JOURNAL OF OCULAR PHARMACOLOGY AND THERAPEUTICS ORIGINAL ARTICLE

Volume 00, Number 00, 2017

ª Mary Ann Liebert, Inc.
DOI: 10.1089/jop.2017.0079

Improvement in Ocular Bioavailability and Prolonged

Delivery of Tobramycin Sulfate Following Topical
Ophthalmic Administration of Drug-Loaded
Mucoadhesive Microparticles Incorporated
in Thermosensitive In Situ Gel

Shagufta Khan, Sonali Warade, and Dilesh J. Singhavi

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Abstract

Purpose: Conventional topical delivery in hyperacute bacterial conjunctivitis and endophthalmitis is associated
with low drug bioavailability due to rapid precorneal clearance. Hence, in the present investigation, an attempt
has been made to enhance ocular bioavailability of tobramycin sulfate by formulating drug-loaded micropar-
ticles dispersed in thermosensitive in situ gel.
Methods: Microparticles prepared by emulsion–ionic gelation technique were characterized for drug loading,
entrapment efficiency, particle size, surface morphology, and in vitro drug release. Consequently microparticles
(F2 prepared with 1.5%w/v chitosan, 0.2%w/v tripolyphosphate, and drug, 30%w/w of polymer) with high drug
loading and encapsulation efficiency were dispersed in thermosensitive in situ gel containing poloxamer 407
and varying percentage of chitosan. In situ gel containing drug-loaded microparticles were evaluated for
gelation temperature, rheological behavior, mucoadhesive strength, in vitro drug release, in vitro permeation,
ocular irritation, and bioavailability in aqueous humor of rabbits.
Results: Formulation containing 17%w/v poloxamer 407 and 0.5%w/v chitosan (P2) gelled at 32C – 1.5C
gave pseudoplastic behavior. In vitro permeability of tobramycin from the formulation P2 was found 2-folds
greater than eye drops. It also gave significantly higher aqueous humor concentration of tobramycin compared
with eye drops with no signs of ocular irritation.
Conclusion: Thus, the formulation possesses high potential for treating ocular infections.

Keywords: chitosan, mucoadhesive, poloxamer, in vitro permeability, aqueous humor, ionic gelation

Introduction drug was dispersed in poloxamer 407 and poloxamer 188

containing in situ gel. However, the foremost concern with
colloidal drug delivery systems is low drug entrapment ef-
T opical administration of conventional dosage forms,
such as eye drops results in very poor bioavailability due
to blinking, reflex tearing, dilution, nasolacrimal drainage etc.1,2
ficiency. Above and beyond the problem of drug entrapment
percentage, retention of these particles in the conjunctival
Several drug delivery systems, such as hydrogels, mi- pouch is a crucial consideration. As a consequence of very
celles, microparticles, nanoparticles, nanoemulsions, and small size (10–1,000 nm), their clearance through the na-
implants have been developed to produce sustained release solacrimal duct is similar to ophthalmic solutions.5
with greater bioavailability of drugs and reduced side ef- Microparticles are attractive carriers which can encapsulate
fects. Zhang et al.3 formulated nanoparticles of rapamycin the drug and can provide sustained release of drug for pro-
using poly(e-caprolactone)-poly(ethylene glycol)-poly(e- longed time at the target site. Choy et al. formulated mu-
caprolactone) block copolymer to achieve sustained release. coadhesive microparticles of poly(lactic-co-glycolic acid) and
Ammar et al.4 observed superior pharmacodynamic ac- poly(ethylene glycol) for prolonged residence on the surface
tivity of dorzolamide when nanoemulsion loaded with the of the eye.6 Microparticles show slower elimination kinetics

Institute of Pharmaceutical Education and Research, Wardha, Maharashtra, India.

1
 2 KHAN, WARADE, AND SINGHAVI

from the precorneal compartment than nanoparticles.7 Fur- India, Chitosan (90% degree of deacetylation and average
thermore, microparticles show greater encapsulation, lower molecular weight between 100,000 and 150,000 g/mole) was
burst release, and better sustained delivery compared with the gift from Nitta Gelatin India Ltd., Kerala, India, and po-
nanoparticles.8 However, their size must be controlled below loxamer 407 was purchased from Signet Chemical Corpora-
10 mm to be tolerated. Mucoadhesive microparticles remain tion Pvt. Ltd., Mumbai, India. All other chemicals were of
adherent to the ocular surface for an extended time with re- either HPLC or analytical grade.
duced loss by tear drainage.6 So far, chitosan microspheres
were mainly prepared by emulsion crosslinking or ionic ge-
lation method. Emulsion crosslinking method involves harsh Formulation of chitosan microparticles
crosslinking agent, such as glutaraldehyde, which may induce
Chitosan microparticles containing TS were prepared by
chemical reaction with active ingredients, whereas thus far
emulsification–ionic gelation method.12 Chitosan was dis-
ionic gelation technique entails external gelation where
persed into 2% v/v aqueous acetic acid using an orbital shaker
chitosan solution is dropped into tripolyphosphate (TPP)
(200 rpm for12 h), TS was then dissolved into it, and the pH
solution. With external gelation, it is difficult to get small,
was made 4.5 with 2% v/v aqueous acetic acid. The aqueous
uniform-sized microparticles, as clusters of ionically linked
phase was dispersed into paraffin oil containing 1% of span
regions may create inhomogeneities. In the present investi-
20 by stirring at 500 rpm (chitosan solution: paraffin oil was
gation, it is attempted to produce low-sized, uniform micro-
30/70 [v/v]). After 15 min of emulsification, TPP (pH 5.0
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particles with high entrapment efficiency by controlled ionic

