des cyclosporine a nanoemulsion formulation chapter...

DES Cyclosporine A Nanoemulsion Formulation

DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM 130

Chapter 6

Jamia Hamdard

6.1. Introduction

Ocular drug delivery system requires a series of specified characteristics according to the

physiological structure of the eye. Eye is a unique and challenging organ for therapeutic drug

delivery on to the surface as well as in the interior part of ocular structure. Many of its

anatomical and physiological makeup interfere with the fate of the administered drug and

bioactives. Tears permanently wash the surface of the eye and exert an anti-infectious activity

by the lysozyme and immunoglobulins they contain. In addition, drug may bind to tear

proteins and conjunctival mucin. To treat the local ophthalmic diseases, liquid eye drop is

the most desirable dosage form when considering convenience of administration and clinical

compliance of the patients. However, conventional eye drops, most of which present in the

drug solution form, usually have quite a limited therapeutic efficiency due to the low

bioavailability. In clinical use of eye drops, frequent instillations are often required to get the

expected therapeutic effect, and this leads to rising inconvenience and adverse effects.

Typically, less than 5% of the drug applied penetrates the cornea/sclera and reaches the

intraocular tissue, with the major fraction of the dose applied often absorbed systemically

through the conjunctiva and nasolacrimal duct. On the other hand, corneal and conjunctival

epithelia of human eye, along with the tear film, construct a compact barrier preventing the

drug absorption into the intraocular area resulting into low bioavailabilty and undesirable

systemic side effects (Lang., 1995). So, drug delivery in ocular therapeutics is a challenging

concern and is a subject of interest to scientists working in the multidisciplinary areas

pertaining to the eye (Bourlais et al., 1998). In general, the major problem in ocular

therapeutics is to maintain an effective drug concentration in ocular tissue or at the site of

action for a significant period of time, in order to achieve the expected therapeutic response.

Ophthalmic drug delivery, probably more than any other route of administration, may get

benefit from the uniqueness of Nanoapproaches -based drug delivery (Sahoo et al., 2003).



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The use of nanocarriers provides attractive prospect for topical ocular drug delivery, mainly

because of their capacity to protect the encapsulated molecule, along with its facilitated

transport to the different compartments of the eye (Losa et al., 1993; Kayseri et al., 2005).

Various nano systems were adopted for the corneal retention and bioavailabilty enhancement

colloidal systems such as liposomes (Pleyer et al., 1993; Bochot et al., 1998) and

nanoparticles (Losa et al., 1991; De Campos et al., 2004) and nanocapsules (Losa et al.,

1993; De Campos et al., 2003) and nanoemulsions. Nanoemulsions (NEs) are defined as the

dispersions of water and oil in the presence of combination of surfactant and co-surfactant

(Smix) in a manner to reduce interfacial tension. On the basis of nature of dispersion and

disperse phase, NEs were classified as: o/w, w/o & bi-continous type. These systems are

usually characterized by clear appearance, higher thermodynamic stability, small droplet size

(< 200 nm), high drug solubility, and drug reservoir for lipophilic and hydrophilic drugs

(Ansari et al., 2008). Nanoemulsions, particularly, oil/water nanoemulsions have received

the greatest attention because of their small size may provide a promising alternative. From

the point of view of production and sterilization, nanoemulsions are relatively simple and

inexpensive because they are thermodynamically stable. Nanoemulsions are also used to

formulate poorly water-soluble drugs since their structure allows solubilization of lipophilic

drugs in the oil phase. Moreover, nanoemulsions achieve sustained release of a drug applied

to the cornea and higher penetration into the deeper layers of the ocular structure and the

aqueous humor than the native drug. These systems offer additional advantages including:

low viscosity, a greater ability as drug delivery vehicles and increased properties as

absorption promoters. Earlier successful nanoemulsions of pilocarpine,timolol maleate,and

chloramphenicol have been formulated (Akhter et al, 2011). Extensive investigations carried

out over the last decade support the view that cyclosporin A (CYA), a hydrophobic peptide

with powerful immunosuppressive action, is effective in the treatment of extraocular



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disorders such as keratoconjunctivitis sicca and dry eye disease and for the prevention of

corneal allograft rejection (Power et al., 1993). However, the poor aqueous solubility of

CYA (6.6_g/ml) is a limiting factor for the formulation of solutions intended for ocular

administration (Lallemand et al., 2003). Despite the evidence that the target sites for the

treatment of these diseases are the cornea and conjunctiva (Gunduz and O zdemir, 1994;

Acheampong et al., 1999), the CYA delivery systems investigated so far (i.e., oils,

emulsions, collagen shields, liposomes and nanocapsules) have not been successful.

Collagen shields were found to provide a sustained delivery of CYA to the surface of the eye.

However, the use of such a system is limited by the ocular irritation and blurring of vision

that it causes (Dua et al., 1996). The most popular oil-based vehicles have serious limitations

that include the slow partition rate of CYA into the corneal epithelium (Acheampong et al.,

1999), the intraocular and/or systemic absorption of CYA (Foets et al., 1985; Bellot et al.,

1992), and the local side effects associated with the use of oils (symptoms of irritation,

blurred vision, itching, transient epithelial keratitis and toxic effects at corneal level (Kaswan

et al., 1989). In 2002, a CYA 0.05% lipid emulsion (RestasisTM, Allergan, Irvine, USA)

received FDA approval as the first and only therapy for patients with keratoconjunctivitis

sicca, whose lack of tear production is presumed to be due to ocular inflammation. However,

as the corneal concentration achieved with dosing four times a day is insufficient to prevent

immunologic graft reactions, RestasisTM is not effective in preventing rejection after corneal

allograft (Price and Price, 2006). Previous attempt by Calvo et al have been carried out to

improve the ocular penetration of CYA by developing CYA loaded poly-o-caprolactone

nanocapsules. These new delivery systems were efficient at improving the transcorneal

transport of CYA while reducing systemic absorption but they did not provide significant

CYA at the ocular mucosa for extended periods of time (Calvo et al., 1996). However

polymeric nanosystem release the entrapped drug in controlled fashion over extended period



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of time but in such cases we can’t get the therapeutic concentration at a time and sustained

the same for e longer period. So, particularly in case of infectious and immunological

diseases of eye, it is desirable that the delivery system should release the bioactive molecule

fast the elicit the immunosuppressive action. Moreover, improving the tissue penetration and

ocular retention further improve the management of diseases condition with a small dosing

regimen. Consequently, the design of a system with improved drug delivery properties to the

ocular surface would be a promising step towards the management of external ocular

diseases, such as keratoconjunctivitis sicca or dry eye disease. Taking into account this

information and also the fact that the cornea and conjunctiva have a negative charge, it was

thought that the use of mucoadhesive polymers which might interact intimately with these

extra-ocular structures would increase the concentration and residence time of the associated

drug. Mucoadhesive nanoemulsion system as a carrier for CYA, may play a key role and

fulfilled the desirable characteristic required for effective CYA delivery. Among the

mucoadhesive polymers investigated until now, the cationic polymer chitosan (CH) has

attracted a great deal of attention because of its unique properties, such as acceptable

biocompatibility and biodegradability (Knapczyk et al., 1989; Hirano et al., 1990) and

ability to enhance the paracellular transport of drugs (Artursson et al., 1994). Moreover, CH

has recently been proposed as a material with a good potential for ocular drug delivery. More

specifically, CH solutions were found to prolong the corneal residence time of antibiotic

drugs (Felt et al., 1999), whereas CH coated nanocapsules were more efficient at enhancing

the intraocular penetration of some specific drugs (Calvo et al., 1997a; Genta et al., 1997).

More recent work has shown the interaction and prolonged residence time of CH

nanoparticles at the ocular mucosa after their topical administration to rabbits (De Campos et

al., 2009). Based on these consideration, chitosan based mucoadhesive nanoemulsion

encapsulate with 3 % w/v of CYA were developed and characterised for in-vitro



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performance. In-vivo study to validate the corneal retention (by γ-scintigraphy technique) and

biodistribution by UPLC/Q-TOF-MS/MS method were also evaluated.

6.2. Materials and methods

6.2.1. Chemicals

Cyclosporin A (CYA) was kindly gifted by Ranbaxy Laboratories Ltd. (Gurgaon, Haryana,

India). Chitosan (CH, deacetylation degree >80%) was received as a gift sample from India

Sea Foods (India). Tween 80, tween 20, oleic acid, isopropyl merestate (IPM), olive oil,

tricetin, castor oil, triton X 100, poly ethylene glycol (PEG 200 & PEG 400), propylene

glycol, span 20, Transcutol P were purchased from CDH, Delhi , India.

Labrafil M, labrafac, labrosol, and Lauroglycol 90 were obtained from Gattefose, France.

Sefsol 218 was gifted from Nikko chemicals (Japan). Milli-Q water was produced in the

laboratory by Milli-Q water purification system (MA, USA), whereas, LCMS grade

acetonitrile was obtained from Qualigens Fine Chemicals (Mumbai, India). All other

chemicals and reagents used were of analytical grade and were purchased from Merck Ltd.

(Mumbai, India).

6.3. Methods

6.3.1. UPLC/Q-TOF-MS/MS Quantification Method for CYCLOSPORIN A

UPLC was performed with a Waters Acquity™ UPLC system (Serial No# F09 UPB 920M;

Model Code# UPB; Waters, MA, USA) equipped with a binary solvent delivery system, an

auto-sampler, column manager and a tuneable MS detector (Serial No# JAA 272; Synapt;

Waters, Manchester, UK). Chromatographic separation was performed on an Acquity UPLC

BEH C18 (100mm×2.1mm, 1.7μm) column at 35±2ºC. The mobile phase consisting of

Acetonitrile, Water and Formic Acid ( 0.1% v/v) in the ratio of 45:45:10 with a flow rate of

0.5 ml / min was employed for a total run time of 3 min. Data acquisition, data handling and

instrument control were performed by Empower Software v1.0.



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Mass spectrometry was performed on a Waters Q-TOF Premier (Micromass MS

Technologies, Manchester, UK) mass spectrometer. The nebulisation gas was set to 500 L/h,

the cone gas was set to 50 L h-1 and the source temperature was set to 100ºC. The capillary

voltage and sample cone voltage were set to 3.0 KV and 40 V, respectively. The Q-TOF

Premier™ was operated in V mode with a resolution over 8500 mass with 1.0 min scan time,

and 0.02 s inter-scan delay. The accurate mass and composition for the precursor ions and for

the fragment ions were calculated using the MassLynx V 4.1 software incorporated in the

instrument. Argon was employed as the collision gas at a pressure of 5.3 х 10-5 torr.

Quantitation was performed using Synapt mass spectrometer (Q-TOF) of the transitions of

m/z 1225.1764→1113.0751 for with a scan time of 1.0 min, and 0.02 s inter-scan per

transition. The optimum values for compound-dependent parameters like trap collision

energy and transfer collision energy were set to 13.2 and 80 eV, respectively for

fragmentation.

