1
2
THESIS/DISSERTATION APPROVED BY
Alekha K. Dash, Ph.D.
3
i
PREPARATION, CHARACTERIZATION, AND IN VITRO EVALUATION OF
MULTIPLE EMULSION FOR TOPICAL APPLICATION OF LAWSONE AND
DIHYDROXYACETONE
___________________________________
By
Anne Grana
___________________________________
A THESIS
Submitted to the faculty of the Graduate School of the Creighton University in Partial
Fulfillment of the Requirements for the degree of Master of Science in the Department of
Pharmaceutical Sciences
_________________________________
Omaha, NE
(November 25, 2014)
ii
© Anne Grana, 2014
iii
ABSTRACT
Dihydroxyacetone (DHA) is the primary ingredient in most sunless tanners.
Lawsone is a primary component of red henna, used in henna tattooing and reversed
tattooing. DHA initiates a reaction called the Maillard reaction which results in the
browning of the skin. Application of lawsone results in the production of colored
keratin-bound fluorescent polymers called melanoidins. DHA alone gives some
protection in the UVA spectrum, with minimal UVB. When lawsone is applied after the
application of DHA, a different set of melanoidins are produced, giving UVB and even
greater UVA protection.
In this study, a multiple emulsion (W1/O/W2) containing 0.035% (w/w) lawsone
and 1% (w/w) DHA were prepared using the two step process by Matsumoto et al. The
first step was the production of the primary (W1/O) emulsion. The primary emulsion,
making up 40% of the final volume of the emulsion, was then added to the secondary
aqueous phase (W2). A combination emulsion was prepared by combining doubled
concentrations of lawsone and DHA multiple emulsions (W1/O/W2) in a 1:1 ratio through
trituration. The structure of the multiple emulsions was determined by light and
fluorescent microscopy. The stability of the prepared emulsions was characterized after
storage at 25˚C, 32˚C, and 40˚C and over a period of 28 days. DHA and DHA in
combination showed a significant decrease in % entrapment efficiency to 37.41 ± 6.03%
and 5.41 ± 3.82% after 28 at 40˚C, showing physical/ chemical instability of emulsions at
increased temperature. Chemical instability of the emulsions was confirmed by varying
decrease in pH at 32˚C and 40˚C of DHA emulsion and combination emulsion. Structural
instability of emulsions at 40˚C was confirmed by increase of zeta potential and
iv
rheological tests using flow and oscillatory stress/time sweeps. No significant changes in
particle size suggest no coalescence occurring. Spreadability remained within the range
of ≈ 20 cm²/g to 35cm²/g respectively. Characterization testing suggest the stability of
lawsone and DHA emulsions were best when kept separate at 25˚C. In vitro surface
release and Franz diffusion studies verified and confirmed drug release were more
effective when lawsone and DHA emulsions were kept separate.
v
Dedicated to my family
vi
ACKNOWLEDGEMENTS
I would first and foremost like to express my immense gratitude to my advisor
and mentor Dr. Alekha Dash for his encouragement, guidance, patience, and support
throughout the duration of the program. I am thankful to him for providing me the
opportunities and skills in lab to think critically and scientifically in order to better myself
professionally and personally. I extend my gratitude to my committee members, Dr.
Somnath Singh and Dr. Justin Tolman for their valuable suggestions in my project and
manuscript.
I would also like to thank my lab members: Sneha Dhapare, Shantanu Chandratre,
Swasti Pandey, Dr. Igor Meerovich for their help and scientific input during lab meetings.
I especially would like to thank Dan Munt for his knowledge, guidance, and
encouragement on this project, of which he initiated. I extend my gratitude to Taunya
Plater, Dawn Trojanowski, and the entire Graduate School and Department of
Pharmaceutical Sciences for their support throughout the past two years. A special
thanks goes to all my fellow graduate students, both past and present, who have been my
family and moral support here in Omaha. You have made my years here memorable.
My never ending gratitude goes to my parents, Mr. Salvador Grana and Mrs. Eden
Grana, for their immense love and constant support. I would especially like to thank my
brothers, Albert and Alvin Grana, for their support and inspiration throughout the
duration of my program. A special thanks goes to all my friends and family members at
home who have been my source of encouragement from afar. Finally, I would like to
thank God for all the blessing he has bestowed upon me to get me to this point in my life.
vii
TABLE OF CONTENTS
Page Number
Abstract iii
Table of Contents vi
List of Figures xi
List of Tables xiv
List of Equations xv
CHAPTER 1: Introduction 1
1.1 Topical application of sunscreen 2
1.2 UV and Skin Pigmentation 4
1.3 Maillard Reaction 7
1.4 DHA and Lawsone 10
1.5 Mulitple Emulsion 12
1.6 Objective, Hypothesis, and Specifications 13
CHAPTER 2: Formulation of Multiple Emulsion System Incorporating
Lawsone and DHA 15
2.1 Introduction 16
2.2 Materials 18
2.3 Methods 18
viii
2.3.1 Formulation of water-in-oil-in-water multiple emulsion 18
2.3.2 Verification of W1/O/W2 multiple emulsion 20
2.3.2.1 Light Microscopy 20
2.3.2.2 Fluorescence Microscopy 21
2.3.3 % Entrapment Efficiency 21
2.3.3.1 Chromatography 22
2.4 Calculations 22
2.5 Statistical data analysis 23
2.6 Results and Discussion 23
2.6.1 Fluorescence Microscopy 23
2.6.2 Light Microscopy 24
2.6.3 % Entrapment Efficiency 26
2.7 Conclusion 29
CHAPTER 3: Characterization of Multiple Emulsion Incorporating Lawsone
and DHA 30
3.1 Introduction 31
3.2 Methods 36
3.2.1 Particle Size and Zeta Potential Analysis 36
ix
3.2.2 Determination of pH 36
3.2.3 Spreadability 37
3.2.4 Rheology 38
3.2.5 Statistical Data Analysis 39
3.3 Results 40
3.3.1 Particle Size and Zeta Potential 40
3.3.2 Spreadability 42
3.3.3 pH 43
3.3.4 Rheology 45
3.4 Discussion 52
3.5 Conclusion 56
CHAPTER 4: In vitro Evaluation of Multiple Emulsion Incorporating
Lawsone and DHA 58
4.1 Introduction 59
4.2 Materials 61
4.3 Methods 62
4.3.1 In vitro surface release studies 62
4.3.2 Franz diffusion studies using snake skin 64
x
4.3.3 Statistical Analysis of Data 66
4.4 Results and Discussion 66
4.4.1 In vitro surface release studies 66
4.4.2 Franz diffusion using snake skin 68
4.5 Conclusion 70
CHAPTER 5: Summary and Future Directions 73
5.1 Summary 74
5.2 Future Directions 78
References 80
xi
LIST OF FIGURES
Page number
Figure 1. Anatomy of the skin showing the epidermis, dermis, and
subcutaneous tissue; melanocytes are found within the basal
cells which are in the deepest part of the epidermis 5
Figure 2. Generalized reaction forming a Schiff base 8
Figure 3. Chemical structure of dihydroxyacetone 9
Figure 4. Chemical structure of lawsone 9
Figure 5. Illustration of 3 phases of multiple emulsion droplet: internal water
droplets (W1) dispersed within the oil phase which is also dispersed
into a continuous water (W2) phase 16
Figure 6. Fluorescence microscopy photographs (a and b) of W1/O/W2
emulsion containing rhodamine, diluted 1:100 with deionized
filtered 0.2 µm filtered water, and magnified at 500x 23
Figure 7. Light microscopy photographs (a and b) of W1/O/W2 emulsion
containing DHA, diluted 1:100 with deionized filtered 0.2 µm filtered
water and magnified at 500x 25
Figure 8. Light microscopy photographs of W1/O/W2 emulsion containing
lawsone, diluted 1:100 with deionized filtered 0.2 µm filtered water,
and magnified at a) 200x and b) 500x 26
Figure 9. Entrapment efficiency for lawsone, DHA, and combination
lawsone/DHA at 25˚C, 32˚C, and 40˚C 26
Figure 10. Particle size analysis for lawsone, DHA, and the combination of
lawsone/DHA emulsions at a) 25˚, b) 32˚C, and c) 40˚C after 28 days 40
xii
Figure 11. Zeta potential for lawsone, DHA, and the combination of
lawsone/DHA emulsions at a) 25˚C, b) 32˚C, and c) 40˚C
after 28 days 41
Figure 12. Spreadability (cm²/g) for lawsone, DHA, and the combination
lawsone/DHA emulsions at a) 25˚C, b) 32˚C, and c) 40˚C
after 28 days 43
Figure 13. pH readings for lawsone, DHA, and combination lawsone/DHA
emulsions at a) 25˚C, b) 32˚C, and c) 40˚C after 28 days 44
Figure 14. DHA rheograms for OSS of G’ vs oscillatory stress at a) 25˚C,
b) 32˚C, and c) 40˚C 46
Figure 15. DHA rheogram of flow curves plotting viscosity vs shear rate at
a) 25˚C, b) 32˚C, and c) 40˚C. 48
Figure 16. DHA rheogram flow curves plotting shear stress vs shear rate at
a) 25˚C, b) 32˚C, and c) 40˚C. 49
Figure 17. OTS rheograms for lawsone at a) 25˚C, b) 32˚C, and c) 40˚C, G’ vs
Time(s) 51
Figure 18. OTS rheograms for DHA at a) 25˚C, b) 32˚C, and c) 40˚C, G’
vs Time (s) 52
Figure 19. Schematic of USP Apparatus 5 (Paddle Over Disk) Assembly 62
Figure 20. Picture representation of altered version of USP Apparatus 5
(sample and vessel) and schematic of sampling process/ analysis 63
Figure 21. Illustrated representation of multi-station Franz Diffusion cell system 64
Figure 22. Picture representation of Franz diffusion cell 65
xiii
Figure 23. The in vitro release profiles of a) lawsone and b) DHA multiple
emulsions in comparison with the combination lawsone/DHA
multiple emulsion 66
Figure 24. In vitro release profile from franz diffusion testing as a function
of the square root of time for a) lawsone and b) DHA multiple
emulsions in comparison with the combination of lawsone/DHA
multiple emulsion 69
xiv
LIST OF TABLES
Page number
Table 1. Table of volume percentages of chemicals used in formulation of
W1/O/W2 multiple emulsion and corresponding HLB values 19
Table 2. Parameters for OSS step for analysis of lawsone/ DHA emulsions 38
Table 3. Parameters for SF step for analysis of lawsone/DHA emulsions 39
Table 4. Parameters for OTS for analysis of lawsone/DHA emulsions 39
Table 5. Storage modulus (G’), loss modulus (G”), and tan δ of lawsone,
DHA, and combination lawsone/DHA at temperatures 25˚C,
32˚C, and 40˚C 47
Table 6. Summary of yield stress values and maximum viscosity values
at 25˚C, 32˚C, and 40˚C for lawsone, DHA, and combination
lawsone/ DHA emulsions 50
Table 7. Comparison of thickness, lipid content, and water evaporation
rate between human stratum corneum and shed snake skin 60
xv
LIST OF EQUATIONS
Equation 1. % Entrapment Efficiency 22
Equation 2. Stokes Law 31
Equation 3. Laplace Equation 33
Equation 4. Dampening factor (tan δ ) 35
Equation 5. Area of circle 37
Equation 6. Spreadability 37
Equation 7. Average cumulative amount of drug released (Q) 61
xvi
1
CHAPTER 1
Introduction
2
1.1.Topical applications of sunscreens
Sunscreens are protective agents applied to the skin to reduce the harmful effects of
solar radiation. Solar radiation produced by the sun can be categorized into ultraviolet
radiation (UVR) or visible light. The ultraviolet (UV) spectrum, which lies within
200nm to 400nm, can further be categorized according to its wavelength: UVA (320-
400nm), UVB (280-320nm), and UVC (200-280nm)1. UV radiation from the sun is
strongest between daylight hours of 10:00am to 4:00pm2. The UVR that causes damage
to the skin are mostly UVA and UVB. UVC is not considered as harmful for it is mostly
absorbed and filtered by the ozone layer and is only transmitted artificially through
germicidal or mercury lamps3. UVA and UVB are considered the most common
radiation that can pose a threat when overexposed. UVA delivers less energy to the skin,
therefore is not as considered as effective as UVB. UVA is mainly responsible for
producing suntan and not sunburn3,4
and is considered the cause of photoaging5. UVA
overexposure is more apparent because of its consistency throughout the day, reaching
the earth’s surface twenty times more than UVB3. Photoaging results in extracellular
matrix degradation and dysregulation of collagen metabolism5. The change in
extracellular matrix affects the elasticity of skin and consequently results in the formation
of wrinkles6. UVB, on the other hand, is considered to be the most erythrogenic and
melanogenic because it delivers such a high amount of energy to the stratum corneum
and superficial layers of the skin1,7
. UVB is the main cause of sunburn, suntan and skin
cancers3. In a study linking UVB with skin cancer, it was found that UVB energy was
directly absorbed by DNA, forming photoproducts which block replication and
transcription8. The resulting mutations specifically occur in the tumor suppressor gene
3
p53. P53 mutations have been linked to squamous cell carcinoma (SCC)9. Because of
the potential immediate (i.e. erythema) and long-term (i.e. photoaging,
immunosuppression, and carcinogenicity) harm that both UVA and UVB can cause, the
stress on application of sunscreens has become more apparent2.
The most common sunscreens on the market can be separated into two types: physical
and chemical sunscreens. Physical sunscreens are also referred to either inorganic or
reflectant sunscreens because they tend to use UV filters that act as a physical barrier that
can block transmission of UVR to the skin10
. Some of the most commonly used physical
sunscreens are those containing titanium dioxide, zinc oxide, kaolin, and ferric chloride.
