Method for treating venous malformations

Abstract

This disclosure relates generally to methods of treating venous malformations using particles with a photothermal effects.

Description

METHOD FOR TREATING VENOUS MALFORMATIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims benefit under 35 U.S.C. § 119(e) of the U.S. Provisional Application No. 63 / 513,272 filed July 12, 2023, and No. 63 / 514,656 filed July 20, 2023, the contents of each of which are incorporated herein by reference in their entireties.TECHNICAL FIELD

[0002] This disclosure relates generally to methods of treating venous malformations using particles with a photothermal effects.GOVERNMENT SUPPORT

[0003] This invention was made with government support under grant numbers R35GM131728, K08HL153782, and R01HL117952 awarded by the National Institutes of Health (NIH). The government has certain rights in the invention.BACKGROUND

[0004] Venous malformations (VMs) consists of hugely enlarged and dysmorphic veins that cause significant disfigurement, pain, and complications such as bleeding and coagulopathy. Current treatment for patients with venous malformations is limited by the anatomical location of the venous malformations and entails life-long pharmacotherapy and / or surgical procedures. Current pharmacotherapy solutions (with sirolimus) slow venous malformation growth, but do not reduce or eliminate venous malformations. Additionally, once usage of the pharmaceutical is stopped (often due to systemic toxicity), the venous malformations expand. Regarding surgical solutions, the surgical procedures often need to be repeated throughout the life of the patient. The surgical procedures are also often unsuccessful and can entail severe hemorrhage.

[0005] Presently, there are no treatments for venous malformations that effectively eliminate venous malformations. Therefore, a solution is needed that eliminates or reduces venous malformations, preferably without surgery or life-long pharmaceutical use.SUMMARY

[0006] Embodiments of the invention concern methods of treating venous malformations.

[0007] In some embodiments of the invention, a method of treating venous malformations includes selecting a patient having venous malformations, administering an effective amountof particles to the patient, and irradiating at least one of the venous malformations. The particles are capable of causing a photothermal effect.BRIEF DESCRIPTION OF THE DRAWINGS

[0008] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0009] Disclosed herein are embodiments of systems, apparatuses, and methods pertaining to treating venous malformations. This description includes drawings, wherein:

[0010] FIG. 1 is an overview of systemically injecting near infrared light triggered gold nanoshells for treatment of venous malformations.

[0011] FIG. 2 is a transmission electron micrograph image of gold nanoshells. The scale bar is 100 nm.

[0012] FIG. 3 is a UV-visible spectrum of gold nanoshells suspended in water. Absorbance(in absorbance units) is graphed as a function of wavelength (in nm).

[0013] FIG. 4 is a representative graph demonstrating changes in temperature (in °C) from a baseline as a function of time (in minutes) of gold nanoshells in solution at different concentrations, namely, 25, 50, 100, and 200 pg / mL gold nanoshells, during irradiation with near infrared light (808 nm) at an intensity of 1000 mW / cm2for 10 minutes.

[0014] FIG. 5 is a representative graph demonstrating changes in temperature (in °C) from a baseline as a function of time (in minutes) of gold nanoshells in solution (200 pg / mL), during irradiation with near infrared light (808 nm) at different intensities, namely, 250, 500, 750, and 1000 mW / cm2for 10 minutes.

[0015] FIG. 6 is a representative graph demonstrating changes in temperature (in °C) from a baseline as a function of time (in minutes) of gold nanoshells in solution (200 pg / mL), during and after repeated irradiation events with near infrared light (808 nm) at an intensity of 1000 mW / cm2for 10 minutes. N=4 independent experiments.

[0016] FIG. 7 is a graph of whole blood concentration of elemental gold (in ng gold / pl whole blood) as a function of time after tail vein injection of gold nanoshells, as determined by inductively coupled plasma mass spectrometry (ICP-MS). Measurements were taken at 30 minutes, 4 hours, and 24 hours after injection. Data expressed as means ± SD. N = 4 mice.

[0017] FIG. 8 is a bar chart of concentrations of elemental gold in organs (spleen, liver, lungs, heart, kidney) and venous malformations (in ng gold / mg tissue) 24 hours after tail vein injection of gold nanoshells. Data expressed as means ± SD. N = 4 mice.

[0018] FIG. 9 is a collection of representative hematoxylin and eosin stained sections of the venous malformation at 600X magnification demonstrating passive gold nanoshells aggregate accumulation (red arrow pointing to dark spots) 24 hours after tail injection. V indicates a blood vessel.

[0019] FIG. 10 is an overview of an experimental timeline. SAL indicates saline. AuNS indicates gold nanoshells. HUVEC indicates HUVEC-TIE2-L914F cells.

[0020] FIG. 11 is a collection of representative thermal images of venous malformations following intravenous saline or gold nanoshells injection followed by NIR (near infrared) irradiation (808 nm) at an intensity of 1000 mW / cm2for 10 minutes.

[0021] FIG. 12 is a graph of absolute venous malformation volume (mm3), measured by calipers every other day following treatment, as a function of time, in days. Data expressed as means ± SD. N = 8 venous malformations. Analyzed by t-test (p < 0.001).

[0022] FIG. 13 is an image of a collection of representative murine venous malformation lesion explants at Day 20 of the experiment overviewed in FIG. 10. Grey circles indicate venous malformations that were eliminated.

[0023] FIG. 14 is a graph of venous malformation weights at Day 20 of the experiment overviewed in FIG. 10. Data expressed as means ± SD. N = 8 venous malformations. Analyzed by t-test (p < 0.01).

[0024] FIG. 15 is a collection of representative body surface photographs of venous malformations in situ on Day -1 (prior to gold nanoshells injection), Day 3 (48 hours following irradiation), Day 20 (conclusion of experiment), and upon necropsy (skin reflected). The black arrows indicate venous malformations. The red arrow indicates skin burn. The dotted red arrow indicates a scar.

[0025] FIG. 16 is a bar chart of serum laboratory values of organ function. ALB indicates albumin. ALP indicates alkaline phosphatase. ALT indicates alanine aminotransferase. AMY indicates Amylase. TBili indicates total bilirubin. BUN indicates blood urea nitrogen. CR indicates creatinine. GLU indicates glucose. NA+ indicates sodium. K+ indicates potassium. CA indicates calcium. PHOS indicates phosphorous. Day -1 is prior to gold nanoshells injection, and Day 3 and Day 20 are following gold nanoshells injection. Data expressed as means ± SD. N = 4 mice.

[0026] FIG. 17 is a bar chart of organ weights of animals 20 days following saline (SAL; indicated by red bars) or gold nanoshells (AuNS; indicated by blue bars) injection. Data expressed as means ± SD. N = 8.

[0027] FIG. 18 is a collection of representative hematoxylin and eosin stained sections of heart, lungs, liver, kidney, and spleen 20 days after injection with gold nanoshells injection. N = 4. The scale bar indicates 20 pm. Red arrows indicate gold aggregates.

[0028] FIG. 19 is a collection of images displaying HUVEC-TIE2-L914F cells form blood vessels derived from human cells in immune-deficient mice. Panel A is a representative photo of a venous malformation taken in situ at necropsy with skin reflected. Panels B-E are slides of the same tissue stained differently. Panel B is a representative 15X magnitude image of hematoxylin and eosin stained section of venous malformations. The red outline delineates ectatic blood vessels filled with red blood cells. The scale bar indicates 1000 pm. Panel C is a detail of venous malformation at high power magnification (600X). V indicates a blood vessel. The scale bar indicates 20 pm. Panel D is a representative 15X magnification of a venous malformation tissue section immunostained for human CD31+ cells (brown). The scale bar indicates 1000 pm. Panel E is a detail of venous malformation stained for human CD31 at high power magnification (600X). + indicates vessels surrounded by CD31+ cells. The scale bar indicates 20 pm.

[0029] FIG. 20 is a collection of representative 15X magnification images of residual venous malformations taken at necropsy. Venous malformations are from animals in the gold nanoshells group. Panel A is a hematoxylin and eosin stained section. The red outline delineates ectatic blood vessels filled with red blood cells. Panel B is adjacent venous malformation tissue section immunostained for human CD31 to identify human cells (brown).

[0030] FIG. 21 is a collection of representative 15X magnification images of residual venous malformations taken at necropsy. Venous malformations are from animals in the gold nanoshells and NIR group. Panel C is a hematoxylin and eosin stained section without evidence of perfused blood vessels. Panel D is adjacent venous malformation tissue section immunostained for human specific CD31 without evidence of human derived (CD31+) vessels.

[0031] FIG. 22 is a representative image taken by thermal camera of skin area 1 cm lateral to the venous malformation after injection with gold nanoshells followed 24 hours later by irradiation of the venous malformation with 808 nm NIR laser at an irradiation intensity of 1000 mW / cm2for 10 minutes. Area 1 is not irradiated. Area 2 is irradiated.