6.3.2. Preparation of nanoemulsion formulations

6.3.2.1. Screening & optimization of oil, surfactant and co-surfactants

The most important criterion for screening of components is the solubility of poorly soluble

drug in oils, surfactants and cosurfactants (Akhter et al., 2008). The solubility of

Cyclosporin was determined in different oils viz. oleic acid, isopropyl myristate (IPM),

olive oil, triacetin, jojoba oil, castor oil, safsol, labrafac, babchi oil and soyabean oil. 2 ml of

different oils was taken in small vials and excess amount of the drug was added. The vials

were tightly stoppered and were continuously stirred for 72 hours at 37 ± 0.5o C and samples

were centrifuged at 10,000 rpm for 10 min. The suspension was filtered through a

membrane filter (0.45 μm) and after appropriate dilution with methanol; solubility was

determined by UPLC-MS/MS. Similar method was adopted for solubility determination of

CYA in surfactant and cosurfactant. On the basis of solubility studies, oleic acid was



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selected as the oil phase. Due to slight difference in the solubility profile of drug in different

surfactants and cosurfactants, phase behaviors were studied by taking different surfactants

and cosurfactants. Total eight combinations of surfactant and cosurfactants were prepared.

The combination giving the larger nanoemulsion region was selected for the further study.

For optimization of surfactant, initially cosurfactant (Transcutol P) was kept constant and

different surfactant (Tween 20, 80, Cremophore EL and labrasol) in 1:1 ratio with

cosurfactant was used and the mixture is called as Smix. For each phase diagram, oil and

specific Smix were mixed well in different ratios. Sixteen different combinations of oil and

Smix (1:9, 1:8, 1:7, 1:6, 1:5 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1) were made

for phase diagram construction. The phase diagram was developed by aqueous titration

method. For the optimization of cosurfactant, Tween 20 and cremophore EL were taken as

surfactants. Aqueous titrations were performed by using different cosurfactants (Transcutol P,

PEG 200, PEG 400 and propylene glycol). The ratio of surfactant to cosurfactant (Smix ratio)

was kept constant (1:1 v/v) while oil to Smix ratio was taken 1:9 v/v.

6.3.2.2. Method of Preparation of nanoemulsion

After optimization of oil, surfactant and cosurfactant, nanoemulsion formulations were

developed by using oleic acid as oily phase, tween 20 and cremophore EL as surfactants and

Transcutol P as cosurfactant. For each phase diagram, Smix ratios 1:0, 1:1, 1:2, 2:1, 3:1, 1:3,

4:1, 1:4 was prepared. Oil and specific Smix were mixed well in different ratios. Sixteen

different combinations of oil and Smix (1:9, 1:8, 1:7, 1:6, 1:5 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5,

6:4, 7:3, 8:2 and 9:1) were made for phase diagram construction. For each Smix ratio separate

phase diagram was constructed.

6.3.2.3. Thermodynamic stability testing of drug loaded nanoemulsions

To evaluate the stability, CYA loaded nanoemulsions were subjected to thermodynamic

stability testing, which comprises of heating cooling cycle, freeze thaw cycle and



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centrifugation test. Physical stability was continuously monitored throughout the

experiment. Various aspects like phase separation, turbidity etc. at room temperature were

observed (Akhter et al., 2008).

6.3.2.4. Freeze thaw cycle

Selected nanoemulsions were kept in deep freezer (at -20oC) for 24h. After 24h the

nanoemulsions were removed and kept at room temperature. The thermodynamically stable

nanoemulsions returned to their original form within 2-3 minutes. 2-3 such cycles were

repeated.

6.3.2.5. Centrifugation studies

Nanoemulsions after freeze thaw cycle were subjected to centrifugation studies where they

were made to undergo centrifugation for 30 minutes at 5,000 rpm in a centrifuge. The stable

formulations did not show any phase separation or turbidity.

6.3.2.6. Heating cooling cycle

Nanoemulsions were kept at 37±0.5 o C for 24 hrs. After that the nanoemulsions were kept at

room temperature. The stable nanoemulsion should not show any sign of turbidity, cracking,

creaming during the entire cycle.

6.3.3. Preparation of mucoadhesive chitosan solutions and assessment of mucoadhesive

strength

Different concentrations of chitosan solutions were prepared in the range of 0.1 to 1.0 % w/v

in 0.2 % glacial acetic acid solution. The prepared solutions were evaluated for the

mucoadhesive strength using TA.XTPlus Texture analyzer (Stable Micro Systems, Surrey,

UK). The double-sided tape was placed on the tip of load cell and formulation was placed on

excised goat cornea. Cornea with the formulation was then placed beneath the load cell and

force (0.08 N) was applied by the load cell for 200 s. After this the load cell was pulled back



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and force required to detach the particles from the cornea (by double sided tape) was

determined as the mucoadhesive strength.

6.3.4. Characterization of nanoemulsions

6.3.4.1. Particle size distribution and zeta potential

Globule size of oil droplet in NE was determined by photon correlation spectroscopy that

analyzes the fluctuations in light scattering due to Brownian motion of the particles using a

Zetasizer (Nano-ZS, Malvern Instruments, UK). The formulation (0.1 mL) was dispersed in

50 mL of water in a volumetric flask, mixed thoroughly with vigorous shaking, and light

scattering was monitored at 25°C at a 90°angle. Whereas zeta potential was measured using a

disposable zeta cuvette. For each sample, the mean diameter/zeta potential ± standard

deviation of six determinations was calculated applying multimodal analysis.

6.3.4.2. Transmission electron microscopy (TEM)

Morphology of the NE was studied using TEM (Morgagni 268D SEI, USA) operating at 200

KV and of a 0.18 nm capable of point to point resolution. Combination of bright field

imaging at increasing magnification and of diffraction modes was used to reveal the form and

size of the microemulsion. In order to perform the TEM observations, the diluted

microemulsion was deposited on the holey film grid and observed after drying.

6.3.4.3. Viscosity determination

The viscosity of the NEs was determined using Brookfield DV III ultra V6.0 RV cone and

plate rheometer (Brookfield Engineering Laboratories, Inc., Middleboro, MA) using spindle #

CPE40 at 25 ± 0.5 °C. The software used for the calculations was Rheocalc V2.6.

6.3.4.4. Refractive index and pH measurement

Refractive index of microemulsions was determined using an Abbes type refractometer

(Nirmal International, New Delhi, India). The apparent pH of the formulation was measured

by pH meter (AccumentAB 15, Fisher scientific, USA) in triplicate at 25 ± 1ºC.



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6.3.4.5. Preparation of chitosan mucoadhesive nanoemulsion

Selected nanoemulsion formulation was imparted with mucoadhesive characteristics by using

optimised chitosan solutions. In brief, the selected formulations in their optimised Smix ratio

and oil to Smix ratio were titrated against the chitosan solution (1%w/v in 0.5 % v/v of

glacial acetic solution). The developed mucoadhesive nanoemulsions were further

characterized for particle size and zeta potential.

6.3.4.6. In vitro Release Study

In vitro release studies were performed using standard Franz diffusion cells (FDC-6, LOGAN

Instrument Corp., Somerset, NJ, USA). The diffusion area was 0.75 cm2 and receptor volume

was 5.0 mL. Receptor chambers were filled with 5 ml of PBS (pH 7.4; osmolality 297

mOSm/kg) and constantly stirred by small magnetic bars. The receptor fluid was stirred with

a magnetic rotor at a speed of 600 rpm and the temperature was maintained at 35 ± 0.5°C in

order to mimic the ocular surface temperature. Donor and receptor chambers were separated

by means of activated dialysis membrane bag (molecular weight cut off 12,000 Da). One

milliliters of each formulation were loaded into the donor compartment before occluding the

chamber with Parafilm. Samples were withdrawn at regular intervals (0.025, 0.5, 1, 2 4,8 and

12 hr), filtered through 0.45-µm membrane filter and analyzed for drug content by UPLC/Q-

TOF-MS/MS.

6.3.4.7. In-vivo study

Ocular retention and biodistribution study of CYA in cornea, conjunctiva, aqueous humor

and blood were carried out on New Zealand Albino rabbits (2.25±0.25 kg). The study was

carried out under the guidelines of CPCSEA (Committee for the Purpose of Control and

Supervision of Experiments on Animals, Ministry of Culture, Government of India). The

protocol was approved by Institutional Animal Ethics Committee, Jamia Hamdard, New

Delhi (approval no. 822) and the ARVO guidelines for animal usage were followed. Utmost



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care was taken to ensure that animals were treated in the most humane and ethically

acceptable manner.

6.3.4.7.1. Ocular retention study by Gamma scientigraphy

The precorneal retention of mucoadhesive NE system was assessed by γ- scintigraphy. The

liquid radio labelling of CYA suspension, NE, CH-NE were done using 99mTc as per the

protocol developed by INMAS, New Delhi. Labelling efficiency was determined using

instantaneous thin layer chromatography (ITLC) and was found to be greater than 98% for

more than 6 h in all the cases. Corneal retention of 99mTc labelled mucoadhesive

nanoemulsion was compared with 99mTc labelled NE and CYA suspension. A total of 20 μL

of the labelled formulations were instilled into the cul-de-sac of the left eye of the rabbit, and

the eye was manually blinked three times to distribute the formulation over the cornea. The

right eye of each rabbit served as a negative control. Gamma camera (Millenium VG,

Milwaukee, Wisconsin), autotuned to detect the 140 KeV radiation of Tc-99m, was used for

scintigraphy study. Rabbits were anesthetized to made ease of the study by using ketamine

HCl injection given intramuscularly in a dose of 15 mg/ kg body weight. The rabbits were

positioned 5 cm in front of the probe, and 50 μL of the radiolabeled formulation was instilled

onto the left corneal surface of each rabbit. Recording was started 5 seconds after instillation

and continued for 30 minutes using 128 × 128 pixel matrix. Sixty individual frames (60 × 30

seconds) were captured by dynamic imaging process. Region of interest (ROI) was selected

on one frame of the image, and time activity curve was plotted to calculate the rate of

drainage from the eye. Two minute static images were also taken at 0.5, 1, 2, 4 and 6 h post-

instillation. All the images were recorded on a computer system assisted with the software

Entegra Version-2.

6.3.4.7.2. Biodistribution study of CYA in cornea, conjunctiva, aqueous humor and

blood



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Three groups, each having seven New Zealand Albino rabbits (2.25±0.25 kg), were used for

the ocular study. Each group received, in both the eyes, a single topical instillation (50 µL) of

nanoemulsions (B1), CH coated mucoadhesive nanoemulsions (CH-B1) and suspension in

water (CYA-sus), containing dose equivalent to 0.5% w/v of CYA. At different times post-

instillation (0.5, 6, 12 and 24 h), the aqueous humour was withdrawn from the anterior

chamber with the aid of a 25-gauge needle fitted to an insulin syringe. Prior to sacrifice of

rabbit, blood samples were collected at the above mentioned times from the marginal ear vein

in tubes containing heparin and immediately centrifuged at 37°C, 4500 rpm for 4 min and the

plasma fraction was then quickly separated. In addition, the eyes were proptosed and rinsed

with normal saline. Cornea and conjunctiva were subsequently dissected in situ. Each tissue

was rinsed with normal saline, blotted dry in order to remove any adhering drug and

transferred to pre-weighed counting vials. The vials were re-weighed and the weight of the

tissues was calculated. All the biological samples were then analyzed by UPLC/Q-TOF-

MS/MS.

6.3.4.8. Statistical analysis

Data of in vivo analysis was expressed as mean of experimental value ± S.D. The data was

compared for statistical significance by the one-way analysis of variance (ANOVA) followed

by Tukey–Kramer multiple comparisons test using GraphPad Instat software (GraphPad

Software Inc., CA, USA).

6.4. Result and Discussion

6.4.1. Determination of solubility of cyclosporine A in oils, surfactants and co-

surfactants

Being a moderately lipophilic drug, it was very important to find out an appropriate solvent

to dissolve cyclosporin A, because only the dissolved state of drug in moderately lipophilic

carrier facilitates the drug permeation (Akhter et al., 2008). In order to screen appropriate



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solvent/s for the preparation of NE, the solubility of cyclosporin A in various oils,

surfactants and co-surfactants was measured.

