Selective Cell Targeting With
Light-Absorbing Particles
by
Costas M. Pitsillides
B.S. Physics
Northeastern University, 1997
SUBMITTED TO THE DEPARTMENT OF MECHANICAL ENGINEERING IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE IN MECHANICAL ENGINEERING
AT THE
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
FEBRUARY 2000
©2000 Massachusetts Institute of Technology. All rights reserved.
Signature of Author:
Cerified by:
Wellman Laborat6isMGH/Harvard M dical School
Department of Mechanical Engine . g, MIT
14, 2000
Janu
Peter T. So
Assistant Professor of Mechanical Engineering
Thesis Supervisor
Accepted by:
Ain A. Sonin
Professor of Mechanical Engineering
Chiarman, Committee for Graduate Students
MASSACHUSETTS INSTITUTE
OF TECHNOLOGY
SEP 2 0 2000
LIBRARIES
Selective Cell Targeting With
Light-Absorbing Particles
by
Costas M. Pitsillides
Submitted to the Department of Mechanical Engineering
on January 14, 2000 in Partial Fulfillment of the
Requirements for the Degree of Master of Science in
Mechanical Engineering
ABSTRACT
Pigmented particles can be briefly heated by short laser pulses to create very localized cell
damage. Transient heating of these particles vaporizes a thin layer of fluid in contact with
the particles producing microscopic underwater explosions (cavitation) which can cause
rapid cell death. Experiments were carried out with human leukocytes in vitro, to
investigate the use of laser-pumped microparticles and nanoparticles for selective cell
killing. Using 0.83 pm iron oxide-containing latex microspheres that were bound to the cell
membrane using antibodies (against the CD8 lymphocyte receptor), lethality in 80-90% of
the target cells was achieved after exposure to single or multiple 20 nsec laser pulses (565
nm) at a fluence of 0.4 J/cm 2 . In contrast, up to 10% of the non-targeted cells lost their
viability due to nonspecific killing. Similar results were obtained using 30 nm immunogold
particles against CD8+ lymphocytes, where irradiation with 100 pulses at 0.5 J/cm 2
resulted in over 95% killing of targeted CD8+ T cells and only about 5% non-specific,
CD8- cell death. Efficient cell killing was achieved with an average of 5 microparticles or
500 nanoparticles per cell.
To probe the effect of particle localization on cell death, endothelial cells were incubated
with 0.77 pm microspheres and irradiated at fluences of 0.2 to 0.4 J/cm 2 . Cavitationinduced cell death was shown to be due, in part, to the release of degrading enzymes from
late endosomes and lysosomes where the particles localized following phagocytosis by the
cells. This technique has potential applications in cancer therapy, where selective
destruction of tumor cells or tumor vasculature is the goal and in areas where inactivation of
specific subcellular structures (for example cell surface receptors) is desired.
In experiments employing sublethal laser doses and 20 nm gold particles, cellular functions
such as the permeability of the plasma membrane were successfully modified without
inducing death to targeted cells. Transiently altering the cell membrane permeability to
foreign molecules offers the possibility for light-activated delivery of proteins and genes
into living cells. Potential theiapy based on light-absorbing particles will allow control over
time and location of the treatment with minimal dark toxicity.
Thesis Supervisor: Peter T. So
Title: Assistant Professor of Mechanical Engineering
INTRODUCTION
Development of novel cancer therapies entails the design of techniques to selectively target
and destroy tumor cells while at the same time minimizing damage to healthy tissue. The
use of monoclonal antibodies conjugated to radioisotopes, cytotoxic drugs and other antitumor agents has been championed as a promising new cancer modality [1,2]. However,
the success of monoclonal antibodies as therapeutic agents has been limited mainly because
of problems in delivering the drugs to targeted tissues. These problems include slow
removal from the blood, low localization in tumor combined with high uptake in non-tumor
areas such as the liver and spleen and degradation of the antibody conjugates by the cells of
the body [3,4]. A more indirect approach aims to inhibit the formation of, and destroy, the
vessels that supply tumors with blood [5,6]. Angiogenesis inhibition as a cancer therapy,
although not a new concept, has received a lot of attention in recent years and several
approaches are currently being pursued to target the endothelial tissues that constitute the
tumor vasculature [7,8].
