See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/49668781

A potential new therapeutic system for

glaucoma: Solid lipid nanoparticles containing
methazolamide

Article in Journal of Microencapsulation · March 2011

DOI: 10.3109/02652048.2010.539304 · Source: PubMed

CITATIONS READS

36 210

7 authors, including:

Rui Li "Dongfei" Liu

Nanjing Medical University Harvard University
13 PUBLICATIONS 246 CITATIONS 49 PUBLICATIONS 700 CITATIONS

SEE PROFILE SEE PROFILE

Xinyun Bi
Nanjing Medical University
3 PUBLICATIONS 44 CITATIONS

SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Aerowood View project

Fabrication of ultrahigh drug loaded carriers View project

All content following this page was uploaded by Rui Li on 29 January 2015.

The user has requested enhancement of the downloaded file.

 Journal of Microencapsulation, 2011; 28(2): 134–141
ß 2011 Informa UK Ltd.
ISSN 0265-2048 print/ISSN 1464-5246 online
DOI: 10.3109/02652048.2010.539304

A potential new therapeutic system for glaucoma: solid lipid

nanoparticles containing methazolamide
Rui Li, Sunmin Jiang, Dongfei Liu, Xinyun Bi, Fengzhen Wang, Qing Zhang and Qunwei Xu
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11

School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, People’s Republic of China

Abstract
Methazolamide (MTA) is an antiglaucoma drug; however, there are many side effects of its systemic
administration with insufficient ocular therapeutic concentrations. The aim of this study was to formulate
MTA-loaded solid lipid nanoparticles (SLNs) and evaluate the potential of SLNs as a new therapeutic system
for glaucoma. SLNs were prepared by a modified emulsion–solvent evaporation method and their physi-
cochemical characteristics were evaluated. The pharmacodynamics was investigated by determining the
percentage decrease in intraocular pressure. The ocular irritation was studied by Draize test. Despite a burst
release of SLNs, the pharmacodynamic experiment indicated that MTA–SLNs had higher therapeutic
efficacy, later occurrence of maximum action, and more prolonged effect than drug solution and commer-
cial product. Formulation of MTA–SLNs would be a potential delivery carrier for ocular delivery, with the
advantages of a more intensive treatment for glaucoma, lower in doses and better patient compliance
For personal use only.

compared to the conventional eye drops.

Keywords: Methazolamide, solid lipid nanoparticles, emulsion–solvent evaporation, ocular delivery,
intraocular pressure, ocular irritation

Introduction of MTA administered systemically has poor access to

Glaucoma, one of the leading causes of irreversible ablep-
sia in the world, is estimated to be suffered by 70 million
people. It generally occurs in people aged above 40, but it
may also influence the visual acuity of much younger
people, even children (Sommer et al., 1991). Glaucoma
is the term used for a group of ophthalmic disorders
characterised by an increase in intraocular pressure
(IOP), which results in a damage to the optic disc and
visual field disturbances. Increase in IOP is a consequence
of an imbalance between the production and drainage of
aqueous humour. Carbonic anhydrase inhibitors (CAIs),
as a mainstay in the medical treatment of glaucoma, have
been used since 1954 (Becker, 1954), owing to their ability
to lower IOP by reducing aqueous humour formation in the
ciliary body.
As one of the most common CAIs, methazolamide
(MTA) has been used orally to reduce the IOP for more
than half a century (Zimmerman, 1978). However, most
the aqueous humour. The blood–aqueous and the blood–
retinal barriers prevent MTA from entering into the eye and
reduce convection of MTA molecules (Yasukawa et al.,
2004). Thus, a frequent administration at an extremely
high concentration is usually needed, which limits oral
administration by unpleasant systemic side effects
such as central nervous system depression, renal failure,
diuresis, vomiting, anorexia and metabolic acidosis
(Kaur et al., 2002).
The possibility of the topical administration directly
to the eye was extensively investigated by several
researchers to minimise or eliminate the systemic side
effects. However, negative results had been constantly
obtained initially. The major challenge was to improve
the therapeutic effects, which was quite insufficient
mainly because of MTA’s relatively low solubility in water
(1.7 mg/mL at 25 C; Maren, 1967); the pre-corneal loss fac-
tors such as lacrimal secretion and nasolacrimal drainage
in the eye; and the relative impermeability of the corneal

Address for correspondence: Qunwei Xu, School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing 210029, People’s Republic of China.
Tel/Fax: þ86 025 86862008. E-mail: qunweixu@163.com

