Methods for increasing cytoplasmic membrane permeability and structures suitable for use in the methods

Abstract

This invention relates to a method for increasing the permeability of a cell membrane by directing at least one cell to or near a structure containing particles capable of absorbing electromagnetic radiation. These particles are present at a concentration of 0.001 vol% to 20 vol% and are embedded in the material of the structure. At least 60% of the particles present in the structure are embedded in the material such that the shortest distance L between these particles and the free surface S of the structure is 1 nm to 500 nm. The invention also relates to a structure suitable for permeating cells during photothermal processes and the use of said structure.

Description

Technical Field

[0001] This invention relates to a method for increasing the permeability of the cell membrane by guiding cells to or near a structure containing embedded particles and irradiating the structure. The method of this invention does not require contact between the particles and the cells. This invention also relates to a structure suitable for methods of increasing cell membrane permeability. Background Technology

[0002] Intracellular delivery of exogenous compounds within cells is a common requirement in many biotechnological and biomedical applications. Examples include creating mutant cell lines for basic research, drug screening of biopharmaceutical compounds, and producing cells for cell-based immunotherapies (e.g., CAR-T cells). Regardless of the specific application, a common challenge is overcoming the cell membrane, a major barrier, especially for large molecules like DNA, RNA, or proteins. In recent years, research into novel physical transfection methods in this field has increased significantly, aiming to deliver molecules, particularly large molecules, as efficiently as possible while minimizing cytotoxicity.

[0003] Physical methods for delivering compounds into cells have attracted considerable interest. These methods share the common feature of increased cell membrane permeability, allowing compounds to cross the cell membrane.

[0004] Nanoparticle (NP)-sensitized photoporation is a promising emerging physical method for delivering compounds into cells. In photoporation, a combination of laser irradiation and photoresponsive nanoparticles temporarily makes the cell membrane permeable. Cells are first incubated with nanoparticles (typically gold, iron oxide, or carbon particles) that can adsorb onto the cell membrane. Next, laser irradiation is applied, making the cell membrane permeable through photothermal or photochemical effects (e.g., localized heating, induction of pressure waves, or generation of reactive oxygen species).

[0005] While photoperforation is a promising technique, for example, for producing engineered cells for cell therapy, general safety concerns exist regarding the contact between nanoparticles and cells. In fact, the potential toxicological effects of nanoparticles are generally quite uncertain. Furthermore, plasma nanoparticles, such as gold nanoparticles, tend to fragment into smaller pieces under the intense laser irradiation used in photoperforation. Nanoscale gold particles have been reported to potentially exhibit genotoxicity upon internalization into cells. Given that photoperforation methods require close contact between plasma nanoparticles and cells, the use of photoperforation (e.g., for transfecting cells used in cell therapy) may present nanotoxicological challenges.

[0006] Therefore, it is of current interest to develop methods to avoid direct contact between plasma nanoparticles and cells during photoperforation.

[0007] US 9,957,476 describes a system for perforating cells using plasma nanoparticles. This system uses a laser to create an optical trap to position the nanoparticles near the cells, and uses the laser guided onto the light-trapping particles to cause the particles to disintegrate under laser-induced conditions, resulting in cell perforation. However, the system has limited throughput and does not allow for scaling up to process large numbers of cells. Therefore, this system is unsuitable for producing large quantities of engineered cells for cell therapy.

[0008] US 9,139,416 describes a microfluidic device comprising a substrate having microchannels, wherein the walls of the microchannels are provided with nanowires. When irradiated with a laser, cells can be photoperforated as they flow through the channels. However, this device does not allow adjustment of the distance between the plasma structure and the cells, which is necessary to maximize system performance, especially when processing different cell types. Furthermore, the device is susceptible to nanowire damage, thus having a limited lifespan.

[0009] EP 2272945 describes a cell perforation method in which cells are placed on or near a substrate surface and irradiated with laser pulses. The substrate has a surface structure coated with a thin metal layer (e.g., gold). A drawback of this method is that the cell membrane is permeable on one side (i.e., the side where the cell contacts the substrate surface structure (the bottom side of the cell)), but the compounds required to be delivered into the cell are primarily located on the other side (the top side of the cell). This limits the efficiency with which molecules, especially macromolecules, can enter the cell. Summary of the Invention

[0010] One object of the present invention is to provide a method for increasing the permeability of the cell membrane, thereby avoiding the disadvantages of methods known in the art.

