Photo-mediated ultrasound therapy for cutaneous vascular malformation

Abstract

Provided herein are systems and methods for decreasing blood vessel density, removing blood vessels or capillaries, and / or treating a cutaneous vascular malformation (e.g., port-wine stain) in a subject. The methods comprise applying a laser pulse(s) spatiotemporally synchronized with an ultrasound burst(s) to at least a portion of target area (e.g., an affected skin surface or tissue up to 3 mm from the skin surface).

Description

[0001] PHOTO-MEDIATED ULTRASOUND THERAPY FOR CUTANEOUS VASCULAR MALFORMATION

[0002] FIELD

[0003] Provided herein are systems and methods for decreasing blood vessel density, removing blood vessels or capillaries, and / or treating a cutaneous vascular malformation (e g., port-wine stain) in a subject.

[0004] CROSS REFERENCE TO RELATED APPLICATIONS

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 571,599, filed March 29, 2024, the content of which is herein incorporated by reference in its entirety.

[0006] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0007] This invention was made with government support under EB030875 and EY029489 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0008] BACKGROUND

[0009] Cutaneous vascular malformations (CVM), such as port wine stain (PWS), which occurs in 0.3 to 0.5% of newborns, are often a cause of great concern to patients for both medical and cosmetic reasons. Laser irradiation with flashlamp-pumped pulsed dye lasers, e.g., photothermolysis therapy (PTT), is currently the gold standard treatment for PWS. Most patients with PWS, however, fail to clear completely after PTT. Photodynamic therapy (PDT) offers an alternative method for treatment of PWS. PDT requires the systemic injection of photosensitizers, which necessitates the avoidance of sun exposure for up to a week after treatment, time-dependent infusion, and systemic side-effects with suboptimal treatment effect. Thus, effective treatment methods with limited side effects are needed for PWS and other CVM.

[0010] SUMMARY

[0011] Provided herein are systems and methods for decreasing blood vessel density and / or removing blood vessels or capillaries at a target site in a subject comprising applying to the target site a laser pulse spatiotemporally synchronized with an ultrasound burst and laser pulse. In some embodiments, the ultrasound burst has a peak negative pressure of about 0.1 to about 5 MPa. In some embodiments, the ultrasound burst has a peak negative pressure of 0.5-1.5 MPa. In some embodiments, the ultrasound burst has a peak negative pressure of 0.70-0.75 MPa.

[0012] In some embodiments, the laser pulse is delivered by a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. In some embodiments, the laser pulse is at about 400 to about 1600 nm. In some embodiments, the laser pulse is at about 1064nm. In some embodiments, the laser pulse is at about 532nm.

[0013] In some embodiments, the laser pulse has a laser fluence greater than 1 mJ / cm2. In some embodiments, the laser pulse has a laser fluence greater than 100 mJ / cm2. In some embodiments, the laser fluence is less than 1 J / cm2. In some embodiments, the laser fluence is between 150 mJ / cm2and 1 J / cm2. In some embodiments, the laser fluence is between 500 mJ / cm2and 1 J / cm2. In some embodiments, the laser fluence is about 700 mJ / cm2.

[0014] In some embodiments, the laser pulse length is between 0.1 ns to 100 ns.

[0015] In some embodiments, the method comprises a single synchronized ultrasound burst and laser pulse.

[0016] In some embodiments, the spatiotemporally synchronized laser pulse and ultrasound burst are repeated at a repetition frequency of between 1 Hz and 1000 Hz. In some embodiments, the repetition frequency is about 5 Hz to about 20 Hz. In some embodiments, the repetition frequency is about 15 Hz. In some embodiments, the repetition frequency is about 10 Hz.

[0017] In some embodiments, the applying lasts for at least 10 seconds. In some embodiments, the applying lasts for 1 to 10 minutes. In some embodiments, the applying lasts for 4 to 6 minutes.

[0018] In some embodiments, the target site is a skin surface or tissue up to 3 mm from the skin surface. In some embodiments, the target site is a skin surface or tissue up to 1 mm from the skin surface.

[0019] In some embodiments, the subject has a cutaneous vascular malformation. In some embodiments, the subject has a hypervascular dermal disease. In some embodiments, the subject has port-wine stain (PWS).

[0020] Also provided herein are methods for treating a cutaneous vascular malformation in a subject. In some embodiments, the methods comprise applying a laser pulse spatiotemporally synchronized with an ultrasound burst to at least a portion of an affected skin surface or tissue up to 3 mm from the skin surface. In some embodiments, the affected skin surface or tissue is up to 1 mm from the skin surface.

[0021] In some embodiments, the ultrasound burst has a peak negative pressure of about 0.1 to about 5 MPa. In some embodiments, the ultrasound burst has a peak negative pressure of 0.5-1.5 MPa. In some embodiments, the ultrasound burst has a peak negative pressure of 0.70-0.75 MPa.

[0022] In some embodiments, the laser pulse is delivered by a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. In some embodiments, the laser pulse is at about 400 to about 1600 nm. In some embodiments, the laser pulse is at about 1064nm. In some embodiments, the laser pulse is at about 532nm.

[0023] In some embodiments, the laser pulse has a laser fluence greater than 1 mJ / cm2. In some embodiments, the laser pulse has a laser fluence greater than 100 mJ / cm2. In some embodiments, the laser fluence is less than 1 J / cm2. In some embodiments, the laser fluence is between 150 mJ / cm2and 1 J / cm2. In some embodiments, the laser fluence is between 500 mJ / cm2and 1 J / cm2. In some embodiments, the laser fluence is about 700 mJ / cm2. In some embodiments, the laser fluence is between about 250 mJ / cm2and about 500 mJ / cm2.

[0024] In some embodiments, the laser pulse length is between 0.1 ns to 100 ns.

[0025] In some embodiments, the method comprises a single synchronized ultrasound burst and laser pulse.

[0026] In some embodiments, the laser pulse and ultrasound burst are repeated at a repetition frequency of between 1 Hz and 1000 Hz. In some embodiments, the repetition frequency is about 5 Hz to about 20 Hz. In some embodiments, the repetition frequency is about 15 Hz. In some embodiments, the repetition frequency is about 10 Hz.

[0027] In some embodiments, the applying lasts for at least 10 seconds. In some embodiments, the applying lasts for 1 to 10 minutes. In some embodiments, the applying lasts for 4 to 6 minutes.

[0028] In some embodiments, the subject has a hypervascular dermal disease. In some embodiments, the subject has port-wine stain (PWS).

[0029] Also provided herein are systems comprising one or more devices (e.g., lasers, ultrasound devices, sensors, imaging devices, displays, amplifiers, etc.), and / or control software that conducts any of the above methods or other methods described herein. For example, in some embodiments, the system comprises a computer processor running a program that comprises one or more of the following features: laser activation and deactivation, ultrasound activation and deactivation, laser and / or ultrasound param eter / settings management, sensor monitoring and data collection, and imaging and display control.

[0030] Other aspects and embodiments of the disclosure will be apparent in light of the following detailed description.

[0031] BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGS. 1A-1C show representative treatment outcomes from a chicken wattle treated with Photo-mediated Ultrasound Therapy (PUT). FIG. 1 A shows photographs of the chicken wattle taken before and at different time points (days 1 and 14) after PUT treatment as well as laser- only and ultrasound-only treatments. FIG. IB shows the B-scan OCT-A images overlapped on the B-scan OCT images acquired along the center of the treatment region shown in a.l-a.3. Images were acquired immediately before and at different time points (1 day, 14 days) after PUT treatment. FIG. 1C shows maximum intensity projection images of 3D OCT-A of the areas marked in the red dash squares. The white circles mark the treated area. 3D, three-dimensional; FUS, focused ultrasound; OCT, optical coherence tomography; OCT-A, optical coherence tomography angiography; PUT, photo-mediated ultrasound therapy.

[0033] FIGS. 2A-D show the quantitative assessment of vessel density changes in chicken wattles treated with either PUT, laser only, or ultrasound only. FIG. 2A shows photographs of a chicken wattle with 1 PUT-, 1 ultrasound-only-, and 1 laser-only-treated regions at different time points. FIG. 2B shows maximum intensity projection images of 3D OCT-A (0-150 pm depth in the dermis) of the PUT -treated region at different time points. FIG. 2C shows vessel density maps generated by binarization of the maximum intensity projection images in FIG. 2B. FIG. 2D shows normalized vessel density of all the regions treated with either PUT (n=7), laser only (n=7), or ultrasound only (n=7) from day 0 (before treatment) to day 21 after treatment, represented by mean ± SD. **P < 0.01 for paired t-test between the measurement at each time point and the baseline measurement on day 0. Day 0 (before the treatment), Day 1 after the treatment, Day 7 after the treatment, Day 14 after the treatment, and Day 21 after the treatment. 3D, three-dimensional; FUS, focused ultrasound; OCT, optical coherence tomography; OCT-A, optical coherence tomography angiography; PUT, photo-mediated ultrasound therapy.

[0034] FIGS. 3 A-3D show H&E-stained section of a wattle tissue harvested on day 21 after PUT treatment. FIG. 3 A is an H&E-stained section of a wattle tissue harvested on day 21 after PUT treatment, 10 magnification. The red dash square marks the treated area. FIG. 3B is an enlarged view of a treated area close to the epidermis (0-400 pm depth), X20 magnification. No blood vessels remain in the treated region. FIG. 3C is an enlarged view of a treated area at 400-800 pm depth, X20 magnification. Only large vessels remain. FIG. 3D is an enlarged view of an untreated area where many blood vessels can be seen, X20 magnification. Bv denotes blood vessels, Ep denotes epidermis, and De denotes dermis. PUT, photo-mediated ultrasound therapy.

