Published on: Mar 3, 2016
Transcripts - Nanomedicina8
Lasers in Surgery and Medicine 41:240–247 (2009)
Method for Disruption and Re-Canalization of
Atherosclerotic Plaques in Coronary Vessels With
Photothermal Bubbles Generated Around Gold
Ekaterina Y. Lukianova-Hleb,1 Alexander G. Mrochek,2 and Dmitri O. Lapotko, PhD1*
A.V. Lykov Heat and Mass Transfer Institute, 15 P. Brovka Street, Minsk 220072, Belarus
Republican Scientiﬁc and Clinical Center for Cardiology, 110 R. Luksemburg Street, Minsk 220036, Belarus
Background and Objectives: Rapid and safe re-canal- ance properties of a plaque tissues that were used as optical
ization of totally occluded coronary vessels, especially those absorbers, a re-stenosis of artery walls, and, most impor-
with the calciﬁed plaques, represent a challenge for tant, a high risk of a thermal damage to an artery wall [10–
cardiology. 12]. As a result, the modern laser-based methods were
Study Design/Materials and Methods: We have sug- eventually re-directed from arteries to veins  with their
gested to employ photothermal microbubbles (PTMBs) that current rather limited clinical applications for treating
are generated around injected to plaque or thrombus gold atherosclerotic disease .
nanoparticles with a short laser pulse for selective mechan- Recent developments in light-absorbing (plasmonic) bio-
ical disruption and removal of the plaque tissue and without compatible nanomaterials, such as gold nanoparticles
thermal and mechanical damage to arterial wall. PTMBs (GNPs), improved the localization and efﬁcacy of laser-
were generated in vitro around 30–250 nm gold spheres and induced thermal effects and signiﬁcantly lowered the
with 10 nanoseconds laser pulse at 532 nm in three models: threshold laser energies [14,15]. Also, the advent of
the layer of the living ﬁbroblasts, the epoxy layers, and molecular targeting therapies enhanced the selective
human arteries with plaques. delivery of GNP to speciﬁc targets [16,17]. Using these
Results: In all three models, complete removal of the principles and materials, we have recently developed the
material was observed after 1–10 single laser pulses. The cell-level method for a selective mechanical destruction of
size of cleared zones (20–220 mm) was found to be 500– individual target cells—LANTCET (Laser-Activated Nano-
1,000 times bigger than the size of the nanoparticles Thermolysis as Cell Elimination Technology) [18,19]. The
applied. PTMB generation did not increase the temperature LANTCET uses a mechanical rather than thermal dis-
of the microenvironment outside PTMB and the debris size ruption of individual target cells through the laser pulse-
was below 2 mm. induced photothermal microbubbles (PTMBs), which are
Conclusions: New proposed method for non-thermal generated around GNP. The size and duration of the PTMB
mechanical and localized removal of plaque tissue with are conﬁned by the dimensions of a single cell. The
PTMB can provide safe and rapid re-canalization of totally generation of PTMB excludes signiﬁcant heating of the
occluded and calciﬁed arteries without collateral damage. surrounding media  and the damage to collateral
Lasers Surg. Med. 41:240–247, 2009. normal cells and tissues [18,19,21].
ß 2009 Wiley-Liss, Inc. Laser-induced bubbles were previously employed in the
laser angioplasty [22–27]; nevertheless, the mechanism of
Key words: gold nanoparticles; laser; bubbles; re- generation of the bubbles (using the tissues as optical
canalization absorber and relatively long laser pulses) resulted in
accompanying heating of the collateral normal tissues,
INTRODUCTION relatively big size of the laser-induced bubbles (milli-
Re-canalization of occluded and calciﬁed arteries still meters) and, consequently, in a thermal and mechanical
represents a major challenge when nor catheter can be damage to artery walls [16,17,28,29]. Compared to cardi-
guided through a plaque neither a latter can be rapidly ovascular laser-induced bubbles [16,17,28,29], application
lysed with drugs. That causes signiﬁcant level of lethal of light-absorbing micro- and nanoparticles provided sig-
outcomes . Laser-based methods were applied to this niﬁcant reduction in bubble size and in laser threshold
problem more than 25 years ago [2–4], and they have
demonstrated promising results based on ablation and
vaporization of atherosclerotic plaques and thrombi [5–9]. *Correspondence to: Dmitri O. Lapotko, PhD, A.V. Lykov Heat
and Mass Transfer Institute, 15 P. Brovka Street, Minsk 220072,
However, long and massive efforts did not turn the laser Belarus. E-mail: email@example.com
angioplasty into a successful clinical tool. This was caused Accepted 13 January 2009
Published online in Wiley InterScience
by several principal limitations such as an embolization (www.interscience.wiley.com).
with large debris, low and heterogeneous optical absorb- DOI 10.1002/lsm.20749
ß 2009 Wiley-Liss, Inc.
