Colloid and Interface Science Communications 28 (2019) 41–48

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Colloid and Interface Science Communications

journal homepage: www.elsevier.com/locate/colcom

Chloramphenicol Loaded Microemulsions: Development, Characterization T

and Stability
Muhammad Abid Rashida, , Tehreem Naza, Madeeha Abbasa, Sadia Nazirb, , Nafisa Younasa,
⁎ ⁎

Sana Majeeda, Naseem Qureshic, Muhammad Nadeem Akhtard

a
Department of Chemistry, University of Agriculture, Faisalabad 38040, Pakistan
b
Department of Chemistry, Govt. College Women University, Faisalabad, Pakistan
c
Department of Chemistry, Karakoram International University, Gilgit 15100, Pakistan
d
Faculty of Industrial Sciences & Technology, University Malaysia Pahang, Pahang 26300, Gambang, Malaysia

ARTICLE INFO ABSTRACT

Keywords: This study aimed to formulate and characterize microemulsions containing chloramphenicol. Microemulsions
Microemulsions represent highly biocompatible drug delivery systems due to their potential for increased absorption as well as
Novel carrier high solubilization capacity. MEs were composed of Oleic acid, non-ionic surfactants tween 20/60, 1-propanol
Chloramphenicol and phosphate buffer. The optimum weight ratios of components and MEs areas were determined by pseudo-
Drug location
ternary phase diagram. All formulations were physically characterized by centrifugation, pH, refractive index,
Stability
conductivity, viscosity, surface tension and partition coefficient. The specific residence site of chloramphenicol
was detected by 1H NMR study. It was uncovered that drug is entrapped between the oxyethylene groups of
hydrophilic shell of MEs. So, the drug was screened from bulk water and its stability was enhanced. Thus, all
characterizations have suggested that formulated MEs have potential for ocular application, being able to use as
efficient drug carrier for ocular drug delivery.

1. Introduction contents of chloramphenicol are < 0.25% [6]. So, it is challenging

The eye is unique in its therapeutic challenges [1]. Ocular diseases
are most widely treated by the topical application of ophthalmic solu-
tions in the form of eye drops. The available ophthalmic solutions ac-
count for 90% of conventional dosage forms [2]. One of the major
concern of conventional eye drops (solutions, suspensions, etc.) is the
meager bioavailability (< 5%) due to high tear fluid turnover and
drainage from eyes due to its physiology [3]. Hence the topical eye
drops do not penetrate into the internal segments of eyes [4]. The
corneal barrier and drainage from eyes reduces the worth of a useful
drug, these concerns could be ameliorated by the application of novel
pharmaceutical delivery systems.
Environmental air pollution is source of many bacterial infections
causing irritation, inflammation and burning of eyes. Chloramphenicol
is one of antibacterial agents which are used to treat the ocular infec-
tions. Chloramphenicol is originally isolated from the certain strains of
Streptomyces Venezuelae. It performs its action as bactriostatic antibiotic
and shows broad action spectra against gram-positive and gram-nega-
tive bacteria [5]. Chloramphenicol is easily hydrolyzed into glycol in
conventional eye drops. The eye drops are turned unqualified if the
objective to develop aproficient ocular drug carrier which can offer
enhanced stability, bioavailability and prolonged drug residence time.
In last few decades microemulsions (MEs) are emerged as a pow-
erful drug carrier because of their unique properties [7]. MEs are
transparent, optically isotropic and thermodynamically stable disper-
sions which posses' small droplet size (5-200 nm) and low surface ten-
sion, which may result in improved permeation and high drug ab-
sorption. Hence there is strong possibility to deliver drug to the
posterior segment of eye [8]. Scientifically, microemulsion is a disper-
sion of oil, surfactant and water [9]. MEs form spontaneously and
contain distinct boundary between water phase and oil phase and at the
boundary surfactant is located. MEs can be a good liquid membrane
carrier for the transport of hydrophilic substances across the lipoidal
mediums and to carry the hydrophobic substances through the aqueous
medium [10].
It is critical to select a novel dosage form because a useful drug
becomes worthless due to poor drug carrier. Therefore, in this research
plan efforts have been made to formulate a drug carrier which is cap-
able of targeted delivery with high therapeutic efficacy. Since, new MEs
composed of oleic acid/Tween 20/Tween 60/n-propanol/phosphate

⁎
Corresponding authors.
E-mail addresses: marashid@uaf.edu.pk (M.A. Rashid), sadia_nazir78@yahoo.com (S. Nazir).

