Chapter Six NANOEMULSION PREPARATION, OPTIMIZATION...

Chapter Six

NANOEMULSION PREPARATION,

OPTIMIZATION AND CHARACTERIZATION

Chapter 6: Nanoemulsion preparation, optimization and characterization

6.1. INTRODUCTION: NANOEMULSIONS

Nanoemulsions constitute an interesting group of drug delivery vehicles. They are

part of a broad class of multiphase colloidal dispersions. Although some lyotropic

liquid crystalline phases, also known as micellar phases, mesophases and

microemulsions may appear to be similar to nanoemulsions in composition and

nanoscale structure, such matrices are actually quite different. Nanoemulsions do not

form spontaneously, because an external shear has to be applied to rupture larger

droplets into smaller ones (Mason et al., 2006). They can be prepared by spontaneous

emulsification such as phase inversion temperature (PIT) emulsification or phase

inversion composition, or by using a high shear device (Sonneville-Aubrun et al. ,

2004). Generally, nanoemulsions can be defined as oil in water emulsions with mean

particle size diameters ranging from 20 to 200 nm (Gutierrez et al., 2008). The

particles which are formed exhibit a liquid, lipophilic core separated from the

surrounding aqueous phase by a monomolecular layer of phospholipids (Fig. 37). Due

to their lipophilic interior, nanoemulsions are more suitable for the transport of

lipophilc compounds than liposomes.

Fig. 37: Structure of nanoemulsion droplet

(www.pharmoscorp.com/img/bd_fig2.jpg, access date: 11.2008)

The attraction of nanoemulsions for application in health care as well as in cosmetics

is due to the following advantages (Tadros et al. , 2004):

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http://www.pharmoscorp.com/img/bd_fig2.jpg

^ The very small droplet size causes a large reduction in the gravity force and

the Brownian motion may be sufficient for overcoming gravity. This means

that no creaming or sedimentation occurs on storage.

^ Nanoemulsions are suitable for efficient delivery of active ingredients across

the skin, especially for non polar active compounds. The large surface area of

the emulsions system allows rapid penetration of actives.

^ Nanoemulsions may be applied as a substitute for liposomes and vesicles,

those are less stable.

Although, nanoemulsions are proposed for numerous applications in pharmacy as

drug delivery systems, one of the main problems is the Ostwald ripening, which is

perhaps the most serious problem with nanoemulsions. This results from the

difference in solubility between small and large particles (Solans et al., 2005;

Gutierrez et al., 2008). Ostwald ripening can be overcome for example by addition of

a second disperse phase component or by modification of the interfacial film at the

o/w interface (Tadros et al., 2004).

6.1.1. Why nanoemulsions are chosen for nose to brain drug delivery

Literature survey revealed that intranasal administration of nanoemulsion offers a

practical, noninvasive, alternative route of administration for drug delivery to the

brain (Gladstone & Gawel, 2003; Bigal et al., 2003). Intranasal administration

allows transport of drugs to the brain circumventing BBB, thus providing better

option to target drugs to the brain (Vyas et al. , 2005; . Illum, 2000; Talegaonkar &

Mishra, 2004; Vyas et al., 2004; Illum, 2003).

6.1.2. Challenges in nose to brain drug delivery via Nanoemulsion

1. The main problem in a nanoemulsion application is a high concentration and a

narrow range of physiologically acceptable surfactants and co-surfactants

(Corswant et al., 1998; Aboofazeli et al., 1995).

2. Large surfactant concentration (10-40%) determines their stability (Shinoda &

Kunieda, 1973).

3. Selection of components: if the systems are to be used topically, selection of

components involves a consideration of their toxicity, irritation and sensitivity

(Siebenbrodt & Keipert, 1993).


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4. Nasal congestion due to cold or allergies may interfere with absorption of drug

through nasal mucosa.

5. Delivery is expected to decrease with increasing molecular weight of drug.

6. Some therapeutic agents may be susceptible to partial degradation in the nasal

mucosa or may cause irritation to the mucosa.

7. Concentration achievable in different regions of the brain and spinal cord varies with each agent.

8. Fluidity of interfacial film should be low to promote the formulation of

Nanoemulsion (Attwood, 1994).

6.1.3. Application of Nanoemulsion in Treatment of Epilepsy

Vyas et al. prepared mucoadhesive Nanoemulsion for an antiepileptic drug

clonazepam (Vyas et al., 2005). The aim was to provide rapid delivery to the rat

brain. Brain/blood ratio at all sampling points up to 8h following intranasal

administration of clonazepam mucoadhesive Nanoemulsion compared to i.v. was

found to be 2-fold higher indicating larger extent of distribution of the drug in the

brain. Kwatikar et al. (2009) prepared Nanoemulsion containing valproic acid

showed a fractional diffusion efficiency and better brain bioavailability efficiency.

Hence Nanoemulsions are the promising approach for delivery of valproic acid to the

brain for treatement of epilepsy.

Florence et al. (2009) has prepared clobazam nanoemulsion and mucoadhesive

nanoemulsion. Formulations were assessed for the average onset of seizures in

pentylenetetrazole treated mice. The study demonstrated high brain targeting

efficiency of prepared clobazam mucoadhesive nanoemulsion and delayed onset of

seizures induced by pentylenetetrazole in mice after intranasal administration of

developed formulation. However, clinical evaluation of the developed formulation

may result into a product suitable for the treatment of acute seizures due to status

epileptics and patients suffering from drug tolerance and hepatic impairment on

chronic use in the treatment of epileptics, schizophrenia and anxiety. Shende et al.,

(2007) prepared nanoemulsion of lamotrigine from nose to brain delivery. Intranasal

administration allows transport of the drug to the brain circumventing BBB, thus

providing the better option to target drug to the brain with quick onset of action in

case of emergency in epilepsy.


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Lorazepam (LZM) is a poorly water-soluble drug which can be used as tranquillizers,

muscle relaxant, sleep inducer, sedative and antiepileptic agent (Holvoet et al. , 2005).

However, cosolvent based parenteral formulations suffer from several disadvantages

such as pain and tissue damage at the site of injection and precipitation of the drug on

dilution in several cases (Date & Nagarsenker). Furthermore, parenteral

administration of the organic cosolvents can also cause hemolysis (Yalin et al., 1997).

Amit et al. (Amit & Vandana, 2008) has prepared Lorazepam Nanoemulsions and

investigate that Nanoemulsion have very low hemolytic potential and exhibit good

physical and chemical stability and can be considered as a viable alternative to the

currently marketed Lorazepam formulations.

6.2. PREPARATION, OPTIMIZATION & CHARACTERIZATION of NANOEMULSION

6.2.1. Screening of oil, surfactant and co- surfactant for the Amiloride free base

(AMB) Nanoemulsion Formulation Development:

Optimum selection of components is crucial for a stable nanoemulsion (NE)

formulation development. The method adopted for screening of oils, surfactants and

cosurfactants are discussed below. The observation tables related to following studies

are given and discussed later in results and discussion part of this chapter.

6.2.1.1. Determination of solubility of AMB in oils, surfactants and co

surfactants

Various oils belonging to natural and semi synthetic sources were investigated so that

the oil with optimum solubility could be measured. The solubility of AMB was

determined in different oils e.g. oleic acid, isopropyl myristate (IPM), olive oil,

triacetin, castor oil, Labrafac, Labrafil 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 hrs at 37 ± 0.5°C, and samples were

centrifuged at 2000 rpm for 10 min. The supernatant was separated, filtered and after

appropriate dilution with methanol, solubility was determined. Same method was

adopted for solubility determination of drug in surfactant and cosurfactant.


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6.2.1.2. Optimization of surfactants and co-surfactants

On the basis of solubility studies, oleic acid was selected as the oil phase. Due to

difference in the solubility profile of AMB in different surfactants and co-surfactants

various phase diagrams were constructed by taking different surfactants and co

surfactants. Total eight combinations of surfactant and co-surfactants were prepared,

and the combination which gives larger nanoemulsion region with oleic acid was

selected.

6.2.1.2. a) Selection of Surfactant:

For optimization of surfactant, initially co-surfactant carbitol was kept constant,

different surfactant tween 20, tween 80, cremophore EL and labrasol in (0:3 to 3:0)

ratio with co-surfactant 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. The phase diagram was developed by aqueous titration

method.

