Nanogold-coated stent facilitated non-invasive photothermal ablation of stent thrombosis and restoration of blood flow

In-stent restenosis (ISR) and stent thrombosis (ST) are the most serious complications of coronary angioplasty and stenting. Although the evolution of drug-eluting stents (DES) has significantly restricted the incidence of ISR, they are associated with an enhanced risk of ST. In the present study, we explore the photothermal ablation of a thrombus using a nano-enhanced thermogenic stent (NETS) as a modality for revascularization following ST. The photothermal activity of NETS, fabricated by coating bare metal stents with gold nanorods generating a thin plasmonic film of gold, was found to be effective in rarefying clots formed within the stent lumen in various in vitro assays including those under conditions mimicking blood flow. NETS implanted in the rat common carotid artery generated heat following exposure to a NIR-laser that led to effective restoration of blood flow within the occluded vessel in a model of ferric chloride-induced thrombosis. Our results present a proof-of-concept for a novel photothermal ablation approach by employing coated stents in the non-invasive management of ST.


Introduction
Coronary artery disease (CAD) is the leading cause of mortality worldwide with an estimated 17.9 million people succumbing to it each year. 1,2Ever since the rst successful coronary artery bypass procedure was performed by Rene Favaloro in 1968, it has become a standard of care in patients with signicant coronary atherosclerosis.However, due to this being a major surgery and a highly invasive procedure, angioplasty was developed as a relatively non-invasive substitute.Although balloon angioplasty was effective in the management of coronary stenosis due to atherosclerotic plaque, its use was associated with serious complications.Stents were developed in an attempt to provide a temporary scaffold to tide over limitations associated with balloon angioplasty.Stent implantation eliminated acute vessel recoil and chronic constrictive remodelling, thereby reducing restenosis rates.Use of adjuvant dual antiplatelet therapy also reduced the rates of thrombosis to acceptable levels.In sum, current treatment options for CAD include anti-platelet medications, percutaneous coronary intervention (PCI) and coronary artery bypass graing (CABG).PCI with stenting has become the preferred mode of treatment, especially when the extent of stenosis is more than 50%.2][3] First, there could be thrombosis in the peri-stent region (stent thrombosis or ST), which can occur early (less than 30 days), late (30 days to 1 year) or very late (more than 1 year) following the procedure.The common etiological mechanisms of ST include: (i) exposure of blood to the prothrombotic subendothelial matrix, stent struts and/or polymer before they are covered by re-endothelialization, (ii) persistent slow coronary blood ow, (iii) inadequate suppression of platelet activation by antiplatelet drugs and (iv) systemic prothrombotic states. 4The rates of ST vary from as high as 20% without antiplatelet/anticoagulant drugs 5 to as low as 2% with aggressive dual anti-platelet therapy. 6Second, there could be renarrowing of the vessel lumen (in-stent restenosis or ISR) in 10-30% of the patients [7][8][9] characterized by excessive smooth muscle cell proliferation and migration into the intimal layer.This complication of neointimal hyperplasia is addressed with drugeluting stents (DES), which release anti-proliferative medications like sirolimus and paclitaxel.Although DES remarkably restricted the incidence of ISR to less than 10%, they are ironically associated with a higher risk of late or very late ST than bare metal stents (BMS) due to several reasons. 4First, reendothelialization is signicantly delayed with DES compared to BMS, thus providing a substrate of injured vessel wall for eventual stent thrombosis. 10Second, late stent malapposition as a result of positive vessel wall remodelling away from the stent is more common with DES than BMS. 11Lastly, appearance of new atherosclerotic plaques in the peri-stent area, whose rupture can lead to ST, occurs earlier in DES than BMS. 12,13ST is life-threatening with mortality rates ranging from 11% to 42% and warrants immediate repeat angioplasty or CABG surgery.However, it is important here to emphasize that there has been no development in coronary stent technologies to directly address this complication.
8][29] For example, an attempt has been made to manage obstructive rectal cancer 30 or minimize rat esophageal granulation tissue 31 with heat generated from photothermally active stents.By employing both in vitro assays and a murine model of thrombosis in vivo, we have for the rst time demonstrated that brin clots/thrombi could be destabilized by heat due to dissociation of non-covalently assembled brin monomers, and provided a proof-of-concept to harness photothermal therapy as an effective tool towards targeted thrombolysis. 324][35] The concept introduced a safe and smart approach to revascularization, which minimizes the off-target life-threatening complications associated with the currently employed brinolytic drug regime.In the present study, we have extended this concept to fabricate novel thermogenic stents employing GNRs, which have the ability to generate heat upon irradiation with a NIR-laser from outside.Thermogenic stents implanted in situ can effectively bring about photothermal lysis of in-stent/peri-stent clots that can be washed off from lesion sites by arterial hydrodynamic shear, leading to partial or complete restoration of blood ow.This being the rst study would need further investigation towards clinical validation.