https://doi.org/10.1016/j.colcom.2018.11.006
Received 16 August 2018; Received in revised form 3 November 2018; Accepted 20 November 2018
Available online 27 November 2018
2215-0382/ © 2018 Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
M.A. Rashid et al.
buffer were developed for enhancing the stability and bioavailability of
chloramphenicol. Pseudo-ternary phase diagram was delineated to find
the microemulsion zone. The 1H NMR spectroscopy was used to find the
location of chloramphenicol in microemulsions. In addition physico-
chemical characteristics were also analyzed and it is expected that
microemulsion system may enhance the bioavailability of chlor-
amphenicol and prevent the degradation of it.
2. Materials and Methods
Chloramphenicol drug was purchased from Unesco Pharmaceutical
Industry Lahore, Pakistan. Polyethylene glycol sorbitan monolaurate
(Tween 20: CAS, 9005-64-5) and Tween 60 Polyoxyethylene sorbitan
monostearate (Tween 60: CAS, 9005-67-8) were from Sigma Germany
and both were of analytical grade. Oleic acid (CAS, 112–80-1, EP
Grade), 1-propanol (CAS, 71-23-8, GR Grade 99.5%) and phosphate
buffer standard solution (PB, pH = 7) were purchased from Dae-Jung
Korea.
2.1. Microemulsion Formulation
Pseudo-ternary phase diagram was mapped using oil (Oleic acid;
HLB = 16), surfactant (Tween 20; HLB = 14.5), co-surfactants (Tween
60; HLB = 15) and 1-propanol and phosphate buffe (PB) as an aqueous
phase (pH = 7.0) at room temperature (25°С). In the start, oleic acid
was mixed with Tween 20/Tween 60/1-propanol and then PB was
added drop-wise to obtain the ME. Transparent and Single phase and
transparent mixtures were selected as microemulsions. All the samples
were clear and stable for a period of upto 6 months.
2.2. Incorporation of Drug in Microemulsions
MEs loaded with 5% (w/w) of chloramphenicol were formulated by
dispersing drug in oil phase, then adding in the mixture of surfactants
and co-surfactants. The phosphate buffer was added drop wise to this
mixture followed by constant stirring at ambient temperature. 8 for-
mulations were obtained.
2.3. Characterization of Microemulsions
2.3.1. Optical Brightness
The optical isotropy and homogeneity of pure and drug loaded MEs
was examined by a polarimeter(Code 36-222 made in UK, Bellingham
Stanley limited) and visual evaluation at RT (25°С).
2.3.2. Stability Study
Thermodynamic stability of pure and drug-loaded MEs was de-
termined by centrifugation at 3000 rpm for 20 min using 80–2 CENT-
RIFUGE.
2.3.3. pH
The pH values of all the formulated MEs were measured with Digital
Multimeter fitted with a pH electrode. All the values were taken at
25°С.
2.3.4. Refractive Index
The refractive indices of all formulated MEs were evaluated by using
a refractometer (ATAGO, RX-5000) at wavelength approximate to D-
line(589.0 nm) and 25°С.
2.3.5. Conductivity Measurements
The values of conductivities were measured by a Conductivity Meter
(HANNA HI 9811-5) fitted with an electrode (HI 1285-5) at 25°С to
determine the type MEs.
42
Colloid and Interface Science Communications 28 (2019) 41–48
2.3.6. Surface Tension Measurement
Stalagmometerwas used to determine the Surface tension values by
drop number method [11]. Number of drops of water and each for-
mulated MEs were counted, than the surface tension was calculated by
using this formula
n2d1
1 = 2
n1d2 (1)
γ1 = surface tension of microemulsion γ2 = surface tension of
water.
n1 = no. of drops of microemulsion n2 = no. of drops of water.
d1 = density of microemulsion d2 = density of water.
2.3.7. Viscosity measurements
NDJ-8S rotary viscometer was used to measure dynamic viscosities
of all formulated MEs at 25°С.
2.3.8. Partition coefficients
Oil/buffer partition coefficients were inspected by dissolving 20 mg
chloramphenicol in 7 mL isopropyl myristate. Buffer was added in it
(1:1 ratio v/v). The mixture was shaken well for 10 min and centrifuged
for 2 h. The two layers were separated and the content of chlor-
amphenicol in aqueous layer (PB) was assessed by UV–visible spectro-
photometer at 278 nm (λmax of drug). The two layers were assessed by
Perkin Elmer Lambda 25UV/VIS spectrophotometer at 278 nm [12].
2.3.9. 1H NMR Spectroscopy
1
H NMR experiments were performed on a Bruker spectrometer at
600 MHz. Chemical shifts of all microemulsions determined by using
D2O as solvent.
3. Results and Discussion
In present study the MEs system is composed of oleic acid as oil
phase, tween 20/60 surfactants and 1-propanol as cosurfactant. The
choice of dispersed phase for ocular drug delivery is very important
because it governs both the solubilization of drug and existence of
microemulsion. The oil phase also influences the curvature, so it is
necessary to select an appropriate oil phase. Therefore oleic acid is
selected as oil phase because of its solubilization potential and it is well
tolerated by the eye [13]. Selection of surfactant is also a critical step to
design a ME system. There should be appropriate lipophilic character of
surfactant to facilitate the right curvature at the interface of ME droplet.
Generally, for O/W MEs the surfactant with high HLB (> 12) are sui-
table [14]. Non ionic surfactants are widely used in pharmaceutical
preparations and these are also nontoxic and irritations free [15].
Therefore, Tween 20 (HLB = 14.5) and tween60 (HLB = 15) were
chosen to formulate these MEs. The flexibility of interface is the major
consideration in development of MEs. If the interfacial film is rigid, the
formation of microemulsion is prevented. For this reason co-surfactants
are often combined with surfactants [10].The requirement of additional
input of energy is overcome by short chain alcohols [16] and alcohols
also influence the activity of nonionic surfactants in micellar state [17].
An oil solution without aqueous phase was formed when a mixture
of oil, surfactant and cosurfactant was blended. 1-propanol an alcohol
interacts with the ethoxylated head region of tween 20 and tween 60
and develops hydrogen bonding which controls the packing para-
meters. When aqueous phase is added to this oily solution the hydrated
head groups of tween 20 are organized into a polar core so 1-poropanol
prevents the lyotropic crystal formation. In the start water in oil mi-
croemulsion are formed which then turned into bicontinuous phase and
then on further dilution converted into oil in water microemulsion
without any phase separation.
The relationship between the phase behaviour of a mixture and its
composition can be captured with the aid of a phase diagram. The
pseudo ternary phase diagram was mapped and area of existence of ME
M.A. Rashid et al. Colloid and Interface Science Communications 28 (2019) 41–48