[0035] FIGS. 4A-4D show H&E-stained section of a wattle tissue harvested on day 7 after PUT treatment. FIG. 4A is an H&E-stained section of a wattle tissue harvested on day 7 after PUT treatment, X10 magnification. The red dash square marks the treated area. FIG. 4B is an enlarged view of a treated area close to the epidermis (0-400 pm depth), X20 magnification. The vessel lumens are filled with erythrocytes, endothelial cells have degenerated, and the overlying epidermis is intact. FIG. 4C is an enlarged view of a treated area at 400-800 pm depth, x20 magnification. FIG. 4D is a further enlarged view of a treated area at 800 pm depth, X40 magnification. PUT, photo-mediated ultrasound therapy.

[0036] FIGS. 5A-5F show H&E-stained sections of wattle tissues. Shown are wattle tissues harvested on day 1 (FIG. 5 A), day 7 (FIG. 5B), day 14 (FIG. 5C), and day 21 (FIG. 5D) after PUT treatment, 10 magnification. FIG. 5E is an H&E-stained section of a wattle tissue harvested on day 21 after laser-only treatment, X10 magnification. FIG. 5F is an H&E-stained section of a wattle tissue harvested on day 21 after ultrasound-only treatment, x 10 magnification. In each one, the treated area is marked by the red dash square. PUT, photo-mediated ultrasound therapy.

[0037] FIGS. 6A-6D show safety evaluation on day 3 after PUT treatment. FIG. 6A is a CD31- stained section of a wattle tissue, including a treated area. FIG. 6B is a CD31 -stained section of a wattle tissue that was untreated. FIG. 6C is an H&E-stained section of the same wattle tissue in FIG. 6A, including a treated area. FIG. 6D is an H&E-stained section of the same wattle tissue in FIG. 6B that was untreated. PUT, photo-mediated ultrasound therapy.

[0038] FIGS. 7A-7D show safety evaluation on day 3 after PUT treatment. FIG. 7A is caspase- 3-stained section of a wattle tissue, including a treated area. FIG. 7B is caspase-3 -stained section of a wattle tissue that was untreated. FIG. 7C is an MTC-stained section of the same wattle tissue in FIG. 7A, including a treated area. FIG. 7D is an MTC-stained section of the same wattle tissue in FIG. 7B that was untreated. MTC, Masson’s trichrome; PUT, photo-mediated ultrasound therapy.

[0039] FIGS. 8 A and 8B are a data processing schematic. Two cases are presented for the chicken wattle tissue immediately before PUT treatment (FIG. 8A) and 14 days after PUT treatment (FIG. 8B). The left hand top images (a.1 and b.l) show the B-scan OCT-A images overlapped on the B-scan OCT images along the center of treatment regions marked in the center image. The left hand middle images (a.2 and b.2) show the surface of the skin and the area of the epidermis segmented from the B-scan OCT images. The left hand lower images (a.3 and b.3) show the segmented layer with 0-150 pm depth in the dermis for analyzing the OCT-A signals. The middle images (a.4 and b.4) show en face projections of the OCT-A signals in the segmented layer. The right hand images (a.5 and b.5) show binarization results of the en face projections of the OCT-A signals in the segmented layer. OCT, optical coherence tomography; OCT-A, optical coherence tomography angiography; PUT, photo-mediated ultrasound therapy.

[0040] FIGS. 9A-9D show a system schematic and experiment setup for PUT treatment of chicken wattle. FIG. 9A is a detailed schematic of the integrated ultrasound and laser system for PUT treatment of chicken wattle. FIG. 9B is a photo of the experimental setup showing positions of an agar-gelatin gel cone and a PDMS pad. FIG. 9C is a photo of a chicken wattle positioned between the PDMS and the gel cone during the treatment. FIG. 9D is a pretreatment picture of a chicken wattle, with marked circles indicating the spots for treatment. FUS, focused ultrasound; PDMS, polydimethylsiloxane; PUT, photo-mediated ultrasound therapy.

[0041] FIGS. 10A-10C show the theoretical modeling of the pre-existing bubble dynamics under different light fluence and ultrasound pressure during PUT treatment of a blood vessel. FIG. 10A, left (a.1), shows a simulated photoacoustic (PA) wave near the center of a blood vessel with a diameter of 0.1 mm when illuminated by a 3-ns light pulse at 1064 nm wavelength. FIG. 10A, center (a.2), shows concurrently applied ultrasound burst with 0.25 MHz frequency and 1 MPa pressure amplitude. FIG. 10A, right (a.3), shows a combined PA waveform and ultrasound wave, where the PA wave is synchronized at the negative phase of an ultrasound cycle. FIG. 10B shows simulated bubble size evolution under different combinations of treatment parameters, including 0.6 MPa ultrasound and 100 mJ / cm2light fluence (FIG. 10B, left (b. l)), 0.8 MPa ultrasound and 120 mJ / cm2light fluence (FIG. 10B, center (b.2)), and 1.0 MPa ultrasound and 120 mJ / cm2light fluence (FIG. 10B, right (b.3)). R / R0 stands for dynamic bubble size R over its initial size R0. FIG. 10C, left, shows a simulated rectified diffusion threshold (e.g., cavitation threshold) for different initial bubble sizes and at different levels of light fluence. FIG. 10C, right, shows a simulated dynamics of a 100-nm pre-existing bubble under the treatment of different combinations of ultrasound rarefaction pressure and light fluence, where the bubble dissolves (e.g., no treatment effect) in the blue region and grows (e.g., effective treatment of blood vessel) in the yellow region. The dashed red line indicates the ultrasound pressure for cavitation threshold which decreases with the increased light fluence.

[0042] FIGS. 11A-11C show in situ validation of cavitation activity in a blood vessel phantom treated by PUT. FIG. 11 A is a schematic of the experimental setup used in the phantom study. FIG. 1 IB is representative ultrasound imaging frames when the applied ultrasound pressure was fixed at 0.6 MPa while the light fluence was changed from 0 to 200 mJ / cm2. FIG. 11C shows the cavitation probability measured from the blood vessel phantom treated with different levels of ultrasound pressure (0-1.4 MPa) and light fluence (0-200 mJ / cm2).

[0043] FIG. 12 shows the setup of in vivo PUT treatment of the blood vessels in chicken wattle. The block at the top right corner shows the strategy of PUT treatment of deep vessels, which is to deliver increased ultrasound pressure in deep skin to compensate for the light attenuation in overlying tissues.

[0044] FIGS. 13A-13C show representative treatment outcome from a chicken wattle treated with PUT. FIG. 13 A is polarized dermoscope images of the chicken wattle with three PUT treatment areas (labelled by white dash circles) taken before and at different time points (Day 1 and Day 7) after the treatment. FIG. 13B is maximum intensity projection images of 3D OCT-A of the three treatment areas and one untreated area (control) on the same chicken wattle. Scale bar: 1 mm. FIG. 13C is a graph of normalized vessel density measured by OCT-A of the regions treated with PUT (n = 12) and untreated regions (n = 4) at Day 1 and Day 7 after treatment, represented by mean ± SD. ***P < 0.001 for paired t-test between the measurements from untreated regions vs. treated regions, indicating that the treatment reduced the vessel density at both the top and the bottom sides of the wattles.

[0045] FIGS. 14A-14C show representative H&E histology (FIG. 14A), CD31 immunohistochemistry (FIG. 14B), and RMP histochemistry (FIG. 14C) photos from a chicken wattle harvested at Day 7 after PUT treatment.

[0046] FIGS. 15A-15E show histopathological analyses across the entire section of a chicken wattle conducted at Day 7 post-treatment. FIG. 15A is an H&E stained section, including the original photo and the zoom-in photos from two treated areas (a.2 from the top and a.3 from the bottom of the treated region) vs. an untreated area (a.l) as the control. Endothelial necrosis can be seen in both the top (a.2) and the bottom (a.3) of the treated region. FIG. 15B is CD31 stained IHC from an adjacent section, including the original photo and the zoom-in photos from the same three areas (b.2 and b.3 from the top and the bottom of the treated region, and b.l from the untreated area). In the original photo, the endothelial cells are highlighted in red. In the zoom-in photos, original brightfield images demonstrate CD31 negative on treated vessels. FIG. 15C is RMP histochemical stain from an adjacent section, including the original photo and the zoom-in photos from the same three areas (c.2 and c.3 from the top and the bottom of the treated region, and c.1 from the untreated area). Thrombus in vessel lumens (bright red) in the treated areas can be seen, while no obvious change in collagen (yellow) is noticed. FIG. 15D is quantified vascular densities in the treated tissues vs. the untreated tissues by analyzing the IHC CD31 results. p<0.05 when comparing the two groups via a paired t-test (n=10 for each group). FIG. 15E is quantified collagen densities in the treated tissues vs. the untreated tissues by analyzing the RMP results. No statistically significant difference is noticed when comparing the two groups (n=6 for each group). For original photos, scale bar=500 pm. For zoom-in photos, scale bar=50 pm.

[0047] DETAILED DESCRIPTION

[0048] Provided herein are methods for treatment of cutaneous vascular malformations utilizing effective and highly selective anti-vascular therapy techniques, termed Photo-mediated Ultrasound Therapy (PUT). PUT is based on microcavitations in blood vessels produced by synergistically applied laser pulses and ultrasound bursts. As shown herein, PUT is capable of eliminating cutaneous vessels without causing unwanted collateral damage to the surrounding skin tissue (as shown in a clinically relevant chicken wattle model of PWS in vivo), offering an alternative solution for treatment. Additionally, PUT can precisely remove cutaneous vessels without damaging surrounding skin tissue. As demonstrated herein, using a laser fluence of only 0.707 J / cm2at a wavelength of 1064 nm combined with ultrasound bursts, achieved a decrease in blood vessel density in the chicken wattle by 73.23% with a treatment depth of up to 1 mm from the skin surface after a single PUT treatment without causing collateral thermal damage in surrounding tissues. Thus, PUT technique facilitates targeted and precise anti-vascular therapy while reducing the treatment burden and side effects.