Physical sunscreens are generally stable, but are sometimes cosmetically unappealing
because of their often thick and opaque consistency can be difficult to apply.11
Because
of its grainy texture, application can be sandy and quite messy. Despite their unappealing
texture, physical sunscreens tend to not be easily washed off, appealing to those who
have laborious jobs that cause them to sweat or for those who work in the water such as
lifeguards3. In order to provide superior UV protection and improve on cosmetic
appearance, UV filters like zinc oxide and titanium dioxide have been incorporated into
nanoparticle delivery systems12
. Toxicity and percutaneous penetration of the nano-
materials through the skin are still debated subjects of concern.
The second type of sunscreen available on the market is chemical sunscreens.
Chemical sunscreens are commonly referred to as organic or absorbent sunscreens
because they reduce the amount of UVR that reaches the stratum corneum by absorbing
the harmful radiation1. Chemical sunscreens have been mainly UVB absorbers. They
have shown to a selectively absorb the photons of sunburn-producing (UVB) radiation
4
while still allowing for the tanning reaction to occur because they don’t completely block
the transmission of UVA radiation1. Some of the common chemical sunscreen active
ingredients include: octocrylene, avobenzene, cinnamates, octinoxate, octisalate,
oxybenzone, homosalate, helioplex, mexoryl SX and XL, salicylates, benzophenomes,
and uvinul T150 and A Plus. Chemical sunscreens are colorless and more cosmetically
accepted, often found in moisturizers or mineral makeup. After application of chemical
sunscreens, irritation to the skin or eyes may occur. Chemicals such as salicylates,
cinnamates, anthranilates, and benzophenones tend to be surface agents and do not adhere
to the skin13
. Therefore, reapplication becomes necessary in order to provide adequate
and effective protection.
1.2.UV and Skin pigmentation
When it comes to sun protection, it is important to understand the basics of what
happens to the skin when exposed to UV radiation. UV exposure on the skin results in
two defense mechanisms produced from the skin. The skin first undergoes an epidermal
thickening and then increases in production of melanin, the polymer responsible for
pigmentation of the skin14
. In Figure below, melanocytes can be seen within the basal
layer of the epidermis of the skin. The melanocytes then transfer melanosomes,
organelles that contain melanin, to surrounding keratinocytes which would result in the
various tanned color of the skin after sun exposure15
Melanin has two types of chemical
compositions: eumelanin and pheomelanin. Eumelanin is a brown polymer and
pheomelanin is a yellow-reddish pigment. Whether eumelanin or pheomelanin
predominates is usually dependent on polygenic inheritance15
.
5
Figure 1. Anatomy of the skin showing the epidermis, dermis, and subcutaneous tissue;
melanocytes are found within the basal cells which are in the deepest part of the
epidermis16
Most Caucasians of European decent are thought to have less melanin content in
their skin, as seen in a high frequency of skin cancer occurring within that population.
On the other hand individuals with darkly pigmented skin are thought to have
significantly more epidermal melanin content, of which protects the skin by acting as free
radical scavengers, preventing from the incidence of cancer.17
When the skin is exposed to UV radiation, increased pigmentation from melanin
occurs in a three stages:
1. IPD- Immediate pigment darkening
2. PPD- Persistent pigment darkening
3. DT- Delayed tanning
6
IPD occurs within minutes after exposure to the sun. The skin appears grayish in color
and gradually fades to a brown over minutes or days, depending on the amount of UV
exposure and the skin color of the individual18
. IPD is not caused by the synthesis of new
melanin, but results from the photoxidation of preexisting melanin and the movement of
melanosomes to a peripheral dendritic location19
. The second phase is the PPD which
occurs after UV exposure and persists at least 3 to 5 days20,21
. This phase results in a tan
to brown color which is a result of oxidation of melanin and with exposure to UVA more
so than UVB20
. The final stage is the DT phase, which results from an increase of
melanocyte activity and new melanin formation in response to UVB8. The DT effect is
considered to be mildly photoprotective, with a sun protective factor (SPF) of about 318
.
According to Jimbow et al, melanin pigmentation can be associated with the following14
.
1. Resistance to sunburn, solar degradation, and skin cancer
2. Thermoregulation by enhancement of absorption of solar radiation
3. Regulation of vitamin D3 biosynthesis by influences on penetration of UV light
into skin
4. Protection of vital metabolites from photodestruction
Although melanin can aid in prevention of UV damage, there are still plenty of fair
complexioned people who lack the necessary amounts of melanin for its benefits.
Therefore, another viable option for UV protection is the use of keratin-bound
melanoidins. In this form of photoprotection, the application of dihydroxyacetone
(DHA) initiates a reaction called the Maillard reaction which results in the browning of
the skin from its combination with keratin amines (filaggrin). This reaction is followed
by the addition of lawsone, also known as henna, resulting in the production of brown
7
colored keratin-bound fluorescent polymers called melanoidins.17
Melanoidins produced
from DHA alone gives some photoprotection in the long UVA and Soret band of the
electromagnetic spectrum with minimal UVB protection17,22
. In a study by Fusaro et al,
it was shown that when lawsone is applied after the application of DHA, a different set of
melanoidins are produced, giving high UVB SPF protection and even greater UVA
protection than when DHA is used alone17,23
. The Maillard reaction and the properties of
Lawsone and DHA will be further explained in the following sections.
1.3. Maillard Reaction
The Maillard reaction was named after its founder Louis-Camille Maillard in France
in 1912. Maillard’s goal was to explain the reaction when amino acids react with sugars
at elevated temperatures during biological protein synthesis. Instead, his explanation was
used in food science to explain the browning process which occurs in food when being
cooked (i.e. in baked bread or barbequed meat). In 1953, John E Hodge, a chemist who
worked for the U.S. Department of Agriculture, took Maillard’s concepts and further
established a chemical mechanism of the reaction. Hodge broke down the reaction in the
following parts24
:
1. Initial Stage: (products colorless, without absorption in the ultraviolet (≈ 280
nm))
a. Reaction A: Sugar-amine condensation
b. Reaction B: Amadori rearrangement
2. Intermediate Stage: (products colorless or yellow, with strong absorption in
the ultraviolet)
8
a. Reaction C: Sugar Dehydration
b. Reaction D: Sugar fragmentation
c. Reaction E: Amino acid degradation (Strecker degradation)
3. Final Stage: (products highly colored)
a. Reaction F: Aldol Condensation
b. Reaction G: Aldehyde- amine condensation and formation of
heterocyclic nitrogen compounds
Hodge’s mechanism of explaining Maillard’s reaction showed how truly complex
the process of browning can be. To summarize, the Maillard reaction starts off with the
condensation reaction between a sugar and amine forming a Schiff base and liberating a
water molecule, which can be seen in the Figure below:
Figure 2. Generalized reaction forming a Schiff base25
The Schiff base then undergoes the Amadori rearrangement generating a
ketoamine product also known as a Heyns product. The Heyns product undergoes a
series of dehydration, degradation, and condensation reactions to form the final products
of the non-enzymatic browning called melanoidins. Melanoidins are different form the
melanin that is produced from enzymatic browning24
. Maillard’s reaction is not only
used in food chemistry, but also has implications in the field of medicine, especially in
the skin. For the past 50 years, dihydroxyacetone (DHA) has been used as sunless tanner.
9
Figure 3. Chemical structure of dihydroxyacetone 26
The corneocytes in the upper layers of the stratum corneum contain a large concentration
of amino acids that are produced from the degradation of filaggrin, the filament
associated protein which binds to the keratin fibers in epithelial cells27
. Filaggrin in the
stratum corneum is responsible for the skin barrier function28
. When DHA is applied to
the skin, the carbonyl group on the DHA (Figure 3) reacts with the amines in the keratin
layer, resulting in water and the Schiff base17
. Because the water content of the
epidermis increases the deeper it goes, the production of free radicals from this part of the
Maillard reaction ceases at the lower edge of the keratin layer as the law of mass action
reverses the equilibrium of the chemical reaction 17,27
. Therefore, the free radicals
produced by the Maillard reaction stops at the keratin layer of the skin. The Schiff base
and the intermediate products of the Maillard reaction progress to form non-enzymatic
covalent bonding into the skin protein, forming the keratin bound browning polymer
known as melanoidins17,29
. Like in food, melanoidins on the skin produces a brown tone
that stains the skin.
Figure 4. Chemical structure of lawsone30
10
Lawsone (Figure 4), the principle coloring agent of henna dye, also is said to produce
melanoidins on the skin. This can be especially evident after applying henna as a
temporary tattoo where the skin is designed and dyed with a brown stain. According to
Forestier et al, lawsone reacts with keratin through either the hydrosulfide group on
keratin or by forming a Schiff base by binding the keto-carbon on lawsone with the free
amine group on keratin31,32
. The formation of a Schiff base is said to be more likely to
occur, resembling the same type of reaction as DHA32
. The melanoidins produced by
DHA alone are said to give some photoprotection in the long UVA and Soret band of the
electromagnetic spectrum with minimal UVB protection. The melanoidins induced from
lawsone, on the other hand, are thought to produce a high UVB SPF protection and an
even greater UVA photoprotection than DHA17
.
1.4.DHA and Lawsone
DHA is used in cosmetic products to darken the skin chemically, providing sunless
tanning 33
. Store bought sunless tanners containing DHA are usually within the range of
3% to 5% in concentration, with professional products usually ranging between 5% and
15% depending on extent of tanning needed by the consumer. DHA based sunless
tanning has been recommended as a safer alternative to sun exposed tanning by the Skin
Cancer Foundation, the American Academy of Dermatology Association, the Canadian
Dermatology Association, and the American Medical Association34
. In the 1970s, DHA
was added to the United States Food and Drug Administration’s (FDA) list of approved
cosmetic ingredients. Studies by Faurschou et al demonstrated that the use of DHA
provides modest SPF in humans depending on the concentration used. DHA was shown
to shield against longwave UVA, visible (blue) light, and short wave UVB, delaying
11
photocarcinogenesis in hairless mice35,36
. In addition to its versatility and photoprotective
benefits, DHA binds covalently to the amines in the keratin layer, thus is not easily
washed off after application. Even after harsh physical activity, the melanoidins formed
from DHA will remain on the skin for 1 to 2 weeks or until the stratum corneum sloughs.
Henna is a member of the Lythraceae family that is cultivated in warm dry climates.
The dried leaves of the plant are usually crushed to a powder to produce the pigment
called lawsone37
. Lawsone has been used for centuries as hair dye or an expression of
traditional decorative body art in many Arab or Hindu cultures. Some consumer products
that currently contain henna include shampoos, conditioners, hair dyes, and body washes.
The safety of use for lawsone has always been a constant debate. The Scientific
Committee for Consumer Products (SCCP) of the European Union evaluated the use of
lawsone in hair dyes in 2002 and claimed that use of lawsone caused mutagenic and
clastogenic results in both in vitro and in vivo experiments. It was then concluded that
lawsone was not suitable for use as hair dye. Then in 2005, the SCCP concluded that the
mutagenicity data was insufficient to assess the safe usage of lawsone in hair dye. A
study by Kraeling et al initiated studies of lawsone related shampoos and pastes in order
to determine the extent of absorption when applied to human skin38
. In determining the
amount of lawsone that has possibly been absorbed, an important factor to analyze was
the amount of lawsone still remaining on the skin after diffusion studies were conducted.
Kraeling determined that the amount of lawsone still remaining was isolated within the
skin reservoir, or the stratum corneum, and was not available for systemic skin
absorption. Extended studies, spanning 48 and 72 hours, were also performed to verify
the lack of systemic absorption of lawsone. Although an increase of lawsone was seen
12
within the receptor fluid after the 72 hour time span for 3 out of 4 of the products, the
amount would still not be considered absorbed.
When lawsone and DHA are combined together forming the keratin-bound
melanoidin sunscreen, they work symbiotically to protect the skin from both UVA and
UVB regions. In the initial patent produced by Fusaro et al, the application of the
mixture of lawsone and DHA would be done separately39
. First the carbonyl compound
(DHA) would be applied to prepare the skin for reception of the subsequently applied
quinone (lawsone). Then there is a15 minute wait time before application of lawsone and
the two step application is repeated in order to enhance efficacy. In preliminary data
produced in by Fusaro et al in 1966, it was shown that the combination of lawsone and
DHA within the same formulation caused decomposition of the drugs within one to
several days when stored at temperatures of 25˚C or above23
. Combination of the two
drugs just prior to application was the most effective way in inducing the sunscreen filters
within the skin. Because of the melanoidin’s ability to combine to the keratin of the skin,
constant reapplication after every 3 hours or after sweating/ swimming is unnecessary
with lawsone/DHA sunscreens. Application of DHA followed by lawsone at bedtime
would suffice without any reapplication during the daytime.
1.5.Multiple Emulsion
In the current study, lawsone and DHA were both formulated into a multiple
emulsion water-in oil-in-water (W/O/W) system in an attempt to create a formulation
containing both drugs. A W/O/W system is composed of initial emulsion (W/O)
emulsified into an addition water (W) phase. According to DeLuca et al, a three phase
13
emulsion is recommended for the extended delivery of an active material because the
material would have to pass through two interfaces than an single interface of a two-
emulsion system40,41
. This was the intention when formulating a multiple emulsion for
lawsone and DHA. The various phases of the multiple emulsion would act as inherent
barrier between drugs, therefore protecting the drugs from possible decomposition as
seen from Fusaro et al. A benefit in formulating a three phase W/O/W emulsion is its
ability to create creams and lotions with the desired consistency of the external water
phase42
. A W/O/W emulsion also is able to enhance the solubility of compounds which
otherwise are only slightly soluble in hydrocarbons or water and the slow release of
active substance from the emulsion droplet40
. Because of the two water phases within the
system, another advantage includes the ability of placing two incompatible hydrophilic
materials in the same emulsion, one in the internal aqueous phase and the other in the
external phase40
. With these integral benefits of a multiple emulsion system in mind, a
formulation incorporating both lawsone, DHA, and combination of lawsone/DHA were
created and characterized in the following study.
