Post on 25-May-2020
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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^ 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).
Chapter 6: Nanoemulsion preparation, optimization and characterization
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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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
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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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
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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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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:
Chapter 6: Nanoemulsion preparation, optimization and characterization
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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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Chapter 6: Nanoemulsion preparation, optimization and characterization
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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Chapter 6: Nanoemulsion preparation, optimization and characterization
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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Chapter 6: Nanoemulsion preparation, optimization and characterization
RatioOil:Smix
Oil^ L
Surfactant (Smix) ^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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Chapter 6: Nanoemulsion preparation, optimization and characterization
RatioOil:Smix
Oil^ L
Surfactant (Smix) ^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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Chapter 6: Nanoemulsion preparation, optimization and characterization
RatioOil:Smix
Oil^ L
Surfactant (Smix) ^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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Chapter 6: Nanoemulsion preparation, optimization and characterization
RatioOil:Smix
Oil^ L
Surfactant (Smix) ^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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Chapter 6: Nanoemulsion preparation, optimization and characterization
RatioOil:Smix
Oil^ L
Surfactant (Smix) ^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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Chapter 6: Nanoemulsion preparation, optimization and characterization
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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Chapter 6: Nanoemulsion preparation, optimization and characterization
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
Chapter 6: Nanoemulsion preparation, optimization and characterization
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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
132
Chapter 6: Nanoemulsion preparation, optimization and characterization
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)
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
137
Chapter 6: Nanoemulsion preparation, optimization and characterization
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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
139
Chapter 6: Nanoemulsion preparation, optimization and characterization
Fig. 41: Pseudo-ternary Phase Diagram Construction with Tween 20 & Carbitol for Amiloride
Nanoemulsion optimization.
140
Chapter 6: Nanoemulsion preparation, optimization and characterization
Fig. 42: Pseudo-ternary Phase Diagram Construction with Labrasol & Carbitol for Amiloride
Nanoemulsion optimization.
141
Chapter 6: Nanoemulsion preparation, optimization and characterization
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
System Formulation
Oil(%)
Smix(%)
AQ(%)
Globule Size (nm) ± SEM
Zeta potential (mV) ± SEM
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
System Formulation
Oil(%)
Smix(%)
AQ(%)
Globule Size (nm) ± SEM
Zeta potential (mV) ± SEM
Transmittance(%) ± SEM
Thermodynamicstability
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
System Formulation
Oil(%)
Smix(%)
AQ(%)
Globule Size (nm) ± SEM
Zeta potential (mV) ± SEM
Transmittance(%) ± SEM
Thermodynamicstability
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
System Formulation
Oil(%)
Smix(%)
AQ(%)
Globule Size (nm) ± SEM
Zeta potential (mV) ± SEM
Transmittance(%) ± SEM
Thermodynamicstability
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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;
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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;
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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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
Fig. 43: Texture Analyzer (TA) graph of in vitro bioadhesion test.
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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).
Chapter 6: Nanoemulsion preparation, optimization and characterization
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)
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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).
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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
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Chapter 6: Nanoemulsion preparation, optimization and characterization
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
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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.
Chapter 6: Nanoemulsion preparation, optimization and characterization
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).
Chapter 6: Nanoemulsion preparation, optimization and characterization
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