FEASIBILITY OF THE PTMB GENERATED AROUND GNP 241
energies, required for the generation of such bubbles [30–
32]. The advantage of the GNP that possesses maximal
optical absorbance compared to any other materials is their
relative safety . That was recently demonstrated by us
with the LANTCET method that allows to generate micro-
bubbles selectively in speciﬁc target cells, where GNP
clusters have been formed. Besides, no bubbles are
generated in the irradiated volume of relatively trans-
Fig. 1. Principle of plaque disruption for re-canalization of
parent media (including blood and tissues cells), where
occluded arteries with nanoparticle-generated photothermal
there are no large GNP clusters, thus precluding a
microbubbles (PTMB): (a) gold nanoparticles are injected from
collateral damage. The aim of our work was to evaluate
the catheter to the plaque site, (b) PTMBs are generated
the feasibility of the PTMB generated around GNP for the
around gold nanoparticles upon irradiation with short laser
selective disruption of the atherosclerotic and calciﬁed
pulse at the wavelength of plasmon resonance, (c) PTMBs
plaques so as to overcome the limitations of the previously
mechanically disrupt and remove plaque tissue thus creating
developed laser angioplasty methods.
the channel in the plaque.
MATERIALS AND METHODS
ically disrupts the plaque tissue. The GNP’s size may vary
Generation of Vapor Bubbles from 30 to 250 nm. They may be delivered to the plague by
Generation of vapor bubbles involves several steps: (1) the two ways (Fig. 1a). The direct mechanical delivery
absorbance of optical energy at speciﬁc wavelength (visible assumes the injection of the buffer suspension of GNP with
and near-infrared) by GNP through the plasmon resonance the catheter located at the close proximity to the plaque.
mechanism that provides 4–6 orders of magnitude higher This creates and maintains the necessary concentration
optical absorbance comparing to that of any biomolecules; of GNP at the plaque surface. Further increase of the
(2) rapid thermalization of GNP to the temperatures selectivity and efﬁcacy of a PTMB generation can be
exceeding evaporation thresholds of a surrounding tissue; realized through the selective formation of large GNP
(3) vaporization of a surrounding tissue within 0.1–1 clusters at the plaque site. Earlier we have demonstrated a
nanosecond, expansion and collapse of the resulting bubble GNP clusterization at the cell level [21,34] by using target-
within 1–10 microseconds. A rapidly expanding bubble speciﬁc antibodies conjugated to GNP and also by the
creates a high local tension that causes a mechanical endocytosis. In the case of the calciﬁed plaque, the other
disruption and fragmentation of a surrounding tissue. Due mechanisms of the GNP aggregation may be employed. It is
to the optical and thermal origin of such vapor bubbles and important that a GNP has little or no toxicity as it was
keeping in mind their dimensions, we deﬁned them as the determined during their in vitro and in vivo studies
PTMBs to distinguish from bigger laser-induced tissue- [19,33,34]. Therefore, we expect no signiﬁcant adverse of
generated bubbles and from artiﬁcial gas-ﬁlled micro- GNP-related effects during their injection. Next, GNPs are
particles. PTMB diameter can be considered as its main exposed to the laser pulse (Fig. 1b) with the ﬂuence being
tissue-damaging parameter and, as we have shown pre- above PTMB generation threshold (0.1–10 J/cm2 depending
viously [18,19,21], it can be controlled in the range of 1– on GNP size). PTMB can be generated with a single pulse or
100 mm by varying the ﬂuence of a laser pulse and the size of with a pulse train. The lifetime of PTMB is proportional to
GNP or of their clusters. It should be emphasized that the its maximal diameter, which can be controlled by varying
bubble generation threshold of the laser energy (ﬂuence) laser pulse ﬂuence. The laser pulse can be delivered through
depends upon GNP size: increasing the GNP diameter an optical ﬁber catheter [22,23,35,36] and through optically
signiﬁcantly lowers the PTMB threshold ﬂuence [21,34]. transparent media between the catheter tip and the plaque
Thus, the laser ﬂuence can be lowered to the levels that (such as saline buffer) because blood cells would scatter and
PTMB will be generated only around the biggest clusters of decrease the optical energy of the pump laser pulse. As the
GNP in the sites of their maximal concentration and will PTMBs remove a plaque tissue, the process should be
not be generated in other sites exposed to the laser pulse repeated until the re-canalization is achieved (Fig. 1c). The
(including artery wall, cells, and even the areas occupied by diameter of the cleared space can be controlled by varying
the occasional single GNP). Also GNPs with speciﬁc the laser ﬂuence and the number of pulses.
