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i
Photophysical and Chemical Approaches
to Cellular Biophysics
Victor Akpe
Licentiate Thesis in Biological Physics
Stockholm, Sweden 2008
ii
TRITA-FYS 2008:54
ISSN 0280-316X
ISRN KTH/FYS/--08:54--SE
ISBN 978-99-7415-200-5
KTH AlbaNova University Center
Cell Physics
SE-106 91 Stockholm
SWEDEN
Akademisk avhandling som med tillstånd av Kungl Tekniska högskolan framlägges till
offentlig granskning för avläggande av teknologie licentiatexamen i biologisk fysik
torsdagen den 18 december 2008 klockan 11.00 i FA32, AlbaNova Universitetscentrum,
Roslagstullsbacken 21, Stockholm.
© Victor Akpe, 2008
Tryck: Universitetsservice US AB
iii
Abstract
The central theme in this thesis is reversibility. Two main attempts has been made to
approach reversibility in cellular systems from both chemical and physical points of view.
Reversibility of immunolabeling of proteins on the cell surface has been adressed by
development of new fluorescent substances optimized for CALI (Chromophore-Assisted
Laser Inactivation of protein). Aluminum phthalocyanine (AlPc) is here identified to be a
good candidate for a new generation of fluorophores for efficient hydroxyl radical
generation. It is shown that cells can be reversibly labeled with antibody-AlPc conjugates.
In experiments on living cells the AlPcs were not only active as classic fluorophores but
also as photocatalytic substances with destaining properties.
Reversibility of cell immobilization is also reported, where cells cultured in
microstructures were immobilized and 3D supported using hydrogels. Hydrogel
formulation and application was optimized to achieve a system where both viability and
ease of use was satisfied. Gel reversibility was actualized with pH and enzyme treatment.
The developped method offers the possibility of stop flow culturing cells in controlled
and reusable 3D environments.
iv
TABLE OF CONTENTS
1 Introduction 1
2 Materials 2 2.1 Fluorescent Dyes ................................................................................................................................................ 2 2.2 Gels .................................................................................................................................................................. 4 2.3 Gelatin ............................................................................................................................................................. 4 2.4 Hydrogels .......................................................................................................................................................... 5 2.5 Cell cultures ...................................................................................................................................................... 6
3 Experimental Setups 7 3.1 Setup for Chromophore- Assisted Laser Inactivation (CALI) Technique ............................................................ 7 3.2 Confocal microscopy ........................................................................................................................................... 8 3.3 Setup for immobilized dyes in gelatin using a FRAP based approach .................................................................. 9 3.4 Setup for Time Resolved Laser Flash Photolysis ............................................................................................... 10 3.5 Setup for singlet oxygen and photodegradation quantum yields ........................................................................... 11 3.6 Setup for the chemical synthesis ......................................................................................................................... 12
4 Results and Discussion 13 4.1 Summary of Paper 1........................................................................................................................................ 13 4.2 Summary of paper 2 ........................................................................................................................................ 15
5 Conclusions 21
6 Acknowledgements 22
7 References: 24
v
This thesis is based on the following studies:
I. Victor Akpe, Tebello Nyokong, Hjalmar Brismar. Photophysics and
Photochemistry of Zinc, Aluminum and Zinc octakis (benzylthio)
phthalocyanine. TRITA-FYS 2008 :44, KTH, (2008).
II. Victor Akpe, Erik Vernet, Torbjörn Gräslund, Hjalmar Brismar. Characterization
studies of Aluminum Phthalocyanine Binding to antibodies from SKBR 3 Cell
line. TRITA-FYS 2008:45, KTH, (2008).
III. Thomas Liebmann, Susanna Rydholm, Victor Akpe, Hjalmar Brismar.
Self- assembling Fmoc dipeptide hydrogel for in situ 3D cell culturing.
BMC Biotechnology. 7:88 (2007).
Contributions by the author
The contributions of Victor Akpe to the publications listed above include:
I. Experimental design, characterization, physicochemical measurements,
interpretation of the data and writing.