[0032] FIG. 23 is a collection of photographs taken of animal skin (area 2 of FIG. 22). The photograph on the left was taken immediately following irradiation and the photograph on the right was taken 5 days post irradiation, demonstrating no skin injury.

[0033] FIG. 24 is a graph of body weights (in grams) of animals following injection of saline (red line with circle markers) or gold nanoshells (blue line with triangle markers) as a function of time (in days). Weights were obtained every other day. N = 8 mice.

[0034] FIG. 25 is a TEM (transmission electron microscopy) image of gold nanoparticles. The bar represents 20 nanometers.

[0035] FIG. 26 demonstrates thermal characteristics of gold nanoparticles that were irradiated with near infrared light (808 nm) in vitro with an irradiation intensity of 1000 mW / cm2. Increases in temperature (in °C) are graphed as a function of time (in minutes) for concentrations of gold nanoparticles at 15, 30, 60, and 120 pg / mL.

[0036] FIG. 27 demonstrates thermal characteristics of gold nanoparticles that were irradiated with near infrared light (808 nm) in vitro with various irradiation intensities, namely, 250, 500, 750, and 1000 mW / cm2. Increases in temperature (in °C) are graphed as a function of time (in minutes) for gold nanoparticles.

[0037] FIG. 28 is a graph of temperature (in °C) of gold nanoparticles as a function of time (in minutes) for gold nanoparticles that were repeatedly irradiated with near infrared light (808 nm) with an intensity of 1000 mW / cm2to generate heat. The red lines above indicate the duration of irradiation.

[0038] FIG. 29 is a representative histology of passive gold nanoparticle accumulation (arrows) within venous malformations at 6X magnification.

[0039] FIG. 30 is a representative histology of passive gold nanoparticle accumulation (arrows) within venous malformations at 40X magnification.

[0040] FIG. 31 is a graph of venous malformation volume (mm3) as a function of time (days) throughout a representative animal experiment, in which venous malformations were measured over time following systemic gold nanoparticle injection. The blue line with square markers represents animals that did not receive irradiation (hv) at the venous malformation. The red line with circle markers represents animals that did receive hv (808 nm near infrared light at an intensity of 1000 mW / cm2for 15 minutes) at the venous malformation 24 hours following intravenous administration of gold nanoparticles.

[0041] FIG. 32 is a collection of representative photos from the experiment of FIG. 31 demonstrating venous malformation size at Day 0 (prior to gold nanoparticle injection), Day 3 (48 hours following hv), and Day 40 (conclusion of the experiment).DETAILED DESCRIPTION

[0042] The present invention is described with reference to the attached figures. The figures are not drawn to scale, and they are provided merely to illustrate the instant invention. Several aspects of the invention are described below with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the invention. One having ordinary skill in the relevant art, however, will readily recognize that the invention can be practiced without one or more of the specific details or with other methods. In other instances, well- known structures or operations are not shown in detail to avoid obscuring the invention. The present invention is not limited by the illustrated ordering of acts or events, as some acts may occur in different orders and / or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the present invention.Definitions

[0043] As used herein, the term “comprising” means that other elements can also be present in addition to the defined elements presented. The use of “comprising” indicates inclusion rather than limitation.

[0044] As used herein the term “consisting essentially of’ refers to those elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0045] The term “consisting of’ refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0046] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0047] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.”

[0048] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within one or more than one standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value.

[0049] Where a range of values is provided, each numerical value between the upper and lower limits of the range is contemplated and disclosed herein.

[0050] The terms “composition” and “formulation” are used interchangeably.

[0051] A “patient” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult, or senior adult)) and / or other non-human animals, for example, mammals (e.g., primates (e.g., cynomolgus monkeys, rhesus monkeys); commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs) and birds (e.g., commercially relevant birds such as chickens, ducks, geese, and / or turkeys). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal. In jurisdictions that forbid the patenting of methods that are practiced on the human body, the meaning of “administering” to a human patient shall be restricted to prescribing a controlled substance that will be administer to the patent by any technique (e.g., orally, inhalation, topical application, injection, insertion, etc.). The broadest reasonable interpretation that is consistent with laws or regulations defining patentable subject matter is intended. In jurisdictions that do not forbid the patenting of methods that are practiced on the human body, the “administering” includes both methods practiced on the human body and also the foregoing activities.

[0052] The terms “administer,” “administering,” or “administration,” refers to implanting, absorbing, ingesting, injecting, inhaling, or otherwise introducing a compound, or a pharmaceutical composition thereof. Particles described herein can be administered by any appropriate route known in the art including, but not limited to, oral or parenteral routes, including intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary, nasal, rectal, and topical (including buccal and sublingual) administration. Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, administration will generally be systemic. In some other embodiments, administration will be local, e.g., the particles can be administered directly at site of venous malformation.

[0053] In some embodiments, the particles are administered parenterally, i.e., parenteral administration. The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection, infusion and other injection or infusion techniques, without limitation..

[0054] In some embodiments, administration is subcutaneous administration. As used herein, the term “subcutaneous administration” refers to administration just below the skin.

[0055] In some embodiments, administration is intravenous administration. As used herein, “intravenous,” as used herein, refers to taking place within, or administered into, a vein or veins.

[0056] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a “pathological condition” (e.g., a disease, disorder, or condition, or one or more signs or symptoms thereof) described herein. In some embodiments, treatment may be administered after one or more signs or symptoms have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or condition. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.

[0057] The terms “condition,” “disease,” and “disorder” are used interchangeably.

[0058] An “effective amount” of an agent described herein refers to an amount sufficient to elicit the desired biological response. In certain embodiments, an effective amount is an amount of particles sufficient to elicit the desired biological response upon irradiation of the particles.An effective amount of particles described herein may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the particles, the condition being treated, the mode of administration, and the age and health of the subject. In certain embodiments, an effective amount is a therapeutically effective amount. In certain embodiments, an effective amount is a prophylactic treatment. In certain embodiments, an effective amount is the amount of particles described herein in a single dose. In certain embodiments, an effective amount is the combined amounts of particles described herein in multiple doses. When an effective amount of a composition is referred herein, it means the amount is prophylactically and / or therapeutically effective, depending on the subject and / or the disease to be treated. Determining the effective amount or dosage is within the abilities of one skilled in the art.

[0059] In some embodiments, the effective amount of particles administered to the subject is from about 10 mg / kg to 200 mg / kg. For example, the effective amount of particles administered to the subject is from about 10 mg / kg to about 150 mg / kg. In some embodiments, the effective amount of particles administered to the subject is from about 10 mg / kg to about 100 mg / kg. For example, the effective amount of particles administered to the subject is from about 10 mg / kg to about 50 mg / kg. In some embodiments, the effective amount of particles administered to the subject is from about 20 mg / kg to about 40 mg / kg. For example, the effective amount of particles administered to the subject is from about 30 mg / kg to about 40 mg / kg. In some embodiments, the effective amount of particles administered to the subject is about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 65 mg / kg, about 70 mg / kg, about 75 mg / kg, about 80 mg / kg, about 85 mg / kg, about 90 mg / kg, about 95 mg / kg, or about 100 mg / kg. For example, the effective amount of particles administered to the subject is about 25 mg / kg, about 26 mg / kg, about 27 mg / kg, about 28 mg / kg, about 29 mg / kg, about 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg, about 37 mg / kg, about 38 mg / kg, about 39 mg / kg, about 40 mg / kg, about 41 mg / kg, about 42 mg / kg, about 43 mg / kg, about 44 mg / kg or about 45 mg / kg. In some preferred embodiments, the effective amount of particles administered to the subject is about 30 mg / kg, about 31 mg / kg, about 32 mg / kg, about 33 mg / kg, about 34 mg / kg, about 35 mg / kg, about 36 mg / kg, about 37 mg / kg, about 38 mg / kg, about 39 mg / kg, or about 40 mg / kg.

[0060] A “therapeutically effective amount” of particles described herein is an amount sufficient to provide a therapeutic benefit in the treatment of a condition or to delay or minimize one or more symptoms associated with the condition. A therapeutically effective amount of particles means an amount of particles, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition. In certain embodiments, a therapeutically effective amount is an amount of particles, alone or in combination with other therapies, which provides a therapeutic benefit in the treatment of the condition, upon irradiation of the particles. The term “therapeutically effective amount” can encompass an amount that improves overall therapy, reduces or avoids symptoms, signs, or causes of the condition, and / or enhances the therapeutic efficacy of another therapeutic agent. In certain embodiments, a therapeutically effective amount is an amount of particles sufficient to elicit the desired biological response, upon irradiation of the particles.