1.6.Objective, hypothesis, and specific aims
The objective of this study was to formulate and characterize a multiple emulsion
for topical delivery of both lawsone and dihydroxyacetone and to evaluate its
compatibility under in vivo conditions. The underlying hypothesis of this investigation
was that a stable multiple W/O/W emulsion containing both lawsone and
dihydroxyacetone (DHA) can be prepared and characterized and can be effective as a
topical application. The specific aims were the following:
14
I. Development of multiple emulsion system incorporating both lawsone and
DHA
II. Characterization of the lawsone/ DHA multiple emulsion under various
conditions to determine its stability through time and temperature change
III. Evaluation of lawsone/ DHA multiple emulsion through in vitro studies to
verify compatibility under in vivo conditions
15
CHAPTER 2
Formulation of Multiple Emulsion System Incorporating Lawsone and DHA
16
2.1. Introduction
A multiple emulsion is a delivery system that incorporates both water-in-oil
(W/O) and oil-in-water (O/W) emulsions simultaneously. Multiple emulsions can also be
seen as a heterogeneous system of one immiscible liquid dispersed in another in the form
of droplets, which usually have diameters greater than 1µm43
. Figure 5 is an illustration
representing the phases of a multiple emulsion system.
Figure 5. Illustration of 3 phases of multiple emulsion droplet: internal water droplets
(W1) dispersed within the oil phase which is also dispersed into a continuous water (W2)
phase44
A major issue concerning multiple emulsions is the stability of the two
thermodynamically unstable interfaces (i.e. W/O interface of the primary emulsion and
O/W interface of multiple emulsion)45,46
. Therefore, when formulating a W1/O/W2
emulsion, it is necessary to utilize two emulsifiers within the formulation. When choosing
17
an emulsifier, it is necessary to consider its hydrophilic-lipophilic balance (HLB) value.
According to Griffin¸ a low HLB value is attributed to lipophilic surfactants, whereas
hydrophilic surfactants are thought to possess higher HLB values47
. In a study done by
Matsumoto et al, the emulsion process occurs in two steps48
. The first step is the
production of the primary W1/O emulsion, of which utilizes an emulsifier between the
range of 3 and 8. After proper homogenization of the primary emulsion, it is then added
to the secondary aqueous phase (W2), making up 40% of the final volume of the emulsion
while the remaining volume would be the continuous phase. A hydrophilic surfactant
within the range of 9 and 12 would then be incorporated into the external aqueous phase.
In addition to choosing the right hydrophilic and lipophilic emulsifiers, other
factors that are imperative to formulating a stable multiple emulsion are the type of
equipment used for mixing, the nature of the oil phase, and the phase volume ratio. In
mixing the primary emulsion, a homogenizer would allow the droplets to properly
disperse into the continuous phase. On the other hand, the second emulsification stage
would require gentler mixing, for high shear rates would cause the primary emulsion
droplets to rupture. Generally, the oil phase chosen for a multiple emulsion should be
nontoxic. The use of vegetable oils (soybean, sesame, peanut, etc.), refined hydrocarbons
(i.e. liquid paraffin, squalene, etc.) and mineral oils have often been used in multiple
emulsions. In comparison, mineral oil based multiple emulsions were said to form more
stable emulsions than those produced with vegetable oil49
. The phase volume is the ratio
of aqueous to oil phase which influence the stability of a multiple emulsion. The phase
volume of the primary emulsion doesn’t hold as much importance as the phase volume
ratio of the secondary phase50
. According to Matsumoto et al, stable multiple droplets
18
are produced at low volume fractions only48
. However, De Luca et al reported that stable
multiple emulsions can be obtained with a high volume ratio of 70% to 90% 41
.
The present study utilized all the aforementioned considerations to develop a
multiple W1/O/W2 emulsion incorporating lawsone, DHA, and combination of
lawsone/DHA. In this chapter, the formulation and development of the multiple
emulsions, as well as the drug entrapment of lawsone, DHA, and combination of the two
drugs will be addressed and analyzed.
2.2. Materials
Lawsone was obtained from Sigma Aldrich (St. Louis, MO). Dihydroxyacetone
was manufactured by Pfaltz & Bauer (Waterbury, Ct) and purchased through Fisher
Scientific (Pittsburg, PA). Sorbitan monooleate (Span 80) and polyoxyethylene sorbitan
monooleate 80 (Tween 80) were provided by Spectrum (New Brunswick, NJ). Mineral
oil was obtained from PCCA (Houston, TX). Coconut oil was provided by Carrington
Farms (Closter, NJ). Beeswax was obtained from Acros (New Jersey) and stearic acid
was obtained from Fisher Scientific (Pittsburg, PA). Soy lecithin was provided by a The
Herbarie (Prosperity, SC).
2.3. Methods
2.3.1. Formulation of water-in-oil-in-water multiple emulsion
A multiple emulsion was prepared using a two-step procedure as stated by
Matsumoto et al.48
The first step is the preparation of the primary (W1/O) emulsion. The
second step is obtained by dispersing 40% of the primary emulsion into the final aqueous
19
solution containing a hydrophilic emulsifier. The mass (g) of the each chemical in Table
1 was determined using their densities, which was then used to accurately weigh the
volume percentages of each phase.
Table 1. Table of volume percentages of chemicals used in formulation of W1/O/W2
multiple emulsion, and corresponding HLB values
% (w/w) Material HLB Value
Water (W1)
14.10 Phosphate Buffer pH
7.4/ Deionized Water -
0.035% / 1% Lawsone/ DHA -
Oil (O)
13.20 Mineral oil 10.5
6.00 Beeswax 12.0
4.32 Coconut oil 8.0
0.48 Stearic Acid 15.0
1.10 Soy Lecithin 4.0
0.80 Span 80 4.3
Water (W2) 0.25 Tween 80 15.0
59.75 Deionized Water -
Water (W1) and oil (O) phases were weighed in separate beakers and were heated
at the same time until both simultaneously reached 65˚C to 70˚C, or until the oil phase
has melted in the oil phase. While remaining heated at 70˚C, a hand homogenizer (Omni
TH) was used to homogenize the mixture as W1 phase was incorporated drop by drop
into the oil phase. The emulsion was kept under the highest speed of the homogenizer for
15 minutes. Meanwhile, the final water phase (W2) was weighed and heated on a
separate hot plate. After homogenization, 40% (≈40 g) W1/O was weighed and returned
to the hot plate to remain in liquid state for easy pouring (≈ 65˚C to 70˚C). Using an
overhead mechanical stirrer (Caframo RZR1) with a paddle attachment (speed setting 2),
W1/O was added drop by drop into the external aqueous phase (W2). Once incorporated,
20
temperature was decreased until hot plate was turned off. Vigorous mixing was
continued until water-in –oil-in –water (W1/O/W2) multiple emulsion achieved right
consistency and was fully cooled.
In the internal aqueous (W1) phase of the multiple emulsion, DHA was combined
with deionized water which was filtered using a 0.2µm filter. Lawsone multiple
emulsion’s internal aqueous phase (W1) was comprised of the lawsone mixed with
phosphate buffer pH 7.4 (PB). Deionized water in the laboratory was within the range of
pH 5 to 5.5, decreasing the solubility of lawsone. Therefore, a slightly more alkaline PB
was used in replacement of the deionized water. Combination of lawsone/DHA multiple
emulsion was comprised of a doubled concentration of lawsone (0.07% w/w) and DHA
(2% w/w) multiple emulsions combined in a 1:1 ratio (w/w) through trituration until
homogenous.
2.3.2. Verification of W1/O/W2 multiple emulsion
2.3.2.1 Light microscopy
In order to inspect the dispersion state of the inner W1/O emulsion containing
both lawsone and DHA, the W1/O/W2 emulsion was diluted (1:100) by weight with 0.2
µm filtered deionized water and sonicated in a bath sonicator for 5 minutes until
emulsion is fully in the continuous phase. The dispersion was placed onto a microscope
slide and visualized with light microscopy, using Leica DM2500M at 200x and 500x
settings. Photographs were taken to record results.
21
2.3.2.2 Fluorescence microscopy
To confirm the structure of the internal W1/O of the W1/O/W2 emulsion,
rhodamine was incorporated into the internal water (W1) phase. The oil (O) and
secondary water phase (W2) was prepared and incorporated as previously stated into the
multiple emulsion. The emulsion was diluted (1:100) by weight with 0.2 µm filtered
deionized water and sonicated in a bath sonicator for 5 minutes until emulsion was fully
in the continuous phase. The dispersion was placed onto a microscope slide and
visualized with fluorescence setting on Leica DM2500M. Photographs were taken to
record results.
2.3.3. % Entrapment Efficiency
Lawsone (0.035% w/w) was added into the internal water (W1) phase of a
multiple emulsion. DHA (1% w/w) was added to the internal water (W1) of a separate
emulsion. A combination of lawsone (0.07% w/w) and DHA (2% w/w) was prepared by
forming two separate W1/O/W2 emulsions and combining the two separate emulsions by
triturating both emulsions until homogenous in a 1:1 ratio (by weight). To determine
drug content, a biphasic liquid-liquid extraction was performed for all three emulsions
(lawsone, DHA, and combination lawsone/DHA). The emulsion (0.2g) was weighed in
an Eppendorff tube. At a ratio of 1:1, 1.5 mL of methylene chloride and sodium
phosphate buffer (pH 7.4) was added to Eppendorff tube. The samples were vortexed
(Vortex Genie 2) at speed 8 for approximately 10 seconds, until emulsion was in solution.
Samples were centrifuged (Accu-spin MicroR, Fisher Scientific) for 5 minutes at 13,000
rpm, until emulsion was separated into biphasic layers. Because both lawsone and DHA
22
are miscible in sodium phosphate pH 7.4, the aqueous layer was extracted and filtered
with 0.2 µm syringe filter. Samples were analyzed using UPLC method in the following
section. Entrapment efficiency was calculated using Equation 1:
% Entrapment Efficiency =DrugTotal−DrugFree
DrugTotal× 100 (1)
2.3.3.1. Chromatography
A Ultra Performance Liquid Chromatography (UPLC) method was performed on
the lawsone and DHA samples using a reversed phase Waters Acquity system (Waters,
Milford, MA) equipped with a quaternary solvent pump, auto sampler, and photodiode
array detector. The chromatographic separation of lawsone and DHA was obtained by
isocratic elution on a Waters Acquity BEH C18 (1.7 µm, 2.1 ×50 mm) column. The
mobile phase consisted of 0.2 µm filtered 0.1M acetic acid was mixed with methanol in a
ratio of 70:30 (v/v). Prior to usage on UPLC, 0.1M acetic acid was degassed in a bath
sonicator for 5 minutes. The flow rate was maintained at 0.5 mL/minute and the column
effluents were monitored at the detector wavelength range of (250 - 400 nm). The
injection volume was 10 µL, with a total run time of 6 minutes.
2.4. Calculations
The standard solution for lawsone (0.05% (w/v)) was prepared by dissolving
25mg of lawsone in 50 mL of sodium phosphate buffer pH 7.4. DHA (5% (w/v))
standard solution was prepared by dissolving 0.5g of DHA with 10 mL of sodium
phosphate buffer pH 7.4. A series of 15 serial dilutions were made for both lawsone and
DHA standard solution. The standard curve was obtained by plotting the peak height of
23
the standards to their corresponding concentrations. The unknown concentrations of
lawsone, DHA, and the combination of lawsone/DHA were determined by interpolating
from the regression equation relating to the peak height obtained from the standard curve.
2.5. Statistical data analysis
The statistical analysis of this experimental data for the purpose of comparison
was performed using a 2 tailed Student’s T-Test. Data was considered statistically
significant if p<0.05.
2.6 Results and Discussion
2.6.1. Fluorescence Microscopy
Figure 6. Fluorescence microscopy photographs (a and b) of W1/O/W2 emulsion
containing rhodamine, diluted 1:100 with deionized filtered 0.2 µm filtered water, and
magnified at 500x
In order to visualize and observe if the W/O/W formulation was able to
incorporate a hydrophilic drug within the internal (W1) phase without secretion into the
surrounding (O) oil and (W2) external aqueous phase, rhodamine was incorporated into
a)
) 0102030405060708090100
020406080100120140
% T
b)
24
the W1 phase of the multiple emulsion. Rhodamine is a water soluble fluorescent dye and
therefore is able to dissolve into the (W1) initial aqueous phase quite easily. After
completing the emulsion preparation process, the final emulsion was perceived to be a
deep pink color. To determine whether or not rhodamine was able to remain in the
internal (W1) phase, the emulsion was diluted 1:100 and dispersed into the continuous
phase (W2) with deionized water. By doing so, it was easier to observe the internal
(W1/O) phase. In Figure 6a and 6b, the rhodamine dye was able to fluoresce under the
fluorescent microscope, of which it can be discerned that the dye was able to remain
within the internal aqueous phase (W1) of the multiple emulsion. It can also be observed
that the rhodamine dispersion was shaped in a spherical form which seemed uniform in
size and structure. This also verifies that no other phase of the multiple emulsion, (O) oil
or (W2) external aqueous phase, contained any of the rhodamine dye. Figure 6a and 6b
is a visual confirmation of the feasibility of incorporating a hydrophilic drug into the
internal (W1) aqueous phase of a multiple W1/O/W2 emulsion.
