effect of plant derived saponin on the structure and ...4460/datastream... ·...
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Effect of Plant Derived Saponin on the Structure and Stability of Lipid
Membranes in the Absence of Cholesterol
A Thesis
Submitted to the Faculty
of
Drexel University
by
Amanda Rose Decker
in partial fulfillment of the
requirements for the degree
of
Master of Science
June 2014
© Copyright 2014 Amanda Rose Decker. All Rights Reserved
ii
DEDICATION
For my friends and family, especially my parents, Joe and Karen Decker,
without whom I would not be the person I am today.
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ACKNOWLEDGEMENTS
First, I offer my greatest and most sincere thanks to Dr. Steven Wrenn for his
constant support and guidance throughout the past three years.
Next, a big thanks to my co-‐workers, Nicole Wallace, An Nguyen, and Jordan-‐
Alexandria Shepard for all of your help, whether it was training on equipment, make
sense of some nonsensical data, or preparing liposomes during busy weeks.
Thank you to Ms. Dolores Conover and Drexel University’s Biomedical
Engineering department for use of their lab space and TECAN equipment.
Finally, I would like to acknowledge Simeon Stoyanov at Unilever, for
providing this inspiration for this project.
iv
TABLE OF CONTENTS
LIST OF TABLES .......................................................................................................................................... v
LIST OF FIGURES ...................................................................................................................................... vi
ABSTRACT ................................................................................................................................................. viii
CHAPTER 1: INTRODUCTION AND BACKGROUND .................................................................... 1
CHAPER 2: MATERIALS AND METHODS ........................................................................................ 5
2.1 Materials ....................................................................................................................................................... 5
2.2 Preparation of Small Unilamellar Vesicles (SUV) ........................................................................ 5
2.3 Calcein Leakage Assay ............................................................................................................................. 8
2.4 Förster Resonance Energy Transfer (FRET) Assay .................................................................... 9
2.5 Fluorescence Spectroscopy ................................................................................................................ 11
2.6 Dynamic Light Scattering (DLS) ....................................................................................................... 11
2.7 Turbidity Measurement ...................................................................................................................... 12
CHAPER 3: RESULTS .............................................................................................................................. 13
3.1 Calcein Leakage Assay .......................................................................................................................... 13
3.2 FRET Assay ................................................................................................................................................ 20
CHAPTER 4: DISCUSSION .................................................................................................................... 28
CHAPTER 5: FUTURE WORK .............................................................................................................. 36
CHAPTER 6: LIST OF REFERENCES ................................................................................................. 37
APPENDIX A: ADDITIONAL CALCEIN DATA ............................................................................... 38
APPENDIX B: ADDITIONAL FRET DATA ....................................................................................... 51
APPENDIX C: FITC-‐Dextran EXPERIMENT ................................................................................... 53
v
LIST OF TABLES
Table 1: Liposome Formulations for All Experiments .............................................................. 6 Table 2: Summary of Particle Size vs. Saponin Level ............................................................... 17 Table 3: Summary of Turbidity vs. Saponin Level (Stand in for Precipitate
Formation) ........................................................................................................................................ 18 Table 4: Summary of Linear Fit for Figure 16 ............................................................................. 26 Table 5: Plate Layout for All Experiments. ................................................................................... 38 Table 6: Turbidity Data for Pure Liposome Solution with No Saponin ........................... 51 Table 7: Liposome Dimensions. ........................................................................................................ 53 Table 8: FITC-‐Dextran Sizes Relative to Weight ........................................................................ 54
vi
LIST OF FIGURES Figure 1: Structure of QuilA Saponin ................................................................................................ 2 Figure 2: Ternary diagram for liposome compositions ............................................................ 7 Figure 3: Liposome compositions overlaid on Demana et al.'s phase diagram ............. 8 Figure 4: % Leakage vs. Time Data for 5 mol% Saponin Samples ..................................... 13 Figure 5: % Leakage vs. Time Data for 15 mol% Saponin Samples ................................... 14 Figure 6: Final % Leakage vs. Saponin Level for 0% Cholesterol Liposomes ............... 15 Figure 7: Final % Leakage vs. Saponin Level for 25% Cholesterol Liposomes ............ 15 Figure 8: Final % Leakage vs. Saponin Level for 50% Cholesterol Liposomes ............ 16 Figure 9: Qualitative Visual Data for Precipitate Formation after 24 hours in 4 °C. .. 18 Figure 10: FRET Curves for Liposome C with 5% DAN .......................................................... 20 Figure 11: FRET Curves for Liposome C with 10% DAN ........................................................ 21 Figure 12: FRET Curves for Liposome F with 5% DAN .......................................................... 22 Figure 13: FRET Curves for Liposome F with 10% DAN ........................................................ 23 Figure 14: FRET Curves for Liposome G with 5% DAN .......................................................... 24 Figure 15: FRET Curves for Liposome G with 10% DAN ....................................................... 25 Figure 16: DHE/DAN Max Emission Peak Ratios vs. Saponin Level for All Liposome
Types and DAN Levels ................................................................................................................. 26 Figure 17: Calcein Fluorescence Intensity vs. Time Data for Liposome A ...................... 38 Figure 18: Calcein Fluorescence Intensity vs. Time Data for Liposome B ...................... 39 Figure 19: Calcein Fluorescence Intensity vs. Time Data for Liposome C ...................... 40 Figure 20: Calcein Fluorescence Intensity vs. Time Data for Liposome D ...................... 41 Figure 21: Calcein Fluorescence Intensity vs. Time Data for Liposome E ...................... 42 Figure 22: Calcein Fluorescence Intensity vs. Time Data for Liposome F ...................... 43
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Figure 23: Calcein Fluorescence Intensity vs. Time Data for Liposome G ...................... 44 Figure 24: Calcein Fluorescence Intensity vs. Time Data for Liposome H ..................... 45 Figure 25: Calcein Fluorescence Intensity vs. Time Data for Liposome I ....................... 46 Figure 26: QuilA Saponin Fluorescence for Calcein Leakage Study .................................. 47 Figure 27: % Leakage vs. Time Data for 0 mol% Saponin Samples ................................... 48 Figure 28: % Leakage vs. Time Data for 40 mol% Saponin Samples ................................ 49 Figure 29: % Leakage vs. Time Data for 75 mol% Saponin Samples ................................ 50 Figure 30: QuilA Saponin Fluorescence for FRET Experiment ............................................ 52 Figure 31: FITC-‐dextran Size Graphical Interpretation of Table 8. ................................... 54 Figure 32: FITC 4K Fluorescence vs. Time for Liposome A .................................................. 55 Figure 33: FITC 250 K Fluorescence vs. Time for Liposome A ............................................ 56 Figure 34: FITC 4 K Fluorescence vs. Time for Liposome B ................................................. 57 Figure 35: FITC 250 K Fluorescence vs. Time for Liposome B ............................................ 58 Figure 36: FITC 4 K Fluorescence vs. Time for Liposome C .................................................. 59 Figure 37: FITC 250 K Fluorescence vs. Time for Liposome C ............................................ 60 Figure 38: FITC 4 K Fluorescence vs. Time for Liposome F .................................................. 61 Figure 39: FITC 250 K Fluorescence vs. Time for Liposome F ............................................ 62 Figure 40: FITC 4 K Fluorescence vs. Time for Liposome G ................................................. 63 Figure 41: FITC 250 K Fluorescence vs. Time for Liposome G. ........................................... 64 Figure 42: QuilA Saponin Fluorescence for FITC Experiment ............................................. 65
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ABSTRACT Effect of Plant Derived Saponin on the Structure and Stability of Lipid Membranes in
the Absence of Cholesterol Amanda Rose Decker
Steven Wrenn, Supervisor, Ph.D. QuilA is a form of saponin that is derived from the inner bark of the plant
Quillaja saponaria. Traditionally, this compound has been used as a detergent-‐like
cleaning agent and an eco-‐toxin for hunting. Now, this and many other forms of
saponin are used as emulsifiers and frothing agents for foods, as well as a growing
list of pharmacological applications such as vaccine adjutant and anti-‐cancer
supplements. The mechanism behind saponin’s interaction with lipid membranes is
still unknown.
