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Drug Dev Ind Pharm, 2014; 40(6): 765773

! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2013.783589

RESEARCH ARTICLE

Liposomal diltiazem HCl as ocular drug delivery system for glaucoma

Mahmoud Mokhtar Ibrahim1,2, Salma A. H. Tawfique1, and Mahmoud M. Mahdy1
1
Department of Pharmaceutics, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt and 2Department of Pharmaceutics, Faculty of Pharmacy,
Najran University, Najran, K.S.A

Abstract Keywords
In this study, unilamellar liposomal vesicles of diltiazem HCl (DH) were prepared using either Cetyl alcohol, cholesterol, liposomes,
reversed phase evaporation (REV) or proliposome methods. Soya phosphatidylcholine (SPC) proliposomes, REV
was used for preparing the liposomes, and the vesicles were rigidified using cholesterol (Chol)
or cetyl alcohol (CA) in different molarities. The major differences in both the entrapment History
efficiency percent (EE%) and drug release were evaluated as a function of the method of
preparation, Chol or CA contents, and charging lipids. Moreover, the morphology of Received 17 December 2012
the vesicles was confirmed by transmission electron microscopy. The effects of Chol or CA Revised 1 March 2013
incorporation into the liposomes were discussed based on thermal analysis. The in vivo Accepted 5 March 2013
evaluation of liposomal DH was assessed using intra-ocular pressure (IOP), reducing effects in Published online 9 April 2013
rabbit eyes. Liposomes prepared via REV exhibited higher EE% and lower release rates when
compared with those prepared from proliposomes. The incorporation of either Chol or CA in
the liposomes enhanced the EE% and decreased the release rates; however, Chol yielded higher
results than CA. In addition, both dicetyl phosphate (DCP; negative charge inducer) and stearyl
amine (SA, positive charge inducer) decreased the EE% and increased the DH release rate. The
in vivo antiglaucoma effects of the liposomes were calculated according to the area above
the IOP/Time curve, the maximum response and the time for the maximum response and
were compared with effects of the DH solution. The results were in the following order:
DH solution5SPC/CA (REV)5SPC/Chol/DCP(REV)5SPC/Chol (proliposomes)5SPC/Chol/
SA(REV)5SPC/Chol (REV).

Introduction number of aqueous compartments6. The location of medication in

the vesicle depends on its physicochemical characteristics and on
Glaucoma is a disease that may progressively damage visibility
the types of lipids used in the preparation of the liposomes7,8.
with a characteristic of a higher level of intra-ocular pressure
Recently, researchers have taken great interest in liposomes as a
(IOP). The average human IOP is 15.5  2.57 mmHg. An IOP
vehicle of choice in ophthalmic drug delivery. Liposomes
equal to or greater than 20.5 mmHg may indicate glaucoma,
conveniently fulfill the need for an ophthalmic drug delivery
whereas an IOP greater than 24 mmHg definitely indicates
system in the form of a drop while localizing and maintaining drug
glaucoma1. The treatment of glaucoma requires prolonged
activity at the site of action9. Liposomes can be easily administered
therapy involving eye medications, and thus, liposomes may be
in liquid dosage forms, such as eye drops, that yield high patient
a useful carrier system for antiglaucoma drugs. diltiazem HCl
compliance. A major advantage of liposomal use is prolonged and
(DH) is a calcium channel blocker and can lower the IOP by
controlled action of the drug at the corneal surface. One of the
reducing the secretion of the aqueous humor2. Mito et al.3
proven mechanisms for this action involves the vesicles preventing
reported that calcium may activate different ion transport systems,
metabolism of the drug from the enzymes present at the tear/
such as the Na-K-ATPase and the Na-K-Cl co-transport,
corneal epithelial surface via the barrier effect9. Moreover, reports
both of which play a key role in the secretion of the aqueous
indicate that liposomal encapsulation of drugs might improve
humor. Moreover, calcium has been observed to modulate the gap
corneal drug absorption for both hydrophilic and lipophilic
junctions located between the pigmented and non-pigmented
drugs10,11. Important factors such as surface charge, lipid compos-
ciliary epithelial cells that are responsible for the functional
ition and bilayer rigidity can alter the behavior of an entrapped drug
coupling of these two layers in the aqueous humor formation4,5.
and its affinity for the corneal surface of the eye12,13. The charge
Liposomes are colloidal particulates that are nearly spherical
inducer additionally confers high physical stability to the liposomal
microscopic vesicles and are composed of one or more lipid
vesicles by inhibiting their fusion and by giving them a high zeta
bilayers arranged in a concentric fashion enclosing an equal
potential. The most commonly used positively charge inducer is
stearyl amine (SA), and the most commonly used negatively charge
Address for correspondence: Mahmoud Mokhtar Ibrahim, Ph.D., Depart- inducer is dicetyl phosphate (DCP).
ment of Pharmaceutics, Faculty of Pharmacy, Zagazig University, Proliposomes are an anhydrous form of concentrated compact
Zagazig, Egypt. Tel: +966537028103. E-mail: mahmoktar@yahoo.com liposomal vesicles that are easily prepared by first heating the
766 M. Mokhtar Ibrahim et al. Drug Dev Ind Pharm, 2014; 40(6): 765773