[0061] A “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient,” as used herein, refers to any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The compositions may also contain other active compounds providing supplemental, additional, or enhanced therapeutic functions

[0062] “Photothermal,” as used herein, refers to having a photothermal conversion function, i.e., absorbing incident radiation and converting the radiation to thermal energy. For example, a photothermal material is a material that absorbs light and generates heat. In some embodiments, the radiation absorbed is in the infrared or near-infrared regions of the electromagnetic spectrum The terms “photothermal material” and “photothermal conversion material” are used interchangeably herein. Exemplary photothermal materials include, but are not limited to metallic materials, semiconductors, carbon-based materials, and conjugated organic materials.

[0063] In some embodiments, the photothermal material is a metal. For example, the photothermal material is a noble metal, e.g., gold, silver, rhenium, ruthenium, rhodium, palladium, osmium, iridium, and platinum. In some embodiments, the photothermal material comprises gold. In some embodiments, the photothermal material comprises silver.

[0064] In some embodiments, the photothermal material is a carbon-based material. For example, the photothermal material is graphite (e.g., exfoliated graphite), carbon black, carbon nanotubes (CNTs), graphene, graphene oxide, or carbon dots.

[0065] In some embodiments, the photothermal material comprises an inorganic semiconductor.

[0066] In some embodiments, photothermal material comprises, one or more polymers. Examples of polymers include, but are not limited to, polyaniline (PANI), polypyrrole (PPy), polythiophene (PTh), polydopamine (PDA), donor-acceptor (D-A) conjugated polymers, and poly(3,4-ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS).

[0067] In some embodiments, the photothermal material is selected from the group consisting of cyanine dyes with one or more sulfate and / or sulfonate groups, compounds having a phthalocyanine skeleton (phthalocyanine dyes), compounds having a naphthalocyanine skeleton (naphthalocyanine dyes), compounds having a squarylium skeleton (squarylium dyes), diimmonium salts, aminium salts, quinacridone, diketopyrrolopyrrole, naphthol -based azo pigments, anthraquinone, acetacetic acid-based and / or an anhydride-based azo pigment, dioxazine, benzotri azole, polymethine dyes (e.g., bis(aminoaryl)polymetbine IR dyes), and any combination thereof.

[0068] In some embodiments, the photothermal material comprises TiCh. In some embodiments, the photothermal material comprises indocyanine green. In some embodiments, the photothermal material comprises metals and / or organic dyes, including but not limited to, silver, platinum, copper sulfide, zinc sulfide, and / or heptamethine cyanine.

[0069] A “sclerosant,” as used herein, refers to a substance, generally comprising an irritant, injected into the lumen of a vein in order to affect the destruction of the target vessel, i.e., any agents or chemical irritants that can be used in sclerosing veins, particularly sclerosant which act by protein denaturation, or a substance which causes tissue irritation and / or thrombosis with subsequent local inflammation and tissue necrosis. Sclerosing agents can be powders, solutions, detergents, acids or bases. Examples of sclerosants include, but are not limited to, absolute ethanol, hypertonic saline, hypertonic glucose, acetic acid, Polidocanol, bleomycin, Picibanil, Sodium tetradecyl sulfate (STS), and sclerosant foam. “Sclerosant” is interchangeable with “sclerosant agent.”

[0070] A “particle,” as used herein, refers to a small mass that can be composed of any material, such as alumina, glass, silica, latex, plastic, agarose, gold, silver, metal, any polymeric material, and be of any size and shape. Stated in another way, a “particle” is not limited to a particular shape and size and can include spherical, rod like, faceted, plates, shells, oviods, or other shapes. Particles can be monodisperse or polydisperse. The size distribution of particles can be characterized by Poly dispersity index (PDI). PDI of particle size distribution isdetermined by methods commonly known by one of ordinary skill in the art, for example, by dynamic light scattering (DLS) measurement. With regard to DLS used for particle size determinations, the common use of second or third order cumulant analyses to fit the autocorrelation function leads to the values of PDI. The absolute value of PDI determined from this method ranges from zero and higher, with small values indicating narrower distributions. For example, PDI ranging from 0 to about 0.3 or from 0 to about 0.4 presents relatively monodisperse particle size distributions. This criterion has been generally accepted in the art of dynamic light scattering for particle size determinations. In some embodiments the particles are monodisperse. For example, the particles have a narrow particle size distribution such as having a poly dispersity index below about 0.5, such as below about 0.4, below about 0.3 or below about 0.2.

[0071] In some embodiments, the size and morphology (e.g., diameter, sphericity, and porosity) of particles may be characterized by techniques including, but not limited to, dynamic light scattering, coulter counter, microscopy, sieve analysis, dynamic image analysis, static image analysis, and laser diffraction.

[0072] In some embodiments, the particle is a microparticle. As used herein, the term “microparticle” refers to particles that are from about 1pm to about 500 pm in size. For example, a microparticle is from about 1pm to about 500 pm in size. In some embodiments, the microparticle is from about 1 pm to about 250 pm, from about 1 pm to about 200 pm, from about 1 pm to about 150 pm or from about 1 pm to about 100 pm in size. For example, the microparticle is from about 1 pm to about 75 pm or from about 1 pm to about 100 pm in size.

[0073] In some embodiments, the particle is a nanoparticle. As used herein, the term “nanoparticle” refers to particles that are from about 1 nm to about 950 nm in size. For example, a nanoparticle is from about 10 nm to about 500 nm in size. In some embodiments, the nanoparticle is from about 10 nm to about 250 nm, from about 25 nm to about 200 nm, from about 50 nm to about 175 nm or from about 75 nm to about 150 nm in size. For example, the nanoparticle is from about 80 nm to about 125 nm or from about 90 nm to about 120 nm in size.

[0074] In some embodiments, the particles have a hydrodynamic diameter of about 20 nm to about 180 nm. For example, the particles have a hydrodynamic diameter of about 20 nm to about 100 nm. In some embodiments, the particles have a hydrodynamic diameter of about 20 nm to about 50 nm.

[0075] Generally, the particle comprises a photothermal material. Exemplary photothermal materials are described elsewhere herein.

[0076] “Irradiating” and “irradiation,” as used herein, refers to exposing a subject to a selected wavelength of electromagnetic radiation, where at least a portion of the supplied electromagnetic radiation is absorbed by at least some of the particles administered to the subject.

[0077] A “venous malformation,” as used herein, refers to a vascular malformation or lesion that results from veins that have developed abnormally, or are widened and / or abnormally shaped.

[0078] A “targeting ligand,” as used herein, refers to a molecule that binds relatively specifically to a target molecule, e.g., a cell surface receptor.Methods of Treatment

[0079] In one aspect provided herein is a method of treating venous malformations. Generally, the method comprises administering to a patient in need thereof an effective amount of particles capable of causing a photothermal effect and irradiating at least one of the venous malformations. For example, the particles may be irradiated to generate heat.

[0080] In some embodiments, the method further comprises a step of selecting a patient in need of treatment for venous malformations. For example, the method comprises selecting a patient having or diagnosed with venous malformations. In some embodiments, selecting a patient in need of treatment comprises diagnosing the patient.

[0081] For targeting to venous malformation, particles can be conjugated with a targeting ligand. For example, the particles can be conjugated with a targeting ligand capable of binding with venous malformation endothelial cell. In some embodiments, the targeting ligand is capable of binding, e.g., bind specifically with endothelial adhesion molecule-1, such as an anti-CD31 antibody.

[0082] Inventors have discovered inter alia that particles that do not comprise a targeting ligand unexpectedly and surprisingly are effective, and intravenous administration of particles is also unexpectedly and surprisingly effective. Accordingly, in some embodiments of the method, the particles are not conjugated with a targeting ligand. For example, the particles are not conjugated with a targeting ligand capable of binding with endothelial adhesion molecule- 1. In some embodiments, the particles are not conjugated with a targeting ligand capable of binding with venous malformation endothelial cell. In some embodiments, the particles are not conjugated with an anti-CD31 antibody.

[0083] In some embodiments, the method further comprises co-administering a therapy or agent known in the art for treating venous malformations. As used herein, the term “coadminister” refers to administration of two or more therapies within a 24 hour period of each other, for example, as part of a clinical treatment regimen. In other embodiments, “coadminister” refers to administration within 12 hours, within 6 hours, within 5 hours, within 4 hours, within 3 hours, within 2 hours, within 1 hour, within 45, within 30 minutes, within 20, within 15 minutes, within 10 minutes, or within 5 minutes of each other. In other embodiments, “co-administer” refers to administration at the same time, either as part of a single formulation or as multiple formulations that are administered by the same or different routes. When the therapies are administrated in different pharmaceutical compositions or at different times, routes of administration can be same or different.

