Formulation and Evaluation of Niosome-Encapsulated

Levofloxacin for Ophthalmic Controlled Delivery

Swati Dikshit 1, Amit Sharma, Niraj Upamanyu1, Navdeep Raghuwanshi2, Amit Dubey2, Ashish Pathak2

1. Department of pharmaceutics, R.K.D.F College of Pharmacy, Bhopal, M.P

2. Department of Pharmaceutics, Sapience Bioanalytical Research Laboratory,
Bhopal, M.P., India-462021.
Introduction:
Drug delivery in ocular therapeutics is a challenging problem and is a subject of interest
to scientists working in the multi-disciplinary areas pertaining to the eye, including
chemical, biochemical, pharmaceutical, medical, clinical, and toxicological sciences [1]
The current treatment of ocular infections requires frequent topical antimicrobial drug
administration in microbial keratitis and repeated injection of antimicrobial drugs into
[2]
the site of infection in endophthalmitis . The therapeutics requires the association of
more than one antimicrobial treatment for several weeks, which often leads to poor
patient compliance, contributing to low therapy efciency[3]. Vesicular drug delivery
systems allows the entrapment of drug molecule into lipid bilayer or surfactant vesicles
and thus increase drug concentration at the site of application with sustained drug
delivery of medicament, which results in improved bioavailability. Such vesicles
(liposome and niosome) acts as carrier for controlled ocular drug delivery by preventing
metabolism of drug from enzymes present at the corneal epithelial surface. Vesicle
entrapped drug can be easily administered in liquid dosage forms such as eye drops with
patient compliance, modulated drug release profile and high drug pay load. Niosomes
can encapsulate both tear results in the rapid removal of the drug from eye lipophilic and
hydrophilic drugs and protect against acidic and enzymatic effects in vivo. They offer
several advantages over liposomes such as higher chemical stability, intrinsic skin
penetration enhancing properties and lower costs.[4]
Levofloxacin is a antibacterial agent of the fluoroquinolone class that exhibits a broad
spectrum of in vitro activity. It has been demonstrated to be effective in the treatment of a
wide range of community and hospital-associated infections.[5] This review presents a
microbiological perspective of the use of levofloxacin for topical treatment of bacterial
external ocular infections, presenting in vitro, pharmacokinetic and clinical trials data.
Levofloxacin eye drops were launched in several European countries in 2002. The
widespread use of levofloxacin in ophthalmology can be attributed to its appropriate
antibacterial spectrum and good corneal penetration. The main indications for treatment
are bacterial blepharitis and conjunctivitis, bacterial keratitis, especially when associated
with contact lenses and endophthalmitis, where it is provided as an additional therapeutic
agent. Furthermore, levofloxacin eye drops are used as prophylaxis to reduce the bacterial
conjunctival flora prior to intraocular surgery. In a healthy eye the conjunctival bacterial
flora primarily consists of Staphylococcus spp. (mainly S. epidermidis), Corynebacterium
spp. and, to a lesser degree, Streptococcus spp. and various Gram-negative rods. [6] The
predominant bacterial organisms isolated from patients with acute bacterial conjunctivitis
are S. aureus, S. pneumoniae and Haemophilus influenzae, the latter being frequently
recovered from children.[7,8]
Material and Method:
Levofloxacin was a gift sample from Renbaxy Labs, Devas, M.P, India, Cholesterol,
Span 60 were gift sample from Zydus Cadila, Ahmdabad, Gujrat. Methanol, dihydrogen
phosphate, disodium hydrogen phosphate were purchased from India Ltd, Mumbai.
Preformulation study: [9]
The FT-IR spectrum of pure drug was analyzed to check the purity of the drug using
Bruker Fourier Transform Spectrophotometer by KBr pellet method. The IR absorption
spectra of the pure drug were taken in the range of 400-4000-1 cm. FT-IR Graph reported
in figure 1.
Preparation of niosomes: [9-10]
The composition of the tested niosomal formulae are reported in Table 1. Niosomes
containing levofloxacin were prepared by thin film hydration technique. Briey,
surfactants, cholesterol and DCP in different molar ratios, were accurately weighed in to
along necked quick fit round-bottom fask and dissolved in 10ml chloroform. The organic
solvent was slowly evaporated at 60C under reduced pressure, using a rotary evaporator
at 150 rpm such that a thin dry film of the components was formed on the inner wall of
the rotating flask. The dried thin lipid film was then hydrated with 10ml of phosphate
buffered saline (PBS, pH7.4), containing 10mg levofloxacin, by rotating the flask in a
water bath using a rotavapor under normal pressure in order to ensure complete hydration
of the film. The niosomal suspension was left to mature overnight at 4C. For sterility, all
the above mentioned steps were done under aseptic conditions. All glassware was
sterilized by autoclaving, phosphate buffered saline was passed through a 0.22m
membrane filter, and the entire procedure was carried out in a laminar flow hood.
Microscopy [10]
The vesicle formation by the particular procedure was confirmed by optical microscopy
in 400x resolution. The niosome suspension placed over a glass slide and fixed over by
drying at room temperature, the dry thin film of niosome suspension observed for the
formation of vesicles.
Entrapment efficiency
The proportion of encapsulated levofloxacin was obtained by ultra-centrifugating 1ml of
the niosomal suspension at 15,000rpm for 1h using a cooling centrifuge at 4C (Remi,
Mumbai, India). The niosomes were separated from the supernatant and were washed
twice, each time with 1ml phosphate buffered saline, and recentrifuged again for 1h. The
amount of entrapped levofloxacin was determined by lysis of the separated vesicles with
isopropanol. A 100 l sample of niosomes was mixed with 1ml of isopropanol, the
volume was completed to 10ml with phosphate buffered saline and covered with para
film to prevent evaporation. The concentration of the drug was determined
spectrophotometrically (Systronic, model UV-1601 PC, Ahmedabad, India) after
[11]
derivatization with o-phthaldialdehyde reagent by Zhangs method .Briey, the o-
phthaldialdehyde reagent was formulated by adding 2.5g o-phthaldialdehyde, 62.5 ml
methanol and 3ml 2-mercaptoethanol to 560 ml sodium borate solution, pH 8. There
agent was stored in a brown bottle in dark chamber for atleast 24h before use, as it is light
sensitive. This reagent could be used only upto 3days. Levofloxacin solution, o-
phthaldialdehyde reagent, and isopropanol (to avoid precipitation of the products formed)
were mixed in similar proportions and stored for 30 min at room temperature. The o-
phthaldialdehyde reagent reacted with levofloxacin amino groups and chromophoric
products were obtained, whose absorbance was measured at 298 nm.
The entrapment efficiency is defined as follows [12]:

Total Drug Diffused Drug

Percent of Entrapmet Efficiency = ---------------------------------------- 100
Total Drug
In-vitro drug release study [13]
In vitro release pattern of niosomal suspension was carried out in dialysis bag method.
1.5 mg equivalent of 0.3 % of niosomal suspension was taken in dialysis bag (Hi media)
and the bag was placed in a beaker containing 250 ml simulated tear fluid (pH7.4
phosphate buffer). The beaker was placed over magnetic stirrer and the temperature was
maintained at 37 1C. 5 ml samples were withdrawn periodically and were replaced by
fresh buffer. The sink condition was maintained throughout the experiment. The
withdrawn samples were analyzed for drug content using U.V. spectrophotometer at 294
nm keeping phosphate buffer pH 7.4 as blank.
Ocular irritance test [14]
The potential ocular irritation and/ or damaging effects of the niosomes under test were
evaluated by observing them for any redness, inflammation (or) increased tear
production. Formulation was tested on six rabbits by dispensing niosomes in the cul-de-
sac of the left eye.
Results and Discussion:
Entrapment Efficiency %EE:
The entrapment efficiencies of optimized niosomal formulations are reported in Table 2.
In order to attain high levofloxacin encapsulation efficiency, several factors, including the
type of surfactant, presence of PEO and the ratio of cholesterol added were evaluated and
optimized. Due to entrapment efficiency we know that Span 20 is the better entrapped. So
that span 20 was optimized for other tests.
Characterization of Levofloxacin Niosomes
Photomicroscopy and Transmission Electron Microscopy
The formation of niosomal vesicles as well as their morphological aspects were evaluated
by using photo and transmission electron microscopy. Various niosomal formulations
were examined under optical microscope and photo-graphed at a magnication of 40, by
means of a fitted camera. Levofloxacin niosomal samples were also examined by
transmission electron microscope at 70kV, after being negatively stained. A saturated
ammonium molybdate aqueous solution was used as the staining agent. The images are
shown in figure 3 & 4 respectively.
In Vitro Release of Nimesulide from Niosomes
niosomes prepared by lipid film hydration method. The comparative release data
indicates that, NF1 is the better release in compare to NF2 and NF3. It is observed that
the release was systemic and sustained action. All the data is shown in table no.3.
Ocular Irritancy Test of Niosomes
It was observed that over the study period (48 h) none of the tested formulae showed any
signs of redness, inflammation or increased tear production. Thus it could concluded that
the non ionic surfactants namely Span 20 [NF1], Span 60 [NF2], and Span 80 [NF3],
used in the niosomal formulations as well as the other excipients were non-irritant to the
eye.
CONCLUSION
The main objective of this study was to design suitable niosome-encapsulated drug
delivery for ocular drug delivery on levofloxacin, to study the in vitro behaviour of the
prepared system and ocular irritancy test in Albino rabbit eye. Finding of all this
investigation conclusively demonstrate prolongation of drug release at a constant and
controlled rate, after encapsulation of levofloxacin. This study suggests that niosomal
formulation can provide consistent and prolonged release of levofloxacin from different
niosomal formulations. It will lead to sustained action of the entrapped drug that reduce
the side effects associated with frequent administration of the drug.

Acknowledgement:
The authors are thanks to Principal, R.K.D.F College of Pharmacy, Bhopal for providing
facilities to carry out this work. Authors are also thanks to Diretor, Sapience Bioanalytical
Reserch Laboratory and also thanks to Renbaxy Ltd., Devas for providing gift sample of
levofloxacin, PEO and Ranchem Levoratory for providing Span 20, 60, 80.

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Tables:

Table I. Formulae Composition (Molar Ratio)

Formula Composition
Span 20 Span 60 Span 80 Cholesterol Dicetayl
Phosphate
F1 1 1 0.1
F2 1.5 1 0.1
F3 2 1 0.1
F4 1 1 0.1
F5 1.5 1 0.1
F6 2 1 0.1
F7 1 1 0.1
F8 1.5 1 0.1
F9 2 1 0.1

Table 2: Optimized of niosomal formulations

Formulation Surfactant Drug:surfactant: Entrapment
Cholesterol Efficiency %EE
NF3 Span20 1:2:1 68.4

NF6 Span60 1:2:1 59.32

NF9 Span80 1:2:1 58.27

Table 3: Comparative In vitro dissolution profile of different formulations
Time % of drug release
(hour)
NF3 NF6 NF9

0 0.00 0.00 0.00

1 13.36 12.05 09.48

2 19.75 18.68 13.06

3 26.67 24.34 23.80

4 35.21 28.72 28.13

5 42.13 38.40 36.12

6 49.36 47.63 44.23

8 54.36 51.54 48.35

10 62.36 61.38 59.58

12 76.57 73.08 71.56

Figure:
Figure 1: FTIR of graph of Levofloxacin
Figure 2. Drug release profile of levofloxacin Niosomes:

Figure 3: Microscopic Image of levofloxacin Niosomes

Fig 4. TEM of Levofloxacin Niosome