phospholipids to melt with other additives and then re-cooling the water bath was maintained at 30  C until a smooth suspension
them to room temperature. Proliposomes are gel-like and can be of unilamellar liposomes was formed. The amount of organic
further used in the preparation of liposomes by simple hydra- solvent used was three times the quantity of the aqueous phase for
tion14. Proliposomes are more stable precursors for liposomes and optimum drug entrapment. To achieve a homogeneous population
can be reconstituted directly before use by the patient15. However, of unilamellar liposomes, the preparations were sonicated again
the Reverse-phase evaporation (REV) method of liposomal with a probe type sonicator for approximately 15 min at 20  C in
preparation is complicated and requires well-trained professionals cycles of 3-minute sonication followed by a 2 min pause to
to produce liposomes. This study investigates the differences prevent heating of the liposomal suspension and to minimize
between both methods of preparing liposomes encapsulating DH. phospholipid destruction. To obtain charged liposomes, 5 mol%
The use of a fatty alcohol is essential to rigidify the vesicles and SA or DCP was used to impart positive or negative charges to the
reduce drug leakage, thereby increasing the entrapment efficiency liposomes of SPC/Chol or CA at a 1:1 molar ratio, respectively.
percent (EE%) of the drug and reducing its release rate. Both Chol The charging lipids with phospholipids and the fatty alcohol were
and CA were tested in this study as the major components of dissolved in diethyl ether with minute quantities of chloroform
liposomal vesicles, and their effects on EE% and drug release as a co-solvent. All the formulations are listed in Table 1.
were investigated. The in vivo performance of the prepared
liposomes with various compositions and charges may demon- Preparation of liposome by proliposome method
strate that liposomal DH is a promising delivery system for the
Proliposomes were prepared according to the method described
treatment of glaucoma.
by Perrett et al. and Rengel et al. with some modifications18,19.
Accurately weighed amounts of phospholipid were mixed in glass
Materials and methods
vials with appropriate amounts of Chol or CA in molar ratios of
Materials 1:0, 9:1, 7:3 and 1:1 (Table 2). Propylene glycol (approximately
200 mg) was added to SPC or SPC/Chol or CA mixtures, and
DH was a gift from the Egyptian Pharmaceutical Industrial
then, the vials were tightly sealed and warmed in a water bath
Company (E.P.I.Co), Egypt. The Epikuron 200 phospholipids
(5560  C) for approximately 10 minutes with stirring until the
(492% soya phosphatidylcholine [SPC]) were a gift from Lucas
complete dissolution of Chol or CA. DH was accurately weighed
Meyer, Hamburg, Germany. Cholesterol (Chol; 99%) and Cetyl
and added to the dissolved SPC/Chol or CA hot mixture. To each
alcohol (CA) were purchased from Sigma Chemical Co., St.
of the formed solutions, approximately 0.16 ml of hot isotonic
Louis, MO. SA and DCP were obtained from Fluka Chemical Co.
phosphate buffer solution (5560  C) was added while warming
(Seelze, Germany). All other chemicals and solvents were
the solution in a water bath for 35 min until a clear or translucent
analytical grade and obtained from the El-Nasr Company for
solution was produced. The lipid/drug mixtures will form
pharmaceutical chemicals, Cairo, Egypt.
proliposomal gels if allowed to cool down at room temperature.
The proliposome mixture was converted into a liposome suspen-
Preparation of liposomes
sion with the addition of approximately 7 ml of warmed isotonic
Reverse-phase evaporation method phosphate buffer (pH 6.8) to each vial and upon shaking for
10 min at room temperature. This liposomal suspension was then
Unilamellar liposomes were prepared using the REV method
described by Mehanna et al. and Nekhil et al., with some
modifications16,17. The required amount of the phospholipid with Table 2. Formulation design of DH liposomes prepared by proliposomal
or without Chol or CA in molar ratios of 1:0, 9:1, 7:3 and 1:1 was method.
accurately weighed and transferred to a 50 ml round bottom flask.
The contents were then dissolved using diethyl ether. The aqueous SPC:Chol SPC:CA Propylene Isotonic
Formulation molar ratio molar ratio DH glycol buffer
phase of DH (40 mg/ml) was prepared using a isotonic phosphate code % % (mg/ml) (mg) (ml)
buffer (pH 6.8) as a vehicle and added to the lipid mixture in an
organic solvent. A w/o emulsion was subsequently formed by the F12 100:0 40 200 0.16
high-energy transfer provided by the ultrasonic probe-type F13 90:10 40 200 0.16
F14 70:30 40 200 0.16
sonicator (Probe ultrasonic processor, Model GE 50, Serial F15 50:50 40 200 0.16
17976 c, Power 50 Watts 50/60 Hz, Frequency 20 KHz, Hamburg, F16 90:10 40 200 0.16
Germany) for 34 min. The organic solvent was then removed F17 70:30 40 200 0.16
from the emulsion using a Buchi Rotary Evaporator Cole-Parmer F18 50:50 40 200 0.16
T-1602-21 (Taito-ku, Tokyo, Japan) under reduced pressure, and

Table 1. Formulation design of DH prepared by REV method.