6.2.1.2. b) Construction of phase diagram

The pseudo ternary phase diagram of oil/surfactant/co-surfactant was developed by

the water titration method. Aliquots of each surfactant and co-surfactant mixture

(Smix) were mixed with the oil at ambient temperature. For each phase diagram, the

ratio of oil to the Smix was varied as 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 (v/v).

Water was added drop-wise to each oil- Smix mixture under vigorous stirring. After

equilibrium, the samples were visually checked for clarity of NE. No heating was

conducted during the preparation. Phase diagrams were constructed using Chemix

software. The phase diagram with different ratios of surfactant and cosurfactant with

different oils were constructed to explore the NE region. The area of the monophasic

region was used as a tool for the selection of suitable surfactant and co-surfactant

mixture.


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Fig. 38: Design tree for optimization of nanoemulsion with respect to balancing Oil:Smix ratio by

keeping one constant and varied another and titrated with water to obtain a clear system having

thermodynamic stability.

6.2.2. Preparation of Nanoemulsion:

AMB nanoemulsion (ANE) was prepared by titration method using oleic acid as oil,

carbitol as co-surfactant and tween20/labrasol/cremophore EL as surfactant and

purified water as continuous phase.

Oil phase was mixed with Smix of a particular ratio, Oil and Smix ratio (0-3:3-0)

were taken in various ratios (1-9:9-1) and finally titrated with purified water was

added to drug loaded internal phase in dropwise manner under continuous stirring.

The compositions which were optically clear were evaluated further by constructing

pseudo ternary phase diagrams.

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6.2.3. Optimization of Nanoemulsion

Formulation of AMB containing different % of oil (2.5%, 5% and 7.5% v/v),

surfactant-cosurfactant mixture (20%, 30%, 40% and 50% v/v) and water was

tabulated in Table 23. NEs were evaluated for Globular size (G), Zeta potential (Z),

percentage transmittance (%T) and dilution characteristic. Consider the amount and

solubility of drug to be incorporated in the NE for the selection of formulation. The

final composition of NE was optimized based on GS, ZP, %T and dilution

characteristics.

6.2.4. Nanoemulsion Optimization Chart:

As discussed above following mentioned chart has been used to capture the

compositional observations with respect to various levels of Oil: Smix: Water

proportion in the NE System:


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Table 23: Titration Chart to find out Nanoemulsion region

RatioOil:Smix

Oil Surfactant (Smix) ^L

W ater^ L

W aterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

W ater%

10 90 10 10 110 9.09 81.82 9.0910 90 20 10 120 8.33 75.00 16.6710 90 25 5 125 8.00 72.00 20.0010 90 35 10 135 7.41 66.67 25.9310 90 45 10 145 6.90 62.07 31.0310 90 55 10 155 6.45 58.06 35.4810 90 65 10 165 6.06 54.55 39.39

1:910 90 80 15 180 5.56 50.00 44.4410 90 100 20 200 5.00 45.00 50.0010 90 120 20 220 4.55 40.91 54.5510 90 150 30 250 4.00 36.00 60.0010 90 185 35 285 3.51 31.58 64.9110 90 235 50 335 2.99 26.87 70.1510 90 300 65 400 3 23 7510 90 400 100 500 2.00 18.00 80.0010 90 550 150 650 2.00 14.00 8510 90 900 350 1000 1.00 9.00 90.0010 90 2000 1100 2100 0.48 4.29 95.24

20 80 10 10 110 18.18 72.73 9.0920 80 20 10 120 16.67 66.67 16.7620 80 25 5 125 16.00 64.00 20.00

2:8 20 80 35 10 135 14.81 59.26 25.93(1:4) 20 80 45 10 145 13.79 55.61 31.03

20 80 55 10 155 12.90 51.61 35.4820 80 65 10 165 12.12 48.48 39.3920 80 80 15 180 11.11 44.44 44.4420 80 100 20 200 10.00 40.00 50.0020 80 120 20 220 9.09 36.36 54.5520 80 150 30 250 8.00 32.00 60.0020 80 185 35 285 7.02 28.07 64.9120 80 235 50 335 5.97 23.88 70.1520 80 300 65 400 5.00 20.00 75.0020 80 400 100 500 4.00 16.00 80.0020 80 550 150 650 3.08 12.31 84.6220 80 900 350 1000 2.00 8.00 90.0020 80 2000 1100 2100 0.95 3.81 95.24

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Ratio Oil Surfactant Water Water Total Oil Surfactant WaterOil:Smix ^ L (Smix) ^L ^ L Added

^ L^ L % Smix % %

30 70 10 10 110 27.27 63.64 9.0930 70 20 10 120 25.00 58.33 16.6730 70 25 5 125 24.00 56.00 20.0030 70 35 10 135 22.22 51.85 25.93

3:7 30 70 45 10 145 20.69 48.28 31.03(1:2.3) 30 70 55 10 155 19.35 45.16 35.48

30 70 65 10 165 18.18 42.42 39.3930 70 80 15 180 16.67 38.89 44.4430 70 100 20 200 15.00 35.00 50.0030 70 120 20 220 13.64 31.82 54.5530 70 150 30 250 12.00 28.00 60.0030 70 185 35 285 10.53 24.56 64.9130 70 235 50 335 8.96 20.90 70.1530 70 300 65 400 7.50 17.50 75.0030 70 400 100 500 6.00 14.00 80.0030 70 550 150 650 4.62 10.77 84.6230 70 900 350 1000 3.00 7.00 90.0030 70 2000 1100 2100 1.43 3.33 95.24

40 60 10 10 110 36.36 54.55 9.0940 60 20 10 120 33.33 50.00 16.6740 60 25 5 125 32.00 48.00 20.0040 60 35 10 135 29.63 44.44 25.9340 60 45 10 145 27.59 41.38 31.03

4:6(1:1.5)

40 60 55 10 155 25.81 38.71 35.4840 60 65 10 165 24.24 36.36 39.3940 60 80 15 180 22.22 33.33 44.4440 60 100 20 200 20.00 30.00 50.0040 60 120 20 220 18.18 27.27 54.5540 60 150 30 250 16.00 24.00 60.0040 60 185 35 285 14.04 21.05 64.9140 60 235 50 335 11.94 17.91 70.1540 60 300 65 400 10.00 15.00 75.0040 60 400 100 500 8.00 12.00 80.0040 60 550 150 650 6.15 9.23 84.6240 60 900 350 1000 4.00 6.00 90.0040 60 2000 1100 2100 1.90 2.86 95.24

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RatioOil:Smix

Oil^ L

Surfactant (Smix) ^L

Water^ L

WaterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

Water%

50 50 10 10 110 45.45 45.45 9.0950 50 20 10 120 41.67 41.67 16.6750 50 25 5 125 40.00 40.00 20.0050 50 35 10 135 37.04 37.04 25.93

5:5 (1:1) 50 50 45 10 145 34.48 34.48 31.0350 50 55 10 155 32.26 32.36 35.4850 50 65 10 165 30.30 30.30 39.3950 50 80 15 180 27.78 27.78 44.4450 50 100 20 200 25.00 25.00 50.0050 50 120 20 220 22.73 22.73 54.5550 50 150 30 250 20.00 20.00 60.0050 50 185 35 285 17.54 17.54 64.9150 50 235 50 335 14.93 14.93 70.1550 50 300 65 400 12.50 12.50 75.0050 50 400 100 500 10.00 10.00 80.0050 50 550 150 650 7.69 7.69 84.6250 50 900 350 1000 5.00 5.00 90.0050 50 2000 1100 2100 2.38 2.38 95.24

60 40 10 10 110 54.55 36.36 9.0960 40 20 10 120 50.00 33.33 16.6760 40 25 5 125 48.00 32.00 20.0060 40 35 10 135 44.44 29.63 25.93

6:4 60 40 45 10 145 41.38 27.59 31.03(1:0.7) 60 40 55 10 155 38.71 25.81 35.48

60 40 65 10 165 36.36 24.24 39.3960 40 80 15 180 33.33 22.22 44.4460 40 100 20 200 30.00 20.00 50.0060 40 120 20 220 27.27 18.18 54.5560 40 150 30 250 24.00 16.00 60.0060 40 185 35 285 21.05 14.04 64.9160 40 235 50 335 17.91 11.94 70.1560 40 300 65 400 15.00 10.00 75.0060 40 400 100 500 12.00 8.00 80.0060 40 550 150 650 9.23 6.15 84.6260 40 900 350 1000 6.00 4.00 90.0060 40 2000 1100 2100 2.86 1.90 95.24