II. Experimental design, major part of the contribution for the characterization
of the antibody-dye conjugates, interpretation of the data and writing.
III. Design and supervision of the chemical synthesis.
1 Introduction
1
1 Introduction
Dyes are used in many biological applications, especially in their applications as
fluorescent probes in bioassays [1, 2], cell biology [3], in vivo imaging [4], flow cytometry
[5, 6], and as reactive dyes [7, 8]. Reactive dyes in recent years have also been seen to
advance in the photodynamic therapy treatment (PDT) of cancer [9-13], in the CALI
(Chromophore-Assisted Laser Inactivation) technique [14-16], which can be used to
remove protein at the cell surface. This method offers specific and non-invasive approach
for protein inactivation at both cellular and molecular level.
Cellular biophysics involves the application of biophysical approaches to address the
mechanisms of biological processes. This thesis therefore borrows backgrounds in the
Biological, Chemical, Physical, and Mathematical Sciences. Thus, the studies in this thesis
include:
• Synthesis and Characterization of novel Zinc, Aluminum and Tin Octakis
(Benzylthio) phthalocyanines (paper 1-technical report 1).
• Photophysics and photochemistry of Zinc, Aluminum and Tin Octakis
(Benzylthio) phthalocyanines (paper 1-technical report 1).
• The use of CALI protocol to study reversible antibody staining, where the
system investigated consists of HER 2 antibody coupled to AlPc dye and
Alexa fluorophore (paper2-technical report 2).
• Evaluation of the mechanism that is involved in the laser inactivation of
proteins (paper 2-technical report 2).
• Immobilization of the free dyes (MPc, Alexa, and Rh6G) in commercial gel
to study the: 1) phtotostability of the dyes; 2) the triplet state dynamics of the
dyes (paper 2-technical report).
• Lastly, the synthesis of self- assembling Fmoc dipeptide hydrogel and
application in 3D cell culturing. (Paper 3).
Victor Akpe
2
2 Materials
All the chemicals used were either purchased from Merck, Sigma-Aldrich or Fluka. The
metallopthalocyanine dyes were either synthesized or modified while alexa 488
fluorophore, rhodamine 6G and alexa antibody were purchased from Invitrogen
(molecular probes). Two gel types were used in this thesis; commercially obtained edible
gel and prepared synthetic gel from the laboratory. The detailed materials used are
contained in each separate publication. Also the two cell types, SKBR 3 and MDCK cell
cultures are contained in the different papers. All other materials not mentioned have
been explained in the different papers.
2.1 Fluorescent Dyes
Fluorescent dyes are used routinely in the Life sciences to trace the presence of
biomolecules in cells and other biological systems. They are also used in many areas of
research in the applied sciences, such as reactive dyes mentioned earlier, fluorescence
recovery after photobleaching (FRAP) [17], Fluorescence Resonance Energy Transfer
(FRET)[18], Fluorescence activated cell sorting (FACS) [19, 20], Time Resolved
Fluorescence (TRF)[21], Fluorescence Polarization (FP)[22], Fluorescence Correlation
Spectroscopy (FCS)[23] and many more.
Aluminum phthalocyanine (AlPc) has been used in paper 2, particularly for its
photodriven properties. Like in most phthalocyanines, a sulphonyl chloride (SO2Cl) can
easily be introduced into the ringstructure of the molecule, thereby increasing the
possibility for coupling to antibodies. Previously reported is the drug delivery application
of a stable sulphonamide bond [24-26]. It is for this reason that metallophthalocyanine
dyes are commonly used as reactive dyes rather than dyes with carboxylic ends which are
mostly used for cell staining. Other phthalocyanine (Pc) dyes synthesized are also
reported in paper 1 where the characterization, photophysical and photochemical
properties have been discussed. Figure 2-1 shows the chemical structures of some of the
Fluorescent dyes used.
2 Materials
3
Figure 2-1: Chemical structure of the dyes used in the thesis.