Previous studies have shown that cholesterol is an important player in the
saponin/lipid interaction. This study explores the effect of QuilA on lipid
membranes in the absence of cholesterol. The fluorescent dye release and FRET
analysis experiments of this study have shown that minimal leakage and membrane
reorganization are possible without cholesterol.
In the absence of cholesterol, the data from this study suggest that the
saponin is not incorporated into the membrane. Instead, the saponin acts to isolate
individual lipids from the membrane and facilitate a “vesicle-‐to-‐micelle” transition.
This process results in membrane restructure but with limited release of the
solution encapsulated within the liposome.
The lipid composition of the membrane also appears to have a significant
affect on the saponin/lipid interaction. Lipid membranes with higher compositions
ix
of saturated lipids experience lower leakage and slower membrane reorganization.
Conversely, lipid membranes with higher compositions of unsaturated lipids
experience higher leakage and faster membrane reorganization.
1
CHAPTER 1: INTRODUCTION AND BACKGROUND
Saponins are biological compounds that are found in a wide variety of plants,
bacteria, and lower animals [1]. Structurally, saponins contain an aglycone and a
sugar head group. Saponins are utilized in a wide variety of applications, such as
emulsifiers, surfactants, and pesticides [1]. Traditional uses of soap root as cleaning
agents and fish poisons for hunting were made possible by the saponins present in
the plant.
More recently, pharmacological applications of saponin are being developed.
Once such application is as a vaccine adjutant [2]. Vaccine adjutants are compounds
that help stimulate an immune response by the body, so the vaccine is more
effective. Other uses include anti-‐cancer treatment [1] and cholesterol-‐lowering
supplements [1, 3]. Saponins are also widely used in the food industry, as
emulsifiers and cholesterol removing agents in foods and foaming agents in
beverages [4].
However, before saponins are applied for pharmaceutical uses, there needs
to be a better understanding behind the mechanism of saponin/lipid membrane
interactions. Because there are so many forms of saponin and so many factors that
can influence the structure and stability of a lipid membrane, the exact mechanism
or mechanisms, for saponin/lipid interaction is still unknown.
This study focused on a form of saponin Quillaja. Quillaja is derived from the
plant Quillaja saponaria, better known as soapbark tree, a plant commonly found in
South America and China [4]. Quillaja is a mixture of up to 100 slightly different
forms of saponin. However, these isomeric forms share two important structural
2
features, a fatty acid domain and a triterpene aldehyde [4]. In this study, one
particular isomer, QuilA, was isolated and used as the saponin compound.
Figure 1: Structure of QuilA Saponin. The section labeled A identifies the two-‐glucose moieties that signify α-‐hederin. Section B is the entire hydrophilic sugar head group. Section C is the hydrophobic aglycone group.
All saponin molecules contain a hydrophilic glycoside (sugar) head group
and a hydrophobic aglycone structure [5]. The QuilA form of saponin is called an α-‐
hederin [2, 6]. There are multiple classifications of hederin, mainly characterized by
the number and organization of the sugar moieties in the head group [2]. α-‐hederin
saponins contain a two-‐glucose group.
Previous studies [2, 6, 7] have looked into lipid membrane reorganization
using fluorescent and visual microscopy. This study examined the structural
3
changes of liposomes due to saponin interactions by spectrophotometry. Two
experiments were conducted as part of this study.
The first was a calcein leakage assay, which examined the rate and amount of
release of a fluorescent dye from the liposome due to saponin interactions. This
experiment utilized a dequenching assay, where the fluorescent dye was
encapsulated inside the liposome at concentrations high enough to block
fluorescence. Once the dye was leaked from the liposome, the dye fluoresced. A
total of nine liposome compositions were created, each of which corresponded to
one of three subsets depending on the cholesterol content; 0%, 25%, 50%. These
liposomes were then subjected to a range of saponin molar concentration, 0%, 5%,
15%, 40%, and 75%.
The second experiment was Förster resonance energy transfer (FRET)
analysis. This experiment aimed to examine the reorganization of the lipid
molecules as a result of saponin interactions. FRET is used to measure the
fluorescence intensity of interacting fluorescent probes. The two fluorescent probes
used in this study were dehydroergosterol (DHE) and dansylated lecithin (DAN) [8].
In FRET, the donor probe DHE, is excited to a higher energy state by light. This
energy can then released in one of two ways; DHE could directly release the energy
in the form of light with a maximum at 385 nm, or transfer the energy to an adjacent
acceptor probe, DAN. The acceptor probe is then subsequently excited and releases
that energy in the form of light with a maximum at 515 nm. In this way, two
fluorescent wavelengths are produced. The intensity of the fluorescence is directly
correlated to the proximity of the donor and acceptor probes. If the donor and
4
acceptor probes are very close, more energy will be transfer from DHE than
released from DHE as light. If the probes are separated, more energy goes into DHE
light emission and the DHE fluorescence intensity increases, while less energy goes
into DAN light emission and the DAN fluorescence intensity decreases. Therefore, a
simultaneous increase of DHE fluorescence and decrease of DAN fluorescence is
indicative of FRET alleviation, or that the probes are being separated.
The FRET analysis included only the liposome types that did not contain any
cholesterol. These three liposomes types were treated with the 4 levels of saponin
described in the calcein leakage experiment (5%, 15%, 40%, and 75%).
The data collected from this study were compared against Damara et al.’s
paper “Pseudo ternary phase diagrams of aqueous mixtures of QuilA, cholesterol,
and phospholipid prepared by the lipid-‐film hydration method’, which examined the
structures formed for a wide range of liposome compositions.
The research described herein suggests that lipid membranes without
cholesterol form small micelles slowly by isolating lipids from the membrane, rather
than forming pores or larger micelle structures that require a large structural
change. The effect of saponin is highly dependent on the presence of cholesterol and
the composition of the lipid membrane.
5
CHAPER 2: MATERIALS AND METHODS
2.1 Materials The liposome membrane formulations consisted of monounsaturated lipid
1,2-‐Dioleoyl phosphatidyl-‐choline (DOPC), saturated lipid dipalmitoylphosphatidyl-‐
choline (DPPC), and cholesterol. Dansylated lecithin (DAN) and dehydroergosterol
(DHE) were used as fluorescent probes for FRET analysis.
Calcein dye, phosphate buffered saline powder (PBS), Sephadex G50, ergosta-‐
5,7,9(11),22-‐tetraen-‐3β-‐ol (DHE) and cholesterol were purchased from Sigma-‐
Aldrich Chemical Company (Sigma-‐Aldrich, St. Louis, MO).
1, 2-‐Dioleoyl-‐sn-‐glycero-‐3-‐phosphocholine (DOPC), 1, 2-‐Dipalmitoyl-‐sn-‐
glycero-‐3-‐phosphocholine (DPPC), and 1-‐Myristoyl-‐2-‐[12-‐[(5-‐dimethylamino-‐1-‐
naphthalenesulfonyl)amino]dodecanoyl]-‐sn-‐glycero-‐3-‐phosphocholine (DAN) were
purchased from Avanti Polar Lipids, Inc. (Alabaster, AL).
Chloroform for liposome preparation was purchased from Fischer-‐Scientific
(Pittsburgh, PA).
Quillaja A saponin (QuilA) was supplied by Unilever. The partially purified
powder (27.8 wt% saponin) was rehydrated using 1x PBS to a final saponin
concentration of 1.61 e-‐5 mol/mL.