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RatioOil:Smix

Oil^ L


Water^ L

WaterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

Water%

70 30 10 10 110 63.64 27.27 9.0970 30 20 10 120 58.33 25.00 16.67

7:3 70 30 25 5 125 56.00 24.00 20.00(1:0.43) 70 30 35 10 135 51.85 22.22 25.93

70 30 45 10 145 48.28 20.69 31.0370 30 55 10 155 45.16 19.35 35.4870 30 65 10 165 42.42 18.18 39.3970 30 80 15 180 38.89 16.67 44.4470 30 100 20 200 35.00 15.00 50.0070 30 120 20 220 31.82 13.64 54.5570 30 150 30 250 28.00 12.00 60.0070 30 185 35 285 24.56 10.53 64.9170 30 235 50 335 20.90 8.96 70.1570 30 300 65 400 17.50 7.50 75.0070 30 400 100 500 14.00 6.00 80.0070 30 550 150 650 10.77 4.62 84.6270 30 900 350 1000 7.00 3.00 90.0070 30 2000 1100 2100 3.33 1.43 95.24

80 20 10 10 110 72.73 18.18 9.0980 20 20 10 120 66.67 16.67 16.6780 20 25 5 125 64.00 16.00 20.0080 20 35 10 135 59.26 14.81 25.9380 20 45 10 145 55.17 13.79 31.03

8:2 80 20 55 10 155 51.16 12.90 35.38(1:0.25) 80 20 65 10 165 48.48 12.12 39.39

80 20 80 15 180 44.44 11.11 44.4480 20 100 20 200 40.00 10.00 50.0080 20 120 20 220 36.36 9.09 54.5580 20 150 30 250 32.00 8.00 60.0080 20 185 35 285 28.07 7.02 64.9180 20 235 50 335 23.88 5.97 70.1580 20 300 65 400 20.00 5.00 75.0080 20 400 100 500 16.00 4.00 80.0080 20 550 150 650 12.31 3.08 84.6280 20 900 350 1000 8.00 2.00 90.0080 20 2000 1100 2100 3.81 0.95 95.24

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RatioOil:Smix

Oil^ L


Water^ L

WaterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

Water%

90 10 10 10 110 81.82 9.09 9.0990 10 20 10 120 75.00 8.33 16.67

9:1 90 10 25 5 125 72.00 8.00 20.00(1:0.1) 90 10 35 10 135 66.67 7.41 25.93

90 10 45 10 145 62.07 6.90 31.0390 10 55 10 155 58.06 6.45 35.4890 10 65 10 165 54.55 6.06 39.3990 10 80 15 180 50.00 5.56 44.4490 10 100 20 200 45.00 5.00 50.0090 10 120 20 220 40.91 4.55 54.5590 10 150 30 250 36.00 4.00 60.0090 10 185 35 285 31.38 3.51 64.9190 10 235 50 335 26.87 2.99 70.1590 10 300 65 400 22.50 2.50 75.0090 10 400 100 500 18.00 2.00 80.0090 10 550 150 650 13.85 1.54 84.6290 10 900 350 1000 9.00 1.00 90.0090 10 2000 1100 2100 4.29 0.48 95.24

20 40 6 6 66 30.30 60.61 9.0920 40 11 5 71 28.17 56.34 15.4920 40 15 4 75 26.67 53.33 20.0020 40 20 5 80 25.00 50.00 25.0020 40 26 6 86 23.26 46.51 30.23

1:2 20 40 33 7 93 21.51 43.01 35.4820 40 40 7 100 20.00 40.00 40.0020 40 50 10 110 18.18 36.36 45.4520 40 60 10 120 16.67 33.33 50.0020 40 75 15 135 14.81 29.63 55.5620 40 90 15 150 13.33 26.67 60.0020 40 112 22 172 11.63 23.26 65.1220 40 140 28 200 10.00 20.00 70.0020 40 180 40 240 8.33 16.67 75.0020 40 240 60 300 6.67 13.33 80.0020 40 340 100 400 5.00 10.00 85.0020 40 540 200 600 3.33 6.67 90.0020 40 1140 600 1200 1.67 3.33 95.00

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RatioOil:Smix

Oil^ L


Water^ L

WaterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

Water%

20 60 10 10 90 22.22 66.67 11.1120 60 14 4 94 21.28 63.83 14.89

1:3 20 60 20 6 100 20.00 60.00 20.0020 60 27 7 107 18.69 56.07 25.2320 60 35 8 115 17.39 52.17 30.4320 60 43 8 123 16.26 48.78 34.9620 60 54 11 134 14.93 44.78 40.3020 60 66 12 146 13.70 41.10 45.2120 60 80 14 160 12.50 37.50 50.0020 60 98 18 178 11.24 33.71 55.0620 60 120 22 200 10.00 30.00 60.0020 60 149 29 229 8.73 26.20 65.0720 60 187 38 267 7.49 22.47 70.0420 60 240 53 320 6.25 18.75 75.0020 60 320 80 400 5.00 15.00 80.0020 60 455 135 535 3.74 11.21 85.0520 60 700 245 780 3 8 9020 60 1520 820 1600 1.25 3.75 95.00

20 70 10 10 100 20.00 70.00 10.0020 70 16 6 106 18.87 66.04 15.0920 70 23 7 113 17.70 61.95 20.3520 70 30 7 120 16.67 58.33 25.00

1.35 20 70 39 9 129 15.50 54.26 30.2320 70 49 10 139 14.39 50.36 35.2520 70 60 11 150 13.33 46.67 40.0020 70 74 14 164 12.20 42.68 45.1220 70 90 16 180 11.11 38.89 50.0020 70 110 20 200 10.00 35.00 55.0020 70 135 25 225 8.89 31.11 60.0020 70 168 33 258 7.75 27.13 65.1220 70 210 42 300 6.67 23.33 70.0020 70 270 60 360 5.56 19.44 75.0020 70 360 90 450 4.44 15.56 80.0020 70 510 150 600 3.33 11.67 85.0020 70 810 300 900 2.22 7.78 90.0020 70 1710 900 1800 1.11 3.89 95.00

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RatioOil:Smix

Oil^ L


Water^ L

WaterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

Water%

20 100 14 14 134 14.93 74.63 10.4520 100 22 8 142 14.08 70.42 15.49

1:5 20 100 30 8 150 13.33 66.67 20.0020 100 40 10 160 12.50 62.50 25.0020 100 53 13 173 11.56 57.80 30.6420 100 65 12 185 10.81 54.05 35.1420 100 80 15 200 10.00 50.00 40.0020 100 100 20 220 9.09 45.45 45.4520 100 120 20 240 8.33 41.67 50.0020 100 147 27 267 7.49 37.45 55.0620 100 180 33 300 6.67 33.33 60.0020 100 225 45 345 5.80 28.99 65.2220 100 280 55 400 5.00 25.00 70.0020 100 360 80 480 4.17 20.83 75.0020 100 480 120 600 3.33 16.67 80.0020 100 680 200 800 2.50 12.50 85.0020 100 1100 420 1220 1.64 8.20 90.1620 100 2300 1200 2420 0.83 4.13 95.04

20 120 16 16 156 12.82 76.92 10.2620 120 25 9 165 12.12 72.73 15.1520 120 35 10 175 11.43 68.57 20.0020 120 47 12 187 10.70 64.17 25.1320 120 60 13 200 10.00 60.00 30.00

1:6 20 120 76 16 216 9.26 55.56 35.1920 120 93 17 233 8.58 51.50 39.9120 120 115 22 255 7.84 47.06 45.1020 120 140 25 280 7.14 42.86 50.0020 120 172 32 312 6.41 38.46 55.1320 120 210 38 350 5.71 34.29 60.0020 120 260 50 400 5.00 30.00 65.0020 120 330 70 470 4.26 25.53 70.2120 120 420 90 560 3.57 21.43 75.0020 120 560 140 700 2.86 17.14 80.0020 120 800 240 940 2.13 12.77 85.1120 120 1260 460 1400 1.43 8.57 90.0020 120 2700 1440 2840 0.70 4.23 95.07