Formulation SPC:Chol SPC:CA SA DCP DH Diethyl ether Isotonic buffer

code molar ratio % molar ratio % molar % molar % (mg/ml) (ml) (ml)
F1 100:0 40 10 3
F2 90:10 40 10 3
F3 70:30 40 10 3
F4 50:50 40 10 3
F5 90:10 40 10 3
F6 70:30 40 10 3
F7 50:50 40 10 3
F8 47.5:47.5 5 40 10 3
F9 47.5:47.5 5 40 10 3
F10 47.5:47.5 5 40 10 3
F11 47.5:47.5 5 40 10 3
DOI: 10.3109/03639045.2013.783589
allowed to sonicate in a probe-type sonicator for approximately
15 min at 20  C in cycles of 3 min sonication followed by a 2 min
pause to prevent heating of the liposomal suspension and to
minimize phospholipid destruction.
The liposomes prepared either by REV or the proliposome
method were left to hydrate for 1 h at 2  C. The liposomes were
formulated to yield a final lipid concentrations of 60 mmol/ml, and
the final volume was adjusted to 10 ml by the same buffer to all
the liposomal formulations. All steps were performed under
aseptic production procedures20.
Transmission electron microscopic examination of
liposomes
The vesicular structures were examined using a JEOL JEM-1010
electron microscope (JEOL Korea, Seoul, Korea). The samples
were prepared using a carbon coated grid and were negatively
stained using uranyl acetate.
Thermal analysis
The thermal properties of various liposomal formulations were
studied using simultaneous differential thermal analysis (TA-50
thermal analyzer, Shimadzu, Kyoto, Japan). The samples were
heated from 0  C up to 400  C at a rate of 6  C/min.
Determination of EE% of DH into liposomes
The EE% of DH in liposomes was determined using the dialysis
method21. The unentrapped free drug was removed by placing
1 ml of the liposomal suspension into a glass tube that was open
on one end and to which a cellophane membrane was attached at
the other. The glass tube was suspended in a 100 ml isotonic
phosphate buffer solution (pH 6.8), which was agitated in a shaker
water bath (Servewell Instruments and Equipment, Pvt. Ltd.,
Bangalore, India) at a speed of 100 rpm and maintained at
ambient temperature. Every hour, the entire medium (100 ml) was
replaced with fresh medium until the absorbance reached a
constant reading, indicating that no drug was available in an
unentrapped form. The drug content was determined using a UV-
Vis spectrophotometer (Shimadzu-UV-1201) at 236 nm with an
isotonic phosphate buffer as a blank. The amount of entrapped
drug was determined by subtracting the amount of the
unentrapped drug from the total drug incorporated22. The EE%
was calculated according to the following equation:
% Encapsulation efficiency
amount of drug encapsulated
 100
total amount of drug
In vitro release of DH from different liposomal
formulations
The in vitro release of DH from different liposomal preparations
was determined using a simple dialysis method23. 1 ml of the
liposomal formulation was simply placed in a hollow cylinder
glass tube that was previously covered on one end with a semi-
permeable cellophane membrane (which was previously soaked in
an isotonic phosphate buffer (pH 6.8)) and served as a donor
compartment. The glass tube was placed in a beaker containing
100 ml of a receptor compartment (isotonic phosphate buffer pH
6.8). The temperature of the receptor medium was maintained at
37  0.1  C and the medium was agitated at a speed of 100 rpm
using a shaker water bath. Approximately 3 ml samples were
withdrawn at specified time intervals of 15, 30, 60, 120, 180, 240,
300 and 360 min and replaced with equal volumes of fresh
isotonic buffer solution (pH 6.8) to maintain a constant volume for
Drug delivery systems 767
the receiving medium during the experiment. The collected
samples were analyzed spectrophotometrically at 236 nm using an
isotonic phosphate buffer (pH 6.8) as a blank.
In vivo study of DH as anti-ocular pressure liposome
Albino rabbits (22.5 Kg) were used for this experiment. The
rabbits were obtained from the Faculty of Veterinary Medicine,
Zagazig University, Animal Breeding Center, Egypt and were
treated according to the standards of the Ethical Committee of
Animal Handling at Zagazig University ECAHZU. DH lowers
the IOP of the rabbits eye; therefore, bioavailability was assessed
by measuring the IOP using a Schiotz tonometer (Riester,
Germany). Isotonic benoxinate HCl (0.4%) solution was dropped
into the rabbits eyes to anaesthetize the cornea. One drop (50 ml) of
ophthalmic DH solutions or liposomal formulations were instilled
into the right eye, where the non-medicated formulations (serving
as a control) were instilled in the left eye. During instillation, the
upper eyelid was slightly raised and the lower eyelid was gently
pulled away from the globe. At certain time intervals, the IOP was
measured before and after ocular administration of both the control
and test formulations. The mean of three consecutive tonometric
measurements was calculated for each sample. Each preparation
was tested in a group of four rabbits. The measurements were made
for each formulation until the IOP returned to the basal line to
determine the following parameters: (a) the area above the curve
measured using the trapezoidal method and (b) the maximum
response and time of maximum response24.
Statistical analysis
The results were statistically evaluated using a one-way ANOVA
test followed by a LSD test at a level of significance of p50.001.
The data were reported as a mean  SD. Each experiment was
repeated four times (n 4).
Results and discussion
Physical properties of liposomal vesicles
The DH liposomes were examined using transmission electron
microscopy using the negative staining method. All the liposomal
vesicles appeared spherical or oval in shape with the average size
varying depending on the method used and the incorporated
components (from the images, it is clear that the size ranged from
30 to 500 nm). The liposomes containing Chol were observed to
be larger than the CA-containing liposomes (Figure 1). In
addition, the proliposome method produced vesicles of larger
sizes compared with the REV method. The use of sonication in
both methods of preparation is thought to yield a smaller size of