Materials and methods
Alexa Fluor 488conjugated brinogen was purchased from Invitrogen.Human brinogen, thrombin and gold nanorods (GNRs) (axial diameter: 9.0-11.0nm; longitudinal diameter: 36.9-45.1 nm), methyl methacrylate (MMA), butylmethacrylate (BMA), methylbenzoate, polyethylene glycol 400, benzoyl peroxide and N,N-dimethyl-p-toluidine were procured from Sigma, while methylene blue, benzoyl peroxide and Drabkin's solution were products of Merck, Thomas baker and Linear Chemicals S.L.U., respectively.The rest of the chemicals were either from Sigma or Merck.All reagents were of analytical grade.Type I deionized water (18.2MU cm, Millipore) has been used throughout the experiment.

Thin-layer coating of NIR-active nanomaterials on the stent surface
Thin layer coating of gold on the bare metal stent surface by employing photothermally active gold nanorods was achieved in three steps: (a) activation step: prior to coating, the stent surface was activated by treatment with HCl (0.1 M) solution for 1-2 h.This was followed by washing with type I deionized water and short bath sonication (Branson 2510 Ultrasonic Bath, 40 Hz) to remove excess acid.The washing was repeated 3 times and the stent was dried in a vacuum oven (Anil Scientico) at 80 °C for 20 min.(b) Coating step: next, assembly of the nanomaterial on the stent surface was accomplished by bath sonicating the stent in the presence of gold nanorods (30 mg ml −1 ) for 10 min each time with 10 min intervals for up to 3 h, which was followed by stirring in GNR solution with an Eppendorf Thermomixer (model 5350) for 4 h.(c) Annealing step: nally, the thin plasmonic lm of gold on the stent was achieved by placing the stent in a vacuum oven at 350 °C under an inert (nitrogen gas) environment for 3 h.The coating and annealing steps were repeated for 4 cycles to achieve a dense layer of gold coating on the stent surface.

Characterization of nano-enhanced thermogenic stents (NETS)
Surface morphologies of stents following coating were characterized by scanning electron microscopy (SEM) and energydispersive X-ray spectroscopy (EDS or EDX) coupled with SEM (M/S FEI quanta 200).The bare metal and coated stents were mounted on carbon tape adhered on a sample holder (stub) and SEM and EDX images were captured. 36The stent underwent additional characterization through a comprehensive analytical approach utilizing Raman spectroscopy and Fourier-transform infrared spectroscopy (FTIR) in attenuated total reection (ATR) mode (detailed information in the ESI section †).
The photothermal efficiency of NETS was evaluated by irradiating the stents with a 3 W solid-state continuous diode laser (808 nm) (M/S Changchun New Industries, China) at 1.05 W cm −2 and the rise in temperature was recorded with an infra-red thermal camera (M/S FLIR, USA) (ESI Fig. 1 †).In order to investigate the stability of the coating on the stent surface when exposed to hydrodynamic shear of perpetual blood ow as may occur within the coronary artery, a buffer was allowed to ow over the coated stents employing a high-resolution peristaltic pump (Bio-Rad).The superfusion of the buffer upon the stent was maintained consistently for 6 to 8 h each day for 30 days.The stability of the coating was then evaluated from the uniformity in heat generated on different days when the stent was irradiated with a laser.