(Received 8 Jun 2010; accepted 1 Nov 2010)

http://www.informahealthcare.com/mnc
134

epithelial membrane (Le Bourlais et al., 1998; Sultana et al.,
2006). To overcome these problems, various ophthalmic
vehicles such as -cyclodextrin or its derivative
(Fridriksdottir et al., 1997; Jonathan et al., 1996), and
ocular implants (Chang, 2006) had been investigated.
These dosage forms offered numerous advantages over
conventional oral forms, yet they were not devoid of draw-
backs including poor patient compliance (discomfort, dif-
ficulty of insertion and blurring of vision), as ocular
implants and tissue irritation, as well as damage and toxi-
cological complications caused by cyclodextrin (Kaur et al.,
2004).
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11
Considering these points, solid lipid nanoparticles
(SLNs) as a drug delivery system were utilised in
our study for formulating MTA as ocular eye drops.
The potential use of SLNs as ocular drug carriers is followed
by the development of various colloidal dispersion systems
such as liposomes (Hathout et al., 2007), microemulsion
(Vandamme, 2002), nanoemulsion (Ammar et al., 2009),
polymer nanoparticles (or nanosuspension; Pignatello
et al., 2002; Kassem et al., 2007) and nanoparticles
(Gupta et al., 2010) that many published articles have
reported as effective ophthalmic drug carriers. SLNs
are defined as particles with the average particle size of
10–1000 nm, composed of lipids which are solid both at
body and room temperatures, being stabilised by a suitable
For personal use only.
surfactant. To develop efficient drug ocular delivery sys-
tems, various matrices like natural or synthetic solid
lipids, phospholipids and polymers (Muller et al., 1995)
can be used. SLNs combine the advantages and avoid the
disadvantages of other colloidal dispersion systems, and
are regarded as their alternative for ophthalmic use.
The advantages of the SLNs include low irritation, high
penetration into the deeper layers of the ocular structure
and aqueous humour, ease of sterilisation as well as
patient-friendly use. All these properties make SLNs
a promising avenue to achieve therapeutic action with
a smaller dose and fewer systemic and ocular side effects.
This study focused on the preparation, characterisation
of SLNs, in vitro and in vivo performance of MTA-loaded
SLNs; and some comparison studies were also performed
with reference eye drops.
Materials and methods
Materials
MTA, of 99% purity, was purchased from Hangzhou
Aoyipollen Pharmaceutical Co. Ltd. (Hangzhou, China);
1% w/v brinzolamide eye drops (AZOPTÕ ) were purchased
from Alcon (Puurs, Belgium). Glyceryl palmitostearate
powder (Precirol ATO5Õ ) was a kind gift from Gattefosse
sas Co. Ltd (Saint-Priest, France). Phospholipids (Lipoid
S100) were provided by Lipoid (Ludwigshafen, Germany).
Tween 80 was purchased from JiuJiu Biotech. Co. Ltd.
(Jiangsu, China). Polyethylene glycol 400 (PEG 400) was
supplied by the Dow Chemical Company (Shanghai,
China). HPLC grade methanol was obtained from Jiangsu
A potential new therapeutic system for glaucoma 135
Hanbon Science and Technology Co., Ltd. (Jiangsu, China).
Distilled water was purified by Hitech-K flow Water
Purification System (Hitech Instruments Co. Ltd.,
Shanghai, China). All other chemicals and solvents were
of analytical grade or higher.
Animals
New Zealand albino rabbits of either sex, weighing
2.5–3.0 kg with normotensive eyes, were used in this
study. They were provided by Animal Experimental
Center of Nanjing Medical University. Rabbits were kept
in individual cages with free access to standard food and
water, under controlled environmental conditions
(Ta ¼ 24 C; RH ¼ 60%; 12 : 12 h light : dark cycle). All the
procedures were conducted in accordance with the
Principles of Laboratory Animal Care (NIH publication
no. 92-93, revised in 1985) and approved by the local
ethics committees for animal experimentation.
Quantitative determinations
The quantitative determinations were performed by HPLC
using a LC-10 ATvp pump, a SPD-10Avp UV–Vis
detector (Shimadzu Co., Tokyo, Japan) with a Shim-pack
VP-ODS-2C18 column (5 mm, 4.6  150 mm2; Shimadzu
Co., Tokyo, Japan) at 40 C. The mobile phase was a mixture
of methanol andwater (30 : 70, v/v) with a flow rate at
1.0 mL/min. The detection wavelength was set at 290 nm.
Aliquots of 20 mL of each sample were injected into the
column and MTA could be detected at a retention time
of about 5.0 min. Each sample was analysed in triplicate.
The MTA calibration curve was linear from 1 to 30 mg/mL
(r ¼ 1.0000). The limits of detection and quantisation were
10 and 30 ng, respectively. No interference from the com-
position of the formulation was observed. All samples were
filtered through a 0.45 mm pore size membrane filter to
protect the column.
SLNs preparation
The MTA-loaded SLNs were prepared according to
a modified emulsion–solvent evaporation method
(Ye et al., 2008). In brief, MTA, Precirol ATO5
(lipid component) and phospholipids (emulsifier) were
dissolved in ethanol at 70 C to form an oil phase. The aque-
ous phase (0.5% Tween 80 and 1.0% PEG 400 used as sur-
factant and co-surfactant, w/v) was heated to the same
temperature of oil phase. The oil phase was instilled into
the hot aqueous phase under mechanical stirring at