plasmon resonances (nanorods and nanoshells) allow to
shift a laser wavelength into the near-infrared region Experimental Set-Up
where the natural absorbance by any tissue is minimal. The samples were treated with a pulsed laser (model LS-
2132; Lotis TII, Minsk, Belarus) and analyzed by the
Vessel Re-Canalization With PTMB inverted optical microscope Nikon 200 (Nikon Instruments,
We consider the totally occluded (and calciﬁed) plaques Inc., NY). The pulsed laser beam (532 nm, 10 nanoseconds)
as the main targets. The re-canalization of such target was directed vertically down on the sample and was
includes several steps: the delivery of GNP to the plaque, scanned across its surface (Fig. 2a). The PTMBs were
the irradiation of the GNP with a short laser pulse (1– imaged with the two time-resolved techniques of trans-
10 nanoseconds), and generation of PTMB, which mechan- illumination (white light) and a side scattering of the pulsed
242 LUKIANOVA-HLEB ET AL.
Fig. 2. Fibroblast model: (a) experimental scheme of nanoparticle and laser delivery,
(b) microscopic image of the intact layer of the ﬁbroblasts, (c) photothermal microbubbles
generated around gold nanoparticles during exposure to laser pulse (532 nm, 10 ns), (d) treated
area showing removed by PTMB cells and clear bottom, (e) thermal ablation of the cells with
high-ﬂuence laser pulses: black area shows ablated ﬁbroblasts and melted plastic bottom of the
culture well. Scale bar is 75 mm.
illuminating source (pulsed probe laser (model LT-2211 Ti- Experimental Models
Sa laser; Lotis TII) at 750 nm) that are described in detail in We have employed three experimental models. The
Ref. . The PTMBs were optically detected with the horizontal layer of human living ﬁbroblast cells has been
additional continuous probe laser (model LGN-223-1; used to study in vitro the inﬂuence of the PTMB on a tissue.
PLASMA, Russian Federation) beam (633 nm) that was The cells were grown on a transparent plastic until they
directed coaxially with the pump laser onto the sample and formed a continuous layer (Fig. 2a,b). Then 30 nm GNP
its axial intensity was monitored with a photodetector (#15706; Ted Pella, Inc., Redding, CA) were added on top of
(PDA10A; Thorlabs. Inc., Newton, NJ). The PTMB caused the layer in the concentration of 2.0Â1011 GNP/ml. The
scattering of the probe beam and the output amplitude of laser treatment was applied after 20 min of sedimentation
the photodetector signal decreased thus indicating the of GNP on the cell layer. After the generation of the PTMB
bubble generation. This method also referred as a ‘‘thermal at speciﬁc laser pulse ﬂuence, we have measured the
lensing’’ as well as the whole experimental set-up were diameter of residual zone that became free of the cells. Prior
previously described by us in detail Ref. . The thermal to laser treatment the cells represented a solid layer
lensing technique was used for measuring a PTMB lifetime without clear spots (Fig. 2).
and for monitoring a relative temperature of the tissue The second model was employed to study the effect of
volume that was exposed to the pump laser. Any laser- location of GNP (surface/volume) and of the hardness of
induced heating causes the deviation of the signal from the sample. Bio-tissues are mainly soft compared to calciﬁed
baseline (Fig. 3). The PTMB-induced removal of the plaques and are also very heterogeneous. To exclude these
materials in the sample was detected optically and was factors, we have studied a transparent epoxy resin layers at
quantiﬁed by (1) measuring an optical density of the the different stages of its polymerization: from liquid to
irradiated area with CCD image detector (Luca DL-658 hard solid phases. Thus, we have modeled different
M; Andor Technology, Ireland) before and after the mechanical conditions of the tissues while excluding the
generation of the PTMBs and (2) by measuring the size of effect of the heterogeneity. GNP of 250 nm diameter
the cleared zone in post-treatment images of the samples. (#15714; Ted Pella, Inc.) were applied on top (Fig. 4a) or
distributed through the whole volume of the sample that
was prepared as 170 mm horizontal and transparent layer.