Victor Akpe
4
2.2 Gels
Gel is an apparent solid-like material formed from a colloidal solution. Different types of
gel exist depending on the application it is intended for. In this thesis, two types of gels
have been used, commercially obtained gelatin which is used as food ingredients and the
synthesized hydrogel used for the 3D cell culturing. The interesting thing about gels in
general is that they are superabsorbant and have the propensity for taking in water.
2.3 Gelatin
The decision to use edible gelatin in this study is based on its ready availability and
convenience in preparation. The different dyes (AlPc, Alexa and Rhodamine) were stirred
in the gelatin prepared and stored in the fridge at 4oC for 10 minutes. For a longer time
and a lower temperature could impede the jelly-like nature of the gelatin. Gelatin exists in
most cases as an amphoteric protein with isoionic point between 5 and 9 depending on
the process method-acid or alkaline pretreatment. The protein is made up of peptide
triplets of glycine-x-y, where proline has the preference for the x position and
hydroxyproline the y position. Gelatin has several uses, particularly as animal glue, gelling
binder in gummy products and are also known to have thermally reversible gelling
properties with water. In order to study the photobleaching property of the different
dyes, the free dyes were immobilized in gelatin. This enabled the study of the recovery
profiles observed for the different dyes. For more reading on gelatin, the reader is
directed to Dr Coles (Univ. Pretoria) extensive website on the subject [27].
NN
N N
N
H
CH
CH3
C
O
N
H
CH
H
C
OC
O
N
H
CH
O
CH2
CH2
CH2
NH
C NH2+
NH2
C
H
CH C
O
N
H
H
CH
CH2
CH2
C O
O-
C
OOH
C
O
N
H
CH
H
C
O C
O
Figure 2-2: Typical gelatin structure that consists of polypeptide chains of (-Ala-Gly-Pro-
Arg-Gly-Glu-4Hyp-Gly-Pro-) units.
2 Materials
5
2.4 Hydrogels
Hydrogels are water-swollen, cross- linked polymeric structures obtained by reactions of
monomers or by hydrogen bonding. They tend to mostly absorb a great volume of water
as compared to dry weight. Like gelatin, a hydrogel has a three dimensional insoluble
polymeric networks. Hydrogels are used in numerous biomedical applications including,
transdermal drug delivery [28, 29], wound healing [30, 31], bioadhesive carriers [32, 33],
capsules in the encapsulated form [34, 35], implantation coatings [36] . In this thesis, the
procedure employed by Xu et al [37] was similarly used to produce 9-fluorenyl methyl
carbamate (Fmoc). The application enabled the compartmentalization of the
immobilized cells in the microfluidic chamber [38].
Figure 2-3: Firm Fmoc hydrogel formed in a conical shape to demonstrate
morphological stability. Hydrogel layering is illustrated by colored dyes in dispersion
media.
Victor Akpe
6
2.5 Cell cultures
Two cell culture types were used in this thesis. SKBR 3 and MDCK. The SKBR 3 cell
line was obtained from American Type Culture Collection (Manassas, VA). All cell culture
reagents were from Invitrogen (Carlsbad, CA). The MDCK cell line (renal epithelial cell
line, originally obtained from African Green Monkey embryos) was prepared in the
laboratory according to standard protocols. The SKBR 3 cells were cultivated in RPMI
1640 medium supplemented with 15 % PBS and 1% Antibiotic-Antimycotic in 5% CO2
at 37°C.