[0084] In some embodiments, a therapy or agent known in the art for treating venous malformations is not co-administered to the patient.

[0085] In some embodiments, a sclerosant is not co-administered to the patient. For example, one or more of absolute polidocanol, ethanol, hypertonic saline, hypertonic glucose, acetic acid, bleomycin, picibanil, Sodium tetradecyl sulfate (STS), sclerosant foam, or any other suitable sclerosant is not co-administered to the subject.

[0086] In some embodiments, the administering is intravenous. However, the administering need not be intravenous and may be accomplished by oral ingestion, instillation, non-intravenous injection, or any other suitable means. In some embodiments, the administering is by injection into one or more venous malformations. In some embodiments, the administering is through an intravascular catheter into a feeder vessel. In some embodiments, the administering is intravenous and the particles are not conjugated with a targeting ligand capable of binding with venous malformation endothelial cell (e.g., a targeting ligand capable of binding with endothelial adhesion molecule- 1), optionally the targeting ligand is an anti-CD31 antibody.

[0087] In some embodiments, the administering is intravenous and the particles are conjugated with a targeting ligand capable of binding with venous malformation endothelial cell. For example, the administering is intravenous and the particles are conjugated with a targeting ligand capable of binding with endothelial adhesion molecule-1, e.g., the particles are conjugated with an anti-CD31 antibody.

[0088] In some embodiments, the particles are substantially free of a sclerosant. The particles may be microparticles, nanoparticles, or any other suitable particle. According tosome embodiments, the particles are substantially spherical. However, this is not required and the particles may be of any suitable shape, such as rods, shells, ovoids, etc.

[0089] According to some embodiments, the particles have a poly dispersity index (PDI) below about 0.5. Optionally, the PDI is measured by dynamic light scattering (DLS). The PDI may be below 0.4, below 0.3, below 0.2, and / or below 0.1. In some embodiments, the PDI is approximately 0.5, greater than 0.5, greater than 0.6, greater than 0.7, greater than 0.8, and / or greater than 0.9.

[0090] In some embodiments, the particles are made of, in whole or in part, a photothermal material. For example, the particles may be made of, in whole or in part, one or more metals. According to some embodiments, the particles are made of, in whole or in part, one or more noble metals. For example, the particles may be made of, in whole or in part, one or more of rhenium, ruthenium, rhodium, palladium, silver, osmium, iridium, platinum, and / or gold. In some embodiments, the particles are made of, in whole or in part, an inorganic semiconductor. In some embodiments, the particles are made of, in whole or in part, one or more polymers. Examples of polymers include polyaniline (PANI), polypyrrole (PPy), polythiophene (PTh), poly dopamine (PDA), donor-acceptor (D-A) conjugated polymers, and poly (3,4- ethylenedioxythiophene):poly(4-styrenesulfonate) (PEDOT:PSS). It is contemplated that the particles are made of, in whole or in part, graphene and / or graphene oxide.

[0091] According to some embodiments, the irradiating occurs for at least 15 minutes. In some embodiments, the irradiating occurs for at least 20 minutes, for at least 25 minutes, for at least 30 minutes, for at least 35 minutes, for at least 40 minutes, for at least 45 minutes, for at least 50 minutes, for at least 55 minutes, and / or for at least 60 minutes after administering the particles. In some embodiments, irradiating occurs at least 5 minutes, at least 15 minutes, at least 30 minutes, at least one hour, at least two hours, at least three hours, at least four hours, at least six hours, at least eight hours, at least twelve hours, at least sixteen hours, and / or at least twenty-four hours after administering.

[0092] In some embodiments, the irradiating is at a wavelength between about 600 nm and about 2500 nm, between about 600 nm and about 1500 nm, between about 600 nm and about 1000 nm, between about 700 nm and about 900 nm, between about 775 nm and about 825 nm, and / or is at a wavelength of about 808 nm. In some embodiments, the effective amount of particles is from about 10 mg to about 200 mg of particles per kilogram of the patient, from about 10 mg to about 150 mg of particles per kilogram of the patient, from about 10 mg to about 100 mg of particles per kilogram of the patient, from about 10 mg to about 50 mg ofparticles per kilogram of the patient, from about 20 mg to about 40 mg of particles per kilogram of the patient, from about 30 mg to about 40 mg of particles per kilogram of the patient, and / or is about 36 mg of particles per kilogram of the patient.

[0093] According to some embodiments, the particles have a hydrodynamic diameter of about 20 nm to about 180 nm, about 20 nm to about 100 nm, and / or about 20 nm to about 50 nm. In some embodiments, the particles accumulate within abnormal vasculature of the tissue of one or more venous malformations. The size of the particles affects particle accumulation within the venous malformations.

[0094] In some embodiments, the irradiating occurs for at least one minute, for at least two minutes, for at least three minutes, for at least five minutes, for at least fifteen minutes, for at least thirty minutes, and / or for at least thirty minutes. According to some embodiments, the administering and irradiating steps are repeated. In some embodiments, the administering and irradiating steps are repeated at least two times, at least three times, at least four times, at least five times, and / or at least six times. In some embodiments, the administering and irradiating steps are repeated until a venous malformation is reduced in size and / or eliminated.

[0095] In some embodiments, the method further includes, after the irradiating step, (a) waiting for a period of time and (b) repeating the irradiating step. Waiting does not include irradiating. The period of time may be at least five seconds, at least ten seconds, at least fifteen seconds, at least thirty seconds, at least one minute, at least five minutes, at least ten minutes, at least fifteen minutes, at least thirty minutes, at least one hour, at least two hours, at least four hours, at least eight hours, at least one day, and / or at least one week. In some embodiments, the method further includes repeating steps (a) and (b) once, twice, thrice, and / or four or more times.

[0096] In some embodiments, the irradiating is at an intensity of at least 150 mW / cm2. In some embodiments, the irradiating is at an intensity of about 200 mW / cm2to about 2000 mW / cm2, about 200 mW / cm2to about 1000 mW / cm2, about 300 mW / cm2to about 800 mW / cm2, and / or about 400 mW / cm2to about 600 mW / cm2. In some embodiments, the particles are in a composition including the particles and, optionally, a pharmaceutically acceptable carrier or excipient.

[0097] Some exemplary aspects of the disclosure are described by one or more of following numbered Embodiments:

[0098] Embodiment 1 : A method of treating venous malformations comprising: selecting a patient having venous malformations; (b) administering an effective amount of particles tothe patient, wherein the particles are capable of causing a photothermal effect; and (c) irradiating at least one of the venous malformations.

[0099] Embodiment 2: The method of Embodiment 1, wherein the particles are not conjugated with a targeting ligand capable of binding with venous malformation endothelial cell (e.g., a targeting ligand capable of binding with endothelial adhesion molecule- 1), optionally the targeting ligand is an anti-CD31 antibody.

[0100] Embodiment 3: The method of Embodiment 1 or 2, wherein a sclerosant is not coadministered to the patient, optionally the sclerosant is absolute polidocanol, ethanol, hypertonic saline, hypertonic glucose, acetic acid, bleomycin, picibanil, Sodium tetradecyl sulfate (STS), or sclerosant foam.

[0101] Embodiment 4: The method of any one of Embodiments 1-3, wherein the administering is intravenous.

[0102] Embodiment 5: The method of any one of Embodiments 1-4, wherein the particles are substantially free of a sclerosant.

[0103] Embodiment 6: The method of any of Embodiments 1-5, wherein the particles are microparticles.

[0104] Embodiment 7: The method of any of Embodiments 1-6, wherein the particles are nanoparticles.

[0105] Embodiment 8: The method of any one of Embodiments 1-7, wherein the particles are substantially spherical.

[0106] Embodiment 9: The method of any one of Embodiments 1-8, wherein the particles have a poly dispersity index (PDI) below about 0.5, optionally the PDI is measured by dynamic light scattering (DLS).

[0107] Embodiment 10: The method of any one of Embodiments 1-9, wherein the particles comprise a photothermal material.

[0108] Embodiment 11 : The method of any of Embodiments 1-10, wherein the particles comprise one or more metals.

[0109] Embodiment 12: The method of any of Embodiments 1-11, wherein the particles comprise one or more noble metals.

[0110] Embodiment 13: The method of any of Embodiments 1-12, wherein the particles comprise gold.

[0111] Embodiment 14: The method of any of Embodiments 1-13, wherein the particles comprise silver.

[0112] Embodiment 15: The method of any of Embodiments 1-14, wherein the particles comprise an inorganic semiconductor.

[0113] Embodiment 16: The method of any of Embodiments 1-15, wherein the particles comprise one or more polymers.

[0114] Embodiment 17: The method of any of Embodiments 1-16, wherein the administering comprises injecting into one or more venous malformations.