2.2 Preparation of Small Unilamellar Vesicles (SUV) SUVs were prepared in a three step process; dehydration, rehydration, and
pressure extrusion. Stock solutions of DOPC, DPPC, DHE, and DAN were prepared in
6
chloroform. Each SUV formulation was prepared by pipetting the lipid solutions
into a round bottom flask, then dried under a vacuum rotary evaporator for two
hours. Table 1 shows the SUV formulations for each type. Each batch of liposomes
contained a total of 1.0 e-‐5 moles, where the volumes of lipids were calculated
according to this final mole value and the lipid percentage in the membrane. The
films were rehydrated in buffers appropriate for the experiment (see sections 2.3 –
2.4), sonicated for five minutes, then mixed on a stir plate for 30 minutes at 60 °C.
The lipid solution was then passed through two stacked polycarbonate filters
(Nuclepore, Whatman Inc., Clifton, NJ) five times (200 nm) and seven times (100
nm) to yield SUVs with a mean liposome diameter of 120 nm. After extrusion, FRET
SUVs were stored in 4 °C until use. Calcein SUV solutions were put through size
exclusion chromatography (SEC) in a Sephadex G50-‐packed column to remove
unencapsulated dye. The column was eluted with a 1x PBS solution.
Table 1: Liposome Formulations for All Experiments. Mole percents are in terms of 1.0e-‐5 total moles.
Liposome(Type Test(Use Cholesterol((mol%) DOPC((mol%) DPPC((mol%) DHE((mol%) DAN((mol%) Rehydrated(with(A Calcein 50 50 0 0 0 70,mM,CalceinB Calcein 50 0 50 0 0 70,mM,CalceinC Calcein 0 50 50 0 0 70,mM,CalceinD Calcein 25 10 65 0 0 70,mM,CalceinE Calcein 26 65 10 0 0 70,mM,CalceinF Calcein 0 100 0 0 0 70,mM,CalceinG Calcein 0 0 100 0 0 70,mM,CalceinH Calcein 25 37.5 37.5 0 0 70,mM,CalceinI Calcein 50 25 25 0 0 70,mM,CalceinC FRET 0 47.5 47.5 5 0 1x,PBSF FRET 0 95 0 5 0 1x,PBSG FRET 0 0 95 5 0 1x,PBSC FRET 0 45 45 5 5 1x,PBSF FRET 0 90 0 5 5 1x,PBSG FRET 0 0 90 5 5 1x,PBSC FRET 0 42.5 42.5 5 10 1x,PBSF FRET 0 85 0 5 10 1x,PBSG FRET 0 0 85 5 10 1x,PBS
7
Figure 2 shows the placement of all nine liposome types on a ternary
diagram. This diagram maps out the concentration of unsaturated lipid (DOPC),
saturated lipid (DPPC), and cholesterol.
Figure 2: Ternary diagram for liposome compositions. The bottom edge corresponds to the percentage of DPPC (saturated lipid). The left side corresponds to the percentage of DOPC (unsaturated lipid). The right side corresponds to the percentage of cholesterol. The shaded region in the middle corresponds to a two-‐phase system where the membrane components are not miscible in one another and segregate into distinct domains.
Figure 3 below shows where the liposome compositions from this
experiment fall on the ternary diagram created from Demana et al.’s data.
8
Figure 3: Liposome compositions overlaid on Demana et al.'s phase diagram [7]. The left edge corresponds to the saponin level. The right edge corresponds to the cholesterol level. The bottom edge corresponds to the lipid level. As the lipid composition in this phase diagram is not specified, the arrows denote the path along the saponin levels from 0% to 75%.
2.3 Calcein Leakage Assay Leakage from liposomes was measured by the dequenching of self-‐quenched
calcein encapsulated in the SUV. When calcein is in concentration of 70 mM, the
fluorescent probe is self-‐quenched [9]. Once released, the concentration decreases
sufficiently for the calcein to fluoresce. The fluorescence of the encapsulated, self-‐
quenched calcein was recorded and subtracted from the leaked fluorescence
intensity to account for fluorescence as a result of unleaked calcein. The calcein was
excited at a wavelength of 488 nm and the intensity of the emitted light at
wavelength 527 nm was recorded.
A, I, B E, H, D F, C, G
9
Leakage was calculated from fluorescence data with respect to the Triton
release and unleaked, pure liposome sample for each liposome type:
% Leakage =
!!"#$%&!!!"#$ !"#$%$&'!!"#$%&!!!"#$ !"#$%$&' !
!!"#$%&!!!"#$ !"#$%$&'!!"#$%&!!!"#$ !"#$%$&' !"# !"# !"" !"#$%$&' !"#$%
(1)
where i denotes the individual liposome type and I represents fluorescence
intensity. The subscript ‘sample’ differentiates among the different saponin
concentrations for each liposome type. The numerator in equation 1 was calculated
for each data point (liposome type, saponin level, time). Liposome type B resulted
in the largest value for all saponin levels. Therefore, the maximum fluorescence of
liposome B for each saponin level (5%, 15%, 40%, 75%) was set as the maximum
for all liposome types, and was used as the denominator in equation 1.
2.4 Förster Resonance Energy Transfer (FRET) Assay FRET SUVs of liposome types C, F, and G were created according to section
2.2. Each composition was made three times with constant levels of DHE (5%) and
varying levels of DAN (0%, 5%, 10%). All liposomes were rehydrated using 1x PBS.
All liposomes were tested within 3 days of preparation. Each liposomes type was
subjected to 0%, 5%, 15%, 40%, or 75% mol saponin.
Fluorescence was measured by an excitation wavelength of 300 nm and
recording emission wavelength intensity from 330 nm – 550 nm. This range of
wavelength encompasses both the DHE and DAN fluorescence. The maximum
10
emission wavelength for DHE and DAN were taken to be 385 nm and 515 nm
respectively. Fluorescence data were recorded over the course of 2 hours.
The FRET data had to be corrected to account for the saponin fluorescence at
these wavelengths and the scattering effect of the spherical liposomes using
equation 2.
I!"##$!%$& = I!"#×10! − I!"#$%&% (2)
where turbidity data (τ) for the pure liposomes were recorded according to
section 2.7. The corrected data were then plotted corrected intensity versus
emission wavelength (Figures 10 -‐ 15).
The peak ratios of DHE and DAN maximum emission intensities were also
used with equation 3 to further describe the effect of saponin levels on FRET for
each liposome type and DAN level.
Peak Ratio =!!"##$!%$& !"#
!!"##$!%$& !"# !"!#!$%!!"##$!%$& !"#
!!"##$!%$& !"# !"!#!$%
(3)
where Icorrected DHE and Icorrected DAN correspond to the maximum emission
wavelength intensity of 385 nm and 515 nm respectively for each saponin level.
Likewise, Icorrected DHE initial and Icorrected DAN initial correspond to the emission wavelength
intensities for the initial fluorescence level, or 0% saponin. These peak ratios were
then plotted versus the saponin levels (Figure 16).
11
2.5 Fluorescence Spectroscopy Fluorescence measurement were taken with a TECAN Infinite ® 200 PRO.
Separate programs files were created, specifically for each test (sections 2.3 – 2.4).
All of the programs contained some common steps. The instrument mixed each
sample for 1 second before each data measurement. The temperature of the
instrument was maintained between 19 – 23 °C. Measurements were taken from
the top of the plate well. Each sample set used a clear plastic, 96-‐well plate.
2.6 Dynamic Light Scattering (DLS) Liposome sizes were determined in a separate experiment before and after
saponin addition (separately from the fluorescence measurements), using a
Brookhaven 90Plus dynamic light scattering apparatus. Effective diameter of the
particles were calculated using the Stokes-‐Einstein equation:
𝐷!"" = !!!!!"#
(4)
where kB is the Boltzmann constant, T is the temperature of the system (25°C), ν is
the solvent viscosity (taken to be water), and D is the diffusivity calculated from the
cumulate fit of the data.
12
2.7 Turbidity Measurement The turbidity of the samples, as a result of particulate formation, was
measured using Perkin-‐Elmer Instruments Lambda 40 UV/Vis Spectrometer.
For each calcein liposome formulation (A-‐I), absorbance of light at 527 nm
was measured ever five minutes over the course of two hours after the addition of
saponin. This wavelength of light was selected as it was the emission wavelength of
calcein. The absorbance or scattering of 527 nm light may have explained lower
fluorescence intensity than expected, especially with the higher saponin levels.