3 Experimental Setups
7
3 Experimental Setups
IR spectra (KBr pellets) were recorded on a Perkin-Elmer spectrum 2000 FTIR
spectrometer. H-nuclear magnetic resonance (NMR, 400 MHz) spectra were obtained
using the Bruker EMX 400 NMR spectrometer. UV-visible spectra were recorded on a
Varian 500 UV/visible/NIR spectrophotometer. Fluorescence excitation and emission
spectra were recorded on a Varian Eclipse spectrofluorimeter. Photo-irradiations were
done using a General electric Quartz line lamp (300W). A 600 nm glass cut off filter
(Schott) and a water filter were used to filter off ultraviolet and infrared radiations
respectively. An interference filter 700 nm with a band width of 40 nm was additionally
placed in the light path before the sample. Light intensities were measured with a
POWER MAX 5100 (Molelectron Detector Inc.) power meter. Triplet absorption and
decay kinetics were recorded on a laser flash photolysis system, the excitation pulses were
produced by a Nd: YAG laser (Quanta-Ray, 1.5 J / 90 ns) pumping a tunable dye laser
(Lambda Physic FL 3002, Pyridine 1 dye in methanol). The analyzing beam source was
from a Thermo Oriel xenon arc lamp, and a photomultiplier tube was used as a detector.
Signals were recorded with a two-channel digital real-time oscilloscope (Tektronix TDS
360); the kinetic curves were averaged over 256 laser pulses. The mass spectra were
recorded on MALDI (Matrix Assisted Laser Desorption Ionization) where the mass-to-
charge ratio was recorded on a time of flight device. The Confocal images were recorded
using a LSM 510 META (Carl Zeiss, Jena) with visible and UV laser modules. ND-1000
(Nanodrop, Wilmington, DE) using Nanodrop 2.5.1 software was used to measure the
absorbance of the free dye and the antibody labeled dye. Spectral recordings in paper 2
were performed using a Cary 50 Bio UV-Vis Spectrometer (Varian, Palo Alto, CA) using
Cary Win UV Simple Reads Application 2.0 software and a LS50B Luminescence
Spectrometer (Perkin Elmer, Waltham, MA) using FL Win Lab 3.0 software.
3.1 Setup for Chromophore- Assisted Laser Inactivation (CALI) Technique Chromophore- assisted laser inactivation (CALI) is a technique that selectively inactivates
protein of interest to elucidate their in vivo functions [39]. This technique has a wide
application in biological processes and it also has the adaptive feature of being modified.
Victor Akpe
8
Here, AlPc was modified and labeled with antibody from Mouse c-erbB-2 Ab-2.
Essentially, the principle relies on high level of monochromatic light source, regulated
level of output power and changeable irradiated area, labeled dye molecule at the
physiological pH. Upon irradiation of the labeled dye region with pulsed laser light, the
protein is inactivated. The hydroxyl radicals migrate to the antibody-antigen extracellular
matrix of the cell where protein is inactivated within effective damage distance between
15Å-34Å radius. Figure 3-1 shows a schematic diagram for the CALI principle. The
experimental setup is further explained in paper 2.
Figure 3-1: Illustration of the basic principle for CALI protocol. The protein of interest
is presented as orange that has been inactivated. The choice of the excitation light source
is dependent on the excitation wavelength of the fluorochrome (dye).
3.2 Confocal microscopy Confocal laser scanning microscopy (CLSM) is a powerful tool used in biological
sciences, medicine, materials science, etc. A microscope objective is used to focus a laser
beam onto the specimen where it excites fluorescence. Fluorescent radiation is collected
from the objective lens and efficiently directed to the detector via a dichroic beam
splitter. The emission filter selects the choice of the wavelength and it also blocks the
source of the excitation laser line. To get a sharp and distinct image, focus must be
3 Experimental Setups
9
achieved. Light coming from planes above or below the focal plane is out of focus. It is
therefore important to arrange the pinhole in front of the detector on a plane conjugate
to the focal plane of the objective. For more readings on the principles and applications,
the reader is directed to [40-42].
Figure 3-2: Principles of a confocal microscope (from [43]).
3.3 Setup for immobilized dyes in gelatin using a FRAP based approach The experimental setup for the immobilized dyes in gelatin is essentially the same as in
FRAP (fluorescence recovery after photobleaching) method; only here the cells are not
alive. The dyes are immobilized according to the protocol reported in paper 2. The
essence of immobilization is to restrict the movement of the dye molecules in order to
capture the triplet state dynamics and photostability of the dyes. The setup also help in
clarifying what could be referred to as irreversible photobleached dye [44] and reversible
photobleached dye [45].