[0115] Embodiment 18: The method of any one of Embodiments 1-17, wherein the irradiating occurs at least 15 minutes, e.g., at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes or at least 55 minutes after administering the particles.

[0116] Embodiment 19: The method of any of Embodiments 1-18, wherein the irradiating occurs at least one hour after administering.

[0117] Embodiment 20: The method of any of Embodiments 1-19, wherein the irradiating occurs at least two hours after administering.

[0118] Embodiment 21 : The method of any of Embodiments 1-20, wherein the irradiating occurs at least four hours after administering.

[0119] Embodiment 22: The method of any of Embodiments 1-21, wherein the irradiating occurs at least eight hours after administering.

[0120] Embodiment 23: The method of any of Embodiments 1-22, wherein the irradiating occurs at least sixteen hours after administering.

[0121] Embodiment 24: The method of any of Embodiments 1-23, wherein the irradiating occurs at least twenty -four hours after administering.

[0122] Embodiment 25: The method of any of Embodiments 1-24, wherein the irradiating is at a wavelength between about 600 nm and about 2500 nm.

[0123] Embodiment 26: The method of any of Embodiments 1-25, wherein the irradiating is at a wavelength between about 600 nm and about 1500 nm.

[0124] Embodiment 27: The method of any of Embodiments 1-26, wherein the irradiating is at a wavelength between about 600 nm and about 1000 nm.

[0125] Embodiment 28: The method of any one of Embodiments 1-27, wherein the irradiating is at a wavelength between about 700 nm and about 900 nm.

[0126] Embodiment 29: The method of any one of Embodiments 1-28, wherein the irradiating is at a wavelength between about 775 nm and about 825 nm

[0127] Embodiment 30: The method of any of Embodiments 1-29, wherein the irradiating is at a wavelength of about 808 nm.

[0128] Embodiment 31 : The method of any of Embodiments 1-30, wherein the effective amount is from about 10 mg to about 200 mg of particles per kilogram of the patient.

[0129] Embodiment 32: The method of any of Embodiments 1-31, wherein the effective amount is from about 10 mg to about 150 mg of particles per kilogram of the patient.

[0130] Embodiment 33: The method of any of Embodiments 1-32, wherein the effective amount is from about 10 mg to about 100 mg of particles per kilogram of the patient.

[0131] Embodiment 34: The method of any of Embodiments 1-33, wherein the effective amount is from about 10 mg to about 50 mg of particles per kilogram of the patient.

[0132] Embodiment 35: The method of any of Embodiments 1-34, wherein the effective amount is from about 20 mg to about 40 mg of particles per kilogram of the patient.

[0133] Embodiment 36: The method of any of Embodiments 1-34, wherein the effective amount is from about 30 mg to about 40 mg of particles per kilogram of the patient.

[0134] Embodiment 37: The method of any of Embodiments 1-36, wherein the effective amount is about 36 mg of particles per kilogram of the patient

[0135] Embodiment 38: The method of any of Embodiments 1-37, wherein the particles have a hydrodynamic diameter of about 20 nm to about 180 nm.

[0136] Embodiment 39: The method of any of Embodiments 1-38, wherein the particles have a hydrodynamic diameter of about 20 nm to about 100 nm.

[0137] Embodiment 40: The method of Embodiment 39, wherein the particles have a hydrodynamic diameter of about 20 nm to about 50 nm.

[0138] Embodiment 41 : The method of any of Embodiments 1-40, wherein the irradiating occurs for at least one minute.

[0139] Embodiment 42: The method of any of Embodiments 1-41, wherein the irradiating occurs for at least two minutes.

[0140] Embodiment 43: The method of any of Embodiments 1-42, wherein the irradiating occurs for at least three minutes.

[0141] Embodiment 44: The method of any of Embodiments 1-43, wherein the irradiating occurs for at least five minutes.

[0142] Embodiment 45: The method of any of Embodiments 1-44, wherein the irradiating occurs for at least fifteen minutes.

[0143] Embodiment 46: The method of any of Embodiments 1-45, wherein the administering and irradiating steps are repeated.

[0144] Embodiment 47: The method of any of Embodiments 1-46, wherein the administering and irradiating steps are repeated at least two times.

[0145] Embodiment 48: The method of any of Embodiments 1-47, wherein the administering and irradiating steps are repeated at least three times.

[0146] Embodiment 49: The method of any of Embodiments 1-48, further comprising after the irradiating step: (a) waiting for a period of time (e.g., at least thirty seconds), wherein waiting does not include irradiating; and (b) repeating the irradiating step.

[0147] Embodiment 50: The method of Embodiment 49, further comprising repeating steps (a) and (b).

[0148] Embodiment 51 : The method of any of Embodiments 49 or 50, further comprising repeating steps (a) and (b) twice.

[0149] Embodiment 52: The method of any of Embodiments 49-51, further comprising repeating steps (a) and (b) at least three times.

[0150] Embodiment 53: The method of any one of Embodiments 1-52, wherein the irradiating is at an intensity of at least 150 mW / cm2.

[0151] Embodiment 54: The method of any of Embodiments 1-53, wherein the irradiating is at an intensity of about 200 mW / cm2to about 2000 mW / cm2.

[0152] Embodiment 55: The method of any of Embodiments 1-54, wherein the irradiating is at an intensity of about 200 mW / cm2to about 1000 mW / cm2.

[0153] Embodiment 56: The method of any of Embodiments 1-55, wherein the irradiating is at an intensity of about 300 mW / cm2to about 800 mW / cm2.

[0154] Embodiment 57: The method of any of Embodiments 1-56, wherein the irradiating is at an intensity of about 400 mW / cm2to about 600 mW / cm2.

[0155] Embodiment 58: The method of any of Embodiments 1-57, wherein the particles are in a composition comprising the particles and optionally a pharmaceutically acceptable carrier or excipient.

[0156] Embodiment 59: The method of any of Embodiments 1-58, wherein the administering comprises administering through an intravascular catheter into a feeder vessel.

[0157] Embodiment 60: The method of any of Embodiments 1-59, wherein the administering is intravenous, and wherein the particles are not conjugated with a targeting ligand capable of binding with venous malformation endothelial cell (e.g., a targeting ligandcapable of binding with endothelial adhesion molecule-1), optionally the targeting ligand is an anti-CD31 antibody.

[0158] Embodiment 61 : The method of any of Embodiments 1 or 3-59, wherein the administering is intravenous, and wherein the particles are conjugated with a targeting ligand capable of binding with venous malformation endothelial cell.EXAMPLES

[0159] In order for the present disclosure to be more fully understood, the following examples are set forth. The synthetic and biological examples described in this application are offered to illustrate the compositions, pharmaceutical compositions, methods, and kits provided herein and are not to be construed in any way as limiting their scope.Materials and Methods Used.

[0160] Reagents. Sodium citrate tribasic dehydrate, sodium borohydride, gold (III) chloride trihydrate, polyvinylpyrrolidone (PVP) (average molecular weight 55,000 kDa), and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Cobalt chloride hexahydrate, nitric acid, hydrochloric acid, and Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix were purchased from Thermo Fisher Scientific (Waltham, MA, USA). CD31 / PECAM- 1 Recombinant Monoclonal Antibody was purchased from Bethyl Laboratories Montgomery, TX, USA). EBM Basal Medium and EGM Endothelial Cell Growth Medium SingleQuots Supplements were purchased from Lonza (Basel, Switzerland).

[0161] Synthesis of Gold Nanoshells. Gold nanoshells (AuNS) were synthesized by reduction of Au (III) at the surface of sacrificial cobalt seeds. 38.1 mg of cobalt chloride hexahydrate and 47.1 mg sodium citrate tribasic dehydrate were mixed with 400 mL DI water in a round-bottom flask. Under constant stirring, the solution was purged with nitrogen for 40 minutes with 2.0 mL 1 weight % PVP solution added to the solution following 30 minutes of purging. Subsequently, 0.4 mL ice-cold 1 M sodium borohydride solution was added, immediately reducing the cobalt. The solution was purged for an additional 30 minutes to ensure complete oxidation of the borohydride. 360 mL of the resulting cobalt nanoparticle solution was transferred to a separate flask containing 120 mL DI water and 180 pL 0.1 M gold (III) chloride trihydrate. The mixture was stirred for 90 minutes. The resulting particles were concentrated by centrifugation (15,000 rpm for 15 min), washed three times with DI water, and stored at 4°C.