Characterization of thrombolysis elicited in and around NIR laser-irradiated NETS in vitro
Clot formation (brin polymerization) was induced around the thermogenic stent by addition of 2-5 mM CaCl 2 and 1 U ml −1

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thrombin to puried human brinogen as described. 32Whenever required, a uorescent brin clot was generated by adding 45 ml puried brinogen (1 mg ml −1 ) and 5 ml Alexa Fluor 488conjugated brinogen.Next, the stent was irradiated with an NIR laser and the extent of clot lysis due to the heat generated from the fabricated stents was determined by various assays as already reported. 32(a) Fluorescence-based assay: NETS was irradiated with a NIR laser for 30 min and the supernatant was collected.The presence of uorescent brin in the supernatant was analysed with a uorescence multi-mode microplate reader (BioTek model Synergy H1) (excitation, 488 nm; emission, 519 nm) as a measure of brin released due to thrombolysis.(b) Drabkin's assay: a clot containing trapped RBCs was generated around the stent by mixing 2.5 ml RBCs and 47.5 ml brinogen solution followed by thrombin and CaCl 2 as mentioned above.
Thermal disruption of the thrombus released RBCs, which was quantied using Drabkin's reagent.The supernatant (containing released RBCs) was added to an equal volume of Drabkin's solution (K 2 HPO 4 , 50 mM; KCN, 38 mM; [K 3 Fe(CN) 6 ], 30 mM; surfactant, 2-5% w/v), and kept at RT for 20 min.Absorbance was recorded at 540 nm as a measure of the cyanoderivative of hemoglobin.(c) Methylene blue assay: ablation of the clot was determined from the extent of release of methylene blue from brin mesh around the stent.The clot was induced to form as described above where 0-1 mg ml −1 dye replaced the RBCs.Following laser irradiation of the NETS the supernatant was collected, and the extent of methylene blue released was recorded from the absorbance at 540 nm.(d) Scanning electron microscopy (SEM): the change in surface morphology of the brin clot and thrombus-depleted areas, as well as diameter of brin bres was recorded using an analytical SEM 37,38 as mentioned in the ESI section.† (e) Confocal laser scanning microscopy (CLSM): the ber density and thrombus-depleted areas in the uorescently labelled clots were analysed employing confocal microscopy 37,38 (details in the ESI section †).

Fluorescence recovery aer photobleaching (FRAP) analysis
The altered molecular dynamism within the structure of the instent/peri-stent thrombus following exposure to the NIR laser, reective of brin depolymerization/repolymerization kinetics, was evaluated employing the FRAP analysis tool.A uorescent clot was generated around the stent as stated above.The clotmoulded stent was carefully transferred to a fabricated microscopic slide (bordered with adherent tape as a spacer; depth: 450 mm) as already described. 32,39The in-stent clot was exposed to the NIR laser (808 nm) in a humid chamber for 15 min.FRAP was performed on a confocal microscope (Zeiss, model LSM 700) equipped with a plan-apochromat 1.4 NA oil immersion objective (63×), 488 nm argon ion laser, and LSM acquisition soware.The detector gain and offset were set before collecting FRAP images.The detector gain was set to a level such that very few or no pixels were saturated.Selected areas on the uorescent thrombus were photobleached using the 488 nm laser at 100% output at a rate of 20 ms per pixel.Five pre-bleached images were captured as a reference for the steady-state distribution of uorescent molecules.Images of uorescence recovery were collected at 2% excitation laser power using Zen Imaging soware and analysed using ImageJ soware (National Institutes of Health).Data were analysed with GraphPad Prism soware.

NETS-mediated thrombolysis under arterial shear
In order to evaluate stent thrombolysis against arterial uid shear resembling the physiological milieu, a uorescent thrombus was generated around the stent as described above.
The stent carrying the thrombus was placed within a fabricated silicon tube, and uid was allowed to perfuse over it at arteriolar hydrodynamic shear (1500 s −1 ) precisely regulated with a highresolution peristaltic pump (Bio-Rad) (ESI Fig. 2 †). 32The stent was exposed to the NIR laser for 30 min and the extent of thrombolysis was quantied from the release of the uorescent brin in the uid of the reservoir as described above.
In-stent/peri-stent thrombolysis by the heat generated from a NIR laser-irradiated thermogenic stent graed in the rat common carotid artery in situ (a) Stent angioplasty.Stent implantation in the murine common carotid artery was carried out as described 40,41 with minor modications.Briey, Swiss albino adult rats (250-300 g) (n = 14) were administered aspirin (10 mg kg −1 d −1 ) orally for 5 days. 42Rats were anesthetized using 3% isourane strictly as per recommendations of the Animal Ethical Committee of Banaras Hindu University.Aer careful shaving and proper disinfection of the area a small median incision was made in the ventral neck region with standard surgical tools to isolate the common carotid artery of the rats along with external and internal carotids under a stereo microscope (Zeiss Stemi 2000; magnication: 6.5-50×).The external carotid artery was permanently ligated as distal as possible using a 5/0 silk thread (Merilsilk).The proximal parts of the common and internal carotid arteries were temporarily occluded by binding knots with the 5/0 silk thread to interrupt blood ow (Fig. 6).The exposed vessels were xed or stretched in such a way that the common and external carotid arteries fell in a straight line, which allowed easy stent deployment.A small incision was made in the external carotid artery.The coated stainless steel thermogenic stent (length, 2.5 mm; diameter, 635 mm; strut thickness, 70 mm) was carefully introduced through arteriotomy proximal to ligation, which was further guided to the common carotid artery using a silicone tube.Aer the stent reached the desired location, the silicon tube was pulled back and the stent was allowed to inate within the artery.Next, the temporary sutures were removed from the common and internal carotid arteries to allow blood ow along the implanted NETS.
(b) Photoacoustic (PA) imaging of the implanted stent.The stent was implanted at the desired location in the anesthetized rat aer the surgical procedure described above.The animal was then placed on a Vevo imaging platform and subjected to multi-modal in vivo PA imaging analysis employing a Vevo LAZR-X (FujiFilm VisualSonics Inc.) 43 for high resolution anatomical and functional visualization of the implanted NETS.Anaesthesia was maintained at 2% throughout the imaging