Victor Akpe
10
(1) (2) (Recovery of the
green area )
(Full recovery)
(4)
Figure 3-3: Immobilized AlClPc (SO2Cl)4 dye. Picture captured during FRAP experiment
of the bleached region using 633nm full laser power.
Figure 3-4: Immobilized Alexa-488 dye. Picture captured during FRAP experiment of the bleached region using 488nm full laser power.
3.4 Setup for Time Resolved Laser Flash Photolysis
Flash photolysis a technique generally used for monitoring photochemical reactions. For
reactions with moderate rate decay, flash lamps (typically Xenon lamps) are used since
they provide moderate time response. For faster reactions, lasers are usually used to make
up for the slow decay time of light emission from a flash lamp. Several lasers are available
for this purpose. Here, we used neodymium-yttrium aluminum garnet (Nd-YAG) solid
state laser. It is a synthetic mineral that produces light in the IR region of the spectrum at
1064nm upon excitation by flash lamp. To convert IR light into Visible and UV region, a
special optic is used.
The freshly prepared sample solutions of MPc dyes with absorbance value of 3.0 were
deoxygenated by purging in argon gas for 20 minutes and then excited within the lower
3 Experimental Setups
11
limit and upper limit of the excitation wavelength of the dye. From the experiment, the
triplet lifetimes, triplet quantum yields were calculated.
Nd: YAGLASER Dye LASER
Xenon lamp
Sample cell holder
Monochromator (w ith PMT)
computer
Focusing mirror
Collimating lens
UV-V
is s
pec.
Oscilloscope
Figure 3-5: Diagram of a time resolved laser flash photolysis setup.
3.5 Setup for singlet oxygen and photodegradation quantum yields The setup for singlet oxygen and photodegradation is similar as in photolysis experiment,
but here the laser is excluded. A simple setup is depicted in the schematic diagram below
(Figure 3-6). For more details on the experimental setup, the reader is directed to read
this article [46].
Victor Akpe
12
Figure 3-6: Schematic diagram of the setup. 1= Light source (General electric quartz line
lamp); 2= Lens (convex); 3= Water filter; 4= Schott glass cut-off filter ; 5= Interference
filter; 6= Quartz cell.
3.6 Setup for the chemical synthesis
All the MPc dyes used were either synthesized or modified. The preparation and
characterization are contained in the different papers. The MPc dyes synthesized in
paper 1 are all novel MPcs and have been characterized by known standard techniques
using IR, UV-Vis spectrophotometer, NMR, Mass spectrometer. Most MPcs are mostly
synthesized using high temperature and usually the finished raw products are very
difficult to purify. A lithium pentanoloate route is employed in the synthesis of paper 1.
It requires low heat (< 80oC) in comparison to the traditional method that goes up to
180oC. The traditional method was employed initially during the synthesis but the
method did not give a stable phthalocyanine. The alternative method proved to be a
better choice. Once the LiPc (Lithium phthalocyanine) has formed, the metal of interest
is introduced into the ring of the phthalocyanine, thereby displacing the Lithium metal.
The LiPc is the intermediate stage in the phthalocyanine formation with known
characteristic features of phthalocyanines. The first confirmation of a newly formed
MPc is usually a significant shift in the UV-Vis spectrum position. The photophysical
and photochemical properties of these dyes reveal their potential applications as
photocatalytic and photoactivatable compounds. Attempt to convert the benzene end
positions to thiol groups proved abortive. The thiol end groups in the phthalocyanine
could for instance be used as immobilization support on goal plates, coupling extension
to antibodies. In paper 2, commercial aluminum phthalocyanine was first modified by
dissolving in oleum (which is a mixture of conc. H2SO4 and SO3), followed by several
other steps in the modification. For more on the preparation and subsequent
modification, the reader is referred to paper 2.