Fig. 1. Pseusdo-Ternary phase diagram and microemulsion region of microemulsions composed of oleic acid, tween 20/60/1-propanol and phosphate buffer.

Table 1
Optimized microemulsion formulations.
Microemulsion Oleic acid (w/w%age) Surfactant (Tween Co-surfactanta (w/w%age) Co-surfactantb (w/w%age) Phosphate buffer (PB)
(ME) 20) (w/w%age) (w/w%age)

1 0.1 1 0.42 0.1 8.38

2 0.2 1.21 0.5 0.2 7.89
3 0.3 1.51 0.8 0.4 6.99
4 0.4 1.8 1 0.6 6.2
5 0.5 2.2 0.8 0.61 5.88
6 0.6 2.6 1 0.8 5
7 0.7 2.84 1.2 0.9 4.36
8 0.8 2.05 1.4 1 3.85

a
Tween60.
b
1-Propanol.

consisted of Oleic acid/Tween 20/Tween 60/1-propanol/PB is shown in
Fig. 1. The microemulsion was formed by the spontaneous mixing of all
components at room temperature. Eight formulations were carried out
from one phase isotropic region in Fig. 1 and compositions of selected
microemulsions are described in Table 1.
3.1. Optical Brightness
The homogeneity and optical isotropy of all pure and drug loaded
MEs were examined at 25°C. All the samples were clear and transparent
single phase solution and also all the MEs were remained stable.
formulated MEs have maximum shelf life of six months. The pH values
are adjusted by using phosphate buffer (pH = 7) as aqueous phase, the
buffer solution also enables the eye to maintain the pH within the
physiological range [19]. For topical formulations the ocular comfort
pH ranges from 6.6 to 7.8 due to limiting buffering capacity of eyes
[20]. Since the pH of eye drops applied as therapeutic substance vary
from 3.5 to 8.5, so the pH values of formulated MEs were in the ac-
ceptable and tolerable range and meet the requirements of ophthalmic
delivery system [21].
3.4. Refractive Index

3.2. Stability Study
Adequate physical stability was shown by all the formulated MEs
after centrifugation at 3000 rpm for 20 min. So, it revealed that all the
components of MEs were compatible and there was no discrete altera-
tion in the transparency of all formulations.
3.3. pH
Stability of formulated microemulsions, the activity of product,
comfort safety, drug preservative efficacy and drug bioavailability all
are affected by the pH value [18]. So, the pH of ophthalmic formula-
tions should be adjusted close to the pH of tears. The pH values of all
formulated neat and drug loaded MEs were ranged from 5.49 to 6.49
and 5.65–6.43 respectively. At these pH values all neat and drug loaded
microemulsions were stable and there was no phase separation and all
The refractive index of tear fluid must not be higher than 1.476 with
an optimum value of 1.404 [21]. The refractive indices of pure and drug
loaded MEs were ranged from 1.35433–1.41948 and 1.35379–1.41714
respectively as shown in Table 2. So the refractive indices of the pre-
pared drug loaded MEs were within the tolerable range of tear fluid.
Therefore, after the administration of microemulsion, there will be no
discomfort and any impairment of vision [22].
There was a slight change in the refractive index of each formula-
tion after the drug loading. This slight change indicated that the drug
has successfully loaded on microemulsion system without any phase
changes in this multicomponent system.
3.5. Conductivity Measurements
The human body has hydrophilic physiological environment, so the
chloramphenicol should be incorporated in oil in water microemulsion