[0049] The PUT treatment used in this study has a mechanism of action different from that of traditional PDL treatment. The widely used PDL therapy is based on the principle of selective heating of targets on the basis of the absorption of laser energy, also known as selective photothermolysis. In selective photothermolysis, the selected targets, such as capillaries and postcapillary venules in the case of PWS, absorb laser energy and are destroyed owing to heating without causing any significant damage to the surrounding tissues. In contrast, PUT, which uses a laser pulse spatiotemporal ly synchronized with an ultrasound burst, induces vascular bioeffects through the mechanical effects of cavitation. The nanosecond laser pulse irradiation of a vessel generates a photoacoustic wave, which results in high rarefaction pressure near the center of the vessel. This photoacoustic wave, when superimposed on the peak negative pressure of an ultrasound wave, results in high rarefaction pressure and nucleates air cavities in the blood, also known as the photospallation effect. The nucleated air cavities or bubbles expand and compress inside the blood vessel owing to the simultaneous presence of an ultrasound wave. The PUT- induced cavitation interacts with blood vessels and generates stresses on the vessel wall. The stresses, mainly shear stress, impact the vascular endothelial cell functions and alter the release of vasoactive agents such as nitric oxide and prostacyclin, which ultimately results in vasculature degeneration. Additionally, the laser fluence used in PUT is lower (e.g., 10-100 times lower) than that used in traditional PDL therapy.

[0050] Based on the mechanistic differences, two main potential advantages of PUT over the traditional PDL therapy are apparent for PWS: minimized thermal effect and treatment depth. Unwanted thermal damage cannot be completely avoided in pure laser-based PDL treatment, even with mitigation efforts, owing to the use of high laser fluence and millisecond domain laser pulse durations, necessary to induce sufficient heating in target vessels. As shown herein, the optimized laser fluence was 0.707 J / cm2for the 1064 nm laser with a pulse duration of 3-5 ns. During the treatment, the temperature on the wattle surface increased from an average of 23 to 35 °C during the 4-minute treatment. This increase in temperature during PUT was mainly due to the simultaneously applied ultrasound instead of the laser pulses. A temperature increase <20 °C from the baseline of 23 °C is considered safe because it is still below the denaturation threshold of collagen. Immunohistochemistry and histology analyses shown herein involving CD31, H&E, caspase-3, and MTC stains provide evidence that PUT-induced therapeutic effect is confined and specific to blood vessels only, whereas unwanted collateral damage in other skin tissues such as collagen are avoided.

[0051] The OCT-A images shown herein indicate that almost all the blood vessels within the depth range of 0 to 150 pm in the dermis of the chicken wattle were removed by PUT. However, this depth is not the limitation of the PUT treatment depth but instead the OCT-A imaging depth, which is limited by the extremely dense vasculature in chicken wattle tissue. The histology and immunohistochemistry results further validated that the treatment depth of PUT could reach up to 0.8 mm. Further analysis showed that PUT can effectively penetrate the entire thickness of chicken wattle tissue, which is about 3 mm, and significantly reduce blood vessel density by 45.20% with a light fluence 10-100 times less than the fluence used in traditional PDL therapy. OCT-A imaging showed local blood perfusion was significantly reduced, and the reduced blood perfusion persisted for at least 7 days post-treatment in the treated areas. Histopathological analyses based on H&E, CD31, and Russell-Movat Pentachrome (RMP) stains confirmed effective and selective vascular damage through the entire thickness of chicken wattle without causing collateral thermal damage.

[0052] It is technically possible to achieve a much greater treatment depth in PUT using 1064 nm light, which has the highest penetration depth and lowest melanin absorption among all the laser wavelengths currently used for treatment of PWS. The 1064 nm laser can be safely used in PUT without causing collateral thermal damage in background skin tissues because PUT treatment of blood vessels is not based on the laser-based heating of vessels but instead based on the photoacoustic effect induced by nanosecond laser pulses. When working with spatiotemporally synchronized ultrasound bursts, PUT can eliminate blood vessels when utilizing much lower light energy levels. Apart from the laser wavelength, the treatment depth of PUT also depends on spatial synchronization between ultrasound and laser. The treatment threshold in PUT depends on the combination of ultrasound bursts and laser pulses. Ultrasound can penetrate deeper into the skin without much scattering, whereas laser energy decreases much faster with the depth of penetration owing to the strong scattering and absorption of light in skin tissues. With PUT, the low laser energy at greater dermal depths can be compensated for by focusing the ultrasound energy at specific locations. Hence, a combination of high ultrasound energy and low laser energy at deep dermis and a combination of low ultrasound energy and high laser energy at superficial dermis can both produce cavitation of PWS vessels. This positions PUT as a flexible and effective treatment for a wide variety of these heterogeneous vascular lesions.

[0053] Definitions

[0054] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. However, two or more copies are also contemplated. The singular forms “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0055] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated.

[0056] A “subject” or “patient” may be human or non-human and may include, for example, animal strains or species used as “model systems” for research purposes as described herein. Likewise, patient may include either adults or juveniles (e.g., children). Moreover, patient may mean any living organism, preferably a mammal (e.g., human or non-human) that may benefit from the administration of compositions contemplated herein. Examples of mammals include, but are not limited to, any member of the Mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. Examples of non-mammals include, but are not limited to, birds, fish, and the like. In one embodiment, the mammal is a human.

[0057] As used herein, “treat,” “treating,” and the like means a slowing, stopping, or reversing of progression of a disease or disorder when provided a peptide or composition described herein to an appropriate subject. The term also includes a reversing of the progression of such a disease or disorder to a point of eliminating or greatly reducing the disease. As such, “treating” means an application or administration of the peptides or compositions described herein to a subject, where the subject has a disease or a symptom of a disease, where the purpose is to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease or symptoms of the disease.

[0058] Unless otherwise defined herein, scientific, and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear; in the event, however of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0059] Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present disclosure. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting.

[0060] Methods

[0061] The disclosure provides systems and methods for decreasing blood vessel density and / or removing blood vessels or capillaries at a target site in a subject. In some embodiments, the target site is a skin surface and tissue up to 3 mm from the skin surface. Accordingly, the disclosure also provides methods for treating a cutaneous vascular malformation in a subject.

[0062] Cutaneous vascular malformation are characterized by discolored, e.g., pink, red, blue, or purple, patches on the skin. Usually they are smooth and flat, but may be raised or bumpy. The discoloration may increase or decrease over time with age, as may the topography (e.g., roughness / bumpiness) of the patches. The malformation may be on any skin surface, including the face, neck, scalp, arms, back, shoulder, or legs. The majority of vascular malformations occur as sporadic anomalies. However, some are familial. The cutaneous vascular malformation may be due to abnormally developed or prevalent blood vessels in the affected areas.

[0063] In some embodiments, the subject has or is suspected of having a hypervascular dermal disease (e.g., Sturge-Weber Syndrome, Klippel-Trenaunay-Weber, Parkes Weber syndrome, Servelle-Martorell Syndrome, Proteus Syndrome, CLOVES syndrome). In some embodiments, the subject has port-wine stain (PWS), also known as nevus flammeus.

[0064] The methods comprise applying spatiotemporally synchronized ultrasound burst(s) and laser pulse(s) to the target site. In some embodiments, at least a portion of the laser pulse temporally overlaps with at least a portion of the ultrasound burst. In an alternative embodiment, the laser pulse(s) may be pulsed during a longer burst or continuous operation of the ultrasound burst. In an alternative embodiment, the laser is operated continuously while the ultrasound burst(s) is pulsed during the operation of the laser.

[0065] For spatiotemporally synchronization, the location of the laser pulse(s) also at least partially overlaps with the location of the ultrasound burst(s). The overlap covers or encompasses at least a portion of the target site or affected area, based on the respective focal diameters of the laser pulse(s) and ultrasound burst(s). In some embodiments, the laser pulse(s) completely overlaps with the location of the ultrasound burst(s). In alternative embodiments, the ultrasound burst(s) completely overlaps with the location of the laser pulse(s). In alternative embodiments, the ultrasound burst(s) partially overlaps with the location of the laser pulse(s).

[0066] The ultrasound burst(s) can be delivered to the target site by a therapeutic ultrasound transducer. Any known therapeutic ultrasound transducer configured for providing ultrasound treatment to a shallow tissue region, such as a region comprising an epidermis or dermis may be utilized in the disclosed methods. In some embodiments, the therapeutic ultrasound transducer can be configured for spatial control and / or temporal control by changing the position of transducer, its drive frequency, focal depth, drive amplitude, and timing.

[0067] Each ultrasound burst has a series of positive pressures and negative pressures, each with a respective peak positive pressure and peak negative pressure. The ultrasound burst(s) may have a peak negative pressure of about 0.1 to about 5 MPa. For example, the peak negative pressure may be about 0.1 MPa, about 0.2 MPa, about 0.3 MPa, about 0.4 MPa, about 0.5 MPa, about 0.6 MPa, about 0.7 MPa, about 0.8 MPa, about 0.9 MPa, about 1.0 MPa, about 2.0 MPa, about 2.5 MPa, about 3.0 MPa, about 4.0 MPa, or about 5.0 MPa. In some embodiments, the peak negative pressure is about 0.1 to about 5 MPa. In some embodiments, the peak negative pressure is about 0.5 to about 1 MPa. In select embodiments, the peak negative pressure is about 0.7 mPa, e.g., 0.70-0.75 mPa.