4 Results and Discussion
13
4 Results and Discussion
In this thesis, two approaches were conducted to study reversibility in cellular systems from both chemical and physical points of view. The first study (paper 1) reports on the photophysics and photochemistry of octakis (benzylthio) phthalocyanines, and suggests their potential application as photoactivatable dyes and as photocatalysts. The second study (paper 2) reports on the combination of FRAP technique and the CALI protocol to study and evaluate Aluminum phthalocyanine as a photocleavable agent. Below, a brief summary of the two studies is provided.
4.1 Summary of Paper 1.
Fluorescence emission spectra of the MPc dyes are mirror images of the absorption
spectra, suggesting that the absorbing species is also the fluorescing species. The
photophysical and photochemical parameters of the MPcs are listed in Table 1. The ΦF
values in Table 1 demonstrate the influence of the central metal on the fluorescence
efficiency. The value of ΦF may be influenced by several factors including aggregation
and the heavy atom effect. Fluorescence quantum yield values are expected to be larger
for MPcs containing lighter atoms (e.g. Al), and smaller for those MPcs containing
heavier atoms (e.g. Sn and Zn). Heavy atom effect results in intersystem crossing of the
heavier atoms, hence less fluorescence. The observed trend for the fluorescence quantum
yields is as expected, in this regard with compound 5 having a value almost double that
of the other compounds. Triplet quantum yields (ΦT, Table 1) are correspondingly larger
for compounds 4 and 6 compared to compound 5, due to the heavy atom effect.
Table 1: The influence of the central metal (compound 4,5 and 6) on the fluoresce efficiency (ΦF)
Compounds
)(nmQλ
)(nmFλ
)( sTμ
τ02.0±
Fφ01.0±
Tφ510
dφ 02.0±
Δφ
T
S
φφΔ=
Δ K1−s
d
Zn (4) 710 719 630 0.13 0.53 0.03 0.43 0.81 4.76 ZnPcSmix i 675 682 530 0.14 0.86 13.65 0.72 0.84 2.58 x 103
Al (5 ) 699 707 810 0.31 0.40 0.04 0.36 0.90 4.94 AlPSmix i 685 690 800 0.39 0.52 5.79 0.48 0.92 0.72 x 103
Sn (6) 703 717 610 0.15 0.47 0.33 0.35 0.74 54.1 SnPcSmix i 694 684 120 0.13 0.87 14.01 0.65 0.75 11.7 x 103
Victor Akpe
14
The triplet lifetimes are lower for compounds 4 and 6 compared to compound 5, again
due to the heavy atom effect( Figure 4-1). The triplet quantum yield values (0.40-0.53) are
within the typical range for phthalocyanines in DMSO. Also the long lived triplet states
(610 µs – 810 µs) values may find potential applications as photocatalysts and also for
Photodynamic therapy (PDT). Table 1 shows that ΦIC values are higher for the
compound 4, compared to the other two compounds, suggesting that more electronic
energy of the excited singlet state is lost through internal conversion to the ground
singlet state for compound 4 compared to compounds 5 and 6.
The efficiency of energy transfer from the triplet state of a photosensitizer to ground
state molecular oxygen is quantified by a quantity SΔ (Table 1). SΔ is the fraction of the
triplet state quenched by ground state molecular oxygen. The efficiency of quenching was
fairly high for all the molecules (0.74-0.90), confirming the suitability of these molecules
for application as photocatalysts. Compound 5 showed better quenching ability followed
by compound 4, with compound 6 showing the least quenching ability. The degradation
of the molecules under irradiation is used to study the photostability of the molecules.
This is especially important for those molecules intended for use as photocatalysts.
Table 1 gives the photodegration quantum yields (Φd) for compounds 4 to 6. These
values are generally lower than reported for MPcs, showing that the thiophenyl
substituent imparts a degree of stability onto the MPc compounds.