For each FRET liposome formulation (C, F, G), absorbances for a range of
wavelengths (330 nm – 550 nm) were recorded in 5 nm increments. These
measurements were taken for pure liposomes (no saponin) in order to correct for
the scattering of light due to the spherical liposomes.
13
CHAPER 3: RESULTS
3.1 Calcein Leakage Assay
All fluorescence data was first corrected using equation 1. The corrected
data were then plotted versus time and separated by saponin mole percentage. Due
to precipitation turbidity effects, 40% and 75% saponin results were not considered
representative of the actual calcein leakage.
Figure 4: % Leakage vs. Time Data for 5 mol% Saponin Samples. Each data set corresponds to a liposome type according to table 1 with 5% saponin. All samples were run in triplicate and then averaged together with an overall error of 0.5% (error bars not shown). Raw fluorescence data were corrected using equation 1. Error bars (not shown) are on the order of 0.5% of the
14
Figure 5: % Leakage vs. Time Data for 15 mol% Saponin Samples. Each data set corresponds to a liposome type according to table 1 with 15 mol% saponin. All samples were run in triplicate and then averaged together with an overall error of 0.5% (error bars not shown). Raw fluorescence data were corrected using equation 1.
Figures 4 and 5 indicate that these trends are comparable for both saponin
levels. The end points of liposome leakage group together correlating to cholesterol
content. Liposomes with 50% cholesterol (types A, B, and I) show higher final
percent leakages than liposomes with 25% cholesterol (types D, E, and H), while
liposomes with 0% cholesterol (types C, F, and G) show the lowest final percent
leakages.
Leakage levels were then plotted against saponin levels according to
cholesterol content in order to more clearly observe the differences between
cholesterol levels.
15
Figure 6: Final % Leakage vs. Saponin Level for 0% Cholesterol Liposomes. The final percent leakage values for each liposome type without cholesterol (liposome C, F, and G) and saponin level were plotted.
Figure 7: Final % Leakage vs. Saponin Level for 25% Cholesterol Liposomes. The final percent leakage values for each liposome type with 25% cholesterol (liposome D, E, and H) and saponin level were plotted.
16
Figure 8: Final % Leakage vs. Saponin Level for 50% Cholesterol Liposomes. The final percent leakage values for each liposome type with 50% cholesterol (liposome A, B, and I) and saponin level were plotted.
Figures 6 -‐ 8 indicate that leakage increases dramatically as the cholesterol
content in the lipid membrane increases. Figures 6 -‐ 8 also show that saponin levels
appear to reach a maximum effect at 5% saponin and the effects are approximately
equivalent between 15-‐75%.
Table 2 summarizes the effect of saponin levels and lipid membrane
composition on particle size. Due to the excessive particle sizes after the addition of
saponin, the particles that were being detected were not assumed to be the
liposomes, but instead precipitation. This precipitation could have been as a result
of the saponin/liposome interactions or the impurities in the saponin solution. The
distinction between these two possibilities cannot be made at this time without a
pure saponin compound.
17
Table 2: Summary of Particle Size vs. Saponin Level. Each liposome type (refer to Table 1) was subjected to 5, 15, 40, or 75 mol% saponin. Particle size of each mixture was determined using DLS (section 2.6). Missing entries for liposome A were due a lack of the liposome sample. Empty entries for liposomes F and G were due to the particle size exceeding the measurement capacity of the instrument.
Table 3 summarizes the effect of liposome membrane composition and
saponin levels on precipitate formation. The sample turbidities were taken by
measuring the absorptivity of light at 527 nm. The difference in the incident and
transmitted light of each sample represents the light either scattered or absorbed by
the solution. As neither saponin nor the liposomes absorb or emit light at 527 nm,
the different is attributed to scattering by particles. These data correspond to the
visual observations made, in which precipitation increased with decreasing
cholesterol amounts. A solution of only liposomes would present similar
absorptivity levels. The differences in these data are can be attributed to the
differences in precipitate formation. Liposome types C, F, and G resulted in the
highest amount of precipitation, while liposome types A, B, and I resulted in the
lowest amount.
Sample Liposome+Type/Saponin+Levels 0 0.05 0.15 0.4 0.75A 50:50+Chol:DOPC 264.6 454.0 474.7 @@@ @@@B 50:50+Chol:DPPC 163.6 1173.4 940.7 739.9 464.6C 50:50+DOPC:DPPC 262.9 2146.3 4000.0 3500.0 3500.0D 25:10:65+Chol:DOPC:DPPC 148.6 171.2 232.2 188.3 110.5E 25:65:10+Chol:DOPC:DPPC 143.0 258.2 289.7 292.8 335.7F 100+DOPC 144.7 584.3 5148.1 5558.9 @@@G 100+DPPC 144.6 3177.4 @@@ @@@ @@@H 25:37.5:37.5+Chol:DOPC:DPPC 144.8 231.9 229.5 217.9 203.5I 50:25:25+Chol:DOPC:DPPC 139.6 258.4 268.0 800.4 321.1
Particle+Size+(nm)
18
Table 3: Summary of Turbidity vs. Saponin Level (Stand in for Precipitate Formation). Each liposome type (refer to Table 1) was subjected to 5, 15, 40, or 75 mol% saponin. Particle size of each mixture was determined via section 2.7. Missing entries for liposomes A and C were due a lack of the liposome sample.
Figure 9 below shows the liposome/saponin solutions for liposome B, D, and
G, respectively, after 24 hours in 4 °C. Precipitate levels were directly related to the
cholesterol content in the lipid membrane. Liposomes with 50% cholesterol did not
produce any visible precipitate, while liposomes with 0% cholesterol developed
dark precipitate immediately after saponin addition. Precipitate formation also
increased with saponin levels.
Figure 9: Qualitative Visual Data for Precipitate Formation after 24 hours in 4 °C. Each set of curvettes are arranged in order of increasing saponin level, 0%, 5%, 15%, 40%, and 75%. The first set of cuvettes is liposome type B (50% cholesterol). The second set is liposome type D (25% cholesterol). The third set is liposome type G (0% cholesterol). Note the lack of visible precipitate in liposome B sample, the thin layer of white precipitate in liposome D samples, and the thicker layer of dark precipitate in liposome G samples.
Sample Liposome+Type/Saponin+Levels 0 0.05 0.15 0.4 0.75A 50:50+Chol:DOPC >>> >>> >>> >>> >>>B 50:50+Chol:DPPC 1.207 1.440 1.521 1.506 1.551C 50:50+DOPC:DPPC >>> >>> >>> >>> >>>D 25:10:65+Chol:DOPC:DPPC 1.602 1.786 1.836 1.867 1.930E 25:65:10+Chol:DOPC:DPPC 0.962 1.048 1.054 1.146 1.237F 100+DOPC 2.227 2.654 2.820 2.944 3.020G 100+DPPC 1.680 1.869 1.828 1.871 1.917H 25:37.5:37.5+Chol:DOPC:DPPC 1.493 1.683 1.703 1.769 1.796I 50:25:25+Chol:DOPC:DPPC 1.149 1.319 1.358 1.398 1.464
Absorbtivity
19
These data generally agree with the data from Demana et al., with the
exception of liposomes with no cholesterol (liposomes C, F, and G). Demana et al.
concluded that membranes with no cholesterol form lipic layered structures, which
suggests that the liposomes unfold completely. This mechanism would release all
encapsulated calcein in liposome types C, F, and G, which does not agree with the
above calcein leakage data. The remainder of the study was then focused on further
examining the effects of lipid membrane composition in the absence of cholesterol
(liposome types C, F, and G).
20
3.2 FRET Assay
All fluorescent data were corrected using equations 2 and 3. The corrected
data were then plotted versus emission wavelength and separated by liposome type
and DAN concentration.