1200 rpm (RET Control-visc C, IKA, Germany) at 70 C
for dispersion. After removing the organic solvent, the
pre-emulsion was poured into the cold continuous phase
(2–3 C) under stirring at 1000 rpm and SLNs were obtained
after continual stirring for 2 h. The continuous phase con-
tained certain volume of 5.0% (w/v) mannitol, 1.9% boric
 136 R. Li et al.
acid and 0.01% (w/v) benzalkonium bromide, which were
used as isotonicity agent, pH adjustor and bacteriostatic
agent, respectively. The resulting suspensions were steri-
lised by filtration through a membrane with 0.22 mm pore
size and kept at 4 C for use. Blank SLNs were prepared in
a similar way without the drug.
All glassware was sterilised by autoclaving and the entire
procedure mentioned above was done under aseptic
conditions.
SLNs characterisation
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11
Osmotic pressure and pH
Osmotic pressure was determined by the freezing-point
method using a Model FM-9X Osmometer (Instrumental
Factory of Shanghai Medical University, Shanghai, China).
The pH was determined at 25 C using a Model PHS-3C
pH-meter (Shanghai precision & Scientific Instrument
Co., Ltd, China).
Particle size and zeta potential analysis
The mean particle size and polydispersity index (PI) of
the formulations were determined by photocorrelation
spectroscopy with a Zetasizer 3000 HAS (Malvern
For personal use only.
Instruments Ltd, Malvern, UK). Every sample was
appropriately diluted with distilled water and the reading
was carried out at a 90 . The zeta potentials were deter-
mined by a laser Doppler anemometer using the same
instrument. A suitable amount of sample (50–100 mL)
was diluted with 5 mL of filtered water and injected in
the electrophoretic cell of the instrument, the obtained
electrophoretic mobility values were used to calculate the
zeta potentials. In each case, the experiment was carried
out three times.
Particle morphology
The shape and morphology of SLNs were characterised
using a transmission electron microscopy (TEM, JEM-200
CX, JEOL, Tokyo, Japan) with an accelerating voltage
of 100 kV. One drop of the SLNs suspension was deposited
on a carbon-coated copper grid. The excess staining
solution was removed with filter paper in 60 s and
the sample was allowed to dry before examined.
All the data presented were the mean values of at least
three independent samples produced under identical
production conditions.
Entrapment efficiency and drug loading
In this study, the entrapment efficiency (EE) of MTA-
loaded SLNs was determined by an ultrafiltration
method. One millilitre of SLN dispersion was transferred
to the upper chamber of centrifuge tubes fitted with an
ultrafilter (Amicon Ultra 15, MWCO 100 KD, Millipore,
Ireland). The unit was centrifuged at 21 000  g for 10 min
at 4 C using a Sigma-3k30 Centrifuges (Sigma-Aldrich,
St. Louis, MO). The amount of initial drug for assay and
free non-entrapped drug in the aqueous phase after isola-
tion of the system was detected by the HPLC method. The
amount of incorporated drug was calculated by subtracting
the amount of free drug in the aqueous phase from the total
amount of the drug in the suspension. Drug loading (DL)
was calculated as drug analysed in the nanoparticles versus
the total amount of drug inside the particles and the lipid
matrix (Precirol ATO5 and phospholipids) added during
preparation. The EE and DL of MTA in SLNs were calcu-
lated according to the following equations (Souto et al.,
2004; Luo et al., 2006):
Winitial drug  Wfree drug
EE ð%Þ ¼  100%, ð1Þ
Winitial drug
Winitial drug  Wfree drug
DL ð%Þ ¼  100%, ð2Þ
Winitial drug  Wfree drug þ Wlipid
where ‘‘Winitial drug’’ is the amount of drug used for the
assay and ‘‘Wfree drug’’ is the amount of free drug detected
in the aqueous phase after isolation of the suspension.
‘‘Wlipid’’ stands for the weight of lipid matrix
(Precirol ATO5 and phospholipids).
In vitro release studies
The drug release from SLNs was performed using the
dialysis bag method (Ye et al., 2008) and an RC Drug
Dissolution Tester (Tianjin Medical Instrumental Factory,
Tianjin, China). The diffusion medium used was freshly
prepared simulated tear fluid (STF) which was prepared
using NaCl 0.678 g, NaHCO3 0.218 g, CaCl22H2O 0.0084 g,
KCl 0.138 g and water up to 100 g (Hagerstrom et al., 2000).
The dialysis bag (12 000 MWCO, Sigma) could retain nano-
particles and allow the diffusion of free drug into dissolu-
tion media. The bags were soaked in STF for 12 h before
use. The formulation (2 mL) to be studied were pipetted
into the bag and immersed in 100 mL STF, and stirred at
50 rpm in a 35  0.5 C water bath. Aliquots (4 mL) were
withdrawn at each time interval (5, 10, 20, 30, 40, 60, 120,
180, 240, 360 and 480 min) and immediately replaced with
an equal volume of STF to maintain a sink condition (Huo
et al., 2005). Samples were analysed by the HPLC method
as described above.
The cumulative amount of drug (Qn, mg/mL) was plotted
as a function of time (t, min) and calculated based on the
following equation:
X
n1
Qn ¼ C n  V 0 þ Ci  Vi , ð3Þ
i¼1
where Cn stands for the drug concentration of the dissolu-
tion medium at each sampling time, Ci for the drug con-
centration of the ith sample, and V0 and Vi stand for the
volumes of the dissolution medium and the sample,
respectively.