∆A
time (s)
Figure 4-1: Singlet depletion curve for compound 4 in DMSO. λexc =699nm
The possible photodegradation mechanism was further investigated by studying the
photodegradation behavior in different environments. Figure 4-2 shows the kinetics of
4 Results and Discussion
15
the photodegradation of the compounds in air, oxygen, DABCO (a radical oxygen
scavenger), deuterated DMSO (singlet oxygen is longer lived in DMSO) and nitrogen. A
faster rate was observed in oxygen than in nitrogen, air or deuterated DMSO, suggesting
that oxygen is involved in the photodegradation. Since the compounds were found to be
stable in deuterated DMSO, molecular oxygen is likely not involved in the
photodegadation progress but rather oxygen radicals and/or singlet oxygen. Ab
sorb
ance
0 ≤ t (s) ≤ 800. where t is at 100s interval
Figure 4-2: Kinetic curves for the photodegradation of 5 in DMSO in the presence of (i)
oxygen, (ii) nitrogen, (iii) DABCO and (iv) Deuterated DMSO. Excitation wavelength ~
700nm.
4.2 Summary of paper 2 According to the CALI approach, hydroxyl radical and other oxygen species produced
are products of photochemical processes that occur at the triplet state during charge
transfer of electrons of the organic dye to a nearby target, in this case antigen. Figure 4-3
shows the photochemical pathways that MPc dyes can take during photoactivation with
633nm laser light. Both pathways result in the generation of hydroxyl radical and
hydroxyl ions, where the hydroxyl ions are probably associated in the aqueous
environment. Hydroxyl radical in particular have short-lifetimes in aqueous
environments, giving an effective action radius of approximately 15 Å. Within this action
radius, it is argued that proteins may be denatured and decoupled.
Victor Akpe
16
Figure 4-3: Reaction pathways common to Metallophthalocyanines (MPcs)
Two mechanisms are therefore proposed for the reversible process; one is the case where
the MPc dye is detached from the antibody but the antibody is still attached to the
protein at the surface. This possibility may arise if the hydroxyl radical is short-lived in
the aqueous environment before it gets to the antibody-antigen attachment. When this
happens, the dye area being destained will appear dark indicating that the dye is no longer
in that region but probably been washed away in the aqueous environment (see Figure 4-
4). The other possibility is the migration of the hydroxyl radical to the site of the
antibody-antigen interaction site, where the hydroxyl radical causes the denaturing of the
protein at the cell surface (Figure 4-5).
4 Results and Discussion
17
Figure 4-4: Schematic representation for the detachment of the dye from the antibody
but where the antibody is still attached to the protein at the surface.
Figure: 4-5: Schematic representation of the dye bound antibody linked to a protein at
the cell surface.
Experimentally, the SKBR3 cell line was stained by the Mouse c-erbB-2 Ab-2 without
internalization. Good staining was obtained for both the positive control experiment,
Victor Akpe
18
green fluorescent dye (c-erbB-2 Ab-2 Alexa 488) and red fluorescent dye (c-erbB-2 Ab-2
Aluminum phthalocyanine) (Figure 4-6).
A B
C
D
4 Results and Discussion
19
Figure 4-6 : Cell staining and FRAP. (A) antibody-alexa staining (prebleach). Excitation
source: 488nm, green light; (B) antibody-AlPc(SO2Cl)4 staining (prebleach). Excitation
source: 633nm, red light; (C) antibody-AlPc(SO2Cl)4 staining (prebleach) with excitation
source at 633nm and alexa-antibody staining (prebleach) with excitation wavelength at
488nm; (D) postbleach result from (C), where a loss of the red signal and a fading green
signal is observed. (D) shows fading compared to (A) but with the signal still present.;
(E) Seperation of green signal from (D) using dichroic beam splitter; (F) Seperation of
red signal from (D) using the dichroic splitter. (F) shows a gradual loss of red signal
when compared with (B) at the pre-bleach state.