2.6.2. Light Microscopy
The emulsion containing DHA which was used in Figure 7was diluted (1:100)
and dispersed into the continuous aqueous (W2) phase, aiding in visualizing the internal
(W1/O) phase. This can be observed in both Figure 7a and 7b. With light microscopy
and magnification at 500x, the internal W1 phase containing DHA can be seen within the
oil globules, more so observed in Figure 7a. Assuming that DHA was fully dispersed
into W1 phase during the formulation and preparation process, this can be regarded as a
confirmation that DHA was able to be incorporated into a W1/O/W2 multiple emulsion.
25
Figure 7. Light microscopy photographs (a and b) of W1/O/W2 emulsion containing
DHA, diluted 1:100 with deionized filtered 0.2 µm filtered water and magnified at 500x
The same can be said about the Figure 8a and 8b. Because lawsone is a pigment
and naturally colored, the internal W1/O phase can be clearly observed. In the
photographs above, the lawsone emulsion was also diluted (1:100) and dispersed into the
continuous aqueous (W2) phase, making it easier to envision the internal W1/O phase. In
Figure 8a, the internal W1/O particles can be seen to be far less than 100 µm,
encompassing a spherical, tinted internal (W1) phase within the oil (O) globules. In
Figure 8b, the dispersion was magnified to 500x. The internal (W1) phase can be
observed even clearer, also confirming that lawsone was fully dispersed only into the
internal phase and not into the other (O) oil or (W2) external aqueous phases. The light
microscopy analysis of lawsone at both 200x and 500x magnification validates and
confirms that lawsone was able to be incorporated into W1/O/W2 multiple emulsion.
a)
)
) 0102030405060708090100
020406080100120140
% T
b)
26
Figure 8. Light microscopy photographs of W1/O/W2 emulsion containing lawsone,
diluted 1:100 with deionized filtered 0.2 µm filtered water, and magnified at a) 200x and
b) 500x
2.6.3. % Entrapment Efficiency
Figure 9. Entrapment efficiency for lawsone, DHA, and combination lawsone/DHA at
25˚C, 32˚C, and 40˚C
a)
) 0102030405060708090100
020406080100120140
% T
b)
a)
) 0102030405060708090100
020406080100120140
% T
c)
) 0102030405060708090100
020406080100120140
% T
b)
) 0102030405060708090100
020406080100120140
% T
27
Figure 9 is a graphical representation of the % entrapment efficiency of lawsone,
DHA, and both the drugs in combination. The entrapment efficiency determines the
amount of hydrophilic drug that was entrapped within the internal water phase.
Therefore, the lesser amount of drug detected by the UPLC, the greater the entrapment
within the inner phase of the multiple emulsion. In Figure 9a, the amount of entrapped
lawsone, DHA, and combined drugs were calculated after the emulsions were kept at a
storage temperature of 25˚C. Although there were significant changes observed within
lawsone, DHA, and DHA in combination emulsions, the entrapment efficiency remained
within the range of ≈71.1% to 83%, respectively. The emulsion containing lawsone in
combination showed a significant increase from 93.38% on day 0 and to approximately
99% on day 7 through 28. An important observation to note is that all values remained
within a consistent range within this storage temperature, which means that no drug
leakage is occurring from internal phase. In Figure 9b, the storage temperature
increased to 32˚C. The lawsone emulsion showed a significant decrease starting on day
21 to about 78.65 ± 2.43% entrapment efficiency on day 28. A significant decrease in
entrapment was also observed in DHA and DHA in combination emulsions starting on
day 21, but still remained within a range of ≈ 76% to 65% throughout the full 28 day
duration. On the other hand, lawsone in combination emulsion showed a significant
increase from day 7 to 28, which is similar to the pattern observed at 25˚C where the
entrapment efficiency increased to ≈ 99%. The pattern of consistency observed at 32˚C
was similar to that observed at 25˚C. Figure 9c displays the entrapment efficiency for
the emulsions stored at 40˚C. At day 0, all emulsions started with drug entrapment
efficiency within the range of ≈ 69% to 78%. Lawsone emulsion remained consistent
28
with an entrapment of 69.83 ± 0.53% on day 0 to 70.64 ± 3.02% by day 28. In the
lawsone in combination emulsion, the same pattern as the other storage temperatures was
observed, showing a significant increase in entrapment from ≈ 78.45% at day 0 to
99.58% respectively by day 28. On the other hand, a significant decrease in entrapment
was observed for both DHA and DHA in combination. For the DHA only emulsion, a
significant decrease in entrapment was seen from 69.13% on day 0 to 37.41 ± 6.03 % by
day 28. The DHA in combination showed a significant decrease in entrapment starting
from day 14 through day 28, resulting in a final entrapment of 5.41 ± 3.82 %.
The decrease in entrapment efficiency is correlated to the increase of free drug
detected by the UPLC. A possible explanation for decrease in entrapment may be the
chemical instability of DHA. As reported by Fusaro et al, preliminary stability studies
with DHA and lawsone showed that stock solutions of both drugs kept separately at room
temperature or 4˚C showed more stability as when stored at higher temperatures or in
combination23
. It can be suggested that DHA’s chemical instability in an environment
with increased temperature might have been the reason for its possible diffusion out of
the inner phase to the continuous phase and increased detection of free drug in both DHA
and DHA in combination emulsion. Another possible explanation for the increased
detection of DHA in both emulsions is that the structure of the emulsion at 40˚C might
have been compromised, resulting in complete delivery of the drug into the external
aqueous phase which would cause more of the free drug to be detected. The structural
integrity of the emulsion can only be determined through further characterization, which
can be found in the following chapter.
29
It should be noted that the entrapment efficiency for all emulsions were calculated
using UPLC analysis of drug content by biphasic extraction. After conducting the
extraction process using methylene chloride and phosphate buffer, centrifugation, and
removal of the aqueous phase, it was determined that not all of the hydrophilic drugs
(lawsone and DHA) were fully removed. Instead, the foam layer after centrifugation
housed the residual drugs. Therefore, in order to achieve 100% entrapment efficiency at
day 0, the samples needed a second extraction to remove any remaining drug. It can be
noted that the values presented were an underrepresentation of the true % entrapment
efficiency of the emulsion samples.
2.7 Conclusion
Using light microscopy and fluorescence microscopy, it was visually verified that
a multiple emulsion formulation was successfully produced. By conducting the
entrapment efficiency, the presence of both lawsone, DHA, and a combination mixture of
both drugs was validated. Stability studies and characterization of the multiple emulsion
system were conducted, after storage at 25˚C, 32˚C, and 40˚C, in the following chapter
for further validation.
30
CHAPTER 3
Characterization of Multiple Emulsion Incorporating Lawsone and DHA
31
3.1 Introduction
The stability of pharmaceutical emulsions is characterized by the absence of
coalescence of the internal phases, absence of creaming, and maintenance of elegance
with respect to appearance, odor, color, and other physical properties51
. According to
Martin, problems that may occur in formulating emulsions can be the following51
:
(1) Flocculation and creaming
(2) Coalescence and breaking
(3) Miscellaneous physical and chemical changes
(4) Phase inversion
Flocculation occurs when an attractive force between the internal droplets brings
them together, forming an aggregation of droplets, or clusters, without any increase in
droplet size. Monitoring the electrostatic repulsion of the internal droplet surface
by measuring the zeta potential can therefore help determine if flocculation may be
present. Hence, the greater the electrostatic charges between droplets, the less
likely flocculation would occur.
Creaming is a property related to the Stokes law:
ν =d2(ρs−ρ0)g
18η (2)
In Equation 2, the rate of creaming (𝜈) is proportional to the diameter (d) of the
droplet in cm, the densities of both the dispersed phase (𝜌s) and continuous phase
(𝜌o), the acceleration due to gravity (g), and inversely proportional to the viscosity
of the dispersion medium (𝜂). In a multiple emulsion, the dispersed phase is the
32
internal W1/O phase, which would be relatively lighter in weight, due to the
presence of oils, than the external (W2) water phase. Therefore, an upward or a
negative rate of creaming would be observed in an unstable multiple W/O/W
emulsion. From the Stokes law in Equation 3, the rate of creaming is shown to be
dependent upon the droplet or globule size. By doubling the globule size, the rate of
creaming can increase by a factor of 451
. Also droplet size measurements are a good
indicator of the formulation’s stability. A fast droplet size increase indicates low system
stability52
.
According to Ficheux et al, multiple emulsions instability usually follows two
possible mechanisms 53,54:
Coalescence of the small inner droplets with the globule interface
Coalescence of the inner droplets within the globule
Coalescence usually occurs when the particles of the dispersed phase comes
together to form larger particles40. As larger particles begin to accumulate, breaking
or phase separation usually occurs. Disproportionation, also known as Ostwald
ripening, is a process dependent on the diffusion of disperse phase molecules, or the
chemical components, from smaller to larger droplets to the continuous phase55. It
is a result of the pressure inside a droplet being higher than the pressure outside the
droplet40. The pressure of dispersed material is greater for smaller droplets as
shown by the Laplace equation (Equation 3), where P is the Laplace pressures, 𝛾 is
the surface tension, and r is the droplet radius. Consequently, monitoring the particle
33
size of the internal W/O droplets of a multiple emulsion can help the formulators
determine the stability of the formulation over a period of time.
𝑃 = 2γ
r⁄ (3)
Chemical reactions within the emulsion samples may also occur after being stored
at various temperatures and with an elongated time span. Chemical reactions that may
occur within the emulsion can be relatively determined by monitoring its pH during its
storage time range. By doing so, the emulsion’s stability and any potential chemical
reactions that may compromise the quality of the product could be determined. The pH of
the skin has always been thought to be around pH 5.5 to 6. According to Lambers et al,
the pH of healthy skin is actually closer to 4.7, preserving any resident bacterial flora 56
.
Therefore, by monitoring the pH, it can be determined whether the emulsion is
compatible with the skin or may cause any irritation. Any changes in batches and the
quality of emulsion could also be checked by detecting any variations in pH.
Phase inversion is another obstacle that may occur when formulating an emulsion.
Phase inversion involves the change of emulsion type from O/W to W/O or vice versa51
.
In the phase inversion phenomenon, the phase-volume ratio of the emulsion plays an
important part in its stability. Depending on what type of emulsion (W/O or O/W), the
phase-volume ratio is a term that refers to the volume of water and oil that is added to
emulsion. Theoretically, from the study done by Ostwald and Kolloid, the phase-volume
ratio should not exceed 74% of the total volume of the emulsion. Ostwald and Kolloid
showed that an increase in the phase-volume ratio of oil to water in an O/W emulsion to
above 74% resulted in the oil globules to coalesce and break the emulsion. This limit
34
reached is called the critical point, which can be defined as the concentration of the
internal phase above which the emulsifying agent cannot produce a stable emulsion51
.
When the volume concentration reaches the 74% limit, a change in viscosity
occurs within the emulsion. Viscosity and flow properties of the formulation may be
monitored and assessed through spreadability testing and rheological analysis of the
product. Characterizing the spreadability of the emulsion is important in order to
determine if an even and efficacious dose is being delivered to the target site.
Spreadability, in principle, is related to the contact angle of the drop of the liquid or a
semisolid preparation on a standardized substrate and is a measure of the lubricity, which
is directly related to the coefficient of friction57,58
. Related to spreadability, other factors
such as viscosity, elasticity, and rheology are also important to consider when creating a
consistent formulation. Rheology is the science that studies how materials deform and
flow under the influence of external forces59
. Characterizing the rheological properties of
a system is not only important in the design and application, but also during its
processing and to ensure a long shelf life59
. By undergoing rheological analysis, one can
determine the changes that the formulation experiences when subjected to external forces,
measure the deformation and flow of the system, and learn how to improve the
application properties of the product. In order to fully assess the rheological properties of
the sample after application of stress, the following test could be used:
(1) Oscillatory Stress Sweep
(2) Stepped Flow
(3) Oscillatory Time Sweep
(4) Creep and Recovery
35
Oscillatory stress sweep measures the viscoelastic properties of a material. As
stress is applied during the amplitude sweep, the linear viscoelastic region (LVR) is
determined. The LVR is a measure of the inherent strength of the structure of the
emulsion40
. The storage/elastic modulus (G’) is a measure of the extent of the elastic
component (i.e. crosslinking, aggregation, etc) within the emulsion. The loss modulus
(G”) measures the extent of the viscous component of the emulsion. The strength of the
interaction in the formulation would be calculated by the ratio of G’ and G” (Equation 4),
resulting in the dampening factor (tan δ), where δ is the phase angle. The smaller the
value of tan δ, the more pronounced the elastic character of the formulation.
G"/G′ = tan δ (4)
In the experiments performed, the G’ is plotted as a function of oscillatory stress,
which measures and determines the product’s ability to lose its elasticity after applied
stress. Therefore, the more defined and longer the LVR, the product would be considered
to have retained its elasticity longer. The experimental parameters used on the ARG2
Rheometer for the following experiments are listed in Table 2.
Stepped flow (SF) is used to determine the type of flow (Newtonian or non-Newtonian)
and the viscosity profile of the formulation. Viscosity (Pa/s) is determined solely by the
relationship of two parameters, the shear stress (Pa) and the shear rate (1/s)40
. By
measuring the shear stress and shear rate, an understanding of whether force or
displacement is responsible for the flow of the formulation is determined. The parameters
used in the following experiments for SF can be seen in Table 3.
36
Oscillatory time sweep (OTS) is important when testing materials such as dispersions and
polymers, which may undergo macro-micro structural rearrangements with time60
. OTS
directly provides the necessary information about how a material changes with time, by
plotting the G’ as a function of time when the applied stress is held constant. Thus, any
changes to the structure (i.e. chemical structure, chemical reaction, change in
temperature, or curing) can be determined through OTS40
. The parameters used for
analysis of the emulsion samples can be seen in Table 4.