In vivo studies
IOP measurements
This study was of a single-dose crossover design and was
performed on blank solution, MTA solution (0.1%, w/v),
the commercial product (AZOPTÕ , 1% w/v brinzolamide,
as a reference standard), the blank SLNs in addition
to the MTA-loaded SLNs formulations. Sterility of
formulations was achieved by filtration through sterile
0.22 mm pore size pyrogen-free cellulose filters.
The rabbits were acclimatised before the beginning of
the study. An indentation tonometer (YJI, Suzhou Mingren
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11
Medical Apparatus and Instruments Co. Ltd., Suzhou,
China) was used to determine IOP measurements
after instillation of one drop of 0.2% (w/v) lidocaine
hydrochloride as local anaesthetic. Eye drops were instilled
topically into the lower cul-de-sac of the eye and the
eye was manually blinked three times; one eye received
50 mL of the preparation and the other was not treated
as a control. In all cases, the IOP was measured with
the interval of 1 h after the treatment. All the measurements
were done three times at each interval and mean
values were taken. The percentage decrease in IOP was
determined according to the following equation
(Ammer et al., 2009):
For personal use only.
IOP control eye  IOP dosed eye
%Decrease in IOP ¼  100:
IOP control eye
ð4Þ
The experiments were carried out in the same laboratory
by the same investigator using the same instrument. On
any given day, only a single dose was tested on a single
animal, which was washed out at least 2 days between
experiments. The pharmacodynamic parameters taken
into consideration were maximum percentage decrease
in IOP, time for maximum response (Tmax), area under per-
centage decrease in IOP versus time curve (AUC0–8h) and
mean residence time (MRT). These parameters were
calculated using the 3P87/97 software.
Statistical analysis of the results was performed using
one-way analysis of variance (ANOVA) (Palma et al.,
2009). Statistical analysis was computed with the SPSSÕ
software.
Ocular irritation test
The ocular tolerance and potential irritation of the SLN
dispersions were evaluated according to a modified
Draize test (Draize et al., 1944), following single application
of 50 mL of test products to one eye, while the other eye
served as control was treated with physiological saline or
blank SLNs. Observation of the ocular tissue condition was
performed at the 8 h after application. The congestion,
swelling, discharge and redness of the conjunctiva were
graded on a scale from 0 to 3, 0 to 4, 0 to 3 and 0 to 3,
respectively. Irritation and corneal opacity were graded on
a scale from 0 to 4 (Das et al., 2010).
A potential new therapeutic system for glaucoma 137
Statistical analysis
All experiments were performed in replicates for validity of
statistical analysis. Results were expressed as mean  SD.
ANOVA was performed on the data sets generated by the
SPSSÕ software. Differences were considered significant for
p 5 0.05.
Results and discussion
Characterisation of the SLNs
In this study, we have developed an economical, simple
and reproducible method for the preparation of SLNs.
Some essential characteristics of the SLNs were given
in Table 1.
Osmolality and pH
The pH of ophthalmic preparations can vary from 4 to 9.
A pH exceeding this range is irritant and causes a painful
sensation. The osmolality of lacrimal fluid is between 280
and 293 mOsm/L and solutions with an osmolality
lower than 100 mOsm/L or higher than 640 mOsm/L are
considered as irritants (Ammar et al., 2009). Unsuitable
pH value and osmotic pressure can stimulate tear secretion
and accelerate the outflow of therapeutic substances.
In this study, formulations were prepared without
adding any buffer, because in this case the limited buffer-
ing capacity of the tears was able to adjust the pH to phys-
iological levels on administration and differing pH values
could be tolerated (Fialho and da Silva-Cunha, 2004). The
pH values of the prepared formulations were within the
acceptable range. The osmolality of the prepared SLNs
ranged from 274 to 280 mOsm/L (Table 1).
Particle size and zeta potential analysis
Particle size is a crucial parameter for absorption or perme-
ation through the ocular barriers which should not exceed
10 mm (Zimmer and Kreuter, 1995). The mean particle size
of all batches obtained was about 200 nm (Table 1).
All SLNs exhibited slightly negative zeta potential values
(Table 1). This fact could be explained by the shielding
effect of ions (Hþ, Cl, Naþ and CO2 3 ) present in the
particle dispersion, adsorbing to the particle surface and
compensating for charge.
Particle morphology
Transmission electron micrograph of SLNs showed that the
particles had nearly spherical shape (Figure 1).
EE% and DL%
As shown in Table 1, the EE% increased while DL%
decreased with the reduction of MTA amount. This result
was in good agreement with that reported by Liu et al.
(2010). The solubility of MTA in oil phase and the overall
content of lipid matrix were constant. The decreased
amount of MTA could let less number of drug molecules
incorporate into the lipid matrix, while it served the matrix
 138 R. Li et al.