[0162] Characterization of Gold Nanoshells. Particle size, poly dispersity index, and zeta potential of synthesized AuNS were measured by a Malvern Nano ZetaSizer (Malvern Panalytical, Westborough, MA). The ultraviolet-visible absorption spectra of the AuNS were measured by spectrophotometry (Agilent Cary UV-Vis-NIR spectrophotometer, Santa Clara, CA, USA). Morphology was assessed by JOEL 1200EX transmission electron microscopy (Tokyo, Japan) operating at 80 kV. Samples were deposited onto carbon-coated copper grids without further staining. The concentration of gold was measured by ICP-MS (inductively coupled plasma mass spectrometry) (Agilent, Santa Clara, CA, USA) using standard procedures. To prepare the sample, a small volume (10 pL) of AuNS was dissolved in freshly prepared aqua regia for 24 hours, followed by dilution in 5% HC1.

[0163] Photothermal Profile of Gold Nanoshells. The photothermal profile of AuNS under NIR irradiation was assessed every minute using a thermometer. AuNS were suspended in DI water at different gold concentrations (range 25-200 pg / mL Au) and irradiated by an 808 nm NIR iodine laser at varying intensities (range 250-1000 mW / cm2 ) (LDX Optronics Maryville, TN, USA).

[0164] Biodistribution and in Vivo Effect of Gold Nanoshells plus NIR Irradiation. Male 6-8-week-old nude athymic (nu / nu) mice were purchased (Massachusetts General Hospital, Boston, MA, USA). All experimentation was conducted in accordance with protocols approved by Boston Children’s Hospital Institutional Animal Care and Use Committee.

[0165] Human umbilical vein endothelial cells (HUVECs) with a mutation in TIE2-L914F (HUVEC-TIE2- L914F) were cultured on 0.1% gelatin coated plates with EBM Basal Medium supplemented by EGM Endothelial Cell Growth Medium SingleQuots Supplements and 10% FBS. HUVEC-TIE2-L914F cells were obtained directly from Dr. Elisa Boscolo laboratory.

[0166] Murine VM model was generated as described herein. Briefly, cells (2.5 x 106 HUVEC-TIE2-L914F) were isolated and suspended in Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix. For each VM, 200 pl of the cell suspension was injected subcutaneously with a 26G needle to the dorsal flank of the animal. Two VMs were introduced per mouse, with one on each flank. Mice were monitored for the days following the injections and the size (length and width) of the VMs were measured each day with a digital caliper. Tumor volume was calculated as (Vlf I * w)2.

[0167] To determine pharmacokinetics and biodistribution of AuNS, mice (n = 8) were injected (intravenous) with AuNS (450 pg gold) once VM volumes reached 300 mm3± 100 mm3. Approximately 200 pl of blood was drawn from animals at 30 minutes (n = 4), 4 hours(n = 4), and 24 hours (n = 8) post-injection. At 24 hours post-injection, animals were sacrificed, and their VMs and organs were harvested, weighed, homogenized for digestion in freshly prepared aqua regia for at least 24 hours followed by dilution in 5% HC1. Blood samples were similarly digested. Elemental gold was determined by ICP-MS. The remainder of the VMs and organs were prepared for histology.

[0168] To examine the in vivo effect of photothermal therapy, mice with VMs were randomly assigned to two groups (normal saline or AuNS, n = 8 in each group) once tumor volumes reached 300 mm3± 100 mm3. Animals were intravenously injected with either 200 pl of 0.9% normal saline or AuNS solution (450 pg gold). Twenty-four hours following injection, the mice were anesthetized and placed on their side. One VM was irradiated with NIR (808nm, 1000 mW / cm2) for 10 minutes. The VM on the other side was not irradiated. The VM temperature was captured with a photothermal camera (FLIR Systems, Wilsonville, OR). VM size and mouse body weight were measured every other day.

[0169] Blood was obtained by facial vein, was allowed to clot undisturbed for a period of 30 minutes and then centrifuged at 1,500 g for 10 minutes. Plasma was analyzed for chemistries by a VETSCAN VS2 Chemistry Analyzer (Zoetis, Parsippany-Troy Hills, NJ, USA).

[0170] Histology. VMs and organs were immersed in 10% neutral buffered formalin for at least 24 hours, followed by embedding in paraffin and sectioning. Slides were sent to iHisto (Salem, MA) and were stained with hematoxylin and eosin or with anti-human CD31 / PECAM- 1 Recombinant Monoclonal Antibody followed by peroxidase secondary antibody using standard staining procedures.

[0171] Statistical Analyses. All data shown are expressed as the mean ± standard deviation. All p-values were calculated by the unpaired t-test to compared two groups and by ANOVA to compare more than two groups, p < 0.05 was considered statistically significant. Statistical analysis was conducted using Graph Pad Prism (version 9.5.1).Example 1: Synthesis and Characterization of AuNS

[0172] Gold nanoshells (AuNS) were synthesized by galvanically reducing gold (Au(III)) at the surface of cobalt seeds. Resultant AuNS had a hydrodynamic diameter of 39.9 ± 5.7 nm by dynamic light scattering, zeta potential of -14 mV ± 0.6, and poly dispersity index of 0.301 ± 0.035. Transmission electron microscopy (TEM) micrographs, as shown in FIG. 2, revealed spherical particles. Spectroscopy revealed an absorption peak of -800 nm, as shown in FIG.3. Heating of AuNS in phosphate buffered saline (PBS) under irradiation with 808 nm NIRlaser at 1000 mW / cm2for 10 minutes was concentration dependent, as can be seen in FIG. 4. Irradiation of 200 pg / ml AuNS resulted in a 30 degree temperature increase (from room temperature to 50 °C). Heating of 200 pg / ml AuNS was dependent on the irradiance, as can be seen in FIG. 5. Irradiation of 200 pg / ml AuNS with power intensity of 1000 mW / cm2for 10 minutes resulted in a temperature change of 30 °C. Heating of AuNS could be triggered repeatedly by irradiation at 1000 mW / cm2for 10 minutes, as can be seen in FIG. 6.Example 2: Accumulation of AuNS Following Systemic Injection via EPR Effects

[0173] Nanoparticle accumulation in diseased tissues (and therefore the efficacy of those nanoparticles) can be greatly increased by enhanced permeation and retention (EPR)-like effects, where the leakier abnormal vasculature allows increased nanoparticle egress. EPR has been documented in tumors and EPR-like effects have been demonstrated in animal models of choroidal neovascularization, myocardial ischemia and in bioengineered blood vessels. VMs have abnormalities of the endothelium and vascular smooth muscle, and blood flow is slow or stagnant. The presence of EPR-like nanoparticle accumulation in a well-accepted murine model of VMs has been assessed. Up to 60% of human VMs are associated with activated TIE2 mutations (the most common being p.L914F). This activating TIE2 mutation (p.L914F) can induce human umbilical vein endothelial cells (HUVEC-TIE2-L914F) suspended in a basement membrane matrix to form VMs following injection in immunodeficient mice. Within 10 days, mice injected with these HUVECs develop vascular lesions consisting of hugely enlarged and dysmorphic veins (VMs), as can be seen in FIG. 19, with similar characteristics to those described in human patients. The vasculature of the VMs is fully connected to the host vasculature, allowing for the study of systemically administered therapeutics. After VM volumes reached 300 mm3, VM mice were injected with 15 mg / kg of AuNS (450 pg per mouse), or saline via tail vein. This dose is consistent with other reports, and pilot studies confirmed that doses > 15 mg / kg AuNS did not further improve the therapeutic effect in this model. Elemental gold could be detected in blood (by inductively coupled plasma mass spectrometry (ICP- MS) after extraction from whole blood) 30 minutes after intravenous injection, as can be seen in FIG. 7. By 24 hours, very little elemental gold (< Ing / pL whole blood) was detected. 24 hours following injection, organs and VMs were either homogenized and elemental gold extracted and quantified using ICP-MS or evaluated by histology. The highest levels of elemental gold were in the spleen, liver, and lungs, as can be seen in FIG. 8. These data are consistent with distribution data of other studies using nanoparticles ofthis material, shape, and / or size range, and others. 7.1 ± 9.6 ng elemental gold / mg of tissue were detected in the VMs of animals injected with AuNS. Hematoxylin and eosin (H&E) staining of the VM demonstrates evidence of AuNS aggregates in the VM, as designated by red arrows in FIG. 9. These data confirmed that AuNS accumulation occurred within VMs following intravenous injection.Example 3: Intravenously Injected AuNS Can Eliminate VMs Following Irradiation

[0174] To study the efficacy of photothermal effect with AuNS in a murine model of VMs, one VM was created in each flank of mice (two VMs per mouse; 16 mice, total of 32 VMs). When the lesions reached 300 mm3(Day -1), eight mice each were injected intravenously once with 200 pL of 0.9% normal saline (SAL) or 15 mg / kg AuNS (in 200 pL of SAL). 24 hours after injection (Day 0), the VM on one flank was irradiated (808 nm NIR for 10 min at 1000 mW / cm2). The VM on the other flank was not irradiated, as can be seen in FIG. 10. Thus, there were four treatment protocols (with 8 VMs in each protocol); (1) normal saline without NIR irradiation (SAL), (2) normal saline with NIR irradiation (SAL+NIR), (3) AuNS without NIR irradiation (AuNS), and (4) AuNS with NIR irradiation (AuNS+NIR).