Paper
Nanoscale Advances procedure.Photoacoustic (PA) imaging employs a non-ionizing laser (680-970 nm) to generate ultrasonic waves, which carry information around the stented artery.These waves were sensed by a transducer (MX250; 15-30 MHz) to generate ultrasound and photoacoustic images.The acquired images were then analyzed through Vevo LAB data analysis soware.
(c) Induction of an in-stent thrombus and evaluation of photothermal thrombolysis.5][46][47][48] Vascular injury due to oxidative damage of the endothelial cells triggers thrombus formation in the blood vessels.The stent was irradiated with an 808 nm NIR laser from an overhead laser source for 15 min, and the heat generated was captured with an infrared thermal camera (FLIR).Occlusion of blood ow due to the growing thrombus and subsequent restoration of ow following NIR laser irradiation, as a measure of clot lysis, was assessed by placing a ow probe (1 PRB; PR-series, Transonic system) on the exposed common carotid artery. 49Signals from the artery were collected using a ow meter (Transonic, model T106) through a Doppler ow probe that measures the difference between the transit time of ultrasonic pulses propagating with and against the ow direction.Data were analysed using a PC-driven Pow-erLab data acquisition system employing LabChart Pro soware (ADInstruments).
(d) Histology of stented carotid segments carrying thrombi.Stents carrying thrombi in situ were also subjected to histology to evaluate the extent of thrombolysis.Carotid arteries with implanted NETS, with or without laser irradiation, were harvested post-surgery from the rat and xed in 4% paraformaldehyde for 8 to 12 h.Samples were dehydrated in 70% ethanol and inltrated with the plastic embedding mixtures (MMA solution I, II and III) using a 3-step protocol in 15 ml Borosil glass vials as described. 50Samples were rst dipped in solution I (60 ml methylmethacrylate, 35 ml butylmethacrylate, 5 ml methylbenzoate and 1.2 ml polyethylene glycol 400) on day 1, which was followed by incubation with solution II (100 ml solution I with 0.4 g dry benzoyl peroxide) on day 2. Finally, samples were inltrated with solution III on day 3, which contains 100 ml solution I and 10.8 g dry benzoyl peroxide.Polymerization was initiated by adding 600 ml accelerator (N,Ndimethyl-p-toluidine) to 100 ml cold solution III followed by stirring, which was added to glass vials containing the inltrated artery sample.As the polymerization process is sensitive to oxygen, glass vials were completely lled with the mixture and tightly capped to avoid oxygen contact.Next, the vials were transferred to a deep freezer for 3 days to complete the polymerization process.
Aer polymerization, plastic blocks were carefully removed from the glass vials and were trimmed to the desired size for sectioning.The 3-5 mm thick sections were cut using a rotary microtome (Leica RM2255) by keeping the block and blade moist with 30-40% methanol.Sections were transferred to slides coated with poly-L-lysine and carefully stretched or attened using a rubber roller.Next, the slides were covered with polyethylene and clamped to a slide press, which was followed by incubation at 42 °C for 2 days.The de-plasticization of the sections was performed by employing two changes of dichloromethane and acetone for 20 and 10 min, respectively.Finally, sections were subjected to hematoxylin-eosin staining and analyzed under a light microscope (Nikon, model Eclipse Ti-E) at 4× magnication to evaluate the clot lysis efficacy of implanted NETS following NIR laser irradiation.