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M.A. Rashid et al. Colloid and Interface Science Communications 28 (2019) 41–48

Table 2
Values of the physicochemical parameters characterizing the neat and drug loaded microemulsions.
ME pHa,b Refractive Indexa,b Conductivitya,b (mS) γa,b (mN/m) Viscositya,b (mPa.s)

a b a b a b a b a b

1 6.43 6.49 1.35433 1.35379 1.41 1.99 36.7 37.9 54 51

2 6.24 6.29 1.35933 1.35702 1.49 1.66 36.5 36.6 62 64
3 6.1 6.11 1.36946 1.37177 1.14 1.15 31.5 32.6 74 76
4 5.96 5.88 1.38187 1.38086 0.84 0.77 28.2 30.1 85 88
5 5.83 5.75 1.38471 1.41714 0.67 0.69 28.7 29.9 97 101
6 5.75 5.7 1.40665 1.34549 0.50 0.41 29.5 29.3 107 110
7 5.53 5.67 1.40443 1.38482 0.28 0.37 31.2 26.9 117 121
8 5.65 5.49 1.41948 1.3661 0.17 0.18 28.7 26.2 128 133

a
Neat microemulsions.
b
Drug loaded microemulsions.

for its ophthalmic application. Therefore, it is necessary to inspect the
phase behavior of formulated MEs. The conductivity measurements are
used to determine the type of MEs whether it is water-continuous or oil-
continuous [23]. The conductivity values of pure and drug loaded MEs
were ranged from 1.14–0.17 mS and 1.99–0.18 mS respectively. Such
higher values of conductivities proved that all the formulated micro-
emulsions were oil in water microemulsions. Fig. 2.(a) showed varia-
tion of conductivities of formulated MEs. As the concentration of water
content decreased, the concentration of oil was increased the con-
ductivity goes to decrease because the movement of ions became dif-
ficult to conduct.
ocular bioavailability of chloramphenicol can be increased as compare
to commercial eye drops.
Viscosity values also influence the stability of formulations so op-
timum viscosity values are required for good stability. As viscosity in-
creases there will be reduction of globule's flocculation due to restricted
mobility and hindered Brownain movement, leading to creaming [30].
The results depict that within this viscosity range all the formulated
MEs showed good stability there was no creaming for the period of six
months.
3.8. Partition Coefficient

3.6. Surface tension
Surface tension of pure and chloramphenicol loaded MEs were
varied from 28.2–36.7 mN/m and 26.2–37.9 mN/m respectively
(Table 2) and graphically represented in Fig. 2.(b). There is a de-
creasing trend in the surface tension values from ME-1 to ME-8. It is
revealed from the results that as the concentration of surfactants is
increased in the formulation it reduced the surface tension values of
pure and drug loaded formulations because the increased amount of
surface active agents at interface led to reduce the interfacial tension
[24]. There is modification of interface properties by the preferential
adsorption of surfactants [25] which ultimately form a flexible film
around the droplets. In comparison to the surface tension of human
lachrymal fluid (40–50 mN/m) [26] the values of formulated MEs are
lower which indicate that all formulations can be spread uniformly on
the corneal surface. The uniform distribution of MEs will ultimately
lead to enhancement of permeability of drug. Hence the absorption area
will be enhanced and the bioavailability of chloramphenicol will also
increase.
3.7. Viscosity measurements
Viscosity is a fundamental parameter to assess the efficiency of any
formulation (low viscosity reduces bioavaiability due to immediate
drainage from eye) and its compatibility to eye (high viscosity causes
blinking pain and blurred vision). Viscosity is largely influenced by the
presence of aggregates, their interaction and concentration. It is well
known that by increasing the dispersed phase in microemulsion the
viscosity of the system in increased [27].
For reported system there was an increase in viscosity of ME-1 to
ME-8 by increasing the concentration of dispersed phase as shown in
Table 2 and graphically represented in Fig. 2.(c). It was observed that
the viscosity measurements for all formulations were high than the
viscosity of other ophthalmic solutions (20 mPa.s) [28]. These low
viscosity eye drops cause the spillage of drug and lead to low bioa-
vailability, so, it is required to insert the solution many times in a day
[29]. Thus to reduce the instillation frequency per day these MEs were
developed with high viscosity so that the drug residence time and
Drug transport characteristics, which include distribution, reten-
tion, absorption and elimination, are influenced by partition coeffi-
cients. Since the drugs are transported by blood, they must traverse and
penetrate many cells to reach at the site of action. Hence the site of
action of given compound will be determined by the partition coeffi-
cients.
3.9. Oil/Buffer Partition Coefficients
The logarithmic values of drug solubilities are called partition
coefficients [Log P] and biopharmaceutical properties of drugs are
predicted by it [31]. The partition coefficient (Log P) of chlor-
amphenicol in oil/buffer is 2.4076 ± 0.20. Acocording to Lipinski's
“rule of five” there will be poor drug absorption and permeation if the
partition coefficient is > 5 [32]. The partition coefficients were lower
than 5, it showed that the present formulated system is capable of
loading efficiency and partitioning of chloramphenicol, to improve in-
corporation of drug, to increase the uptake of drug and retention, and
sustained release of drug [33].
The presence of tween 20 (5% in buffer) and co-surfactants (5%-
Tween 60 + 1-Propanol) reduced the partition coefficients to
1.1567 ± 0.15 and 1.2979 ± 0.20 respectively. The concentration of
drug in oil phase was reduced due to the presence of surfactant
(Table 3). The partitioning of drug between aqueous and micellar phase
is governed by the hydrophilic-lipophilic balance (HLB) of surfactant.
The penetration of drug deeply in the micelle is prevented due to the
attraction between drug and surfactant [34]. Therefore the drug re-
mains localized at the interface of micelle. Thus, the partition studies
show that the chloramphenicol may be resided at the interface. The
solubility of chloramphenicol may be increased due to the presence of
drug at the hydrophilic shell of microemulsion droplet. The partition
coefficients were determined by following formula [35]:
A (org ) × Vf (org ) × V (aq)
P=
A (aq) × V (org ) × Vf (aq) (2)
where A (aq) and A (org) is the absorbance of aqueous and organic
layer, V (org) and V (aq) is the volume of aliquot from organic and
aqueous layers, Vf (org) and Vf (aq) is the final volume of sample from