In this work, a novel method for selective cell targeting is introduced, based on the use of
laser-pumped micro- and nanoparticles. These particles offer the distinct advantage of being
toxic to tissue only when irradiated by short laser pulses. Previous studies at the Wellman
Laboratories of Photomedicine have shown that pigmented cells can be selectively targeted
by short laser pulses both in vitro and in vivo. By using laser pulses of a sufficiently short
duration (less or equal to the thermal relaxation time of the particle), at a wavelength that is
strongly absorbed by the target, the pulse energy can be thermally confined. Transient
heating of intracellular pigment particles vaporizes a thin layer of fluid in contact with the
particle surface producing microscopic bubbles (cavitation) which can cause rapid cell
3
death. Cells containing pigment particles cavitate and lose viability, while adjacent non
pigmented cells remain viable [9,10]. This principle has been successfully applied to the
treatment of various skin diseases such as port wine stains (with hemoglobin as the target
absorber) [11], the treatment of macular disease (by selective coagulation of the retinal
pigment epithelium) [12,13] and for laser removal of hair and tattoos [14,15].
In extending the above work, I have investigated the use of exogenous iron oxide
microparticles and gold nanoparticles, rather than endogenous pigments, in order to target
specific, non-pigmented cell populations which are normally transparent to the laser
wavelength. Super paramagnetic iron oxide/latex microparticles are used for magnetic cell
separation while immunogold nanoparticles are used extensively in electron microscopy
due to their high electron density. Both types of particles exhibit strong absorption in the
visible part of the spectrum with iron oxide having a broader absorption range. Gold has a
distinct absorption peak around 520 nm which strongly depends on the size of the particle
as demonstrated by Mie in his theory of absorption and scattering of light by spherical
particles [16] (Fig. 1). To produce localized cell damage, short laser pulses are required
since heat conduction away from the heated particles is minimized during the short
exposure time. For micrometer-sized particles the required pulse duration is on the order of
100 nanoseconds or shorter. For nanoparticles the ideal pulse duration is in the picosecond
domain. In these studies, 20 nanosecond pulses at 532 or 565 nm were used for both the
micro- and nanoparticle experiments. High-speed imaging was employed to investigate the
interaction between the laser pulse and the particle absorbers and gain an understanding of
the mechanism of laser-induced cellular damage. Temperature calculations were performed,
based on a heat transfer model developed by Goldenberg & Tranter [17],
4
to simulate the temperature distribution around the nanoparticle absorbers during the laser
pulse. Calculations indicate that temperature increases are of the order of a thousand
degrees and that the heating is confined within a very small volume surrounding the particle
targets.
The ability of these micro- and nanoabsorbers to inflict localized damage to cells has been
further exploited to transiently modify cellular functions rather than cause cell death. By
adjusting particle numbers and laser energy delivered to the targets it is possible to induce
such changes in cells as altering the permeability of the plasma membrane. The ability to
regulate the plasma membrane permeability opens up a host of possibilities by allowing the
introduction of foreign molecules (such as drugs, genes or proteins) into living cells in a
way that is controllable, reproducible and efficient.
5
MATERIALS AND METHODS
Isolationof lymphocytes (microparticleexperiments)
The mononuclear cell population was initially isolated from whole blood of healthy human
donors, using density gradient centrifugation. 28 ml of Dulbecco's phosphate buffered
solution (PBS - calcium and magnesium free) were added to 10 ml of freshly-drawn blood.
Using a glass pipette the blood was transferred to the surface of the density gradient
Histopaque@ (12 ml, Sigma, St. Louis, MI) and centrifuged for 30 minutes at 400g and 25
'C in a Beckman GPR centrifuge. The mononuclear cells, which separate out as a cloudy
white layer, were transferred to 20 ml of PBS solution and centrifuged for 10 minutes at
300g and 25 C. The pellet was then removed, washed three times (20 ml PBS, 10 min at
300g and 25 C) and resuspended in appropriate volume of Cellgro's RPMI 1640
solution.