Table 1. Essential physicochemical characteristics of the SLNs investigated in the study.a

Batch of SLNs Particle PI Zeta potential Osmotic pH EE (%) DL (%)

size (nm) (mV) pressure
(mOsm/L)

Blank–SLNs 199.5  2.6 0.226  0.015 8.4  2.1 280 5.6 – –

0.05% MTA–SLNs 191.6  3.8 0.211  0.014 8.1  1.9 278 5.4 63.7  3.3 3.42  0.17
0.03% MTA–SLNs 188.2  2.3 0.188  0.009 10.7  2.5 274 5.5 70.4  3.8 2.29  0.12

Note: aThe results are the means  SD (n ¼ 3).

Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11
For personal use only.

Figure 2. In vitro release profiles of MTA solution and MTA from SLNs
(mean  SD, n ¼ 6).

The lipid might start crystallising first and formed an inner

Figure 1. TEM photomicrograph of SLN dispersions containing MTA.
(Bar ¼ 200 nm).
with more space for the incorporation of MTA thus leading
to the consequence of less amount of MTA escaping into
the external phase, that was why DL% decreased and EE%
increased.
In vitro release studies
The obtained release curves (Figure 2) revealed that
MTA–SLNs exhibited slightly sustained drug release behav-
iour compared with that of MTA solution. For MTA solu-
tion, almost all drugs were released within 3 h. The amount
of drug released was about 92.64% to the medium after 1 h
for the solution, compared to 77.34% for the SLNs. Also,
there was a burst release pattern of SLNs which possibly
could be explained by two reasons.
One possible explanation of the burst release was
mainly because of the free drug dispersed in external
phase or the encapsulated drug concentrated in the outer
shell of the SLNs and/or on the surface of the particles.
Such enrichment might occur during the cooling process.
core, leading to more MTA enriching around the lipid
core and aqueous medium than in the core of the lipid
nanoparticles (Zur Muhlen and Mehnert, 1998).
Another reason was that the large specific surface of the
small particles could increase the initial drug release.
In this formulation of SLNs, the presence of phospholipids
and surfactants provided an additional specific surface area
and smaller particle size (Schubert and Muller-Goymann,
2005). Thus, MTA–SLNs had a large total external surface
and high diffusion coefficient allowing for a more intense
interaction with the medium in which they were dispersed,
resulting in a fast drug release, as was demonstrated
by Wakayima et al. (1981).
In vivo studies
IOP measurements
Table 2 and Figure 3 demonstrated the results of therapeu-
tic efficacy after administration of a single dose of MTA–
SLNs, drug solution and the commercial product. It could
be seen that blank solution and blank SLNs were devoid of
any significant change in IOP. MTA–SLNs showed pro-
nounced decrease in IOP in contrast to drug solution
(p 5 0.05). It was also observed that the mean maximum
percentage decrease in IOP occurred 2 h after instillation
 A potential new therapeutic system for glaucoma 139