After performing solubility study in different oils, it was found that cyclosporin A exhibited

maximum solubility in the oleic acid (21.79±0.93 mg/mL) [table 6.1; figure 6.1].

Table 6.1: Solubility of cyclosporin A in different oils

S.No Oils Solubility (mg/ml±S.D)

1 Oleic acid 21.79±0.93

2 Ispropyl myristate (IPM) 3.42±0.14

3 Olive oil 11.21±0.27

4 Triacetin 0.47±0.03

5 Castor oil 09.11±0.81

6 Labrafac 2.80±0.04

7 Soyabean oil 1.23±0.11

8 Safsol 3.25±0.06

Figure 6.1: Solubility of cyclosporin A in different selected oils.



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Therefore oleic acid was chosen as the oil phase. The other advantage with the use of oleic

acid that it has also been reported it is a powerful penetration enhancer for lipophilic barriers

(Rhee et al., 2001), as it increases the fluidity of the intercellular lipid barriers by forming

separate domains which interfere with the continuity of the multi-lamellar corneal epithelial

and induce highly permeable pathways (Akhter et al., 2011) and such mechanism is

supported by the work carried out on transdermal delivery by Puranjoti et al (Puranjoti et

al., 2002).

Based on preliminary solubility studies, the surfactants; polysorbate 20 (Tween 20) (1.16

±0.19 mg/mL), polysorbate 80 (Tween 80) (1.13 ±0.15 mg/mL), polyethoxylated castor oil

(Cremophore EL) (1.50±0.41mg/mL) and caprylocaproyl macrogol- 8-glyceride (Labrasol)

(2.50±0.70 mg/mL) and co-surfactants; Transcutol P (Transcutol P) (2.73±0.41 mg/mL),

PEG 200 (2.15±0.45 mg/mL), PEG 400 (2.68±0.39 mg/mL) and propylene glycol (1.30

±0.40 mg/mL) showing comparable solubility of drug, were chosen for further optimization

of NE formulations (table 6.2; figure 6.2).

Table 6.2: Solubility of cyclosporin A in surfactants and co-surfactants

S. No. Surfactants Solubility (mg/ml)

1 Tween 20 1.16 ±0.19

2 Tween 80 1.13 ±0.15

3 Cremophore EL 1.50±0.41

4 Labrasol 2.50±0.70

5 Span 20 0.62±0.13

7 Lauroglycol 90 0.50±0.10

8 Labrafil M 0.84±0.14

Co-surfactants

9 Transcutol P 2.73±0.41



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10 PEG 200 2.15±0.45

11 PEG 400 2.68±0.39

12 Propylene glycol 1.30 ±0.40

Figure 6.2: Solubility profile of cyclosporin A in different selected surfactants and co-surfactants.

6.4.2. Screening & optimization of surfactant and cosurfactants

The screening of surfactant and cosurfactant on the basis of solubility is difficult because

there is no significant difference in solubility of drug among these surfactant and

cosurfactant. So, in this work, we carried out the selection of surfactant and cosurfactant

was based on formation of larger NE region in the pseudo ternary phase diagram.

Constructed pseudo-ternary phase diagrams are self explanatory about the presence of NE

region which assists easy selection of ingredients proportions for preparation of stable

formulation (Akhter et al., 2008; Eccleston., 1992). Large NE region would also facilitate

the selection of formulation with low surfactant and cosurfactant concentration, desirable for

preparing non irritating formulations particularly to the eyes.

For optimization of surfactant, initially cosurfactant Transcutol P was kept constant,

different surfactant Tween 20, Tween 80, Cremophore EL and Labrasol in 1:1 ratio with



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cosurfactant (Transcutol P) was used. For each phase diagram, oil and specific Smix were

mixed well in different ratios. Sixteen different combinations of oil and Smix (1:9, 1:8, 1:7,

1:6, 1:5 1:4, 1:3.5, 1:3, 3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1) were made for phase diagram

construction. Each combination was titrated with water and the resultant physical state of

NE was marked on a pseudo ternary phase diagram with one axis representing the water,

one representing oil and the third representing a Smix. The combinations is presented in the

table 6.3, the titrating ratios of oil, Smix and aqueous phase are given in table 6.4-6.8 and

pseudo ternary phase diagram for the corresponding titration are presented in figure 6.3-6.6.

Table 6.3: Oil, surfactants cosurfactant and their combination ratios used for the

optimization of surfactant

Oil Phase

Cosurfactant Surfactant Smix (Cosurfactant: Surfactant)

Oil: Smix Ratio

Inference

Oleic Acid Transcutol P

Tween20

1:1

1:9, 1:8, 1:7, 1:6, 1:5, 1:4,

1:3.5, 1:3, 3:7, 1:2, 1:1, 2:3, 6:4, 7:3, 8:2

and 9:1

Large nanoemulsion area

Cremophore EL

Large nanoemulsion area

Tween80 Smaller nanoemulsion area

Labrasol Poor nanoemulsion area

Table 6.4: Nanoemulsion points for the mixture containing Smix ratio 1:1

Oil phase- oleic acid Smix- surfactant: cosurfactant

Surfactant: tween20 Cosurfactant: Transcutol P

S. No. % Oil

(v/v)

% Smix

(v/v)

Ratio (oil: Smix) %Water

(v/v)

1. 6.45 58.06 1:9 35.48



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2. 6.06 54.55 1:9 39.39

3. 5.56 50.00 2:8 44.44

4. 5.00 45 2:8 50

5. 4.55 40.19 2:7 54.55

6. 4.20 40.00 1:5 54.34

7. 4.00 36.00 1:5 60.00

8. 3.51 31.58 1:5 64.00

9. 28.17 56.34 1:5 15.49

10. 20.69 48.28 1:5 31.03

11. 22.22 51.85 1:5 25.93

12. 24.00 56 1:5 20.00

13. 10.00 50.00 1:5 40.00

14. 29.39 62.17 1:3 10.43

15. 26.69 59.07 1:3 14.23

16. 20 60 1:3 20

17. 21.28 63.83 1:3 14.89

18. 15.50 54.26 1:3.5 30.23

19. 16.67 58.33 1:3.5 25

20. 17.70 61.95 1:3.5 20.35

21. 18.87 66.04 1:3.5 15.09

22. 12.90 51.61 1:4 35.48

23. 13.79 55.17 1:4 31.03

24. 14.81 59.26 1:4 25.93

25. 16 64 1:4 20

26. 16.67 66.67 1:4 16.67

27. 10 50 1:5 40

28. 10.81 54.05 1:5 35.14

29. 11.56 57.80 1:5 30.64

30. 12.50 62.50 1:5 25



Chapter 6

Jamia Hamdard

31. 13.33 66.67 1:5 20

32. 7.84 47.06 1:6 45.10

33. 8.58 51.50 1:6 39.91

34. 9.26 55.56 1:6 35.19

35. 10 60 1:6 30

36. 10.70 64.17 1:6 25.13

37. 11.43 68.57 1:6 20

38. 4.55 40.91 1:5 45.45

39. 11.56 57.80 1:5 30.64

40. 13.79 55.17 1:6 31.03

41. 11.56 57.80 16 30.64

42. 14.00 56.43 1:6 30.07

43. 8.13 56.91 1:6 34.96

44. 8.70 60.87 1:6 30.43

45. 9.35 65.42 1:6 25.23

46. 10.00 50.00 1:6 40.00

47. 8.82 60.87 1:7 30.45

48. 9.09 45.00 1:7 45.45

49. 6.12 48.93 1:7 44.95

50. 6.67 53.76 1:7 40.02

51. 7.22 57.76 1:7 35.02

52. 5.56 50.00 1:7 44.44

53. 9.09 81.82 1:8 9.09

6. 4.20 40.00 1:8 54.34

55. 6 45 1:8 49.00

56. 5 35 1:8 60.00

57. 6.90 62.07 1:8 31.03

58. 6.45 58.06 1:8 35.48

59. 6.06 54.55 1:8 39.39



Chapter 6

Jamia Hamdard

60. 5.56 50 1:8 44.44

61. 5 45 1:8 50

Figure 6.3: Pseudoternary phase diagram showing o/w nanoemulsion region for S/ CoS ratio 1:1(Tween20: Transcutol P).

Table 6.5: Nanoemulsion points for the mixture containing Surfactant/ Co-Surfactant ratio 1:1

Oil phase: Oleic acid Surfactant: Cremophore EL Cosurfactant: Transcutol P

S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1. 6.06 54.55 1:9 39.39

2. 5.56 50.00 1:9 44.44

3. 11.11 44.44 1:9 44.44

4. 10.10 40.40 1:9 50.00

5. 9.09 36.36 1:9 54.55

6. 8.00 32.00 1:9 60.00

7. 7.49 37.45 1:9 55.06



Chapter 6

Jamia Hamdard

8. 6.67 33.33 2:8 60.00

9. 5.80 28.99 2:8 65.22

10. 5.00 25.00 2:8 70.00

11. 10.00 60.00 1:5 30.00

12. 9.26 55.56 1:5 35.19

13. 8.58 51.50 1:5 39.91

14. 7.84 47.06 1:5 45.10

15. 8.70 60.87 1:6 30.00

16. 8.13 56.91 1:6 34.96

17. 7.49 52.43 1:6 40.07

18. 6.78 47.46 1:6 45.76

19. 7.75 62.02 1:6 30.23

20. 7.22 57.76 1:6 35.02

21. 6.67 53.33 1:6 40.00

22. 6.12 48.34 1:6 44.45

23. 5.56 44.44 1:6 50.00

24. 9.26 55.56 1:7 35.19

25. 10 60 1:7 30

26. 10.70 64.17 1:7 25.13

27. 6.25 43.75 1:7 50

28. 6.78 47.46 1:7 45.76

29. 7.49 52.43 1:7 40.07

30. 8.13 56.91 1:7 34.96

31. 8.70 60.87 1:7 30.43

32. 9.35 65.42 1:7 25.23

33. 10 70 1:7 20

34. 5 40 1:7 55

35. 5.56 44.44 1:7 50

36. 6.12 48.93 1:7 44.95



Chapter 6

Jamia Hamdard

37. 6.67 53.33 1:7 40

38. 7.22 57.76 1:8 35.02

39. 7.75 62.02 1:8 30.23

40. 8.33 66.67 1:8 25

41. 7.41 66.67 1:8 25.93

42. 6.90 62.07 1:8 31.03

43. 6.45 58.06 1:8 35.48

44. 6.06 54.55 1:8 39.39

45. 5.56 50 1:8 44.44

46. 5 45 1:8 50

47. 4.55 40.91 1:8 54.55

48. 4 36 1:8 60

Figure 6.4: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:1. (Cremophore EL: Transcutol P).



