Methods for selectively making cells permeable and / or fragmenting them

Abstract

The present invention relates to a method for selectively permeabilizing and / or fragmenting cells, the structure comprising a material and particles capable of absorbing electromagnetic radiation, and the cells are brought into close proximity to each other. Some of the particles embedded in the structure are exposed at a free surface of the structure. The structure and in particular the particles in the structure are irradiated with electromagnetic radiation to selectively permeabilize and / or fragment the cells. Furthermore, the present invention relates to a structure for use in a photothermal process for selectively permeabilizing and / or fragmenting cells.

Description

[Technical Field]

[0001] Field of the present invention The present invention relates to a method for selectively making cells permeable and / or fragmenting them, for example, for selectively killing or excising cells. In particular, the present invention relates to a structure comprising particles capable of absorbing electromagnetic radiation, and the use of such a structure in a method for selectively making cells permeable and / or fragmenting them. The structure is suitable for use in nanosurgical procedures, for example, for killing or excising corneal cells. [Background technology]

[0002] Background technology For the treatment of several diseases, including cancer, the elimination of cells on the surface of tissue is required. Despite significant advances in cancer surgery, removing all cancer cells from a tumor remains extremely difficult, especially when tumor cells are intertwined with healthy tissue. This explains why adjuvant therapies such as radiation therapy and chemotherapy are often preferred after surgery.

[0003] In ophthalmology, incomplete tumor removal remains a significant challenge when dealing with advanced cancers of the eye's surface (conjunctiva and cornea). Adjuvant therapy is particularly limited for this type of cancer. Indeed, controlling the deposition and dosage of chemotherapeutic agents on the ocular surface is extremely difficult, while in-situ radiotherapy and cryotherapy also suffer from poor outcomes.

[0004] Photodynamic therapy (PDT) and photothermal therapy (PTT) have proven to be promising approaches for cell death. These techniques offer high spatial precision and can even enable "single-cell death." However, the uncontrolled heat dissipation following laser treatment (surface overheating) can irreversibly damage surrounding tissue.

[0005] In recent years, nanoparticle-sensitized laser irradiation has been investigated as a way to kill cells, such as cancer cells. When irradiated with a laser, cells are damaged or killed through photothermal effects such as localized heating. In this approach, (free) nanoparticles are delivered to cancer cells. Because there is uncertainty about the toxicological effects of nanoparticles, there may be toxicological concerns regarding such nanoparticle-sensitized laser irradiation.

[0006] Therefore, there is current interest in developing methods to selectively kill cells by reducing or avoiding direct contact between plasmonic nanoparticles and cells. [Overview of the project]

[0007] Summary of the present invention The object of the present invention is to provide a method for selectively making cells permeable and / or fragmenting them, thereby avoiding the drawbacks of the prior art. Another object of the present invention is to provide a method for selectively permeable and / or fragmenting cells with high spatial resolution using a structure containing particles capable of absorbing electromagnetic radiation. In particular, an object of the present invention is to provide a method for selectively killing or excising cells with high spatial resolution.

[0008] A further object of the present invention is to provide a method for selectively making cells permeable and / or fragmenting them while limiting the diffusion or accumulation of particles, such as microparticles and / or nanoparticles, or particle materials or fragments into surrounding cells or tissues.

[0009] A further object of the present invention is to provide a method for selectively making cells permeable and / or fragmenting cells without requiring direct contact between the structure and the cells, in particular, without requiring direct contact between particles embedded in the structure and the cells. Methods according to the present invention enable the selective making of cells permeable and / or fragmenting cells using particles such as microparticles and / or nanoparticles, thereby reducing or avoiding the uptake of particles, or particle material or fragments, by cells.

[0010] A further object of the present invention is to provide a method for selectively making cells permeable and / or fragmenting them while limiting the diffusion or accumulation of particles, such as microparticles and / or nanoparticles, or particle materials or fragments into surrounding cells or tissues.

[0011] A further object of the present invention is to provide a method that enables the fragmentation of cells, for example, by using low-fluence laser pulses to kill them. Another object of the present invention is to provide a method for making cells permeable and / or fragmenting them, thereby allowing structures to be easily removed from cells, for example, from tissue, after irradiation (laser treatment).

[0012] A further object of the present invention is to provide a method that enables the removal of superficial tumors. A further objective is to provide a structure containing particles capable of absorbing electromagnetic radiation for use in methods of selectively making cells permeable and / or fragmenting them, for example, in methods of selectively killing cells (e.g., corneal cells).

[0013] According to a first aspect of the present invention, an in vitro or ex vivo method is provided for selectively making cells permeable and / or fragmenting them. The method includes, for example, selectively killing cells or selectively excising them. - providing a structure comprising a material and particles capable of absorbing electromagnetic radiation (the structure defines a volume V and a free surface S. The free surface S has a free surface area A S . The particles are embedded in the material of the structure. At least a portion of the particles is partially exposed at the free surface S. Each of the particles partially exposed at the free surface S defines a particle free surface P within the free surface S. The particle free surface P has a particle free surface area A P . The particles are embedded in the material such that the sum of the particle free surface areas A P of all particles is in the range of 0.0001 to 50% of the free surface area A S .); - providing at least one cell, preferably a plurality of cells; - bringing the structure and the at least one cell to a distance d of less than 100 μm from each other, wherein the distance d is defined as the shortest distance between the at least one cell and the structure, preferably the shortest distance between the at least one cell and the free surface S of the structure; - irradiating the structure with electromagnetic radiation.

[0014] The free surface S of the structure is the entire surface of the structure that is in direct contact with the environment. When the structure is submerged in a fluid, for example a liquid (such as a medium containing (living) cells) or a gas (such as air), and the structure comprises a material that is impermeable to said fluid, the free surface S of the structure can also be defined as the entire surface of the material of the structure that is in contact with or may come into contact with the fluid. The free surface S defines a free surface area A S .

[0015] Preferably, the ratio of the free surface area A S of the structure to the volume of the structure, i.e. the ratio A S / V, is between 10 -2 and 10 2 μm -1 , for example between 10 -1 and 50 μm -1 or between 1 and 10 μm -1It is within the range of [the specified period]. The term "area" refers to the size of a flat surface on a plane, that is, a measurement of the size of a two-dimensional object.

[0016] The term "surface area" refers to a measurement of the size of the surface of a three-dimensional object. The term “free surface area A S " refers to the measured size of the free surface of a structure.

[0017] The concentration of particles capable of absorbing electromagnetic radiation present in a structure according to the present invention is preferably in the range of 0.001 volume% to 20 volume% (particle volume / structure volume). More preferably, the concentration of particles capable of absorbing electromagnetic radiation present in a structure according to the present invention is in the range of 0.01 volume% to 10 volume% or 0.01 volume% to 5 volume%, and for example, 0.05 volume%, 0.1 volume%, 0.2 volume%, 0.5 volume%, 1 volume%, 2 volume%, or 5 volume%.

[0018] As mentioned above, a portion of the particles present in the structure are partially exposed on the free surface S of the structure. Such particles are therefore partially embedded in the material and partially exposed on the free surface S of the structure. It is clear that a structure according to the present invention may include, in addition to particles partially exposed on the free surface S, particles that are completely embedded in the material of the structure.

[0019] Each particle partially exposed to a free surface S has a free surface that is part of the free surface S. The free surface S occupied by such partially exposed particles is called the particle free surface P. The particle free surface P has a particle free surface area A P It holds. The term “particle free surface area A P This refers to the measured size of the particle's free surface P.

[0020] Particles partially exposed on the free surface S of a structure may or may not protrude from the material. Protruding particles protrude from the outer surface defined by the material.

[0021] A structure according to the present invention may include a combination of particles that protrude from the outer surface defined by the material and are partially exposed on the free surface S of the structure, and particles that do not protrude from the outer surface defined by the material and are partially exposed on the free surface S of the structure. The protruding particles, for example, extend to a height of 50 μm when measured perpendicular to the surface of the material, with heights ranging from 0.5 μm to 10 μm.

[0022] Preferably, the particles are in the structure, more specifically in the material of the structure, with the free surface area A of all particles. P The sum of the free surface area A is as shown below. S It is embedded so that it is within the range of 0.0001 to 50%:

number

[0023] In a preferred embodiment, the particles have a particle free surface area A of all particles. P The sum of the free surface area A S Free surface area A between 1% and 25% S Free surface area A between 5% and 25% S The free surface area A of all particles is embedded in the structure so that it is within the range of 15-25%. P The sum of these is, for example, the free surface area A. S It is 20% of that.

[0024] When a structure is irradiated with electromagnetic radiation, particles present within the structure, particularly those partially exposed to the free surface S of the structure, undergo a photothermal effect. This photothermal effect causes localized and transient heating of the material, especially near the irradiated particles, and induces the formation of vapor bubbles, such as vapor microbubbles or vapor nanobubbles. Such vapor bubbles can cause permeabilization or fragmentation of cells at short distances.

[0025] In embodiments of the present invention, the method makes it possible to increase the permeability of cells by bringing cells and structures into contact with each other or bringing them to a short distance from each other, and by irradiating the structure with electromagnetic radiation, in particular by irradiating particles present in the structure that are partially exposed to the free surface S. The method makes it possible to increase the permeability of cells without requiring direct contact between particles and cells.

[0026] In another embodiment of the present invention, the method enables the fragmentation of cells by bringing cells and structures into contact with each other or bringing them to a short distance from each other, and by irradiating the structure with electromagnetic radiation, in particular by irradiating particles present in the structure that are partially exposed to a free surface S. The method enables the fragmentation of cells without requiring direct contact between particles and cells. The method enables the killing or excision of cells by bringing cells and structures into contact with each other or bringing them to a short distance from each other, and by irradiating the structure with electromagnetic radiation, in particular by irradiating particles that are partially exposed to a free surface S. In particular, the method enables the killing or excision of cells without requiring direct contact between particles and cells.