Statistical methods
Data are presented as mean ± SEM of at least 3 independent experiments.Two-tailed Student's t-tests were performed using GraphPad Prism soware to evaluate the signicance in difference between groups.Values were considered signicant when p < 0.05.

Ethical statement
All animal procedures were carried out in strict adherence to the Guidelines for Care and Use of Laboratory Animals of Banaras Hindu University.The experimental protocols were approved by the Animal Ethics Committee of Banaras Hindu University (IAEC-3258).

Characterization of coated thermogenic stents
The thin plasmonic lm of gold on the stent surface achieved by employing GNRs was characterized by SEM and EDX.The SEM images signied the rough surface associated with coated stents as compared to bare metal stents (Fig. 1), which was further examined by EDX analysis (Fig. 2).Chemical microanalysis of the coated stent conrmed the presence of an adequate quantity of the thin plasmonic lm of gold on the thermogenic stent surface (Fig. 2).Raman spectra were used to examine the coating efficacy on the stent by assessing the structural and vibrational characteristics of gold.The image captured using a 50× magnication objective lens (Zeiss) of a Raman instrument showed a dense layer of coating on the stent surface (ESI Fig. 3A †).The observed prominent peak in the Raman spectrum clearly indicated the presence of gold. 51FTIR spectroscopy was performed to examine the coating on the stent surface.The spectrum of the coated stent exhibited characteristic peaks at ∼2930, 2850 and 1430 cm −1 that correspond to the presence of a thin plasmonic lm of gold 52,53 (ESI Fig. 3B †).It may be noted here that annealing at temperatures below 100 °C also yielded satisfactory results.
To investigate the stability of the glossy coating on the stent surface against hydrodynamic shear of perpetual intramural blood ow, the buffer was allowed to perfuse over the coated stents in vitro at the arterial shear rate (1500 s −1 ), which closely simulated uidics in vivo. 32The buffer was allowed to ow over the stent for 6 to 8 h continuously each day for 30 days.The stability of the glossy coating was then veried from the uniformity in heat generated upon irradiation with the NIR laser on different days up to the 30th day.Temperature was recorded to be 60 °C, 61 °C, 58 °C and 59 °C, respectively, on days 0, 10, 20 and 30 (ESI Fig. 4 †), which underscored the lack of erosion of the gold coating by hydrodynamic shear over time.