Chapter 6

Jamia Hamdard

Table 6.6: Nanoemulsion points for the mixture containing S/ CoS ratio 1:1

Oil phase: Oleic acid Surfactant: Tween 80

Cosurfactant: Transcutol P

S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1. 20.69 48.28 1:9 31.03

2. 17.39 52.17 1:9 30.43

3. 15.50 54.26 1:9 30.23

4. 12.90 51.61 1:9 35.48

5. 13.79 55.17 1:4 31.03

6. 10 50 1:4 40

7. 10.81 54.05 1:3.5 35.14

8. 15.56 57.80 1:3.5 33.64

9. 7.84 47.06 1:5 45.10

10. 8.58 51.50 1:5 39.91

11. 9.26 55.56 1:6 35.19

12. 10 60 1:6 30

13. 6.25 43.75 1:7 50

14. 6.78 47.46 1:7 45.76

15. 7.49 52.43 1:7 40.07

16. 8.13 56.91 1:8 34.96

17. 8.70 60.87 1:8 30.43

18. 5 40 1:8 55



Chapter 6

Jamia Hamdard

Figure 6.5: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:1. (Tween 80: Transcutol P)

Table 6.7: Nanoemulsion points for the mixture containing S/ CoS ratio 1:1.

Oil phase: Oleic acid Surfactant: Labrasol


S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1 10 50 1:9 40

2 7.84 47.06 1:9 45.10

3 8.58 51.50 1:4 39.91

4 6.25 43.75 1:5 50

5 6.78 47.46 1:6 45.76

6 7.49 52.43 1:7 40.07

7 5 40 1:7 55

8 5.56 44.44 1:7 50

9 5.56 50 1:8 44.44

10 5 45 1:8 50

11 4.55 40.91 1:9 54.55



Chapter 6

Jamia Hamdard

Figure 6.6: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:1. (Labrasol: Transcutol P).

After studying the results, maximum nanoemulsion areas were obtained with surfactant

tween 20 and cremophore EL, therefore these surfactants were selected for further

optimization of cosurfactant.

Titrations were performed by using different cosurfactants (Transcutol P, PEG 200, PEG

400, propylene glycol) with the selected surfactants. The ratio of surfactant to cosurfactant

(Smix ratio) was kept constant (1:1) while oil to Smix ratio was kept at 1:9 since higher

concentration of Smix is favorable for maximum NE formation (Tenjarla., 1999; Shafiq et

al., 2006). For the further titration, the combination of oil, Smix and water is presented in

table 6.8.



Chapter 6

Jamia Hamdard

Table 6.8: The titrating combination of oil, Smix ratio and number of new points obtained

Oil phase Surfactant Co-

surfactant

Smix (Co-

surfactant:

Surfactant)

Oil :Smix

ratio

No. of

nanoemulsion

points

Oleic acid

Tween 20

Transcutol

P

1:1 1:9

4

PEG 200 2

PEG 400 2

PG 0

Cremophore

EL

Transcutol

P 3

PEG 200 1

PEG 400 1

PG 0

After studying the result, it was found that maximum nanoemulsion region or points were

obtained with Transcutol P and hence Transcutol P was chosen as cosurfactant for

nanoemulsion formulation. Finally, this is inference here that Tween 20 and Cremophore EL

showed maximum formation of NE with Transcutol P as cosurfactant. So, for the

preparation of nanoemulsion formulation oleic acid was selected as oil, Tween 20 and

Cremophore EL as surfactants (to validate the effect of different Smix combination on phase

behavior) and Transcutol P as cosurfactant. For each phase diagram, Smix ratios 1:0, 1:1, 1:2,

2:1, 3:1, 1:3, 4:1, 1:4 was prepared. Oil and specific Smix were mixed well in different

ratios. Sixteen different combinations of oil and Smix (1:9, 1:8, 1:7, 1:6, 1:5 1:4, 1:3.5, 1:3,

3:7, 1:2, 4:6, 5:5, 6:4, 7:3, 8:2 and 9:1) were made for phase diagram construction. For each

Smix ratio separate phase diagram was constructed.

Physical appearance of all NE formulations showed no distinct conversion boundaries from

w/o to o/w at all Smix ratios. The rest of the region on the phase diagram represents the



Chapter 6

Jamia Hamdard

turbid and conventional emulsions based on visual observation. Significant difference was

seen in ternary phase diagrams of NE constructed with different Smix ratio (Table 6.4-6.5

and 6.9-6.19) and (Fig 6.3-6.4 and 6.7-6.17).


Oil Phase: Oleic acid. Surfactant: Tween 20

Cosurfactant: Nil

S. No. % Oil

(v/v)

% Smix

(v/v)

Ratio (oil: Smix)

% Water

(v/v)

1. 40 40 1:9 20

2. 41.67 41.67 1:9 16.67

3. 45.45 45.45 1:4 15.09

4. 32 48 1:4 20

5. 11.56 57.80 1:4 30.64

6. 36.36 54.55 1:4 9.09

7. 25 50 1:4 25

8. 26.67 43.33 1:5 30

9. 13.17 56.34 1:5 41.49

10. 20.30 40.61 1:5 40.09

11. 22.22 51.85 1:5 25.93

12. 24 56 1:5 20

13. 11.56 57.80 1:6 30.64

14. 10.00 60.00 1:6 30.00

15. 8.70 60.87 1:6 30.43

16. 8.13 56.91 1:6 34.96

17. 22.22 66.67 1:6 11.11

18. 7.49 52.43 1:6 40.07

19. 6.25 43.75 1:6 50.00



Chapter 6

Jamia Hamdard

20. 5.62 39.33 1:6 55.06

21. 5.00 35.00 1:6 60.00

22. 8.70 60.87 1:7 30.43

23. 10.56 57.80 1:7 31.64

24. 11.17 58.34 1:7 41.49

25. 18.18 52.73 1:7 32.09

26. 13.33 66.67 1:7 20.00

27. 14.08 70.42 1:7 15.49

28. 3.13 21.88 1:7 10.45

29. 2.50 17.50 1:7 80

30. 1.87 13.12 1:7 85.00

31. 1.25 8.75 1:7 90.00

32. 0.62 4.36 1:7 95.03

33. 10 70 1:8 30.00

34. 10.53 73.68 1:8 15.79

35. 7.75 62.02 1:8 30.23

36. 7.32 57.76 1:8 35.02

37. 6.67 53.33 1:8 40.00

38. 6.12 48.93 1:8 44.95

39. 5.56 44.44 1:8 50.00

40. 5.00 40.00 1:8 55.00

41. 3.88 31.07 1:8 65.05

42. 3.33 26.67 1:8 70.00

43. 2.78 22.22 1:8 75.00

44. 2.22 17.78 1:8 80.00

45. 1.67 13.33 1:8 85.00

46. 1.11 6.89 1:8 90.00

47. 0.56 4.44 1:8 95.00

48. 3.88 31.07 1:8 65.05



Chapter 6

Jamia Hamdard

49. 5.00 40.00 1:8 55.00

50. 5.56 44.44 1:8 50.00

51. 6.06 54.55 1:8 39.39

Figure 6.7: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:0(Tween 20/ Transcutol P).


Oil phase: Oleic acid. Surfactant: Tween 20


S. No. % Oil

(v/v)

% Smix

(v/v)

Ratio(oil: Smix) % Water

(v/v)

1. 11.56 57.80 1:9 30.64

2. 7.84 47.06 1:9 45.10

3. 10.00 60.00 1:9 30.00

4. 9.26 55.56 1:9 35.19

5. 54.55 36.36 1:4 9.09

6. 37.04 37.04 1:4 25.93

7. 40 40 1:4 20

8. 41.67 41.67 1:4 16.67



Chapter 6

Jamia Hamdard

9. 45.45 45.45 1:2.3 9.09

10. 29.63 44.44 1:2.3 25.93

11. 23.26 46.51 1:2.3 30.23

12. 33.33 50 2:3 16.67

13. 20.69 48.28 1:3 31.03

14. 23.26 46.51 1:3 30.23

15. 25 50 1:3 25

16. 20.69 48.28 2:7 31.03

17. 11.56 57.80 2:7 30.64

18. 10.00 60.00 2:7 30.00

19. 20.69 48.28 2:7 31.03

20. 9.26 55.56 2:7 35.19

21. 24 56 1:5 20

22. 6.78 47.46 1:5 45.76

23. 6.25 43.75 1:5 50.00

24. 9.26 55.56 1:5 35.19

25. 10 50 1:5 40

26. 12.50 62.50 1:5 25

27. 13.33 66.67 1:5 20

28. 22.22 66.67 1:6 11.11

29. 14.39 50.36 1:6 35.25

30. 15.50 54.26 1:6 30.23

31. 16.67 58.33 1:6 25

32. 17.70 61.95 1:6 20.35

33. 18.87 66.04 1:6 15.09

34. 5.00 40.00 1:6 55.00

35. 12.90 51.61 1:6 35.48

36. 13.79 55.17 1:6 31.03

37. 6.67 53.33 1:6 40.00



Chapter 6

Jamia Hamdard

38. 7.22 57.76 1:6 35.02

39. 6.12 48.93 1:6 44.95

40. 6.25 43.75 1:7 50

41. 6.78 47.46 1:7 45.76

42. 7.49 52.43 1:7 40.07

43. 8.13 56.91 1:7 34.96

44. 8.70 60.87 1:7 30.43

45. 9.35 65.42 1:7 25.23

46. 10 70 1:7 20

47. 10.53 73.68 1:7 15.79

48. 11.24 78.65 1:7 10.11

49. 8.58 51.50 1:7 39.91

50. 9.26 55.56 1:7 35.19

51. 10 60 1:7 30

52 10.70 64.17 1:6 25.13

53 11.43 68.57 1:6 20

54. 12.12 72.73 1:6 15.15

55. 12.82 76.92 1:6 10.26

56. 6.25 43.75 1:7 50

57. 6.78 47.46 1:7 45.76

58. 7.49 52.43 1:7 40.07

59. 8.13 56.91 1:7 34.96

60. 8.70 60.87 1:7 30.43

61. 9.35 65.42 1:7 25.23

62. 10 70 1:7 20

63. 10.53 73.68 1:7 15.79

64. 11.24 78.65 1:7 10.11

65. 0.56 4.44 1:7 95

66. 5 40 1:8 55



Chapter 6

Jamia Hamdard

67. 5.56 44.44 1:8 50

68. 6.12 48.93 1:8 44.95

69. 6.67 53.33 1:8 40

70. 7.22 57.76 1:8 35.02

71. 7.75 62.02 1:8 30.23

72. 8.33 66.67 1:8 25

73. 8.89 71.11 1:8 20

74. 9.43 75.47 1:8 15.09

75. 10 50 1:8 40

76. 5.56 44.44 1:8 50

77. 6.67 53.00 1:8 40.00

s78. 8 42 1:8 50

79. 7.41 66.67 1:8 25.93

80. 6.90 62.07 1:8 31.03

81. 6.45 58.06 1:8 35.48

82. 5.56 50 1:8 44.44

83. 5 45 1:8 50

84. 3.51 31.58 1:8 64.91

85. 2.99 26.87 1:8 70.15

86. 2.5 22.5 1:8 75

87. 2 18 1:8 80



Chapter 6

Jamia Hamdard

Figure 6.8: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 2:1(Tween20/ Transcutol P).