adjusted with 2% v/v acetic acid) was titrated into chitosan
gelation of the internal chitosan phase using acidified TPP.
solution (rate 0.5 mL/min and volume equal to chitosan so-
Hyperacute bacterial conjunctivitis is a severe, sight-
lution) over a period of 1 h at 2,000 rpm. Chitosan micro-
threatening ocular infection that calls for immediate oph-
particles were washed using 100 mL of isopropyl alcohol with
thalmic follow-up. Similarly, endophthalmitis is a frightening
agitation for 10 min at 200 rpm. Microparticles settled down
condition which occurs in 0.05%–0.5% of routine ocular
after 12 h at 4–8C and the top oily phase layer was removed
surgery.9 Regardless of the use of recent antibiotics, *50%
by aspiration. Subsequent washes were done with isopropyl
of the eyes inflicted with this condition are lost.10 Thus, there
alcohol until no residual oil was observed by optical micro-
is a need to treat this potentially scary condition effectively.
scope. Different batches of microparticles were prepared by
Tobramycin sulfate (TS) is a broad-spectrum aminogly-
varying the concentration of chitosan, TPP, and TS (Table 1).
coside antibiotic used in the treatment of infections caused
Microparticles were sterilized by gamma radiation at a dose
by pseudomonas and a variety of Gram-negative bacilli.10
of 2.5 Mrads gamma rays for 18 h (BRIT, Mumbai, India).
TS is superior to gentamicin and kanamycin against Pseu-
domonas, Staphylococci, and many gram negative organ-
isms.11 However, eye drops of TS require 4–5 times Evaluation of microparticles
administration a day due to rapid clearance, causing patient
incompliance as well as reduced therapeutic efficiency. Percent drug loading and percent encapsulation effi-
Thus, the objective of the present investigation was to ciency. One hundred milligram microparticles were di-
prolong the residence time of TS on the surface of the eye gested in 15 mL simulated tear fluid (STF) with pH 7.4
with slow release to attain an effective drug concentration at (NaCl; 0.670 g, NaHCO3; 0.2 g, CaCl2; 0.008 g in 100 mL
the intended site of action for a sufficient period of time. To water) for 48 h with stirring and subsequently centrifuged at
accomplish the objective, mucoadhesive microparticles for 16,000 rpm to remove polymeric debris. To the 100 mL of
dispersion into in situ gel were designed, which can be in- supernatant, 1 mL 0.2%w/v ascorbic acid in dimethyl sulf-
stilled easily as eye drops and will gel upon instillation. oxide (DMSO) was added, volume was made upto 10 mL
with DMSO, heated for 30 min, and finally analyzed colori-
Methods metrically at 390 nm13 (Double Beam UV Spectrophotometer
2401; Shimadzu Corporation, Japan). Percent drug loading
TS (off-white powder, 1 mg TS was equivalent to 700 mg and encapsulation efficiency (Table 1) were calculated ac-
tobramycin) was gifted by Syntho Pharmaceuticals, Lucknow, cording to the following formula14

Table 1. Composition, Drug Loading, Encapsulation Efficiency, and Diameter of Microparticles

Formulation Chitosan TPP Drug (% w/w Encapsulation
code (%w/v) (%w/v) of polymer) Drug loading (%)a efficiency (%)a Diameter (mm)b
F1 1.25 0.2 20 9.495 – 0.11 68.40 – 1.30 7.5 – 0.14
F2 1.50 0.2 30 18.75 – 0.92 81.69 – 1.22 8.5 – 0.21
F3 1.25 0.4 30 13.00 – 1.12 65.75 – 1.17 4.6 – 0.13
F4 1.25 0.2 30 13.75 – 1.29 71.05 – 1.10 7.0 – 0.11
F5 1.50 0.4 30 16.24 – 1.26 79.73 – 0.82 6.8 – 0.16
F6 1.50 0.4 20 10.25 – 0.91 75.20 – 1.19 8.0 – 0.16
F7 1.25 0.4 20 8.50 – 0.73 63.95 – 1.16 6.0 – 0.09
F8 1.50 0.2 20 11.75 – 0.81 75.36 – 1.04 8.1 – 0.19
a
Mean of 3 observations –SD.
b
Mean of 200 particles –SD.
SD, standard deviation; TPP, tripolyphosphate.
 IN SITU GEL WITH TOBRAMYCIN-LOADED MICROPARTICLES 3

Weight of drug in microparticles microparticles were studied by METTLER differential