Figure 10: FRET Curves for Liposome C with 5% DAN. Liposome C with 5% dansyl (refer to Table 1) was excited with a wavelength of 300 nm. Each curve corresponds to a different saponin level. Raw fluorescence data was corrected using equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
21
Figure 11: FRET Curves for Liposome C with 10% DAN. Liposome C with 10% dansyl (refer to Table 1) was excited with a wavelength of 300 nm. Each curve corresponds to a different saponin level. Raw fluorescence data was corrected using equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
22
Figure 12: FRET Curves for Liposome F with 5% DAN. Liposome F with 5% dansyl (refer to Table 1) was excited with a wavelength of 300 nm. Each curve corresponds to a different saponin level. Raw fluorescence data was corrected using equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
23
Figure 13: FRET Curves for Liposome F with 10% DAN. Liposome F with 10% dansyl (refer to Table 1) was excited with a wavelength of 300 nm. Each curve corresponds to a different saponin level. Fluorescence data was corrected using equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
24
Figure 14: FRET Curves for Liposome G with 5% DAN. Liposome G with 5% dansyl (refer to Table 1) was excited with a wavelength of 300 nm. Each curve corresponds to a different saponin level. Fluorescence data was corrected using equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
25
Figure 15: FRET Curves for Liposome G with 10% DAN. Liposome G with 10% dansyl (refer to Table 1) was excited with a wavelength of 300 nm. Each curve corresponds to a different saponin level. Fluorescence data was corrected using equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
Figures 10 -‐ 15 show the fluorescence intensity of DAN (peak at 515 nm)
decreases while fluorescence intensity of DHE (peak at 385 nm) increases as more
saponin is added. This is indicative of FRET alleviation. To take a closer look at the
effects of saponin on fluorescence, the peak ratios between DHE and DAN were
calculated and plotted versus saponin levels. It should be noted that the 75%
saponin data points for liposome F (both DAN concentrations) were removed from
the data set, as they were determined to be outliers. A least-‐squares analysis was
completed in Excel to determine these outliers.
26
Figure 16: DHE/DAN Max Emission Peak Ratios vs. Saponin Level for All Liposome Types and DAN Levels. FRET fluorescence data was corrected using equations 2 and 3. Peak ratios for each liposome type (refer to Table 1) were plotted against saponin level to track the effect of saponin level on membrane reorganization. A larger peak ratio signifies greater structural changes.
Table 4: Summary of Linear Fit for Figure 16. Each data set from Figure 16 was fit with a linear trend line. The slopes are summarized below. Slopes were related to the rate of lipid membrane reorganization. The larger the slope, the more the membrane was restructured.
Liposome(Type %(DAN SlopeC 5 0.1736F 5 0.1361G 5 0.1352C 10 0.2796F 10 0.3003G 10 0.2076
27
These data show that the amount of saponin and the type of liposome affect
the FRET. The addition of more saponin results in more FRET alleviation. Liposome
F, 100% unsaturated lipids, saw the greatest rate of FRET alleviation, while
liposome G, 100% saturated lipids, saw the slowest rate of FRET alleviation. The
rates of FRET alleviation were taken to be the slope of the plots in Figure 16,
summarized in Table 4, and were assumed to be representative of the extent of
structural reorganization.
Comparing Figures 6 -‐ 8 with Figure 16 shows that increasing the amount of
saponin did not generally increase the calcein fluorescence in the absence of
cholesterol, however, additional saponin continued to reorganize the lipid
membrane structure.
28
CHAPTER 4: DISCUSSION Demana et al.’s work on saponin focused on the effect on liposome structure.
They conclude that liposomes without cholesterol, a pseudo-‐binary system of
saponin and lipids results in lipidic particles and layered structures. After the
addition of saponin, they describe the resulting structures to contain mostly stacked
layers of bilayer membranes, not organized into a spherical liposome form, “QuilA
appears to hinder vesicle formation despite forming bilayers structures with PC,
possibly by altering lipid packing” [7]. The formation of layered structures from
liposomes leads to the conclusion that these layered structures are formed by the
opening of the liposome to form a flat sheet of lipid bilayer. Additional saponin
resulted in the further reorganization of the layered lipid structures into micelles.
This mechanism for saponin/lipid membrane interaction differs greatly from the
liposome types that include cholesterol, which were shown to form ISCOM matrices
and various forms of micelles [7].
Demana et al.’s study focused on long-‐term (1 day to 2 months) effects of
saponin on liposome structure. This study evaluated more immediate effects (up to
2 hours) and used fluorescence instead of microscopy to observe structural changes.
It should also be noted that Demana et al. study used egg yolk, which contains an
unquantified mixture of lipids for the membrane. This study was designed to assess
the effect of saponin as a function of lipid membrane composition, paying
particularly close attention to the types of lipids that make up the membrane.
Figures 4 and 5, as well as Figures 27 and 28 in the Appendix A, show the
calcein fluorescence in terms of leakage for each saponin level over 2 hours. The
29
leakage trends roughly group into each liposome’s composition subset, in that the
liposomes that contain the most cholesterol (types A, B, and I) show the highest
leakage, the liposomes with the least cholesterol (types C, F, and G) show the lowest
leakage, and liposomes with an intermediate amount of cholesterol (types D, E, H)
show an intermediate leakage.
Liposome G demonstrates a negative percent leakage value. Figure 23 in
Appendix A shows that the addition of saponin to liposome G did result in a small
level of fluorescence. However, applying equation 1 to the raw calcein fluorescence
data resulted in a negative value. As demonstrated in Table 3, the 0% cholesterol
liposomes produced significant amounts of precipitate, which scatter the emitted
light, artificially lowering the fluorescence intensity data. It is important to note that
unlike the FRET data, the calcein release data were not corrected for this
precipitation-‐induced scattering. Correction for light scattering in the FRET data
resulted in an overall increase of fluorescence values, but not a change in the trends.
Therefore, it can be assumed that if the calcein release data were corrected for the
precipitation induced light scattering, then the overall trends would not change, but
would be shifted up to higher percent leakage values.
The results for 50% cholesterol and 25% cholesterol liposomes are
consistent with Demana et al.’s data.
According to Demana et al., liposomes containing 50% cholesterol form
mostly ring-‐like micelles, which requires almost complete opening and reformation
of the liposome structure, consistent with a high amount of leakage. Liposomes
containing 25% cholesterol form mostly ISCOM matrices, in which the liposome still
30
retains some form of structure that can trap some level of calcein, which accounts
for the intermediate level of leakage seen in this study. However, the calcein
leakage and Demana’s data for 0% cholesterol liposomes are not consistent. The
formation of lipidic/layered structures requires that the liposome is completely
opened to for the flat bilayer necessary for layering. This mechanism should result
in a high level of calcein leakage, as it does in the formation of ring-‐like micelles.
Instead, liposomes containing 0% cholesterol result in the smallest amount
of calcein leakage. In addition to this inconsistency, the leakage trends for 0%
cholesterol liposome types C, F, and G were very different from each other (Figures
4 -‐ 5). Liposomes C and F showed a small immediate jump in leakage, but while F
stayed constant after that initial change, C leakage slowly increased before reaching
a maximum level. Liposome G showed no initial leakage jump and remained at
essentially 0% leakage for the length of the 2-‐hour experiment.
Another noticeable difference among the liposome types was the amount of
precipitation that formed after the addition of saponin. The 50% cholesterol subset
had little to no visible precipitation, even after several days. The 25% cholesterol
subset had a minimal amount of precipitation, which appeared only after storage at
4 °C overnight. The 0% cholesterol subset formed a significant amount of
precipitation immediately upon saponin formation.
The discrepancy between 0% cholesterol liposomes and the other types, as
well as the discrepancy among the 0% liposome types lead to the narrowing of the
scope of this study to liposome types C, F, and G for the FRET analysis.