[0027] In a further embodiment of the present invention, the method enables the fragmentation of other cells while increasing the permeability of specific cells by bringing cells and structures into contact with each other or bringing them to a short distance from each other, and by irradiating the structures with electromagnetic radiation, in particular by irradiating particles present in the structures that are partially exposed to a free surface S. In particular, the method enables the fragmentation of other cells while increasing the permeability of specific cells without requiring direct contact between particles and cells.

[0028] Bringing cells and structures into contact with each other means that there is direct contact between the cells and the structure. In such cases, the cells are in contact with the free surface S of the structure. The cells may be in direct contact with the material of the structure and / or with one or more particles partially exposed on the free surface S of the structure.

[0029] Bringing cells and structures to a short distance from each other means that the shortest distance d between cells and structures, in particular the shortest distance between cells and the free surface S of the structure, is 100 μm or less. The shortest distance d between cells and structures is, for example, 50 μm, 20 μm, 10 μm, 5 μm, 3 μm, 2 μm, 1 μm, 0.5 μm, or 0.1 μm. Preferably, the shortest distance between cells and particles partially exposed on the free surface S of the structure is 100 μm or less. The shortest distance between cells and particles partially exposed on the free surface S of the structure is, for example, 50 μm, 20 μm, 10 μm, 5 μm, 3 μm, 2 μm, 1 μm, 0.5 μm, or 0.1 μm.

[0030] At least one cell may include, for example, a cell suspension or a tissue of cells. Examples of tissues include eye tissue or skin tissue. Specific examples include tumor boundaries.

[0031] To enable spatially selective permeabilization and / or fragmentation with single-cell resolution, the density of particles at least partially exposed to the free surface S should be chosen such that each cell is sufficiently close to the particles at least partially exposed to the free surface S.

[0032] The density of cells brought to a distance d that is equal to or less than 100 μm is, for example, 1 cell / mm². 2 ~10 5 cells / mm 2 It is within the range of, for example, 10 cells / mm 2 ~10 4 cells / mm 2 During 10 2 cells / mm 2 ~104 cells / mm 2 During, or 10 3 cells / mm 2 ~3.10 3 cells / mm 2 It is within the range of [the specified period].

[0033] The density of particles partially exposed on the free surface S of the structure is preferably 1 particle / 100 μm 2 ~10 particles / 100μm 2 For example, 1 particle / μm 2 ~5 particles / 100μm 2 (For example, 2 particles / μm) 2 , 3 particles / 100μm 2 Or 4 particles / 100μm 2 It is within the range of ).

[0034] Particles embedded in a structure according to the present invention may include any particles that are capable of absorbing electromagnetic radiation and are adapted to generate a photothermal effect when irradiated with electromagnetic radiation.

[0035] As used herein, the term "particle" refers to a particle or a group, aggregate, or cluster of two or more particles having dimensions (more specifically, the maximum dimensions of a particle) ranging from approximately 1 nm to approximately 40,000 nm (40 μm), for example, 1 nm to 10,000 nm (10 μm), 10 nm to 2,000 nm (2 μm), 50 nm to 1,000 nm (1 μm), or 100 nm to 500 nm, for example, 200 nm.

[0036] Dimensions are preferably measured by measuring the maximum dimensions of the particle. The maximum dimensions of a particle correspond to the maximum distance between two points on that particle. The maximum dimensions of a particle refer to, for example, the width (maximum width), height (maximum height), or diameter (maximum diameter) of that particle.

[0037] An alternative way to determine particle size is by equivalent spherical diameter (also called equivalent volume diameter). For irregularly shaped objects, the equivalent spherical diameter (or ESD) is the diameter of a sphere of equivalent volume. In some embodiments, particles may have equivalent spherical diameters ranging from about 1 nm to about 40,000 nm (40 μm). In some embodiments, particles may have equivalent spherical diameters ranging from about 1 nm to about 10,000 nm (10 μm). As an example, particles may have equivalent spherical diameters ranging from about 10 nm to 200 nm (2 μm), 50 nm to 1,000 nm (1 μm), or 100 nm to 500 nm, for example, 200 nm.

[0038] The dimensions of a particle (e.g., particle width, height, or diameter), and in particular the maximum dimensions of a particle (e.g., maximum particle width, height, or diameter) or equivalent spherical diameter, can be determined using a transmission electron microscope (TEM), scanning electron microscope (SEM), or atomic force microscope (AFM). For the purposes of this invention, the terms “group of particles,” “aggregate of particles,” and “cluster of particles” are used interchangeably.

[0039] A particle cluster comprises at least two individual particles, preferably between 10 and 1000 individual particles, for example between 10 and 1000 individual particles. Each individual particle in a cluster is preferably in contact with at least one other individual particle in the cluster or located several micrometers away from another individual particle in that cluster.

[0040] If the particles include clusters, the individual particles of the cluster preferably have dimensions of about 1 nm to about 200 nm, for example, 50 nm to 100 nm (more specifically, the maximum dimensions of the individual particles). The clusters preferably have dimensions (more specifically, the maximum dimensions of the clusters) of about 1000 nm to about 40000 nm (10 μm), for example 10 nm to 2000 nm, 50 nm to 1000 nm, or 100 nm to 500 nm, for example 200 nm.

[0041] As used herein, the term "microparticle" refers to a particle or a group, aggregate, or cluster of two or more particles having dimensions (more specifically, the maximum dimensions of the particle) greater than 1000 nm (>1 μm) and at most 10000 nm (≤10 μm). If the microparticle includes a cluster, the individual particles of such a cluster preferably have dimensions (more specifically, the maximum dimensions of the individual particle) of about 1 nm to about 200 nm, for example, 50 nm to 100 nm.

[0042] The term "nanoparticle" refers to a particle or a group, aggregate, or cluster of two or more particles having dimensions (maximum particle size) of at least 1 nm (≧1 nm) and at most 1000 nm (≦1 μm). If the microparticles include clusters, the individual particles of such clusters preferably have dimensions (more specifically, the maximum size of individual particles) of about 1 nm to about 200 nm, for example, 50 nm to 100 nm.

[0043] Particles with dimensions (maximum particle size) within the range of 100 nm to 1000 nm are also sometimes referred to as "submicron particles." The particles may have any shape. They may be spherical, elliptical, rod-shaped, conical, branched, or have an irregular shape.

[0044] The particles may be solid particles, which may include a shell structure or core-shell structure containing one or more materials. Preferred particles include metal particles, metal oxide particles, carbon or carbon-based particles, particles containing one or more light-absorbing compounds, or particles on which one or more light-absorbing compounds are mounted or functionalized with them.

[0045] Examples of metal particles include gold particles, silver particles, platinum particles, palladium particles, copper particles, and their alloys. Preferred metal particles include gold particles, silver particles, and their alloys. Examples of metal oxide particles include iron oxide, titanium oxide, zirconium oxide, cerium oxide, zinc oxide, and magnesium oxide. Examples of carbon or carbon-based particles include graphene quantum dots, (reduced) graphene oxide, and carbon nanotubes.

[0046] Examples of particles containing one or more light-absorbing compounds, or particles on which one or more light-absorbing compounds are mounted or functionalized, include particles containing, mounted on or functionalized with synthetic organic or inorganic absorbers, as well as particles containing, mounted on or functionalized with naturally occurring absorbers or derivatives thereof. Specific examples include liposomes, solid lipid nanoparticles, and polymer-based particles containing, mounted on, or functionalized with light-absorbing dye molecules such as indocyanine green, inorganic quantum dots (with low fluorescence quantum yield), naturally occurring light absorbers such as dyes (such as melanin, rhodopsin, photopsin, or iodopsin), and synthetic analogs such as polydopamine, or photosensitizers used in photodynamic therapy.

[0047] In embodiments of the present invention, the particles may be biodegradable. In embodiments, the particles may be biocompatible. In embodiments, the particles may be both biodegradable and biocompatible. In embodiments, the particles may contain or consist of clinically approved components. Advantageously, the particles are biodegradable, biocompatible, and contain or consist of clinically approved components.

[0048] A structure according to the present invention may include one type of particle, or a combination of different particles, for example, particles having different sizes, different compositions, and / or different shapes.

[0049] The material of a structure in which particles capable of absorbing electromagnetic radiation are embedded includes, for example, inorganic materials or inorganic-based materials, organic materials or organic-based materials such as silica or silica-based materials or ceramics or ceramic-based materials, carbon or carbon-based materials or polymers or polymer-based materials. The material of the structure may also include composite materials comprising at least one of the materials described above, for example, composite materials comprising organic and inorganic materials.

[0050] Preferred materials for structures include or are based on polystyrene, polycaprolactone, ethylcellulose, cellulose acetate phthalate, polylactic acid, polylactic acid glycolic acid copolymer, cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginate, hyaluronic acid, dextran, starch, polycarbonate, or polyacrylate. In other embodiments, the material for structures includes gels or hydrogels. Gels include non-fluid polymers that are expanded over their entire volume by a fluid. Hydrogels are gels whose swelling agent is water. In particular, hydrogels are three-dimensional network structures of (hydrophilic) polymers that can swell in water and can hold large amounts of water while maintaining their structure due to chemical or physical crosslinking of individual polymer chains. Hydrogels may include homopolymers, copolymers, semi-interpenetrating network structures, and interpenetrating network structures.

[0051] In a preferred embodiment, the structure comprises a surface-modified material, such as a surface-modified polymer material. Surface modification includes, for example, the application of a coating, such as a protein coating, an antibody-containing coating, a lipid-containing coating, a polymer coating, or a combination thereof.

[0052] The material is preferably transparent to visible light. Preferably, the material has a transmittance of at least 50% to visible light having wavelengths in the range of 450 to 650 nm, or at least 60% to visible light having wavelengths in the range of 450 to 650 nm.

[0053] Preferably, the structure comprising the material and particles has a transmittance of at least 25% for visible light having a wavelength in the range of 450 to 650 nm, at least 35% for visible light having a wavelength in the range of 450 to 650 nm, or at least 50% for visible light having a wavelength in the range of 450 to 650 nm.

[0054] Structures containing materials and particles embedded within those materials may include continuous or discontinuous structures. The structure may be rigid or flexible. Flexible structures may be preferable because they can be adapted to the surface of the tissue being treated.