Table 2. Pharmacodynamic parameters after administration of MTA solution, SLNs and the commercial product
(mean  SD).

Formula Pharmacodynamic parameters

Maximum Tmax (h) AUC0–8h MRT

percentage
decrease in IOP

0.1% MTA solution 12.63  3.58 c 2  0.45 46.19  5.82 c 3.99  0.08 d
AZOPTÕ 33.09  1.60 a 2  0.55 148.83  3.64 a 4.08  0.15 b
0.03% MTA–SLNs 35.69  1.98 a,d 3  0.20 196.48  4.17 a,c 5.16  0.14 a,c
0.05% MTA–SLNs 36.66  5.96 a,d 4  0.20 186.11  7.21 a,c 5.28  0.06 a,c

Notes: AUC0–8h: area under the percentage decrease in IOPtime curve); Tmax, time required to reach the peak effect,
MRT: mean residence time.
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11

‘‘a’’ and ‘‘b’’ denote significant difference and no significant difference from the group of MTA solution, respectively
(p 4 0.05).
‘‘c’’ and ‘‘d’’ denote significant difference and no significant difference from the group of AZOPTÕ , respectively
(p 5 0.05).
For personal use only.

Figure 3. Percentage decrease in IOP after administration of MTA–SLNs, drug solution and commercial product (mean  SD, n ¼ 6).

