoparticles in four ratios showed zeta
more than 90% drug diffusing in 70 min.
potential [6 20 mV, suggesting electro-kinetic stability of
A nonlinear fitting of in vitro release data was per-
the formulations. Nevertheless, nanoparticles having zeta
formed using Higuchi square root of time model. Release
potential [30 mV are more stable than those having zeta
of drug from almost all the nanoparticles formulations fol-
potential between 20 and 30 mV. Hence, SLNs were the
lowed Higuchi square root of time equation. For albumin
most stable carriers for ciprofloxacin.
nanoparticles, the release followed the equation F 5
0.0942 t1/220.0087 with an R2 of 0.9789; for gelatin nano-
Drug Entrapment particles, the release followed the equation F 5 0.1028 t1/22
The average percent entrapment efficiency was ranging 0.001 with an R2 of 0.9699; for CS nanoparticles, the release
from 70.79% 6 2.49% to 11.90% 6 1.59% for CS nano- followed the equation F 5 0.1062 t1/220.0076 with an R2 of
particles formulations, 73.07% 6 2.32% to 25.30% 6 0.9809; and for SLNs, the release followed the equation
2.83% for albumin nanoparticles formulations, 66.01% 6
3.09% to 21.15% 6 2.59% for gelatin nanoparticles formu-
lations, and 38.71% 6 2.38% to 8.66% 6 1.64% for SLN
formulations (Figure 4).
The percent entrapment varied with the fraction of drug.
To study this, a correlation between drug:carrier ratio and
percent drug entrapment was established. Equations for
regressed straight lines of this correlation for different for-
mulations are as following: y 5 20.7802x 1 80.279; R2 5
0.8553 (CS nanoparticles), y 5 20.6589x 1 90.03; R2 5
0.9798 (albumin nanoparticles), y 5 20.607x 1 78.975; R2
5 0.9776 (gelatin nanoparticles), y 5 20.4001x 1 45.913;
R2 5 0.9482 (SLNs), where x is the amount of drug per
100 mg of carrier and y is the percent drug entrapment. Up
to 70% drug entrapment was observed for albumin and CS
nanoparticles using a drug-to-carrier ratio of 0.25:1. For
albumin nanoparticles, a drug-to-carrier ratio of 0.6:1
was preferred because it had the maximum zeta potential
of 16.97 6 2.76 mV and a moderate drug entrapment Figure 4. Average percent drug entrapment efficiency of different
of 48.20% 6 3.01%. The 0.5:1 ratio CS nanoparticles nanoparticulate formulations.

Journal of Biomedical Materials Research Part B: Applied Biomaterials

110 JAIN AND BANERJEE
Figure 5. In vitro drug release kinetics of ciprofloxacin hydrochlor-
ide-loaded nanoparticles in PBS (pH 7.4).
F 5 0.1096 t1/220.1002 with an R2 of 0.9575 where F is the
fraction of drug released and t is the time.
DISCUSSION
The discrepancy in the size of CS nanoparticles’ TEM and
PCS may be explained by the swelling of the particles in
the aqueous media. Similar phenomenon was observed by
Aktas et al.16 and Banerjee et al.27 Also, the greater parti-
cle size of CS nanoparticles may be due to aggregation of
nanoparticles due to strong inter or intramolecular hydro-
gen bonding between CS molecules. There was not much
difference in the particle sizes of albumin nanoparticles
regardless of the drug-to-carrier ratios used. Desolvation
process can lead to particles of similar size but in different
concentrations. A similar phenomenon was described by
Weber et al.,28 when the ethanol volume addition was [1.5
fold. The lesser particle size of SLNs may be due to spon-
taneous solidification of the oil droplets of the microemul-
sion formed during the preparation of the SLNs.