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RatioOil:Smix

Oil^ L


Water^ L

WaterAdded

^ L

Total^ L

Oil%

Surfactant Smix %

Water%

20 140 18 18 178 11.24 78.65 10.1120 140 30 12 190 10.53 73.68 15.79

1:7 20 140 40 10 200 10.00 70.00 20.0020 140 54 14 214 9.35 65.62 25.2320 140 70 16 230 8.70 60.87 30.4320 140 86 16 246 8.13 56.91 34.9620 140 107 21 267 7.49 52.43 40.0720 140 135 28 295 6.78 47.46 45.7620 140 160 25 320 6.25 43.75 50.0020 140 196 36 356 5.62 39.33 55.0620 140 240 44 400 5.00 35.00 60.0020 140 300 60 460 4.35 30.43 65.2220 140 375 75 535 3.74 26.17 70.0920 140 480 105 640 3.13 21.88 75.0020 140 640 160 800 2.50 17.50 80.0020 140 907 267 1067 1.87 13.12 85.0020 140 1440 533 1600 1.25 8.75 90.0020 140 3050 1610 3210 0.62 4.36 95.02

20 160 20 20 200 10.00 80.00 10.0020 160 32 12 212 9.43 75.47 15.0920 160 45 13 225 8.89 71.11 20.00

1:8 20 160 60 15 240 8.33 66.67 25.0020 160 78 18 258 7.75 62.02 30.2320 160 97 19 277 7.22 57.76 35.0220 160 120 23 300 6.67 53.33 40.0020 160 147 27 327 6.12 48.93 44.9520 160 180 33 360 5.56 44.44 50.0020 160 220 40 400 5.00 40.00 55.0020 160 335 115 515 3.88 31.07 70.0020 160 420 85 600 3.33 26.67 75.0020 160 540 120 720 2.78 22.22 75.0020 160 720 180 900 2.22 17.78 80.0020 160 1020 300 1200 1.67 13.33 85.0020 160 1620 600 1800 1.11 8.89 90.0020 160 3420 1800 3600 0.56 4.44 95.00

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6.2.5. Characterization of Nanoemulsion

The characterization of NE was an essential step before proceeding for the next

studies. The characterization was performed for predicting the reproducible

characteristics of the prepared formulation. NE’s were characterized for GS, ZP,

Assay, in vitro drug release (drug diffusion), surface morphology and viscosity.

Various techniques for characterization of NE’s included, Photon correlation

spectroscopy (PCS) based on the dynamic light scattering (DLS) for GS and its

distribution. The surface characteristic like charge was examined by measurement of

ZP, surface morphology by transmission electron microscopy (TEM). Amount of drug

present in NE was determined as assay. The in vitro release of the drug from the NE

influenced the in vivo pharmacokinetic and pharmacodynamic behaviour and was

estimated by developed analytical method. Viscosity of NE affects delivery method

and distribution to various organs. Viscosity of NE was determined by Brookfield

Viscometer.

6.2.5.1. Appearance

Appearance of NE of AMB was evaluated against white and black background.

6.2.5.2. Globule size and Zeta potential determination

The GS determination (Kaler & Prager, 1982; Roland et al., 2003) of AMB loaded

NE were determined using photon correlation spectroscopy (PCS) with in-built

Zetasizer (model: Nano ZS, Malvern instruments, UK) at 633 nm.

Measurement conditions for GS were optimized by measuring GS for the dispersions

of different dilutions. The dilution of the NE in water was made in such a way that the

integrity of the globules were maintained with sufficient inter particle space and

minimal multiple light scattering during measurement.

Malvern Zetasizer Nano ZS was used to measure the ZP of the globules based on the

electrophoresis and electrical conductivity of the formed NE. The electrophoretic

mobility (^m/s) of the particles was converted to the ZP by in-built software based on

Helmholtz-Smoluchowski equation. Measurements were performed using small

volume disposable zeta cell.

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6.2.5.3. Transmission Electron microscopy (TEM)

TEM is used as a tool to study the morphology and structure of the delivery systems.

The TEM images of nanoemulsions were taken to get idea about the size of

nanoemulsions (Sheikh & Faiyaz, 2007). The images were taken Tecnai 200 with

CCD camera operating at 200kV (Philips Instruments, Holland) and capable of point

to point resolution. NE were diluted in de-ionized water (1 in 10 dilution). To measure

the morphology and size distribution, a drop of sample was placed onto a 300-MEh

copper grid coated with carbon. Approximately 2 min after deposition, the grid was

tapped with filter paper to remove surface water and air-dried. Staining was

performed using a droplet of 0.5% w/v phosphotungstic acid.

6.2.5.4 Estimation of drug in Nanoemulsion

0.1 mL of ANE was sufficiently diluted with methanol. Estimation of drug in NE was

determined as per the method described in the Analytical section and the results were

recorded.

6.2.5.5. pH Determination

The pH of ANE was measured by digital pH meter at 25° C ± 1°C. The pH was

recorded in triplicate. pH meter was calibrated using buffer solutions prior to use.

6.2.5.6. Viscosity Determination

Viscosity of the formulations was determined using Brookfield cone and plate

Rheometer (Model LVDV III) using CPE spindle at the rotational speed of 5 rpm,

shear rate of 10 at room temperature and the results were recorded.

6.2.5.7. Transmittance

The percentage transmittance of NE was checked against distilled water using UV-

Visible spectrophotometer (UV, 1700, Shimadzu, Japan) at 630 nm.

6.2.6. Thermodynamic stability testing of drug loaded nanoemulsions

To check that the nanoemulsions were stable, the drug loaded nanoemulsions were

subjected to thermodynamic stability testing, which comprises of heating cooling

cycle, freeze thaw cycle and centrifugation tests. Physical stability was continuously

129

monitored over the period of time. Various aspects like phase separation, turbidity

etc. at room temperature were observed and recorded (Sheikh et al., 2007).

a. Freeze thaw cycle

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

nanoemulsions were removed and kept at room temperature. The thermodynamically

stable nanoemulsions returned to their original form within 20-30 minutes. 2-3 such

cycles were repeated.

b. 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.

c. Heating cooling cycle

Nanoemulsions were kept at 37±0.5 ° 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.

6.2.7. Selection of Nanoemulsion Formulations

It is well known that large amounts of surfactants cause skin or mucosal irritation

(Lawrence & Rees, 2000; Warisnoicharoen, 2002; Li et al. , 2005); therefore, it is

important to determine the surfactant concentration properly and use the optimum

concentration of surfactant in the formulation. From pseudoternary phase diagrams,

the formulations in which the amount of oil phase completely solubilized the drug and

which could accommodate the optimum quantity of Smix and distilled water were

selected for the study. However at the same time the other formulation factors like

maximum oil solubility, globular size, stress stability, thermodynamic stability studies

were taken as number of the variable for the final selection of the formulation for in-

vitro permeation studies.

6.2.8. Selection of Mucoadhesive agent for Mucoadhesive Nanoformulations

Various polymers from natural origin like chitosan, sodium alginate, gellan gum or

synthetic derivative like carbopol were used at different concentration to assess their


130

mucoadhesive property using Texture Analyzer (TAXT2i/; Stable Micro Systems,

Surray, UK).

6.2.9. Evaluation of the Mucoadhesive Strength

The mucoadhesive potential of each polymer was determined by measuring the force

required to detach the formulation from nasal mucosal tissue using a modified method

described by (Jones et al., 1997). The goat nasal mucosa has been chosen for its

smooth surface and thinness. Goat nasal mucosa were drawn immediately after the

sacrifice of the animals at the slaughterhouse and then frozen at -20°C. The mucosa

were defrozen and cleaned before the tests, using an isotonic solution (NaCl 0.9%) at

room temperature, cut into discs of 2 cm in diameter and then it was fixed on the

lower support of the tensile tester by cyanoacrylate glue (Duch'ene et al., 1988). Very

thin layers of the Mucoadhesive polymer solution of defined concentration

(0.1/0.2/0.3%) were applied in 1.5 cm disks of electrophoresis foils and then glued on

the upper metal probe. The tests were performed applying a pre-load of 1-10 N for a

time contact of 5 min and raising the upper probe at the constant speed of 5 mm/min.

Five replicates were performed for each type of polymer solution and the average and

standard deviations were then calculated.

6.2.10. Preparation of Mucoadhesive Nanoemulsion (MNE) of Amiloride

(AMNE)

Mucoadhesive nanoemulsions (AMNE) were prepared by addition of mucoadhesive

polymer (showing maximum strength, ref section of selection of mucoadhesive agent)

such as chitosan (Dodane et al., 1999) to optically clear nanoemulsion.