Laser beam has been scanned across the sample and the
size of the residual cavities left after the generation of the
PTMB was accessed with CCD imaging detector.
The third model represented human postmortem artery
slices of 20 mm thickness. These slices included calciﬁed
plaques and arterial wall. We have used thin slice for the
preliminary examination of PTMB in human arteries to
provide optical access to and the transparency of the
sample. This allowed direct control of GNP application,
laser treatment, and of the generated PTMBs. GNPs were
horizontally injected into the plaque as a transverse
Fig. 3. Monitoring of the PTMB generation and thermal state microjet that was directed with the microneedle of 50 mm
of the bulk media in the ﬁbroblast sample with photothermal diameter in the sample plane inside the artery slice
microscopy: (a) PTMB signal without residual heating after (Fig. 5a). Laser beam was applied orthogonally and has
bubble collapse was obtained under conditions as in Figure 2c been scanned across the surface of the sample so as to
for GNP-generated bubbles, (b) PTMB signal with residual irradiate plaque and arterial wall. The diameter of laser
laser-induced heating was obtained under conditions as in beam in the sample plane was 20 mm as was determined
Figure 2e during thermal ablation of the media. with CCD camera.
FEASIBILITY OF THE PTMB GENERATED AROUND GNP 243
their imaging (Fig. 2c) and through monitoring the
intensity of the continuous axial probe laser (Fig. 3a). The
PTMBs generated at the ﬂuence of 1.7 J/cm2 have had the
diameter in the range of 20–50 mm and the lifetime of 340
nanoseconds (the width of the signal at the half of its
maximum at Fig. 3a). After the application of the laser
pulses during 5 seconds at 15 Hz, the area where PTMBs
Fig. 4. PTMBs in epoxy model: (a) experimental scheme for were generated was cleared of the ﬁbroblasts and clean and
surface model shows the location of gold nanoparticles (GNPs), intact bottom of the culture well was observed (Fig. 2d). The
laser beam direction, and transparent layer of the epoxy resin, size of the cleared zone was 180–220 mm and it coincided
(b) microscopic image of the sample after scanning the pump with the region of the generation of the PTMB. No
pulse laser beam and created by PTMB cleared path (black signiﬁcant cell debris remained in this area. Also, the
holes), (c) dependence of the size of cleared zone upon irradiation of the intact layer of the cells that were not
laser pulse ﬂuence for various locations of GNP (surface treated with GNP caused no PTMB and did not inﬂuence
and volume) and various degrees of epoxy polymerization: the viability of the cells as was determined with the trypan
hollow circle—surface-located GNP, polymerization time: 1–2 blue staining of the control (97.0% of viable cells) and
hours (viscous gel); half right circle—surface-located GNP, irradiated (98% of viable cells) cells.
polymerization time 4–5 hours (soft compound); solid circle—
Thermal Impact of PTMB
surface-located GNP, polymerization time 20–24 hours
(hard compound); half right square—volume-located GNP, We have analyzed the laser-induced bulk thermal impact
polymerization time 4–5 hours; solid square—volume-located on the media during the laser treatment of the cell layer by
GNP, polymerization time 20–24 hours. comparing the amplitude levels of the PTMB signals before
and after PTMB (Fig. 3a). There was no detectible residual
All three models provided rather wide range of the bulk heating of the sample immediately after the collapse of
environments for PTMB such as mechanical state of the the bubble (approximately 600 nanoseconds after laser
sample, GNP delivery, and localization. These models were pulse). This result illustrates rather important feature of a
designed for preliminary evaluation of the GNP–PTMB PTMB generation mechanism that we have recently
potential to remove plaque tissues in cases where tradi- studied in detail : PTMBs do not cause the heating of
tional methods fail to penetrate through or/and remove the the media around them. This distinguishes the PTMB laser
plaque due to its calciﬁcation, stenosis, and other compli- method from traditional methods of laser angioplasty that
cations. Also, all these models allowed rather direct control were based on thermal effects of a laser radiation. The
and imaging of the PTMBs, which is important for the very latter mode of a laser–tissue interaction was realized in the
initial evaluation of the new method and cannot be easily same ﬁbroblast model by increasing the laser ﬂuence from
achieved in more complicated three-dimensional in vitro 1.7 to 23 J/cm2 and by irradiating the intact cells (not
(long pieces of the arteries) and in vivo models. treated with the GNP). In this case, we have observed the
destruction of the cell layer (Fig. 2e) but also the damage to
RESULTS the plastic bottom of the culture well then was apparently
melted or burned (black vertical line in Fig. 2e).