[0055] The structure may be flat or planar, or it may be non-flat, for example, curved. The structure may have a flat or non-flat outer surface. The structure may include porous or non-porous structures.

[0056] In some applications, porous structures have a large free surface area. S It is preferable because it has the advantage of having a large surface area that can be exposed to cells introduced on or near the structure according to the method of the present invention. Preferably, such a porous structure has a pore size that allows cells introduced on or near the structure to partially or completely penetrate into the pores. Preferably, the porous structure has a pore size that does not restrict the access of molecules present in the cell culture medium to the cells.

[0057] The porosity of a structure is defined as the ratio of the volume of pores or voids in the structure to the sum of the total volume occupied by the structure, i.e., the volume of the structure V (volume of material and particles embedded in the material) and the volume of the pores or voids in the structure. The porosity can be in the range of 0% to 100%. If the structure includes a porous structure, the porosity of the structure is preferably at least 50%, at least 60%, at least 80%, at least 90%, at least 95%, or at least 99%.

[0058] A first group of structures according to the present invention includes a film or foil containing particles capable of absorbing electromagnetic radiation embedded within the film or foil. Such a film or foil is, for example, non-porous. An example is a polymer film or polymer foil containing particles capable of absorbing electromagnetic radiation embedded within the polymer film or polymer foil. For the purposes of the present invention, the terms “film” and “foil” are used interchangeably.

[0059] Examples of preferred films include polymer films comprising or based on polystyrene, polycaprolactone, ethylcellulose, cellulose acetate phthalate, polylactic acid, polylactic acid glycolic acid copolymer, cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginate, hyaluronic acid, dextran, starch, polycarbonate, or polyacrylate, and having iron oxide particles and / or carbon particles embedded in the polymer film. The polymer film has a thickness that is, for example, between 0.1 μm and 100 μm, for example between 0.1 μm and 10 μm.

[0060] Preferably, such film defines a first surface S1 and a second surface S2 opposite to the first surface S1. The first surface S1 has a first free surface area A S1 Define the second surface S2 as having a free surface area A S2Define the following: The first surface is, for example, the upper surface (defining the upper free surface area), while the second surface is the lower surface (defining the lower free surface area). Alternatively, the first surface is the lower surface (defining the lower free surface area), while the second surface is the upper surface (defining the upper free surface area).

[0061] When irradiated, the radiation collides, for example, on a first surface S1 or a second surface S2. Preferably, the first free surface area A S1 and the second free surface area A S2 They are the same or substantially the same size.

[0062] The structures of the first group have, for example, a thickness t, a length l, and a width w. The thickness is preferably between 0.1 μm and 10 μm, for example, within the range of 0.1 μm to 10 μm. The volume V of the structure is t·l·w and or t·A S1 This corresponds to the length l and width w of the structures in the first group, which are typically substantially greater than the thickness of the structure, and the free surface area A of the structure. S is, A S1 +A S2 It can be estimated as follows: The irradiated free surface typically corresponds to one of the surfaces of the structure (e.g., the first surface S1 or the second surface S2), and A S1 It can be estimated that this is the case. As a result, the ratio of the free surface area of ​​the structure to the volume of the structure, i.e., S / V, corresponds to 1 / t.

[0063] Preferably, at least one of the first surface S1 or the second surface S2 contains particles that are at least partially exposed to the free surface S of the structure. Each of the particles partially exposed to the first surface S1 or the second surface S2 defines a particle free surface P within the first surface S1 or the second surface S2. The particle free surface P has a particle free surface area A P The particles have a free surface area A of all particles that are partially exposed on the first surface S1 or partially exposed on the second surface S2. P The sum of the free surface area AS1 and free surface area A S2 The particles are embedded in the material such that the sum of the particles is within the range of 0.0001 to 50%. More preferably, the free surface area A of all particles that are partially exposed on the first surface S1 or partially exposed on the second surface S2 P The sum of the free surface area A S1 and free surface area A S2 Between 5% and 25% of the sum of or the free surface area A S1 and free surface area A S2 It is embedded in the material so that it is within the range of 15-25% of the sum of the two.

[0064] In a specific embodiment, at least one of the first surface S1 or the second surface S2 includes particles that are at least partially exposed to the free surface S of the structure. Each of the particles partially exposed to the first surface S1 defines a particle free surface P within the first surface S1. The particle free surface P has a particle free surface area A P The particles have a particle free surface area A of all particles that are partially exposed on the first surface S1. P The sum of the free surface areas A S1 It is embedded in the material in a range of 0.0001 to 50%.

[0065] In a preferred embodiment, the free surface area A of all particles partially exposed on the first surface S1 is P The sum of the free surface areas A S1 Between 5 and 25% of the free surface area A S1 It is embedded in the material so that it is within the range of 15-25%.

[0066] A second group of structures according to the present invention comprises a hydrogel containing particles capable of absorbing electromagnetic radiation embedded within the hydrogel.

[0067] Preferred hydrogels include homopolymers, copolymers, semi-interpenetrating network structures, and interpenetrating network structures. Examples of polymers include poly(2-hydroxyethyl methacrylate) (PHEMA), 2-hydroxyethyl methacrylate ((HEMA)), polyethylene glycol (PEG), polyethylene glycol methacrylate (PEG-MA), methacrylic acid (MAA); carboxymethylcellulose (CMC); polyvinylpyrrolidone (PVP), acrylamide / acrylic acid copolymer poly(N-isopropylacrylamide) (PNIPAM), chitosan, acrylate-modified PEG, acrylate-modified hyaluronic acid, heparin, and amine-terminated functionalized 4-armster PEG. Examples of crosslinkers include polyethylene glycol dimethacrylate, triethylene glycol dimethacrylate (TEGDMA), tetra(ethylene glycol) dimethacrylate, and N,N'-methylenebisacrylamide. The particles include, for example, iron oxide particles and / or carbon particles.

[0068] A third group of structures according to the present invention includes porous structures comprising a material and particles capable of absorbing electromagnetic radiation embedded within the material. An example is a porous polymer structure with particles embedded within the porous polymer structure. Examples of porous structures include structures comprising fibers (e.g., polymer fibers), structures comprising microparticles (e.g., polymer microparticles), structures comprising combinations of fibers and microparticles (e.g., combinations of polymer fibers and / or polymer microparticles), and structures comprising foams (e.g., polymer foams). The fibers and / or microparticles may be interconnected or not. Microparticles, such as polymer microparticles, may include spherical microparticles as well as microparticles with irregular shapes. Particles embedded in the porous structure include, for example, iron oxide particles and / or carbon particles.

[0069] The first example of the third group of structures is a structure containing (polymer) fibers. The (polymer) fibers have a fiber diameter, for example, between 0.1 μm and 10 μm, e.g., 0.5 μm or 1 μm. The (polymer) fibers may be interconnected or uninterconnected. The (polymer) fibers can be obtained by any technique known in the art. A preferred technique for producing (polymer) fibers is electrospinning.

[0070] Alternative techniques include wet spinning, melt spinning, extrusion spinning, dry spray wet spinning, emulsification spinning, and suspension spinning. Examples of preferred polymers include polystyrene fibers, polycaprolactone fibers, ethylcellulose fibers, cellulose acetate phthalate fibers, polylactic acid fibers, and polylactic acid glycolic acid copolymer-based fibers. (Polymer) fibers may be surface-modified.

[0071] (Polymer) fibers have a fiber diameter d fibre Corresponding diameter and fiber length L fibre Since it can be considered as a long cylinder of the corresponding length, the volume of the fiber V fibre teeth,

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[0072] A second example of the third group of structures is a structure containing polymer microparticles, such as polymer (micro)spheres. The microparticles have a diameter, for example, between 0.1 μm and 10 μm, e.g., 0.5 μm or 1 μm. The polymer microparticles may be interconnected or uninterconnected. The (polymer) microparticles can be obtained by any technique known in the art. Preferred examples of microparticles include fibers based on polystyrene, polycaprolactone, ethylcellulose, cellulose acetate phthalate, polylactic acid, and polylactic acid glycolic acid copolymers. The polymer microparticles may be surface-modified.

[0073] The fine particles have a diameter of d ms If it is a microsphere, then the volume of the microsphere is V. ms teeth,

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number

[0074] As mentioned above, the method according to the present invention includes the step of bringing a structure and at least one cell to a distance d of less than 100 μm from each other. At least one cell includes, for example, multiple cells, such as a suspension or tissue of cells.

[0075] The structure and at least one cell can be brought to a distance d from each other by bringing at least one cell onto or near the structure, or by bringing the structure near at least one cell. Cells are introduced onto or near a structure, for example, by applying a suspension containing cells onto or near the structure, particularly on or near the free surface S of the structure. Cells can be introduced onto or near the structure continuously or discontinuously.

[0076] The cell concentration in the suspension is preferably 1 to 10 per mL. 6 Between cells, for example 10 3 ~10 5 It is within the space between cells. In a preferred method, the suspension, i.e., the cells in the suspension, are cultured on or near a structure for a certain period of time.

[0077] In an alternative method, the cells are treated by activating the structure by electromagnetic irradiation immediately or some time after their introduction onto or near the structure.

[0078] In an embodiment of the present invention, after introducing at least one cell onto or near the structure, the at least one cell may be incubated for, for example, one minute or more or one hour or more before (i.e., prior to) the irradiation step.

[0079] Alternatively, the structure can be brought close to at least one cell by applying it, for example, on or at a short distance from a tissue containing cells, such that the free surface S of the structure is in contact with the cell or at a distance d from the cell.

[0080] The structure, and in particular particles partially exposed on the free surface S of the structure, are preferably irradiated with electromagnetic radiation, for example, from a pulsed radiation source, but irradiation with a continuous wave radiation source can also be considered.

[0081] The terms “radiation” and “electromagnetic radiation” may be used interchangeably within this specification. The wavelength of the radiation source may be in the range from the ultraviolet region to the infrared region. In a preferred method, the wavelength range of the radiation used is from the visible light region to the infrared region, including the near-infrared region.