The mucoadhesive nanoemulsions were prepared by first preparing a nanoemulsion of

the drug using minimum volume of external phase and then adding the required

volume of concentrated polymer solution to it such that the required final

concentration achieved. AMNE was prepared as described under AMB nanoemulsion

preparation and chitosan was added in a concentration of 0.25%/0.50%/1.0% w/v with

continuous stirring for 30 minutes.


131

6.2.11. Preparation of nasal mucosa membrane

The freshly excised goat nasal mucosa, except septum part was collected from the

slaughter house and was kept in PBS pH 6.5 for 15 min to equilibrate. The superior

nasal conche was identified and separated from the nasal membrane and made free

from adhered tissues. Selective samples of tissues of 0.2 mm thickness were taken for

the studies. The excised nasal membrane was then mounted on Franz diffusion cell.

The tissue was stabilized using phosphate buffer pH 6.5 in both the compartments and

allowed to stir for 15 min on a magnetic stirrer. After 15 min, solution from both the

compartments was removed and the diffusion media was filled in the receptor

compartment. The mounting of the nasal mucosa was done using glue at the brim of

the donor compartment to avoid the leakage of the test sample and supported with

rubber bands crossover the cell. The temperature of the receiver chamber containing

diffusion media was controlled at 37° ± 1° C under continuous stirring with teflon

coated magnetic bar at constant rate, in such a way that the nasal membrane surface

just flushes the diffusion media.

6.2.12. Ex vivo evaluation of Mucoadhesion using curcumin loaded

Nanoemulsion formulations by using Confocal Laser Scanning Microscopy

(CLSM)

Curcumin (Cm) is yellowish food dye and have similar fluorescent properties to

fluorescence sodium. It shows a strong fluorescence signal after excitation at 488 nm

in the spectral region of 550 nm. The fluorescence signal can be detected at 590 nm,

which is far from the background fluorescence (Otberg et al. , 2003). It is an oil

soluble dye and thus was added during the preparation stages of nanoemulsion. The

studies were performed in triplicate. In order to evaluate the retention capacity of Cm-

Nanoformulations into the nasal epithelium, prepared specimen were washed with

normal saline 3 time on every 5 minutes and then these specimens were directly

mounted, mucosal side up, on a glass slide and examined without further tissue

processing by CLSM (Olympus Fluo View FV 1000, Hamburg, Germany). Samples

were excited with green helium neon 543nm laser beam. Images were taken

employing a 20x oil objective, assembled in an integral image processor and

displayed on a digital video monitor. To confirm the penetration of Cm-

Nanofomrulations, stacks of serial 4.4m optical sections were captured along the Z-

axis.


132


6.2.13. In vitro drug permeation study

In-vitro drug permeation study was performed using Franz diffusion cell as discussed

in earlier section (Willimann et al., 1992). In vitro diffusion of formulations is a

valuable tool to predict the behaviour of a particular formulation with respect to drug

transport across the membrane. According to Gemmell & Morrison (1957), in vitro

models may have limitations in terms of prediction of drug transport across the

mucosal membrane nevertheless: under the testing conditions in vitro studies can be

helpful to access the relative drug transport behaviour across the mucosa. Various

parameters pertaining to formulations such as flux, partition coefficient and diffusion

coefficient can be derived using in vitro evaluation techniques. In present study, all

the test formulations were accessed for in vitro diffusion across the goat nasal

mucosa.

In-vitro permeation studies through goat nasal mucosa were performed using an

automated Transdermal Diffusion Cell Sampling System (SFDC6, LOGAN Inst, NJ,

USA) (Fig. 39). Separated nasal mucosa samples were mounted into the diffusion

cells (area 0.75 cm2: effective diffusion area 0. 636 cm2) equilibrated at 37± 0.2° C for

8-10 hrs. PBS pH 6.4 + 30% ethanol was used as a diffusion media for diffusion

study of AMB loaded NEs. 0.1 mL of NE or 0.12 ml of NE was placed in the donor

compartment along with 0.1 mL of diffusion media. Receptor compartment

containing recipient medium was stirred with Teflon coated magnetic bead. Aliquot

(500 ^L) was withdrawn from the recipient compartment of vertical cell at

predetermined time intervals and analyzed by analytical method as discussed in

chapter 4. Each sample removed was replaced by an equal volume diffusion media.

Study was carried for a period of 8 h, during which the drug in receiver chamber

(^g/ml) across the goat nasal membrane calculated at each sampling point. The

formulations were studied in triplicate for diffusion studies and the mean cumulative

values for % drug release, diffusion coefficients and flux were determined of AMB.

Instrumentation

A. Specification of SFDC-6 transdermal diffusion sampling system

Drive position : 6 (3 for vertical cells, 3 for side-by-side cell)

Cell position : 6 (3 for vertical cells, 3 for side-by-side cell)

133


Control zone

Zone 1

Zone 2

Speed

Temperature control

2

drives side-by-side cell

drives vertical cells

600 rpm

25 to 45 °C

B. Description of SFDC-6 Transdermal Diffusion cell Sampling System:

LOGAN SFDC - 6Transdem tiil Diffusion Celt Drive Console

Water bath ( style o f the bath maybe different from time to time) Water manifold assembling

POWER ON/OFF SWITCH

Side by Side Cells Control Switch ertical Cells Control Switch

Fig. 39: SFDC-6 Transdermal Diffusion Cell Sampling System

LOGAN SFDC-6 transdermal diffusion cell drive console was designed to perform

transdermal/transmucosal diffusion testing, up to 3 Franz cells (Fig. 39/40) and 3

side-by-side cells can be tested at the same time. Skin/mucosa was mounted between

the cell cap (donor) and cell body (receptor). The mucosal side of mucosal was bathed

from below by isotonic saline solution injected through a port provided for such

134

purpose. Temperature was maintained at 37°C by thermostatically controlled water,

which entered the lower port of the water jacket surrounding the saline solution

chamber, and circulated out through the upper port. Homogenous distribution of the

temperature in the saline bathing solution was accomplished by agitation motion of a

Teflon-coated magnetic bar. The sample was withdrawn using micropipette and

analyzed for drug content using analytical method.


Fig. 40: A transdermal diffusion cell assembly

Percent drug diffused

The percent drug diffused across the goat nasal mucosa at predetermined sampling

time interval was determined using following formula.

Amount of drug in receptor compartment at time t% Drug diffused = x 100

Amount of drug loaded in the donor compartment

135


Kinetics of release

I n o r d e r t o i n v e s t i g a t e t h e m e c h a n i s m o f d r u g r e l e a s e f r o m t h e f o r m u l a t i o n , t h e

r e l e a s e r a t e s w e r e i n t e g r a t e d i n t o e a c h o f t h e f o l l o w i n g e q u a t i o n a n d t h e r e g r e s s i o n

c o e f f i c i e n t w a s c a l c u l a t e d .

( i ) Z e r o o r d e r e q u a t i o n Q = K 0 t

W h e r e , Q i s a m o u n t o f d r u g r e l e a s e d a t t i m e t ( h ) a n d K 0 i s z e r o o r d e r r e l e a s e r a t e

c o n s t a n t

( i i ) F i r s t o r d e r e q u a t i o n Q = Q 0 e - K 1t

W h e r e Q , i s a m o u n t o f d r u g r e l e a s e d a t t i m e t ( h ) a n d K 1 i s f i r s t o r d e r r e l e a s e r a t e

c o n s t a n t .

( i i i ) H i g u c h i ’ s e q u a t i o n Q = K H x V t

W h e r e , Q i s a m o u n t o f d r u g r e l e a s e d a t t i m e t ( h ) a n d K H i s z e r o o r d e r r e l e a s e r a t e

c o n s t a n t .

( i v ) H i x s o n - c r o w e l l c u b e r o o t l a w Q 01/3- Q t 1/3= K H C t

W h e r e , Q 0 i s i n i t i a l a m o u n t o f t h e d r u g i n t h e f o r m u l a t i o n , Q t i s a m o u n t o f t h e d r u g

r e l e a s e d a t t i m e t ( h ) a n d K H C i s H i x s o n - C r o w e l l r a t e c o n s t a n t .

( v ) K o r s m e y e r - p e p p a s e q u a t i o n M t / M „ = K t ^ n

W h e r e M t - a m o u n t o f t h e r e l e a s e d d r u g a t t i m e t ( h ) , M „ i s t o t a l a m o u n t o f d r u g

r e l e a s e d a f t e r a n i n f i n i t e t i m e , K i s d i f f u s i o n a l c h a r a c t e r i s t i c c o n s t a n t o f d r u g / p o l y m e r

s y s t e m a n d n i s e x p o n e n t t h a t c h a r a c t e r i z e s t h e m e c h a n i s m o f d r u g r e l e a s e .