% Drug loading ¼ · 100 scanning calorimetry (DSC) 30S (Mettler Toledo, Switzer-
Weight of microparticles
(1) land). Approximately 2–10 mg of sample was placed in the
aluminium pan and hermetically sealed. The sample was
heated under a nitrogen flow (30 mL/min) at a scanning rate
Actual drug loading of 10C/min from 30 to 400C (Fig. 1).
% Encapsulation efficiency ¼ · 100
Theoretical drug loading
(2)
Particle size and surface morphology. Particle size was
estimated by the imaging system (Metzar-M; Ark Associ-
ates, Mumbai, India). Microparticles were placed on the
Differential scanning calorimetry. Thermal behavior of glass slide and average particle size of 200 microparticles
TS, physical mixture of TS, and chitosan and drug-loaded was calculated (Table 1).
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FIG. 1. Differential scan-

ning calorimetry of (a) TS,
(b) chitosan, (c) physical
mixture of TS and chitosan,
and (d) chitosan microparti-
cles loaded with TS. TS, to-
bramycin sulfate.
 4 KHAN, WARADE, AND SINGHAVI

FIG. 2 Scanning electron micro-

scopic photograph of (a) single
microparticle and (b) multiple mi-
croparticles.

For surface morphology, microparticles were placed on a Preparation of in situ gel containing drug-loaded
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double-sided adhesive plate, one side of which was stuck to microparticles

the glass slide. Excess microparticles were removed, slide
was kept on the sample holder, and scanning electron mi- Poloxamer 407 was soaked in water and kept overnight in
croscopic photograph was taken15 ( JEOL JSM6380A, Japan) refrigerator for complete hydration.18 Chitosan HCl (0.5%–
(Fig. 2). 1.5% w/v) was added to the poloxamer solution (at 10C)
with stirring. NaCl, 0.9% w/v, was then added and pH was
In vitro drug release. Drug release was studied using 2- maintained between 4.0 and 5.0. The solution was sterilized
chamber jacketed diffusion cell (EDC-02; Electrolab, Chennai, by passing through a 0.2 mm membrane filter (Ultipor, N66;
India) with 15 mm cell diameter and 7.5 mL capacity of receptor Pall Life Sciences Pvt. Ltd., India) and to it TS-loaded mi-
compartment. A piece of dialysis membrane (MW 12000- croparticles previously sterilized by gamma radiation were
14000KDa; Himedia Ltd., India) previously soaked in STF for dispersed aseptically (Table 2).
24 h was mounted on the diffusion cell.16 Microparticles
equivalent to 300 mg tobramycin was placed in the donor com- Evaluation of in situ gel containing drug-loaded
partment, and the receptor compartment contained STF. Tem- microparticles
perature and stirring speed were 35C – 0.5C and 50 rpm, Gelation behavior. The phase transition behavior was
respectively. At predetermined intervals, a 0.5 mL sample was investigated by measuring elastic modulus G¢ and viscous
withdrawn from the receptor compartment, to it 1 mL 0.2%w/v modulus G† upon temperature sweep and time sweep using
ascorbic acid in DMSO was added, volume was made upto 5 mL thermostated oscillatory rheometer (PHYSICA MCR 301;
with DMSO, heated for 30 min, and finally analyzed colori- Anton Paar, Germany) equipped with a cone having 50 mm
metrically at 390 nm. Absorption and TS concentration showed diameter and 1 angle. The oscillatory parameters were eval-
good linearity from 4 to 80 mg/mL in STF with R2 0.998 (Fig. 3). uated over the temperature range of 10–40C at a heating rate
of 0.5C/min (Fig. 5A). Angular frequency was 1 Hz and the
Interaction studies. To examine interaction between TS constant strain amplitude was 1%.19 Time sweep was recorded
and polymer and to study the effect of gamma radiation, TS, at 35C for 10 min (Fig. 5B). Gelation time was determined by
physical mixture of TS and polymer, unsterilized and ster- the crossover time between G¢ and G†.20
ilized TS-loaded microparticles (Fig. 4) were analyzed by
Fourier transform infrared spectroscopy17 in the range of Viscosity and rheological behavior. Viscosity was mea-
400–4,000 cm-1 (Model 84005; Shimadzu Asia Pacific Pvt. sured using Brookfield’s Viscometer (CAP 2000 +; Brook-
Ltd., Singapore). field Engineering Laboratories, Inc.). Two drops of sample
maintained at 10C were placed on the temperature-sensitive
plate and temperature was increased at a rate of 1C/min.
Cone no. 3 was held on the plate and viscosity was noted at
the shear rate 133/s over the period of 30 s21(Fig. 5C).
For rheological behavior, 2 drops of formulation were
placed on the temperature-sensitive plate of the Brookfield
Viscometer. Cone no. 3 was held on the plate and shear
stress was calculated for increasing rate of shear at
35C – 1C (Fig. 5D).