3.2 Methods
3.2.1. Particle Size and Zeta Potential Analysis
The particle size (PS) and zeta potential (ZP) of the formulation were determined
using the zetameter (ZetaPlus, Brookhaven Instruments Corporation, Holtsville, NY).
The PS and ZP of lawsone, DHA, and the combination lawsone/DHA W1/O/W2
emulsions were determined at various temperatures and time points by diluting the
emulsion sample by a ratio of 1:100 with filtered 0.2µm deionized water within a
scintillation vial. The solution was sonicated in a bath sonicator for 5 minutes and shaken
before use. The PS measurements were measured in a series of 10 readings, reporting the
effective diameter (nm) of the particle. The ZP measurements were also measured in a
series of 10 measurements, reporting the mean charge (mV). PS and ZP were of all three
emulsions were taken in triplicate weekly for a total of 28 days at 25˚C, 32˚C, and 40˚C.
3.2.2. Determination of pH
The pH of lawsone, DHA, and combination lawsone/DHA emulsions were
obtained by using Fisher Scientific ™ Accumet™ Basic AB 15 with a glass body
37
combination double junction electrode. The pH was obtained by dipping the electrode in
each emulsion, recording the pH in triplicate weekly for a total of 28 days at 25˚C, 32˚C,
and 40˚C.
3.2.3. Spreadability
Spreadability of each lawsone, DHA, and combination lawsone/DHA was
determined through the parallel plate method. The emulsion was first placed within a 1
cm diameter circle within the center of a glass plate (75 × 50mm) and weighed (g). A
second glass plate was placed on top of the first glass plate, with the addition of a 500 g
weight for a total of 15 seconds. The final diameter was determined (cm) on an x, y, and
z axis. The average diameter (cm) is used to determine the area of the circle by using the
following equation:
𝐴 = 𝜋𝑟2 (cm²) (5)
Spreadability was then calculated using Equation 7:
S500 =A (cm2)
Weight of Emulsion (g) (6)
Spreadability was performed in triplicate weekly for a total of 28 days at 25˚C, 32˚C, and
40˚C
3.2.4 Rheology
Rheology was conducted using ARG2 Rheometer (TA Instruments, LTD, New
Castle, DE). Geometry attachment for rheometer was chosen to be 40mm, 2˚degree
stainless steel cone due to its common use with thicker dispersions such as emulsions or
38
gels. Prior to starting rheological analysis, calibration of inertia and bearing friction was
conducted to determine if rheometer was working at optimal function. Once the
geometry was attached, the gap was zeroed and then raised for application of sample.
The emulsion sample (≈1 to 2 g) was added onto the center of the Peltier plate and
lowered. Excess emulsion was strategically cleaned off at a 90˚ angle so a straight edge
was obtained. The geometry was then lowered further to the zero gap distance (53).
Temperature was sustained at 25˚C. Protocols for oscillatory stress sweep (OSS),
stepped flow test (SF), and oscillating time sweep (OTS) were obtained from TA
Instruments (New Castle, DE) for determination and analysis of rheologically unknown
material. Rheograms for OSS was viewed as elastic modulus G’ (Pa) vs oscillation stress
(Pa). Rheograms for SF was viewed both as viscosity (Pa.s) vs shear stress (Pa) and
shear rate (1/s) vs shear stress (Pa). Rheograms for OTS was viewed as G’ (Pa) vs time
(s). Protocols for each test were as follows:
Table 2. Parameters for OSS step for analysis of lawsone/ DHA emulsions
Oscillatory Stress Sweep
Conditioning Step
Temperature 25˚C
Equilibration time 8 minutes
Deformation Step
Stress Sweep Broad torque range (1-10,000 mN.m)
Frequency 1 Hz
Points per decade 10
Mode Log
39
Table 3. Parameters for SF step for analysis of lawsone/DHA emulsions
Stepped Flow Step
Conditioning Step
Temperature 25˚C
Equilibration time 8 minutes
Stepped Flow Step
Stress Broad torque range (1-10,000 mN.m)
Points per decade 5
Mode Log
Constant time 10 seconds, average last 5 seconds
Table 4. Parameters for OTS for analysis of lawsone/DHA emulsions
Oscillatory Time Sweep
Conditioning Step 1
Temperature 25˚C
Equilibration time 3 minutes
Conditioning Step 2
Pre-shear Value of shear rate when viscosity is
reduced in SF step
Equilibration time 0
Time Sweep Step
Time duration 15 minutes
Frequency 1 Hz
Control Variable 0.5968 Pa
Sampling time 5 seconds
3.2.5. Statistical data analysis
The statistical analysis of this experimental data for the purpose of comparison
was performed using a 2 tailed Student’s T-Test. Data was considered statistically
significant if p<0.05.
3.3 Results
3.3.1. Particle Size and Zeta Potential
40
Figure 10. Particle size analysis for lawsone, DHA, and the combination of
lawsone/DHA emulsions at a)25˚, b)32˚C, and c)40˚C after 28 days
Lawsone, DHA, and combination emulsions were prepared fresh before placing
into the stability chamber at each temperature. By diluting the emulsions with water at a
ratio of 1:100, the internal W1/O droplet was dispersed into the continuous phase of the
multiple emulsion, which was measured by the ZetaPlus particle analyzer. In Figure 10a
and 10c, no significant changes in particle size was seen between all three emulsions at
temperatures of 25˚C and 40˚C. A significant increase in particle size was observed in
the combination lawsone/DHA emulsion (Figure 10b) by day 21 at 32˚C, where the
particle size enlarged from 414.7 ± 13.5nm to 475.97 ± 43nm. Despite the significant
increase at that point, the overall particle size of all emulsions, despite change in
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41
temperature, remained within a diameter size of between 300 nm and 500 nm
respectively.
Figure 11. Zeta potential for lawsone, DHA, and the combination of lawsone/DHA
emulsions at a)25˚C, b)32˚C, and c)40˚C after 28 days
After dispersion into the continuous phase, the zeta potential of the internal W/O
droplets can determine the potential stability of the colloidal system61
. By having an
overly negative or positive zeta potential, the particles will tend to repel each with no
tendency for aggregation or flocculation, remaining generally stable61
. In Figure11a
below, no significant differences were seen from each emulsion after 28 days at 25˚C. At
32˚C (Figure 11b), however, there was a significant increase in zeta potential for DHA
after 28 days. The most significant changes, on the other hand, occurred at 40˚C (Figure
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42
11c) for DHA at 14 and 28 days respectively, where the zeta potential showed an increase
from -20.83 ± 0.99 mV to -14.93 ± 0.97 mV. Combination lawsone /DHA had an
increase in zeta potential to -19.3 mV by day 28. In the lawsone emulsion, an increase
was observed after 7 and 14 days, from -33.7 ±3.10 mV on day 0 to -25.4 ± 0.87 mV by
day 14, but returned back to baseline by day 21. Overall, variations in zeta potential for
all emulsions were seen as the temperature increased to 40˚C, showing relative instability
3.3.2. Spreadability
Spreadability was obtained by measuring the displacement in diameter of the
emulsion after application of a 500g weight when placed between two glass slides. The
area of resulting circular spread (cm²) was then measured and divided by the initial
weight of emulsion used (g). The measured spreadability of each emulsion varied with
temperature and time. At 25˚C (Figure 12a), lawsone exhibited a significant increase in
spreadability after 21 days to 21.6 ± 1.1 cm²/g, and combination lawsone/DHA showed
an increase at day 7 and 28 to about 24.1 ± 1.77 cm²/g. Combination lawsone/DHA also
showed an increase at 21 days at 32˚C (Figure 12b) to a spreadability of 29.5 ± 0.6
cm²/g. In Figure 12c, DHA showed a significant increase in spreadability at day 7 to
about 34.0 ± 1.1 cm²/g, and lawsone showed a significant decrease in spreadabiltiy at day
21, but both returned to baseline. In summary, no obvious spreadability patterns within
the 28 days were observed at each temperature. Although variance in results occurred,
the spreadability values remained within the range of ≈ 20 cm²/g to35cm²/g.
43
Figure 12. Spreadability (cm²/g) for lawsone, DHA, and the combination lawsone/DHA
emulsions at a) 25˚C, b) 32˚C, and c) 40˚C after 28 days
3.3.3. pH
The pH values of the multiple emulsions kept at different storage conditions
(25˚C, 32˚C, and 40˚C) are shown below in Figure 13a, 13b, and 13c. Lawsone and
DHA emulsions were separately prepared, as well as a combination containing both
drugs. They were all stored separately and kept at the various temperatures. For the
freshly prepared lawsone and combination lawsone/DHA emulsions, the pH value ranged
between 5 and 6 respectively. DHA emulsions that were freshly prepared were roughly
within the range of pH of 4 and 5. Values of pH at time zero were used as the standard
and significance level was calculated using Student’s T-Test (p < 0.05). Although the
lawsone emulsion showed a significant decrease at day 7 to pH 5.85 ± 0.12 from 6.27 ±
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0.18 at 25˚C, it remained within a range of its original value of ≈ 6.27 after 28 days. A
significant increase was observed in DHA within days 14 to 28, from a pH of 4.41 ± 0.33
at time zero to a pH of 5.15 ± 0.05. Combination lawsone/DHA showed a fluctuating
increase and decrease within the 28 day time span, with a final pH of ≈ 5.1.
Figure 13. pH readings for lawsone, DHA, and combination lawsone/DHA emulsions at
a)25˚C, b)32˚C, and c)40˚C after 28 days
At 32C, changes in pH were observed for the lawsone emulsion, with a significant
increase to on day 28 to pH ≈ 6.05 after 28 days in the stability chamber. DHA emulsion,
with a pH of 6.03 ± 0.18 after being freshly prepared, showed a significant decrease in
pH weekly throughout the 28 day time span to a final pH of 4.92 ±0.08. The same
pattern was seen with combination lawsone/DHA. At the start of the stability test at time
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45
zero, the emulsion was measured to be a pH of 5.21 ± 0.10, but throughout the 28 days a
significant drop in pH to an average of ≈ 3.88 ensued. This suggests that some chemical
degradation is happening in DHA and more so when both lawsone and DHA is
combined.
Finally at 40C, lawsone emulsions exhibited a significant decrease in pH from
6.83 ± 0.11 to 5.85 ± 0.04 after 28 days. The same pattern was also obvious in the
combination lawsone/DHA where a significant drop in pH from 6.64 ± 0.22 to 3.71± 0.25
occurred. DHA emulsion remained consistent and without any significant changes until
day 28, when the average pH dropped from ≈ 5 to 4.15. The change in pH levels as
temperature increase suggests chemical degradation of the drugs and/or instability of
chemical components of the emulsion.
3.3.4. Rheology
Linear viscoelastic region (LVR) was determined by conducting the oscillatory
stress sweep, which graphed the (elastic/storage modulus) G’ versus the oscillatory stress
(Pa) that was applied to the sample emulsions. Figures 14a, 14b, and 14c below are
examples of rheograms of weekly stability analysis of DHA at 25˚C, 32˚C, and 40˚C.
The LVR region is a measure of the inherent strength of the structure of the emulsion40
.
The elasticity (G’) of the emulsion samples was measured as a function of increasing
stress, until a point at the end of the LVR. By continuing the oscillatory stress applied on
the sample, the structure eventually becomes destroyed and therefore a downward curve
was observed. In Figure 14a and 14b, the LVR for DHA remained consistent at both
25˚C and 32˚C, which can be validated in Table 6 below. The G’ values showed
46
minimal decrease within the 28 day span throughout both temperatures. As the
temperature increased to 40˚C, the G’ values decreased significantly within each week
from 1516 Pa at day 0 to 497.8 Pa by day 28. Also in Figure 14c, the LVR for DHA
emulsion gradually becomes shorter until it underwent a quick decline at day 28. This
pattern suggests that the elasticity of the emulsion decreased, showing a decline in
structure with the increase of temperature. Lawsone and combination lawsone/DHA
emulsions also exemplified the same decline in elasticity at 40˚C. The resulting graphs
could be viewed in the Appendix.
Figure 14. DHA rheograms for OSS of G’ vs oscillatory stress at a) 25˚C, b)32˚C, and
c)40˚C
0 100.0 200.0 300.0 400.0 500.0 600.0
osc. stress (Pa)
0.1000
1.000
10.00
100.0
1000
10000
G'
(P
a)
Osc Stress Sweep DHA 25C OSS DHA DAY 0
OSS DHA DAY 7
OSS DHA DAY 14
OSS DHA DAY 21
OSS DHA DAY 28
0 100.0 200.0 300.0 400.0 500.0 600.0
osc. stress (Pa)
0.1000
1.000
10.00
100.0
1000
10000G
' (
Pa
)
OSS DHA DAY 0
OSS DHA DAY 7
OSS DHA DAY 14
OSS DHA DAY 21
OSS DHA DAY 28
Osc Stress Sweep DHA 32C
0 100.0 200.0 300.0 400.0 500.0 600.0
osc. stress (Pa)
1.000E-3
0.01000
0.1000
1.000
10.00
100.0
1000
10000
G'
(P
a)
OSS DHA DAY 0
OSS DHA DAY 7
OSS DHA DAY 14
OSS DHA DAY 21
OSS DHA DAY 28
Osc Stress Sweep DHA 40C
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LVR
LVR
LVR
47
Table 5 is a summary of results of OSS in terms of tan δ. Tan δ is the value that
measures the overall strength and interaction of the emulsion. Generally, the smaller
value of tan δ would suggest that the emulsion would exhibit more of an elastic character.