44
M.A. Rashid et al. Colloid and Interface Science Communications 28 (2019) 41–48

Fig. 2. a) Variation of electrical conductivity for neat and drug loaded MEs; b) Variation of Surface tension for neat and drug loaded MEs at 25 °С; c) Variation of
viscosity for neat and drug loaded MEs at 25°С.

Table 3 organic and aqueous layer.

Oil/buffer partition coefficient of Chloramphenicol (mean ± S.D). The standard free energy ΔGopcan be calculatedfor the transfer of
System P log P ΔGop = -RTlnP
additive to micelle from bulk aqueous phaseby using following equa-
(kJ/mol) tion [36]:

Oil/buffer 2.5568 × 102 2.4076 ± 0.20 −13.73

Go p = RTlnP (3)
Oil/buffer(5% Tween 20) 1.4346 × 101 1.1567 ± 0.15 −6.60
Here T absolute temperature and R denotes the gas constant.
Oil/buffer(5% Tween 1.9856 × 101 1.2979 ± 0.20 −7.40
60 + 1-Propanol) The spontaneity of solubilization is expressed by this parameter, a
negative value indicates the spontaneous transfer of drug to micelles
[37]. The values of standard free energy ΔGop for chloramphenicol,

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M.A. Rashid et al. Colloid and Interface Science Communications 28 (2019) 41–48

Fig. 3. Structures of (a) chloramphenicol, (b) tween 60 and (c) tween 20.

Table 4
1
H NMR chemical shifts values for surfactants in microemulsion with different concentration.
Functional Group δ(ppm)

δ0 δ1 δ2 δ3 δ4 δ5 δ6 δ7 δ8

ω-CH3 0.7642 0.7773 0.7638 0.7752 0.7756 0.7734 0.7773 0.7792 0.7754
(CH2)n 1.1664 1.1737 1.1715 1.1691 1.1713 1.1092 1.1708 1.1716 1.1684
β1-CH2 1.2173 1.4729 1.4662 1.4633 1.4664 1.3709 1.4639 1.4647 1.4599
α1-CH2 2.2789 2.2120 2.2085 2.2028 2.1191 2.0434 2.2056 2.2085 2.2025
(CH2CH2O)n 3.6303 3.5253 3.5257 3.5228 3.5234 3.5487 3.5264 3.5264 3.5239
α2-CH2 4.1480 4.0395 4.0340 4.0903 4.0366 3.9736 4.0344 4.0402 4.0908

δ0chemical shifts for surfactants of pure microemulsion,δ1-δ8 chemical shifts for surfactants of microemulsion loaded with drug 5–12%wt with respect to oil con-
centration.