T h e o r d e r o f r e l e a s e w a s d e t e r m i n e d b y p e r f o r m i n g t h e r e g r e s s i o n o v e r t h e m e a n

v a l u e s o f p e r c e n t d r u g d i f f u s e d v s . t i m e ( f o r z e r o o r d e r ) , l o g p e r c e n t d r u g d i f f u s e d v s .

t i m e ( f o r f i r s t o r d e r ) , p e r c e n t d r u g d i f f u s e d v s . s q u a r e r o o t o f t i m e ( f o r h i g u c h i o r d e r ) ,

d i f f e r e n c e o f c u b e r o o t o f p e r c e n t t o t a l d r u g a n d p e r c e n t d r u g r e l e a s e d v s . t i m e

( H i x s o n - c r o w e l l c u b e r o o t l a w ) a n d l o g c u m u l a t i v e p e r c e n t a g e o f d r u g r e l e a s e d v s .

l o g t i m e ( K o r s m e y e r - p e p p a s e q u a t i o n ) .

136

Flux

The skin flux was experimentally determined from the following equation (Lee et al. ,

2005) J = (dQ/dt)/A

Where, J is the steady-state flux (^g/cm2/h), A is the diffusion area of skin tissue

(cm2) through which drug permeation takes place, and dQ/dt is the amount of drug

passing through the skin per unit time at a steady-state (^g/h). The cumulative amount

of drug permeating through the membrane was plotted as a function of time.

Diffusion coefficient

The diffusion coefficient of the drug was calculated using the following equation

(Aulton, 2007) D= Jxh/C0

Where, J is Flux, C0 is drug concentration in donor compartment and h is thickness of

the membrane.

6.3. RESULTS AND DISCUSSION:

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

dissolve AMB, because only the dissolved drug can permeate through nasal mucosa.

In order to screen appropriate solvent/s for the preparation of NE, the

solubility/miscibility of AMB in various oils, surfactants and co-surfactants was

measured.

After performing solubility study in different oils, it was found (Table 24) that

Amiloride exhibited maximum solubility in the oleic acid (37.0 ± 0.5 mg/mL).

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

oleic acid is that, it is a powerful permeability enhancer for trans-membrane delivery

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

stratum corneum by forming separate domains which interfere with the continuity of

the multi lamellar stratum corneum and induce highly permeable pathways in the

stratum corneum (Puranjoti et al., 1999; Hadgraft et al., 2001).

Similarly based on solubility of AMB in various surfactant and co-surfactant (Table

25), their screening was done.


137


6.3.1. Solubility study of Amiloride:

Table 24: Solubility of AMH and AMB in different Oil Phases

Oil Phase Solubility (± SD) in mg/ml (n=3)

AMH AMB

Oleic Acid 0.2 ± 0.04 37.0 ± 0.5

Isopropyl Myristate 0.12 ± 0.02 15.0 ± 0.3

Olive Oil 0.14 ± 0.01 17.0 ± 0.3

Triacetin 0.1 ± 0.01 10.0 ± 0.1

Castor Oil 0.12 ± 0.02 14.0 ± 0.2

Labrafac 0.1 ± 0.01 12.0 ± 0.2

Labrafil 0.15 ± 0.02 18.0 ± 0.4

Lauroglycol 90 0.1 ± 0.001 3.0 ± 0.02

Table 25: Solubility of AMH and AMB in different Surfactants & co-surfactants

Oil Phase Solubility (± SD) in mg/ml (n=3)

AMH AMB

Tween 20 1.6 ± 0.03 12.6 ± 0.2

Tween 80 0.5 ± 0.01 16.8 ± 0.3

Labrasol 7.4 ± 0.15 95.0 ± 1.3

Cremophore EL 0.2 ± 0.01 9.0 ± 0.2

Propylene Glycol 167.6 ± 1.2 7.7 ± 0.1

Ethylene Glycol 38.2 ± 1.1 14.9 ± 0.5

Carbitol 16.3 ± 0.19 127.1 ± 2.1

Ethyl alcohol 4.2 ± 0.01 8.1 ± 0.02

PEG 200 17.15 ± 0.5 8.0 ± 0.04

138

6.3.2. Pseudo Ternary Phase diagram construction:

Constructing phase diagrams is time-consuming, particularly when the aim is to

accurately delineate a phase boundary (Eccleston, 1995). Care was taken to ensure

that observations were not made on metastable systems—although the free energy

required to form an emulsion is very low, the formation is thermodynamically

spontaneous (Craig et al., 1995). The relationship between the phase behavior of a

mixture and its composition can be captured with the aid of a phase diagram

(Lawrence & Rees, 2000). Pseudoternary phase diagrams were constructed

separately for each Smix ratio, so that o/w nanoemulsion regions could be identified

and nanoemulsion formulations could be optimized.

6.3.3. Construction of Amiloride (ANE) loaded Nanoemulsion Phase diagram:

Among the selected surfactant and cosurfactant, pseudoternary phase diagrams were

constructed by phase titration method in order to define the extent and nature of

nanoemulsion region and surrounding two & three phase domains. The construction

of pseudoternary phase diagrams was started using surfactant i.e. tween 20, and

labrasol and cosurfactant carbitol in different ratios. It was found that the region of

nanoemulsion existence was higher with tween 20 / carbitol than the labrasol /

carbitol combination. The existence of nanoemulsion region was highest in 3:1 ratio

of tween 20 / carbitol (Fig. 41 & 42) while in contrast not a single nanoemulsion

point was obtained with 1:4 ratios of tween 20 / carbitol. One interesting result was

also seen with this study that with increase of Smix ratio the nanoemulsion region

gets increased up to the certain limit and vice versa.

The selection of surfactant and co-surfactant mixture was on the basis of HLB

values, drug solubility, safety and stability profile. Non-ionic surfactants are known

to be least toxic and chemically highly stable (Williams & Payne, 2001) and hence,

use of non-ionic surfactant for pharmaceutical NE formulation is gradually

increasing. Basis on the maximum nanoemulsion region can be seen with the

Surfactant/cosurfactant Tween-20/carbitol, hence selected for the formulation of

ANE.


139


Fig. 41: Pseudo-ternary Phase Diagram Construction with Tween 20 & Carbitol for Amiloride

Nanoemulsion optimization.

140


Fig. 42: Pseudo-ternary Phase Diagram Construction with Labrasol & Carbitol for Amiloride

Nanoemulsion optimization.

141


6.3.4. Characterization of Developed AMB Nanoemulsion (ANE):

Table 26: Initial Characterization of Developed AMB Nanoemulsion (ANE) and inference from the thermodynamic stress testing

System Formulation

Oil(%)

Smix(%)

AQ(%)

Globule Size (nm) ± SEM

Zeta potential (mV) ± SEM

Transmittance (%) ± SEM

Thermodynamicstability

Smix 1:0 (Tween20:Carbitol)

ANE 1 10 50 40 21.29 ± 11.36 -12.83± 2.45 99.1±0.44 Pass

ANE 2 20 50 30 59.09± 13.36 -14.71± 2.87 96.7±0.63 Fail

ANE 3 30 50 20 120.11±15.28 -17.33± 0.82 91.4±0.40 Pass

ANE 4 40 50 10 102.43 ±11.62 -22.86±0.31 99.1±0.52 Fail

ANE 5 10 70 20 81.33± 4.91 -31.33±1.54 93.2±0.41 Pass

ANE 6 20 65 15 92.62 ± 4.73 -19.72± 2.72 96.2±2.42 Pass

Smix 2:1 (Tween20:Carbitol)

ANE 7 10 50 40 111.21 ± 12.41 -14.32± 2.53 97.1±0.13 Pass

ANE 8 20 50 30 90.13± 12.262 -23.24± 4.98 96.8±0.43 Fail

ANE 9 30 50 20 73.13±11.42 -18.04± 1.68 96.4±0.2 Fail

ANE 10 40 50 10 108.25 ±18.21 -13.08±0.42 98.9±0.4 Fail

ANE 11 10 70 20 102.12± 4.94 -21.32±0.32 95.3±0.6 Pass

ANE 12 20 65 15 55.36 ± 14.57 -34.24± 2.69 94.9±3.21 Pass

142


System Formulation

Oil(%)