Mucoadhesive strength and in vitro duration of mu-

coadhesion. Mucoadhesive strength was measured on a
modified physical balance.22 A section of freshly excised
cornea of an adult goat was fixed onto 2 glass vials with the
mucosal surface facing outside. One vial with membrane
was attached to the balance in inverted position, whereas the
FIG. 3 Drug release profiles of microparticles. second vial was placed on a height-adjustable pan. A 0.1 mL
 IN SITU GEL WITH TOBRAMYCIN-LOADED MICROPARTICLES 5

Duration of mucoadhesion was assessed by applying 1 g

formulation containing 0.1% Blue Lake (Victoria blue R;
Sigma-Aldrich Chemical Pvt. Ltd., Bangalore, India) on the
excised corneal surface (method of corneal separation de-
tailed under in vitro permeation) attached over a polyeth-
ylene plate fixed at an angle of 40 relative to the horizontal
plane. STF maintained at 37C – 1C was pumped over the
tissue (1 mL/min) and the duration for complete washing of
the formulation was recorded.24

In vitro drug release. Drug release from microparticle-

laden in situ gel was done as described previously. One
hundred microliters of formulation containing 300 mg of to-
bramycin was placed in the donor compartment and STF in
the receptor compartment. Temperature and stirring speed
were 35C – 0.5C and 50 rpm, respectively. A 0.5 mL sam-
ple was collected from the receptor compartment at pre-
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determined time intervals over the period of 11 h and analyzed

colorimetrically at 390 nm as described previously (Fig. 6A).

In vitro permeation. For permeation study, the whole

goat eye ball obtained from the local abattoir was placed in
the cold normal saline. The cornea was removed within 1 h
of slaughter, washed with the cold normal saline until the
washings were free of protein, and subsequently mounted on
the 2-chamber diffusion cell as described in the in vitro drug
release study. Formulation equivalent to 300 mg of to-
bramycin was placed on the cornea and STF was filled in the
receptor compartment.25 Samples were collected for 12 h at
predetermined time intervals and were analyzed according
to the method described in the in vitro drug release study of
microparticles (Fig. 6B).

Ocular irritation test and estimation of tobramycin in

aqueous humor. The experimental protocol was approved
by the Institutional Animal Ethics Committee (CPCSEA no.
2014-15/16). All institutional guidelines for the care and use
of laboratory animals were followed, including the Asso-
ciation for Research in Vision and Ophthalmology Statement
for the Use of Animals in Ophthalmic and Visual Research.
Six rabbits (2–3 kg) were used for the study. The left eyes of
animals were marked as control (no sample), whereas right
eyes were marked as test. The test eyes received 100 mL of
formulation twice daily for 7 days. The irritancy was tested at
1, 24, 48, 72 h, and a week after administration, and the eyes
were observed for redness, swelling, and watering.26 The
FIG. 4 Infrared spectra of (a) TS, (b) chitosan, (c) phys- scores were assigned as follows:
ical mixture of TS and chitosan, and (d) microparticles 0: No redness, inflammation, or tearing 1: Mild redness,
loaded with TS. inflammation, and slight tearing 2: Moderate redness, in-
flammation, and excessive tearing 3: Severe redness, in-
formulation was placed onto the membrane of the second flammation, and excessive tearing.
vial and the height was adjusted so that the mucosal surfaces For estimation of drug in aqueous humor, 28 albino rabbits
of both vials came into close contact. The mucosal surfaces (2–3 kg) were divided into 2 groups, group I consisted of 12
were held in contact for 2 min, and then weights were placed animals, whereas group II had 16 animals. Both eyes were
on the pan until vials got detached. Mucoadhesive strength used for the study. Each rabbit was kept separately under a
was calculated by the following equation23 (Table 2): well-controlled environment (21C – 1C, 55% – 1% relative
humidity, 12-h light–12-h dark cycle) with food and water ad
   libitum. In group I 100 mL, (2 drops, with 300 mg of to-
Mucoadhesive strength dynes cm2 ¼ mg=A (3) bramycin) of Toba eye drops (0.3%w/v) was instilled in each
eye, whereas group II received 100 mL (contains 300 mg of
where, m is the weight required for detachment (g), A is the tobramycin) of in situ gel containing TS-loaded microparti-
area of mucosa (cm2), g is the acceleration due to gravity cles (Formulation P2 was selected as it gave highest in vitro
(980 cm/s2). drug permeation). The rabbits were anesthetized by i.v.
 6 KHAN, WARADE, AND SINGHAVI

Table 2. Composition, Gelation Behavior, and Mucoadhesive Property

of Microparticles Dispersed In Situ Gel
Batches
Ingredients P1 P2 P3 P4
a,c
Tobramycin microparticles (mg) 1142.82 1142.82 1142.82 1142.82
Poloxamer 407 (%w/v) 17.0 17.0 17.0 17.0
Chitosan HCl (%w/v) 0.0 0.5 1.0 1.5
Sodium Chloride (%w/v) 0.9 0.9 0.9 0.9
Benzalkonium chloride (%w/v) 0.05 0.05 0.05 0.05
Water (upto mL) 50 50 50 50
Properties
b
Gelation time (s) 48 – 10 40 – 5 35 – 10 30 – 8
b
Gelation temperature (C) 33 – 1.0 32 – 1.5 31 – 1.2 30 – 1.0
b
Mucoadhesive force (dynes/cm2) · 102 8.0 – 0.54 25 – 0.86 32 – 0.75 35 – 0.65
b
Duration of mucoadhesion (h) 4.5 – 0.3 8.85 – 0.25 9.16 – 0.35 9.30 – 0.36
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a
Weight of microparticles equivalent to 150 mg of tobramycin.
b
Results are the mean of 5 observations –SD.
c
Microparticles were prepared according to the composition of F2.