Table 5
Difference of chemical shifts values for surfactants in microemulsion by loading chloramphenicol.
Functional Group δ(ppm)

δ0−δ1 δ0−δ2 δ0−δ3 δ0−δ4 δ0−δ5 δ0−δ6 δ-0δ7 δ0−δ8

ω-CH3 −0.0131 0.0004 −0.011 −0.0114 −0.0092 −0.0131 −0.015 −0.0112

(CH2)n −0.0073 −0.0051 −0.0027 −0.0049 0.0572 −0.0044 −0.0052 −0.002
β1-CH2 −0.2556 −0.2489 −0.2460 −0.2491 −0.1536 −0.2466 −0.2474 −0.2426
α1-CH2 0.0669 0.0704 0.0761 0.0735 0.2355 0.0733 0.0704 0.0764
(CH2CH2O)n 0.1050 0.1046 0.1075 0.1069 0.0816 0.1039 0.1039 0.1064
α2-CH2 0.1085 0.1140 0.1123 0.1114 0.1744 0.1136 0.1078 0.1107

using P, are given in Table 3. The more negative value of ΔGop indicated
that the partitioning of chloramphenicol is spontaneous and the system
is stable.
3.10. 1H NMR Study
More recently, the NMR techniques has been used to characterize
the colloidal systems of pharmaceutical importance e.g. MEs. To de-
termine the locus of drug in the microemulsion interior, we reported
here different results of 1H NMR study. Different proton chemical shifts
were originated due to surrounding electronic environment. So the
change in chemical shifts may serve as a sensitive probe for determi-
nation of drug location and its local interactions. Therefore the possible
residence site of chloramphenicol has been determined by proton NMR
spectroscopy of microemulsion system consisting of Oleic acid (oil
phase), Tween 20 (as surfactant), tween 60 + 1-propanol (as co-
surfactant) and PB (as aqueous phase). The composition of selected ME
was fixed and the concentration of chloramphenicol was varied from 5
to 12% wt with respect to oil contents. In this system oleic acid, tween
20 and tween 60 have many oxyethylene groups and long hydrocarbon
chains. So many functional groups (ω-CH3,α1-CH2, β1-CH2, (CH2)n, α2-
CH2and CH2CH2O)n) (Fig. 3) and many molecules resulted complex
NMR spectra. The results obtained from NMR spectra are summarized
in Tables 4. and 5. It was noted that there was a change in the chemical
shifts of certain groups of surfactants by the addition of chlor-
amphenicol. This study revealed that the chemical shifts of oxyethylene