Smix(%)

AQ(%)



Transmittance(%) ± SEM

Thermod-ynamicstability

ANE 13 10 50 40 46.58 ± 22.80 -33.58± 1.09 97.4±0.3 Fail

ANE 14 20 50 30 109.83±11.21 -14.19± 3.19 95.2±0.2 Fail

Smix 3:1 ANE 15 30 50 20 47.23±18.14 -31.30± 1.08 99.1±0.4 Pass

(Tween20: Carbitol ANE 16 40 50 10 112.16 ±8.86 -30.32±0.19 95.9±0.1 Fail

ANE 17 10 70 20 44.18± 5.22 -16.30±1.14 94.1±0.5 Pass

ANE 18 20 65 15 55.16 ± 14.72 -31.23± 4.19 94.4±1.39 Pass

ANE 19 10 50 40 106.28 ± 15.34 -30.98± 3.31 98.2±0.7 Fail

ANE 20 20 50 30 109.62±11.34 -35.09± 2.17 96.8±0.4 Fail

Smix 4:1ANE 21 30 50 20 60.11±13.28 -23.20± 0.18 95.4±0.5 Pass

(Tween20:Carbitol) ANE 22 40 50 10 118.15±38.16 -22.16±0.19 92.1±0.4 Fail

ANE 23 10 70 20 81.42± 8.92 -28.14±3.01 98.8±0.7 Pass

ANE 24 20 65 15 64.86 ± 24.57 -13.74± 5.22 98.4±2.29 Pass

143


System Formulation

Oil(%)

Smix(%)

AQ(%)





ANE 25 10 50 40 68.11 ± 2.33 -13.58± 2.09 98.2±0.6 Fail

ANE 26 20 50 30 49.29± 16.26 -25.09± 2.37 99.8±0.3 Pass

Smix 1:1 ANE 27 30 50 20 70.21±5.28 -23.41± 1.88 99.4±0.5 Fail

(Tween20: Carbitol ANE 28 40 50 10 104.25 ±8.16 -20.81±0.42 94.2±0.1 Fail

ANE 29 10 70 20 54.17± 6.22 -35.30±1.34 99.5±0.4 Pass

ANE 30 20 65 15 105.06 ± 54.17 -21.84± 3.22 95.3±2.19 Pass

ANE 31 10 50 40 66.18 ± 22.31 -32.13± 3.91 98.1±0.8 Pass

ANE 32 20 50 30 100.09± 16.14 -25.09± 2.42 95.8±0.4 Fail

Smix 1:2ANE 33 30 50 20 52.31±12.18 -23.10± 1.98 99.4±0.6 Pass

(Tween20:Carbitol) ANE 34 40 50 10 88.43 ±12.19 -22.18±1.42 96.4±0.8 Fail

ANE 35 10 70 20 48.18± 6.57 -35.46±1.04 99.9±0.3 Pass

ANE 36 20 65 15 105.96 ± 24.07 -12.54± 4.29 92.4±6.29 Fail

144


System Formulation

Oil(%)

Smix(%)

AQ(%)





Smix 1:0 (Cremophore

EL: Carbitol)

ANE 37 10 50 40 135.28 ± 32.30 -33.08± 4.19 91.3±0.7 Fail

ANE 38 20 50 30 99.91± 14.32 -25.19± 5.17 93.3±0.2 Fail

ANE 39 30 50 20 127.19±25.22 -33.27± 4.18 94.4±0.6 Pass

ANE 40 40 50 10 88.25 ±28.16 -22.38±3.52 94.1±0.4 Fail

ANE 41 10 70 20 104.42± 6.12 -35.40±3.71 99.4±0.3 Pass

ANE 42 20 65 15 77.61 ± 24.57 -33.14± 4.19 96.3±0.9 Pass

Smix 2:1 (Cremophore

EL: Carbitol)

ANE 43 10 50 40 89.48 ± 22.10 -33.58± 2.19 98.2±0.4 Fail

ANE 44 20 50 30 109.13± 15.74 -23.19± 6.30 97.2±0.5 Fail

ANE 45 10 35 55 42.11±15.28 -13.00± 0.98 99.4±0.5 Pass

ANE 46 40 50 10 138.15 ±18.76 -2.08±0.22 97.9±0.3 Fail

ANE 47 10 70 20 64.28± 5.82 -26.40±0.74 99.8±0.5 Pass

ANE 48 20 65 15 75.76 ± 14.57 -32.54± 1.29 99.4±1.29 Pass

145


System Formulation

Oil(%)

Smix(%)

AQ(%)





Smix 3:1 (Cremophore EL: Carbitol)

ANE 49 10 50 40 76.18 ± 12.30 -23.58± 1.09 96.2±0.2 Fail

ANE 50 20 50 30 89.09± 10.24 -15.69± 1.27 99.8±0.6 Fail

ANE 51 30 50 20 120.11±15.28 -13.00± 0.98 98.4±0.5 Pass

ANE 52 40 50 10 138.15 ±18.76 -2.08±0.22 97.9±0.3 Fail

ANE 53 10 70 20 64.28± 5.82 -26.40±0.74 99.8±0.5 Pass

ANE 54 20 65 15 75.76 ± 14.57 -32.54± 1.29 99.4±1.29 Pass

Smix 1:2 (Cremophore EL: Carbitol)

ANE 55 10 50 40 76.18 ± 12.30 -23.58± 1.09 96.2±0.2 Fail

ANE 56 20 50 30 89.09± 10.24 -15.69± 1.27 99.8±0.6 Fail

ANE 57 20 40 40 34.32±25.18 -33.41± 1.98 99.4±0.4 Pass

ANE 58 40 50 10 68.12 ±38.76 -23.81±3.22 96.5±0.7 Fail

ANE 59 10 70 20 104.28± 3.82 -16.43±1.34 97.4±0.3 Pass

ANE 60 20 65 15 65.46 ± 14.17 -34.89± 5.79 97.4±3.29 Pass

146


SystemFormul

ation

Oil

(%)

Smix

(%)

AQ

(%)

Globule Size

(nm) ± SEM

Zeta potential

(mV) ± SEM

Transmittance

(%) ± SEM

Thermod

ynamic

stability

Smix 1:3

(Cremophore

EL:

Carbitol)

ANE 61 10 50 40 54.58 ± 2.30 -31.18± 2.03 94.1±0.4 Fail

ANE 62 20 50 30 109.73± 11.14 -25.10± 3.47 98.4±0.5 Fail

ANE 63 30 50 20 92.01±25.18 -33.10± 1.18 99.4±0.4 Pass

ANE 64 40 50 10 48.15 ±28.73 -32.18±0.23 98.9±0.4 Fail

ANE 65 10 70 20 44.12± 5.12 -34.40±1.14 99.9±0.4 Pass

ANE 66 20 65 15 66.11 ± 15.54 -33.44± 6.24 99.5±2.19 Pass

* Values are represented as mean ± SD, n=3;

147


Table 27: Final Compositions and Characterization of AMB Nanoemulsion (ANE) Selected for In-vitro permeation studies

SystemFormu

lation

Oil

(%)

Smix

(%)

AQ

(%)

Globule

Size (nm) ±

SEM

Zeta

potential

(mV) ±

SEM

Transmittance*

(%) ± SEM

Thermod

ynamic

stability

pH Viscosity PDI

Smix 2:1

(Tween20:

Carbitol

ANE 1 10 50 4021.29 ±

11.36

-12.83±

2.4599.1±0.44 Pass 5.4±0.2 112±11 0.12±0.1

Smix 3:1

(Tween20:

Carbitol

ANE

2620 50 30

49.29±

16.26

-25.09±

2.3799.8±0.3 Pass 5.3±0.1 103±15 0.23±0.2

Smix 1:3

(Labrasol:

Carbitol)

ANE

4510 35 55 42.11±15.28

-13.00±

0.9899.4±0.5 Pass 5.6±0.4 134±15 0.11±0.2

Smix 1:2

(Labrasol:

Carbitol)

ANE

5720 40 40 34.32±25.18

-33.41±

1.9899.4±0.4 Pass 6.1±0.3 114±23 0.16±0.1

* Values are represented as mean ± SD, n=3;

148

6.3.5. Selection of Mucoadhesive agent for Mucoadhesive Nanoformulations

The bioadhesive force, expressed as the detachment stress in dyne/cm2, was

determined from the minimal weights that detached the tissues from the surface of

each formulation using the following equation.