S. No. % Oil

(v/v)

% Smix

(v/v)

Ratio (oil: S mix) % Water

(v/v)

1. 0.48 4.29 1:9 95.24

2. 6.90 62.07 1:9 31.03

3. 8 72 1:9 20.00

4. 9.09 81.82 1:9 9.09

5. 8.5 76.5 1:9 15

6. 5 45 1:9 50

7. 4.5 40.5 1:9 55

8. 6 54 1:9 40

9. 7 63 1:9 30

10. 17.04 37.04 1:4 45.93

11. 10.04 25.04 1:4 65.93



Chapter 6

Jamia Hamdard

12. 20 10 1:3.5 70

13. 18.18 72.73 1:4 9.09

14 10 40 1:4 50

15 15 60 1:4 25

16 17 15 1:4 68

17 19.25 3.75 1:4 77

18. 14.08 15.49 1:5 70.42

19. 14.93 10.45 1:5 74.63

20. 10 30 1:6 60

21. 12.12 15.15 1:6 72.73

22. 12.82 10.26 1:6 76.92

23. 10.53 15.79 1:7 73.68

24. 11.24 10.11 1:7 78.65

25. 8.89 20 1:8 71.11

26. 9.43 15.09 1:8 75.47

27. 10 10.00 1:8 80

28 7.75 30.25 1:8 62

29. 0.48 95.24 1:8 4.29

30. 6.90 31.03 1:8 62.07

31. 8 20.00 1:8 72

32. 9.09 9.09 1:8 81.82

33 8.5 15 1:8 76.5

34 5 50 1:8 45

35 4.5 55 1:8 40.5

36 6 40 1:8 54

37 7 30 1:8 63



Chapter 6

Jamia Hamdard

Figure 6.9: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:2(Tween20/ Transcutol P).




S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1. 48 32 1:0.6 20

2. 50 33.33 1:0.6 16.67

3. 40 40 1:1 20

4. 41.67 41.67 1:1 16.67

5. 45.45 45.45 1:1 9.09

6. 32 48 1:1.5 20

7. 33.33 50 1:1.5 16.67

8. 36.36 54.55 1:1.5 9.09

9. 25 50 1:2 25

10. 26.67 53.33 1:2 20

11. 28.17 56.34 1:2 15.49



Chapter 6

Jamia Hamdard

12. 30.30 60.61 1:2 9.09

13. 20.69 48.28 1:2.3 31.03

14. 22.22 51.85 1:2.3 25.93

15. 24 56 1:2.3 20

16. 25 58.33 1:2.3 16.67

17. 27.27 63.64 1:2.3 9.09

18. 17.39 52.17 1:3 30.43

19. 18.69 56.07 1:3 25.23

20. 20 60 1:3 20

21. 21.28 63.83 1:3 14.89

22. 22.22 66.67 1:3 11.11

23. 15.50 54.26 1:3.5 30.23

24. 16.67 58.33 1:3.5 25

25. 17.70 61.95 1:3.5 20.35

26. 18.87 66.04 1:3.5 15.09

27. 20 70 1:3.5 10

28. 12.90 51.61 1:4 35.48

29. 13.79 55.17 1:4 31.03

30. 14.81 59.26 1:4 25.93

31. 16 64 1:4 20

32. 16.67 66.67 1:4 16.67

33. 18.18 72.73 1:4 9.09

34. 10 50 1:5 40

35. 10.81 54.05 1:5 35.14

36. 11.56 57.80 1:5 30.64

37. 12.50 62.50 1:5 25

38. 13.33 66.67 1:5 20

39. 14.08 70.42 1:5 15.49

40. 14.93 74.63 1:5 10.45



Chapter 6

Jamia Hamdard

41. 7.84 47.06 1:6 45.10

42. 8.58 51.50 1:6 39.91

43. 9.26 55.56 1:6 35.19

44. 10 60 1:6 30

45. 10.70 64.17 1:6 25.13

46. 11.43 68.57 1:6 20

47. 12.12 72.73 1:6 15.15

48. 12.82 76.92 1:6 10.26

49. 6.25 43.75 1:7 50

50. 6.78 47.46 1:7 45.76

51. 7.49 52.43 1:7 40.07

52. 8.13 56.91 1:7 34.96

53. 8.70 60.87 1:7 30.43

54. 9.35 65.42 1:7 25.23

55. 10 70 1:7 20

56. 10.53 73.68 1:7 15.79

57. 11.24 78.65 1:7 10.11

58. 5 40 1:8 55

59. 5.56 44.44 1:8 50

60. 6.12 48.93 1:8 44.95

61. 6.67 53.33 1:8 40

62. 7.22 57.76 1:8 35.02

63. 7.75 62.02 1:8 30.23

64. 8.33 66.67 1:8 25

65. 8.89 71.11 1:8 20

66. 9.43 75.47 1:8 15.09

67. 10 80 1:8 10

68. 9.09 81.82 1:9 9.09

69. 8.33 75 1:9 16.67



Chapter 6

Jamia Hamdard

70. 8 72 1:9 20

71. 7.41 66.67 1:9 25.93

72. 6.90 62.07 1:9 31.03

73. 6.45 58.06 1:9 35.48

74. 6.06 54.55 1:9 39.39

75. 5.56 50 1:9 44.44

76. 5 45 1:9 50

77. 4.55 40.91 1:9 54.55

78. 4 36 1:9 60

79. 3.51 31.58 1:9 64.91

80. 2.99 26.87 1:9 70.15

81. 2.5 22.5 1:9 75

82. 2 18 1:9 80

83. 1.54 13.85 1:9 84.62

84. 1 9 1:9 90

85. 0.48 4.29 1:9 95.24

Figure 6.10: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 3:1 (Tween20/ Transcutol P).



Chapter 6

Jamia Hamdard

Table 6.13: Nanoemulsion points for the mixture containing S/ CoS ratio 1:3. Oil Phase: Oleic acid Surfactant: Tween 20


S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1. 36.36 54.55 1:1.5 9.09

2. 48 32 1:0.6 20

3. 63.64 27.27 1:0.42 9.09

4. 28.17 56.34 1:2 15.49

5. 30.30 60.61 1:2 9.09

6. 24 56 1:2.3 20

7. 25 58.33 1:2.3 16.67

8. 27.27 63.64 1:2.3 9.09

9. 21.28 63.83 1:3 14.89

10. 22 66.67 1:3 11.11

11. 18.87 66.04 1:3.5 15.09

12. 20 70 1:3.5 10

13. 18.18 72.73 1:4 9.09

14. 14.08 70.42 1:5 15.49

15. 14.93 74.63 1:5 10.45

16. 10 60 1:6 30

17. 12.12 72.73 1:6 15.15

18. 12.82 76.92 1:6 10.26

19. 10.53 73.68 1:7 15.79

20. 11.24 78.65 1:7 10.11

21. 8.89 71.11 1:8 20

22. 9.43 75.47 1:8 15.09

23. 10 80 1:8 10.00



Chapter 6

Jamia Hamdard

24. 0.48 4.29 1:9 95.24

25. 6.90 62.07 1:9 31.03

26. 8 72 1:9 20.00

27. 9.09 81.82 1:9 9.09


Table 6.14: Nanoemulsion points for the mixture containing S/ CoS ratio 4:1 Oil phase: Oleic acid. Surfactant: Tween 20


S. No. % Oil

(v/v)

% Smix

(v/v)

Ratio (oil: Smix)

% Water

(v/v)

1. 40 40 1:1 20

2. 41.67 41.67 1:1 16.67

3. 45.45 45.45 1:1 9.09

4. 32 48 1:1.5 20

5. 33.33 50 1:1.5 16.67

6. 36.36 54.55 1:1.5 9.09

7. 25 50 1:2 25



Chapter 6

Jamia Hamdard

8. 26.67 53.33 1:2 20

9. 28.17 56.34 1:2 15.49

10. 30.30 60.61 1:2 9.09

11. 22.22 51.85 1:2.3 25.93

12. 24 56 1:2.3 20

13. 25 58.33 1:2.3 16.67

14. 27.27 63.64 1:2.3 9.09

15. 20 60 1:3 20

16. 21.28 63.83 1:3 14.89

17. 22.22 66.67 1:3 11.11

18. 16.67 58.33 1:3.5 25

19. 17.70 61.95 1:3.5 20.35

20. 18.87 66.04 1:3.5 15.09

21. 20 70 1:3.5 10

22. 14.81 59.26 1:4 25.93

23. 16 64 1:4 20

24. 16.67 66.67 1:4 16.67

25. 18.18 72.73 1:4 9.09

26. 13.33 66.67 1:5 20

27. 14.08 70.42 1:5 15.49

28. 14.93 74.63 1:5 10.45

29. 11.43 6.57 1:6 20

30. 12.12 72.73 1:6 15.15

31. 12.82 76.92 1:6 10.26

32. 9.35 65.42 1:7 25.23

33. 10 70 1:7 20

34. 10.53 73.68 1:7 15.79

35. 11.24 78.65 1:7 10.11

36. 8.33 66.67 1:8 25



Chapter 6

Jamia Hamdard

37. 8.89 71.11 1:8 20

38. 9.43 75.47 1:8 15.09

39. 10 80 1:8 10

40. 9.09 81.82 1:9 9.09

41. 8.33 75 1:9 16.67

42. 8 72 1:9 20

43. 7.41 66.67 1:9 25.9

44. 6.90 62.07 1:9 31.03

45. 9.09 81.82 1:9 9.09

46. 8.33 75 1:9 16.67

47. 8 72 1:9 20

48. 7.41 66.67 1:9 25.93

49. 6.90 62.07 1:9 31.03

50. 6.45 58.06 1:9 35.48

51. 6.06 54.55 1:9 39.39




Chapter 6

Jamia Hamdard

Table 6.15: Nanoemulsion points for the mixture containing S/ CoS ratio 1:0 Oil Phase: Oleic acid

Surfactant: Cremophore EL Cosurfactant: Nil

S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1. 36.36 54.55 1:1.5 9.09

2. 48 32 1:0.6 20

3. 63.64 27.27 1:0.42 9.09

4. 28.17 56.34 1:2 15.49

5. 30.30 60.61 1:2 9.09

6. 24 56 1:2.3 20

7. 25 58.33 1:2.3 16.67

8. 27.27 63.64 1:2.3 9.09

9. 21.28 63.83 1:3 14.89

10. 22 66.67 1:3 11.11

11. 18.87 66.04 1:3.5 15.09

12. 20 70 1:3.5 10

13. 18.18 72.73 1:4 9.09

14. 14.08 70.42 1:5 15.49

15. 14.93 74.63 1:5 10.45

16. 10 60 1:6 30

17. 12.12 72.73 1:6 15.15



Chapter 6

Jamia Hamdard

Figure 6.13: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:0. (Cremophore EL/Transcutol P).

Table 6.16: Nanoemulsion points for the mixture containing S/ CoS ratio 1:2 Oil phase: Oleic acid.