31
The FRET data from liposome types C, F, and G indicated a clear alleviation of
energy transfer (Figures 10 -‐ 15). Further manipulation of the peak intensities
revealed that Liposome F experienced the greatest rate of alleviation, followed by C,
then G. Figure 16 and table 4 summarize the results. The rate of FRET alleviation
was taken to be the slope of the peak ratios versus saponin level. These data mirror
the calcein leakage data, in that the liposome type with the least saturated lipids saw
the greatest structural change (largest final peak ratio) and underwent that change
faster (larger slope).
There is a distinct discrepancy between the calcein leakage and FRET
analysis data for liposome G. FRET suggests that liposome G undergoes a not
insignificant structural change, yet calcein leakage shows that this structural change
must result in minimal leakage. Therefore, the explanation for the structural change
is not likely to be pore formation.
One possible mechanism for saponin lipid membrane reorganization is a
budding of the outer membrane [2]. This budding phenomenon may explain the
structural change without calcein leakage. However, according to Lorent et al., this
budding phenomenon is only seen with δ-‐hederin saponin molecules, or
triterpenoid molecules with a one-‐sugar head group [2] and QuilA saponin is
designated as an α-‐hederin [2, 6]. Previous studies on α-‐hederin saponin show that
these forms of saponin mostly interact with lipid membranes by associating with
cholesterol to form highly curved regions of the membrane. These regions first form
pores, and then continue to curve to form rolled lip structures while continually
increasing the pore diameter [2]. This kind of structural change is not likely in the
32
case of liposome type G, as continually growing pores would release encapsulated
calcein efficiently. It is worth noting that the Lorent study used GUVs (giant
unilaminar vesicles) that contained cholesterol, which differed greatly from the
SUVs (small unilaminar vesicles) without cholesterol in this study. In the absence of
cholesterol, an α-‐hederin saponin such as QuilA could promote a different
mechanism for lipid membrane rearrangement.
FRET can also be used to indicate a vesicle-‐to-‐micelle transition [8]. Vesicle-‐
to-‐micelle transition is the phenomenon when a single, large vesicle (in this case, the
liposome) undergoes a molecular reorganization to form many, smaller micelles,
which contain approximately the same composition of lipids as the original
liposome. However, these micelles have a much larger surface area-‐to-‐volume
ratios and curvatures compared to the liposome, thus increasing the distance
between probes. The micelles can also diffuse through the solution away from the
parent vesicle. This increases the distance between micelles and therefore, the
probes.
Previous studies on saponin’s effect on lipid membranes all agree that
cholesterol is vital to facilitate the interaction between saponin and the lipid
membrane. Cholesterol appears to be necessary for the saponin to physically get
into the membrane. The resulting structural change is due to the structure of the
saponin molecule. The two-‐sugar head group of the saponin with the linear
aglycone creates wedge-‐like molecules, which induces curvature of the membrane.
This curvature can then result in multiple forms, such as pores [2], ISCOM matrices,
ring-‐like, and worm-‐like micelles [7]. In the absence of cholesterol, the saponin is
33
prevented from inserting itself directly into the membrane, but can still alter the
structure of the lipid membrane.
The extent of the structural change appears to be highly dependent on the
lipid membrane composition. The calcein release data indicates that leakage
happens fastest for liposomes, without cholesterol, with pure unsaturated lipids, or
liposome F. Leakage for liposomes with pure saturated lipids, liposome G, showed
no leakage at all. Liposome C, which contained 50% saturated and 50% unsaturated
lipids, fell between the two limiting cases. These results correlate to the FRET data.
Pure unsaturated liposomes saw the greatest structural change, pure saturated
liposomes saw the least, but not negligible, change, and mixed-‐lipid liposomes fell in
between.
The relatively low calcein leakage results for the 0% cholesterol liposomes
but still significant structural changes suggest that the saponin/lipid mechanism in
the absence of cholesterol is likely a vesicle-‐to-‐micelle transition.
Liposome F, pure unsaturated lipids, probably undergoes a quick, immediate
micellization. This accounts for the initial jump in fluorescence followed by a
constant fluorescence. An immediate micellization, which requires multiple
phospholipids being pulled out at once, could happen too fast for the membrane to
“heal” itself fast enough to completely prevent encapsulated calcein leakage. The
liposomes were not completely transformed into micelles, because the maximum
calcein leakage was far below that of the control liposomes that were completely
micellized by a detergent, Triton (Figure 23 in Appendix A). Therefore, the saponin
must have reached a limiting constrain on how many micelles could be formed.
34
Liposome G, pure saturated lipids, probably experiences a very slow
micellization process. The saturated lipids appear to greatly impede vesicle-‐to-‐
micelle transformation. The phospholipids are removed slow enough that the
phospholipid diffusion across the membrane to repair fill in the void is faster than
the calcein diffusion through those sparse, small openings. This results in
membrane structure reorganization but little to no calcein leakage.
Liposome C, a mixture of unsaturated and saturated lipids, appears to
undergo a steady rate of micellization. Unlike liposome F, which micellizes to its
fullest extent quickly, the fraction of saturated lipids appears to slow down the
micellization process. Instead of an immediate micellization of the liposome, the
lipids are constantly pulled from the membrane, resulting in fluorescent dye flux
that slows down as the micellization limit is reached, analogous to the maximum
micellization achieved by liposome F.
A vesicle-‐to-‐micelle transition will result in liposomes of smaller sizes than
the starting liposome. Specifically for the calcein release assay, the diffusion of
calcein from inside the liposome to the outside is normally dependent on the size of
the vesicle. Decreasing the volume of a vesicle increases the overall surface area-‐to-‐
volume ratio. A smaller volume for a spherical vesicle decreases the distance that a
particle would need to diffuse before being released from the liposome. However, in
the calcein release assay, the concentration of calcein inside the liposome is
sufficiently large compared to the concentration outside in the bulk fluid that this
vesicle-‐to-‐micelle transition can be assumed to be reaction rate limited. The calcein
encapsulated in the liposomes is assumed to immediately diffuse at a steady rate
35
into the bulk fluid upon the formation of an opening in the membrane. It should also
be noted that the calcein does not diffuse through the entirety of the surface of the
liposome. In order to be transported from the inside of the liposome to the bulk
solution, the calcein must pass through an opening. Therefore, the overall increase
in surface area as volume decreases affects only the rate at which the saponin
interacts with the membrane. Because the interaction of saponin with lipid
membranes in the absence of cholesterol is so slow, it can be assumed that the slight
increase in surface area does not have a significant effect on the rate of opening
formation or calcein release.
The difference between the saturated and unsaturated lipids was expected.
The unsaturated lipids used in this study (DOPC) have a melting point of -‐17 °C [10],
while the saturated lipids (DPPC) have a melting point of 40 °C [10]. As these
experiments were conducted between 19 – 23 °C, the saturated lipids were in a
semi-‐solid gel phase while the unsaturated lipids were much more fluid. The
difference between these “oil” and “butter” lipid phases can support that conclusion
that the saponin has an easier time interacting with the free unsaturated lipids than
with the rigid saturated lipids.
In the biological lipid membrane, cholesterol is necessary for creating a
highly fluid environment, in order to facilitate imbedded protein transport around
the membrane. In the absence of cholesterol, the pure unsaturated lipids provide
the environment that most closely resembled a biological lipid membrane, so the
saponin is likely to have a greater structural effect.
36
CHAPTER 5: FUTURE WORK
Further analysis is necessary to determine the cause of precipitation in the
samples. As the QuilA provided contained only 27.8% saponin, the solution used
consisted of a majority of unknown impurities. There was a strong correlation
between precipitation and cholesterol content in the lipid membrane. This
relationship would be interesting to further examine. However, it must first be
determined that the precipitation is caused by the interaction of saponin and lipid
membrane, and not due to some kind of impurity from the saponin.
FRET analysis on the other liposome types (A, B, D, E, H, and I) may provide
some more information on pore formation versus micellization. Liposome types
similar to D, E, H, and I are known to form pores [2], so obtaining a FRET response
from those liposome types may help distinguish between pore formation and other
structural changes.