[0175] It was hypothesized that irradiation at the VM immediately following AuNS injection (when AuNS are circulating in the blood) may exacerbate skin bums. In an effort to minimize off-target effects, animals were irradiated 24 hours after injection of AuNS (or SAL), when AuNS stopped circulating. During irradiation at the VM, skin temperature was recorded with a photothermal camera. In animals injected with SAL, irradiation increased the skin temperature over the VM by 2.5 °C in 10 minutes. In animals injected with AuNS, irradiation increased skin temperature change by 12.7 °C in 10 minutes (to 41.1°C), as can be seen in FIG.11

[0176] In animals injected with SAL, VM size increased approximately 3 -fold over 3 weeks, with VMs increasing in size from 300 mm3to 900-1000 mm3, whether or not they were irradiated, as can be seen in FIG. 12. After AuNS treatment, VM size increased 2.2 fold from 300 mm3to 700 mm3in the absence of irradiation. There was no statistically significant difference in VM size among these groups (SAL, SAL+NIR, AuNS). In contrast, in the AuNS+NIR group, VMs began regressing ~48 hours after irradiation, and by Day 11, were reduced in size > 6-fold, from 300 mm3to a mean of 17.49 ± 18.78 mm3(including two VMs that were completely eliminated); which was statistically significant compared to SAL, SAL+NIR, and the AuNS groups (p < 0.0004 or lower), as can be seen in FIG. 12. At the timeof necropsy on Day 20, all of the VMs in the SAL, SAL+NIR, and AuNS groups were significantly larger than VMs in the AuNS+NIR group, and appeared more vascularized (turgor, deep purple color, identifiable feeder vessels). 2 out of the 8 VMs in the AuNS+NIR group were eliminated, as can be seen in FIG. 13. In the remaining VMs in the AuNS+NIR group, histological evaluation demonstrated a marked reduction in size and in only one of those VMs were there any perfused blood vessels of human origin, as can be seen in FIGS. 20-21. Mean VM weight in the AuNS+NIR group was 8.1 ± 9.0 mg which was 40-60 fold less than the VM weights in the other groups: SAL ( 377.8 ± 258.8 mg; p = 0.0012 compared to AuNS+NIR), SAL+NIR (465.0 ± 384.9 mg; p = 0.0047), or AuNS (310.5 ± 226.4 mg; p = 0.0012), as can be seen in FIG. 14. While photothermal therapy significantly reduced or eliminated VMs, skin burns occurred in all 8 animals in the AuNS+NIR group. Thermal injury was not evident immediately after irradiation but was most notable on Day 3, as can be seen in FIG. 15. The bums were fully healed as early as 13 days after irradiation. In all animals, burns were healed by Day 20, with minor residual scarring. In AuNS injected animals, irradiation of skin 1 cm away from the VM did not affect skin temperature or cause skin burns, as can be seen in FIGS. 22-23, suggesting AuNS accumulation in the VM was critical to inducing the photothermal effect. None of the animals in the SAL, SAL+NIR, or AuNS groups developed significant skin burns.Example 4: Biocompatibility of Intravenously Administered AuNS for Photothermal Therapy

[0177] To evaluate biocompatibility of intravenously administered PTT, animal body weight was evaluated following injection with SAL or AuNS, which were similar throughout the duration of the study, as can be seen in FIG. 24, suggesting the animals were not systemically ill. To verify organ function was not affected by intravenous gold administration, laboratory data assessing liver function (alanine transaminase, alkaline phosphatase, total bilimbin, and albumin), renal function (blood urea nitrogen (BUN) / creatinine), pancreatic function (amylase), and other important serum analytes (glucose, sodium, potassium, calcium, phosphorus) were obtained prior to (Day -1) and following AuNS administration (Day 3 and Day 20). No evidence was found of organ dysfunction or electrolyte abnormality by laboratory data, as can be seen in FIG. 16. (All p values > 0.05). On Day 20, organs from animals injected with SAL or AuNS were weighed. Organ weights in the SAL group were similar to those in the AuNS group. (Heart: p = 0.82, value, Lungs: p = 0.65, Liver: p= 0.46, Kidney: p = 0.53, spleen: p = 0.12., as can be seen in FIG. 17). Organs were evaluated by hematoxylin and eosinstaining. There was no evidence of injury or inflammation in remote tissues, as can be seen in FIG. 18. Despite accumulation in organs, AuNS were not associated with organ dysfunction or obvious tissue injury.Examples Overview and Summary

[0178] It has been demonstrated that intravenously injected AuNS can accumulate within VMs and, upon irradiation, can induce a photothermal effect that can eliminate VMs. This is important because at present there is no effective treatment for venous malformations. Current therapies often require recurrent surgeries or exposure to life-long pharmacologic therapy. Pharmacological treatments are limited in that the drugs used are not highly effective and that the doses required for effect entail considerable toxicity.

[0179] A system has been designed, as described herein, that will accumulate in VMs after systemic administration. Currently, there is no known specific target that would allow for specific nanoparticle targeting to the abnormal VM vasculature. While greater than 60% of human VMs are associated with activated TIE2 mutations, all vessels express the TIE2 receptor. Recently, gold nanorods have been used for PTT after direct injection into VMs. However, intralesional administration may be technically challenging in some cases, and may not be clinically useful except in small VMs.

[0180] NIR light allows for deep tissue penetration (compared to shorter wavelengths of light) and may be suitable for VMs that extend a few centimeters from the surface. However, VMs can be massive — tens of centimeters across. In such cases, repeated administration of AuNS and / or irradiations may be required. Even in cases where irradiation events would only ablate a portion of a VM, this strategy could facilitate surgical resection, as resecting scar tissue might have a lesser risk of massive hemorrhage than resecting a large vascularized VM.

[0181] Clinical trials using PTT to treat patients with cancer have had promising results. These trials, using intravenously administered gold nanoshells, reported no serious adverse events and long term follow up has identified no toxicity. These results are consistent with pre-clinical studies indicating gold nanoshells remain in the liver and spleen, perhaps indefinitely, but are not associated with toxicity by histopathology. In the examples described herein, evidence of organ dysfunction by serum analysis or evidence of inflammation on histological evaluation was not found despite evidence of AuNS accumulation in organs.

[0182] One limitation of this proof-of-principle study was the potential for skin thermal injury. It is possible that skin burns could be eliminated by optimizing parameters such asAuNS accumulation in the VM and or irradiation conditions (lower power intensity for longer time). Skin bums may also be less likely in humans as murine skin is much thinner and has fewer epidermal layers. Treatment of deeper VMs, where hyperthermia would be remote from the skin, would likely not cause skin injury. In some cases, skin bums may be unavoidable, even with nanoparticle and irradiation optimization, for example when the VMs involve the skin. In such cases, skin grafting would likely be necessary anyways. As in all medical procedures, the clinicians would have to weigh the risks and benefits. In massive VMs where surgical resection is often accompanied by massive hemorrhage that may be hard to control, treatment with PTT may be preferable, even if it entails subsequent plastic surgery because of burns.