the prepared liposomes. A small particle size of the eye
preparations is essential to avoid irritation to the eye. In addition
to affecting bioavailability, the size of the particles plays an
important role in the irritation potential of the formulation; hence,
it is recommended that particles of an ophthalmic solution be less
than 10 m to minimize irritation to the eye25.
Thermal analysis
Figure 2(a) reveals a single sharp endothermic peak at 207.55  C
and another broad one at 245.50  C for the thermogram of DH
alone. The sharp peak corresponds to the melting point of the
drug while the broad one corresponds to its decomposition26. In
addition, a strong, sharp endothermic peak of Chol appeared at
147.81  C (Figure 2b). However, CA produced a sharp endother-
mic peak at 48.78  C (Figure 2c), and SPC produced a weak and
broad peak at 232.38  C, which corresponds to its decomposition
(Figure 2d). In addition, Figure 2(e and f) and (g and h)
768 M. Mokhtar Ibrahim et al.
Figure 1. Transmission electron microscope photomicrograph of liposomes by different methods of preparation. Magnification power 100 000.
demonstrate the effect of incorporation of both Chol and CA into
liposomal vesicles at molar ratios of 9:1 and 1:1 for SPC/Chol and
SPC/CA, respectively. The results reveal the complete disappear-
ance of the DH peak for all the liposomal formulations. In
addition, the decomposition peak of phoshatidylcholine was
abolished. The appearance of weak endothermic peaks at
100  C indicates the presence of water in the liposomal compos-
ition. Moreover, the peaks of Chol and CA disappeared, which
indicates that both Chol and CA could abolish the gel to liquid
transition temperature of the liposome27,28, which suggests less
leaky and more stable liposomal vesicles. In addition, these results
demonstrate the presence of DH in an amorphous rather than
crystalline form.
Encapsulation efficiency of DH into liposomal vesicles
The encapsulation efficiency of liposomes is the capability of the
vesicles to retain drug molecules in their aqueous core or in their
bilayer membranes29. The preparation method and the hydro-
philic/lipophilic properties of the drug are among the factors that
affect drug encapsulation in liposomes. Table 3 lists the EE% of
various liposomal formulations for DH with SPC/Chol or CA in
1:0, 9:1, 7:3 and 1:1 molar ratios. The results indicate that all the
liposomal formulations prepared by REV produced higher
encapsulation efficiencies for DH than those prepared using the
proliposome method. It is possible that the relatively higher
encapsulation efficiency of REV compared with the proliposome
method could be related to liposomes being prepared using the
Drug Dev Ind Pharm, 2014; 40(6): 765773
REV method producing large unilamellar vesicles. In addition,
because of the larger aqueous fluid volume of the resulting
vesicles, these types of vesicles are thought to exhibit higher
encapsulation efficiency for hydrophilic drugs16,30. The sonic-
ation process reduced the size of the large unilamellar vesicles
however, the entrapped aqueous compartment could be divided
between the newly formed vesicles due to sonication, and only a
small volume could escape to the external compartment. This
explanation may account for the higher EE% recorded for DH in
liposomes prepared using REV compared with the proliposome
method. The lower EE% of DH when using proliposome method
may be attributed to the increased surface of dry lipids in the
proliposomes while maintaining the low aqueous volume. Hence,
this method is thought to be ideal for preparations where the
material to be entrapped is incorporated into a lipid membrane.
Thus, the method produced lower entrapment compared with the
REV method with DH, which has high aqueous solubility and
tends to escape to the aqueous compartment outside the liposomal
vesicles.
The incorporation of Chol or CA with phospholipids led to an
increase in EE% of DH into liposomal vesicles either prepared by
REV or proliposomal methods for all molar ratios used and
attained a maximum value at SPC/Chol or SPC/CA at a 1:1 molar
ratio. The incorporation of Chol into the bilayers of the liposomes
is known to affect vesicle stability and permeability31,32. Chol was
observed to induce the surface expansion of the phospholipid
molecules, resulting from the reorientation of phospholipid head
groups, which then require access to a larger area of water to
DOI: 10.3109/03639045.2013.783589 Drug delivery systems 769
35
physiological temperatures . As a result, an increase in the
transition temperature of Chol leads to a decrease in the fluidity of
the chains, which in turn decreases the permeability of the bilayer
lipid membranes and leads to the formation of less leaky vesicles
than CA. In addition, the effects of charge-inducing agents on the
EE% of DH in liposomal vesicles of SPC and Chol or CA in a 1:1
molar ratio and prepared using the REV method were examined.
The incorporation of either negatively charged (DCP) or posi-
tively charged (SA) decreased the EE% of DH into vesicles more
than neutral liposomes. The EE% was approximately
55.18%  0.14 and 51.14%  0.67 for SA and DCP containing
liposomes of SPC/CA, respectively, where neutral liposomes of
SPC/CA yielded 61%  0.63 DH entrapment. In addition, the
EE% was 55.29%  0.46, 54.75%  0.93 and 69%  0.05 for SA,
DCP and a neutral liposome prepared from SPC/Chol, respect-
ively. The reduction in the EE% of DH upon the addition of SA
may be related to the slight electrostatic repulsive force occurring
between the partial positive center on the drug molecules acquired
from the hydration medium and SA. However, DCP carries
an opposite charge to that of the DH molecules, and its use in
place of SA also decreased the EE% of the drug. This result could
be explained by the electrostatic induced chain tilt and the
subsequent changes in the lateral packing of the bilayers caused
by the effect of the charge-inducing agents, which may affect the
internal aqueous volume of the vesicles and result in a decreasing
EE% of DH36.