[0082] Electromagnetic radiation may be generated by a laser such as a pulsed laser. Laser irradiation, such as irradiation with pulsed lasers, for example pico-, femto- and / or nanosecond pulsed lasers, in combination with a structure according to aspects of the present invention, can efficiently permeabilize cellular barriers, for example by laser-induced vapor nanobubble generation. While laser irradiation may be advantageous, irradiation with another (intense) light source is not necessarily excluded to achieve the same or similar effect.

[0083] The wavelength of the radiation source may be in the range from the ultraviolet region to the infrared region. In a preferred method, the wavelength range of the radiation used is from the visible light region to the infrared region, including the near-infrared region.

[0084] When a pulsed radiation source is used, the pulses preferably have a duration in the range of 1 fs to 1 ms, for example in the range of 1 fs to 100 μs, in the range of 10 fs to 10 μs, in the range of 100 fs to 1 μs, or in the range of 1 ps to 100 ns. The laser pulses may consist of 1 to 1000 laser pulses, such as 1 to 500 laser pulses, 1 to 100 laser pulses, 1 to 20 laser pulses, or 1 to 10 laser pulses (per cell or per cell sample). The number of laser pulses may depend, for example, on the type of photoresponsive organic particles and the type of cells.

[0085] The fluence per pulse of the radiation source (electromagnetic energy delivered per unit area) is preferably 0.0001 J / cm 2 ~1000 J / cm 2 , for example in the range between 0.001 J / cm 2 ~100 J / cm 2 , between 0.01 J / cm2 ~10 J / cm 2 between, 0.1 J / cm 2 ~10 J / cm 2 between, for example 0.1 J / cm 2 , 1 J / cm 2 or 5 J / cm 2 .

[0086] An advantage of the method according to the present invention is that fragmentation or release of particles embedded in the structure is reduced or avoided. ICP-MS analysis demonstrated that no detectable amount of particle material was released. Another advantage of the method according to the present invention is the spatial resolution of the method, which makes it possible to permeabilize and / or fragment single cells.

[0087] A further advantage of the method according to the present invention is that no direct contact between the structure and the cells, in particular between the particles and the cells, is required to permeabilize and / or fragment the cells, whereby the uptake of the particles or fragments of the particles by the cells is reduced or avoided.

[0088] A still further advantage according to the method of the present invention is the simplicity of fabrication of the structure. The method as taught herein may likewise be useful for selectively permeabilizing and / or fragmenting cell(s) of the human or animal body.

[0089] According to a second aspect of the present invention, there is provided a structure as described above for use in a photothermal process for selectively permeabilizing and / or fragmenting cells that are in contact with or in proximity to the structure. The structure comprises a material and particles capable of absorbing electromagnetic radiation. The structure defines a volume V and a free surface S. The free surface S has a free surface area A S . The particles are embedded in the material. At least a portion of the particles is partially exposed at the free surface S. Each of the particles partially exposed at the free surface S defines a particle free surface P within the free surface S. The particle free surface P has a particle free surface area AP The particles have a free surface area A of all particles. P The sum of the free surface areas A S It is embedded in the material in a range of 0.0001 to 50%.

[0090] The photothermal process is - A step of bringing the structure and at least one cell to a distance d of less than 100 μm from each other, where distance d is the shortest distance between at least one cell and the structure, in particular the shortest distance between at least one cell and the free area surface S of the structure; - The process includes irradiating a structure with electromagnetic radiation to selectively permeate and / or fragment at least one cell.

[0091] Structures according to the present invention are particularly suitable for use in photothermal processes in methods of treatment of a subject, which selectively make the target cells permeable and / or fragmented, and the method is - A step of bringing the structure to a distance d of less than 100 μm from at least one cell of the object, where distance d is the shortest distance between at least one cell of the object and the structure, in particular the shortest distance between at least one cell and the free area surface S of the structure; - Including irradiating a structure with electromagnetic radiation to selectively permeate and / or fragment at least one cell.

[0092] At least one cell preferably comprises a cell suspension or a tissue of cells. Examples of tissues include eye tissue or skin tissue. Specific examples include tumor boundaries. The treatment methods include, for example, nanosurgical procedures. Specific procedures include killing corneal cells or excising corneal cells.

[0093] The structures and processes taught herein enable treatments, such as laser-assisted therapy, for diseases or conditions in a subject. The terms “subject,” “individual,” or “patient” may be used interchangeably herein and typically and preferably refer to humans, but references to non-human animals, preferably warm-blooded animals, and more preferably mammals may also be included.

[0094] Any of the structures described above is intended for use in photothermal processes in therapeutic methods of a subject, which selectively make the target cells permeable and / or fragmented, and the method includes the following: Electromagnetic radiation may be generated by any of the sources described above. Preferably, the electromagnetic radiation includes pulsed radiation generated by a laser.

[0095] In a preferred method, the structure according to the present invention is brought to a close distance from the cells, for example, from the cell tissue, before irradiation. Such a method is referred to as “structure on cells.” In such a method, the structure can be easily removed after irradiation. In a particularly preferred method, the film, for example, a polymer film containing particles capable of absorbing electromagnetic radiation, is brought to a close distance from the cells, for example, from the cell tissue, before irradiation ("film on the cells").

[0096] In an alternative method, cells are applied to a structure before irradiation and, for example, grow on the structure. Such a method is referred to as "cells on a structure." In a particularly preferred method, the film, cells are applied to the film, for example, a polymer film containing particles capable of absorbing electromagnetic radiation, before irradiation ("cells on film"). The cells are grown, for example, on the film, for example, a polymer film containing particles capable of absorbing electromagnetic radiation.

[0097] Simple description of the drawing The present invention will be discussed in more detail below with reference to the attached drawings. [Brief explanation of the drawing]

[0098] [Figure 1] Figure 1a provides a schematic description of a structure for selectively permeable and / or fragmenting cells according to the present invention. Figure 1b shows a cross-sectional view of the structure shown in Figure 1a, with cells located at a distance d from the structure. [Figure 2-3] Figures 2 and 3 schematically illustrate a method for selectively making cells permeable and / or fragmenting cells according to the present invention, which involves cells growing on a film as shown in Figure 2 ("cells on film") and the film being applied to a cell layer as shown in Figure 3 ("film on cells").

[0099] [Figure 4] Figure 4 illustrates the method by which a particle-containing structure is applied to the cornea of ​​a cow's eye before being irradiated with a pulsed laser. [Figure 5-6] Figure 5 shows the average cluster size of particles in the PLA film as a function of IONP concentration. Figure 6 shows the cluster density (number of particle clusters / 100 μm²) in the PLA film as a function of IONP concentration.

[0100] [Figure 7-8] Figure 7 shows images obtained by dark-field microscopy before and after irradiation of PLA-IOC films (0.1% ION, 2% PA) with single laser pulses of 0.3 J / cm² and 1.6 J / cm², respectively. Figure 8 shows the number of clusters per Hela cell grown on PLA-IOC films with different concentrations of IONP.

[0101] [Figure 9-10]Figure 9 shows the uptake of FD500, cell viability, and rMFI (relative mean fluorescence intensity) of HeLa cells grown on a PLA film with 0.1% IONP. Figure 10 shows images obtained by confocal microscopy showing the uptake of FD500 grown on a PLA film with 0.1% IONP irradiated with a single laser pulse of 0.3 J / cm2.

[0102] [Figure 11-12] Figure 11 shows the cell viability of HeLa cells grown on PLA-IOC film (with or without 0.1% IONP) as a function of laser fluence after a single irradiation. Figure 12 shows the cell viability of HeLa cells grown on PLA-IOC film (0.1% IONP) irradiated with 1 or 2 0.3 J / cm2 laser pulses.

[0103] [Figure 13-14] Figure 13 shows the selective irradiation of a given area (a circle with a diameter of 6.5 mm) of a PLA-IOC film (2% PLA, 0.1% IONP) with a single pulse at various laser fluences (scale bar is 1000 μm). Figure 14 shows the percentage of cells acquired after selective irradiation as shown in Figure 13, expressed as a function of various laser fluences.

[0104] [Figure 15-16] Figure 15 shows the ratio of living cells (stained with calcein AM) to dead cells (stained with propidium iodide (PI)) after selective irradiation as shown in Figure 13, expressed as a function of various laser fluences. Figure 16 shows selective cell death upon local irradiation of a PLA-IOC film (2% PLA, 0.1% IONP) (scale bar is 10 μm).

[0105] [Figure 17-18]Figure 17 shows the success rate of single-cell excision for cells grown on a PLA-IOC film (2% PLA, 0.1% IONP) irradiated with a single laser pulse (0.3–1.3 J / cm2). Thirty single-cell excision experiments were performed for each laser fluence value (n=30). Figure 18 shows spatially selective laser-induced death of HeLa cells covered with a PLA-IOC film (2% PLA, 0.1% IONP).

[0106] [Figure 19-21] Figure 19 shows the illumination results of PLA-IOC (2% PLA, 0.1% IONP)-covered HeLa cells, as shown in Figure 18, by illuminating them with a pulsed laser following a predetermined pattern (Ghent University logo) of calcein AM, PI-stained and merged photographs. The scale bar is 1000 μm. Figures 20 and 21 show the ratio of live cells to dead cells and the total amount of cells, respectively, in the treated area as shown in Figure 18, following a series of laser pulses using a laser fluence of 1.6 J / cm2.

[0107] [Figure 22-23]Figure 22 shows a schematic diagram (left) of pore formation during cell death and the resulting FD500 uptake by HeLa cells (right) after irradiation (1.6 J / cm2; 4 consecutive times) with the PLA-IOC film positioned over the cells (as shown in Figure 18). Figure 23 shows ICP-MS analysis (iron content) of HeLa cells following 4 consecutive irradiations (1.6 J / cm2) of the PLA-IOC film; “Cells” refers to untreated cells; “IONPs” and “IONPs + Laser” refer to cells in the presence of free IONP (1 g / L) with or without laser treatment, respectively; and “PLA-IOC” and “PLA-IOC + Laser” refer to cells covered with the PLA-IOC film with and without laser treatment, respectively. Data are shown as mean ± SD. Statistical significance: Student's t-test; * indicates p<0.05, ** indicates p<0.01, and ns indicates no significance.