Labeling of CD8+ T cells with 0.83 pm superparamagneticiron oxide particles
The 0.83 [tm streptavidin coated super paramagnetic iron oxide/latex microspheres (Bangs
Laboratories, Fisher, IN) were first washed 3 times with PBS solution (calcium and
magnesium free) containing 0.1% bovine serum albumin (BSA) by suspending them in an
Eppendorf tube and letting them sit in a magnetic separation rack (Dynal, Lake Success,
NY) for 1 minute. The washed spheres were then added to biotin conjugated monoclonal
anti-human CD8 mouse IgG (Sigma, St. Louis, MI) in an Eppendorf tube and left to
incubate for 40 minutes at room temperature. Captured antibody was magnetically
separated by suspending in PBS-BSA solution and placing in the separation rack for 3
minutes (repeated 3 times). The ligand-bound microspheres were added to a mixed
population of human lymphocytes, together with R-Phycoerythrin conjugated monoclonal
6
anti-human CD8 mouse IgG (Sigma, St. Louis, MI) and incubated for 45 minutes at room
temperature. Excess, unbound antibody was removed by suspending in 1.5 ml of PBSBSA (0.1%) solution and centrifuging three times at 300g. The cells were resuspended in
25 R1 of PBS-BSA solution and plated out on a Lab-Tek8 glass 2-well chamber slide for
about 45 minutes at 37 'C. The chamber slide was then flooded with 1 ml of RPMI 1640
medium (10% FCS) containing the live cell probe calcein-AM (concentration 0.5 [tg/ml,
Molecular Probes, Eugene, OR) and the cells were irradiated approximately half an hour
later.
Isolation of lymphocytes (nanoparticleexperiments)
The mononuclear cell population was initially isolated from whole blood of healthy human
donors, as in the microparticle experiments. However, the mononuclear cells (in RPMIFCS medium) were then plated out in a six-well tissue culture dish and incubated for 2 hrs
at 37 C. Since the monocytes tend to adhere to the culture plate, the cell population
remaining in the medium was monocyte-depleted. The lymphocyte population was then
washed from the well and resuspended in appropriate volume of RPMI 1640 (with 10%
FCS).
Labeling of CD8+ T Cells with 30 nm immunogold particles
The human lymphocytes (suspended in RPMI-FCS medium) were incubated with
unconjugated monoclonal anti-human CD8 mouse IgG and R-phycoerythrin conjugated
monoclonal anti-human CD8 mouse IgG (both from Sigma, St. Louis, MI) for 45 minutes
at room temperature. Excess, unbound antibody was removed by suspending in 1 ml of
PBS-BSA (0.1%) solution and centrifuging three times at 400g. The cells were
resuspended in 25 [d of PBS-BSA solution and plated out on a Lab-Tek@ glass 2-well
7
chamber slide for about 45 minutes at 37 C together with the 30 nm gold anti-mouse IgG
conjugate (Nanoprobes, Stony Brook, NY). The cells were incubated in 1 ml of RPMIFCS medium containing calcein-AM (0.2 tg/ml) for half an hour and were then irradiated.
Incubation of bovine aorta endothelialcells with 0 . 77 pm iron oxide particles
Primary cultures of bovine aorta endothelial cells where grown on Lab-TekO glass 16-well
chamber slides in full culture medium. Super paramagnetic iron oxide/latex microspheres
(0.77 [m, non-coated) where added to the cells and incubated overnight at 37 0C. To
investigate localization of the particles, the cells were loaded with a 50 nM LysoTracker
solution ( Molecular Probes, Eugene, OR), which localizes in late endosomes and
lysosomes, and incubated for 2 hours at 37 C prior to irradiation. To quantify cell death
due to the laser, cells incubated overnight with microspheres where irradiated using a
specially built scanning irradiation set-up and examined at one- and six- hour time points.
Cell necrosis was determined using a standard trypan blue exclusion method, while cell
apoptosis was examined using either TUNEL staining (ONCOR ApopTag kit) or using
double staining with propidium iodide and Annexin V solutions (to distinguish between
necrotic and apoptotic cells). Apoptosis could also be distinguished by the distinct
morphology of apoptotic nuclei which were visualized by staining formalin-fixed cells with
propidium iodide (10 tg/ml). To study the role of proteases in cavitation-induced cell
death, the cells were treated, prior to irradiation, with two cysteine protease inhibitors (a
cell membrane permeable inhibitor, E64d and a membrane impermeable one, E64), the
serine protease inhibitor TLCK and the caspase inhibitor Z-VAD-fmk.
Labeling offibroblasts with cationic gold particles
Mouse fibroblasts of the NS47 cell line were grown on Lab-Tek@ glass 2- and 4-well
8
chamber slides in full culture medium. Prior to experiments, the cells were suspended in a
phosphate buffered solution (calcium and magnesium free) and incubated, for 20 minutes,
with 20 nm gold particles coated with poly-L-lysine (BBI International, UK). Poly-Llysine is a highly positively charged amino acid chain and is attracted to the net negative
surface charge from anionic plasma membrane components. The incubation (100K
particles/cell) was carried out at 4 'C to inhibit phagocytosis of the gold. 10 kDa
fluorescein-dextran conjugate (100 stM) or propidium iodide solution (50 Rg/ml) was then
added as a probe for membrane permeabilization, and the cell suspension was irradiated
using a scanning irradiation set-up. The cells were washed about 15 minutes following
irradiation and the probe solution replaced with culture medium in order to minimize
background fluorescence and allow imaging of the probe uptake in the cells. In the case of
the fluorescein-dextran probe, propidium iodide (10 Rg/ml) was added in the culture
medium, following washing of the cells, to assay cell viability.