The zeta potential has a substantial influence on the sta-
bility of suspensions, the interaction of nanoparticles with
charged drugs, and also on the adhesion of drug delivery
systems onto biological surfaces. The CS nanoparticles
were charged positively; this indicates that only a part of
the amino groups are neutralized during nanoparticle for-
mation. The residual amino groups would be responsible
for the positive zeta potential. Moreover, these groups are
freely accessible for interaction with drugs as well. The
relation of particle stability with zeta potential is an empiri-
cal one. However, nanoparticle formulations showing zeta
potential [ 6 30 mV are considered stable, and those with
zeta potentials between 5 and 15 mV are considered to ex-
hibit limited flocculation.29 Thus, apart from SLNs, the al-
bumin nanoparticle and CS nanoparticle formulations
showing zeta potential 10–17 mV and 21–25 mV, respec-
tively may be considered to have a reasonable stability. In
this regard, we can classify ciprofloxacin hydrochloride-
loaded albumin, CS and SLNs as electro-kinetically stable
formulations. The SLNs are, however, the most stable for-
mulations based on their higher zeta potential.
Percent entrapment of the drug was found to be
decreased with increasing amount of the drug. Good per-
centage entrapment of ciprofloxacin hydrochloride in albu-
min nanoparticles was obtained. This could be attributed to
the adequate protein binding capacity of ciprofloxacin
hydrochloride.30 Human serum albumin nanoparticles pos-
sess more lysyl residues on the particle surface than gelatin
nanoparticles. To prepare a drug carrier system by covalent
attachment of the drug to the colloidal system, the amount
of available amino groups on the particle surface is one of
the important parameters.28 The lesser encapsulation effi-
ciency observed in CS nanoparticles may be due to the par-
tial repulsion of CS and ciprofloxacin hydrochloride due to
the positive charge of both of them. The lesser entrapment
of the drug with SLNs may be due to the hydrophilic na-
ture of the ciprofloxacin hydrochloride.
The release of the drug from nanoparticles in the sur-
rounding environment (i.e., PBS, pH 7.4) at 378C 6 28C
may be caused by a dissociation mechanism. The drug
could be associated to the nanoparticles in three different
states: at the nanoparticle surface, in the core as a reversi-
ble complex, or in the core as irreversible complex.16 Gen-
erally, drug release follows more than one type of
mechanism. In case of release from the surface, drug
adsorbed on the surface of nanoparticles dissolves instanta-
neously when it comes in contact with the release medium.
The early phase of the release corresponds to the release of
drugs physically bound to the surface of the nanoparticles
and the delayed phase due to the release of entrapped drug
due to diffusion of drug from the rigid matrix structure.
Ocular infections like corneal ulcers and keratitis are
frequently treated by topical application of the ciprofloxa-
cin 0.3% solutions as eye drops. However, due to lachry-
mal drainage, blinking reflex, and systemic absorption in
the conjunctiva, only a small fraction of drug is absorbed
into the eye and reaches the anterior segments but hardly
reaches the posterior segments. Another drawback associ-
ated with eye drops is pulse kinetics, that is the concentra-
tion of the drug rises sharply then followed by a sharp
decline. This necessitates frequent doses of administration,
which poses a potential risk of toxicity. The need for fre-
quent dosing can be reduced by the use of nanoparticles
for these applications. The nanoparticulate delivery systems
can be instilled as eye drops but will have a prolonged du-
ration of action. After administration, the nanoparticles can
remain at the site of action for prolonged duration with
sustained release of the drug. CS can prolong the corneal
residence time due to its mucoadhesive nature. In endop-
thalmitis the use of ciprofloxacin is restricted due to its
decreased levels in the vitreous. The nanoparticles developed
Journal of Biomedical Materials Research Part B: Applied Biomaterials
 NANOPARTICLES FOR DRUG DELIVERY
in this study can be used to achieve a greater penetration of the
drug in the vitreous. Similar albumin nanoparticles loaded