[0068] In some embodiments, the duty cycle of the therapeutic ultrasound transducer is 1-10%. In some embodiments, the duty cycle is about 1%, about 2 %, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10%. In some embodiments, the duty cycle is 1-5%. In select embodiments, the duty cycle is about 2%. In some embodiments, a single ultrasound burst can be used.

[0069] In some embodiments, the therapeutic ultrasound transducer may provide ultrasound burst(s) at central frequency of 100-1000 kHz. In some embodiments, the central frequency is about 100 kHz, about 150 kHz, about 200 kHz, about 250 kHz, about 300 kHz, about 350 kHz, about 400 kHz, about 450 kHz, or about 500 kHz. In some embodiments, the central frequency is 200-300 kHz. In select embodiments, the central frequency is about 250 kHz.

[0070] The laser pulse(s) may be delivered by any appropriate laser which operates in the desired wavelength range (e.g., 400 to 1600 nm). In some embodiments, the laser pulse(s) is delivered by a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser. In some embodiments, the laser emits light at 1000 to 1100 nm e.g., 1064 nm. In some embodiments, the laser emits light at about 1064nm. In some embodiments, the laser emits light at 500 to 600 nm e.g., 532nm.

[0071] The surface fluence may be monitored and controlled during application using one or more sensors and / or cameras. The laser fluence may be greater than 1 mJ / cm2or less than 1 J / cm2, e.g., at the surface. In some embodiments, the laser fluence is between 150 mJ / cm2and 1 J / cm2. In some embodiments, the laser fluence is between about 250 mJ / cm2and about 500 mJ / cm2. For example, the laser fluence may be about 150 mJ / cm2, about 200 mJ / cm2, about 250 mJ / cm2, about 300 mJ / cm2, about 350 mJ / cm2, about 400 mJ / cm2, about 450 mJ / cm2, about 500 mJ / cm2, about 550 mJ / cm2, about 600 mJ / cm2, about 650 mJ / cm2, about 700 mJ / cm2, about 750 mJ / cm2, about 800 mJ / cm2, about 850 mJ / cm2, about 900 mJ / cm2, or about 1 J / cm2. In select embodiments, the laser fluence is about 700 mJ / cm2.

[0072] The laser pulse length may be between 0.1 ns to 100 ns. In some embodiments, the laser pulse length is 0.1 ns to 10 ns, 0.1 ns to 20 ns, 0.1 ns to 30 ns, 0.1 ns to 40 ns, 0.1 ns to 50 ns, 0.1 ns to 60 ns, 0.1 ns to 70 ns, 0.1 ns to 80 ns, 0.1 ns to 90 ns, 0.5 ns to 10 ns, 0.5 ns to 20 ns, 0.5 ns to 30 ns, 0.5 ns to 40 ns, 0.5 ns to 50 ns, 0.5 ns to 60 ns, 0.5 ns to 70 ns, 0.5 ns to 80 ns, 0.5 ns to 90 ns, 0.5 ns to 100 ns, 1 ns to 10 ns, 1 ns to 20 ns, 1 ns to 30 ns, 1 ns to 40 ns, 1 ns to 50 ns, 1 ns to 60 ns, 1 ns to 70 ns, 1 ns to 80 ns, 1 ns to 90 ns, 1 ns tolOO ns, 10 ns to 20 ns, 10 ns to 30 ns, 10 ns to 40 ns, 10 ns to 50 ns, 10 ns to 60 ns, 10 ns to 70 ns, 10 ns to 80 ns, 10 ns to 90 ns, 10 ns to 100 ns, 20 ns to 30 ns, 20 ns to 40 ns, 20 ns to 50 ns, 20 ns to 60 ns, 20 ns to 70 ns, 20 ns to 80 ns, 20 ns to 90 ns, 20 ns to 100 ns, 30 ns to 40 ns, 30 ns to 50 ns, 30 ns to 60 ns, 30 ns to 70 ns, 30 ns to 80 ns, 30 ns to 90 ns, 30 ns to 100 ns, 40 ns to 50 ns, 40 ns to 60 ns, 40 ns to 70 ns, 40 ns to 80 ns, 40 ns to 90 ns, 40 ns to 100 ns, 50 ns to 60 ns, 50 ns to 70 ns, 50 ns to 80 ns, 50 ns to 90 ns, 50 ns to 100 ns, 60 ns to 70 ns, 60 ns to 80 ns, 60 ns to 90 ns, 60 ns to 100 ns, 70 ns to 80 ns, 70 ns to 90 ns, 70 ns to 100 ns, 80 ns to 90 ns, 80 ns to 100 ns, or 90 ns to 100 ns,

[0073] The methods may comprise one spatiotemporally synchronized laser pulse and ultrasound burst, so-called single shot. Alternatively, the methods may comprise repeated spatiotemporally synchronized laser pulses and ultrasound bursts. For example, spatiotemporally synchronized laser pulses and ultrasound bursts repeated at a repetition frequency of between 1 Hz and 2000 Hz. In some embodiments, the laser pulses and ultrasound bursts may be operated at a repetition frequency of between 1 Hz and 1000 Hz. In some embodiments, the laser pulses and ultrasound bursts may be operated at a repetition frequency of about 1 Hz, about 2 Hz, about 4 Hz, about 6 Hz, about 8 Hz, about 10 Hz, about 15 Hz, about 20 Hz, about 30 Hz, about 40 Hz, about 50 Hz, about 60 Hz, about 70 Hz, about 80 Hz, about 90 Hz, about 100 Hz, about 200 Hz, about 300 Hz, about 400 Hz, about 500 Hz, about 600 Hz, about 700 Hz, about 800 Hz, about 900 Hz, about 1000 Hz, about 1100 Hz, about 1200 Hz, about 1300 Hz, about 1400 Hz, about 1500 Hz, about 1600 Hz, about 1700 Hz, about 1800 Hz, about 1900 Hz, or about 2000 Hz. The repetition frequency may be between 5 Hz and 20 Hz, between 5 Hz and 50 Hz, between 5 Hz and 100 Hz, between 5 Hz and 200 Hz, between 5 Hz and 300 Hz, between 5 Hz and 400 Hz, between 5 Hz and 500 Hz, between 5 Hz and 600 Hz, between 5 Hz and 700 Hz, between 5 Hz and 800 Hz, between 5 Hz and 900 Hz, between 5 Hz and 1000 Hz, between 5 Hz and 1500 Hz, or between 5 Hz and 2000 Hz. In select embodiments, the repetition frequency is between 5 Hz and 20 Hz. In select embodiments, the repetition frequency is 15 Hz. In some embodiments, the repetition frequency is about 10 Hz.

[0074] The application may be repeated over the course of any length of time to decrease blood vessel density, remove blood vessels or capillaries, and / or reduce discoloration of the target site. In some embodiments, the duration of the application is at least 10 seconds. For example, the duration of the application may be about 10 seconds, about 20 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes, about 90 minutes, or about 120 minutes. In some embodiments, the duration of the application is 1 to 10 minutes. In select embodiments, the duration of the application is 4 to 6 minutes.

[0075] The method may result in any decrease to blood vessel density or any removal of blood vessels or capillaries. In some embodiments, the blood vessel density is decreased by about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60% , about 70%, about 80%, about 90%, or more. In some embodiments, the methods disclosed herein remove about 5%, about 10%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60% , about 70%, about 80%, about 90%, more of the blood vessels or capillaries at the target site.

[0076] As described above, the methods may be used to treat cutaneous vascular malformation or hypervascular dermal diseases which often result in skin discoloration. The methods disclosed herein may decrease overall discoloration of the skin, may decrease overall area of discoloration, or a combination thereof.

[0077] The methods may be repeated over a series of treatment sessions, e.g., separated by weeks or months. For example, after a desired affected area or target site is treated in a first treatment session, a second treatment session may be one month, two months, three months, four months, five months, six months, or more after the first treatment session. Subsequent treatment sessions may follow with a similar or increasing time interval until the desired results are achieved. In some embodiments, the methods disclosed are repeated in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) treatment sessions. In some embodiments, the methods are repeated after initial treatment for maintenance treatment sessions (e.g., every few years) after desired treatment level is achieved.

[0078] EXAMPLES

[0079] MATERIALS AND METHODS

[0080] Experimental setup A detailed schematic of an exemplary PUT system is shown in FIG.

[0081] 9. The PUT system combines a focused ultrasound (FUS) system and a pulsed laser system. The laser source was a Q-switched (Minilite II, Continuum Electro-Optics), which emitted 1064-nm wavelength pulses with 5-7 ns pulse width. The laser repetition rate and pulse energy in the range of 0-15 Hz and 0-50 mJ, respectively, were controlled through a delay generator (DG535, Stanford Research Systems). The ultrasound bursts were produced by a FUS transducer with 0.25 MHz central frequency (H-l 17, Sonic Concepts). The transducer had a radius of curvature of 63.2 mm with a focal depth and focal width of 6 and 40 mm, respectively. The sine wave burst for FUS was produced by a function generator (DG345, Stanford Research Systems) and was amplified by a power amplifier (2100L, Electronics & Innovation) before being passed to the FUS transducer through an impedance-matching circuit. A delay generator was used to trigger both the FUS system and the laser system to temporally synchronize the ultrasound bursts and laser pulses over the treatment area.