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Characterization of thrombolysis induced by the heat generated from NIR laser-irradiated thermogenic stents in vitro In order to study the efficiency of NETS in inducing photothermal ablation of clots, a thrombus was allowed to form around the stent placed within a glass cuvette followed by irradiation with the NIR laser for 30 min, which raised the temperature up to 50 °C.The extent of thrombolysis was evaluated by three standard assays as described earlier. 32Photothermal brinolysis was found to be 57% higher in the uorescence-based assay whereas the rise was 73% each in Drabkin's and methylene blue assays, respectively (Fig. 3A-E).
In order to evaluate stent thrombolysis under arterial hydrodynamic shear resembling the physiological milieu, the uid was allowed to perfuse over the stent carrying a uorescent thrombus at a shear rate of 1500 s −1 (ESI Fig. 2 †).The extent of thrombolysis as quantied from the release of Alexa 488-labeled brin was found to be 35% (Fig. 3F), which thus conrmed photothermal ablation of stent thrombosis.The heat-induced alteration in brin clot structural morphology following 30 min NIR laser irradiation was evaluated using orthogonal tools like SEM and CLSM.The SEM images were consistent with a signicant 12-fold increase in thrombus-depleted areas (ESI Fig. 5A-C †) associated with a drop (by 5%) in brin diameter (ESI Fig. 5D and E †).In agreement, confocal images too demonstrated a nearly 5 times expansion in areas of thrombus depletion examined at different  Thermal destabilization of in-stent/peri-stent thrombi following 15 min NIR-laser exposure was further validated employing FRAP analysis.FRAP characterizes the dynamism of brin monomers/oligomers within the clot by quantifying brin mobility and turnover kinetics. 32The in-stent clot was focused on and observed under a microscope (Fig. 4A-C).A narrow region within the uorescent thrombus was photobleached using a 488 nm laser (at 100% power) and uorescence recovery in that region was recorded over time at 2% excitation laser power in both control (Fig. 4D-F), as well as NIR laser-irradiated (Fig. 4G-I) thrombi.The speed of uorescence recovery in thermally loosened brin strands was reective of the extent of brin mobility within destabilized thrombi, which amounted to 13.4% mobile brin fractions in NIR laser-irradiated stents as Fig. 3 Photothermal ablation of in-stent/peri-stent clots exposed to the NIR laser for 30 min.Clots were generated by addition of 2-5 mM CaCl 2 and 1 U ml −1 thrombin to a solution of fibrinogen (1 mg ml −1 ) carrying the thermogenic stents, followed by irradiation with the NIR (808 nm) laser at a power density 1.05 W cm −2 .Whenever required, the fluorescent fibrin clot was generated by addition of Alexa Fluor 488conjugated fibrinogen (10% v/v) to the above solution.Thus, results from this study underscore enhanced brin dynamism within the in-stent thrombi following photothermal heat generation that could lead to thrombus rarecation.
Photothermal lysis of in-stent thrombi and partial restoration of blood ow upon NIR laser irradiation of implanted NETS Rats medicated with oral aspirin (10 mg kg −1 d −1 ) for 5 days 42 were anesthetised.The coated thermogenic stents (length, 2.5 mm; diameter, 635 mm; strut thickness, 70 mm) were carefully guided into and deployed in the murine common carotid artery (Fig. 5 and 6A), as described.The anatomical and optoacoustic imaging of the implanted stent was carried out in B (ultrasound)-mode and PA (photoacoustic)-mode by employing a photoacoustic imaging platform (Fig. 6B and C).The thrombus was induced locally by topical application of 10% FeCl 3 , which was followed by irradiation with a 3 W solid-state continuous diode laser (808 nm) for 15 min.Temperature around the stent was found to be elevated up to 47 °C following laser exposure as captured by a thermal camera.Blood ow was assessed by placing a Doppler ow probe on the exposed artery, the signal from which was collected through a transonic ow probe and further analysed.Restriction in blood ow due to the growing thrombus was found to be 98%, which was partially restored (by 30%) upon laser irradiation (Fig. 6D and E), underscoring the applicability of the novel NETS as an essential tool against stent-thrombosis.The hematoxylin-eosin-stained sections of stented carotid segments revealed a signicant reduction in size of the in-stent thrombus in laser-treated rats

Discussion
In this report we demonstrate the potential of 'thermogenic' stents (NETS) in bringing about relief from 'stent thrombosis', a life-threatening complication that warrants urgent interventions like repeat angioplasty and/or CABG surgery.Notably, none of the existing stent technologies permits lysis of the instent thrombus, thus underlining the need for novel approaches to counter the threat of scaffold thrombosis.Subjects carrying coated NETS can be irradiated frontally from an external NIR laser source upon diagnosis of stent thrombosis.The heat generated from the deployed stent would result in ablation/rarecation of the thrombus in and around the implant leading to restoration of blood ow that can be monitored with color Doppler.Although we have observed ∼30% restoration in blood ow following 15 min irradiation with the laser, the duration of laser exposure can be expanded to achieve greater relief from occlusion.The occluded stent can thus become functional again in the absence of any painful invasive procedure or expensive setup and the patient can be discharged early.A coating of a NIR-active material on the stent surface was found to be stable against uid shear and can coexist with the drug-eluting polymer.Photothermal therapy (PTT) is a well-established but burgeoning eld of medical research that has been widely employed in wound healing, [14][15][16][17] treatment of bacterial infection, [18][19][20][21] pain relief, 22,23 drug delivery [24][25][26][27] and for selective thermal ablation of malignant tissues [54][55][56][57][58] at temperatures above 40 °C, which ensues protein denaturation and disruption of target cell membranes.3][34][35] These studies opened newer areas of application for thermal therapy against mural thrombosis in addition to its well reported application against tumors.
The effect of temperature on tissues surrounding the deployed stent is the subject of future investigation.Temperature at the 'heat focus' area (maximum ∼47 °C) is signicantly quenched by the ongoing blood ow (2 mm s −1 ), thus restricting the temperature in the surrounding blood from exceeding ∼43 °C. 59Temperatures in the intimal layer and subendothelium in direct contact with the stent drop nonlinearly with depth that is further compensated by the blood ow. 59onsidering the thickness of intima to be ∼600 mm, 60 conduction of heat to cardiac tissues is tolerated without seriously compromising cell viability and primarily localized to the intima.
][63] Hyperthermia, too, has successfully been used for controlled site-specic drug delivery around the implanted magnetizable stent when applied under a uniform magnetic eld. 64There have been several approaches in the past to expose the stent in situ to local heat though each procedure has its share of limitations.These include invasive procedures like insertion of a special thermal catheter into the deployed stent, 65 or guiding the NIR beam through the optical ber to endovascular locations, 59,66,67 as well as non-invasive methods like induction (eddy-current) heating of stents using electromagnetic elds. 62,68,69Non-invasive attempts have been made like induction (eddy-current) heating of stents using electromagnetic elds, 62 or forming a radiofrequency resonant circuit. 70PTT and photothermal effect-induced drug release have also been explored previously as a modality to prevent in-stent restenosis by employing gold nanoparticle-coated multifunctional stents. 59he penetration depth of the NIR laser at 808 nm is about 2 cm, 36,69 which can be further enhanced by switching to longer wavelength NIR (1000-1350 nm; bio window II), 57,71 thus allowing non-invasive photothermal ablation of stent thrombosis when irradiated from an external source.Alternatively, the occluded stent can be irradiated with an optical ber-guided NIR laser 72,73 though guiding the NIR beam to endovascular locations for PTT still remains challenging.In conclusion, this study for the rst time presents a proof-of-concept in favour of deployment of thermogenic coronary scaffolds as a guard against stent thrombosis and requires further investigation towards clinical validation.