[0108] [Figure 24-26] Figure 24 shows laser-induced cell death in a bovine cornea covered with a PLA-IOC film (2% PLA, 0.1% IONP). The cornea was extracted and stained with propidium iodide. The entire cornea of ​​the extracted bovine eye was irradiated four times with a laser pulse of 1.6 J / cm2. Figure 25 shows the number of stained cells (dead cells) resulting from the laser-induced cell death shown in Figure 24, using the PLA-IOC film, compared to a control experiment (PLA film without IOC). Figure 26 shows laser-induced cell death in a bovine cornea covered with a PLA-IOC film (2% PLA, 0.1% IONP). The cornea was extracted and immersed in FD500 solution. IOC film (2% PLA, 0.1% IONP). The entire cornea of ​​the extracted cow's eye was illuminated four times with a laser pulse of 1.6 J / cm2.

[0109] [Figure 27-28]Figure 27 shows the FD500 uptake resulting from laser-induced cell death shown in Figure 26, using a PLA-IOC film compared to a control experiment (PLA film without IOC). Figure 28 shows bovine eye corneal epithelium stained with calcein, isolated and placed on a PLA-IOC film, followed by four illuminations to single cells at fluences of 1.6 J / cm2 and 4.5 J / cm2. [Modes for carrying out the invention]

[0110] Description of the manner The present invention is described in terms of specific embodiments and with reference to certain drawings, but the invention is not limited thereto and is limited only by the claims. The drawings are schematic and non-limiting. For illustrative purposes, the sizes of some elements in the drawings may be exaggerated and not drawn to scale. Dimensions and relative dimensions do not correspond to actual reductions in practice of the invention.

[0111] When referring to the endpoints of a range, the values ​​at the endpoints of the range are included. In describing the present invention, terms used shall be interpreted according to the following definitions unless otherwise specified.

[0112] The terms “first,” “second,” etc., used in the specification and claims are used to distinguish between similar elements and do not necessarily indicate order in time, space, sequence, or any other form. It should be understood that such terms are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein may be applied in any order other than those described or explained herein.

[0113] When listing two or more items, the term "and / or" means that any one of the listed items can be adopted on its own, or any combination of two or more of the listed items can be adopted.

[0114] The term "area" refers to the size of a flat surface on a plane, that is, a measurement of the size of a two-dimensional object. The term "surface area" refers to a measurement of the size of the surface of a three-dimensional object.

[0115] The term "cell" refers to all types of living cells, including eukaryotic and prokaryotic cells. Cells may also refer to human cells, animal cells, and plant cells. The term "fragmentation" refers to any way of dividing, cutting, or separating something into fragments. Cell fragmentation refers to any way of dividing, cutting, or separating a cell into fragments, and includes cell death and cell excision.

[0116] The terms “to increase permeability,” “to make permeable,” “to make permeable,” and “permeabilization” refer to any way of altering, at least partially or locally, the permeability of a cell, particularly the permeability of the cell membrane or barrier, such as the cell’s plasma membrane. After permeabilization, the membrane or barrier, such as the cell’s plasma membrane, is altered to be more permeable to one or more compounds, such as molecules, macromolecules, particles, or nanoparticles.

[0117] The term “perforate,” “perforation,” or “perforation” refers to any way in which one or more openings, holes, or pores are provided in a membrane or barrier, such as the plasma membrane of a cell. By perforating a membrane or barrier, such as the plasma membrane of a cell, an opening is created in the membrane or barrier, such as the plasma membrane of a cell, allowing for the transport of compounds such as molecules, macromolecules, particles, or nanoparticles across the membrane or barrier, such as across the plasma membrane of a cell.

[0118] For the purposes of this invention, the terms “to increase the permeability of ~,” “to make permeable,” “to make permeable,” and “permeation” and the terms “to perforate,” “to perforate,” and “perforation” may be used interchangeably. Similarly, the terms “opening,” “hole,” and “pore” may be used interchangeably.

[0119] The term "vapor bubble generation" encompasses the expansion of vapor bubbles, the collapse of vapor bubbles, or a combination of both expansion and collapse of vapor bubbles, as well as secondary effects that may result from the expansion and collapse of bubbles, such as pressure waves and the flow of the surrounding medium.

[0120] The terms "vapor bubble" or "bubble" refer to vapor nanobubbles and vapor microbubbles. Preferably, the terms "vapor bubble" or "bubble" refer to vapor bubbles having a diameter in the range of 10 nm to 100 μm. Vapor bubbles contain water vapor, but are not limited to this.

[0121] Experimental results Example 1 Figure 1a shows an embodiment of a structure 10 according to the present invention, which includes a polymer material 11 and a film containing particles 12 capable of absorbing electromagnetic radiation. A portion of the particles is partially exposed on one surface, for example, the upper surface 13 of the structure. The free surface area A of all particles P The sum of the free surface areas A S Between 0.0001% and 50% of the comfortable free surface area A S Between 5 and 25% of the free surface area A S The particles are embedded so that they are within the range of 15-25% of the total particle free surface area A. More specifically, the particles are embedded so that the free surface area A of all particles is within the range of 15-25% of the total particle free surface area A of all particles. P The sum of the upper free surface area A S1 Between 0.0001 and 50% 、 Comfortable free surface area A S1 Between 5 and 25% of the free surface area A S1 They are embedded so that the percentage is within the range of 15-25%.

[0122] Figure 1b shows the structure of Figure 1a with a cell 15 or more cells 15 located at a short distance d from the structure 10. The example described below includes polylactic acid (PLA) as a structural material and iron oxide particles as particles capable of absorbing electromagnetic radiation.

[0123] It is clear that other materials and other particles can be considered in a similar manner. Two types of experiments were performed to evaluate whether vapor bubbles generated on the surface of such a structure can make permeable and / or fragment cells that are in contact with or at a short distance d from them (Figure 1b): - Cells were grown on a structure, and the structure (more specifically, particles within the structure) was irradiated with (pulsed) electromagnetic radiation ("cells on a structure") (Figure 2); - The structure was applied to a cell layer, and the structure (more specifically, the particles within the structure) was irradiated with (pulsed) electromagnetic radiation ("structure on cells") (Figure 3).

[0124] Furthermore, a particle-containing structure was applied to the cornea of ​​a cow's eye and irradiated with a pulsed laser (Figure 4).

[0125] 1. Materials and Methods 1.1 Materials Iron oxide (Fe3O4) nanoparticles (IONP) with a size of 50-100 nm, polylactic acid (PLA) with a molecular weight (Mw) of 80 kDa, FITC-dextran with a molecular weight of 500 kDa, and chloroform were all purchased from Sigma-Aldrich (Belgium). Calcein AM and propidium iodide (PI) were purchased from Fisher Scientific (USA). Celltiter-Glo® was purchased from Promega (USA).

[0126] 1.2 Preparation of PLA-IOC (iron oxide cluster) film PLA films with and without IONP were prepared by a one-step spin-coating method. The films were prepared on a square coverslip (22 mm × 22 mm) placed in the center of the spin-coating device. Briefly, IONP was dispersed in PLA solutions (2%, 4%, and 8% (w / v) in chloroform); the concentration of IONP in the PLA solution was varied between 0.01%, 0.1%, and 0.2% (w / v).

[0127] The IONP-PLA dispersion was first sonicated at 10% amplitude for 1 minute using a tip sonicator (Branson Digital Sonicator, Danbury, USA). Next, 0.5 mL of the dispersion was applied onto a coverslip placed in the center of the spin-coating device. The speed and time for spin-coating were set to 2000 rpm and 20 seconds, respectively. After spin-coating, the film was placed overnight in a 50°C oven to allow for the evaporation of any remaining organic solvent.

[0128] To prepare the fluorescent PLA-IOC film, PLA (2%, w / v) and rhodamine B (0.01% w / v) (Sigma-Aldrich, Belgium) were dissolved overnight in chloroform. Then, iron oxide nanoparticles (0.1% w / v) were added to the PLA-rhodamine B solution. Next, the PLA-rhodamine B film was prepared as described in the paragraph above.

[0129] 1.3 Surface Morphology of PLA-IOC Films The surface morphology of PLA-IOC films was characterized by scanning electron microscopy (SEM, FEI Quanta 200F (Thermo Scientific)). SEM images were acquired at an accelerating voltage of 20 kV. It should be noted that iron oxide clusters (IOCs) of varying sizes may be observed depending on the concentration of IONP.

[0130] Since the iron oxide particles are arranged within iron oxide particle clusters (IOCs), the film is referred to as a PLA-IOC film (instead of a PLA-IONP film). The size and distribution of the IOCs were calculated based on SEM images.

[0131] 1.4 Laser-induced vapor bubble (VNB) formation and VNB threshold To evaluate whether PLA-IOC films have the ability to generate VNBs, they were irradiated with a pulsed laser (HE 355 LD laser, OPOTEK Inc.; 7 ns, 561 nm). VNBs scatter light for the duration of their existence and can therefore be easily visualized by dark-field microscopy. Dark-field images of the films (covered with a thin layer of water) were recorded before and during irradiation of the films with laser pulses at various fluences. The "VNB threshold," which is the fluence of a single laser pulse at which 90% of the IOCs form VNBs, was determined by plotting the number of bubbles as a function of laser fluence.

[0132] 1.5 Cell culture Hela cells (ATCC® CCL-2®) were cultured in DMEM / F-12 (Gibco-Invitrogen) supplemented with 10% fetal bovine serum (FBS, Biowest), 2 mM glutamine, and 100 U / mL penicillin / streptomycin (Gibco-Invitrogen) at 37°C and 5% CO2. For cell division, cells were first washed with Dulbecco's phosphate-buffered saline (DPBS) and then detached using trypsin-EDTA solution (Gibco-Invitrogen).

