Lasers in Surgery and Medicine 41:240–247 (2009)

Method for Disruption and Re-Canalization of
Atherosclerotic Plaques ...
FEASIBILITY OF THE PTMB GENERATED AROUND GNP                                             241

energies, required for the g...
242                                               LUKIANOVA-HLEB ET AL.

                 Fig. 2. Fibroblast model: (a)...
FEASIBILITY OF THE PTMB GENERATED AROUND GNP                                                   243

244                                               LUKIANOVA-HLEB ET AL.

Therefore, the laser PTMB method, proposed by us,...
FEASIBILITY OF THE PTMB GENERATED AROUND GNP                                             245

previous results, we have mo...
246                                              LUKIANOVA-HLEB ET AL.

ature in any other cell or tissue (including arter...
FEASIBILITY OF THE PTMB GENERATED AROUND GNP                                                 247
23. Brinkmann R, Hansen C...
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  1. 1. 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 Nanoparticles Ekaterina Y. Lukianova-Hleb,1 Alexander G. Mrochek,2 and Dmitri O. Lapotko, PhD1* 1 A.V. Lykov Heat and Mass Transfer Institute, 15 P. Brovka Street, Minsk 220072, Belarus 2 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 [10] 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 [13]. 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 [20] 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 [1]. 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: 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 ( with large debris, low and heterogeneous optical absorb- DOI 10.1002/lsm.20749 ß 2009 Wiley-Liss, Inc.
  2. 2. 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 [33]. 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
  3. 3. 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. [21]. 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. [21]. 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.
  4. 4. 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 [20]: 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.
  5. 5. 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 Tissue-Generated Bubbles 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
  6. 6. 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 [21] 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 [39], 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-
  7. 7. 246 LUKIANOVA-HLEB ET AL. ature in any other cell or tissue (including artery wall) will 2. Lee C, Ikeda RM, Kozina J, Mason DT. Laser dissolution of coronary atherosclerotic obstruction. Am Heart J 1981;102: be significantly lower than that in red blood cell. This 1074–1075. estimate as well as difference in the bubble generation 3. Abela CS, Normann S, Cohen D, Feldman RL, Geiser EA, thresholds for GNP and cells implies that the PTMB Conti CR. Effects of carbon dioxide, Nd-YAG, and argon laser radiation on coronary atheromatous plaques. Am J Cardiol generation should be selective and hence safe process 1982;50:1199–1205. without collateral mechanical and thermal damage. 4. Choy DSJ, Stertzer SH, Rotterdam HZ, Bruno MS. Laser The selectivity of the tissue disruption and removal was coronary angioplasty: Experience with 9 cadaver hearts. Am studied for the postmortem samples of human arteries. We J Cardiol 1982;50:1209–1211. 5. Borst C. Percutaneous recanalization of arteries: Status and have never observed the disruption of the artery walls that prospects of laser angioplasty with modified fibre tips. Lasers were exposed to the same levels of laser fluence as the Med Sci 1987;2:137–151. sample areas occupied with the plaque tissue (Fig. 5). Also 6. Benett W, Broughton K, Celliers PM, Da Silva LB, Esch V, London RA, Visuri SR. Opto-acoustic recanalization delivery the viability of the fibroblasts exposed to laser pulses system. US Patent 6368318, April 9, 2002. without cell pre-treatment with GNP was not compromised 7. Ginsburg R, Geschwind HJ. Laser Angioplasty, 2nd edition. at the fluence levels that provided the removal of the GNP- Mount Kisco: Futura Publishing, Inc.; 1992:205–215. treated fibroblast layers (Fig. 2). We consider these results 8. Deckelbaum LI. Cardiovascular applications of laser tech- nology. Lasers Surg Med 1994;15:315–341. as the first indication for the selectivity of the proposed 9. 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