injection of ketamine (30 mg/kg) and 100 mL of aqueous upto 5 mL with distilled water and allowed to stand for
humor was aspirated through the limbus with a 26 gauge 15 min. Twenty microliter sample was injected in the chro-
needle at 0.25, 0.5, 1, 2, 4, and 8 h from animals in group I matograph ( JASCO LC-2000 Plus; JASCO Corporation,
and at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h from animals in Japan). The eluents were detected by the fluorescence detec-
group II. Two animals (4 eyes) were used for each time point tor with the wavelength of excitation fixed at 390 nm and that
for each formulation. The aqueous humor was vortexed for of emission fixed at 480 nm. The column was HiQ Sil C18
30 s, and then centrifuged (15 min at 2,000 rpm). To the su- HS (250 · 4.6 mm i.d., particle size 5 mm; KYA Tech Cor-
pernatant, 2 mL borate buffer (pH 8.5) was added followed poration, Japan). The mobile phase (methanol:water [60:40,
by 1.5 mL Fluorescamine (0.01%w/v), and volume was made v/v]) was pumped at the flow rate of 1 mL/min.27

FIG. 5 (A) Temperature dependence of elastic modulus (G¢) and viscous modulus (G†) for formulation P2. (B) Time
dependence of elastic modulus (G¢) and viscous modulus (G†) at 35C for formulation P2. (C) Viscosity of various in situ
gels containing drug-loaded microparticles under increasing temperature. (D) Rheological behavior of in situ gel containing
drug-loaded microparticles.
 IN SITU GEL WITH TOBRAMYCIN-LOADED MICROPARTICLES 7

crosslinking of chitosan in the internal phase during emul-

sification–ionic gelation process. As pH greatly influences
the particle size and size distribution, in the trial batches, pH
of chitosan solution varied from 3.5 to 6.0 (3.5, 4.0, 4.5, 5.0,
5.5, 6.0) and that of TPP from 3.5 to 9.0 (3.5, 4, 4.5, 5.0, 5.5,
6.0, 7.0, 9.0), respectively, to decide on the pH condition
that will facilitate the formation of small-sized, homoge-
neous microparticles. With pH of TPP solution 5.0 and
chitosan solution 4.5, desired-size (<10 mm) microparticles
were obtained (Table 1).
Drug loading ranged from 8.50 – 0.73 to 18.75% – 0.92%
w/w, whereas percent encapsulation efficiency ranged from
63.95 – 1.16 to 81.69 – 1.22 (Table 1). Percent drug loading
was found to depend on the percent of drug by weight of
polymer, concentration of chitosan, and TPP. With increase
in drug level and chitosan concentration, encapsulation ef-
ficiency also increased, whereas, both drug loading and
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encapsulation efficiency decreased with increase in TPP

concentration.
DSC of TS showed a melting endotherm at 273C, whereas
chitosan showed an endothermic peak at about 97C due to
dehydration, whereas an exothermic peak from 274C to
332C due to degradation.28 Physical mixture of chitosan
and TS showed a broad endothermic peak at 99.5C and the
melting endotherm of the drug at 272C (Fig. 1). The DSC of
TS-loaded microparticles showed an endotherm near 99C
attributed to dehydration and a shift in the melting peak of
drug to 255C (Fig. 1).
Microparticles were sized less than 10 mm (Table 1) and
FIG. 6 (A) Drug release profiles of various in situ gels were almost spherical (Fig. 2). Particles >10 mm is not
containing TS-loaded microparticles. (B) Permeation pro- suitable for ophthalmic delivery as they cause irritation and
files of TS from Toba eye drops and various in situ gel reflex tearing.29,30 The size of microparticles varied with
containing drug-loaded microparticles. polymer concentration and extent of crosslinking. Size in-
creased with chitosan concentration, whereas it decreased
Tobramycin was estimated from the calibration curve with the level of TPP.
prepared using spiked aqueous humor from concentration 20 Formulations F1–F8 showed sustained drug release,
to 200 ng/mL. ranging from 69.32% to 94.07% in 8 h (Fig. 3). Release rate
Pharmacokinetic parameters of Toba eye drops and depended upon the concentration of chitosan and TPP. All
formulation P2 in the aqueous humor were calculated using the formulations showed maximum R2 value for zero-order
Kinetica (4.4.1; Thermo) by noncompartment analysis kinetics (>0.99) and n values for Korsmeyer–Peppas equa-
(Table 3). tion were between 0.88 and 0.91
Comparison was done between the mean data of the 2 IR spectroscopic analysis depicted retention of principal
formulations using unpaired (2-tailed) t-test. The level of peaks of TS at 3,253, 3,276, 3,323, 3,336, and 3,348 cm-1
significance was set at P < 0.05. due to N-H stretching and at 3,500, 3,504, and 3,510 cm-1
because of O-H stretching groups in the physical mixture of
Results TS and polymer and in the IR spectrum of microparticles
after sterilization (Fig. 4). No irradiated product was formed
Homogeneous, low-sized microparticles with high en- in the microparticles after sterilization by gamma radiation.
trapment of tobramycin were obtained by controlled ionic Because of high drug content, percent entrapment effi-
ciency, and better control in drug release with more than
90% drug release in 8 h, formulation F2 was selected for
Table 3. Pharmacokinetic Parameters dispersion into the in situ gel.
of Tobramycin in Aqueous Humor of Rabbit’s Eyes To find appropriate in situ gelling composition, concen-
Parameters Toba eye drops P2 tration of poloxamer 407 was varied from 15%–20%w/v in
the trial batches. At 20%w/v poloxamer concentration, ge-
Cmax (lg=ml) 2.25 – 0.55 19.44 – 2.27a lification was noticed below 27C, whereas 15% w/v po-
Tmax (h) 2 1 loxamer solution was liquid at the physiological temperature
AUC0-tlast (lg
ml :h) 10.99 – 3.02b 269.76 – 28.23a,b (35C). Therefore, 17%w/v poloxamer solution, which
t1/2 (h) 1.66 – 0.63 6.38 – 0.15a showed phase transition at 33C was selected and combined
MRT (h) 3.53 – 0.06 10.66 – 0.13a with different concentrations of chitosan. Temperature and
a
P < 0.0001 (unpaired t-test). time dependence of elastic modulus (G¢) and viscous mod-
b
tlast was 8 h for Toba eye drops (the time till it could be ulus (G†) was evaluated to determine gelation temperature
detected) and 24 h for P2. and gelation time. Below 30C, the elastic modulus shows
 8 KHAN, WARADE, AND SINGHAVI