Generation of Photothermal Microbubbles Around In this case, the PTMBs were also generated as it was
Gold NPs and Tissue Removal seen from their characteristic signals (Fig. 3b), but those
A layer of the ﬁbroblasts (Fig. 2b) has been scanned with a signals also indicated signiﬁcant heating of the irradiated
pulsed pump laser in orthogonal plane (Fig. 2a). The volume as the amplitude of the signal after the bubble
generation of the PTMB was detected optically through collapse remained signiﬁcantly higher than the base level.
Fig. 5. Human plaque model: (a) microscopic image of intact line) and laser-treated (solid line) sample, left part corresponds
plaque, black arrow shows the jet of gold nanoparticles, black to the clear glass and the right part corresponds to the plaque
circle shows the pump laser beam (532 nm, 10 nanoseconds), tissue, (d) optical side scattering images of the PTMB obtained
(b) microscopic image of the same sample after exposure simultaneously with their generation and for the same sample.
to laser pulses, (c) optical density proﬁles of the intact (dashed Scale bars are 20 mm.
244 LUKIANOVA-HLEB ET AL.
Therefore, the laser PTMB method, proposed by us, acts — using small and functionalized GNPs that would
through the mechanical, non-thermal removal of the tissue. aggregate selectively into the clusters only at the
plaque sites; at the low ﬂuence level the bubble will
Nanoparticle-Generated PTMBs Versus
therefore be generated only around the GNP clusters
and will not emerge around single GNP that may
We have evaluated the selectivity and safety of the PTMB incidentally get into the normal tissues or into artery
generation by comparing the thresholds and probabilities wall.
of the bubble generation around gold NPs, red blood cells
(maximal natural optical absorbers at 532 nm), and
ﬁbroblasts (normal tissue). These parameters were meas- Mechanical Effect of the PTMB
ured as the function of the ﬂuence of the single laser pulse at Mechanical effect of the PTMB naturally depends upon
532 nm (Fig. 6a). Each sample has yielded similar type of the mechanical properties of the tissue and upon the
transition from no-bubble state at sub-threshold ﬂuence to location of the GNPs that act as PTMB sources. It is difﬁcult
the 100% generation of the bubbles at the above-threshold to provide homogeneous mechanical properties of the
ﬂuence. The thresholds were determined as the ﬂuencies plaque and to precisely control the location of the GNP.
that have matched bubble generation probability value to We have employed optically transparent epoxy resin
be 0.5. It can be seen that at the ﬂuence level required for phantoms of the tissue with the GNPs being distributed
the generation of the PTMB around GNPs, no bubbles are on its surface and, in the separate experiment, in the
generated in normal cells, including the red blood cells that volume of the epoxy layer of 170 mm thickness (Fig. 4a).