Fluorescencemicroscopy and laser irradiation
Microparticle and nanoparticle-labeled lymphocytes were irradiated using a self-built
imaging system employing stroboscopic illumination to detect transient cavitation at <125
nsec following the laser pulse (Fig. 2). The strobe pulse was produced by using a beam
splitter to direct a small fraction of the irradiation beam through a time delay fiber imaged
on the sample. The delay was variable but high speed images were typically taken at 100
nsec. Images were captured and digitized using a COHU 4910 CCD camera and a PC
equipped with a frame-grabber board. Fluorescence images of the cells were taken before,
immediately after and 1 hr following irradiation. In both the microparticle and nanoparticle
experiments, cells were irradiated with 20 nsec, 565 nm pulses from a rhodamine dye cell
pumped with the second harmonic output (532 nm) of a Q-switched Nd:YAG laser.
9
Calcein-AM fluorescence was excited using a 488 nm CW Argon laser while Rphycoerythrin (PE) was excited using a 532 nm CW Nd:YAG laser.
Endothelial cells were irradiated using 20 nsec, 532 nm pulses from a Q-switched Nd:YAG
laser. High-speed imaging was used to detect microcavitation in the LysoTracker-labeled
cells and fluorescent images were taken before and following irradiation to determine
leakage of the dye from damaged lysosomes. Cell viability of irradiated cells was assayed
using propidium iodide fluorescence (excited using a 532 nm CW Nd:YAG laser). In the
experiments to quantify cell necrosis and apoptosis, with and without the protease
inhibitors, the cells were again irradiated with 20 nsec, 532 nm pulses but this time a larger
(5 mm) beam spot was used to permit simultaneous irradiation of large numbers of cells.
Mouse fibroblasts labeled with the 20 nm cationic gold particles were irradiated using a
scanning irradiation system employing 20 nsec, 532 nm pulses from a Q-switched
Nd:YAG laser. The set-up permitted uniform irradiation of the 2- and 4-well chamber
slides using a 2 mm laser beam spot. Fluorescence images of the membrane
permeabilization marker were taken using the same imaging system as in the cell killing
experiments, about 15 minutes following irradiation (fluorescein excitation at 488 nm,
propidium iodide at 532 nm). Cell viability was assayed using propidium iodide
fluorescence.
10
RESULTS
Microparticletargeting of lymphocytes
To investigate cell lethality using laser-pumped microparticles, lymphocytes were incubated
with anti-CD8 antibody bound to 0.83 sim iron oxide microspheres. Cells were doublelabeled with anti-CD8 conjugated to R-phycoerythrin (PE) as a fluorescent probe to identify
the CD8+ T cells (see Fig. 3). Irradiating with 565 nm, 20 nsec laser pulses led to the rapid
heating of the particles and the fluid surrounding them, producing transient cavitation
bubbles around the CD8+ cells. The bubbles, which expand and collapse on the
nanosecond scale, were imaged using high-speed microsopy. Fig. 4 shows the results of
the cell viability assay, 1 hour after microparticle-labeled cells were irradiated by laser
pulses. Viability was assessed by fluorescence microscopy, using calcein-AM as cell
viability indicator. For single pulse exposure at 0.35 J/cm 2 , 77% of the CD8+ T cells lost
viability, whereas only 2% of the CD8- cells were killed. For 20 pulses both specific and
nonspecific cell killing increased slightly: 80% of the CD8+ and 6% of CD8- cell lost
viability. The experiment was performed with an average particle-to cell ratio of 5 which
was found to be optimal. Higher particle to cell ratios resulted in more unbound particles in
the solution and higher nonspecific killing. Lower particle to cell ratios resulted in less
efficient targeting.
Nanoparticletargeting of lymphocytes
In the nanoparticle experiments, lymphocytes were first incubated with anti-CD8 mouse
IgG and then with 30nm gold particles conjugated to anti-mouse IgG antibody. Cells were
double-labeled with anti-CD8-R-phycoerythrin (PE) probe to distinguish CD8+ T cells.