The values of G’, G” and tan δ which are represented were taken at time zero and day 28.
The values of tan δ at day 28 that are highlighted in orange displayed a decrease in value,
signifying an increase in elasticity which might have occurred from possible swelling.
The tan δ values at day 28 that are highlighted in yellow showed an opposite increase in
the value, signifying a decrease in elasticity of the emulsion. This may have occurred
from possible expulsion of the internal water droplets into the continuous medium after
swelling occurred or after having applied stress onto the samples during the oscillatory
stress sweep.
Table 5. Storage modulus (G’), loss modulus (G”), and tan δ of lawsone, DHA, and
combination lawsone/DHA at temperatures 25˚C, 32˚C, and 40˚C
Lawsone DHA Combination
Law/DHA
˚C Day G’ G” Tan δ G’ G” Tan δ G’ G” Tan δ
25 0 2060 157.8 0.077 1447 164.4 0.114 2040 104.6 0.051
28 1576 186.7 0.118 1311 60.3 0.046 1107 166.9 0.151
32 0 3510 707 0.201 1516 175.9 0.116 1905 97.4 0.051
28 1878 212.1 0.113 1408 157.8 0.112 1245 127.7 0.102
40 0 3510 707 0.201 1516 175.9 0.116 1905 97.4 0.051
28 2408 307.1 0.127 497.8 91.6 0.184 1437 208.8 0.145
During the SF tests, all emulsions were evaluated by two sets of rheograms. One
set of rheograms was presented as viscosity vs shear rate, and the other presented as shear
stress vs shear rate. In Figures 15a, 15b and 15c below, the non-Newtonian, shear
thinning behavior, or pseudoplastic flow, of the DHA emulsion can be observed. As
48
stress rates increased, viscosity of the emulsions decreased almost instantaneously. The
yield stress, or the minimum shear stress required to initiate flow, was also determined.
By the presence of yield stress values and its shear thinning behavior, it can be suggested
that the emulsion can be considered viscoelastic, containing both viscous properties of a
liquid and elastic properties of a solid. Viscoelasticity is especially common for cosmetic
semisolids such as lotions and creams.
Figure 15. DHA rheogram of flow curves plotting viscosity vs shear rate at a) 25˚C, b)
32˚C, and c) 40˚C.
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49
Figure 16. DHA rheogram flow curves plotting shear stress vs shear rate at a)25˚C,
b)32˚C, and c) 40˚C.
The shear stress (Pa) vs shear rate (1/s) curves, such as those found in Figure
16a,16b, and 16c above, was used to determine the inherent flow of the formulation. By
increasing the shear rate (i.e. the consumer started to rub onto the skin), the DHA
emulsion’s shear stress gradually increased as well, meaning minimal structural loss was
seen at 25˚C and 32˚C. The viscosity remained relatively stable even during stress. On
the other hand, at 40˚C, a decrease in viscosity was observed throughout each week until
day 28. The increase in temperature could have resulted in structural breakdown as the
weeks passed until it reached its lowest viscosity at 406.4Pa.s on day 28. In Table 6
below, a decrease in viscosity was observed for lawsone emulsions stored at higher
temperatures of 32˚C and 40˚C, which may be attributed to structural breakdown or phase
0 25.00 50.00 75.00 100.0 125.0 150.0 175.0 200.0 225.0
shear rate (1/s)
0
100.0
200.0
300.0
400.0
500.0
600.0
sh
ea
r s
tr
es
s (
Pa
)
Flow DHA 25C Stepped Flow DHA DAY 0
Stepped Flow DHA DAY 7
Stepped Flow DHA DAY 14
Stepped Flow DHA DAY 21
Stepped Flow DHA DAY 28
0 25.00 50.00 75.00 100.0 125.0 150.0 175.0 200.0 225.0
shear rate (1/s)
0
100.0
200.0
300.0
400.0
500.0
600.0
sh
ea
r s
tr
es
s (
Pa
)
Flow DHA 32C Stepped Flow DHA DAY 0
Stepped Flow DHA DAY 7
Stepped Flow DHA DAY 14
Stepped Flow DHA DAY 21
Stepped Flow DHA DAY 28
0 25.00 50.00 75.00 100.0 125.0 150.0 175.0 200.0
shear rate (1/s)
0
100.0
200.0
300.0
400.0
500.0
600.0
sh
ea
r s
tr
es
s (
Pa
)
Flow DHA 40C Stepped Flow DHA DAY 0
Stepped Flow DHA DAY 7
Stepped Flow DHA DAY 14
Stepped Flow DHA DAY 21
Stepped Flow DHA DAY 28
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50
separation may have occurred after 28 days. For combination lawsone/DHA an increase
in viscosity was detected. This may suggest swelling may have occurred within the
emulsion, increasing the rigidity of the second interface by progressive migration of the
lipophilic surfactant, which corroborated with the data observed during the OSS testing
for the same samples62
.
Table 6. Summary of yield stress values and maximum viscosity values at 25˚C, 32˚C,
and 40˚C for lawsone, DHA, and combination lawsone/ DHA emulsions
Lawsone DHA Lawsone/DHA
˚C Day
Yield
Stress
(Pa)
Viscosity
(Pa.s)
Yield
Stress
(Pa)
Viscosity
(Pa.s)
Yield
Stress
(Pa)
Viscosity
(Pa.s)
25 0 37.7 381.2 94.6 373.2 59.7 622.9
28 37.7 1353.0 94.6 298.1 37.7 2839.0
32 0 59.7 3111.0 37.7 2946.0 59.7 872.2
28 37.7 496.2 37.7 3061.0 37.7 3138.0
40 0 59.7 3111.0 37.7 2946.0 59.7 872.2
28 94.6 552.7 37.7 406.4 37.7 4272.0
The OTS rheograms for lawsone and DHA can be seen below in Figures 17a,
17b, and 17c. In the following rheograms, G’, or the elasticity modulus, is plotted along
with time in order to determine the extent of elasticity change after 15 minutes at each
weekly time point at 25˚C, 32˚C, and 40˚C. The rheogram for lawsone showed a
decrease in elasticity (G’) on day 28 at 25˚C and day 14 at 40˚C. The decrease in
elasticity may have resulted in a change or a loss of structure at that time point, which
may be due to a number of reasons such as a chemical reaction or just a change in
temperature. In Figure 18a and 18b, the G’ for DHA emulsion remained consistent at
both 25˚C and 32˚C storage temperatures, showing a minimal change in G’ within the 28
51
days span. In Figure 18c, there was an observed variance in G’ values at 40˚C, more
specifically a drastic decrease was detected on day 28. A change in elasticity and
structure for DHA may have been due to an increase in temperature for a prolonged
period of time, which is also consistent with the results obtained during the OSS tests
above. The results from OTS tests for combination lawsone/DHA showed minimal
changed throughout the 28 day span at all 3 temperatures, of which can be seen in the
Appendix.
Figure 17. OTS rheograms for lawsone at a) 25˚C, b) 32˚C, and c) 40˚C, G’ vs Time (s)
0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0
time (s)
100.0
1000
10000
G'
(P
a)
Osc Time Sweep LAWSONE 25C OTS LAW DAY 0
OTS LAW DAY 7
OTS LAW DAY 14
OTS LAW DAY 21
OTS LAW DAY 28
0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0
time (s)
100.0
1000
10000
G'
(P
a)
Osc Time Sweep LAW at 32C OTS LAW DAY 0 32C
OTS LAW DAY 7 32C
OTS LAW DAY 14 32C
OTS LAW DAY 21 32C
OTS LAW DAY 28 32C
0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0
time (s)
100.0
1000
10000
G'
(P
a)
Osc Time Sweep LAWSONE 40C OTS LAW DAY 0
OTS LAW DAY 7
OTS LAW DAY 14
OTS LAW DAY 21
OTS LAW DAY 28
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52
Figure 18. OTS rheograms for DHA at a)25˚C, b)32˚C, and c) 40˚C, G’ vs Time (s)
3.4 Discussion
Measuring the zeta potential of the internal droplets of a multiple emulsion is a
possible way of determining the attractive forces that may cause aggregation or
flocculation to occur. During flocculation, the droplets tend to aggregate without the
potential increase in droplet size. Zeta potential results showed an increase in zeta
potential for DHA at day 28. Despite that observation, lawsone, DHA, and combination
lawsone/DHA emulsions exhibited overall stability at 25˚C and 32˚C. At 40˚C, all
emulsions showed varying values of zeta potential. Significant increases in zeta potential
for all emulsions were observed. Therefore, the electrostatic repulsion of the internal
0 943.32time (s)
100.0
1000
10000
G'
(P
a)
Osc Time Sweep DHA 25C OTS DHA DAY 0
OTS DHA DAY 7
OTS DHA DAY 14
OTS DHA DAY 21
OTS DHA DAY 28
0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0
time (s)
100.0
1000
10000
G'
(P
a)
Osc Time Sweep DHA 32C OTS DHA DAY 0
OTS DHA DAY 7
OTS DHA DAY14
OTS DHA DAY 21
OTS DHA DAY 28
0 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 1000.0
time (s)
100.0
1000
10000
G'
(P
a)
Osc Time Sweep DHA 40C OTS DHA DAY 0
OTS DHA DAY 7
OTS DHA DAY 14
OTS DHA DAY 21
OTS DHA DAY 28
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53
droplets’ surface may be less, which may lead to a greater chance of flocculation to
occur.
Monitoring particle size within the multiple emulsion may determine the presence
of creaming and coalescence. The results for particle size showed an increase for at 32˚C
for combination of lawsone/DHA. Despite this one increase, the particle size remained
consistent throughout all temperatures and days, lying within the range of 300nm to 500
nm. Therefore, in terms of creaming or coalescence, the emulsions were stable at varying
times and temperatures.
Determination of varying pH at various times and temperature can help determine
any possible chemical reactions that may occur with a multiple emulsion. At 25˚C,
lawsone experienced a pH drop on day 7 but remained within the pH range of its original
value about 6.22 after 28 days. A significant increase was observed within the DHA
emulsion from day 14 to day 28 to 5.1. As for combination lawsone/DHA emulsion, it
showed a fluctuating increase and decrease within the 28 time span, resulting in a final
pH of 5.1. At 32˚ C, lawsone showed a significant increase to a pH of 6.05 by day 28. A
drop in pH was observed for DHA emulsion from 6.03 at day 0 to 4.92 by day 28, and in
combination of lawsone/DHA to a pH of 3.88 from 5.2 at day 0. The same drop in pH
was also observed at 40˚C for combination lawsone/DHA, with a change in pH from 6.64
to 3.71. Drastic changes in pH of DHA and combination of lawsone/DHA suggest
chemical interactions may be occurring. This notion corroborates with the data from the
entrapment efficiency studies presented in the previous chapter and observations by
Fusaro et al, where DHA emulsions showed chemical instability when stored in elevated
temperatures and when in combination with lawsone23
. Hence, it was thought best if both
54
drugs were kept separate and stored at either room temperature or 4˚C, only combining
the two drugs before use.
In order to monitor any changes in viscosity, elasticity, and structure, measuring
spreadability and conducting rheological analysis may be necessary. Spreadability
results showed a significant increase in both lawsone and combination lawsone/DHA
multiple emulsions at 25˚C. At 32˚C, combination lawsone/DHA showed an increase to
29.5cm²/g on day 21. While at a stability temperature of 40˚C, a slight variance was seen
in the DHA emulsion with an increase in spreadability to 34cm²/g. Despite these varying
values, no obvious patterns were observed within each batch or temperature. Generally,
the spreadability of each emulsion remained rather consistent, remaining within the range
of 20cm²/g to 35cm²/g.
Rheological assessment of each emulsion is pertinent in knowing the structural
integrity and flow of the emulsions. The decrease in the LVR region in Figure 14c
suggests that at higher storage temperatures the elasticity of the emulsion decreases,
compromising its overall structure. In Table 5, the orange boxes showed a decrease in
value of tan δ for lawsone at 32˚C and 40˚C and for DHA at 25˚C and 32˚C suggesting an
increase in elasticity which may have happened from possible swelling. According to
Geiger et al, the swelling of the oil globules would lead to the increase in elasticity of the
system62
. Concurrently, the yellow boxes in Table 5 showed an increase in tan δ,
signifying a decrease in elasticity after 28 days. The decrease in elasticity can somewhat
be expected at elevated storage temperatures of 40˚C with any emulsion due to its use of
lipids or waxes. The decrease in elasticity may have occurred from possible expulsion of
55
the internal water droplets into the continuous medium after swelling or after having
applied stress onto the samples during the stress sweep.
Stepped flow curves showed all emulsion displayed a non-Newtonian,
pseudoplastic flow behavior. The presence of yield stress values, as seen in Table 6, and
shear thinning behavior of emulsions suggest viscoelastic flow properties. In Table 6,
the values in orange (lawsone and DHA emulsions) showed a decrease in viscosity
suggesting phase separation may have occurred for lawsone and DHA emulsions left at a
storage temperature of 32˚C and 40˚C after 28 days. This observation validated the
observed results of the orange boxes in Table 5. Since swelling of the oil globules may
have occurred, it may have eventually progressed to phase separation. The yellow boxes
in Table 6, primarily all of combination lawsone/ DHA emulsions left at 25˚C, 32˚C, and
40˚C, showed an increase in viscosity after 28 days, suggesting swelling may have
occurred. Swelling may have led to an increase in the rigidity of the second interface by
the progressive migration of the lipophilic surfactant. In the second step of the of the
multiple emulsion preparation, lipophilic surfactant molecules can diffuse from the first
to the second interface, where they produce a synergistic effect resulting in membrane
strengthening62
. This therefore explains how and why the viscosity possibly increased,
also validating the results found in the yellow boxes in Table 5.