[0082] Treatment procedure The study was carried out on 7 male Leghorn chickens. The first 2 chickens were used to titrate the PUT treatment parameters. Three chickens were used to perform treatments using either PUT, ultrasound only, or laser only with longitudinal OCT observation and histology evaluations. The remaining 2 chickens were used to perform PUT treatment and then killed on day 3 after treatment for additional immunohistochemistry evaluations.

[0083] Before the treatment, the chickens were anesthetized by injecting a mixture of ketamine (15 mg / kg) and xylazine (0.6 mg / kg) intramuscularly. After anesthesia, the chicken wattle inner surface was marked with black circular marks at 6-8 different spots to indicate the treatment regions, as shown in FIG. 9D. For the treatment, the wattle was placed between an agar gelatin cone and a polydimethylsiloxane (PDMS) pad such that the ultrasound energy could be delivered to the wattle through the gel cone and the laser energy could be delivered to the wattle through the PDMS pad (FIG. 9B). The purpose of PDMS was to reduce ultrasound energy deposition at the wattle surface. The ultrasound coupling gel was applied at the cone-wattle and the wattle- PDMS interfaces to ensure efficient ultrasound delivery to the wattle. Moreover, a slight pressure was applied through the PDMS pad to restrict the wattle movement during the treatment. When positioning the wattle with respect to the transducer, the laser pulse was emitted on the target area to generate a photoacoustic signal, which was captured by the transducer, received by the amplifier, and shown on the oscilloscope. Then, the position of the transducer was adjusted by a 3D stage until the photoacoustic signal reached its maximum amplitude to ensure that the ultrasound beam and the laser beam irradiated over the same region on the wattle. The PUT treatment at each spot was performed for <5 minutes, and the total treatment time for each chicken wattle was <40 minutes. During the entire treatment, a noncontact thermal temperature sensing device (NF-521, Noyafa) was used to monitor the temperature on the wattle surface in real time.

[0084] Data collection, processing, and statistical analyses To evaluate the treatment outcome, the treatment areas on the chicken wattle were imaged by a Canfield skin imaging camera (Canon EOS camera-based, Canfield Scientific) and a laboratory, nonclinical spectral domain OCT system (TEL 321 Telesto, Thorlabs). The images were taken at different time points, which were before the treatment (control) and on day 1 (short term), day 7, day 14, and day 21 (longterm) after the treatment. Photos taken by the skin imaging camera were used to assess the changes in skin color after white balance was matched by the histomatch algorithms using MATLAB. Quantitative analyses of the changes in vessel density after treatment were conducted using the imaging results from the spectral domain OCT system, which has an image depth of 2.6 mm in water. Both OCT and OCT-A data were collected during the spectral domain OCT imaging using the software provided by Thorlabs. OCT-A data were generated using a speckle variance analysis over 3 repeated B-scans per position. To quantitatively analyze the vascular density changes, the process detailed in FIG. 8 was conducted. First, skin surface detection was done using OCT volume data. After acquiring a single layer at the top surface, the OCT-A data were depth encoded. Then, the OCT-A signals in the dermis between 0 and 150 mm were extracted, and the maximum intensity projection was used to create a two-dimensional map. To create a vascular density map, a binarization of this two-dimensional perfusion map was performed. On the binarized two-dimensional map, mean vessel densities within and outside the treatment region were measured to establish normalized vessel densities. Statistical analyses were performed using GraphPad Prism 9.0.0 (GraphPad Software) and presented as mean SD in FIG. 2D. P-values were derived from paired t-tests comparing normalized vessel densities at different time points, and P < .05 was determined as statistically significant.

[0085] Histology and immunohistochemistry analysis Chicken wattle samples were collected after the chickens were killed at different time points (days 1, 3, 7, 14, and 21) after the treatment. The tissues for histology and immunohistochemistry were stained with H&E, CD31 antibodies, caspase-3 antibodies, and MTC, which were performed by the In Vivo Animal Core in the Unit for Laboratory Animal Medicine at the University of Michigan (Ann Arbor, MI).

[0086] Paraffin processing and sectioning Briefly, formalin-fixed tissues were processed through graded alcohols and cleared with xylene followed by infiltration with molten paraffin using an automated VIP5 or VIP6 tissue processor (Tissue-Tek, Sakura- Am ericas). After paraffin embedding using a Histostar Embedding Station (Thermo Fisher Scientific), tissues were then sectioned on an M 355S rotary microtome (Thermo Fisher Scientific) at 4-mm thickness and mounted on glass slides.

[0087] Full thickness of the tissue of chicken wattle is composed of epidermis on both sides, with dermis underneath also on both sides, and a scant amount of subcutis in the center. The depth of the lesions was measured from the surface of the epidermal necrosis, crossing dermis and subcutis, to the bottom of the lesion which might be at dermis on the other side of the tissue. The ratio was calculated as the depth of the pathology divided by the full thickness of the tissue in micrometer (pm).

[0088] H&E staining After deparaffmization and hydration with xylene and graded alcohols, formalin-fixed, paraffin-embedded slides were stained on an automated histostainer (Autostainer XL, Leica Biosystems) with Harris hematoxylin (Thermo Fisher Scientific, catalog number 842); differentiated with Clarifier (Thermo Fisher Scientific, catalog number 7401); blued with bluing reagent (Thermo Fisher Scientific, catalog number 7301); stained with eosin Y, alcoholic (Thermo Fisher Scientific, catalog number 832), then dehydrated and cleared through graded alcohols and xylene, and coverslipped with Micromount (Leica Biosystems, catalog number 3801731) using a Leica CV5030 automatic coverslipper.

[0089] MTC staining All staining reagents were obtained commercially from Rowley Biochemical (Danvers, MA) unless otherwise noted. Briefly, after deparaffmization and hydration with xylene and graded alcohols, formalin-fixed, paraffin-embedded slides were mordanted in Bouin’s Fixative (F-367-1) for 1 hour at 56 °C. After a thorough rinse in water, slides were placed in Biebrich Scarlet-Acid Fuchsin (F-367-3) for 15 minutes at room temperature, phosphotungstic-phosphomolybdic acid (F-367-4) for 15 minutes at room temperature, and aniline blue stain (F-367-5) for 8 minutes at room temperature. Slides were then dehydrated and cleared through graded alcohols and xylene and coverslipped with Micromount (Leica Biosystems, catalog number 3801731) using a Leica CV5030 automatic coverslipper.

[0090] Immunohistochemical staining After deparaffinization and hydration with xylene and graded alcohols, formalin-fixed, paraffin-embedded slides were subjected to heat-induced epitope retrieval in Diva Decloaking Buffer, pH 6.2 (Biocare Medical), in a pressure cooker (Biocare Medical). Immunohistochemical staining was performed on an automated IntelliPATH FLX immunohistochemical Stainer (Biocare Medical) and included blocking for endogenous peroxidase and nonspecific binding, primary antibody incubation, detection using a horseradish peroxidase biotin-free polymer-based system, disclosure with diaminobenzidine chromogen, and nuclear counterstaining with hematoxylin.

[0091] Specific to cleaved caspase-3 (Cell Signaling Technology, catalog number 9664), the rabbit monoclonal primary antibody was diluted to 1 :500 in DaVinci Diluent (Biocare Medical, catalog number PD900) and incubated for 30 minutes followed by detection using Rabbit on Rodent HRP -Polymer (Biocare Medical, catalog number RMR622) for 30 minutes. Negative control samples consisted of naive serum (Universal Negative, Biocare Medical) applied in place of the primary antibody, under the same conditions.

[0092] Specific to CD31 (Dianova, number SZ31), rat monoclonal primary antibody was diluted to 1 :50 in DaVinci Diluent (Biocare Medical, catalog number PD900) and incubated for 60 minutes, followed by detection using a Rat-on-Mouse HRP -Polymer (Biocare Medical, catalog number RT517) 2-step probe-polymer incubation for 10 and 30 minutes, respectively. The diaminobenzidine was enhanced with diaminobenzidine Sparkle (Biocare Medical, catalog number DS830). Negative control samples consisted of naive rat sera (NC915, Innovex Biosciences) applied in place of the primary antibody, under the same conditions.

[0093] Histochemical stain- Russell-Movat Pentachrome (RMP) Slides were deparaffinized, hydrated, and stained in elastic stain for 20 minutes. Differentiation was performed in Ferric Chloride, 2% for 15-20 dips, with progress monitored microscopically. Slides were then placed in sodium thiosulfate, 5% for 1 minute to remove excess iodine, and acetic acid, 1% for 2 minutes to equilibrate tissue. They were then stained with Alcian blue, 1%, pH 2.5 for 25 minutes, and Biebrich Scarlet-acid Fuchsin for 2 minutes. Differentiation was performed in phosphotungstic acid, 5% for 3 minutes, twice, with progress monitored microscopically. Slides were then placed in acetic acid, 1% for 1 minute, and Yellow Stain Solution for 15 minutes. Finally, slides were dehydrated, cleared in xylene, and mounted with Micromount.

[0094] Digital slide assessment and quantitation Slides were digitized on a Leica Aperio AT2 digital slide scanner (Leica Biosystems) at a resolution of 0.25 pm / pixel (40x objective). Quantitative assessment of immunohistochemical staining was performed using the open-source program QuPath v0.5.1 (github.com / qupath / qupath). Detection parameters were optimized using the positive and negative control slides. Images were first annotated manually. Then, the annotated area was subjected to a pixel thresholder, which was either trained via Artificial Neural Network or by default, to measure the area of the positive objects, as well as to count the positive objects. Microsoft Excel and Graphpad Prism 10 were then used for data organization, visualization, and statistical analysis.