of drug solution, or the commercial product compared

Table 3. Data for Draize eye irritation test.
to MTA–SLNs which occurred at 3–4 h. With respect to
Degree of irritationa the duration of drug action, it was evident that the effect
of SLNs continued over 8 h, while those of the drug solution
AZOPTÕ MTA–SLNs
and the commercial product lasted for only 6 and 8 h,
Control Test Blank SLNs Test respectively (Figure 3). This would indicate that
SLNs exhibited a more prolonged effect compared to
Cornea
Corneal opacity 0 2 0 0 drug solution and commercial product.
Iris Table 2 demonstrated some pharmacodynamic param-
Irritation value 0 0 0 1 eters for tested formulations. Concerning the parameter of
Conjunctiva area under the percentage decrease in IOP–time curve
Degree of flare 0 1 0 1
Degree of swelling 0 0 0 0
(AUC0–8h), MTA–SLNs showed higher values compared to
Degree of redness 0 0 0 0 that of drug solution and commercial product (p 5 0.05).
Congestion 0 0 0 0 It can be seen from Table 2 that SLNs had a longer MRT for
Secretion (discharge) 0 2 0 0 percentage decrease in IOP in contrast to drug solution and
commercial product (p 5 0.05). This would also indicate
Notes: aIrritation and corneal opacity were graded on a scale from 0 to 4.
Congestion, flare, swelling, discharge and redness of the conjunctiva were that SLNs exhibited a more prolonged effect compared
graded on a scale from 0 to 3, 0 to 4, 0 to 4, 0 to 3 and 0 to 3, respectively. to drug solution and commercial product.
 140 R. Li et al.
Although the release of SLNs was fast in a burst stage
under the present formulation composition, they could still
enhance the extent of ophthalmic drug absorption
as well as the intensity of drug action. From a therapeutic
standpoint, a burst release profile could be considered as
an advantage that a sufficient amount of MTA rapidly
released from the SLNs to the cornea to exert an initial
therapeutic effect. SLNs’ colloidal carrier properties might
account for this phenomenon. SLNs might be applied in
liquid form like eye drop solutions with extremely small
size, whereupon they could make intimate contact between
the drug and the absorbing tissue, thereby decreasing tear-
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11
ing and drainage of instilled dose, locating drug at its site of
action and increasing the probability of ocular drug
absorption. Then, their interaction with the glycoprotein
of the cornea and conjunctiva could form a pre-corneal
depot resulting in prolonged duration of the bound drug.
Another hypothesis to be further verified was the enhance-
ment of corneal penetration of the drug caused by phos-
pholipids and surfactants presented on the SLNs surface;
they could modulate the interfacial properties of the
carrier, thereby, positively influencing the mucoadhesion
and improved drug permeation.
Ocular irritation test
Ophthalmic irritation is a common drawback in the
For personal use only.
development of ophthalmic drugs and in their clinical
use. Ophthalmic irritation may decrease patient
compliance and even be a reason for patients to stop
their medication. In vivo ocular irritancy towards
AZOPTÕ and MTA–SLNs was determined following a mod-
ified Draize test (Table 3). The in vivo results showed
almost no sign of irritation effects to ocular tissues in
rabbit eyes. The scores for conjunctival swelling and iris
hyperaemia were always zero for both formulations.
Corneal opacity and discharge scores were zero for
SLNs which was better than those of commercial formula-
tion. Therefore, the potential clinical interest of MTA–SLNs
is supported because of the absence of irritant effects
in vivo.
Conclusions
In this preliminary study, the developed MTA–SLNs pro-
vided appropriate particle size with better tolerability, good
IOP-lowering intensity as commercial formulation in lower
doses and longer duration in IOP reduction. It suggested
that SLNs have great potential as a new therapeutic system
for topical administration to treat glaucoma.
In further study, SLNs will be modified by bioadhesive to
alert charge and prolong retention at the corneal site. The
cytotoxicity also should be evaluated during further
experiences.
Declaration of interest
This study was financially supported by the Jiangsu Natural
Science Funds (project no. BK2009420).
References
Ammar HO, Salama HA, Ghorab M, Mahmoud AA. Nanoemulsion as
a potential ophthalmic delivery system for Dorzolamide
Hydrochloride. AAPS Pharm Sci Tech, 2009;10:808–19.
Becker B. Decrease in intraocular pressure in man by a carbonic anhydrase
inhibitor, diamox: A preliminary report. Am J Ophthalmol, 1954;37:13–5.
Chang JN. Apparatus and methods for implanting particulate ocular
implants. 2006. US Patent 2007/0293873 A1, 19 June.
Das S, Suresh PK, Desmukh R. Design of Eudragit RL 100 nanoparticles
by nanoprecipitation method for ocular drug delivery. Nanomedicine:
NBM, 2010;6:318–23.
Draize JH, Woodard G, Calvery HO. Method for the study of irritation and
toxicity of substances applied topically to the skin and mucous mem-
branes. J Pharmacol Exp Ther, 1944;82:377–90.
Fialho SL, da Silva-Cunha A. New vehicle based on a microemulsion for
topical ocular administration of dexamethasone. Clin Exp Ophthalmol,
2004;32:626–32.
Fridriksdottir H, Loftsson T, Stefansson E. Formulation and testing of
methazolamide cyclodextrin eye drop solutions. J Control Release,
1997;44:95–9.
Gupta H, Aqil M, Khar RK, Ali A, Bhatnagar A, Mittal G. Sparfloxacin-
loaded PLGA nanoparticles for sustained ocular drug delivery.
Nanomedicine: NBM, 2010;6:324–33.
Hagerstrom H, Paulsson M, Edsman K. Evaluation of mucoadhesion for
two polyelectrolyte gels in simulated physiological conditions using
a rheological method. Eur J Pharm Sci, 2000;9:301–9.
Hathout RM, Mansour S, Mortada ND, Guinedi AS. Liposomes as an ocular
delivery system for Acetazolamide: In vitro and in vivo studies. AAPS
Pharm Sci Tech, 2007;8:E1–E12.
Huo D, Deng S, Li L, Ji J. Studies on the poly (lactic-co-glycolic) acid micro-
spheres of cisplatin for lung-targeting. Int J Pharm, 2005;89:63–7.
Jonathan CJ, Norman BJ, McDonnell P. 1996. Topical compositions for the
eye comprising a -cyclodextrin derivative and a therapeutic agent.
US Patent 5494901, 27 February.
Kassem MA, Abdel Rahman AA, Ghorab MM, Ahmed MB, Khalil RM.
Nanosuspension as an ophthalmic delivery system for certain glucocor-
ticoid drugs. Int J Pharm, 2007;340:126–33.
Kaur IP, Garg A, Singla AK, Aggarwal D. Vesicular systems in ocular deliv-
ery: An overview. Int J Pharm, 2004;269:1–14.
Kaur IP, Smitha R, Aggarwal D, Kapil M. Acetazolamide: Future perspective
in topical glaucoma therapeutics. Int J Pharm, 2002;248:1–14.
Le Bourlais C, Acar L, Zia H, Sado PA, Needham T, Leverge R. Ophthalmic
drug delivery systems–recent advances. Prog Retin Eye Res,
1998;17:35–58.
Liu DF, Jiang SM, Shen H, Qin S, Liu JJ, Zhang Q, Li R, Xu QW. Diclofenac
sodium-loaded solid lipid nanoparticles prepared by emulsion/solvent
evaporation method. J Nanopart Res, 2010; doi: 10.1007/s11051-010-
9998-y.
Luo YF, Chen DW, Ren LX, Zhao XL, Qin J. Solid lipid nanoparticles
for enhancing vinpocetine’s oral bioavailability. J Control Release,
2006;114:53–9.
Maren TH. Carbonic anhydrase: Chemistry, physiology, and inhibition.
Physiol Rev, 1967;47:595–781.
Muller RH, Mehnert W, Lucks JS, Schwarz C, Zur Muhlen A, Weyhers H,
Freitas C, Rühl D. Solid lipid nanoparticles (SLN): An alternative colloi-
dal carrier system for controlled drug delivery. Eur J Pharm Biopharm,
1995;41:62–9.
Palma SD, Tartara LI, Quinteros D, Allemandi DA, Longhi MR,
Granero GE. An efficient ternary complex of acetazolamide with HP-
ß-CD and TEA for topical ocular administration. J Control Release,
2009;138:24–31.
Pignatello R, Bucolo C, Spedalieri G, Maltese A, Puglisi G. Flurbiprofen-
loaded acrylate polymer nanosuspensions for ophthalmic application.
Biomaterials, 2002;23:3247–55.
Schubert MA, Muller-Goymann CC. Characterisation of surface-modified
solid lipid nanoparticles (SLN): Influence of lecithin and nonionic emul-
sifier. Eur J Pharm Biopharm, 2005;61:77–86.
Sommer A, Tielsch JM, Katz J, Quigley HA, Gottsch JD, Javitt JC,
Martone JF, Royall RM, Witt KA, Ezrine S. Racial differences in the
cause-specific prevalence of blindness in east Baltimore. New Engl J
Med, 1991;325:1412–17.
Souto EB, Wissing SA, Barbosa CM, Muller RH. Development of a con-
trolled release formulation based on SLN and NLC for topical clotrima-
zole delivery. Int J Pharm, 2004;278:71–7.
Sultana Y, Jain R, Aqil M, Ali A. Review of ocular drug delivery. Curr Drug
Deliv, 2006;3:207–17.