By plotting G’ as a function of time as in the OTS tests, any structural changes of
the multiple emulsion can be monitored. OTS determines if and how the material
properties change after being loaded onto the rheometer, by monitoring certain
viscoelastic parameters as time advances. In this case, the control variable was 0.5968Pa
which was within the LVR of all the emulsions. Therefore, at this control variable and a
56
constant frequency of 1Hz, evaluation of the multiple emulsion’s behavior with time was
monitored directly. The OTS results showed a decrease in G’ for lawsone emulsion at
25˚C after 28 days (398 Pa). Also, a gradual decrease in G’ was observed at 40˚C for
DHA after a span of 28 days to 490.4Pa, which also exhibited a decrease in emulsion
elasticity through time. This observation validates the previous speculation made when
measuring the entrapment efficiency of DHA emulsions. In previous observations, the
DHA emulsion stored at 40˚C exhibited a decrease in entrapment efficiency, correlating
to the increase of free drug detected by the UPLC. It was previously speculated that the
structure of the emulsion at 40˚C might have been compromised, resulting in the
complete delivery of the drug into the external aqueous phase and consequently more free
DHA to be detected. The results from the OTS tests confirmed this speculation that the
structural integrity and loss of elasticity did occur over a period of time and increased
temperature, which may have been the reason for the decreased entrapment of DHA
within the internal aqueous phase.
3.5 Conclusion
In summary, the overall integrity of lawsone and DHA emulsions remained
generally stable within the storage temperatures of 25˚C for all characterization tests.
With increased storage temperatures of 32˚C and 40˚C, the emulsions have a greater
chance in experiencing flocculation, chemical instability, decrease in structural elasticity,
and possible swelling of the internal globule of the multiple emulsion. As for the
multiple emulsion combining both lawsone and DHA, pH issues suggest a chemical
degradation may be occurring at elevated temperatures which is in agreement with
previous stability studies conducted by Fusaro et al23
. Also, because the combination
57
emulsion was a physical mixture of two multiple emulsions combined in 1:1 ratio,
structural integrity and elasticity was compromised. When combining the two drugs in
that manner, an increased incidence of swelling was observed at all storage temperatures.
In conclusion, optimum stability was observed when lawsone and DHA emulsions were
kept at a storage temperature of 25˚C as separate entities and not in combination.
58
CHAPTER 4
In vitro Evalution of Multiple Emulsion Incorporating Lawsone and DHA
59
4.1. Introduction
In vitro drug release testing is a measure of the active pharmaceutical ingredient
(API) from the drug delivery system, which in this case is a multiple emulsion.
Evaluation of drug release profiles is pertinent to drug development and quality control.
It involves subjecting the dosage form to a set of conditions that will induce a drug
release and the measuring or quantifying of the amount of drug released under those
conditions. By simulating in vitro conditions to that of in vivo conditions, a better
understanding is gained of how the drug product will react within the body’s
environment. Typically, release profiles are determined using a Franz diffusion
apparatus, where the topical drug would spread over a suitable membrane and placed
application side up onto a diffusion cell. In the following experiments, the release profile
of lawsone, DHA, and combination lawsone DHA were determined conventionally by
use of the Franz diffusion apparatus using a snake skin membrane, as well as determined
without any interposed membrane. Because the emulsions were comprised of multiple
interphases with the drug embedded within internal most phase, preliminary release tests
were conducted in order to determine the amount of drug that actually diffused through
the system without the additional barrier of a membrane. By using a modified version of
USP Apparatus 5, the amount of drug released through the multiple emulsion system was
then determined.
Additional release studies were conducted using the vertical diffusion cell (VDC)
method, also known as the Franz diffusion cell. Diffusive communication between the
delivery system and the reservoir takes place through an inert, highly permeable support
membrane63
. In this study, the membrane used was shed snake skin. Shed snake skin is a
60
nonliving pure stratum corneum with no hair follicles64,65
. Snakes shed their skin
periodically, leaving their old stratum corneum behind, which makes it possible to obtain
multiple shed skins from the same individual snake65
. Unlike human stratum corneum
which is made up of 10 to 20 layers of α-keratin-rich intracellular layer and a lipid-rich
intracellular layer, shed snake skin consists of 3 distinct layers66,67
. The three layers are a
β-keratin-rich outermost beta layer, α-keratin and lipid rich intermediate mesos layer, and
α-kertain-rich innermost alpha layer68,69
. The meso layers shows three to five layers of
multilayer structure with cornified cells surrounded by intercellular lipids, which is
similar to the human stratum corneum. Further comparisons between human stratum
corneum and shed snake skin from Itoh et al can be seen below:
Table 7. Comparison of thickness, lipid content, and water evaporation rate
between human stratum corneum and shed snake skin
Human Stratum Corneum Shed Snake Skin
(Elaphe absoleta)
Thickness
13-15µm66,67
, 10µm66
10-20µm
Lipid Content 2.0-6.5%
3.0-6.8%70
6.0%
Water Evaporation Rate
0.1-0.8mg/cm²hr
0.34mg/cm²/hr
Ca. 0.2 mg/cm²hr
0.15-0.22 mg/cm²hr
With the similarities to human stratum cornuem listed above, shed snake skin was
thought to be a viable option as a substitute for actual human cadaver skin. Permeation
of a chemical through the stratum corneum is a process where active transport does not
apply. The layer with the highest resistance to diffusion is the rate-limiting membrane.
61
For many compounds, the lipophilic stratum corneum is the primary rate limiting
barrier71
. On the other hand, the rate limiting barrier for topically applied lipophilic
compounds would be the hydrophilic epidermis and dermis. When conducting in vitro
release testing (IVRT), the formulation is applied to the donor chamber with full contact
with the membrane that is in contact with the receiver liquid, in this case phosphate
buffer pH 7.4. The receiving medium should provide a “diffusional sink” for the active
ingredient released from the semisolid formulation72
. The receiving medium is sampled
as a function of time, and the API is obtained quantitatively to determine permeation
/flux profile. The cumulative amount (Q) release per surface was calculated by
Equation 7 below72
. The relationship of Q (cumulative amount released) versus √T
(square root of time) is derived from the Higuchi model with the assumption that there is
a reservoir of the drug always available to diffuse through72,73
.
Q =CnV+ ∑ Ci
n−1i=1 S
A (7)
The average cumulative amount of drug released was determined from Equation
7 where Cn is the concentration (mg/mL) determined at nth sampling interval, V is the
volume of individual Franz diffusion cell, ∑ 𝐶𝑖𝑛−1𝑖=1 is the sum of concentrations (mg/mL)
determined at sampling intervals 1 through n-1, S is the volume sampling aliquots
(0.1ml), and A is the surface area of the sample well (0.64cm²).
4.2. Materials
Python snake skin used for the Franz diffusion studies was provided by the
generous donations of Henry Doorly Zoo (Omaha, NE). Snake skin was obtained after
62
ecdysis, or sloughing of the skin, which is dependent on various aspects such as species,
age, nutrition etc.
4.3. Methods
4.3.1. In vitro Surface Release Studies
Figure 19. Schematic of USP Apparatus 5 (Paddle Over Disk) Assembly63
Under USP guidelines for measuring drug release from topical and transdermal
products, Apparatus 5 (Paddle Over Disk) assembly, which can be seen in Figure 19,
was to be used. Although the laboratory was equipped with a dissolution apparatus, it
was not equipped with the disk assembly necessary for the release study. Therefore, to
remedy the lack of supplies, an altered version of USP Apparatus 5 was assembled.
Instead of using a large volume dissolution vessel, either 150 mL or 1000 mL, a 20 mL
glass scintillation vial was instead used. Emulsion samples (lawsone, DHA, and
combination lawsone/DHA) were carefully filled into HPLC vial caps, which were used
as a replacement version of the disk assembly of USP Apparatus 5.
63
Figure 20. Picture representation of altered version of USP Apparatus 5 (sample and
vessel) and schematic of sampling process/ analysis
Three sets of each emulsion were weighed and excess emulsion was removed and
smoothened to provide a uniform release surface. The caps filled with emulsions were
carefully maneuvered into the glass scintillation vials. Approximately 10 mL of sodium
phosphate buffer pH 7.4 was used as dissolution medium and was placed into the
scintillation vial with the samples. The release assemblies were placed into an orbital
shaking incubator for a total of 120 hours at a speed of 100 rpm and kept at a temperature
of 32˚C. Samples, about 2% (0.2 mL) of the total volume of dissolution medium, were
removed at various time points using a needle and syringe and analyzed using the UPLC
analysis method previously stated. Time points for sampling were taken in hours (0, 1, 2,
3, 6, 12, 24, 48, 72, 96, and 120). Sodium phosphate buffer pH 7.4 (0.2mL) was used to
replace the volume that was extracted. Figure 20 is a picture representation of the altered
USP Apparatus 5 assembly that was created, which also displays the sampling process
and analysis through UPLC. Release studies were performed in triplicate (n=3).
64
4.3.2. Franz Diffusion using Snake Skin
The Franz diffusion apparatus, also known to the USP as the vertical diffusion
cell (VDC), was used for measuring the drug release from lawsone, DHA, and a
combination of lawsone/DHA multiple emulsions. An illustrated representation of the
multi-station Franz diffusion cell system which was used in this experiment can be seen
in the Figure 2174
.
Figure 21. Illustrated representation of multi-station Franz Diffusion cell system
The Franz diffusion cells, as represented in Figure 22, used for this experiment
were the standard jacketed cells with a flat ground joint, an internal 9 mm (≈ 5 mL)
receptor chamber, and a detachable donor assembly. The glass jacket surrounding the
cells allowed it to be connected to a circulator/heater that kept the temperature consistent
at 32˚C. A stir bar was placed within each cell and kept at a speed of 600 rpm. Before
use in the diffusion apparatus, snake skin was first prepared. The skin used was a large
portion, often over 3 feet long, of sloughed snake skin. Initially, the skin was soaked in
water to be cleaned. It was then dried overnight. The dried skin was cut according to
either the dorsal or ventral regions of the snake. In this particular experiment, dorsal
samples which were color and scale matched were utilized. Lastly, the skin was cut into
65
small (≈ 1 inch²) pieces prior to use. For ease of application, a small circle with the
diameter of the donor chamber was drawn as a reference for applying the emulsions.
The prepared skin squares where then further soaked in sodium phosphate buffer pH 7.4
overnight. The skin was weighed before and after application of the emulsions. Three
sets of approximately 0.2g to 0.4g of lawsone, DHA, and combination of lawsone/DHA
emulsions were weighed on top of the snake skins. To ensure full contact with the skin,
the emulsions were carefully rubbed onto the skin with a cotton tip applicator. Sodium
phosphate buffer pH 7.4 was placed into the receptor chamber and used as the diffusion
medium. The skin/emulsion samples were then positioned on top of the opening of the
receptor chamber. The donor assembly was maneuvered on top of the skin/emulsion
samples, ensuring the excess emulsions were kept within the donor chamber. The sample
(≈ 0.1 mL) aliquots were removed through the sampling port by needle and syringe.
Time points for sampling were taken in hours (0, 1, 2, 3, 6, 12, 24, 48, 72, 96, and 120).
Samples were analyzed using the UPLC method. Diffusion experiments were done in
triplicate.
Figure 22. Picture representation of Franz diffusion cell
66
4.3.3. Statistical Analysis of Data
The statistical analysis of this experimental data for the purpose of comparison
was performed using a 2 tailed Student’s T-Test. Data was considered statistically
significant if p<0.05.
4.4. Results and Discussion
4.4.1. In vitro Surface Release Studies
Figure 23. The in vitro release profiles of a) lawsone and b) DHA multiple emulsions in
comparison with the combination lawsone/DHA multiple emulsion
a) b)
67
Multiple emulsions are composed of two interphases. In this experiment, lawsone
and DHA are incorporated into the internal W1/O phase, while the external phase is the
continuous aqueous (W2) phase. Initial in vitro surface release studies were conducted to
determine the amount of drug that was able to release from the internal phase by
exposing only one portion of the emulsion to the phosphate buffer pH 7.4 medium.
Phosphate buffer pH 7.4 was used as well as kept at a temperature of 32 ͦC to mimic in
vivo skin conditions. In Figure 23 a and b, the graphs show comparative representation
between release profiles of multiple emulsions containing lawsone and DHA alone versus
in a combination multiple emulsion containing both drugs. The combination
lawsone/DHA multiple emulsion was comprised by combining two separate emulsions
double in concentration in a 1:1 ratio and triturated until homogenous. In Figure 23a¸ the
lawsone multiple emulsion obtained a final release of 85.3 ± 3.95%, while the
combination emulsion only obtained 56.4%, with significant differences (p <0.05)
starting from 72 hours until 120 hours. In Figure 23b, DHA only emulsion resulted in a
final release of 49.9 ± 2.08% while the combination lawsone/DHA emulsion had a final
release of about 40.7 ± 2.57%. Multiple emulsions have previously been considered a
controlled release drug delivery system, with the internal phase holding the hydrophilic
drug content. When incorporating two emulsions together as in the combination
lawsone/DHA emulsion, there are not only two interphases, but multiple interphases in
which the drug has to release from since two emulsions were combined to make one.