low values indicating that the formulations were fluidic and

were characterized as viscous sols with higher G† (Fig. 5A
shown for formulation P2). A sharp rise in elastic modulus
between 32 and 33C for P2 indicates formation of gel with
elastic behavior. At gelation temperature, G¢ crosses G† and
becomes higher than G†.20 Formulations gelled rapidly and
the elastic modulus G¢ was higher than G† in less than a
minute for all formulations (Fig. 5B shown for P2).
Chitosan was found to modulate both gelation temperature
and time. The effect was apparent at higher concentration
(Table 2). Formulation P2 and P3 containing 0.5 and 1.0% w/v
chitosan in addition to 17%w/v poloxamer showed insignifi-
cant difference in gelation temperature compared with P1 with
17% w/v poloxamer (P > 0.05, repeated measures ANOVA FIG. 7 Aqueous humor tobramycin concentration–time
with Friedman post test). With higher chitosan concentration profile from eye drops and formulation P2.
(1.5%w/v), gelation temperature reduced significantly (P < 0.05)
from 33C – 1.0C (for P1) to 30C – 1.0C (for P4).
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Formulations showed sudden rise in viscosity above 30C tively. Whereas, the Cmax (19.44 – 2.27 mg/mL) for formu-
due to temperature-mediated gelation (Fig. 5C) and showed lation P2 was 38.3, 97, and 194 times the MIC of E. coli, P.
pseudoplastic behavior. Addition of chitosan resulted in aeruginosa, and S. aureus, respectively. The AUC0-tlast of to-
greater rise in viscosity above 30C. bramycin in the aqueous humor from formulation P2 (269.76 –
In situ gel containing chitosan (P2–P4) showed greater 28.23 mg$h/mL) was significantly higher than (P < 0.05, Stu-
mucoadhesive strength than in situ gel without chitosan dent’s t-test) Toba eye drops (10.99 – 3.02 mg$h/mL).
(P1). With the increase in chitosan concentration from 0.5
to 1.5% w/v (P2–P4), both mucoadhesive strength and Discussion
duration increased.
Microparticle-laden in situ gels (P1–P4) displayed slower Degree of protonation of chitosan essentially depends
drug release rate compared with the drug release rate from upon pH, since it is a weak polycationic polymer with a pKa
the microparticles F2 (Fig. 6A). Zero-order release property 6.5. When the pH of chitosan solution was less than 4.5,
was observed for all formulations as correlation coefficient microparticles could not be formed because of the strong
was maximum (>0.99) for zero-order model. Slope of electrostatic repulsion between chitosan chains with highly
Korsmeyer–Peppas equation ranged from 0.86 to 1.06 in- protonated amino groups, whereas above pH 5.0, large-sized
dicating non-Fickian anomalous to case II transport. microparticles resulted because chitosan chains were less
Permeation of TS was 39.45% in 12 h from Toba eye extended. TPP solution had high concentration of tripoly-
drops, whereas from microparticle-laden in situ gel P1 phosphoric ions along with hydroxide ions at the pH of 9.0.
containing poloxamer, it was 59.91% in 12 h. However, in The tripolyphosphoric ions and hydroxide ions competi-
the same duration, drug permeation was 84.82% from mi- tively react ionically with the protonated amine groups of
croparticles dispersed in P2 having poloxamer along with chitosan. Hydroxide ion preferentially binds to protonated
0.5% chitosan (Fig. 6B). Permeability rate of tobramycin amine of chitosan because of greater mobility, which con-
from P2 was 1.5-fold greater than that from P1and 2-folds sequently caused high degree of intermolecular hydrophobic
that of Toba eye drops. interaction generating large-sized particles. When the pH of
The formulation was well tolerated with no sign of red- TPP solution was reduced, there was partial neutralization
ness and inflammation (Score 0). Very small size of mi- of hydroxide ions with consequent low effect on the degree
croparticles (<10 mm) with a layer of in situ gel surrounding of protonation of chitosan and the protonated amine groups
them was responsible for better tolerance and absence of were linked to tripolyphosphoric ions. With pH of TPP so-
irritation in the eyes. lution 5.0 and chitosan solution between 4.5 and 5.0, re-
The aqueous humor tobramycin concentration versus time spectively, controlled interaction between amino groups and
profiles of Toba eye drops and formulation P2 containing tripolyphosphoric acid could be accomplished within the
TS-loaded microparticles dispersed into in situ gel is depicted internal phase, resulting in low-sized, uniform particles.
in Fig. 7. Therapeutically effective concentration of tobramycin Formulations prepared with high percentage of TS showed
against Pseudomonas aeruginosa (minimum inhibitory con- greater loading because of ample accessibility of drug to be
centration [MIC] 0.2 mg/mL) and Staphylococcus aureus (MIC entrapped in the microparticles. With increase in chitosan
0.1 mg/mL)31,32 was achieved within 30 min and effective con- concentration, entrapment efficiency also increased because
centration against all the sensitive microorganisms, including of high crosslink density, which prevented the rapid loss of
E. coli (MIC 0.5 mg/mL)31,32 was achieved within 1 h and was the drug.33 High level of TPP resulted in greater constriction
maintained for around 4 h. Greater tobramycin concentration of microparticles and reduction in free volume spaces within
in the aqueous humor was obtained by formulation P2 com- the microparticles owing to crosslinking with consequent
pared with eye drops. Therapeutically effective concentration squeezing out of TS in the surrounding medium.33
against all the microorganisms was attained within 15 min Size of microparticles increased with chitosan concen-
and high level was recovered in the last sample at 24 h. The tration in the range 1.25–1.5%w/v, because plenty of chit-
Cmax for Toba eye drops was found to be 2.25 – 0.55 mg/mL, osan molecules were involved in the crosslinking. At low
which was nearly 4 times the MIC of E. coli, and 11 and 21 chitosan concentration, the intermolecular distance in-
times that of MIC of P. aeruginosa and S. aureus, respec- creases, which decreases intermolecular crosslinking density
 IN SITU GEL WITH TOBRAMYCIN-LOADED MICROPARTICLES 9