A second leakage experiment using encapsulated FITC-‐dextran could be used
to determine the pore/opening size distribution. The leakage can be directly
correlated to pore size by comparing the fluorescence intensities of various sizes of
FITC-‐dextran molecules. This experiment could be accomplished by either a
dequenching assay like the calcein leakage experiment (encapsulated FITC is self-‐
quenched and fluoresces upon release) or a quenching assay (encapsulated FITC
fluoresces and is quenched upon release) by controlling the pH of the bulk solution
in addition to a quenching agent trypan blue [11].
37
CHAPTER 6: LIST OF REFERENCES
[1] Ö. Güçlü-‐Üstündağ, “Saponins: Properties, Applications and Processing,” Cric. Rev. in Food Sci. and Nutr., vol. 47, no. 3, pp. 231-‐258, 2007 [2] J. Lorent, “Induction of Highly Curved Structures in Relation to Membrane Permeabilization and Budding by the Triterpenoid Saponins, α-‐ and δ-‐Hederin,” J. Biol. Chem., vol. 288, pp. 14000-‐14017, Mar. 25 2013 [3] S. Mitra, “Micellar Properties of Quillaja Saponin. 1. Effects of Temperature, Salt, and pH on Solution Properties,” J. Agric. Food Chem., vol. 45, pp. 1587-‐1595, 1997 [4] S. Resnik, “Quillaia Extracts,” Chem. And Tech. Ass., vol. 61, pp. 1-‐9, 2004 [5] T. Blijdenstein, “On the link between foam coarsening and surface rheology: why hydrophobins are so different,” Soft Matter, vol. 6, pp. 1799-‐1808, 2010 [6] J. Lorent, “Domain Formation and Permeabilization Induced by the Saponin α-‐Hederin and Its Aglycone Hederagenin in a Cholesterol-‐Containing Bilayer,” Langmuir, vol. 30, no. 16, pp. 4556-‐4569, 2014 [7] P. Demana, “Pseudo-‐ternary phase diagrams of aqueous mixtures of Quil A, cholesterol and phospholipid prepared by the lipid-‐film hydration method,” Int. J. of Pharm., vol. 270, pp. 229-‐239, 2004 [8] S. Wrenn, “Characterization of model bile using fluorescence energy transfer from dehydroergosterol to dansylated lecithin,” J. of Lipid Res, vol. 42, pp. 923-‐934, 2001 [9] Q. Yan, “Reconstitution of Transporters” in Membrane Transporters: Methods and Protocols, vol. 227, Totowa, NJ, Humana Press Inc., 2003, pp. 146 [10] Avanti Polar Lipids Inc., “Phase Transition Temperatures for Glycerophospholipids,” 2014, Available FTP: http://avantilipids.com/index.php?option=com_content&id=1700&Itemid=419 [11] S. Sahlin, “Differentiation between attached and ingested immune complexes by a fluorescence quenching cytofluorometric assay,” J. of Immuno. Meth., vol. 60, no. 1-‐2, pp. 115-‐124, May 1983 [12] TdB Consultancy, “FITC-‐Dextran” Dec 2010, Available FTP: www.tdbcons.se/tdbcons2/attachment/fitcdextran2.pdf
38
APPENDIX A: ADDITIONAL CALCEIN DATA
Table 5: Plate Layout for All Experiments. Template for 96-‐well plate for a single test. Each test was run in triplicate, in which 200 μL of liposome was added to different amounts of detergent (Triton) or saponin. Three tests were conducted at once using 1 plate.
Figure 17: Calcein Fluorescence Intensity vs. Time Data for Liposome A. Calcein fluorescence intensity (raw data) for liposome type A over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
Row/Column 1 2 3 4 5 6 7 8 9 10 11 12A 200#μl#Liposome 200#μl#Liposome 200#μl#LiposomeB 200#μl#Liposome#+#200#μL#1%#Triton 200#μl#Liposome#+#200#μL#1%#Triton 200#μl#Liposome#+#200#μL#1%#TritonC 200#μl#Liposome#+#0.387#μL#Saponin#(5%) 200#μl#Liposome#+#0.387#μL#Saponin#(5%) 200#μl#Liposome#+#0.387#μL#Saponin#(5%)D 200#μl#Liposome#+#1.10#μL#Saponin#(15%) 200#μl#Liposome#+#1.10#μL#Saponin#(15%) 200#μl#Liposome#+#1.10#μL#Saponin#(15%)E 200#μl#Liposome#+#4.14#μL#Saponin#(40%) 200#μl#Liposome#+#4.14#μL#Saponin#(40%) 200#μl#Liposome#+#4.14#μL#Saponin#(40%)F 200#μl#Liposome#+#18.6#μL#Saponin#(75%) 200#μl#Liposome#+#18.6#μL#Saponin#(75%) 200#μl#Liposome#+#18.6#μL#Saponin#(75%)GH
39
Figure 18: Calcein Fluorescence Intensity vs. Time Data for Liposome B. Calcein fluorescence intensity (raw data) for liposome type B over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
40
Figure 19: Calcein Fluorescence Intensity vs. Time Data for Liposome C. Calcein fluorescence intensity (raw data) for liposome type C over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
41
Figure 20: Calcein Fluorescence Intensity vs. Time Data for Liposome D. Calcein fluorescence intensity (raw data) for liposome type D over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
42
Figure 21: Calcein Fluorescence Intensity vs. Time Data for Liposome E. Calcein fluorescence intensity (raw data) for liposome type E over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
43
Figure 22: Calcein Fluorescence Intensity vs. Time Data for Liposome F. Calcein fluorescence intensity (raw data) for liposome type F over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
44
Figure 23: Calcein Fluorescence Intensity vs. Time Data for Liposome G. Calcein fluorescence intensity (raw data) for liposome type G over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
45
Figure 24: Calcein Fluorescence Intensity vs. Time Data for Liposome H. Calcein fluorescence intensity (raw data) for liposome type H over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
46
Figure 25: Calcein Fluorescence Intensity vs. Time Data for Liposome I. Calcein fluorescence intensity (raw data) for liposome type I over 2 hours. Calcein dye was excited at 488 nm and fluorescence of 527 nm was measured. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
47
Figure 26: QuilA Saponin Fluorescence for Calcein Leakage Study. An analogue to Figures 17-‐26, but with 1x PBS instead of liposome. The fluorescence of the saponin (at the respective mole percentages) was recorded over 2 hours. The solutions were excited at 488 nm and fluorescence was measured at 527 nm. The fluorescence intensities are small enough to be considered negligible to the calcein fluorescence data. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size).
48
Figure 27: % Leakage vs. Time Data for 0 mol% Saponin Samples. Each data set corresponds to a liposome type according to table 1 with 0 mol% saponin. All samples were run in triplicate and then averaged together with an overall error of 0.5% (error bars not shown). Raw fluorescence data were corrected using equation 1.
0.00#
0.10#
0.20#
0.30#
0.40#
0.50#
0.60#
0.70#
0.80#
0.90#
1.00#
0.00# 20.00# 40.00# 60.00# 80.00# 100.00# 120.00# 140.00#
%"Leakage""
Time"(min)"
0.00%"Saponin"
Liposome#A#
Liposome#B#
Liposome#C#
Liposome#D#
Liposome#E#
Liposome#F#
Liposome#G#
Liposome#H#
Liposome#I#
49
Figure 28: % Leakage vs. Time Data for 40 mol% Saponin Samples. Each data set corresponds to a liposome type according to table 1 with 40 mol% saponin. All samples were run in triplicate and then averaged together with an overall error of 0.5% (error bars not shown). Raw fluorescence data were corrected using equation 1. Due to the inconsistencies of liposome B data (which served as the standard) this data was determine unusable for further analysis. Inconsistencies are likely due to light scattering effects due to precipitation formation.
50
Figure 29: % Leakage vs. Time Data for 75 mol% Saponin Samples. Each data set corresponds to a liposome type according to table 1 with 75 mol% saponin. All samples were run in triplicate and then averaged together with an overall error of 0.5% (error bars not shown). Due to the inconsistencies of liposome B data (which served as the standard) this data was determine unusable for further analysis. Inconsistencies are likely due to light scattering effects due to precipitation formation.