with ganciclovir have been found to be well tolerated and
effective in CMV retinitis.19,31
Community acquired complicated skin and skin structure
infections like pyodermas, wound infections secondary to
trauma can also be treated with flouroquinolones.3 In such
conditions ciprofloxacin-loaded nanoparticles can be applied
either directly (e.g., mucoadhesive CS nanoparticles) or by
suspending in skin lotions or by entrapment in the hydrogels
or transdermal patches for prolonged release of the antibiotic
at the site of action. SLNs are known to act as physical
sunscreens protecting against UV radiations.32,33 Lower re-
spiratory infections can be treated with ciprofloxacin-loaded
nanoparticles as inhalation therapy. SLNs can be used as
oral delivery systems for such applications. Similar SLNs
have been proven to be effective for oral drug delivery for
tuberculosis.34
Ciprofloxacin exhibits an in vitro minimum inhibitory
concentration (MIC) of 0.1 lg/mL or less against most
strains of the aerobic gram negative microorganisms,
whereas aerobic gram positive bacteria are inhibited at rela-
tively higher concentrations.35 It is well tolerated after topi-
cal or systemic administration36 and has an elimination
half-life of 3–5 h.35 Disease recurrence is commonly
observed with gram negative bacterial infections. A dose of
500 mg BD of ciprofloxacin is recommended for a period
of 1–2 weeks for treatment of typhoid, urinary tract infec-
tion, skin and skin-related infections, bone, joint, and soft
tissue infections, intra-abdominal infections, and so forth to
obtain clinical cure.35 Because the sustained release of anti-
biotic for prolonged durations can be obtained with nano-
particulate delivery systems, such systems may be used to
achieve the MICs for extended durations. The formulations
developed may be used as twice weekly doses due to the
sustained release of drug leading to a prolonged duration of
action. Accordingly, the dose of the antibiotic to be admin-
istered can be modulated using the nanoparticles on the ba-
sis of disease condition, duration of therapy, elimination
half-life, and MIC of the drug for the particular bacterial
species responsible for the disease. The added benefit of
increased elimination half-life by bypassing the reticuloen-
dothelial system can also be achieved using these nanopar-
ticulate formulations.
Thus, the nanoparticles of ciprofloxacin hydrochloride
developed using proteins, lipid, and CS carriers have the
potential to act as long-acting antibiotic release systems in
various regional and systemic infectious states.
CONCLUSION
In this study, we have developed ciprofloxacin hydrochlor-
ide-loaded nanoparticles using four different natural carrier
materials, and compared them on the basis of particle size,
electro-kinetic stability, entrapment efficiency, and drug
Journal of Biomedical Materials Research Part B: Applied Biomaterials
111
release. Implication of the different profiles suggests that
gelatin nanoparticles are unstable because of their very low
zeta potential due to a tendency of aggregation of the par-
ticles. At 0.5:1 ratio, CS nanoparticles and at 0.6:1 ratio,
albumin nanoparticles appeared to be promising formula-
tions due to their high zeta potentials and relatively high
drug entrapment of 35.01% 6 2.66% and 48.20% 6
3.01%, respectively. In case of SLNs, 0.25:1 ratio was the
preferred formulation due to maximum drug encapsulation
of 38.71% 6 2.38% and 228 6 1 mV zeta potential.
SLNs were found to be smaller in size than other carriers.
Nanoparticle formulations were found to be capable of
releasing the drug for prolonged durations up to 120 h (al-
bumin nanoparticles), 96 h (gelatin and CS nanoparticles),
80 h (SLN), showing that the nanoparticulate drug delivery
systems can overcome the burst effect of the free drug,
which released in around 70 min. Drug release from the
nanoparticles followed Higuchi kinetics. Overall, CS and
SLNs were found to be promising formulations for pro-
longed release of ciprofloxacin particularly for local deliv-
ery in ocular and skin infections.
The authors acknowledge Dr. Ashok Omray, G.M. (R&D)
Cadila Pharmaceuticals, Ahmedabad, India for providing Cipro-
floxacin hydrochloride.
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