between chitosan and TPP. However, when the concentra- the corneal tear film. The ocular shear rate ranges from 0.03/s
tion of TPP increased, smaller particles resulted due to in- during interblinking to 4,250–28,500/s during blinking.41
creased crosslinking density between chitosan and TPP With Newtonian systems, if viscosity is kept high (to re-
favoring formation of compact, small-sized particles. duce drainage), blinking becomes painful, followed by reflex
Microparticles were spherical, almost smooth, with few tearing, because Newtonian fluid shows increase in shear
microscopic pores on the surface. The spherical shape may stress with increasing shear rate. However, pseudoplastic
be because of high degree of crosslinking.12 systems exhibit a decrease in viscosity with increasing shear
There was no interaction between TS and chitosan as rates, thus they do not cause pain and discomfort.
melting peak of TS was retained at 272C in the DSC of Poloxamer is reported to have negligible mucoadhesive
physical mixture. However, the shift in the melting endo- strength, yet formulation P1 had fair mucoadhesive strength.
therm of TS to the lower temperature (255C) and the In fact, chitosan microparticles suspended in the gel hy-
broadness of the peak in the DSC of microparticles clearly drates and consequently interact with mucin. Formulation
indicates that the drug was successfully entrapped in the P2–P4 had stronger mucoadhesive strength as a conse-
microparticles and there was decrease in its crystallinity upon quence of mucoadhesion conferred by chitosan containing
entrapment. Slowest drug release from formulation F5 might gel and microparticles together. Microparticles were pre-
be because of poor diffusion of drug through the highly pared by bringing about limited interactions between amino
compact microparticles formed due the presence of high level groups and phosphoric ions, thus ample free amino groups
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of both chitosan and TPP in it. Abundant chitosan molecules remain to interact with the mucin in addition to the chitosan
involved in the crosslinking resulted in slow drug release. present in the gel. Stronger electrostatic interaction between
Drug release occurred due to diffusion as well as chain re- positively charged amino group and negatively charged si-
laxation attributable to waning crosslinking with time. alic acid residue of mucin owing to plentiful involvement of
IR spectroscopy confirms the absence of interaction be- chitosan chains at high concentrations yielded increased
tween TS and chitosan and formation of any irradiated mucoadhesive strength and duration.42
product following sterilization. Slower TS release from the microparticles dispersed in
Microparticles were incorporated into the in situ gel to get a gel is attributable to the slower diffusion of TS released
soothing effect, better retention, and permeation. Poloxamers from microparticles through the viscous gel layer sur-
are widely employed as thermosensitive in situ gel-forming rounding them. Increased chitosan concentration in the gel
polymers; however, poloxamer gels have weak mechanical resulted into slower TS release rate because of the increased
strength and they tend to erode rapidly.34 Therefore, chitosan viscosity of the surrounding diffusion layer. Furthermore,
was added to improve the mechanical strength and prolong chitosan reinforces and improves mechanical property of
the contact time of the formulation with the ocular surface. poloxamer gel, enhances poloxamer chain entanglement,
Chitosan has excellent ocular compatibility in addition to the and reduces gel erosion which caused slow drug release.
mucoadhesive and permeability-enhancing property.35,36 Tobramycin is very polar (logp; -5.8)43 and like other
Preferably the formulation should gel within a short time aminoglycoside antibiotic, such as gentamicin, it has very
upon administration to prevent quick removal from the poor corneal permeability.31 Permeability was highest from
precorneal space. With higher concentration of chitosan, P2 possibly due to greater availability of positively charged
viscosity of the sol increased enough to facilitate the mo- amino group to interact with the negatively charged sites on
lecular entangling and packaging of poloxamer chain. Also, the cornea. The inner part of tight junctions is greatly hy-
at high concentration, the chitosan chains were less ex- drated and contains fixed negative charges. Any modifica-
tended favoring quick gelation. tion in the relative concentration of specific ionic species in
Gelation temperature should most preferably be between the pore volume would result in alteration in tight junction
30C and 35C (the precorneal temperature). If gelification resistance, which might lead to loosening or opening of the
occurs below 30C, the already gelled formulation will be pore.44 P1 demonstrated lesser permeability than P2, as it
difficult to be administered and if gelation occurs above had chitosan microparticles dispersed in simply poloxamer
35C, the formulation would rapidly drain out from the solution, and availability of protonated amine group merely
precorneal space. from microparticles may be scarce since they were involved
The decrease in gelation temperature with higher con- in crosslinking. However, 0.5% chitosan in poloxamer sol in
centration level of chitosan may be attributed to the greater addition to dispersed chitosan microparticles in P2 were
dehydration and entanglement of poloxamer chain due to responsible for providing more protonated amine group and
ample viscosity increase by chitosan, which is corroborated greater permeability. Further increasing chitosan concentration
by high viscous modulus G†. Chitosan could have facilitated in poloxamer sol as in P3 and P4 did not increase the perme-
the accommodation of unbound water derived from micelle ability, which indicates the saturation in the permeability-
core dehydration.37–39 increasing capacity of chitosan. The rate of permeability rather
At low temperature, poloxamer chains were in hydrated decreased in these formulations perhaps because of the slower
condition, whereas at temperatures above 30C, the dehy- drug release rate.
dration of polypropylene oxide fraction of poloxamer chain High aqueous humor concentration of tobramycin from
with subsequent chain entanglement occurred. Chitosan fa- formulation P2 was first due to high retention of the for-
cilitates poloxamer chain entanglement and packing within mulation in the cul de sac. The decreased loss of formulation
the micelles by holding the unbound water produced by de- is due to gelification on administration and mucoadhesion
hydration of polypropylene core. Greater chain entanglement by chitosan present in the in situ gel as well as microparti-
resulted in greater viscosity.37,40 Pseudoplastic behavior cles dispersed in it. Second, higher aqueous humor con-
shown by formulations is the desirable property, as formu- centration was also because of the penetration-enhancing
lation should not affect the pseudoplastic characteristics of effect of chitosan on the cornea. Chitosan is demonstrated to
 10 KHAN, WARADE, AND SINGHAVI