In vitro release study (pH 6.8)

In vitro release tests are generally performed to predict how a
delivery system might work in ideal situations, which may
Figure 2. Thermal analysis, (a) DH alone, (b) Chol alone, (c) CA alone, provide some indications of its performance. Hence, a compara-
(d) SPC alone, (e) SPC/Chol (9:1), (f) SPC/Chol (1:1), (g) SPC/CA (9:1), tive drug release study was performed using the drug solutions
(h) SPC/CA (1:1). and various liposomal formulations and is represented in Figure 3.
As a general observation, all the liposomal formulations
decreased the percent DH released when compared with the
solution form that released approximately 90% of DH after three
Table 3. The effect of method of preparation as well as the Chol or CA hours. After 6 h, the percentage DH released from the liposomal
concentrations on the EE% of DH in various liposomal formulations. vesicles prepared from SPC/Chol in 1:0, 9:1, 7:3 and 1:1 molar
ratios using the REV method was 42.99%  0.42, 19.85%  0.93,
EE% of DH 14.75%  0.09 and 10.96%  0.73, respectively. Using the proli-
Lipid composition (mole ratio) REV Proliposome method posome method, these values were 56.84%  2.55, 34.26%  2.22,
22.87%  0.39 and 15.74%  0.12, respectively. The percent
SPC/Chol or CA (1:0) 48  0.62 27  0.15
SPC/Chol (9:1) 57  0.38 35  1.07
DH released from the liposomal vesicles prepared from
SPC/Chol (7:3) 67  0.28 38  0.55 SPC/CA with the same molar ratios of 1:0, 9:1, 7:3 and 1:1
SPC/Chol (1:1) 69  0.05 41  0.38 SPC:CA was 42.99%  0.42, 40.93%  0.44, 33.99%  3.58
SPC/CA (9:1) 50  1.17 29  0.36 and 30.75%  1.67, respectively, using the REV method
SPC/CA (7:3) 56  0.20 35  0.89 and 56.84%  2.55, 51.88%  0.66, 46.99%  0.57 and
SPC/CA (1:1) 61  0.63 40  0.11 42.66%  3.66, respectively, using the proliposome method after
6 h (Figure 3). From these results, it was observed that the release
of DH from liposomes was high if the liposomes were prepared
become fully hydrated17,33,34. The same results were observed using the proliposomal method. The REV method produced lower
with CA when incorporated into liposomal vesicles. A fatty DH release rates, which may account for the produced unilamellar
alcohol, such as CA, can affect the membrane fluidity. In addition, vesicle with a large internal aqueous core that can entrap a large
a fatty alcohol can interact by H bonding with the phospholipid in amount of a water-soluble drug in its internal core and hence
a similar manner to Chol, thus yielding easy and stable bilayer control its release from its bilayer16,30. The proliposome method
formation and high encapsulation efficiency28. By comparing the was devised to increase the surface of the lipid while maintaining
EE% of DH into liposomal preparations prepared using Chol or a low internal aqueous volume to entrap a smaller amount of the
CA, it was observed that Chol has a more significant effect than hydrophilic drug and yield higher release rates for DH. In
CA in increasing the percent DH entrapped. This result may be addition, by referring to the methods, DH was solubilized in the
due to the lower transition temperature of CA (48.78  C) lipids with the aid of the alcohol; hence, it is believed that the
compared with Chol (147.81  C). Lipids of Tc greater than drug was packed in the lipid bilayer structure after hydration.
37  C make the liposome bilayer membrane more rigid at the This fact could account for the decreased drug entrapment after
physiological temperature and less leaky. In contrast, liposomes hydration, where the drug left the bilayer for the external water
composed of low-Tc lipids (Tc537  C) are more susceptible to the compartment, which may create porous type vesicles, where
leakage of drugs encapsulated to the external aqueous phase at at 37  C the drug leaves the vesicles to the sink more rapidly.
770 M. Mokhtar Ibrahim et al.
Figure 3. In vitro release of DH from liposomes prepared by different methods. (a) liposomes of SPC/Chol (REV), (b) liposomes of SPC/CA (REV),
(c) liposomes of SPC/Chol (proliposome method) and (d) liposomes of SPC/CA (proliposome method). Total lipid was adjusted to 60 mmol/ml. Each
result is the mean  S.D. (n 3).
It has also been previously reported that when the encapsulation
efficiency of the drug increases, a slight decrease in the drug
release rate is observed37. In addition, the incorporation of Chol
or CA into the bilayer membranes of various liposomal vesicles
greatly reduced the release rates of the drug, and as the amount of
incorporated Chol or CA increased, the release of the drug
decreased. This result may be attributed to the addition of Chol to
liposome decreasing the fluidity of the lipid bilayer and improv-
ing the stability of the bilayer membrane, which becomes less
leaky and results in the inhibition of the release of drug molecules
from the liposomes3840. CA produced the same effect as Chol by
decreasing the DH release rate; this result can be explained by the
effect of a fatty alcohol, such as Chol, on the membrane fluidity,
thus offering stable bilayer formation as mentioned previously28.
In comparing the release profile of liposomes prepared from Chol
and CA, it is observed that Chol has more effects in decreasing
DH release than CA, which may be due to the lower transition
temperature of CA as discussed before. Another reason may be
the shorter CA carbon chain (C16) than that of Chol (C27), which
may lead to the formation of more leaky vesicles. However, the
incorporation of both fatty alcohols resulted in decreasing DH
release when compared with liposomal vesicles prepared using
pure phospholipids. The release of DH from neutral, negatively
charged and positively charged liposomes prepared via the REV
method in a 1:1 molar ratio revealed that the percentage of DH
released after 6 h from neutral liposomes containing Chol or CA
was 10.96% and 30.75%, respectively. However, positively
Drug Dev Ind Pharm, 2014; 40(6): 765773
charged liposomes produced 13.22% (SPC/Chol) and 34.81%
(SPC/CA), and negatively charged liposomes produced 14.99%
(SPC/Chol) and 42.46% (SPC/CA), respectively (Figure 4). It was
observed that both SA and DCP exhibited higher DH release rates
compared with the neutral vesicles of the same lipid composition.
This result may be due to the electrostatic induced chain tilt and
the subsequent changes in the lateral packing of the bilayers
caused by the effect of the charge-inducing agents.
In vivo study of DH incorporated into different liposomal
formulations
For the in vivo performance of DH using different liposomal
formulations as drug delivery systems, the drug activity was
evaluated and compared with the solution forms. The drug
activity was investigated based on the IOP reducing effects
produced by the Ca-channel blocking activity of the drug.
Liposomes encapsulating DH (16 mg/ml) and prepared from
either SPC:Chol or SPC:CA in a 1:1 molar ratio and a 60 mmol/ml
total lipid concentration were compared based on the IOP
decreasing effect of the drug. Moreover, the effect of charged
lipid incorporation in SPC/Chol liposomal vesicles either with
positive charge or negative charge was also evaluated. All the
formulations were prepared using the REV method in addition to
liposomes of SPC/Chol being prepared using the proliposomal
method. All the formulations were selected for the in vivo study
according to the highest in vitro EE% results and slower DH
DOI: 10.3109/03639045.2013.783589 Drug delivery systems 771

