DES Cyclosporine A Nanoemulsion Formulation ... - Shodhganga

DES Chapter 6

Cyclosporine A Nanoemulsion Formulation

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).

DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM Jamia Hamdard 130

DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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.


DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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.


DES Chapter 6


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/QTOF-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


DES Chapter 6


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

99m

Tc 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

99m

Tc 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 postinstillation. 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


DES Chapter 6


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 postinstillation (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-TOFMS/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 cosurfactants 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


DES Chapter 6


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.


DES Chapter 6


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


DES Chapter 6

Cyclosporine A Nanoemulsion Formulation 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 cosurfactants. 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


DES Chapter 6


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

Tween20 Cremophore EL

Oleic Acid

Transcutol P

1:1 Tween80

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

Labrasol

Inference

Large nanoemulsion area Large nanoemulsion area Smaller nanoemulsion area 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.

1.

% Oil

% Smix

(v/v)

(v/v)

6.45

58.06

Ratio (oil: Smix)

%Water (v/v)

1:9

35.48


DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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

% Smix

Ratio (oil: Smix)

%Water

(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

(v/v)


DES Chapter 6


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


DES Chapter 6


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).


DES Chapter 6


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

% Smix

Ratio (oil: Smix)

%Water

(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

(v/v)


DES Chapter 6


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 Cosurfactant: Transcutol P S. No.

% Oil

% Smix

Ratio (oil: Smix)

%Water

(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

(v/v)


DES Chapter 6


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.


DES Chapter 6


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

Oil phase

Surfactant

Cosurfactant

Smix (Cosurfactant: Surfactant)

Oil :Smix ratio

Transcutol

2

PEG 400

2

Transcutol

1:1

P Cremophore EL

points

PEG 200

PG

Oleic acid

nanoemulsion

4

P Tween 20

No. of

1:9

0 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 DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM Jamia Hamdard 154

DES Chapter 6


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). Table 6.9: Nanoemulsion points for the mixture containing S/ CoS ratio 1:0 Oil Phase: Oleic acid. Surfactant: Tween 20 Cosurfactant: Nil

S. No.

% Oil

% Smix

Ratio (oil: Smix)

% Water

(v/v)

(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

(v/v)


DES Chapter 6


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


DES Chapter 6


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).

Table 6.10: Nanoemulsion points for the mixture containing S/ CoS ratio 2:1 Oil phase: Oleic acid. Surfactant: Tween 20 Cosurfactant: Transcutol P

S. No.

% Oil

% Smix

(v/v)

(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


DES Chapter 6 9.

Cyclosporine A Nanoemulsion Formulation 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


DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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


S. No.

% Oil

% Smix

Ratio (oil: S mix)

% Water

(v/v)

(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

(v/v)


DES Chapter 6


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


DES Chapter 6


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


S. No.

% Oil

% Smix

Ratio (oil: Smix)

%Water

(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

(v/v)


DES Chapter 6


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


DES Chapter 6


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


DES Chapter 6


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).


DES Chapter 6


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

S. No.

% Oil

% Smix

(v/v)

(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

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


DES Chapter 6


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

% Smix

Ratio (oil: Smix)

% Water

(v/v)

(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

(v/v)


DES Chapter 6

Cyclosporine A Nanoemulsion Formulation 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


DES Chapter 6


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



DES Chapter 6


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

% Smix

(v/v)

(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

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


DES Chapter 6


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

% Smix

(v/v)

(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


DES Chapter 6


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

Figure 6.14: Pseudoternary phase diagram showing o/w nanoemulsion region for surfactant/ cosurfactant ratio 1:2. (Cremophore EL/Transcutol P). DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM Jamia Hamdard 173

DES Chapter 6


Table 6.17: Nanoemulsion points for the mixture containing S/ CoS ratio 2:1 Oil Phase: Oleic acid Surfactant: Cremophore EL Cosurfactant: Transcutol P

S. No.

% Oil

% Smix

(v/v)

(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

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


DES Chapter 6


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 6.18: Nanoemulsion points for the mixture containing S/ CoS ratio 1:3 Oil Phase: Oleic acid Surfactant: Cremophore EL Cosurfactant: Transcutol P

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


DES Chapter 6


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



DES Chapter 6


Table 6.19: Nanoemulsion points for the mixture containing S/ CoS ratio 3:1 Oil phase: Oleic acid. Surfactant: Cremophore EL Cosurfactant: Transcutol P

S. No.

% Oil

% Smix

Ratio (oil: Smix)

% Water

(v/v)

(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

(v/v)


DES Chapter 6


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


DES Chapter 6



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


DES Chapter 6


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).


DES Chapter 6


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 DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM Jamia Hamdard 181

DES Chapter 6


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

Freeze thaw

Centrifugation

Heating

Inference

Smix ratio

cycle

studies

cooling cycle

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


DES Chapter 6


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×). DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM Jamia Hamdard 183

DES Chapter 6


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.


DES Chapter 6


Table 6.21: Selected Nanoemulsions, mucoadhesive nanoemulsion formulation and their characterization parameters Formulation

Percent w/w of

code

components

Characterisation parameters

Oil

Smix

Aqueous

Mean

Zeta

Viscosity

Maximum

(%)

(%)

phase

Droplet

Potential

(mP±SD)

Drug

(%)

Size

(mV±SD)

Release

(nm± SD)

(%±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 DESIGN AND DEVELOPMENT OF NANO SIZED OCULAR DRUG DELIVERY SYSTEM Jamia Hamdard 185

DES Chapter 6


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.


DES Chapter 6


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:


DES Chapter 6


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 Tecnetium99m 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.


DES Chapter 6


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.


DES Chapter 6


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.


DES Chapter 6


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 CH-B1 Image No.

B1

Time (sec)

Counts

Counts/sec

AUC

%Activity Remaining

0

0

0

0

0

0

1

5

380

76

190

100

2

10

374

74.8

377

98.421

3

15

372

74.4

373

4

20

363

72.6

5

25

357

6

30

7

35

8

Log % AR

Counts

Counts/sec

AUC

%Activity Remaining

Log % AR

0

0

0

0

2

130

26

65

100

2

1.9931

124

24.8

127

95.385

1.97948

97.895

1.9908

121

24.2

122.5

93.077

1.96884

367.5

95.526

1.9801

117

23.4

119

90.000

1.95424

71.4

360

93.947

1.9729

114

22.8

115.5

87.692

1.94296

357

71.4

357

93.947

1.9729

112

22.4

113

86.154

1.93527

356

71.2

356.5

93.684

1.9717

111

22.2

111.5

85.385

1.93138

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


DES Chapter 6


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