Therefore, this may be the reasoning behind the decreased release in the combination
lawsone/DHA emulsions for Figures 23a and 23b. The lawsone multiple emulsion’s
internal (W1) aqueous phase was comprised of phosphate buffer pH 7.4 instead of
68
deionized water which was used for the DHA emulsion. The literature value of the
distribution coefficient (log Doct/wat) for lawsone at pH 7.4 is -1.39 75
, while for DHA had
a log D at pH 7.4 of -1.0976
. The log D generally is a measurement of the hydrophobicity
of the drug, which affects how easily the drug can reach its intended target in the body,
how strong an effect it will have once it reaches the target, and how long it will remain in
the body in an active form77
. A higher value of log D would result in a more
lipophilic/hydrophobic drug, and vice versa. Therefore, at pH 7.4, lawsone’s low value
of log D of -1.39 would make it more hydrophilic which would allow more partition
through the oil layer and into the continuous aqueous (W2) phase, explaining the greater
release of lawsone into the distribution medium as opposed to DHA.
4.4.2 Franz Diffusion using Snake Skin
a)
69
Figure 24. In vitro release profile from franz diffusion testing as a function of the square
root of time for a) lawsone and b) DHA multiple emulsions in comparison with the
combination of lawsone/DHA multiple emulsion
The average rate of release is calculated by determining the relationship between
the cumulative amount released (Q) (mg/cm²) versus the square root of time (√T) (hr½
).
The average flux of release is obtained by the slope of the regression line where √T is the
x-axis and Q is the y-axis. In Figure 24a and 24b, the graphs presented compared the
lawsone and DHA multiple emulsions respective to the amount of drug released when
combined into one emulsion. Figure 24a shows a significant increase (p < 0.05) in the
average flux of release of lawsone (0.015 mg/cm²/hr½)
) when incorporated into a solo
emulsion as opposed to when in a combined multiple emulsion with both lawsone and
DHA (0.0014 mg/cm²/hr½). The opposite was observed in Figure 24b, displaying an
increased average flux of release of DHA from the combination multiple emulsion
(1.3326 mg/cm²/hr½) as opposed to the multiple emulsion with just DHA alone (0.7807
mg/cm²/hr½). As established prior, the combination lawsone/DHA multiple emulsion
was composed of two separate lawsone and DHA emulsions, triturated and homogenized
b)
70
to make one final emulsion. Hence, it was expected to see a slower rate of release from
the combined emulsion due to the increase in viscosity and the presence of multiple
interfaces that would act as barriers for release. An opposite reaction was observed for
DHA and its rate of release from the combined emulsion. As established in the previous
chapter in Table 6, combination lawsone/DHA multiple emulsions was shown to have a
tendency to increase in viscosity when stored at 32 ͦC which may be attributed to a
swelling reaction which may occur. Generally, an increase in viscosity would cause a
decrease in average flux release rate as seen in Thakker et al72
. But as discussed
previously, swelling had a possibility of occurring within combination lawsone/DHA
emulsion at temperatures of 32 ͦC and higher, which may be due to the migration of the
lipophilic surfactant to the second interface during the initial preparation process. In this
case, the lipophilic surfactant surrounding the external continuous phase would be the
cause for an increase in attraction to the lipophilic nature of the snake skin. Since the
combination lawsone/DHA emulsion is composed of two multiple emulsions together,
the amount of lipophilic surfactant would be double the amount of a single multiple
emulsion. Also, as discussed in the previous section, the distribution coefficient of DHA
(-1.09) is slightly greater than lawsone (-1.39) at a pH 7.4 resulting in a less hydrophilic
nature and may also be causing an affinity to the lipophilic nature of the snake skin more
so than lawsone. In order to further assess the reasoning behind the increased rate of
flux for DHA in combination, additional studies need to be conducted.
4.5. Conclusion
Lawsone, DHA, and combination lawsone/DHA multiple emulsions were
evaluated to determine their compatability in in vivo conditions. A preliminary release
71
study was conducted to determine if and how much of the drugs were able to release
without the addition of diffusion membrane. By utilizing an altered version of USP
Apparatus 5, the surface release profiles for lawsone, DHA, and combination
lawsone/DHA multiple emulsions were obtained. DHA and DHA in combination with
lawsone both showed comparable release profiles. Lawsone, on the other hand, showed a
cumulative % release of 85.3 ± 3.95% after 120 days as opposed to only 56.4 ± 0.45% for
lawsone in combination. This was mainly due to its inherent log D when at a pH 7.4.
Because lawsone’s log D was lesser (-1.39) than DHA (-1.09), it allowed more partition
through the oil layer to the external aqueous buffer medium and resulting in a greater
release of drug. The multiple interphases, which formed a barrier of sorts for the
combination emulsions, allowed no more than 40.7 ± 2.57% to 56.4 ± 0.45% release of
DHA and lawsone.
The IVRT for the emulsions using snake skin showed a significant increase in the
rate of release for lawsone when compared to the combination multiple emulsion. This
also correlates with the results obtained in the surface release studies. A greater
cumulative amount released (Q) was observed in DHA in combination than when DHA is
alone in the multiple emulsion. This may be due to the fact that at temperatures greater
than 32˚ C, combination lawsone/DHA multiple emulsions was previously shown to
swell, causing the lipophilic surfactant to migrate to the second interface during the
preparation process. Also, the distribution coefficient (log D) of DHA was slightly
higher than that of lawsone. Because of this and the lipophilic nature of the external
phase of the combination multiple emulsion after swelling, we can assume that a greater
affinity to the lipophilic nature of the snake skin was established. Consequently, a greater
72
cumulative amount of drug released would be observed for DHA in combination. In
order to confirm these results, further studies of the partitioning behavior of the multiple
emulsions need to be conducted.
73
CHAPTER 5
Summary and Future Directions
74
5.1. Summary
A multiple emulsions containing 0.035% lawsone (w/w) and 1% DHA (w/w)
were prepared using a two-step emulsion procedure provided by Matsumoto et al. The
first step of emulsion was process was the preparation of the primary (W1/O) phase where
the hydrophilic drugs were incorporated into the internal (W1) phase and was slowly
incorporated into the oil (O) phase through by hand homogenizer. The second step was
the addition of the primary (W1/O) phase to the external aqueous phase (W2) by over-
head stirrer with a paddle attachment. The combination lawsone/DHA emulsions were
prepared by formulating the lawsone and DHA emulsions separately with double the
concentration and triturating both in a 1:1 ratio until homogenous. The existence of all
interphases within the multiple emulsion was verified and visualized using light and
fluorescence microscopy. Entrapment efficiencies of lawsone and DHA within each
emulsion and in combination were determined through a biphasic extraction using a 1:1
ratio of methylene chloride and phosphate buffer (PB) pH 7.4. Entrapped drugs were
analyzed using a UPLC method. The entrapment efficiency at 25˚C showed a consistent
percent entrapment for lawsone, DHA, and DHA in combination multiple emulsions,
lying within the range of ≈ 71.1% to 83% entrapment within the internal (W1) water
phase after 28 days of storage. Meanwhile, the lawsone in combination multiple
emulsion showed an entrapment of ≈ 99%. At 32˚C, about the same patterns were
observed, except a slight decrease in entrapment efficiency for DHA and DHA in
combination emulsion to about ≈ 76% to 65% after 28 days. At 40˚C, lawsone emulsion
remained consistent at 70.64 ± 3.02% after 28 days and lawsone in combination
displayed a drastic increase in entrapment to 99.58 ± 0.07% by day 28. DHA emulsion
75
decreased by day to 37.41 ± 6.04 % entrapment efficiency and DHA in combination
emulsion showed a drastic decrease in entrapment to about 5.41 ± 3.82% after 28 days.
Therefore, less entrapment for DHA and DHA in combination was a clear indication of
the emulsion’s instability at 40˚C, which were later confirmed by characterization and
stability studies.
Lawsone, DHA, and a combination of lawsone/DHA were all stored at 25˚C,
32˚C, and 40˚C and characterized at various time points throughout a 28 day time span to
determine the emulsion’s chemical and structural stability. Particle size for all three
emulsions remained with a consistent range of 300 to 500nm suggesting no drastic
creaming or coalescence occurred despite temperature and time change. Regardless of
the varying values obtained through spreadability testing, the emulsions remained rather
consistent and remained within the range of ≈ 20cm²/g and 35cm²/g with time and
temperature change, suggesting no noticeable change in viscosity or elasticity would be
observed after application of the emulsions. Zeta potential displayed overall stability at
25˚C and 32˚C without any significant changes occurring. However at 40˚C, zeta
potential for DHA emulsion showed a significant increase (p<0.05) from -20.80 ±
0.98mV on day 14 to -14.93 ± 0.97 mV by day 28 and combination lawsone/DHA also
showed a significant increase to -19.3 ± 3.68mV. The variance in zeta potential at this
temperature implies that a greater chance of flocculation may happen at an increased
temperature of 40˚C. Chemical instability was determined by monitoring pH changes in
the emulsions. At increased temperatures of 32˚C and 40˚C, drops in pH were observed
for both DHA and combination lawsone/DHA multiple emulsions. At 32˚C, DHA
observed a significant decrease in pH from 6.03 ± 0.18to 4.92 ± 0.08 and combination
76
lawsone/DHA with a drop of ≈5.2 to 3.88 ± 0.05 after 28 days in the stability chamber.
At 40˚C, pH drops drastically from pH 6.64 ± 0.22 to 3.71 ± 0.26 after 28 days for
combination lawsone/DHA was observed. Drastic changes in pH of DHA and
combination of lawsone/DHA suggest chemical interactions occurred. This notion
corroborates with the data from the entrapment efficiency studies and observations by
Fusaro et al, where DHA emulsions showed chemical instability when stored in elevated
temperatures and when in combination with lawsone23
. Hence, it was thought best if both
drugs were kept separate and stored at either room temperature or 4˚C, only combining
the two drugs before use. This notion was later confirmed by rheological assessment of
the structure of the emulsions. Rheological studies, specifically the OTS tests, suggest
that structural integrity of the multiple emulsion containing DHA and combination DHA
may also have been the reason for the complete delivery of the DHA to the external (W2)
phase, hence leading to the decrease in entrapment efficiency. Stepped flow step showed
an increased in viscosity of combination lawsone/DHA when stored at all temperatures
after 28 days, which may indicate that swelling may have occurred. Swelling would have
occurred when the lipophilic surfactant molecules may have diffused form the first to the
second interface, which would have led to membrane strengthening and an increase in
viscosity of the emulsion. Therefore, in accordance with Fusaro et al, the individual
lawsone and DHA multiple emulsions may be best stored as separate emulsions until
application, with an optimum storage temperature of 25˚C or cooler.
Evaluation of the in vitro surface release profiles of lawsone, DHA, and both
lawsone/DHA in combination multiple emulsions was used to determine if and how
much of the drugs were able to release out of the emulsion without the added barrier of a
77
diffusion membrane. DHA and DHA in combination with lawsone emulsions had
comparable release profiles with a cumulative % release after 120 hours. DHA emulsion
had a cumulative drug release of about 49.9 ± 2.08% after 120 days while, DHA in
combination had a drug release of 40.7 ± 2.57% with no significant difference (p<0.05)
between the two formulations. However, lawsone multiple emulsion showed an increase
in cumulative release (85.3%) as opposed to lawsone in combination, the reason for this
being the distribution coefficient (log D) of lawsone at a pH of 7.4. Because the log D
for lawsone (-1.39) was less than DHA (-1.09), lawsone was allowed more partition
through the oil layer to the external aqueous buffer medium, therefore resulting in a
greater release of the drug. In the diffusion release studies using snake skin, a significant
increase in the average flux of release was observed in lawsone (0.015 mg/cm²/hr½)
multiple emulsion when compared to lawsone in combination (0.0014 mg/cm²/hr½).
Because the combination emulsion was composed of two emulsions integrated together
to make a homogenous formulation, it was expected to see a slower flux of release
especially from the increase in viscosity and the presence of multiple interfaces which
would act as barriers for release. The opposite reaction was seen when comparing the
flux release of DHA in combination and DHA multiple emulsions. The emulsion
containing DHA in combination indicated a higher average flux (1.3326 mg/cm²/hr½)
than DHA alone (0.7807 mg/cm²/hr½). Although the reasons for this reaction can be
speculated, further studies are needed in order to do a full evaluation of diffusion kinetics
of the multiple emulsion. In summary, evaluation of the multiple emulsions through in
vitro studies showed that the multiple emulsions containing lawsone, DHA and a
combination of both lawsone/DHA are capable to diffuse through the interphases of the
78
emulsion itself. Also, the studies exemplified how multiple emulsions can be a viable
drug delivery system for topical application of lawsone, DHA, and/or a combination of
both lawsone and DHA.
5.2. Future Directions
The present study elucidated the formulation and stability characterization of
multiple emulsions containing lawsone, DHA and a combination of lawsone and DHA.
Due to the variance in viscosity and decreased structural elasticity over a period of time
as storage temperatures increased as seen from the rheological assessment of the
emulsions, further fine tuning of the formulation is needed to ensure thermodynamic
stability. One approach to improving stabilization of a multiple emulsion is by stabilizing
the inner (W1/O) phase by addition of various emulsifier combinations78
. Also,
stabilization of the oil phase can be enhanced by choosing a suitable oil type and addition
of proper carriers, complexants, and viscosity builders may be another approach. By
doing so, the solubility and polarity of the oil phase would be modified, making it less
water soluble79
. Finally, stabilization of the external aqueous phase by increasing the
viscosity of the outer aqueous layer may be another way to enhance stability80
(tedajo).
After a new formulation is established, full extraction of the drug by possibly a double
extraction method would be needed in order to not under estimate the full drug load of
the multiple emulsions. In addition, further in vitro studies are needed to determine the
permeability and diffusion kinetics of the multiple emulsions. Concurrently, human
cadaver skin may also be used instead of snake skin in order to determine a truer
representation of skin diffusion. Finally, in vivo studies can be conducted to correlate the
results obtained from the in vitro studies by using actual human volunteers to determine
79
the efficacy of using lawsone and DHA in a multiple emulsion drug delivery system as a
form of sun protection.
80
References
81
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