During multiple irradiation using different wavelengths (488/633 nm), the antibody-AlPc
conjugate was specifically removed from the cell surface while the antibody-Alexa 488
conjugate remained (Figure 4-6). Experiments were further performed with free dyes in
gel under short time periods. The AlPc dye shows a fast recovery profile in intensity
(Figure 4-7), which clearly indicates a non-permanent photobleaching at the triplet state
while the controls show little or no recovery profile (or gain in intensity) indicating a
permanent photobleaching.
(2) 0 100 200 300 400 500 600
80
100
120
140
160
180
200
220
240
time (µs)
Inte
nsity
(A.
u.)
Region1
Region2
Region3
Figure 4-7 : FRAP profile for AlClPc (SO2Cl) 4 showing full signal recovery.
The absence of the red signal intensity in Fig 4-4 D indicates the dye is cleaved from the
extracellular matrix cell. This leaves out two possibilities for the mechanism. One is the
case where the MPc dye is detached from the antibody but the antibody is still attached
to the receptor in an inactivated form. This possibility may arise if the hydroxyl radical is
Victor Akpe
20
short-lived in the immediate environment and is usually the case before it gets to the
antibody-antigen attachment. When this happens, the dye area being destained will
appear dark indicating that the dye is no longer in that region but probably been washed
away. The other possibility is the migration of the hydroxyl radical to the site of the
antibody-antigen interaction site, where the hydroxyl radical causes the denaturing of the
protein at the cell surface.
The results from the experiments suggest there is a multiple interaction of forces taking
place, such as covalent or ionic interaction forces, which is dependent on the site of
bonding between the antibody and the antigen. In the present study, it may be proposed
that hydroxyl can cause the cleavage of the sulphonamide bond such that the dye is
detached from the antibody but where the antibody-antigen interaction are held together
by ionic forces. In order to validate the two proposed mechanisms, the cell was washed
in PBS several times and restained with red dye, MPc-antibody. After 60 minutes of
incubation, the area that previously was destain, showed a restaining (Figure 4-8). The
restaining of the dye suggests that the MPc-antibody could have been cleaved off and the
antibody-protein interaction could also have been separated by the release of hydroxyl
radical before being short-lived in the aqueous environment.
Figure 4-8: Recovery of the signal after several washouts followed by incubation of the
MPc-antibody.
5 Conclusions
21
5 Conclusions
In this thesis, the photophysics and photochemistry of octakis (benzylthio) phthalocyanines is reported and the potential of using octakis (benzylthio) phthalocyanines as a photoactivatable dyes is discussed. In addition, the combination of CALI protocol and the FRAP technique was employed to investigate the possible photocleavage mechanism of antibody that is conjugated to phthalocyanine using SKBR3 cell line. We show that using reversible staining it is possible to characterize cellular differentiation without any long term effects on the cells or remaining contamination of the fluorophores. For the CALI approach, the differentiation state of the cell is identified by the unique protein that is expressed in order to allow for sensitive and specific detection of proteins on the cell surface.
Victor Akpe
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6 Acknowledgements
I would like to thank the Swedish Institute for the scholarship support, for providing the
opportunity of continuing my studies in Sweden. I am indeed grateful to Professor
Hjalmar Brismar; he is truly an exceptional supervisor. He was able to link my chemical
background to the reversible staining project and also for sourcing for additional
financial support for my studies at KTH. Rick Rogers is also acknowledged for not just
his wonderful contributions to the project but also, for introducing me to the CALI
principle. A big thank to the Cell physics department - of both former co-workers and
the present people: Jacob, Susanna, Gustav, Erland, Padideh, Mårten, Linda, Gerald,
Bjorn, Tove, Thomas, and Athanasia. Special thanks to Jacob and Padideh, for their help
with the administration of the thesis. A very special thank you is extended to Dr Aman
Russom for last minute help with the text. Lastly, I would like to thank my brother,
Emeka for the supportive role played throughout my education and my family; Ronke,
Shaddai and Jesimiel for their patience and understanding.
6 Acknowledgements
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Victor Akpe
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