[0095] EXAMPLE 1

[0096] During the titration of parameters for PUT treatment of chicken wattle, the initial set of treatment parameters was based on work in a rabbit ear model (Wang et al, Lasers Surg Med 2020;52(10):984-92). When tested in the chicken wattle model, this initial set of treatment parameters did not lead to an efficient treatment response. Therefore, the negative peak pressure of the ultrasound bursts was gradually increased to 0.72 MPa, and the duty cycle was also increased from 0.2 to 2%, which led to a reduction of blood perfusion in the dermis observable on OCT-A. After that, the laser fluence was gradually increased to 707 mJ / cm2until PUT could completely stop the flow in all the blood vessels inside the treatment area, as indicated by OCT- A. Additional adjustments were made to the treatment duration (4 minutes) and the pulse repetition frequency (15 Hz). These optimized treatment parameters, as shown in Table 1, were determined to minimize thermal damage and maximize blood vessel density reduction that could be achieved by PUT treatment of chicken wattle in vivo and were used later in the treatment of the remaining 5 chickens. Table 1. Laser and ultrasound parameters for PUT treatment of blood vessels in chicken wattle.

[0097] FIG. 1A shows the wattle skin photographs taken using the skin imaging camera immediately before treatment and on day 1 (immediately after) and day 14 after the treatment, comparing 3 treatment spots on the same wattle: one with PUT, one with laser only, and one with ultrasound only. In these wattle skin photographs, a clearly visible change in color from red to white was observed on day 14 for the area treated by PUT. However, no such color change was observed in the areas treated by laser only or ultrasound only. Besides using the skin imaging camera, the treatment outcome from PUT shown in FIG.

[0098] 1 was also evaluated by OCT-A. The red square shows the OCT scanning region on the wattle. The images in FIG. IB were obtained by overlapping the B-scan OCT-A images presenting the blood perfusion information over the B-scan OCT images presenting the tissue structural information. The B-scan was through the middle of the treatment spot marked by a dash blue line. As one can see in the OCT-A images, the blood perfusion was stopped the next day after PUT treatment (FIG. IB, day 1). This cessation in blood perfusion induced by PUT persisted until at least 14 days after the treatment (FIG. IB, day 14), which was the end of the observation period and when the chicken was killed for histopathology analysis. FIG. 1C shows the maximum intensity projection images presenting the blood vessels within the depth range of 0 to 150 mm in the 3D OCT-A results. The scanned areas are marked by the red dash squares. The cessation of blood perfusion after the treatment can be observed as an area with reduced OCT-A intensity in the maximum-intensity projection images. This area with reduced OCT-A intensity has a diameter of around 3 mm, which is the same as the size of the treatment laser beam. Again, the cessation of blood perfusion was observed at least 14 days after the treatment, as shown in (FIG. 1C, day 14).

[0099] Assessment of PUT treatment outcome was performed by quantitatively analyzing the vessel density map generated from the 3D OCT-A images acquired before and at different time points after the treatment, as shown in FIG. 2. In total, 7 treatments (n = 7) were carried out for each treatment group, including PUT, laser only, and ultrasound only, on a total of 3 chickens with the optimized PUT parameters. The maximum-intensity projection images of 3D OCT-A (0-150 pm depth in the dermis) of a PUT -treated region at different time points are shown in FIG. 2B, and corresponding vessel density maps generated by the binarization of the maximum intensity projection images are shown in FIG. 2C. This imaging processing procedure allowed quantitative and volumetric measurement of the change in vessel density in chicken wattle in vivo as a direct and fast assessment of the treatment efficacy. FIG. 2D shows the normalized vessel density of all the regions treated with either PUT, laser only, or ultrasound only from day 0 (before treatment) to day 21 after treatment. Each data point shows the mean ± SD over 7 treatments in each group. For the PUT treatment group, the vessel density reduced by 73.23% on day 1 (immediately after the treatment) and remained at a similar level until day 21. The quantified measurement of normalized vessel density on each time point after treatment was compared with the baseline measurement before treatment by a paired t-test, and statistically significant changes in vessel density were noticed in the PUT treatment group. For either ultrasound-only treatment group or the laser-only treatment group, no significant reduction in vessel density was observed.

[0100] FIGS. 3-5 show the H&E-stained histology results. FIG. 3A is a section of wattle tissue harvested on day 21 after the PUT treatment, and the treatment area is marked with a red dash square. The blood vessel lumens completely disappeared at a depth of 0 to 400 pm from the wattle surface, as shown in FIG. 3B. In addition, most of the capillary lumens are not visible in the 400-800 pm depth, except for 2 large blood vessels at the depth of 800 pm (FIG. 3C), indicating a treatment depth of 700 pm. The untreated region had abundant blood vessels, as illustrated in FIG. 3D. In the untreated region, multiple vessel lumens ranging from 10 to 50 pm can be observed close to the epidermis as well as in the deep dermis up to the depth of 1000 pm. FIG. 4A is a section of wattle tissue harvested on day 7 after the PUT treatment. This section, together with the magnified photographs in FIGS. 4B-4D, confirms that the vessels in the wattle at a depth up to 800 pm from the wattle surface were filled with red blood cells and degenerated endothelial cells after the treatment, whereas the overlying epidermis was left intact.

[0101] FIG. 5 shows H&E-stained sections of wattle tissues treated with either PUT (FIGS. 5A- 5D), laser-only (FIG. 5E), or ultrasound only (FIG. 5F). The tissue sections showing the outcome from PUT were harvested on days 1, 7, 14, and 21 after the treatment. On days 1 and 7 after the PUT treatment, the vessels were occluded with red blood cells, whereas on days 14 and 21 after the PUT treatment, the vessel lumens in the treated regions disappeared. For comparison, in the sections treated with either ultrasound only or laser only, all blood vessels were completely intact on day 21 after the treatment.

[0102] Additional immunohistochemistry evaluations of the treatment outcome were conducted on 2 chickens killed on day 3 after PUT treatment, and both treated and untreated wattle tissues were analyzed and compared. Tissue sections with 100-pm intervals were stained with CD31, H&E, caspase-3, and Masson’s trichrome (MTC) so that tissues in the same vicinity could be subjected to different evaluations. As shown in FIGS. 6A-6B, CD31-positive endothelial cells with clear vessel lumens can be seen in untreated tissue, whereas most endothelial cells in the treated area were found to be CD31 negative, indicating the desired vascular damage induced by PUT treatment. The H&E-stained histology results from the treated and untreated tissues in FIGS. 6C-D are comparable with those in FIG. 4. The key findings in the treated area include clusters of red blood cells obstructing the blood vessel lumen and epidermis necrosis. FIGS. 7A and 7B show caspase-3 negative in both the untreated area and the dermis of the treated area. Some localized activations of caspase-3 were found in the epidermis of the treated area, which however were very minor. As shown by the MTC-stained histology results in FIGS. 7C and 7D, show the collagen inside the treated area has structure and morphology similar to those in the untreated area, indicating no collateral collagen damage induced by the treatment.

[0103] EXAMPLE 2

[0104] PUT was employed to induce cavitation in blood vessels by using different light fluence and ultrasound pressure combinations. Theoretical modeling and in vitro experiments were first conducted to validate and optimize parameters for PUT treatment targeting deep vasculature. An established theoretical model, described in detail below, was used to investigate how light fluence and ultrasound PNP influence pre-existing bubbles during PUT by implementing the light and ultrasound parameters utilized in current experiments.

[0105] FIG. 10 A, left (a.l), shows the simulated photoacoustic waveform observed near the center of a blood vessel with a diameter of 0.1 mm when illuminated by a 3-ns light pulse at 1064 nm wavelength. FIG. 10A, center (a.2), shows the concurrently applied ultrasound burst with 0.25 MHz frequency and 1 MPa pressure amplitude. The light pulse and the ultrasound burst are synchronized with a precisely controlled delay so that the light-induced photoacoustic pulse is superimposed onto the negative phase of an ultrasound cycle, as shown in FIG. IB, right (a.3). An ultrasound pressure of 0.6 MPa combined with a light fluence of 100 mJ / cm2does not reach the rectified diffusion threshold, resulting in the dissolution of the pre-existing bubble as shown in FIG. 10B, left (b.l). In contrast, an ultrasound pressure of 0.8 MPa combined with a light fluence of 120 mJ / cm2surpasses the rectified diffusion threshold, resulting in an explosive growth of the bubble as shown in FIG. 10B, center (b.2). Furthermore, an ultrasound pressure of 1.0 MPa combined with a light fluence of 120 mJ / cm2causes the bubble to expand sufficiently to induce period doubling as shown in FIG. 10B, right (b.3).

[0106] Pre-existing bubble size may vary, FIG. 10C, left (c. l) illustrates the rectified diffusion threshold (e.g., cavitation threshold) in terms of ultrasound pressure for different levels of light fluence and different initial bubble sizes. The result shows that the increased light fluence can reduce the ultrasound pressure needed for cavitation. Assuming a pre-existing bubble with a radius of 100 nm, the dynamics of the bubble under the treatment with different combinations of ultrasound rarefaction pressure (0-1 MPa) and light fluence (0-200 mJ / cm2) were simulated, as shown in FIG. 10C, right (c.2). The result shows a region where the pre-existing bubble grows under PUT treatment. In this region, the bubble, continuously driven by the ultrasound burst, can cause effective and selective treatment of the blood vessel. FIG. 10C, right (c.2) also shows a region (highlighted with purple color) where the pre-existing bubble dissolves under PUT treatment. In the region, there will be no treatment effect to the blood vessel. A dashed red line between these two regions indicates the rectified diffusion threshold in terms of the ultrasound pressure as a function the light fluence. At the skin surface where the light fluence is strong, less ultrasound pressure is needed to produce cavitation; while in deep skin where the light fluence is weaker due to the strong optical attenuation in overlying tissues, the likelihood of cavitation can be increased by applying relatively higher ultrasound pressure.