have the highest optical absorbance among all natural We have generated the PTMBs in the epoxy layers at the
tissue elements. Also, the thresholds of the PTMBs were different stages of the epoxy polymerization that cover
determined as: GNP—0.6 J/cm2, red blood cells—3.7 J/cm2, the range of mechanical states from a highly viscous ﬂuid
intact ﬁbroblasts—26.6 J/cm2. Therefore, we may conclude (1–2 hours), soft (4–5 hours), and solid (20–24 hours)
that GNP–PTMB mechanism may provide sufﬁcient safety tissues. At the latter stage, this phantom is similar to
and selectivity of the bubble generation. calciﬁed plaque. We have optically detected the residual
We have also studied the inﬂuence of the main GNP changes induced by the PTMBs generated with the single
parameter—its size—upon PTMB generation threshold laser pulses (Fig. 4b) of variable ﬂuence. At the ﬂuencies
and their lifetime (which characterize main damaging above the PTMB threshold, each single pulse has produced
parameter of PTMB—its maximal diameter) around the a characteristic hole in the phantom. The diameter of the
single nanoparticle with the diameter of 30 nm, their hole was averaged by 30 pulses as the laser beam was
clusters, and a single GNP with the diameter of 250 nm scanned across the sample (Fig. 4b). We have analyzed the
(Fig. 6b). Increase of NP diameter or their clusterization PTMB effect as the dependence of the whole diameter upon
has signiﬁcantly decreased the bubble generation thresh- the laser pulse ﬂuence, polymerization time, and GNP
old and has increased bubble lifetime. This allows location (Fig. 4c). Due to the general dependence of the
approaches to improve the selectivity and safety of PTMB diameter upon the laser ﬂuence we have observed a
proposed method: consistent increase of the residual clearing size with the
increase of the laser pulse ﬂuence (Fig. 4c). It should be
— using relatively large GNP so as to minimize laser emphasized that single short laser pulses have produced
pulse ﬂuence and to prevent bubble generation in any the holes within microsecond time and with the diameters
cells including red blood cells and blood clots; up to 50 mm though the diameter of the employed GNP was
300 times smaller. Further increase of the size of the
cleared volume can be reached not through the increase of
laser ﬂuence but through the GNP clusterization and using
pulse trains instead of the single pulses. A surface
application of GNP resulted in better clearing effect
compared to the model with internally distributed par-
ticles. This result conﬁrms the method proposed by us of
GNP delivery that does not require a penetration of the
GNP deep inside the plaque. The ultimate hardening of the
sample material has decreased the clearing effect of the
PTMB (Fig. 4c). However, the effect of PTMBs in a
Fig. 6. Bubbles parameters as function of the ﬂuence of the relatively hard phantom after 6 hours of its polymerization
single laser pulse at 532 nm: (a) probabilities of the bubble was very similar to that obtained in a semi-liquid sample,
generation around gold nanoparticles with the diameter of 250 especially at higher ﬂuencies of laser pulses (Fig. 4c).
nm (*), red blood cells (&) (maximal natural optical absorbers
at 532 nm), and ﬁbroblasts (~) (normal tissue); (b) bubble Human Plaque Model
lifetime around the single gold nanoparticles with the We have applied PTMBs for the removal of the human
diameter of 30 nm (&), their clusters (&), and single gold plaque tissue. Postmortem human plaque samples were
nanoparticle with the diameter of 250 nm (*). prepared as the slices of 20 mm thickness. Based on the
FEASIBILITY OF THE PTMB GENERATED AROUND GNP 245
previous results, we have modiﬁed the GNP (30 nm microchannel without the damage to arterial wall.
spheres) delivery method from static into dynamic: the Obtained results are rather preliminary and will be used
microjet of GNP suspension was directed orthogonally into in future (1) for the design of intra-vascular laser ﬁber
the plaque boarder (Fig. 5a). The laser beam (532 nm, 10 probe for GNP injection and for the delivery of laser
nm, 3.5 J/cm2) was applied from the top as the train of radiation and (2) in experiments with three-dimensional
pulses at 15 Hz frequency and was scanned from the plaque calciﬁed arteries.