Surfactant: Cremophore EL Cosurfactant: Transcutol P

S. No.

% Oil

(v/v)

% Smix

(v/v)

Ratio (oil: Smix)

% Water

(v/v)

1. 40 40 1:1 20

2. 41.67 41.67 1:1 16.67

3. 45.45 45.45 1:1 9.09

4. 32 48 1:1.5 20

5. 33.33 50 1:1.5 16.67

6. 36.36 54.55 1:1.5 9.09

7. 25 50 1:2 25

8. 26.67 53.33 1:2 20

9. 28.17 56.34 1:2 15.49

10. 30.30 60.61 1:2 9.09

11. 22.22 51.85 1:2.3 25.93

12. 24 56 1:2.3 20



Chapter 6

Jamia Hamdard

13. 25 58.33 1:2.3 16.67

14. 27.27 63.64 1:2.3 9.09

15. 20 60 1:3 20

16. 21.28 63.83 1:3 14.89

17. 22.22 66.67 1:3 11.11

18. 16.67 58.33 1:3.5 25

19. 17.70 61.95 1:3.5 20.35

20. 18.87 66.04 1:3.5 15.09

21. 20 70 1:3.5 10

22. 14.81 59.26 1:4 25.93

23. 16 64 1:4 20

24. 16.67 66.67 1:4 16.67

25. 18.18 72.73 1:4 9.09

26. 13.33 66.67 1:5 20

27. 14.08 70.42 1:5 15.49

28. 14.93 74.63 1:5 10.45

29. 11.43 6.57 1:6 20

30. 12.12 72.73 1:6 15.15




Chapter 6

Jamia Hamdard



S. No. % Oil

(v/v)

% Smix

(v/v)


(v/v)

1. 36.36 54.55 1:1.5 9.09

2. 48 32 1:0.6 20

3. 63.64 27.27 1:0.42 9.09

4. 28.17 56.34 1:2 15.49

5. 30.30 60.61 1:2 9.09

6. 24 56 1:2.3 20

7. 25 58.33 1:2.3 16.67

8. 27.27 63.64 1:2.3 9.09

9. 21.28 63.83 1:3 14.89

10. 22 66.67 1:3 11.11

11. 18.87 66.04 1:3.5 15.09

12. 20 70 1:3.5 10

13. 18.18 72.73 1:4 9.09

14. 14.08 70.42 1:5 15.49

15. 14.93 74.63 1:5 10.45

16. 10 60 1:6 30

17. 12.12 72.73 1:6 15.15

18. 12.82 76.92 1:6 10.26

19. 10.53 73.68 1:7 15.79

20. 11.24 78.65 1:7 10.11

21. 8.89 71.11 1:8 20

22. 9.43 75.47 1:8 15.09

23. 10 80 1:8 10.00



Chapter 6

Jamia Hamdard

24. 0.48 4.29 1:9 95.24

25. 6.90 62.07 1:9 31.03

26. 8 72 1:9 20.00

27. 9.09 81.82 1:9 9.09




S. No. % Oil (v/v) % Smix (v/v) Ratio (oil: S mix) % Water(v/v)

1. 36.36 54.55 1:1.5 9.09

2. 48 32 1:0.6 20

3. 63.64 27.27 1:0.42 9.09

4. 28.17 56.34 1:2 15.49

5. 30.30 60.61 1:2 9.09

6. 24 56 1:2.3 20

7. 25 58.33 1:2.3 16.67

8. 27.27 63.64 1:2.3 9.09



Chapter 6

Jamia Hamdard

9. 21.28 63.83 1:3 14.89

10. 22 66.67 1:3 11.11

11. 18.87 66.04 1:3.5 15.09

12. 20 70 1:3.5 10

13. 18.18 72.73 1:4 9.09

14. 14.08 70.42 1:5 15.49

15. 14.93 74.63 1:5 10.45

16. 10 60 1:6 30

17. 12.12 72.73 1:6 15.15

18. 12.82 76.92 1:6 10.26

19. 10.53 73.68 1:7 15.79

20. 11.24 78.65 1:7 10.11

21. 8.89 71.11 1:8 20

22. 9.43 75.47 1:8 15.09

23. 10 80 1:8 10.00




Chapter 6

Jamia Hamdard

Table 6.19: Nanoemulsion points for the mixture containing S/ CoS ratio 3:1 Oil phase: Oleic acid.


S. No. % Oil

(v/v)

% Smix

(v/v)

Ratio (oil: Smix)

% Water

(v/v)

1. 40 40 1:1 20

2. 41.67 41.67 1:1 16.67

3. 45.45 45.45 1:1 9.09

4. 32 48 1:1.5 20

5. 33.33 50 1:1.5 16.67

6. 36.36 54.55 1:1.5 9.09

7. 25 50 1:2 25

8. 26.67 53.33 1:2 20

9. 28.17 56.34 1:2 15.49

10. 30.30 60.61 1:2 9.09

11. 22.22 51.85 1:2.3 25.93

12. 24 56 1:2.3 20

13. 25 58.33 1:2.3 16.67

14. 27.27 63.64 1:2.3 9.09

15. 20 60 1:3 20

16. 21.28 63.83 1:3 14.89

17. 22.22 66.67 1:3 11.11

18. 16.67 58.33 1:3.5 25

19. 17.70 61.95 1:3.5 20.35

20. 18.87 66.04 1:3.5 15.09

21. 20 70 1:3.5 10

22. 14.81 59.26 1:4 25.93

23. 16 64 1:4 20



Chapter 6

Jamia Hamdard

24. 16.67 66.67 1:4 16.67

25. 18.18 72.73 1:4 9.09

26. 13.33 66.67 1:5 20

27. 14.08 70.42 1:5 15.49

28. 14.93 74.63 1:5 10.45

29. 11.43 6.57 1:6 20

30. 12.12 72.73 1:6 15.15

31. 12.82 76.92 1:6 10.26

32. 9.35 65.42 1:7 25.23

33. 10 70 1:7 20

34. 10.53 73.68 1:7 15.79

35. 11.24 78.65 1:7 10.11

36. 8.33 66.67 1:8 25

37. 8.89 71.11 1:8 20

38. 9.43 75.47 1:8 15.09

39. 10 80 1:8 10

40. 9.09 81.82 1:9 9.09

41. 8.33 75 1:9 16.67

42. 8 72 1:9 20

43. 7.41 66.67 1:9 25.9

44. 6.90 62.07 1:9 31.03



Chapter 6

Jamia Hamdard


Physical appearance of all NE formulations with Tween 20 and Cremophore EL as surfactant

showed no distinct conversion boundaries from w/o to o/w at all of their Smix ratios with

Transcutol P as cosurfactant. The rest of the region on the phase diagram represents the turbid

and conventional emulsions based on visual observation. Significant difference was seen in

ternary phase diagrams of NE constructed with different Smix ratio.

It was observed, when Tween 20 and Cremophore EL was used alone without Transcutol P

(Smix ratio 1:0), very low amount of oleic acid could be solubilized at high concentration

(>55% w/w) of polysorbate 20 (Table 6.9, Table 6.15 and Figure 6.7. Figure 6.13). This

could be attributed to the fact that transient negative interfacial tension and fluid interfacial

film is rarely achieved by the use of single surfactant, usually necessitating the addition of a

cosurfactant (Lawrence et al., 2000, Akhter et al., 2008, Akhter et al., 2011).

At equal amounts of surfactants and Transcutol P (Smix ratio 1:1), the NE region in

the phase diagram increased significantly (Figure. 6.4 and Figure 6.5) compared to

that obtained at Smix ratio 1:0 (Figure 6.7 and Figure 6.13). The presence of

Transcutol P (cosurfactant) decreases the bending stress of interface and makes the

interfacial film sufficiently flexible to take up different curvatures required to form



Chapter 6

Jamia Hamdard

NE over a wide range of compositions (Kawakami et al., 2002). However, when

concentration of Transcutol P with respect to surfactants was increased (Smix ratio

1:2 and 1:3) the NE area was decreased (Figure 6.9, Figure 6.14 and Figure 6.11,

Figure 6.16 respectively) compared to Smix ratio 1:1 (Figure. 6.4 and Figure 6.5).

The decrease in the NE area is possibly due to presence of low concentration of

surfactant which reduces the amount of micelles and consequently decreases the

solubilisation capacity of NE (Yuan et al., 2008). Moreover, NEs formed at Smix

ratio 1:3 were unstable and showed phase separation within 24 h. More particularly,

as compared to the Tween 20, Cremophore EL as surfactant showed lesser phase area

at all the studied Smix. With further increase in Transcutol P concentration (Smix

ratio 1:4), not a single NE point was found in both the cases. In contrast to this, when

concentration of Transcutol P with respect to polysorbate 20 and Cremophore EL was

decreased (Smix ratio 2:1), the NE area was increased (Figure 6.8 and Figure 6.15)

compared to their respective Smix ratio 1:1 (Figure. 6.4 and Figure 6.5). However,

at further lower Transcutol P concentrations (Smix ratio 3:1 and 4:1), the NE area was

decreased, it was because Transcutol P is a polar solvent with the tendency to highly

incorporate into water, and the relatively lower Transcutol P content in the NE system

decreases the hydrophilicity of the Smix, so the area of o/w NE was decreased. In

brief, Nanoemulsion system at Smix ratio 2:1 formed large isotropic NE region than

the systems at other Smix ratios with both the surfactants. NEs formation behaviour of

Tween 20 is significantly larger than the Cremophore EL with Transcutol P as

cosurfactant. Moreover, at Smix ratio 2:1 and 3:1 of Cremophore EL with Transcutol

P produces larger number of NEs gel form with is not suitable for the topical solution

delivery in eyes. Such phase behaviour is commonly seen with polyethylene glycol

ethers of castor oil like Cremophore EL (Akhter et al., 2008; Akhter et al., 2011).



Chapter 6

Jamia Hamdard

After extensively evaluating the phase behaviour of Tween 20 and Cremophore EL

we concluded here that among the surfactant, Tween 20 is best for the development of

nanoemulsion formulation with Transcutol P as the cosurfactant. So, for the further

study, we selected nanoemulsion formulation from the different phase ratios of Tween

20 and Transcutol P. The selected components for the nanoemulsion formulation

development have appropriate features considering for the topical ocular drug

delivery. Oleic acid (oil phase) is a well known permeation enhancer, itself the

integral part of corneal lipids and nontoxic and considered to be safe for ophthalmic

drug delivery. Tween 20 being a nonionic surfactant and Transcutol P as cosurfactant

are non irritant and effective corneal permeation enhancers (Li et al., 2008; Liu et al.,

2006).

6.4.3. Selection of nanoemulsion formulations from different Smix ratios

Following criteria were chosen for the selection of formulations:

• The oil and Smix concentration should be such that it solubilises the single dose of the

drug and give the final concentration of 3% w/v.

• Formulations with Smix ratio 1:3 and 4:1 were not selected because of the presence of

Nanoemulsion is very less in number and such formulation required very high surfactant

concentration (>50%). Nanoemulsion from phase diagram of Smix ratio 4:1 showed high

viscosity as well as high surfactant were required to develop the nanoemulsion system.

• High concentration of surfactant which might cause ocular irritation. So, we restrict our

formulation selection upto the Smix concentration of 35% from the selected phase

diagram. Moreover at these Smix ratios the area of NE isotropic region was

comparatively small.

Based on these set principal, four Smix ratios 1:0 (A1, A2), 1:1 (B1, B2), 1:2 (C1, C2), 2:1

(D1, D2) and 3:1 (E1, E2) were selected for the thermodynamic stability testing. All these



Chapter 6

Jamia Hamdard

selected formulations have equal Smix concentration i.e 30% and 35% for their first and

second formulations respectively.

6.4.4. Thermodynamic stability testing of drug loaded nanoemulsions

Nanoemulsions are thermodynamically stable systems and are formed at a particular

concentration of oil, surfactant and water, with no phase separation, creaming or cracking. It

is the thermo-stability which differentiates nano or microemulsions from emulsions that have

kinetic stability and will eventually phase separate (Lawrence and Rees, 2000). Thus, the

selected formulations were subjected to different thermodynamic stability by using heating

cooling cycle, centrifugation and freeze thaw cycle stress tests. Those formulations, which

survived thermodynamic stability tests, were taken for characterization with different

physiochemical attributes. Table 6.20 given here showed the result of thermodynamic

stability testing of drug loaded nanoemulsions.