[0133] For the "Cells on Top" experiment (Figure 2), HeLa cells were seeded on a PLA-IOC film located at the bottom of the wells in a 6-well plate; the HeLa cell concentration was 1 × 10⁻⁶. 6The cells were divided into cells / mL; 2 mL of cells were applied to each well. After 1 day of incubation, the PLA-IOC film to which the cells were adhered was removed from their original wells and placed into new wells (of a 6-well plate); this was to remove cells that had not been in contact with the film but were simply adhered to the plastic bottom of the wells.

[0134] For the experiment in which PLA-IOC film was placed on top of cells ("Structure on top of cells") (Figure 3), HeLa cells were placed in 0.5 × 10⁶ wells of a 6-well plate. 6 Cells were seeded at a concentration of cells / mL (2mL). The following day, the Hela cells in the wells were covered with PLA-IOC film. To maximize contact between the cells and the film, 90% of the cell medium was removed before applying the film so that only a thin layer of medium remained over the cells.

[0135] 1.6 Cell death by PLA-IOC film exposed to pulsed light laser As described above, cells were either seeded onto a PLA-IOC film or covered with a PLA-IOC film.

[0136] The PLA-IOC film was then irradiated with a nanosecond pulsed laser (pulse duration <7 ns, 532 nm wavelength). A galvanometer scanner was used to enable high-speed scanning of the laser beam so that each area received essentially a single laser pulse (or twice in overlapping areas between adjacent spots); a single scan of the entire surface of the PLA-IOC film in the wells of a 6-well plate took approximately 2 minutes. In some cases, the film was scanned multiple times as indicated in the text.

[0137] The degree of cell death by PLA-IOC film exposure to light pulses was evaluated by measuring cellular metabolic activity using the CellTiter-Glo® assay. The assay was performed as recommended by the manufacturer, with some minor modifications. Briefly, cells on the PLA-IOC film were treated with light pulses, followed by 2 hours in a cell incubator, allowing sufficient time for cell death.

[0138] After 2 hours, the cell medium was removed and replaced with 1 mL of fresh cell medium (DMEM / F-12) to which 1 mL of CellTiter-Glo solution had been added. The well plate was then placed on a shaker at 120 rpm for 10 minutes at room temperature. Finally, 150 μL of the supernatant was transferred into the wells of a 96-well plate, and the absorbance was measured using a microplate reader (Victor 3, PerkinElmer).

[0139] 1.7 Measuring cell membrane permeabilization To evaluate the extent to which applying laser pulses to PLA-IOC films made cell membranes permeable, cells were seeded onto PLA-IOC films or covered with PLA-IOC films as described above. If cells grew on the film, 1 mL of DMEM / F-12 cell culture medium containing FITC-dextran (molecular weight 500 kDa; FD500) at a concentration of 2 mg / mL was added. If the film was applied on top of the cells, 300 μl of DMEM / F-12 cell culture medium containing FD500 (2 mg / mL) was added to the cells before applying the film. The entire surface of the film was scanned with a laser beam (as described above; at a fluence of 0.3 J / cm² or 1.6 J / cm²), and the cells were thoroughly washed several times with DPBS to remove excess FD500.

[0140] The cells were then trypsinized (using a 0.25% trypsin solution) and neutralized with cell culture medium. The cell suspension was collected and washed several times by centrifugation (500 g; 5 min). Finally, the pellet was resuspended in flow buffer (DPBS, 1% bovine serum albumin (BSA) and 0.1% NaN3). Cytosolic delivery of FD500, which occurs only after membrane permeabilization, was measured by flow cytometry (Cytoflex, Beckman Coulter, Krefeld, Germany); the fluorescent dye was excited at 488 nm, while the fluorescence intensity was detected at 530 nm (bandpass filter 530 / 30).

[0141] 1.8 Spatially Selective Cell Death by PLA-IOC Film Exposed to Pulsed Laser Light In the first experimental setup, HeLa cells were seeded on a PLA-IOC film (0.1% IONP) in a 6-well plate. Laser pulses with increasing fluence were applied to the film within a designated zone. After laser irradiation, the cells on the PLA-IOC film were placed in an incubator (37°C, 5% CO2) for 2 hours. Then, 1 μl of calcein AM (0.5 mmol) and 10 μl of propidium iodide (1 mg / ml) were added to each well. Calcein AM was used to stain living cells, and propidium iodide was used to stain dead cells. After 10 minutes of incubation, the cells were imaged using a confocal microscope (C1-si, Nikon, Japan). The degree of cell death within the treated zone was evaluated.

[0142] In the second experimental setup, PLA-IOC film (with or without 0.1% IONP) was applied over HeLa cells seeded as described above. Four consecutive laser irradiations at 1.6 J / cm² were performed according to a predetermined pattern (similar to the Ghent University logo). After laser scanning, 2 mL of DMEM / F-12 was added to the wells, and the film was carefully removed from the cells. After 30 minutes, 2 μl of calcein AM (0.5 mmol) and 20 μl of propidium iodide (1 mg / ml) were added to stain both live and dead cells.

[0143] 1.9 Single cell death by PLA-IOC film exposed to pulsed laser light. To evaluate the extent to which pulsed laser irradiation of PLA-IOC films enables the specific death of selected target cells ("single-cell death"), cells were seeded as cells on the PLA-IOC film as described above. First, (live) cells were stained by adding 2 μl of calcein AM (0.5 mmol) to each well. Single cells were then randomly selected as targets and imaged by fluorescence microscopy.

[0144] Next, a single laser pulse (of varying fluences) was applied to the target cells. After 30 minutes, 10 μL of propidium iodide (1 mg / ml) was added to each well, and the cells were imaged again using fluorescence microscopy. The red and green fluorescence of the cells was analyzed using ImageJ software. The cells in the wells were also imaged using transmission microscopy; the video was recorded and processed using ImageJ software.

[0145] 1.10 Inductively coupled plasma mass spectrometry (ICP-MS) measurement Hela cells were seeded in a 6-well plate as described above. In the absence of laser treatment, either cell culture medium (1.5 ml), a suspension of free IONP (1.5 ml; 1 g / l), or a PLA-IOC film (maintaining a very thin layer of medium between the film and the cells) was added to / applied to the cells in the wells. After 20 minutes of incubation (37°C, 5% CO2), the cell medium / free IONP (or) film in the wells was removed. Then, 1.5 mL of nitrate (65%) was added to each well for 10 minutes to digest the cells. After 10 minutes, 1 mL was collected from each well for ICP-MS analysis.

[0146] To measure the effect of the laser treatment, each well was scanned four times (1.6 J / cm2) 20 minutes after applying cell medium / free IONP / PLA-IOC film (see paragraph above). After laser treatment, the film was removed and the wells were gently washed twice with fresh cell culture medium. Subsequently, 1.5 mL of 65% nitric acid was added to each well for 10 minutes. After cell digestion, 1 mL of the solution was collected from each well for ICP-MS analysis.

[0147] The (ultra)trace elemental determination of iron (Fe) was performed using an Agilent 8800 ICP-MS / MS instrument (ICP-QQQ, Agilent Technologies, Japan). The sample introduction system consisted of a concentric nebulizer (400 μL min) mounted on a Peltier-cooled (2°C) Scott-type spray chamber. -1 This instrument features a tandem mass spectrometer configuration (Q1-CRC-Q2) consisting of two quadrupole units (Q1 and Q2) and a collision / reaction cell (CRC) located between both quadrupole mass filters. As a result of much better control over intra-cell chemistry (chemical resolution), the MS / MS mode provides an additional means of dealing with spectral overlap in a simpler way compared to the use of existing single-quadrupole ICP-MS instruments.

[0148] In this study, to overcome spectral interferences that significantly hinder the determination of (ultra)trace elements of Fe, such as the signals of polyatomic interference between 40ArO+ and 40CaO+, and the overlap between them of the most abundant Fe isotope (56Fe+, 91.7%) at a mass-to-charge ratio -m / z- 56, CRC was pressurized with an NH3 / He mixture (10% NH3 in He). To overcome spectral overlap, 3.0 mL min -1 When NH3 / He is introduced, 56Fe + Ions are 56Fe(NH3) 2+ The mass shift was transferred to the reaction product ion. After the mass shift, the newly created reaction product ion (56Fe(NH3)) 2+ With an m / z ratio of m / z=90, 56Fe shows no spectral interference. + We were able to detect it.

[0149] For ICP-MS / MS analysis, only high-purity reagents were used. Ultrapure water (low efficiency 18.2 MΩ cm) was obtained from a Milli-Q Element water purification system (Millipore, France). Professional analysis purity level 14M HNO3 (Chem-Lab, Belgium), further purified by sub-boiling distillation, and ultrapure 9.8M H2O2 (Sigma Aldrich, Belgium) were used for sample digestion. 1g L -1 Appropriate dilutions of single-element standard solutions of Fe and Ga (Inorganic Ventures, USA) were used for method development, optimization, and calibration purposes. For quantitative elemental determination of Fe, calibration approaches (0, 0.5, 1.0, 2.5, 5.0, 10, and 20 μg L) were used. -1 External calibration was trusted for Fe. 5.0 μg L -1 The Ga value was used as an internal standard to compensate for instrument instability, signal drift, and matrix effects.

[0150] Prior to sample preparation for ICP-MS / MS analysis, all samples were transferred from Eppendorf tubes to Teflon Savillex beakers that had been pre-washed with HNO3 and then rinsed with HCl and subsequently Milli-Q water. After evaporation to dry (80°C), the samples were digested on a hot plate at 110°C for approximately 18 hours via acid digestion with a mixture of 750 μL of 14 M HNO3 and 250 μL of 9.8 M H2O2.

[0151] Prior to ICP-MS / MS analysis, digested samples were appropriately diluted in Milli-Q water or HNO3 (final acid concentration within the range of 0.35–0.70 M HNO3). To avoid contamination, only metal-free test tubes were used for standards and sample preparation (15 or 50 mL polypropylene centrifuge tubes, VWR, Belgium). The complete sample preparation procedure, including digestion and proper dilution, was performed in a Class 10 cleanroom.