Irradiating cells with 100 laser pulses (565 nm, 20 nsec) at 0.5 J/cm 2 resulted in loss of
11
viability, as determined by calcein-AM fluorescence and by direct observation of cell
swelling and changes in nuclear morphology. Stroboscopic imaging during irradiation did
not capture any transient cavitation events. However, cavitation bubbles which are smaller
than the resolution of our optical system (about 0.5 [tm), are likely to be present. Cell
lethality for target cells increased from 54% with 100 Au particles/cell to 95% with 500 Au
particles/cell. Lethality in untargeted cells (CD8-) varied from 5% to 8% over the same
particle/cell range (Fig. 5).
Effect of particlelocalization on cell death
To investigate whether the location of the particle (membrane bound vs lysosomal
association) during the laser pulse had an effect on cell death, bovine aorta endothelial cells
were incubated overnight with 0.77 [tm non-coated super paramagnetic iron oxide/latex
microspheres. The particles were phagocytosed by the cells and localized in late endosomes
and lysosomes as revealed by staining with the lysosomal marker LysoTracker (Fig. 6).
Irradiating the particle-loaded cells at 532 nm at a fluence above the threshold for
microcavitation (about 0.1 J/cm 2 for the iron oxide microparticles) produced violent
bubbles which caused leakage of the LysoTracker from lysosomal bodies. Irradiating at
sub-threshold fluence did not produce cavitation or LysoTracker leakage (Fig. 7).
Irradiation of endothelial cell cultures with single and multiple 20 nsec, 532 nm laser pulses
resulted in both necrotic and apoptotic death of the particle-loaded cells. Irradiation of cells
that did not contain any particles did not result in cell death. The efficiency of irradiationinduced damage was dependent on the number of particles added (5-20 particles/cell), the
number of laser pulses delivered (1-100 pulses) and the fluence of the laser pulse (0.2-0.4
J/cm 2 ). The rates of both necrotic and apoptotic death increased with increasing particle to
cell ratio, number of pulses, laser fluence and time after irradiation (1 hr v. 6 hrs), with
12
necrosis dominating over apoptosis for the same parameters. The most damage was
observed at the high fluence (0.4 J/cm 2), at which nearly 100% of irradiated cells died by
either necrosis or apoptosis, while up to 80% of cells died at a fluence of 0.3 J/cm 2 . At the
lowest fluence used (0.2 J/cm 2), death rates were 20% or less, for both necrosis and
apoptosis. These findings are consistent with previous studies which have examined cell
vialbility following lysosomal injury [18]. To futher examine the role of lysosomal injury
and release of lytic enzymes, irradiated cells were pre-treated with inhibitors of lysosomal
hydrolases (proteases) and an inhibitor of apoptosis-mediating caspases. The hydrolases
inhibitors, but not the caspase inhibitor, partially inhibited both necrosis and apoptosis
indicating a role for these lysosomal proteases in inducing cell death through a caspaseindependent pathway.
Membrane permeabilizationof mouse fibroblasts using gold nanoparticles
To study the feasibility of using light-pumped nanoparticles to transiently alter the
permeability of the plasma membrane of cells, cultured mouse fibroblasts were labeled with
20 nm gold particles coated with the positively charged amino acid chain, poly-L-lysine.
The cells were irradiated with 20 nsec, 532 nm laser pulses at a fluence of 0.5 J/cm 2 and in
the presence of the membrane-impermeable dye propidium iodide (668 MW). Irradiated
cells took up the fluorescent probe in varying concentrations as imaged by fluorescence
microscopy (Fig. 8). In these experiments, propidium iodide served both as a membrane
permeabilization probe and as a marker for cell death. Transiently permeabilized cells had a
uniform fluorescence distribution and exhibited low levels of nuclear staining. In dead cells
however, which have a permanently damaged plasma membrane, propidium iodide
accumulated in the nucleus where fluorescence intensity was greatly increased. Uptake of
13
propidium iodide increased in cells that were given 10 pulses vs. cells exposed a to single
laser pulse. Induction of cell death was minimal and was also found to be pulse-dependent.
Experiments were also performed with a larger membrane permeabilization marker, the 10
kDa fluorescein-dextran conjugate. Irradiated cells were imaged by fluorescence
microscopy and uptake of the fluorescent dextran was observed (Fig. 9). Viability was
assayed by double staining with propidium iodide (added after irradiation of the cells) and
cell death was again found to be minimal.