[0183] The examples described herein demonstrate that the accumulation of intravenously administered nanoparticles in VMs and intravenous nanoparticulate photothermal therapy is an effective treatment for VMs. Nanoparticles and nanoparticulate drug delivery systems have the potential to greatly improve therapeutic outcomes with VMs by enhancing local accumulation of therapy.REFERENCES1. Limaye, N.; Vikkula, M. Hemangiomas and Vascular Malformations, An Atlas of Diagnosis and Treatment.; 2009; pp 99-108.2. Hochman, M.; Adams, D.; Reeves, T. D. Current Knowledge and Management of Vascular Anomalies, II: Malformations. Arch Facial Plast 5wr 2011, 13, 425-433.3. Enjolras, O.; Ciabrini, D.; Mazoyer, E.; Laurian, C.; Herbreteau, D. Extensive Pure Venous Malformations in the Upper or Lower Limb: A Review of 27 Cases. J Am Acad Dermatol 1997, 36, 219-225.4. Mazoyer, E.; Enjolras, O.; Bisdorff, A.; Perdu, J.; Wassef, M.; Drouet, L. 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M.; Vikkula, M.; Limaye, N.; Eklund, L. Common and Specific Effects of TIE2 Mutations Causing Venous Malformations. Hum. Mol. Genet. 2015, 24 (22), 6374- 6389. https: / / doi.org / 10.1093 / hmg / ddv349. Lee, S.; Shieh, M. Platinum(II) Drug-Loaded Gold Nanoshells for Chemo-Photothermal Therapy in Colorectal Cancer. https: / / doi.Org / 0.1021 / acsami.9bl8855. Bailly, A.-L.; Correard, F.; Popov, A.; Tselikov, G.; Chaspoul, F.; Appay, R.; Al- Kattan, A.; Kabashin, A. V.; Braguer, D.; Esteve, M.-A. In Vivo Evaluation of Safety, Biodistribution and Pharmacokinetics of Laser- Synthesized Gold Nanoparticles. Sci. Rep. 2019, 9 (1), 12890. https: / / doi.org / 10.1038 / s41598-019- 48748-3. Tong, R.; Chiang, H. H.; Kohane, D. S. Photoswitchable Nanoparticles for in Vivo Cancer Chemotherapy. Proc. Natl. Acad. Sci. U. S. A. 2013, 110 (47), 19048- 19053. https: / / doi.org / 10.1073 / pnas.1315336110. Yu, N.; Liu, T.; Zhang, X.; Gong, N.; Ji, T.; Chen, J.; Liang, X.-J.; Kohane, D. S.; Guo, S. 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[0184] In the claims articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The disclosure includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The disclosure includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0185] Furthermore, the disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should it be understood that, in general, where the disclosure, or aspects described herein, is / are referred to as comprising particular elements and / or features, certain embodiments described herein or aspects described herein consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those embodiments have not been specifically set forth in haec verba herein.

[0186] It is also noted that the terms “comprising” and “containing” are intended to be open and permit the inclusion of additional elements or steps. Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments described herein, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0187] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein.

[0188] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A method of treating venous malformations comprising: selecting a patient having venous malformations; administering an effective amount of particles to the patient, wherein the particles are capable of causing a photothermal effect; and irradiating at least one of the venous malformations.

2. The method of claim 1, wherein the particles are not conjugated with a targeting ligand capable of binding with venous malformation endothelial cell (e.g., a targeting ligand capable of binding with endothelial adhesion molecule- 1), optionally the targeting ligand is an anti-CD31 antibody.

3. The method of claim 1 or 2, wherein a sclerosant is not co-administered to the patient, optionally the sclerosant is absolute polidocanol, ethanol, hypertonic saline, hypertonic glucose, acetic acid, bleomycin, picibanil, Sodium tetradecyl sulfate (STS), or sclerosant foam.

4. The method of any one of claims 1-3, wherein the administering is intravenous.

5. The method of any one of claims 1-4, wherein the particles are substantially free of a sclerosant.

6. The method of any of claims 1-5, wherein the particles are microparticles.

7. The method of any of claims 1-6, wherein the particles are nanoparticles.

8. The method of any one of claims 1-7, wherein the particles are substantially spherical.

9. The method of any one of claims 1-8, wherein the particles have a poly dispersity index(PDI) below about 0.5, optionally the PDI is measured by dynamic light scattering (DLS).

10. The method of any one of claims 1-9, wherein the particles comprise a photothermal material.

11. The method of any of claims 1-10, wherein the particles comprise one or more metals.

12. The method of any of claims 1-11, wherein the particles comprise one or more noble metals.

13. The method of any of claims 1-12, wherein the particles comprise gold.

14. The method of any of claims 1-13, wherein the particles comprise silver.

15. The method of any of claims 1-14, wherein the particles comprise an inorganic semiconductor.

16. The method of any of claims 1-15, wherein the particles comprise one or more polymers.

17. The method of any of claims 1-16, wherein the administering comprises injecting into one or more venous malformations.

18. The method of any one of claims 1-17, wherein the irradiating occurs at least 15 minutes, e.g., at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes or at least 55 minutes after administering the particles.

19. The method of any of claims 1-18, wherein the irradiating occurs at least one hour after administering.

20. The method of any of claims 1-19, wherein the irradiating occurs at least two hours after administering.

21. The method of any of claims 1-20, wherein the irradiating occurs at least four hours after administering.

22. The method of any of claims 1-21, wherein the irradiating occurs at least eight hours after administering.

23. The method of any of claims 1-22, wherein the irradiating occurs at least sixteen hours after administering.

24. The method of any of claims 1-23, wherein the irradiating occurs at least twenty-four hours after administering.

25. The method of any of claims 1-24, wherein the irradiating is at a wavelength between about 600 nm and about 2500 nm.

26. The method of any of claims 1-25, wherein the irradiating is at a wavelength between about 600 nm and about 1500 nm.

27. The method of any of claims 1-26, wherein the irradiating is at a wavelength between about 600 nm and about 1000 nm.

28. The method of any one of claims 1-27, wherein the irradiating is at a wavelength between about 700 nm and about 900 nm.

29. The method of any one of claims 1-28, wherein the irradiating is at a wavelength between about 775 nm and about 825 nm30. The method of any of claims 1-29, wherein the irradiating is at a wavelength of about 808 nm.

31. The method of any of claims 1-30, wherein the effective amount is from about 10 mg to about 200 mg of particles per kilogram of the patient.

32. The method of any of claims 1-31, wherein the effective amount is from about 10 mg to about 150 mg of particles per kilogram of the patient.

33. The method of any of claims 1-32, wherein the effective amount is from about 10 mg to about 100 mg of particles per kilogram of the patient.

34. The method of any of claims 1-33, wherein the effective amount is from about 10 mg to about 50 mg of particles per kilogram of the patient.

35. The method of any of claims 1-34, wherein the effective amount is from about 20 mg to about 40 mg of particles per kilogram of the patient.

36. The method of any of claims 1-34, wherein the effective amount is from about 30 mg to about 40 mg of particles per kilogram of the patient.

37. The method of any of claims 1-36, wherein the effective amount is about 36 mg of particles per kilogram of the patient.

38. The method of any of claims 1-37, wherein the particles have a hydrodynamic diameter of about 20 nm to about 180 nm.

39. The method of any of claims 1-38, wherein the particles have a hydrodynamic diameter of about 20 nm to about 100 nm.

40. The method of claim 39, wherein the particles have a hydrodynamic diameter of about 20 nm to about 50 nm.

41. The method of any of claims 1-40, wherein the irradiating occurs for at least one minute.

42. The method of any of claims 1-41, wherein the irradiating occurs for at least two minutes.

43. The method of any of claims 1-42, wherein the irradiating occurs for at least three minutes.

44. The method of any of claims 1-43, wherein the irradiating occurs for at least five minutes.

45. The method of any of claims 1-44, wherein the irradiating occurs for at least fifteen minutes.

46. The method of any of claims 1-45, wherein the administering and irradiating steps are repeated.

47. The method of any of claims 1-46, wherein the administering and irradiating steps are repeated at least two times.

48. The method of any of claims 1-47, wherein the administering and irradiating steps are repeated at least three times.

49. The method of any of claims 1-48, further comprising after the irradiating step:(a) waiting for a period of time (e.g., at least thirty seconds), wherein waiting does not include irradiating; and(b) repeating the irradiating step.

50. The method of claim 49, further comprising repeating steps (a) and (b).

51. The method of any of claims 49 or 50, further comprising repeating steps (a) and (b) twice.

52. The method of any of claims 49-51, further comprising repeating steps (a) and (b) at least three times.

53. The method of any one of claims 1-52, wherein the irradiating is at an intensity of at least 150 mW / cm2.

54. The method of any of claims 1-53, wherein the irradiating is at an intensity of about 200 mW / cm2to about 2000 mW / cm2.

55. The method of any of claims 1-54, wherein the irradiating is at an intensity of about 200 mW / cm2to about 1000 mW / cm2.

56. The method of any of claims 1-55, wherein the irradiating is at an intensity of about 300 mW / cm2to about 800 mW / cm2.

57. The method of any of claims 1-56, wherein the irradiating is at an intensity of about 400 mW / cm2to about 600 mW / cm2.

58. The method of any of claims 1-57, wherein the particles are in a composition comprising the particles and optionally a pharmaceutically acceptable carrier or excipient.

59. The method of any of claims 1-58, wherein the administering comprises administering through an intravascular catheter into a feeder vessel.

60. The method of any of claims 1-59, wherein the administering is intravenous, and wherein the particles are not conjugated with a targeting ligand capable of binding with venous malformation endothelial cell (e.g., a targeting ligand capable of binding withendothelial adhesion molecule-1), optionally the targeting ligand is an anti-CD31 antibody.

61. The method of any of claims 1 or 3-59, wherein the administering is intravenous, and wherein the particles are conjugated with a targeting ligand capable of binding with venous malformation endothelial cell.

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