Vandamme TF. Microemulsions as ocular drug delivery systems: Recent
developments and future challenges. Prog Retin Eye Res,
2002;21:15–34.
Wakiyama N, Juni K, Nakano M. Preparation and evaluation in vitro of
polylactic acid microspheres containing local anaesthetics. Chem
Pharm Bull, 1981;29:3363–8.
Yasukawa T, Ogura Y, Tabata Y, Kimura H, Wiedemann P, Honda Y. Drug
delivery systems for vitreoretinal diseases. Prog Retin Eye Res,
2004;23:253–81.
Journal of Microencapsulation Downloaded from informahealthcare.com by National Library of Health Sciences on 11/26/11
For personal use only.
View publication stats
A potential new therapeutic system for glaucoma 141
Ye J, Wang Q, Zhou X, Zhang N. Injectable actarit-loaded solid lipid nano-
particles as passive targeting therapeutic agents for rheumatoid arthritis.
Int J Pharm, 2008;352:273–9.
Zimmer A, Kreuter J. Microspheres and nanoparticles used in ocular drug
delivery systems. Adv Drug Deliv Rev, 1995;16:61–73.
Zimmerman TJ. Acetazolamide and methazolamide. Ann Ophthalmol,
1978;10:509–10.
Zur Muhlen A, Mehnert W. Drug release and release mechanism of pred-
nisolone loaded solid lipid nanoparticles. Pharmazie, 1998;53:552–5.