14
DISCUSSION
Interaction of micro/nanoparticleabsorberswith short laserpulses
The events that take place following the absorption of the laser pulse energy by the particle
target depend on the duration of laser exposure. Long pulses which exceed the thermal
relaxation time, tr (where tr=d 2/27k for uniform spheres of diameter d and thermal
diffusivity k), of the target cause uniform heating of the particle and the surrounding media
as heat diffuses from the hot target to the cooler surroundings. If the laser pulse duration is
equal to or less than tr then the energy can be thermally confined within the target causing
rapid heating of the target itself. This extreme temperature rise can induce explosive
vaporization of a thin layer of fluid surrounding the target. Cavitation is initiated and the
bubble expands as the high vapor pressures created overcome the surface tension of the
fluid. The vapor in the highly unstable bubble then cools and condenses to cause violent
collapse of the bubble [19]. Cavitation has been previously observed in studies employing
short-pulsed laser to target micrometer size melanin particles in pigmented cells [9].
The interaction of both nanoparticles and microparticles with the laser pulse was
investigated by nanosecond time-resolved imaging. Cavitation from the laser-heated
microparticles was clearly observed as the dominant mode of cellular damage. However,
the gold nanoparticles used in the cell targeting experiments cannot be resolved by optical
microscopy and laser-induced cavitation was not observed during stroboscopic imaging
(although cavitation has been detected in single 200 nm gold particles irradiated with
nanosecond pulses - see Fig. 10). Nanoparticle cavitation bubbles can be imaged when the
particles are clustered together thus behaving like a larger particle when irradiated by a short
15
laser pulse. In experiments using 5 nm gold particles, cells of the mouse macrophage J774
cell line were loaded with approximately half a million particles each and incubated
overnight at 37 C. The particles were packaged closely together in lysosomes and upon
irradiation, micrometer-size bubbles (microcavitation) were observed (Fig. 11).
The 20 nsec duration of the laser pulse used in the cell targeting experiments exceeds the
thermal relaxation time of the nanoparticles (tr< 1 nsec for 30 nm Au spheres) and the
energy is therefore not thermally confined within the target particle. Temperature
calculations were performed, based on a heat transfer model of a heated homogeneous
sphere embedded in an infinite homogeneous medium [17], to characterize the temperature
distribution in the gold nanoparticles and the surrounding fluid. For the 30 nm Au particle
(with an absorption coefficient, Q=2 at 532 nm), temperature rise of the particle is of the
order of 2500 degrees during the 20 nsec pulse (at a fluence of 0.5 J/cm 2 ). However, the
temperature falls rapidly to l/e of the peak temperature at a distance approximately
equivalent to one radius from the particle surface (Fig. 12). Such a temperature profile
suggests that a thin layer of the surrounding fluid can in fact vaporize, creating cavitation
bubbles which expand and collapse rapidly, presenting a potent physical insult to
associated cells. Experiments using anti-CD8-R-phycoerythrin (PE) fluorescent probe on T
lymphocytes showed clustering of cell surface receptors (capping) following binding,
which would effectively increase the size, and efficiency of cell killing, of the bound
particle absorbers (Fig. 13). The explosive nature of the events taking place at the particle
sites during the laser pulse was confirmed by electron microscopy imaging of irradiated
CD4+ T cells that were previously labeled with 30 nm Au particles. Cells irradiated with
532 nm pulses at 0.2 J/cm 2 contained highly localized fragments of gold nanoparticles (Fig.
14), the consequence of either the heating to such extreme temperatures or the high
16
pressures created by the collapse of cavitation bubbles [19] or both.
Dependence of cell damage on particlelocalization
Cavitation produced by exposure of the particle targets to short laser pulses is the likely
mechanism of cell damage. Heating of cellular material is likely to be confined within the
thin layer of heated fluid surrounding the particles and its effect is clearly secondary to the
photomechanical effects associated with cavitation bubbles that can reach several
micrometers in diameter. The type of cavitation-induced cell injury will ultimately depend
on the location of the particle during laser exposure. The gold and iron oxide particles
employed in the lymphocyte targeting studies were directed against receptors of the cell
membrane. The most likely mechanism of cellular damage is thus the disruption of the cell
membrane due to cavitation of the bound particles. However, the presence of particles in
cytosolic vesicles, as revealed by electron microscopy studies of gold-labeled lymphocytes,
suggests that other mechanisms, such as lysosomal damage, may be involved.