Table 6.20: Thermodynamic stability testing result of different selected nanoemulsion formulations

Formulation with

Smix ratio

Freeze thaw

cycle

Centrifugation

studies

Heating

cooling cycle

Inference

A1 (1:0) x x x Failed

A2 (1:0) x x x Failed

B1 (1:1) √ √ √ Passed

B2 (1:1) √ √ √ Passed

C1 (1:2) √ √ √ Passed

C2 (1:2) √ √ √ Passed

D1 (2:1) √ √ √ Passed

D2 (2:1) √ √ √ Passed

E1 (3:1) √ √ √ Passed

E2 (3:1) √ √ √ Passed



Chapter 6

Jamia Hamdard

Results showed that nanoemulsion of Smix ratio 1:0 (A1 and A2) does not tolerated any stage

of stability testing. Absence of cosurfactant which is required to create a suitable HLB value

and stable flexible film for the stable NE system may be the rationale behind the instability of

the NE of Smix ratio of 1:0 (V/V).

6.4.5. Preparation and assessment of mucoadhesive strength of chitosan solutions

The optimized chitosan concentration (1% w/v) of chitosan solution showing the strongest

and desirable mucoadhesion (0.153N) among the test concentration (chapter 5.3) was

selected for imparting the mucoadhesive characteristic in the optimized nanoemulsion (B1).

6.4.6. Characterization of non mucoadhesive and mucoadhesive nanoemulsions

In this work, the influence of concentration of NE components on the characteristics of NE

was studied. The NEs and mucoadhesive were colloidal nano-dispersions as determined by

TEM (figure 6.18 A and B). The NEs were seems to have spherical drug loaded globules that

is uniformly distributed. Mucoadhesive NE (CH-B1) showed increased in size of the droplets

that may be due to the adsorption and interaction of chitosan with the oleic acid and

surfactant of the interface.

Figure 6.18: TEM microphotograph of CYA loaded nanoemulsion by TEM A) formulation

B1 B) formulation CH-B1 (mucoadhesive nanoemulsion) (100,000×).



Chapter 6

Jamia Hamdard

Globules size distribution profile obtained from Zetasizer (Nano-ZS, Malvern Instruments,

UK) for the optimized formulation having the smallest globular size is illustrated here in

figure 6.19.

Figure 6.19: Globule size distribution of CYA loaded nanoemulsion before and after CH

addition; A) formulation B1 B) formulation CH-B1.

Composition of selected nanoemulsion formulations and their different characterization

parameters vis. Droplet size distribution, zeta potential and viscosity are presented in table

6.21. Mean droplet size of formulation A1, A2 (A: 1:1), B1, B2 (B; 1:2) and C1, C2 (C: 2:1)

were 36.10±1.97nm, 39.02±1.46nm, 18.92±1.03nm, 23.05±1.17nm, 42.30±3.30nm and

55.55±3.50nm respectively. Their zeta potential were varied from -14.3± 2.53mV – (-

31.3±1.54) mV. The viscosities of the formulations are low as expected for the o/w type

nanoemulsion. The viscosities of the formulations (A1-C2) were varied from 29.35±1.92mP -

132.67±3.54mP. The lowest viscosity was found for the formulation B1 (Smix; 1:2) which is

29.35±1.92mP.



Chapter 6

Jamia Hamdard

Table 6.21: Selected Nanoemulsions, mucoadhesive nanoemulsion formulation and their

characterization parameters

Formulation

code

Percent w/w of

components

Characterisation parameters

Oil

(%)

Smix

(%)

Aqueous

phase

(%)

Mean

Droplet

Size

(nm± SD)

Zeta

Potential

(mV±SD)

Viscosity

(mP±SD)

Maximum

Drug

Release

(%±SD)

A1 (1:1) 5.0 30 65 36.10±1.97 -22.8±0.31 61.00±2.40 89.7±1.2

A2 (1:1) 7.0 35 58 39.02±1.46 -31.3±1.54 73.40±2.95 87.54±2.1

B1 (1:2) 4.0 30 66 18.92±1.03 -14.3± 2.53 29.35±1.92 99.10±1.9

B2 (1:2) 6.0 35 59 23.05±1.17 -19.7± 2.72 37.30±2.67 98.3±2.8

C1 (2:1) 4.0 30 66 42.30±3.30 -18.0± 1.68 98.67±2.13 78.31±3.1

C2 (2:1) 7.0 35 58 55.55±3.50 -22.8±0.31 132.67±3.54 72.98±1.8

CH-B1 4.0 30 66* 41.70±1.15 +37.2±1.54 52.91±1.92 99.49±1.5

Values of polydispersity index (PI), which is a measure of uniformity of droplet size within

the formulation, were also calculated. All the NE formulations exhibited a narrow size

distribution (PI < 0.181). The results of particle size analysis were in agreement with the

droplet size measured by TEM photograph. In case mucoadhesive nanoemulsion (CH-B1),

there were increased in the droplet size (41.70±1.15nm) and zeta potential was positive

(+37.2±1.54mV). The value of zeta potential clearly favour here that the selected formulation

and the mucoadhesive CH-B1 having good dispersion stability due to fair charge repulsion. It

is hypothetically described that at the optimum Smix ratio (1:2 for present study) the

Transcutol P was exactly inserted into the cavities between the tween 20 molecules, causing



Chapter 6

Jamia Hamdard

the interfacial film to condense and stabilize, resulting in smallest droplet diameters

(18.92±1.03) with lowest polydispersity value (0.125 ± 0.05). In addition, particle size

analysis revealed that with increase in oil concentration the droplet size of NE increases,

irrespective of the Smix ratio. Large droplet size of oil rich formulations could be attributed

to the fact that the higher concentration of Smix is required to solubilise the oil phase in the

aqueous phase resulting an increase in size (Chen et al., 2006). As a result of viscosity

measurements, a similar behavior, as for droplet size was obtained. Viscosity of all the NE

formulations was very low as expected for o/w emulsion. When formulations with different

Smix ratios were compared, the minimum viscosity values were obtained for B1 formulations

(29.35±1.92mP). The low viscosity may be due to the presence of low amount of tween 20 (a

fatty acid polyhydric alcohol ester having high intrinsic viscosity) compared to Transcutol P (a

short chain alcohol having low intrinsic viscosity) (Akhter et al., 2008). All the drug free or

drug loaded NE formulations had pH values ranging from 6.7 to 6.81, favourable for topical

ocular application. It was observed that incorporation of drug did not significantly affect the

pH values of NEs.

6.4.7. In-vitro CYA Release Study

The release profile of the selected NE formulations (A1-C2), mucoadhesive NE (CH-B1) and

control formulations (Smix and oil) are presented here in figure 6.20. The maximum

percentage of CYA release for the tested NE and mucoadhesive NE (CH-B1) are presented in

Table 6.12.



Chapter 6

Jamia Hamdard

Figure 6.20: % drug release Vs time profile of selected formulations and controls for the

period of 12hrs.

As expected for the NEs, fast drug release behaviour were observed due to the enhanced

dissolution and spreading of the oil micellar solubilisation due to the optimized bend of

surfactant and cosurfactant that developed the optimum HLB.

Maximum percentage release of CYA after 12h was found to be highest (99.10±1.9nm) for B1

(Smix: 1:2). Moreover, chitosan coated formulation (CH-B1) also showed the similar release

profile having the equivalent maximum percentage of drug release (99.49±1.5).

Although, all the formulations contain equal drug amount, low release rate as observed for

controls, provided that concentration gradient is not a single factor affecting the rate of

permeation.

Enhanced drug release with NEs could be explained on basis of the several mechanisms:



Chapter 6

Jamia Hamdard

1) Continuous and spontaneous fluctuating stable interfaces of NE enable high drug mobility

and might enhance the drug diffusion process.

2) High solubilisation of CYA in NE resulting in high thermodynamic activity of the drug

providing significant driving force for its release.

6.4.8. In-vivo study

6.4.8.1. Ocular retention study by Gamma scientigraphy

Gamma scintigraphy is a well-established technique for in vivo evaluation of the ocular

retention study of ophthalmic drug delivery. The precorneal clearance of the optimized CYA

non mucoadhesive (B1) and CH-coated mucoadhesive NEs formulation (CH-B1) were

monitored using γ-scientigraphy.

CYA mucoadhesive NEs formulation (CH-B1) and non mucoadhesive formulation (B1) was

radiolabeled with radionuclide Tc-99m. They were instantaneously labeled with Tecnetium-

99m with good labeling efficiency (≥ 95%) and less number of colloids (≤ 5%). After

administration of the radio labeled ophthalmic formulation, a good spreading was observed

over the entire precorneal area. Identifying ROIs and defining them as ocular allowed for

monitoring the transit of the formulation, and subsequently, quantifying the remaining

activity in these regions at different time points. Gamma scintigraphic dynamic images of

whole-body of rabbit for first 30 minutes after administration of the formulations and the

control and the radioactivity remaining in the ROI on dynamic images (radioactivity

remaining Vs time profile) over period of 30 min are illustrated here in figure 6.21 and figure

6.22 respectively.



Chapter 6

Jamia Hamdard

Figure 6.21: Gamma scintigraphic dynamic images of whole-body of rabbit for first 30 minutes (30 sec per frame) after 5 minutes of administration CH-B1.



Chapter 6

Jamia Hamdard

Figure 6.22: Gamma scintigraphic dynamic images of whole-body of rabbit for first 30 minutes (30 sec per frame) after 5 minutes of administration B1.

Percentage of radioactivity remaining at different time point, Log % activity remained, AUC

of activity of CYA mucoadhesive NEs formulation (CH-B1) as compared to non

mucoadhesive formulation (B1) is presented in table 6.22 and table 6.23.



Chapter 6

Jamia Hamdard

Table 6.22: % Activity remaining, AUC0→30 min for CYA mucoadhesive NEs formulation (CH-B1) as compared to non mucoadhesive formulation (B1) after topical application onto

the rabbit eye

Image No.

CH-B1

B1

Time (sec)