[0107] The schematic of the experiment setup is illustrated in FIG. 11 A. The nanosecond light pulses with a 3 nm pulse width and pulse repetition rate of 10 Hz were from an Nd:YAG laser (Continuum PowerliteDLS8010, Santa Clara, CA) working at 1064 nm wavelength. The light beam was directed to the center of a focused ultrasound transducer with a focal length of 39.49 mm, a focal width of 6.04 mm, and working at a center frequency of 0.25 MHz (H-l 17, Sonic Concepts, Bothell, WA). In this study, the transducer generated 2 millisecond long ultrasound bursts with a 2% duty cycle. The transducer was powered by a radio-frequency power amplifier (2100L, ENI, Rochester, NY) through an impedance matching network provided by Sonic Concepts. A pulse delay generator (Model DG355, Stanford Research Systems) was employed to synchronize the triggers for the laser and the ultrasound systems, ensuring that the light pulse reached the target during one of the negative phase of the ultrasound burst. The target was a blood vessel mimicking phantom which was a soft and optically transparent silicone tube (inner diameter: 0.3 mm, outer diameter: 0.6 mm, Liveo™ Silicone Laboratory Tubing, Fisher Scientific) filled with human whole blood obtained from the University of Michigan Blood Center. Driven by a pump, the blood was circulated through the tube at a speed of 1 cm per second. During the measurement, the tube was immersed in a water bath filled with degassed water. For real-time active cavitation detection during PUT, the blood vessel was imaged continuously using an ultrasound imaging system (ZS3, Zonare Medical Systems, Inc., Mountain View, CA) in the B-mode working with a 10 MHz linear probe.

[0108] The imaging result from the ultrasound imaging system confirmed the cavitation activities generated by the concurrently applied light pulses and ultrasound bursts during PUT. The representative results in FIG. 1 IB were acquired when the ultrasound pressure was fixed at 0.6 MPa while the light fluence was swept from 0 to 200 mJ / cm2. When the light fluence increased, the detected cavitation activities also increased. FIG. 11C shows the measured cavitation probability from the blood vessel phantom treated by a combination of different levels of ultrasound pressure and light fluence. The quantified cavitation probability at each point in this map was calculated by counting the number of frames with detected cavitation bubbles from a total of 100 frames of ultrasound images. The points along the dashed line in FIG. 11C have the same level of cavitation probability. For instance, the light fluence of 20 mJ / cm2plus the ultrasound pressure of 1.4 MHz yields a similar cavitation probability as the light fluence of 200 mJ / cm2plus the ultrasound pressure of 0.8 MPa. This experimental result from the phantom study indicates again that similar cavitation activities can be achieved with weaker light fluence in combination with stronger ultrasound pressure (the situation in deep skin) or stronger light fluence in combination with weaker ultrasound (the situation in superficial skin), which is consistent with the finding in FIG. 10 from the theoretical modeling.

[0109] The experiment setup employed for in vivo PUT treatment is shown in FIG. 12. While the focused ultrasound system was the same as the phantom study, additional adjustments were applied to ensure treatment efficiency on chicken wattle. A 3D-printed holder designed to fit the focused transducer was utilized to hold gel and degassed water for ultrasound coupling from the transducer to the target tissue. A central hole in the gel was reserved for passing of the light beam. This design facilitated that the light beam and the ultrasound wave for PUT could be delivered from the same side. Two layers of ultrasound coupling materials, e g., gel and degassed water, were used to reduce the pathlength of the light beam in water which has a relatively strong optical absorption at the wavelength of 1064 nm. To verify the acoustic coupling from the transducer to the target tissue, light was turned on and the transducer collected photoacoustic signals, which were then amplified by a pulser / receiver (PR 5072, Olympus, Japan) and shown on an oscilloscope. The amplitude of the photoacoustic signal was used as a control point for

[0110] 5 treatment at each site, with similar signal amplitude indicating similar acoustic coupling efficiency.

[0111] Table 2. PUT parameters for treatment of blood vessels in chicken wattle in vivo

[0112] The light and ultrasound parameters used during PUT treatment of blood vessels in

[0113] 10 chicken wattle are listed in Table 2, which were initially selected based on previous in vivo studies and further adjusted according to the theoretical modeling and in vitro phantom studies for treatment of deep vessels. Via a 6 mm diameter beam, the light pulse energy delivered to the top surface of the wattle was 80 mJ, leading to a light fluence of 283 mJ / cm2. After optical attenuation through the chicken wattle, the light pulse energy at the bottom of the wattle

[0114] 15 measured using a laser energy meter was 16 mJ. With an estimated beam size of 6 mm in diameter, the light fluence at the bottom of the wattle was 57 mJ / cm2. To promote the treatment of deep vessels, the ultrasound wave was focused at the bottom of the wattle. Based on the precalibration in a water tank using a calibrated needle hydrophone (Model, Onda), the ultrasound pressure arriving at the top surface of the wattle was 0.91 MPa, while the ultrasound

[0115] 20 pressure arriving at the bottom surface of the wattle was 1.36 MPa. The -6 dB focal width of the ultrasound beam was 6 mm.

[0116] To confirm that vessels at various depths receive optimal treatment, the synchronization between the light pulse and the ultrasound burst was swept across the entire 4-minute treatment session. This sweeping was achieved by adjusting the triggering delay by 500 ns every minute.

[0117] 25 For instance, the light pulse was synchronized to the negative peak of an ultrasound cycle at the skin surface during the first minute of treatment. During the second minute, the light pulse was delayed by an additional 500 ns. In this way, the light pulse was synchronized to the negative peak of an ultrasound cycle at the depth of 500 ns - 1.5 mm / ps = 0.75 mm beneath the skin surface. This synchronization between the light pulse and the negative peak of an ultrasound

[0118] 30 cycle would shift to 2.25 mm depth during the 4thminute of the treatment session. Using the parameters shown in Table 2 and the setup shown in FIG. 12, PUT treatment was conducted on four chicken wattles with three treatment spots on each wattle. Before and during the entire course of the treatment, chickens were anesthetized with an intramuscular injection of a cocktail of ketamine (15 mg / kg) and xylazine (0.6 mg / kg). The 3D-printed holder was sealed with a plastic membrane at the bottom, and ultrasound coupling gel was applied between the membrane and the chicken wattle to ensure good ultrasound delivery to the wattle.

[0119] To evaluate treatment outcomes, all treated areas on the chicken wattles were imaged using a polarized dermoscope (Canon EOS camera-based, Canfield Scientific) and a spectral domain optical coherence tomography (OCT) system (TEL 321 Telesto, Thorlabs). For comparison, an additional untreated area on each wattle was also imaged using the OCT system. OCT-angiography (OCT-A) images were generated using speckle variance analysis over three repeated B-scans per position. Images were taken at multiple time points, including before treatment (control), and at Day 1 and Day 7 after treatment. With the OCT-A images acquired at different time points, quantitative analyses of vascular density and its change in response to the treatment were conducted.

[0120] To further investigate treatment efficacy (including depth) and safety, wattle tissues were harvested after the chickens were euthanized at Day 7 post-treatment. The tissues for histology and immunohistochemistry (IHC) were stained with hematoxylin and eosin (H&E), CD31 antibodies, and Russell-Movat Pentachrome (RMP). H&E stained slides were used to assess treatment depth by measuring the depth of inflammation and vascular necrosis performed by a pathologist. CD31 stained slides were used to quantify treatment efficacy by comparing the vascular densities inside and outside the treated area. RMP stains were utilized to assess the safety of the treatment by measuring the collagen densities inside and outside the treated area.

[0121] FIG. 13A displays the photographs of wattle skin taken using a skin imaging camera immediately before treatment (Day 0), immediately post treatment (Day 1), and at Day 7 posttreatment. All the three regions marked by the white dashed circles received the same PUT treatment. A clearly visible change in color can be observed at Day 1 from both the top and the bottom sides of the wattle, suggesting that the treatment effect penetrated the entire chicken wattle.

[0122] To validate blood flow changes as the treatment outcomes from PUT, OCT-A images acquired from both the top and the bottom sides of the treated areas were compared with an untreated area as the control. As shown in FIG. 13B, blood perfusion ceased immediately after PUT treatment in all the treated regions, and this cessation of blood perfusion persisted for the entire observation period of 7 days post-treatment. Quantitative assessment of PUT treatment outcomes was performed by analyzing the vessel density map generated from the 3D OCT-A images that were acquired before and at various time points post-treatment, as shown in FIG. 13C. Each data point represents the mean ± standard deviation (SD) for each group. For the PUT treatment group, vessel density was significantly less than that in untreated regions at both the bottom and the top sides of the wattles at different time points post-treatment. At Day 7, the vessel densities at the top and the bottom sides of the wattles were reduced by 45.20% and 36.06%, respectively, when compared with the untreated regions, indicating good efficacy and depth of PUT in removing the vessels.

[0123] To further evaluate the efficacy and safety of PUT treatment, histopathological analyses were performed across the entire sections of chicken wattles at Day 7 post-treatment. Adjacent tissue slides were stained with H&E, IHC CD31, and histochemical RMP. FIG. 14 shows the representative photos of H&E, CD31, and RMP stained slides. The unique architecture of chicken wattle can be seen in FIG. 14A as well as FIG. 15A. The full thickness of the tissue comprises epidermis on both sides, with dermis underneath on both sides, and a very scant amount of subcutis in the center. As a result of PUT treatment, acute vascular necrosis and mild inflammation were noted in multiple capillary layers in the superficial dermis at both sides, leading to vascular occlusion. PUT treatment depth in the chicken wattle, which has a very dense vasculature, is up to 3 mm, given the treatment effect observed across the full thickness of the harvested chicken wattles (n=12, thickness of mean ± SD = 2.51 ± 0.21 mm).