In a separate set of experiments, the mechanism involved in microcavitation-induced, in
vitro cell killing was investigated using bovine aorta endothelial cells which were incubated
for 24 hrs with 0.77 gm non-coated coated super paramagnetic iron oxide/latex
microspheres. Particles were shown to internalize by phagocytosis and localize in late
endosomes and lysosomes. Irradiation by 20 nsec, 532 nm laser pulses from a Q-switched
Nd:YAG laser led to death of the cells that contained microparticles, by both necrosis and
apoptosis. This cell death was shown to be, at least in part, due to the release, into the
cytoplasm, of hydrolases following microcavitation-induced disruption of late endosomes
and lysosomes containing the phagocytosed particles.
17
Comparisonsto other methods
Selective cell targeting was demonstrated in vitro using both iron oxide microparticles and
gold nanoparticles. The laser fluence required for cell lethality is moderate compared to the
fluences currently used in clinical treatment of pigmented skin lesions (up to 5 J/cm 2 due to
scattering by skin). Nanoparticles are expected to be more effective for in vivo applications
because of better tissue penetration and longer retention time. The feasibility of using lightactivated nanoparticles to deliver foreign molecules into living cells has also been suggested
by these experiments. By varying the particle to cell ratio, number of pulses and laser
fluence it should be possible to efficiently deliver molecules of different sizes while
minimizing cell toxicity.
Photodynamic therapy (PDT) involves the absorption of light by a photosensitizer and the
production of chemically reactive species such as singlet oxygen and superoxide radicals
which are toxic to cells. Although similar to PDT, the cell targeting method investigated in
this work is mediated by a photothermal/photomechanical (transient localized heating and
cavitation) rather than by a photochemical mechanism. As a result, it does not depend on
tissue oxygenation, a major limitation of PDT therapy. Furthermore, while photosensitizers
are excited using optical wavelengths shorter than 800 nm, micro- and nanoparticles can be
excited using any wavelength that is selectively absorbed by the particles. Thus, longer
wavelenghts can be employed which can provide for better light penetration into tissue.
Cell ablation using focused microbeams requires targeting the laser on individual cells, an
approach which is both slow and limited, in spatial resolution, by the focus spot size (on
the order of an optical wavelength - about 0.5 [tm). The micro/nanoparticle targeting
method presented in this work does not require such precision aiming on single cells but,
18
instead, large laser spot sizes can be used to simultaneously target multiple sites in a tissue.
Furthermore, with nanoparticles one can potentially create subcellular damage zones which
are smaller than the wavelength of light. The use of even smaller particles (in the range of
1-5 nm) offers the prospect of targeting specific cellular components (e.g. to inactivate a
plasma membrane receptor) rather than whole cells.
AKNOWLEDGEMENTS
The author would like to thank Drs. Charles P. Lin, R. Rox Anderson and Dariusz
Leszcynski, whose guidance and support during the undertaking of this research have been
invaluable. The above work was carried out at the Wellman Laboratories of Photomedicine
of the Massachusetts General Hospital and Harvard Medical School, and was supported by
the US Department of Defense Medical Free Electron Laser Program under Grant No.
N00014-94-1-0927. Partial support was provided by the Alexander S. Onassis
Foundation.
19
20
APPENDIX A. TEMPERATURE CALCULATIONS
For a homogeneous sphere of radius R embedded in an infinite homogeneous medium and
generating heat for time t > 0 at the constant rate A per unit time per unit volume, the heat
conduction equations are:
T= T (sphere)
T2= T (surrounding
medium)
The equations that describe the problem are
Ti
T2)
2 7
A
+K1
Ti
r
0<r<R
r>R
r{T2
2
with the boundary conditions,
T= T,
at
t= 0
and
K 1 -T
( ar
j
=K1
at
aT)
2
21
r=R