boarder to the right as the plaque was cleared (Fig. 5b). An
observed effect of PTMB was similar to that obtained in the DISCUSSION
described above experiments: the plaque tissues have been
removed within several seconds of the laser treatment and Nanoparticle-Generated Bubbles Versus Tissue-
the diameter of the cleared volume (25 mm) was comparable Generated Bubbles and Thermal Ablation Methods
to that of the area of the intersection of the jet (100–150 mm) Method and experimental results, reported above, are
and the laser beam (20 mm). the very ﬁrst and preliminary demonstration of the
To quantify the efﬁcacy of the PTMB method, we have potential of laser-induced vapor bubbles generated around
compared the optical density proﬁle of the initial sample GNP for the destruction of arterial plaques. Further
(dashed line in Fig. 5c) with that of the GNP- and laser- success of the proposed method depends upon the scientiﬁc
treated sample (solid line in Fig. 5c). The proﬁles were and engineering developments of the several key compo-
obtained for the plaque-free area (it corresponds to the left nents of the method: (1) delivery of GNP, including a
parts of the Fig. 5a–c) and for the irradiated plaque. We selective concentration of the GNP (clusterization) at the
have observed almost complete removal of the plaque tissue atherosclerotic target, (2) laser irradiation of the arterial
from the treated area. Also we have used our imaging set-up vessel, and (3) controlling the PTMB generation process in
to monitor the size of the debris that has been produced terms of its efﬁcacy and safety. The mechanism of a plaque
during the PTMB generation. The maximal size of plaque destruction with the PTMB is principally different from the
debris did not exceed 2 mm. After irradiation of the plaque previously employed mechanisms of a revascularization: it
tissue, we have irradiated the artery wall under the same uses the mechanical, not thermal impact, the bubbles are
conditions of the laser pulse and without the GNP treat- generated selectively only around GNP and are not
ment. We have observed no generation of the PTMB and no generated in the volume exposed only to the laser radiation,
optically detectible damage to the wall tissue at the equal the levels of the laser radiation ﬂuence and the
laser ﬂuence and exposure time. treatment time can be signiﬁcantly decreased relatively
Finally, we have evaluated the optical scattering proper- to those used in previous methods of the laser angioplasty
ties of PTMB for the optical guidance of the process of the because GNP convert the optical energy into heat much
plaque removal. Under the real conditions, the threshold of more efﬁciently compared to any cells, dyes, and tissues.
the PTMB generation and PTMB size signiﬁcantly depend Thus, the PTMB-based method may potentially help to
upon local conditions: the laser ﬂuence, GNP aggregation overcome the major limitation of the previous laser
state. Thus, an optical real-time guidance of the revascula- angioplasty methods that failed to provide sufﬁcient
rization process would help to control its efﬁcacy and safety. selectivity of laser-induced processes that were previously
We have recently demonstrated that the laser-induced employed for plaque and thrombi removal. PTMB-based
PTMB can be used as cellular optical probes  by using method is principally different from the ultrasound
additional pulsed probe laser beam that orthogonally methods that use pre-fabricated bubbles (gas-ﬁlled micro-
illuminates the sample. Any side-scattered light is collected particles of micrometer size) for thrombolitic and athero-
by the microscope objective and forms the image of sclerotic therapies [37,38]. It is much easier to deliver and
scattering objects. A time-resolved scattering imaging of to target the smaller and safer GNP compared to relatively
the PTMB generation in the plaque samples was realized large and less stable microparticles. Also, the ultrasound
with the pulsed probe laser beam (750 nm, 10 nanoseconds) mechanisms cannot provide the spatial selectivity of
that was delayed relatively to the pump laser pulse for 150 mechanical impact that was demonstrated with PTMBs.
nanoseconds to allow the PTMB to expand. The optical
scattering image in Figure 5d was obtained for the sample Selectivity and Safety of the GNP-Generated PTMB
shown in Figure 5a during its exposure to the pump laser. for Collateral Tissues
PTMB-speciﬁc signals (bright small white spots in Fig. 5d) Using our model of photothermal interactions of pulsed
have yielded high amplitude and spatially have coincided laser radiation with the cells , we have estimated
with the location of the cleared zone, as shown in Figure 5d. maximal laser-induced temperatures in the optically
Light scattering by PTMB was detected in our experiment absorbing cells that may present in the volume that is
with the microscope. Also the light, scattered by PTMB irradiated by the pump pulse (532 nm, 10 nanoseconds).
(from the pump laser pulses or from the additional probe Again, we have used red blood cells as the cells with
laser), can be collected by optical ﬁbers that are used in maximal optical absorbance so as to estimate maximal
modern laser catheters for the intra-vascular delivery of possible. For the bubble generation threshold (250 nm
the laser radiation [37,38]. The described experiment with GNP) of 0.6 J/cm2, we estimated initial (maximal) laser-
human plaque sample has demonstrated the potential of induced temperature in red blood cells to be 19 K, which is
the combined action of GNP and PTMB for creating the within the safe range. Naturally, the laser-induced temper-
246 LUKIANOVA-HLEB ET AL.
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