[0152] 1.11 Death of superficial corneal cells by PLA-IOC film exposed to pulsed light laser. Freshly extracted bovine eyes were collected from a local slaughterhouse (Flanders Meat Group, Zele). After extraction, the eyes were kept in a chilled CO2-independent medium (Gibco-Invitrogen) during transport and processed within one hour. Excess tissue was removed, and the eyes were washed in chilled DMEM.

[0153] PLA films (2% PLA) with or without 0.1% IONP (w / v) were used. The PLA-IOC films were placed in 55 mm glass-bottom dishes (Cellvis, USA) with 30 mm microwells, and filled with 3 mL of cell culture medium. Subsequently, the eyes were moved into the dishes and fixed with Parafilm to ensure that the cornea was in close contact with the PLA-IOC film. FD500 was added to the cell culture medium (4 mg / mL) to determine whether the application of laser pulses had made the corneal cell membranes permeable.

[0154] The entire cornea, 1.6 J / cm² 2 The eyes were repeatedly scanned (4 times) with a pulsed laser at fluence. After irradiation, the eyes were washed three times with DPBS. To assess the kill efficiency, the eyes were placed in a fresh dish, supplemented with fresh DMEM / F-12 medium containing propidium iodide (0.01 mg / ml), and incubated at room temperature for 15 minutes.

[0155] Finally, a 12 mm button was driven into the cornea using a trefin blade (Beaver Visitec International, Abingdon, UK). The thus isolated corneal tissue was placed epithelial side down on a glass-bottom dish and imaged using a confocal microscope (Nikon A1R). Z-stacks were recorded at 1 μm intervals, and 3D images were obtained using available software.

[0156] For the spatially selective cell death experiment, excess tissue was removed from fresh bovine eyes, and the eyes were washed in DPBS. To stain living epithelial cells, a solution of calcein AM (5 μg / ml) was applied at the corneal level, and the eyes were incubated at room temperature for 10 minutes. The eyes were then washed three times in DPBS, and the corneas were isolated using a treffin blade. The corneas were then placed on a film, which was a PLA-IOC film. Epithelial cells could be observed by fluorescence microscopy, and single cells were targeted before irradiation with a laser (561 nm; <7 ns).

[0157] 2. Results 2.1 Physicochemical Characterization of PLA-IOC Films To enable targeting of each single cell in a cell culture (or tissue), ideally, each single cell should be in contact with / near at least one IOC. Therefore, we investigated the well-controlled distribution of IOCs in PLA films. SEM imaging of the PLA films revealed that IONP was embedded within the film as clusters (IOCs); some IOCs partially protruded outside the film. The average size and distribution of IOCs in the film depended on the IONP concentration. Indeed, higher IONP concentrations (relative to the concentration of immobilized PLA) resulted in larger clusters (Figure 5). The cluster density was equal at 5.6 ± 2.4 IOC / 100 μm² at a 0.1% IONP concentration, while it was lower at higher IONP concentrations (Figure 6), which can be explained by the formation of larger clusters.

[0158] 2.2 Vapor bubble formation by laser-induced PLA-IOC film To evaluate whether the application of pulsed laser light to PLA-IOC films resulted in the formation of vapor bubbles (VNBs) on the film surface, PLA-IOC films were irradiated with single laser pulses of 0.3 J / cm² and 1.6 J / cm², respectively. Irradiation of PLA-IOC films (0.1% ION, 2% PA) with such laser pulses produced VNBs, as localized flashes of (scattered) light were detected by dark-field microscopy (Figure 7). No VNBs were observed in PLA films without IOCs. At lower fluences, the number of VNBs was significantly lower than at higher fluences.

[0159] Next, the number of VNBs was measured as a function of the laser pulse fluence, and the VNB threshold for a PLA-IOC film (0.1% IONP; 2% PLA), commonly defined as the laser fluence (T90) where 90% of the IOC forms VNBs, was determined to be 0.56 J / cm2. T10, corresponding to the laser fluence where 10% of the IOC results in VNB formation, was determined to be 0.1 J / cm2. At fluences lower than T10, heat generation was dominant (heating mode); between T10 and T90, both confined heat and bubbles were generated; and at fluences higher than T90, VNB formation was the primary photothermal effect (bubble mode). For the subsequent experiment, we selected a PLA film containing 0.01% IONP. This PLA-IOC film was also appealing because it was as transparent as a PLA film without IOC.

[0160] 2.3 Cell death by PLA-IOC film exposed to pulsed light laser Next, we evaluated the ability of the PLA-IOC film to kill HeLa cells grown on its surface ("cells on top") (Figure 2). The IOCs in the film were easily observable because they strongly scatter light (the refractive index of iron oxide is 2.9). Based on images of cells grown on the PLA-IOC film, it was estimated that when the concentration of IONP in the film was equal to 0.1%, each cell was in contact with 4.6 ± 0.5 IOCs (Figure 8); and at lower IONP concentrations (0.01%), each cell was in contact with 1.2 ± 0.4 IOCs.

[0161] Figure 9 shows that (most) cells grown on a PLA-IOC film with 0.01% IONP survived when the film was scanned with a laser at a low fluence (single scan; 0.3 J / cm2) (as measured by flow cytometry; calcein AM staining). A significant number of cells were killed (cell viability 52.8 ± 12.6%) at higher concentrations of IONP in the PLA film (0.1%) and at the same fluence. Unaccompanied It should also be noted that all cells grown on the PLA film survived the laser treatment.

[0162] To visualize changes in cell membrane permeability following laser irradiation (which can lead to cell death), fluorescent dextran (FD500) was added to cells. Due to their large size (approximately 30 nm), FD500 does not diffuse across the cell membrane and is therefore reported to be sufficiently suitable for assessing significant changes in plasma membrane permeability. As shown in Figures 9 and 10, FD500 entered HeLa cells following laser pulse irradiation of PLA-IOC films; it should be noted that FD500 did not enter cells in PLA films without IONP.

[0163] Next, we evaluated how cell death could be maximized by using higher laser fluence and repeated laser scans on the film, respectively. As shown in Figure 11, increasing the laser fluence improved the cell death capability of the PLA-IOC film: cell viability dropped to 30% at a laser fluence of 0.8 J / cm2. This was not the case for PLA films without IOC, which did not induce any cell death even at an extremely high laser fluence of 1.6 J / cm2.

[0164] Since it was observed that the IOC survived a single laser pulse (i.e., was not fragmented), we hypothesized that applying a second pulse could further enhance the film's cell-killing ability. Because approximately 50% of cells remained viable after a single pulse of 0.3 J / cm², we selected a (low) fluence of 0.3 J / cm² to confirm that the effect of a second pulse would be observable. The film was therefore scanned twice consecutively with a fluence of 0.3 J / cm². As shown in Figure 12, cell viability decreased from 46 ± 6% (single scan) to 25 ± 3% (two consecutive scans).

[0165] 2.4 Spatially Selective Cell Removal and Cell Death by PLA-IOC Film Exposed to Pulsed Laser Light Given (i) the fact that the density and size of IOCs in the film can be controlled, and (ii) the inherent ability to scan specific areas within the film, we evaluated to what extent bubble-films can enable target cell killing with high spatial precision. Therefore, a predetermined circular area within a PLA-IOC film was scanned with laser pulses at increasing fluence (Figure 13).

[0166] As observed by the red fluorescence of propidium iodide (PI), which penetrates only dead cells, cells within a designated area (a circle with a diameter of 6.5 mm) were selectively killed. However, at a fluence of 1.3 J / cm2, a significant decrease in the total number of cells (live cells plus dead cells) within the treated area was observed (Figure 14), indicating that cells were "lost" (excised) during laser treatment, likely due to the mechanical force exerted by the vapor bubbles. As shown in Figure 15, staining cells with calcein AM and propidium iodide allowed for the determination of the ratio of live cells to dead cells remaining within the treated area; clearly, this ratio gradually decreased with increasing laser fluence.

[0167] Figure 16 shows single-cell death (scale bar is 10 μm) upon localized irradiation of a PLA-IOC film with a pulsed laser. Single-cell death was observed, as could be seen by the red fluorescence of propidium iodide (PI), which penetrates only dead cells. The transmission image of the cells on the film after selective cell treatment is 1.3 J / cm². 2 Single-cell excision was observed at fluence levels. VNB could be induced using fluence levels between 0.3 and 0.8 J / cm², but cell excision did not occur.

[0168] The success rate of single-cell resection increased with increasing fluence (Figure 17). Furthermore, it was observed that a fluence of 0.3 J / cm² did not enable single-cell resection, while a fluence of 1.3 J / cm² consistently resulted in successful single-cell resection.

[0169] In all the experiments above, cells were grown on a PLA-IOC film. To further explore the clinical potential of particle-containing structures according to the present invention, the cell-killing ability of the film when placed on a cell layer (Figure 18) was evaluated. This mimics the intended use of a film covering the surface of a target tissue.

[0170] As outlined in the Experiment section, after seeding cells in a 6-well plate, the cell medium was removed, except for a thin layer of medium left on top of the cells; then a PLA-IOC film was placed on top of the cells. The distance between the cells and the film was estimated to be 40 μm. A designated area within the film (Ghent University logo) was then exposed to the laser (Figure 19). A higher fluence of 1.6 J / cm² was used because a greater distance between the film and cells was estimated compared to the experiment in Figure 13 where the cells were cultured directly on the film.

[0171] As shown in Figure 20, cells were killed, although four pulses were required to achieve a ratio of approximately 25% live cells to dead cells. It was also noted that the total number of cells did not change (Figure 21), likely due to the longer distance between the cells and the IOC, which reduced the mechanical force exerted by the VNB on the cells.

[0172] To evaluate whether cell membrane perforation also contributes to cell death when a PLA-IOC film is positioned over the cells (Figure 22 (left)), cells were incubated with FD500 and then covered with the film. The film was then irradiated (1.6 J / cm2; 4 consecutive times). As shown in Figure 22 (right), green (FD500) and red (PI) fluorescence could be observed within the cells, while fluorescence was much lower in cells covered with a PLA film without IOC and irradiated with laser pulses.