The solutions for the temperature field inside and outside the heated sphere are
y2 t
fNo
T1=K
K1 +
S
3 K2
2
eXp
2 bR
6
r
I
{sin(y) - y cos(y)) sin
-
y
2
d
b2 2Rn2()dy
(c sin(y) - y cos(y)) 2 + b2 2 sin2
L0
T
R A K, 2
rK1 3 K2 jr
INoe
{sin(y) - ycos(y)) (bysin(y) cos(wy) - (c sin[y] - y cos[y]) sin(wy)}
- ycos(y)) 2 + b 2 y2 sin 2(y)
y3c{Csin(y)
0
where a =
,
b
K
,
c =12-
22
K,
and
=)r-~-:z
6.0
4.0
...... .8........ 80nm Qsca
/4
- - A- -
A
2.0
80nm Qext
--
80nm Qabs
A
.4'
6
A
-1
I
1A
0.0
0.4
0.5
0.6
0.7
O.E
(A)
wavelength
5.0
4.0
-
3.0
2.0-
Qsca
........ *.....---
200nm
-- a--
200nm Qabs
At
1.0 -I
A
0.0
0.4
200nm Qext
0.5
0.6
0.7
0.8
(B)
wavelength
Figure 1. Absorption/scattering cross section of (A) 80 nm and (B) 200 nm gold particles in water
23
100 nsec Time Delay
Optical Fiber
White Light or
Stroboscopic Illumination
Sample Stage
40x Objective
CCD Camera
Z
47
Monitor and PC
Equipped with
Frame-Grabber Board
y
15x Objective
AL
4
I
L
\
Dye Cell
CW Ar+
488 nm
(Excitation)
CW Nd:YAG
532 nm
(Excitation)
Figure 2. Microscope Set-up
25
Q-switched
Nd:YAG
532 nm
Figure 3. (A) T lymphocytes labeled with anti-CD8 Ab + 0.83 mm iron oxide microspheres
(5 particles/cell) and double labeled with anti-CD8 phycoerythrin (PE) fluorescent probe (B). Particles
can be seen to cluster mostly around PE labeled (CD8+) cells. (C) High-speed image taken ~100 nsec
after irradiation with a 565 nm, 20 nsed laser pulse at a fluence of 0.35 J/cm 2 . Calcein-AM fluorescence
before (D) and 1 hr (E) following irradiation indicates loss of viability of the CD8+ lymphocytes
100
-F
9080
70
60
40
30
20
10
0
1
20
No. of Iasr pulsee
Figure 4. Viability of T lymphocytes labeled with anti-CD8 Ab + 0.83 sm iron oxide microspheres (5
particles/cell) following irradiation with 1 or 20 pulses (20 nsec, 565 nm pulses at 0.35 J/cm 2 ). Viability
assayed 1 hr after irradiation using calcein-AM fluorescence.
27
100,-
80
60
40
20-
0
100
250
500
Au particles per cell
Figure 5. Viability of T lymphocytes labeled with 30nm anti-CD8 immunogold particles, irradiated with
100 laser pulses (565 nm, 20 nsec) at 0.5 J/cm2. Calcein-AM fluorescence was used to assay viability 1 hr
following irradiation.
Figure 6. Co-localization of the 0.77 Rm iron oxide microparticles and lysosomal marker LysoTracker in
late endodomes and lysosomes of endothelial cells
29
Figure 7. Endothelial cells loaded with 0.77 gm microparticles are stained with LysoTracker (A), and
irradiated at either sub-threshold (B) or above-threshold fluence, which produces microcavitation bubbles
(C). 15 minutes after laser pulse, intensity of LysoTracker fluorescence decreases only in the cavitating
cell (D) which loses viability as its nucleus stains with propidium iodide (E)
31
Figure 8. Irradiated mouse fibroblasts
(A) take up the membrane-impermeable
dye propidium iodide (B)
Figure 9. Mouse fibroblasts labeled with 20 nm cationic Au particles (A) take up the 10 kDa fluoresceindextran conjugate following irradiation at 532 nm (B)
33
Figure 10. (A) Bovine aorta endothelial cells with 200 nm gold particles. (B) Laser-induced cavitation
from 200 nm Au particles irradiated by a 532 nm, 20 nsec pulse at a fluence of 0.2 J/cm 2 (2x threshold)
Figure 11. J774 sarcoma cells incubated with 5 nm Au particles (-5x105 particles/cell). (A) Before
irradiation, and (B) 100 nsec following a 565 nm, 20 nsec laser pulse at a fluence of 0.4 J/cm 2
35
4
3000
2500
2000
S-41500
----
*100
20 ns
10 ns
1 ns
500
0
x Radius (r)
Figure 12. Theoretical temperature rise in a 30 nm gold particle (Q=2) irradiated with a 20 ns, 532 nm
laser pulse at a fluence of 0.5 J/cm 2
Figure 13. Image of an uncapped (A) and a capped (B) T lymphocyte labeled with anti-CD8
phycoerythrin (PE) fluorescent probe
37
Figure 14. Transmission electron micrographs of CD4+ lymphocytes labeled with 30 nm immunogold
(8x104 particles/cell) and irradiated with 532 nm laser pulses at 0.2 J/cm2 . (A) Unstained section at 39000x
of fragmented and intact particles both on the membrane and in the cell cytoplasm. (B) Stained section at
52000x of fragmented and intact gold associated with cytoplasmic vacuoles
39
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