Figure 4. Release of DH from neutral, positively and negatively charged liposomes. (a) liposomes (REV) of SPC/Chol (1:1 molar ratio), (b) liposomes
(REV) of SPC/CA. Total lipid concentration was 60 mmol/ml total lipid. Each result is the mean  S.D. (n 3).

Table 4. Values for area above the IOP/time curve, maximum response, contribute to the enhancement of the bioavailability of drugs.
and the time of maximum response in solution and in different liposomal Moreover, a significant increase in the maximum response of DH
formulations.
could be achieved by dispersion of liposomal phospholipids.
From these results, it should be noted that all the liposomal
Parameters of activity
formulations of the drug produced a greater effect on the
Area above Maximum Time of parameters of drug action compared with the solution form.
the curve response maximum SPC/Chol prepared by the REV method yielded the highest
Formulations (mmHg.h) (mmHg)* response (h)
enhancement in the parameter of DH activity compared with the
Solution 14.43 4.2 1 same formula prepared using the proliposomal method and also
SPC/Chol (REV) 54.59 6.67 5 with the all the other used formulations. This result could be due
SPC/Chol/SA (REV) 48.44 6.29 4 to a higher encapsulation efficiency; however, this formulation
SPC/Chol (proliposomal) 46.87 6.47 4
also produced the lowest release rate, which could lead to a larger
SPC/Chol/DCP (REV) 39.59 6.47 4
SPC/CA (REV) 30.42 5.7 3 area above the curve and increased duration of action. Moreover,
all the formulations containing Chol produced greater activity
*The difference between the maximum IOP of solution or liposomal compared with those containing CA. These results can be
formulation and control. explained on the basis that Chol incorporation into the phospho-
lipid bilayers of liposomes strongly controls drug release and
increases the fluidity of the stratum corneum, which would help in
release rates. The area above the IOP/time curve, the maximum providing higher drug penetration into ocular tissues4143.
response and the time for maximum response were the parameters Although CA can affect the membrane fluidity in a similar
considered in evaluating the formulation activity (Table 4). For all manner to Chol, it produced a lower encapsulation efficiency than
the liposomal formulations, instillation of either an isotonic the formulations incorporating Chol. This lower activity result
phosphate buffer or empty liposomes (as negative controls) may be caused by the lower transition temperature of CA
resulted in no effect on the IOP measurements. The time courses compared with Chol, which results in a higher release rate and
of the IOP of the rabbits eye after instillation of the drug in smaller area of the IOP/time curve. Concerning the effect of a
solution or liposomal forms are presented in Figure 5. The area charged lipid, it was observed that incorporation of charge
above the IOP/time curve for each formulation was calculated, resulted in lower activity than SPC/Chol prepared using the REV
and the results are presented in Table 4. Statistical analysis of the method. This finding could be due to the excess drug release from
data revealed highly significant differences (p50.001) between the charged vesicles, as previously discussed, and the subsequent
the solution and different liposomal formulations. The area above washout mechanism by the eye, which could reduce the drug
the IOP/time curve, and hence the bioavailability of DH, can be effects. At the same time, the SA-containing vesicles produced
arranged in the following ascending order: DH solution5SPC/CA better IOP reducing activities than the DCP-containing liposomes.
(REV method)5 SPC/Chol/DCP (REV method)5SPC/Chol This result could be caused by the positively charged liposomes
(proliposome method)5SPC/Chol/SA (REV method)5SPC/ being of opposite charge compared with the corneal surface and
Chol (REV method). Concerning the duration of the IOP reducing their good corneal adhesion and permeability, which would help
effect of DH formulated either in solution or in liposomal forms, it in providing higher drug penetration into ocular tissues44.
was observed that all the liposomal formulations could prolong
the duration of action to 12 h, whereas the action of the drug was
Conclusions
terminated completely only after 4 h from application of the
solution form. All the liposomal formulations have been In summary, all the liposomal formulations prepared with REV
demonstrated to contribute significantly (p50.001) to the could yield higher EE% and lower release rates than those
prolongation of the drug effect. These results clearly indicate prepared using the proliposomal method. The EE% and in vitro
that the use of liposomes as controlled release vehicles could release rates of DH from liposomes were greatly affected by the
772 M. Mokhtar Ibrahim et al. Drug Dev Ind Pharm, 2014; 40(6): 765773