Nanoscale Advances Paper
of Scientist-C position from ICMR under CAR.We gratefully Paper Nanoscale Advances
Fig.3Photothermal ablation of in-stent/peri-stent clots exposed to the NIR laser for 30 min.Clots were generated by addition of 2-5 mM CaCl 2 and 1 U ml −1 thrombin to a solution of fibrinogen (1 mg ml −1 ) carrying the thermogenic stents, followed by irradiation with the NIR (808 nm) laser at a power density 1.05 W cm −2 .Whenever required, the fluorescent fibrin clot was generated by addition of Alexa Fluor 488conjugated fibrinogen (10% v/v) to the above solution.Drabkin's assay (A) and (B) and methylene blue assay (C) and (D) were carried out in order to evaluate the extent of clot lysis.(E) In-stent or peri-stent clot lysis analyzed employing an Alexa Fluor 488-labeled thrombus.(F) Lysis of a fluorescently labeled stent thrombus exposed to arterial hydrodynamic shear (1500 s −1 ).Data are representative of five different sets of experiments (mean ± SEM).*P < 0.05; **P < 0.01; ****P < 0.0001, vs. control.

Fig. 4
Fig. 4 FRAP analysis of a laser irradiated in-stent thrombus.(A-C) Confocal images of an in-stent fluorescent clot (10×) representing fluorescent (A), differential interference contrast (DIC) (B) and merged (C) images.(D-I) Pre-bleached, post-bleached and fluorescence recovery images (63×) of control and NIR laser-irradiated samples, as stated.Arrows indicate the regions of interest (ROI) on the fluorescent thrombus.(J) Kinetics of fluorescence recovery in control and NIR laser-treated samples.

Fig. 5
Fig.5Scheme for grafting a thermogenic stent in the common carotid artery of a rat, induction of a thrombus and photothermal thrombolysis.

Fig. 6
Fig. 6 Photothermal lysis of an in-stent thrombus and restoration of the blood flow.(A) Photograph of NETS implanted in the rat common carotid artery; (B) and (C) photoacoustic imaging of implanted NETS in B-mode (ultrasound) and PA-mode (photoacoustic), respectively.The arrow indicates the position of the stent.(D) Measurement of blood flow velocity in the rat common carotid artery with the implanted stent with a flow meter showing occlusion of blood flow due to the growing thrombus and subsequent restoration of flow following NIR laser irradiation.(E) Scatter dot plots representing the corresponding flow velocities after 15-20 min irradiation with the NIR laser or controls without laser exposure (n = 7 in each group).Each dot in the scatter plots represents an independent observation.**P < 0.01 vs. control.(F) and (G) Light microscopy of hematoxylin-and eosin-stained transverse sections of stented carotid segments harvested post-surgery from sacrificed animals with or without irradiation with the NIR laser (the arrow indicates reduction in thrombus size; scale, 100 mm).