be able to transiently open the tight junctions when applied sification/internal gelation technique. Eur. J. Pharm. Sci. 25:
to the confluent cell layer.45 TS being polar, could have been 31–40, 2005.
transported paracellularly through the tight junctions opened 13. Pesez, M., and Bartos, J. Colorimetric and Fluorimetric
by chitosan to get access in the aqueous humor. Analysis of Organic Compounds and Drugs. New York:
Verlag Marcel Dekker, Inc.; 1974.
Conclusion 14. Sairam, M., Babu, V.R., Naidu, B.V.K., and Aminabhavi,
T.M. Encapsulation efficiency and controlled release
TS-loaded microparticles dispersed in thermosensitive in characteristics of crosslinked polyacrylamide particles. Int.
situ gel gave sustained drug release together with enhanced J. Pharm. 320:131–136, 2006.
permeation and greater tobramycin concentration in the 15. Wang, L.Y., Ma, G.H., and Su, Z.G. Preparation of uniform
aqueous humor compared with eye drops. Because of the sized chitosan microspheres by membrane emulsification
rapid attainment and prolonged maintenance of therapeutically technique and application as a carrier of protein drug. J.
effective concentration against all the important organisms, this Control. Release. 106:62–75, 2005.
formulation holds promise for effective treatment of en- 16. Ceulemans, J., and Ludwig, A. Optimisation of carbomer
viscous eye drops: an in vitro experimental design approach
dophthalmitis and bacterial conjunctivitis.
using rheological techniques. Eur. J. Pharm. Biopharm. 54:
41–50, 2002.
Author Disclosure Statement 17. Khan, S., Ali, A., Singhavi, D., and Yeole, P. Controlled
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ocular delivery of acyclovir through rate controlling ocular

No competing financial interests exist. insert of eudragit: a technical note. AAPS PharmSciTech. 9:
169–173, 2008.
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