Figure 5. The IOP (mmHg) of rabbits eye after instillation of 16 mg/ml DH in solution form and in different liposomal formulations. The values in the
figures represent the mean  S.D. (n 4 rabbits).

formulation processing variables such as the method of prepar-
ation, Chol or CA contents, and charged lipids. Moreover, Chol
produced better results in increasing the EE% and decreasing the
release rates of DH compared with CA. Incorporation of Chol or
CA increased the stability of the liposomal vesicles by increasing
the EE% (decreasing drug leakage) and decreasing the release
rates of DH. The in vivo studies proved that the liposomal
formulations have enhanced the DH IOP reducing activity when
compared with the solution form of the same concentration.
Finally, the results indicated that the liposomes containing SPC/
Chol in a 1:1 molar ratio and prepared via the REV method are
the most stable among the tested formulations.
DOI: 10.3109/03639045.2013.783589
Acknowledgements
The authors would like to express their heartfelt thanks to the head of the
Department of Histology, Faculty of Medicine, Zagazig University for his
technical support in transmission electron microscopy. Also, we would
like to thank Dr Walid Barakat in the Department of Pharmacology,
Faculty of Pharmacy, Zagazig University, for providing his support in
measuring the rabbits IOP.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
References
1. Chiang CH. Ocular drug delivery systems of antiglaucoma agents. J
Med Sci 1991;12:15770.
2. Santafe J, Martinez de Ibarreta MJ, Segarra J, Melena J. A long
lasting hypertensive effect of topical diltiazem on the intra-ocular
pressure in conscious rabbits. Naunyn-Schmiedebergs Arch Pharm
1997;355:64550.
3. Mito T, Delamere NA, Coca-Prados M. Calcium dependent regu-
lation of action transport in cultured human non pig-mented ciliary
epithelial cells. Am J Physiol 1993;264:51926.
4. Morrison JC, Freddo TF. Anatomy, microcirculation, and ultrastruc-
ture of the ciliary body. In: Ritch R, Shields MB, Krupin T, eds. The
glaucomas. 2nd ed. St. Louis (MO): Year book Inc.; 1996:12538.
5. Edelman JL, Sachs G, Adorante JS. Ion transport asymmetry and
functional coupling in bovine pigmented and nonpig-mented ciliary
epithelial cells. Am J Physiol 1994;266:121021.
6. Bangham AD, Standish MM, Watkins JC. Diffusion of univalent
ions across the lamellae of swollen phospholipids. J Mol Biol 1965;
13:23852.
7. Gregoriadis G. The carrier potential of liposomes in biology and
medicine. New Engl J Med 1976;295:70410.
8. Fendler JH, Romero A. Liposomes as drug carriers. Life Sci 1977;
20:110920.
9. Achouri D, Alhanout K, Piccerelle P, Andrieu V. Recent advances in
ocular drug delivery. Drug Dev Indus Pharm 2012;16:119.
10. Singh K, Mezei M. Liposomal ophthalmic drug delivery system
II.Dihydrostreptomycin sulfate. Int J Pharm 1983;16:33944.
11. Meisner D, Pringle J, Mezei M. Liposomal ophthalmic drug delivery
III. Pharmacodynamics and biodisposition studies of atropine. Int J
Pharm 1989;55:10513.
12. Li VHK, Lee VHL, Robinson JR. Influence of drug properties and
routes of administration on the design of sustained and controlled
release system. In: Robinson JR, Lee VHL, eds. Controlled drug
delivery. 2nd ed. New York: Marcel Dekker; 1987:361.
13. Abd El-Motaleb ME. Performance of propranolol hydrochloride in
certain ophthalmic formulation [M.Sc. Pharm. thesis]. Egypt:
Faculty of Pharmacy, Assiut University; 1991.
14. Payne NL, Browning I, Hynes CA. Characterization of prolipo-
somes. J Pharmceut Sci 1986;75:3303.
15. Wang S, Ye T, Yang B, et al. 7-Ethyl-10-hydroxycamptothecin
proliposomes with a novel preparation method: optimized formula-
tion, characterization and in-vivo evaluation. Drug Dev Indus Pharm
2013;39:393401.
16. Mehanna MM, Elmaradny HA, Samaha MW. Mucoadhesive
liposomes as ocular delivery system: physical, microbiological,
and in vivo assessment. Drug Dev Indus Pharm 2010;36:10818.
17. Dhoot NO, Wheatley MA. Microencapsulated liposomes in
controlled drug delivery: strategies to modulate drug release and
eliminate the burst effect. J Pharm Sci 2003;92:67989.
18. Perrett S, Golding M, Williams WP. A simple method for the
preparation of liposomes for pharmaceutical applications: charac-