[0173] ICP-MS experiments were performed to determine the amount of intracellular iron after laser irradiation of PLA-IOC films covering cells. As shown in Figure 23, exposure of cells to free IONP (1 g / L, equivalent to the iron concentration in the film) leads to higher iron content in the cells. Laser irradiation of free IONP further increases intracellular iron levels. This is likely due to (i) fragmentation of IONP into smaller fragments and (ii) the perforation of the cell membrane by VNB (because it is produced by free IONP), thereby promoting iron uptake. In cells covered with PLA-IOC film (without laser treatment), the iron content remained as low as in untreated cells. When the PLA-IOC film was irradiated, an increase in intracellular iron content was observed. However, the intracellular iron content remained far below that of cells treated with free IONP and laser.

[0174] 2.5 Death of superficial corneal cells by PLA-IOC film exposed to pulsed light laser Since efficient cell death was achieved when the film was positioned over the cultured cell layer (Figure 18), the next step was to evaluate whether superficial cells in the tissue (cornea) could be killed by the structure according to the present invention. As described in Figure 24, a bovine eye was positioned on the film; the entire cornea was then illuminated four times consecutively at 1.6 J / cm2. The cornea was then excised, stained with PI, and imaged by confocal microscopy.

[0175] The first finding, somewhat unexpectedly, was the presence of PI-positive cells in the control eyes, i.e., eyes without PLA film or covered with PLA film without IOC. This was likely because (i) ex vivo bovine corneal epithelial cells had already begun dying a few hours after sampling, and (ii) epithelial cell death is known to occur physiologically, for example, due to shear forces caused by blinking. However, confocal microscopy images confirmed a significant increase in PI-positive cells in corneas covered with PLA-IOC film irradiated with four 1.6 J / cm2 laser pulses, compared to control experiments using PLA film without IOC (Figure 25).

[0176] To confirm membrane perforation in corneal cells, the experiment was repeated in the presence of green fluorescence FD500. Confocal microscopy images showed a clear difference in green fluorescence in corneas covered with PLA-IOC film and treated with four 1.6 J / cm2 pulses compared to a control experiment using PLA film without IOC. This finding indicates that perforation of the corneal cell membrane actually occurred. Equally important, the green fluorescence (FD500) did not co-localize with the red fluorescence of spontaneously dead cells, indicating that pore formation was attributable to laser irradiation of the PLA-IOC film covering the cornea. It was observed that FD500 did not penetrate untreated corneal cells.

[0177] To check whether spatially selective cell death was achieved at the corneal level, epithelial cells were stained with calcein AM (5 μg / ml). The stained corneas were then placed on a film with the epithelial side down (Figure 28). A region of interest was selected, and laser irradiation (<7 ns; 561 nm) was applied four times. After four irradiations at a fluence of 1.6 J / cm², a localized decrease in fluorescence could be observed, and the irradiated cells lost their staining. At higher fluences (4.5 J / cm², four times), cells within the targeted area were no longer visible, suggesting that excision occurred. For both fluences, surrounding cells (i.e., located outside the laser beam) did not lose their staining after laser irradiation, suggesting that they were left untouched.

[0178] Example 2 A second embodiment of a structure according to the present invention includes a porous structure comprising nanofibers and particles capable of absorbing electromagnetic radiation embedded within the nanofibers, wherein a portion of the particles is partially exposed on the free surface S of the structure, particularly on the free surface of the nanofibers. An example described below includes polycaprolactone as the material of the structure and iron oxide nanopowder as the particles capable of absorbing electromagnetic radiation. It is clear that other materials and other particles can be considered in a similar manner.

[0179] 1. Materials and Methods 1.1 Materials The following materials are used for the synthesis of nanofiber networks: ● Polycaprolactone (PCL, Mw ≈ 70,000 g / mol); ● N,N-dimethylformamide (DMF); ● Tetrahydrofuran (THF); ● Iron oxide (Fe3O4) nanopowder (IONP) (#MKBW3262, Sigma-Aldrich, Belgium); ● Poly(allylamine hydrochloride) (PAH, Mw=17,560 g / mol, #MKBZ2824V, Sigma-Aldrich, Belgium); ● Concentrated sulfuric acid solution (96%) (Sigma-Aldrich); ● Collagen I rat protein (Thermo Fisher Scientific, #A1048301, Gibco®, Belgium).

[0180] 1.2 Preparation of structures containing nanofibers and particles IONP was redispersed in 1:1 DMF / THF solutions to which PCL was added at various concentrations ranging from 0% to 1.15% by volume. The mixture thus obtained was used to produce nanofibers by electrospinning. The nanofibers were collected on a microscope slide glass (#1000912, Marienfeld, Germany) mounted on a ground-level rotating collector. IONP was embedded in nanofibers, with a portion of the particle partially exposed to the free surface S of the nanofiber.

Claims

1. An in vitro or ex vivo method for selectively making cells permeable and / or fragmenting cells in non-human animals, - A structure containing a material and particles capable of absorbing pulsed laser light, wherein volume V and free surface S are defined, and the free surface S is the free surface area A Ｓ The particles have a free surface area A, and at least a portion of the particles is partially exposed to the free surface S, and each of the particles partially exposed to the free surface S defines a particle free surface P, where the particle free surface P is the particle free surface area A Ｐ It has the particle free surface area A of all particles. Ｐ The sum of the free surface area A Ｓ A structure is provided, wherein the particles are embedded in the structure such that they are within the range of 0.0001 to 50%; - A step in which at least one non-human animal cell is provided; - A step in which the structure and at least one cell are brought to a distance d of less than 100 μm from each other, where d is the shortest distance between the cell of at least one non-human animal and the free surface S of the structure; - A step in which pulsed laser light is irradiated onto the structure. The method, including the method described above.

2. An in vitro or ex vivo method for selectively making cells permeable and / or fragmenting cells in isolated tissue or isolated cells, - A structure is provided comprising a material and particles capable of absorbing pulsed laser light, wherein a volume V and a free surface S are defined, the free surface S having a free surface area AS, at least a portion of the particles being partially exposed to the free surface S, each of the partially exposed particles defining a particle free surface P, the particle free surface P having a particle free surface area AP, and the particles being embedded in the structure such that the sum of the particle free surface areas AP of all particles is in the range of 0.0001 to 50% of the free surface area AS; - A step in which the structure and at least one cell are brought to a distance d of less than 100 μm from each other, where d is the shortest distance between at least one cell in the isolated tissue or isolated cell and the free surface S of the structure; - A step in which pulsed laser light is irradiated onto the structure. The method, including the method described above.

3. The method according to claim 1 or 2, wherein at least some of the particles partially exposed on the free surface S protrude from the material.

4. The method according to any one of claims 1 to 3, wherein the particles have dimensions in the range of 1 nm to 40,000 nm.

5. The method according to any one of claims 1 to 4, wherein particles are arranged within a cluster, and the cluster contains between 2 and 1000 particles.

6. The method according to any one of claims 1 to 5, wherein at least one cell comprises a cell suspension or a cell tissue.

7. The density of particles partially exposed on the free surface S of the structure is 1 to 10 particles / 100 μm 2 The method according to any one of claims 1 to 6, which is within the range of 1 to 6.

8. The method according to any one of claims 1 to 7, wherein the particles include particles selected from the group consisting of metal particles, metal oxide particles, carbon or carbon-based particles, particles containing one or more light-absorbing compounds, and particles on which one or more light-absorbing compounds are mounted or functionalized.

9. The method according to any one of claims 1 to 8, wherein the material comprises an inorganic material or an inorganic-based material, a ceramic or a ceramic-based material, an organic material or an organic-based material, a hydrogel, or a composite material comprising at least one of these materials.

10. The pulsed laser light is irradiated onto the structure with a pulse duration in the range of 1 fs to 1 ms and / or 0.001 to 10 J / cm². 2 The method according to any one of claims 1 to 9, comprising irradiation using a pulsed radiation source having pulses with a pulse-per-pulse fluence within the range of .

11. The structure has a first free surface area A Ｓ１ a first surface S1, and a second free surface area A on the side opposite to the first surface Ｓ２ a second surface S2, the structure comprising a film defining the same, wherein the particles are embedded in the structure such that the sum of the particle free surface areas A of all the particles partially exposed on the first surface S1 Ｐ is within a range of 0.0001 to 50% of the free surface area A Ｓ１ The method according to any one of claims 1 to 10, wherein the particles are embedded in the structure such that the ratio is in the range of 0.0001 to 50%.

12. The method according to any one of claims 1 to 11, wherein the structure comprises a porous structure including fibers, fine particles, a combination of fibers and fine particles, or a foam, and the particles are embedded in the fibers, fine particles, or foam.

13. A structure for use in photothermal processes that selectively permeate and / or fragment cells in human or non-human animal cells, wherein the structure comprises a material and particles capable of absorbing pulsed laser light, the structure defines a volume V and a free surface S, and the free surface S is a free surface area A Ｓ The particles have a free surface area A, and at least a portion of the particles is partially exposed to the free surface S, and each of the particles partially exposed to the free surface S defines a particle free surface P, where the particle free surface P is the particle free surface area A Ｐ It has the particle free surface area A of all particles. Ｐ The sum of the free surface area A Ｓ The particles are embedded in the structure such that they are within the range of 0.0001 to 50%, and the photothermal process is performed. - A step of bringing the structure and at least one cell to a distance d of less than 100 μm from each other, where d is the shortest distance between at least one cell and the free surface S of the structure; - A step of irradiating a structure with pulsed laser light to selectively permeate and / or fragment at least one cell. The structure including the said structure.

14. The structure according to claim 13, wherein the photothermal process is for a method of treating a target, wherein the photothermal process selectively makes the target cells permeable and / or fragments them.

15. The structure according to claim 13 or 14, wherein the cells in the photothermal process include a cell suspension or a cell tissue.

16. The structure according to claim 14, wherein the treatment method includes nanosurgical procedures.

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