46
M.A. Rashid et al.
(CH2CH2O)n) and methylene (α2-CH2) are sensitive to the addition of
chloramphenicol. The changes in the chemical shifts of other groups
such as ω-CH3,α1-CH2, β1-CH2 and (CH2)n, were very small. So, it was
then deduced that chloramphenicol is not resided and solublized in
palisade oil layer but it is located in the hydrophilic shell of micro-
emulsion droplets consisting of many oxyethylene groups. The chemical
shifts of oxyethylene groups (CH2CH2O)n) and methylene (α2-CH2)
were moved to upfield due to screening effect of benzene ring [29].
Therefore, the drug is encapsulated in hydrophilic shell. So, this loca-
tion of chloramphenicol in microemulsion formulation enables it to be
screened from bulk aqueous phase and its stability enhanced dramati-
cally.
Commercial eye drops are the solution of chloramphenicol in water;
thus it is not stable and the contents of glycols are higher in eye drops as
compared to the stabilized chloramphenicol in microemulsion for-
mulations. Besides the long-live stabilization of chloramphenicol in ME-
systems, the other effects are expected, such as better penetrability and
sustained effect.
4. Conclusion
New microemulsion systems were developed to encapsulate the
ocular drug chloramphenicol. Pseudo-ternary phase diagrams were
constructed using Oleic acid (as oil phase), tween 20 (as surfactant),
tween 60 and 1-propanol (as cosurfactant) and phosphate buffer (as
aqueous phase). This study established that the formulated micro-
emulsion systems were thermodynamically stable and transparent
single phase solutions indicated by optical brightness and centrifuga-
tion study. The physicochemical properties such as pH and refractive
index were within the tolerable range for ocular application. The con-
ductivity measurements proved that all MEs were O/W microemul-
sions, so these systems are compatible with the hydrophilic physiolo-
gical environment of human body. Surface tension and viscosity studies
indicated that the bioavailability, contact time and absorption area will
be enhanced. The possible residence site of chloramphenicol in micro-
emulsion droplet was determined by the partition coefficient, which
indicated that the drug may reside at the interface of hydrophilic shell
of ME. This indication coincides with the results of 1H NMR study. It is
inferred from the results of 1H NMR that the chloramphenicol should be
located and solublized in the hydrophilic shell which is composed of
many oxyethylene groups of surfactants. So, this enables chlor-
amphenicol to be screened from bulk aqueous phase and its stability
enhanced dramatically. Hence, it leads to conclusion that the MEs are a
protective carrier due todrug entrapment ability and shield drug from
oxidation, decomposition and hydrolysis. So, these are a proficient drug
carrier for targeted drug delivery without toxic effects.
Acknowledgements
Financial support from Higher Education Commission (HEC)
Pakistan is gratefully acknowledged. (HEC scholarship for T. Naz (213-
56141-2PS2-007), HEC grants, NRPU 4923 & 6403 for M. A. Rashid).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.colcom.2018.11.006.
References
[1] T.F. Vandamme, Microemulsions as ocular drug delivery systems: recent develop-
ments and future challenges, Prog. Retin. Eye Res. 21 (1) (2002) 15–34.
[2] C. Peng, L. Bengani, H. Jung, J. Leclerc, C. Gupta, A. Chauhan, Emulsions and
microemulsions for ocular drug delivery, J. Drug Deliv. Sci. Technol. 21 (1) (2011)
111–121.
[3] R. Kumar, V. Sinha, Preparation and optimization of voriconazole microemulsion
for ocular delivery, Colloids Surf. B: Biointerfaces 117 (2014) 82–88.
47
Colloid and Interface Science Communications 28 (2019) 41–48
[4] A. Urtti, Challenges and obstacles of ocular pharmacokinetics and drug delivery,
Adv. Drug Deliv. Rev. 58 (2006) 1131–1135.
[5] A. Musa, I. Yakasai, M. Garba, B. Mathias, In vitro availability of chloramphenicol
in the presence of cadmium and lead salts, J. Basic Clin. Pharm. 1 (2) (2010) 97.
[6] F.F. Lv, N. Li, L.Q. Zheng, C.H. Tung, Studies on the stability of the chloramphenicol
in the microemulsion free of alcohols, Eur. J. Pharm. Biopharm. 62 (3) (2006)
288–294.
[7] J. Lakshmi, B.A. Kumar, S. Gupta, Investigation of microemulsion as a potential
carrier for advanced transdermal delivery: an overview, Int. J. Pharm. Sci. Rev. Res.
20 (2013) 51–59.
[8] M.J. Lawrence, G.D. Rees, Microemulsion-based media as novel drug delivery
systems, Adv. Drug Deliv. Rev. 45 (1) (2000) 89–121.
[9] G.R. Dixit, V.B. Mathur, Microemulsions: platform for improvement of solubility
and dissolution of poorly soluble drugs, Asian J. Pharm. Clin. Res. 8 (5) (2015)
7–17.
[10] R.R. Hegde, A. Verma, A. Ghosh, Microemulsion: new insights into the ocular drug
delivery, Int. Sch. Res. Notices 2013 (2013) 1–11.
[11] G. Venkateshwarlu, M. Sabath, G. Venugopal, M. Sravanprasad, S. Ghosh,
S. Bharathbhushanreddy, Determination of surface tension of various formulation
by using straube's stalagnometer, Asian J. Pharm. Ana 2 (2) (2012) 36–38.