terization of liposomes. J Pharm Pharmacol 1991;43:15461.
19. Rengel RG, Barisic K, Pavelic Z, et al. High efficiency entrapment
of superoxide dismutase into mucoadhesive chiston-coated lipo-
some. Euro J Pharm Sci 2002;15:4418.
20. Hathout RM, Mansour S, Mortada ND, Guinedi AS. Liposomes as
an ocular delivery system for acetazolamide: in vitro and in vivo
studies. AAPS PharmSciTech 2007;8:113.
Drug delivery systems 773
21. Udupa N, Chandraprakash KS, Umadevi P, Pillai GK. Formulation
and evaluation of methotrexate niosomes. Drug Dev Ind Pharm
1993;19:133142.
22. Aggarwal D, Kaur IP. Improved pharmacodynamics of timolol
maelate from a mucoadhesive niosomal ophthalmic drug delivery
system. Int J Pharm 2005;290:1559.
23. El-Badry M. Performance of propranolol hydrochloride in certain
ophthalmic formulations [M.Sc. thesis]. Egypt: Faculty of
Pharmacy, Assiut University; 1991.
24. Gibaldi M. Biopharmaceutics and clinical pharmacokinetics. In:
Delivery of drugs: dosage form and their evaluation. 4th ed.
Philadelphia, London: Lea & Febiger; 1991:4173.
25. Kaur IP, Singh M, Kanwar M. Formulation and evaluation of
ophthalmic preparation of acetazolamide. Int J Pharm 2000;199:
11927.
26. Mazzo DJ, Obetz CL, Shuster J. Diltiazem hydrochloride. In: Britain
HG, ed. Analytical profiles of drug substances and excipients. Vol.
23. San Diego: Academic Press; 1994:5498.
27. New RRC, ed. Liposomes: a practical approach. Oxford: IRL Press
at Oxford University Press; 1990.
28. Bandyopadhyay P, Neeta NS. Evidence for vesicle formation 1:1
nonionic surfactant span 60 and fatty alcohol mixtures in aqueous
ethanol: potential delivery vehicle composition. Colloids Surf B:
Biointerfaces 2007;58:3058.
29. El-Nabarawi MA, Bendas ER, El Rehem RTA, Abary MYS.
Formulation and evaluation of dispersed paroxetine liposomes in
gel. J Chem Pharm Res 2012;4:220922.
30. Tiwari Sandip B, Udupa N, Rao BSS, Uma Devi P. Thermosensitive
liposomes and localised hyperthermia an effective bimodality
approach for tumour management. Indian J Pharmacol 2000;32:
21420.
31. Rogerson A, Cummings J, Florence AT. A driamycin loaded
niosomes: drug entrapment, stability and release. J Microcapsule
1987;4:3218.
32. Gregoriadis G. Liposome technology. 2nd ed., Vols 13. Boca Raton
(FL): CRC Press; 1993.
33. Johnson, SM. The effect of charge and cholesterol on the size and
thickness sonicated phospholipid vesicles. Biochim Biophys Acta
1973;307:2741.
34. McIntosh TJ. The effect of cholesterol on the structure of
phosphatidylcholine bilayers. Biochem Biophys Acta 1978;513:
4358.
35. Gabizon A, Papahadjopoulos D. Liposome formulations with
prolonged circulation time in blood and enhanced uptake by
tumors. Proc Natl Acad Sci USA 1988;85:694953.
36. Jahnig F, Harlos K, Vogel H, Eible H. Electrostatic interactions at
charged lipid membranes: electrostatically incuced tilt.
Biochemistry 1979;18:14608.
37. Glava-Dodov M, Fredro-Kumbarad E, Calis S, et al. Formulation
and characterisation of 5-fluorouracil loaded liposomes. Bull
Chemists Technologists Macedonia 2004;23:1318.
38. Katragadda A, Bridgman R, Betageri G. Effect of liposome
composition and cholesterol on the cellular uptake of stavudine by
human monocyte/macrophages. Cell Mol Biol Lett 2000;5:48393.
39. Duxbury MS, Whang EE. RNA interference: a practical approach.
J Surg Res 2004;117:33944.
40. Cocera M, Lopez O, Coderch L, et al. Permeability investigation of
phospholipids liposomes by adding cholesterol. Colloids Surf 2003;
221:917.
41. Barber RF, Shek PN. Liposomes as a topical ocular drug delivery
system. In: Rolland A, ed. Pharmaceutical particulate carrier. New
York: Marcel Dekker; 1993:120.
42. Jousma H, Talsma H, Spies F, et al. Characterization of liposomes.
The influence of extrusion of multilamellar vesicles through
polycarbonate membranes on particle size, particles size distribution
and number of bilayers. Int J Pharm 1987;35:26374.
43. Abdalla RR, Mahdy MA, Abd El-Rhman MM, Megrab NA. Ocular
controlled delivery of brimonidine using liposomes as a particulate
carrier. Zag J Pharm Sci 2001;2:305.
44. Paul S, Mondol R, Ranjit S, Mti S. Antiglaucomatic niosomal
system: recent trend in ocular drug delivery research. Int J Pharm
Pharm Sci 2010;2:9751491.
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