[0124] FIG. 14B demonstrates a closer look of the CD31-positive endothelial cells formed blood vessel wall found in the untreated area, while CD31 -negative area could be found in the PUT treated area. Additionally, as shown in FIG. 15B, on the same slide, CD31-positive endothelial cells with clear vessel lumens observed in the untreated tissues, whereas endothelial cells in the treated area were CD31 -negative on both sides of the chicken wattle, indicating the vascular damage across the entire wattle induced by PUT treatment. The RMP stained collagen in yellow and thrombus in pink are illustrated in FIG. 14C. FIG. 14C along with FIG. 15C, show that, in the RMP stained sections, the collagen within the treated area retained a structure and morphology similar to those in the untreated area, indicating no collateral collagen damage induced by PUT treatment. Bright red areas were found in vessel lumens in both the top and the bottom layers of the wattle, suggesting thrombus formation after treatment. The CD31 results and the RMP results were further utilized to quantify the vascular densities and the collagen densities, respectively, in both the untreated and the treated tissues. The quantified vascular densities in the treated vs. the untreated wattle tissues are shown in FIG. 15D, which indicates that a single session of PUT treatment led to an average reduction of 43.5% in vascular density. p<0.05 was achieved when comparing the treated and the untreated tissues via a paired t-test (n=10 for each group). The quantified collagen densities in the treated vs. the untreated wattle tissues are shown in FIG. 15E. No statistically significant difference was noticed when comparing the treated and the untreated tissues via a paired t-test (n=6 for each group), confirming that there was no collateral collagen damage induced by PUT treatment. p0(r) = r / zaF(r) (10)

[0125] To study the subsequent bubble dynamics under simultaneous light and ultrasound extinction, the previously established theoretical model based on the Keller-Miksis, which has the form in equation (1), was used. In this equation, R is the bubble radius, dots denote time derivatives, t is time, c is the sound speed in the surrounding medium, p is the density of the surrounding medium, pxis the pressure at infinity, and PB is the pressure at the surrounding medium side of the interface between the medium and the bubble.

[0126] Equation (2) gives the formula of PB, where o is the surface tension coefficient, p is the viscosity of the fluid, and pgis the pressure inside the bubble. To consider the rectified diffusion, pgcan be calculated from Eller and Flynn’s zero-order solution to the diffusion equation, expressed as equation (3), where D is the diffusion constant of the gas in the liquid, no is the number of moles of gas initially present in the bubble, and r is the nonlinear time defined using equation (4-6). In this formulation, TJ is the polytropic exponent of the gas, Co is the saturation concentration of the gas in the liquid, Ci is the initial concentration of gas in the liquid far from the bubble, Ro is the initial equilibrium radius of the bubble, and Ro» is the time-varying equilibrium bubble radius calculated using ideal gas law expressed in equation (7), where Rgis the universal gas constant and Tais the absolute temperature. Pgis a function of n, and, therefore, equations (3) and (6) need to be coupled and solved simultaneously.

[0127] In equation (3), the term p (t + is the applying pressure, which is composed of the applied ultrasound pressure and the photoacoustic (PA) pressure produced by a light pulse. As the light pulse is considered a Gaussian temporal profile, the distribution of initial pressure produced by the light pulse can be calculated by equation (8), where T is the light pulse width defined at the full width of half maximum, and ps is the initial pressure distribution of the absorbing object. Considering the attenuation of light radiant exposure across a blood vessel, the pressure response for a delta heating of an arbitrary absorbing object is given by equation (9), where pocould be calculated using equation (10).

[0128] In all simulations, an air bubble was assumed in blood, and values for the constants are: r = 0.2,a= 10 c = 1500 m s-1, p = 1000 kg m3, p, = 1.01 x IO5Pa, o = 0.0725 N m-1, p = 0.005 Pa s, D = 2 x 109m2s = 1-4, CpCo = 1, Rg= 8.3145 J (mol • K)’1, and T= 293.15 K. To study cavitation in the bloodstream, a given initial equilibrium radius of the cavitation nuclei Ro (50-200 nm) was assumed.

[0129] The scope of the present invention is not limited by what has been specifically shown and described hereinabove. Those skilled in the art will recognize that there are suitable alternatives to the depicted examples of materials, configurations, constructions, and dimensions. Variations, modifications, and other implementations of what is described herein will occur to those of ordinary skill in the art without departing from the spirit and scope of the invention.

[0130] Numerous references, including patents and various publications, are cited and discussed in the description of this invention. The citation and discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entirety.

Claims

CLAIMSWhat is claimed is:

1. A method for decreasing blood vessel density and / or removing blood vessels or capillaries at a target site in a subject comprising: applying to the target site a laser pulse spatiotemporally synchronized with an ultrasound burst, wherein the laser pulse has a laser fluence greater than 1 mJ / cm2.

2. The method of claim 1, wherein the ultrasound burst has a peak negative pressure of about 0.1 to about 5 MPa.

3. The method of claim 1 or 2, wherein the ultrasound burst has a peak negative pressure of 0.5-1.5 MPa.

4. The method of any of claims 1-3, wherein the ultrasound burst has a peak negative pressure of 0.70-0.75 MPa.

5. The method of any of claims 1-4, wherein the laser pulse is delivered by a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser.

6. The method of any of claims 1-5, wherein the laser pulse is at about 400 to about 1600 nm.

7. The method of any of claims 1-6, wherein the laser pulse is at 1064nm.

8. The method of any of claims 1-6, wherein the laser pulse is at about 532nm.

9. The method of any of claims 1-8, wherein the laser fluence is less than 1 J / cm2.

10. The method of any of claims 1-9, wherein the laser fluence is between 150 mJ / cm2and 1 J / cm2.

11. The method of any of claims 1-10, wherein the laser fluence is between 500 mJ / cm2and 1 J / cm2.

12. The method of any of claims 1-11, wherein the laser fluence is about 700 mJ / cm2.

13. The method of any of claims 1-10, wherein the laser fluence is between about 250 mJ / cm2and about 500 mJ / cm2.

14. The method of any of claims 1-13, wherein the laser pulse length is between 0.1 ns to 100 ns.

15. The method of any of claims 1-14, wherein the method comprises a single synchronized ultrasound burst and laser pulse.

16. The method of any of claims 1-14, wherein the laser pulse and ultrasound burst are repeated at a repetition frequency of between 1 Hz and 1000 Hz.

17. The method of claim 16, wherein the repetition frequency is about 5 Hz to about 20 Hz.

18. The method of claim 16 or 17, wherein the repetition frequency is about 10 Hz.

19. The method of claim 16 or 17, wherein the repetition frequency is about 15 Hz.

20. The method of any of claims 1-19, wherein the applying lasts for at least 10 seconds.

21. The method of any of claims 1-20, wherein the applying lasts for 1 to 10 minutes.

22. The method of any of claims 1-21, wherein the applying lasts for 4 to 6 minutes.

23. The method of any of claims 1 -22, wherein the target site is a skin surface or tissue up to 3 mm from the skin surface.

24. The method of any of claims 1-23, wherein the target site is a skin surface or tissue up to 1 mm from the skin surface.

25. The method of any of claims 1-24, wherein the subject has a cutaneous vascular malformation.

26. The method of any of claims 1-25, wherein the subject has a hypervascular dermal disease.

27. The method of any of claims 1-26, wherein the subject has port-wine stain (PWS).

28. A method for treating a cutaneous vascular malformation in a subject comprising: applying a laser pulse spatiotemporally synchronized with an ultrasound burst to at least a portion of an affected skin surface or tissue up to 3 mm from the skin surface.

29. The method of claim 28, wherein the laser pulse is delivered by a neodymium-doped yttrium aluminum garnet (Nd:YAG) laser.

30. The method of claim 28 or 29, wherein the laser pulse is at about 400 to about 1600 nm.

31. The method of any of claims 28-30, wherein the laser pulse is at 1064nm.

32. The method of any of claims 28-30, wherein the laser pulse is at about 532nm.

33. The method of any of claims 28-32, wherein a laser fluence of the laser pulses is 1 mJ / cm2to 1 J / cm2.

34. The method of claim 33, wherein the laser fluence is about 700 mJ / cm2.

35. The method or claim 33, wherein the laser fluence is between about 250 mJ / cm2and about 500 mJ / cm2.

36. The method of any of claims 28-35, wherein the laser pulse length is between 0.1 ns to 100 ns.

37. The method of any of claims 28-36, wherein the ultrasound burst has a peak negative pressure of about 0.1 to about 5 MPa.

38. The method of any of claims 28-37, wherein the ultrasound burst has a peak negative pressure of 0.5 -1.5 MPa.

39. The method of any of claims 28-37, wherein the ultrasound burst has a peak negative pressure of 0.70-0.75 MPa.

40. The method of any of claims 28-39, wherein the method comprises a single synchronized ultrasound burst and laser pulse.

41. The method of any of claims 28-39, wherein the laser pulse and ultrasound burst are repeated at a repetition frequency of between 1 Hz and 1000 Hz42. The method of claim 41, wherein the repetition frequency is about 5 to about 20 Hz.

43. The method of any of claims 28-42, wherein the applying lasts for at least 10 seconds.

44. The method of any of claims 28-43, wherein the applying lasts for 1 to 10 minutes.

45. The method of any of claims 28-44, wherein the subject has a hypervascular dermal disease.

46. The method of any of claims 28-45, wherein the subject has port-wine stain (PWS).

Images