[12] M.F. Nazar, A.M. Khan, S.S. Shah, Microemulsion system with improved loading of
piroxicam: a study of microstructure, AAPS PharmSciTech 10 (4) (2009)
1286–1294.
[13] N. Subramanian, S.K. Ghosal, A. Acharya, S.P. Moulik, Formulation and physico-
chemical characterization of microemulsion system using isopropyl myristate,
medium-chain glyceride, polysorbate 80 and water, Chem. Pharm. Bull. 53 (12)
(2005) 1530–1535.
[14] J. Jiao, Polyoxyethylated nonionic surfactants and their applications in topical
ocular drug delivery, Adv. Drug Deliv. Rev. 60 (15) (2008) 1663–1673.
[15] M. Fanun, Water solubilization in mixed nonionic surfactants microemulsions, J.
Dispers. Sci. Technol. 29 (8) (2008) 1043–1052.
[16] R.G. Alany, T. Rades, S. Agatonovic-Kustrin, N.M. Davies, I.G. Tucker, Effects of
alcohols and diols on the phase behaviour of quaternary systems, Int. J. Pharm. 196
(2) (2000) 141–145.
[17] B.H. Lee, Effects of various alcohols and salts on the mixed micellization of cationic
surfactant (CPC) with nonionic surfactant (TX-100), Colloid. Interf. Sci. Commun.
19 (2017) 1–4.
[18] H. Fawzia, E.M. Mona, M. Shaheer, Ocular drug deliver and the importance of
microemulsion as a potential delivery system, Int. J. Pharm. Chem. Sci. 1 (2) (2012)
723–737.
[19] M.M. Van Ooteghem, Formulations of ophthalmic solutions and suspensions.
Problems and advantages, in: P. Edman (Ed.), Biopharmaceutics of Ocular Drug
Delivery, CRC, Boca Raton, 1993, pp. 31–32.
[20] A. López-Alemany, R. Montes-Mico, M. Garcia-Valldecabres, Ocular physiology and
artificial tears, J. Am. Optom. Assoc. 70 (7) (1999) 455–460.
[21] A. Radomska-Soukharev, J. Wojciechowska, Microemulsions as potential ocular
drug delivery systems: phase diagrams and physical properties depending on in-
gredients, Acta Pol. Pharm. 62 (6) (2005) 465–471.
[22] S.L. Fialho, A. da Silva-Cunha, New vehicle based on a microemulsion for topical
ocular administration of dexamethasone, Clin. Exp. Ophthalmol. 32 (2004)
626–632.
[23] M. D'Angelo, D. Fioretto, G. Onori, L. Palmieri, A. Santucci, Dynamics of water
containing sodium bis(2-ethylhexyl)sulfosuccinate (AOT) reverse micelles: a high-
frequency dielectric study, Phys. Rev. E 54 (1996) 993–996.
[24] L.B. Vieira, M.W. Casimiro, R.G. Santos, Surface tension of aqueous Amoxicillin+
Peg systems, Colloid. Interf. Sci. Commun. 24 (2018) 93–97.
[25] M. Kahlweit, G. Busse, B. Faulhaber, H. Eibl, Preparing nontoxic microemulsions,
Langmuir 11 (11) (1995) 4185–4187.
[26] A. Silva-Cunha, G.R. da Silva, W.V. de Castro, S.L. Fialho, Evaluation of the phar-
macokinetics and ocular tolerance of a microemulsion containing tacrolimus, J.
Ocul. Pharmacol. Ther. 30 (1) (2014) 59–65.
[27] L. Djordjevic, M. Primorac, M. Stupar, D. Krajisnik, Characterization of capryloca-
proylmacrogolglycerides based microemulsion drug delivery vehicles for an am-
phiphilic drug, Int. J. Pharm. 271 (1–2) (2004) 11–19.
[28] H.O. Ammar, H.A. Salama, M. Ghorab, A.A. Mahmoud, Nanoemulsion as a potential
ophthalmic delivery system for dorzolamide hydrochloride, AAPS PharmSciTech 10
(2009) 808–819.
[29] F.F. Lv, L.Q. Zheng, C.H. Tung, Phase behavior of the microemulsions and the
stability of the chloramphenicol in the microemulsion-based ocular drug delivery
system, Int. J. Pharm. 301 (1–2) (2005) 237–246.
[30] C.M. Jadhav, S.M. Shinde, V.K. Kate, S.A. Payghan, Investigating application of non
aqueous microemulsion for drug delivery, Asian J. Biomed. Pharm. Sci. 4 (29)
(2014) 1–9 2014.
[31] H. Devalapally, A. Chakilam, M.M. Amiji, Role of nanotechnology in pharmaceu-
tical product development, J. Pharm. Sci. 96 (2007) 2547–2565.
[32] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and compu-
tational approaches to estimate solubility and permeability in drug discovery and
development settings, Adv. Drug Deliv. Rev. 46 (2001) 3–26.
[33] H.L. Wong, R. Bendayan, A.M. Rauth, H.Y. Xue, K. Babakhanian, X.Y. Wu, A me-
chanistic study of enhanced doxorubicin uptake and retention in multidrug resistant
breast cancer cells using a polymer-lipid hybrid nanoparticle system, J. Pharmacol.
Exp. Ther. 317 (2006) 1372–1381.
[34] S. Noor, N. Younas, M.A. Rashid, S. Nazir, M. Usman, T. Naz, Spectroscopic, con-
ductometric and biological investigation of [Ni (phen) 3] F2. EtOH. MeOH. 8H2O
complex in anionic micellar media, Colloid. Interf. Sci. Commun. 27 (2018) (2018)
26–34.
M.A. Rashid et al.
[35] S.P. Songca, B. Mbatha, Solubilization of meso-tetraphenylporphyrin photo-
sensitizers by substitution with fluorine and with 2, 3-dihydroxy-1-propyloxy
groups, J. Pharm. Pharmacol. 52 (2000) 1361–1367.
[36] H. Kawamura, M. Manabe, Y. Miyamoto, Y. Fujita, S. Tokunaga, Partition coeffi-
cients of homologous. omega.-phenylalkanols between water and sodium dodecyl
48
Colloid and Interface Science Communications 28 (2019) 41–48
sulfate micelles, J. Phys. Chem. 93 (14) (1989) 5536–5540.
[37] M. Shakeel, K. Mehmood, M. Siddiq, Aggregation properties of levofloxacin in
water and ethanol and its interaction with sodium dodecyl sulphate: a thermo-
dynamic study, J. Chem. Sci. 127 (11) (2015) 2073–2079.