51
APPENDIX B: ADDITIONAL FRET DATA Table 6: Turbidity Data for Pure Liposome Solution with No Saponin. The absorbance of pure liposome samples (types C, F, and G from Table 1) were recorded for wavelengths from 350-‐550 nm. These values were then used in equation 2 to correct for light scattering due to the liposomes.
Wavelength Liposome0C Liposome0F Liposome0G350 0.863 0.882 1.65355 0.796 0.794 1.55360 0.762 0.754 1.497365 0.737 0.726 1.458370 0.712 0.701 1.421375 0.696 0.683 1.389380 0.664 0.651 1.342385 0.633 0.619 1.282390 0.615 0.601 1.253395 0.598 0.583 1.223400 0.582 0.567 1.195405 0.568 0.552 1.169410 0.555 0.538 1.144415 0.542 0.524 1.12420 0.529 0.511 1.097425 0.508 0.493 1.068430 0.505 0.49 1.054435 0.494 0.478 1.033440 0.483 0.467 1.013445 0.466 0.449 0.987450 0.463 0.447 0.975455 0.454 0.437 0.957460 0.443 0.427 0.938465 0.435 0.418 0.92470 0.426 0.409 0.902475 0.417 0.4 0.886480 0.41 0.392 0.871485 0.402 0.384 0.856490 0.394 0.377 0.841495 0.386 0.369 0.825500 0.379 0.362 0.809505 0.37 0.353 0.79510 0.362 0.345 0.772515 0.355 0.338 0.757520 0.348 0.331 0.742525 0.34 0.324 0.728530 0.333 0.317 0.715535 0.327 0.311 0.702540 0.321 0.306 0.691545 0.315 0.3 0.68550 0.309 0.294 0.669
Turbidity
52
Figure 30: QuilA Saponin Fluorescence for FRET Experiment. Saponin in 1x PBS instead of liposome was excited at 300 nm and the fluorescence from 350-‐550 nm was recorded. The fluorescence intensity of the saponin at these wavelengths is not negligible compared to the FRET fluorescence. These fluorescence intensities were then used to correct the FRET data in equation 2. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
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APPENDIX C: FITC-‐Dextran EXPERIMENT
A preliminary FITC-‐dextran experiment was accomplished with conflicting
results. The ideal experimental set up would have been to either use a dequenching
assay where the FITC was self-‐quenched within the liposome (analogous to the
calcein leakage assay) or use a combination of pH and quenching agent to quench
the fluorescence outside of the liposome.
However, due to time and material restrictions, a dilution assay was used. In
this assay, the FITC was encapsulated into the liposome at a non-‐self-‐quenched
concentration. Theoretically, leakage outside of the liposome would result in a
lowered fluorescence due to a dilution effect.
This experiment used two sizes of FITC-‐dextran, 4 kDa (4K) and 250 kDa
(250K) as limiting cases. Each FITC compound was encapsulated into the liposome
at a concentration of 0.0089 mM. These two types of FITC-‐dextran were selected
due to their sizes. 4K has a radius of 1.4 nm, which is slightly larger than calcein at
0.6 nm. 250K has a radius of 11.2 nm. The liposomes used in this study had a
diameter of 120 nm and it is unreasonable to expect a pore size large enough to
allow for the diffusion of the 250K FITC.
Table 7: Liposome Dimensions. The measured liposome diameter by DLS is 120 nm. In accounting for a 10 nm membrane, the inner diameter is 110 nm. The volume of the liposome was calculated using (4/3)πr3
Liposome(Diameter((nm)( 120$Effective(Inner(Diameter((nm)( 110$Effective(Inner(Radius((nm)( 55$
Inner(volume((nm3)( 230.4$!
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Table 8: FITC-‐Dextran Sizes Relative to Weight [12]. Summary of FITC-‐dextran radii as a function of molecular weight. The given data in Angstroms was converted to nm for ease of comparison to the liposome volume.
Figure 31: FITC-‐dextran Size Graphical Interpretation of Table 8. The data in table 8 was plotted radius vs. molecular weight. The resulting fit was then used to calculate the radii of FITC sizes not directly stated in the original source [12].
Fluorescence data from the FITC experiments did not support the original
hypothesis. In this experiment, lower fluorescence intensities indicate leakage from
the liposome. Liposome A (Figure 31) and B (Figure 33) did not appear to result in
Dextran(MW((kDa) Stokes(radius((A) Radius((nm)4 14 1.410 23 2.320 33 3.340 45 4.570 60 6150 85 8.5250 112.0 11.2
55
leakage of 4K, as the fluorescence intensities were greater than that of the Triton
control. However, liposome B (Figure 34) suggest that the 250K FITC was released.
It is unlikely that 250K FITC-‐dextran could be leaked while the 4K FITC would not.
Liposome C, F, and G (Figure 35 -‐ 40) all appeared to leak the FITC-‐dextran,
regardless of the sizes. These data indicate an experimental design error.
Figure 32: FITC 4K Fluorescence vs. Time for Liposome A. FITC-‐dextran fluorescence intensity (raw data) for liposome type A over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence above that of the Triton control indicate no leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
56
Figure 33: FITC 250 K Fluorescence vs. Time for Liposome A. FITC-‐dextran fluorescence intensity (raw data) for liposome type A over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence above that of the Triton control indicate no leakage Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
57
Figure 34: FITC 4 K Fluorescence vs. Time for Liposome B. FITC-‐dextran fluorescence intensity (raw data) for liposome type B over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence above that of the Triton control indicate no leakage Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
58
Figure 35: FITC 250 K Fluorescence vs. Time for Liposome B. FITC-‐dextran fluorescence intensity (raw data) for liposome type B over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence at or below the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
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Figure 36: FITC 4 K Fluorescence vs. Time for Liposome C. FITC-‐dextran fluorescence intensity (raw data) for liposome type C over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence below that of the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
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Figure 37: FITC 250 K Fluorescence vs. Time for Liposome C. FITC-‐dextran fluorescence intensity (raw data) for liposome type C over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence below that of the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
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Figure 38: FITC 4 K Fluorescence vs. Time for Liposome F. FITC-‐dextran fluorescence intensity (raw data) for liposome type F over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence below that of the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
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Figure 39: FITC 250 K Fluorescence vs. Time for Liposome F. FITC-‐dextran fluorescence intensity (raw data) for liposome type F over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence below that of the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
63
Figure 40: FITC 4 K Fluorescence vs. Time for Liposome G. FITC-‐dextran fluorescence intensity (raw data) for liposome type F over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence below that of the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
64
Figure 41: FITC 250 K Fluorescence vs. Time for Liposome G. FITC-‐dextran fluorescence intensity (raw data) for liposome type G over 2 hours. FITC dye was excited at 490 nm and fluorescence of 520 nm was measured. In this dilution assay, fluorescence below that of the Triton control indicates FITC leakage. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
65
Figure 42: QuilA Saponin Fluorescence for FITC Experiment. Saponin in 1x PBS instead of liposome was excited at 490 nm and the fluorescence at 520 nm was recorded. The fluorescence intensity of the saponin at these wavelengths is negligible compared to the FITC fluorescence. Each sample tested in triplicate and averaged together for plotted data. Error bars (not shown) are on the order of 101 or 0.5% of fluorescence intensity (smaller than marker size)
It is more plausible to conclude that the inconsistencies in these data were as
a result of poor experimental design. The “dilution” assay is not a proper assay, and
the results may have been skewed if the initial FITC-‐dextran concentration was
close to the self-‐quenching concentration. It is possible that for samples A and B, the
4K was actually partially self-‐quenched, so the leakage of the FITC-‐dextran actually
resulted in an increase in fluorescence, not a decreased as expected.
This kind of experiment would produce value information regarding the size
of pore formation or the extent of the vesicle-‐to-‐micelle transition and should be
repeated with a better assay.