Detachment Stress (dyne/cm2) = m g/A

Where, m is the weight added to the balance in grams; g is the acceleration due to

gravity taken as 980 cm/s; and A is the area of tissue exposed. Measurements were

repeated thrice for each of the gel preparations, but before each measurement a fresh

smooth gel surface was created.

Low Molecular weight Chitosan (ChitoClear; 92% Deacetylation value) were selected

basis on maximum bonding strength to Goat nasal mucosa, which can be seen in the

Fig. 43.


Fig. 43: Texture Analyzer (TA) graph of in vitro bioadhesion test.

149

6.3.6. Globular morphology (by TEM) and Particle Size Distribution:

In the TEM positive image, the nanoemulsion appeared dark and the surroundings

were bright (Fig. 44). Some droplet sizes were measured, as TEM is capable of

point-to-point resolution. These sizes were in agreement with the droplet size

distribution measured using Dynamic light scattering method (DLS) with Malvern

zetasizer (Fig. 45).


AIF-JHU

Fig. 44: TEM images for (a) Amiloride Loaded nanoemulsion (ANE), and (b) mucoadhesive

nanoemulsion (AMNE)

Fig. 45: Globular Size distribution for Amiloride Loaded nanoemulsion (ANE) and mucoadhesive

nanoemulsion (AMNE)

150


6.3.7. In -Vitro Permeation Studies:

In- vitro permeation studies across Goat nasal mucosa were performed to compare

the release of drug from selected nanoemulsion formulations of Amiloride (e.g. ANE

1, ANE 26, ANE 45 and ANE 57) and all having the same quantity of drug. In vitro

permeation across nasal mucosa was highest in formulation ANE 1 and lowest for

ANE 26 (Fig. 46 & Table 28). The formulation ANE 45 and ANE 57 showed an

intermediate permeation profile. The nasal permeation profile of ANE 1 was

significantly different when compared with that of ANE 26 (P <0.05). The

significant difference in Amiloride permeation between nanoemulsion formulations

was probably due to the mean size of internal phase droplets, which were

significantly smaller in nanoemulsions. The maximum release in ANE 1 could be

due to having the lowest droplet size and lowest viscosity of all the nanoemulsions.

As a next step the highest permeate formulation ANE 1 was prepared as

mucoadhesive nanoemulsion and evaluation for three different level of chitosan

concentration (e.g. for Amiloride ANE 1, AMNE 0.25%, AMNE 0.50%, AMNE

1.0%) for the selected one formulation and measure the permeability parameters

across nasal mucosa. Results (Fig. 47 & Table 29) indicate that chitosan increases

the permeability of the AMB in linear manner along with shortening of initial drug

release time (drug release lag phase). This could be attributed to the chitosan’s

property for opening the tight junctions of mucosal cells. The mucoadhesive

properties of chitosan are an important factor in their retention and action in the

nasal mucosa. Chitosan, which is a positively charged polymer with a strong

mucoadhesive property, is frequently used in nasal application of

micro/macromolecules (Lubben et al., 2001; Wong, 2009). Mucoadhesion is

achieved by the ionic interaction of positively charged amine groups of D -

glucosamine units of chitosan with negatively charged sialic acid groups of musin or

other negatively charged groups of the mucosal membrane (Henriksen et al., 1996).

The effect of chitosan that enhances penetration has been associated with its

mucoadhesive property as well as its ability to transiently open the tight junctions in

the nasal mucosa. This increased permeation across nasal mucosa due to temporary

cell junction opening is a reversible phenomenon. It has been reported that chitosan

does not lead to any histological changes in the nasal mucosa (Schipper et al., 1997;

Lehr et al., 1992; Artursson et al., 1994).

151


Fig. 46: In-vitro permeation release profile of AMB from ANE1, ANE26, ANE45 and ANE46

Table 28: Comparative results of the various parameters calculated from In-vitro

permeation profile

In-vitro Permeation study parameters

ANE1 ANE26 ANE45 ANE57

Flux (^g/cm2/min) 10.61 7.24 9.95 8.20

Permeability Coefficient Pb x 103(cm2/min) 3.21 2.19 3.01 2.48

152


Fig. 47: In-vitro permeation profile of AMB from ANE1, AMNE (0.25%), AMNE (0.50%) and

AMNE (1.0%).

Table 29: Comparative results of the various parameters calculated from In-vitro

permeation profile of Mucoadhesive ANE

In-vitro Permeation study parameters ANE

AMNE

(0.25%Ch)

AMNE

(0.5%Ch)

AMNE (1%

Ch)

Flux (^g/cm2/min) 10.61 12.64 14.84 18.36

Permeability

Coefficient Pb x103(cm2 /min)

3.21 3.83 4.50 5.37

153

6.3.8. Qualitative Nasal Mucosa retention studies of NEs using CLSM:

In order to elucidate the disposition of nanoemulsions in the nasal mucosa, we

examined cross-sections of the nasal mucosa by CLSM. The confocal images of

different cross-sections of the goat nasal mucosa post washing with buffer solution

exposed to the Cm-nanoemulsion. Qualitative assessment of confocal images revealed

intense blue colored fluorescent areas located in between and inside the mucosal cells.

Due to the mucoadhesive nature, it was observed that mucoadhesive formulation

AMNE showed bluer colored intese areas as compared to non-mucoadhesive

nanoemulsion ANE as shown in Fig. 48.


Fig. 48: CLSM images of nanoemulsion and mucoadhesive nanoemulsion of AMB (ANE/AMNE)

Nanoemulsions of Amiloride were characterized for their appearance, globule size,

zeta potential, drug content, pH, viscosity, and transmittance, and the results were

recorded. NE formulations had globules in less than 150 nm. Low polydispersity

index values suggested narrow size distribution. ZP were lesser than -8.0 mV

indicating stability against globule-globule aggregation (Salim et al., 2011). The pH

of the formulations was found in the range of 4.5 to 6.5, which is compatible with

nasal mucosa. Viscosities of the developed formulation were recorded in the range

of 100-200cps. The percentage transmittance of NE was found to be more than 99%

and shows that the prepared ANE are isotropic in nature. TEM image (Fig. 44) is in

agreement with the globule size distribution measured by PCS (DLS) (Fig 45). The

in vitro diffusion study through excised Goat mucosa was performed with an aim to

154

assess the drug release through a biological membrane simulating the actual in vivo

barrier to drug permeation. The % cumulative drug permeated across nasal mucosa

from Amiloride loaded nanoformulations and mucoadhesive nanoformulations were

calculated and shown in Fig. 46 & 47 respectively. The results showed that flux and

permeability coefficients for Amiloride nanoemulsion were in the order of ANE 1 >

ANE 45 > ANE 57> ANE 26. Incorporation of mucoadhesive agent chitosan at

various concentrations increased the permeability of the formulation as

concentration increased which clearly confirmed the permeation improvement with

NE systems in the presence of mucoadhesive agent. Mucoadhesive nanoemulsions

(MNEs) permit drug loading at saturation solubility and increase their

thermodynamic activity favouring partition/permeation into biological membrane.

Also, amount of surfactants in NE might lead to tight epithelial junction opening in

nasal membrane thereby increasing net flux. However toxicity of NE on nasal

epithelial membrane needs to be evaluated. NE demonstrated lower flux than MNE,

MNE systems showed more drug permeation at the initial time point showing drug

release required for onset of action indicating suitability for nasal delivery

particularly for epilepsy disorders. MNE was composed of chitosan as mucoadhesive

agent which is natural abundant polymer, favouring permeation into biological

membrane without affecting normal functioning. CLSM studies revealed the

retention of mucoadhesive formulation (AMNE) post washing on nasal mucosa as

compared to non-mucoadhesive formulation (ANE).

6.4. CONCLUSION

Nanoemulsions and mucoadhesive nanoemulsions of AMB were successfully

prepared by aqueous titration method. NEs of AMB have very small glouble size

(~50nm) and negative zeta potential, while MNEs have slight bigger glouble size

(~100nm) and positive zeta potential. The spherical surface of NE and MNE was

confirmed from TEM. pH (4-6) of NEs and MNEs was compatible with nasal fluid

and viscosity (50-150cps) of NEs and MNEs was suitable for nasal administration.

In vitro release of NE and MNE system in nasal mucosal membrane demonstrated

prompt and effective release with more than 75 % of drug release in 4 h. The NE and

MNE were further subjected to stability studies according to ICH guidelines

(Chapter 7).


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