Counts Counts/sec AUC %Activity Remaining

Log % AR

Counts Counts/sec AUC %Activity Remaining

Log % AR

0 0 0 0 0 0 0 0 0 0 1 5 380 76 190 100 2 130 26 65 100 2 2 10 374 74.8 377 98.421 1.9931 124 24.8 127 95.385 1.97948 3 15 372 74.4 373 97.895 1.9908 121 24.2 122.5 93.077 1.96884 4 20 363 72.6 367.5 95.526 1.9801 117 23.4 119 90.000 1.95424 5 25 357 71.4 360 93.947 1.9729 114 22.8 115.5 87.692 1.94296 6 30 357 71.4 357 93.947 1.9729 112 22.4 113 86.154 1.93527 7 35 356 71.2 356.5 93.684 1.9717 111 22.2 111.5 85.385 1.93138 8 40 352 70.4 354 92.632 1.9668 110 22 110.5 84.615 1.92745 9 45 350 70 351 92.105 1.9643 109 21.8 109.5 83.846 1.92348 10 50 349 69.8 349.5 91.842 1.9630 107 21.4 108 82.308 1.91544 11 55 349 69.8 349 91.842 1.9630 107 21.4 107 82.308 1.91544 12 60 346 69.2 347.5 91.053 1.9593 106 21.2 106.5 81.538 1.91136 13 65 345 69 345.5 90.789 1.9580 105 21 105.5 80.769 1.90725 14 70 341 68.2 343 89.737 1.9530 104 20.8 104.5 80.000 1.90309 15 75 340 68 340.5 89.474 1.9517 103 20.6 103.5 79.231 1.89889 16 80 340 68 340 89.474 1.9517 102 20.4 102.5 78.462 1.89466 17 85 338 67.6 339 88.947 1.9491 100 20 101 76.923 1.88606 18 90 336 67.2 337 88.421 1.9466 99 19.8 99.5 76.154 1.88169 19 95 335 67 335.5 88.158 1.9453 99 19.8 99 76.154 1.88169 20 100 327 65.4 331 86.053 1.9348 98 19.6 98.5 75.385 1.87728 21 105 327 65.4 327 86.053 1.9348 98 19.6 98 75.385 1.87728 22 110 326 65.2 326.5 85.789 1.9334 98 19.6 98 75.385 1.87728 23 115 321 64.2 323.5 84.474 1.9267 97 19.4 97.5 74.615 1.87283 24 120 320 64 320.5 84.211 1.9254 96 19.2 96.5 73.846 1.86833 25 125 319 63.8 319.5 83.947 1.9240 96 19.2 96 73.846 1.86833 26 130 319 63.8 319 83.947 1.9240 96 19.2 96 73.846 1.86833 27 135 316 63.2 317.5 83.158 1.9199 95 19 95.5 73.077 1.86378 28 140 316 63.2 316 83.158 1.9199 95 19 95 73.077 1.86378 29 145 315 63 315.5 82.895 1.9185 93 18.6 94 71.538 1.85454 30 150 315 63 315 82.895 1.9185 93 18.6 93 71.538 1.85454 31 155 312 62.4 313.5 82.105 1.9144 87 17.4 90 66.923 1.82558 32 160 311 62.2 311.5 81.842 1.9130 86 17.2 86.5 66.154 1.82056 33 165 305 61 308 80.263 1.9045 85 17 85.5 65.385 1.81548 34 170 303 60.6 304 79.737 1.9017 83 16.6 84 63.846 1.80513 35 175 303 60.6 303 79.737 1.9017 80 16 81.5 61.538 1.78915 36 180 297 59.4 300 78.158 1.8930 78 15.6 79 60.000 1.77815 37 190 592 59.2 593 77.895 1.8915 154 15.4 155 59.231 1.77255 38 200 591 59.1 591.5 77.763 1.8908 152 15.2 153 58.462 1.76687 39 210 593 59.3 592 78.026 1.8922 151 15.1 151.5 58.077 1.76400 40 220 589 58.9 591 77.500 1.8893 148 14.8 149.5 56.923 1.75529 41 230 587 58.7 588 77.237 1.8878 146 14.6 147 56.154 1.74938 42 240 585 58.5 586 76.974 1.8863 147 14.7 146.5 56.538 1.75234 43 250 583 58.3 584 76.711 1.8849 144 14.4 145.5 55.385 1.74339 44 260 582 58.2 582.5 76.579 1.8841 142 14.2 143 54.615 1.73731 45 270 580 58 581 76.316 1.8826 141 14.1 141.5 54.231 1.73425 46 280 581 58.1 580.5 76.447 1.8834 139 13.9 140 53.462 1.72804



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Jamia Hamdard

47 290 579 57.9 580 76.184 1.8819 137 13.7 138 52.692 1.72175 48 300 575 57.5 577 75.658 1.8789 135 13.5 136 51.923 1.71536 49 320 1148 57.4 1149 75.526 1.8781 267 13.35 268.5 51.346 1.71051 50 340 1147 57.35 1147.5 75.461 1.8777 265 13.25 266 50.962 1.70724 51 360 1145 57.25 1146 75.329 1.8770 262 13.1 263.5 50.385 1.70230 52 380 1146 57.3 1145.5 75.395 1.8773 261 13.05 261.5 50.192 1.70064 53 400 1143 57.15 1144.5 75.197 1.8762 258 12.9 259.5 49.615 1.69562 54 420 1141 57.05 1142 75.066 1.8754 254 12.7 256 48.846 1.68883 55 440 1138 56.9 1139.5 74.868 1.8743 252 12.6 253 48.462 1.68540 56 460 1135 56.75 1136.5 74.671 1.8732 250 12.5 251 48.077 1.68194 57 480 1133 56.65 1134 74.539 1.8724 251 12.55 250.5 48.269 1.68367 58 500 1130 56.5 1131.5 74.342 1.8712 247 12.35 249 47.500 1.67669 59 520 1129 56.45 1129.5 74.276 1.8709 245 12.25 246 47.115 1.67316 60 540 1131 56.55 1130 74.408 1.8716 242 12.1 243.5 46.538 1.66781 AUC0-t = 69197.0 AUC0-t = 11833.5

As expected, the drainage of the CYA mucoadhesive NEs formulation was fast, and it was

detectable in the stomach and rectum of the rabbit at the end of the monitoring period. The

poor performance of the CYA NEs (B1) during the drainage study could be attributed to lack

of mucoadhesion with the cornea (Akhter et al., 2011). In contrast, the CYA mucoadhesive

NEs formulation system (CH-B1) was retained on the ocular surface significantly longer

(P<0.05) than B1 (Fig. 6.21 and 6.21 and table 6.22). The AUC for the retained activity of

CYA mucoadhesive NEs formulation system (CH-B1) over the ocular surface is 69197.0 as

compare to the NEs formulation system (CH-B1) which is only 11833.5 (Table 6.22). These

findings suggest that the presence of chitosan may serve to prolong the retention. Possible

mechanisms of interaction of chitosan with the negatively charged mucin over the cornea are

diagrammatically presented here in figure 6.23.



Chapter 6

Jamia Hamdard

Figure 6.23: Hypothesized mechanism describing the mucoadhesion of CYA

mucoadhesive NEs formulation system (CH-B1) due to the presence of chitosan.

A possible explanation for this could be the interactions of chitosan with the mucin of cornea.

Another possible reason behind the longer retention could be its positive zeta potential that

could favour the attachment of the nano-systems onto the negatively charged ocular surface.

Moreover, uniform and spherical large surface area increased the spreading and contact time

over corneal surface subsequently enhanced the corneal retention.

6.4.8.2. Biodistribution study of CYA in cornea, conjunctiva, aqueous humor and blood

The UPLC/Q-TOF-MS/MS method showed satisfactory determination of CsA in rabbit

cornea, conjunctiva, plasma and aqueous humor for the pharmacokinetic study of CsA

suspension (CsA-Sus), CsA nanoemulsion (B1) and mucoadhesive CsA nanoemulsions (CH-

B1). Clinically pharmacokinetic study in ocular tissues is relevant as they might act as a



Chapter 6

Jamia Hamdard

reservoir for CsA. So the pharmacokinetic study was conducted in different tissues like

cornea and conjunctiva. The concentration–time profiles for CsA after topical instillation of

CsA-sus, B1 and CH-B1 to rabbit eyes and their biodistribution in cornea, conjunctiva,

plasma and aqueous humor are presented in figure 6.24, 6.25, 6.26 and 6.27.

Figure 6.24: CyA concentration from B1, CH-B1 and CYA suspension at different time

point in cornea.



Chapter 6

Jamia Hamdard


point in conjunctiva.


point in plasma.



Chapter 6

Jamia Hamdard


point in aqueous humor.

It was also interesting to note that for all three formulations, the corneal and conjunctival

levels showed a maximum concentration at 0.5 h post-administration and decreased gradually

afterwards. The key difference was, however, that at 24 h post-instillation CsA-SUS,

concentration of CsA in corneal and conjunctiva descended to subtherapeutic levels whereas,

using CH-B1, these levels were sufficiently high to adequately modulate the local immune

response and to suppress inflammatory processes (50–300 ng/g levels; Kaswan, 1988;

Acheampong et al., 1999) during 24 hr post-administration. Treatment with CH-B1 Showed

highest concentration of drug over the period of 24 hrs as compared to other formulations in

cornea.

This result might be explained on the following basis:

Nano droplets settle down to close contact with the cornea and a large amount of

inner oleic acid carrying drug might penetrate within the cornea.



Chapter 6

Jamia Hamdard

Permeation of drug carrying nano-sized droplets through cornea without NE fusion

and subsequent drug release as SUS here in the study showing insignificant corneal

deposition.

Continuous and spontaneous fluctuating stable interfaces of NE enable high drug

mobility and might enhance the drug diffusion process.

High solubilisation of drug in NE resulting in high thermodynamic activity of the

drug providing significant driving force for its release and permeation.

Presence of chitosan enhances the corneal retention as well as helpful in opening of

the cellular tight junction may also contribute as the driving force for permeation.

Figure 6.25 showed CsA concentrations in the conjunctiva after topical administration of the

three CsA formulations. CsA levels in the conjunctiva decreased much faster than in the

cornea. Concentration of drug in conjunctiva was found to be highest for CH-B1 as compared

to B1 and CsA-SUS in 24hrs of post instillation. Consequently, using the CsA-loaded CH-

B1, therapeutic concentrations of CsA were maintained in the cornea and conjunctiva

throughout the duration (24 hr) of the study. This suggests that these clinically relevant ocular

tissues may act as a reservoir for CsA-loaded CH-B1 nanoemulsions.

The reason for this improved interaction of CH-B1 with the cornea and the conjunctiva could

be found in the mucoadhesive properties of CH (Lehr et al., 1992). However, the limited

effectiveness of the SUS and CYA-B1 as compared to the CH-B1 indicates that the fact that

CH plays a key role in improving their interaction with the ocular surface. In fact, this

facilitated interaction of the CH with the cornea and the conjunctiva has recently been

visualized by confocal microscopy (Jain et al., 2011). Moreover, it is our hypothesis that it

might not be the mucoadhesive character of CH molecules but the electrostatic interaction

between the positively charged CH and the negatively charged corneal and conjunctival cells

that is the major force responsible for the prolonged residence of CsA in these epithelia. The



Chapter 6

Jamia Hamdard

surface of cornea and conjunctiva is covered by a thin fluid layer called mucus film (Ludwig,

2005). The primary component of mucus is mucin, a high molecular mass glycoprotein which

is negatively charged at physiological pH. Thus, the positively charged CH coating layer can

provide a binding force to the eye surface. Nevertheless, the bioadhesion of chitosan is not

exclusively determined by the positive charge. It could also be promoted by the presence of

free amine and hydroxyl groups of chitosan molecules which form hydrogen bonds to the eye

surface. This might explain that the CH could achieve a prolonged retention though its

positive charge was limited at neutral pH. Indeed, the systemic absorption of CsA cannot be

discarded; however, according to the results shown in Fig. 6. 26, the blood levels observed

for the three formulations tested are much below the described toxic levels (300 ng/ ml;

Bowers and Canafax, 1984). Moreover, the plasma concentrations were found to be least in

case of CH-B1 as compared to B1 and CsA-sus during the study period. Finally, Figs. 6. 27

indicating the CsA concentrations in the aqueous humour after topical administration of the

three formulations. Only minimal intraocular concentrations were attained throughout the

duration of the study and CsA levels in aqueous humour went down below the limit of

detection at 24 h post-administration. The extremely low CsA levels at these inner ocular

structures are justified by the limited intraocular penetration of free CsA, because of its

retention in the corneal stroma (BenEzra and Maftzir, 1990). Nevertheless, these results also

suggest that the Chitosan coated nanoemulsions do not enhance the penetration of CsA,

which remains on the ocular surface. Indeed, the higher CsA levels in the aqueous humour, at

0.5 h post-administration, achieved for B1 should be attributed to lipophilic carrier of the

formulation. Thereby confirming that chitosan favour the accumulation of CsA on the

external tissues without compromising intraocular structures due to its mucoadhesive

characteristics. Therefore, we can conclude that a selective and prolonged CsA delivery was



Chapter 6

Jamia Hamdard

achieved using CH-B1, without compromising inner ocular tissues and minimizing systemic

drug absorption.

des cyclosporine a nanoemulsion formulation chapter...

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