[0011] Another object of the present invention is to provide a method for increasing cell permeability through a photothermal process, particularly by irradiation with electromagnetic radiation such as laser radiation.

[0012] Another object of the present invention is to provide a method for increasing the permeability of the cell membrane by using a structure containing particles embedded therein, guiding cells to or near the structure, and irradiating the structure with electromagnetic radiation.

[0013] Another object of the present invention is to provide a method for increasing cell permeability, thereby limiting or even avoiding direct exposure of cells to particles, such as nanoparticles or components thereof.

[0014] Another object of the present invention is to provide a method for increasing cell permeability using pulsed laser irradiation (e.g., nanosecond pulse irradiation).

[0015] Another objective is to provide a method for increasing cell permeability, which enhances the efficiency of intracellular delivery of cell-impermeable substances, particularly for the intracellular delivery of macromolecules.

[0016] Another objective is to provide a structure comprising particles, such as nanoparticles, capable of absorbing electromagnetic radiation to increase the permeability of the cell membrane, thereby retaining, preferably entirely, the particles and their components within the structure upon laser activation.

[0017] In addition, one objective is to provide a method for increasing cell permeability, applicable to drug screening, cell therapy, immunotherapy, gene therapy, cell labeling, and the production of engineered cells.

[0018] One object of the present invention is to provide a structure suitable for photothermal processes to make cells guided to or near the structure permeable.

[0019] Another object of the present invention is to provide a structure that can be activated repeatedly by laser irradiation, thereby repeatedly increasing the permeability of cells.

[0020] According to a first aspect of the present invention, a method for increasing the permeability of a cell membrane is provided. The method includes the following steps:

[0021] - A structure is provided comprising a material and including particles capable of absorbing electromagnetic radiation. The particles have an average equivalent sphere diameter d and are embedded in the material. The structure defines a volume V and a free region surface S. The concentration of the particles in the structure is from 0.001 vol% to 20 vol% (particle volume / structure volume). At least P% of the particles present in the structure are embedded in the material such that the shortest distance L between the P% of particles and the free region surface S of the structure is from 1 nm to 500 nm; P is at least 60%.

[0022] - Guide at least one cell to or near the structure, preferably at a distance of less than 100 μm from the structure;

[0023] - Irradiate the structure with electromagnetic radiation.

[0024] When a structure is irradiated with electromagnetic radiation, particles present within the structure—that is, particles embedded in the structure—cause a photothermal effect, resulting in localized and temporary heating of the structure and its free surface region S. The photothermal effect particularly causes localized and temporary heating of the material near the irradiated particles. Therefore, the temperature of the free surface region near the particles will increase. The localized and temporary heating caused by irradiation can lead to permeability or perforation of membranes or barriers (e.g., cell membranes) in contact with or near the structure.

[0025] The temperature at the point closest to the particle on the surface S of the free region will reach, for example, a temperature at least 10°C higher than its initial temperature, or at least 20°C or 30°C higher than its initial temperature, and at least 1 nm 2 The region lasts for at least 1 ns. Clearly, the temperature rise at the point closest to the particle on the surface S of the free region can be even higher, for example, at least 50°C or at least 100°C higher than its initial temperature and lasts for at least 1 nm. 2 The region lasts for at least 1 ns.

[0026] The temperature at the point closest to the particle on the surface of the free region S reaches, for example, 60°C, which is considered the temperature at which the cell membrane becomes permeable. Therefore, cells in contact with or near this locally heated area of ​​the structure, i.e., cells in contact with or near the free region surface closest to the irradiated particle, will become permeable. Clearly, the temperature at the point closest to the particle on the surface of the free region S may be higher than 60°C and may reach, for example, higher than 100°C.

[0027] The method of this invention increases the permeability of cells guided to or near a structure without requiring direct contact between the particles and the cells. Guiding cells to a structure means guiding the cells so that the cells, or at least a portion of the cells, come into contact with the structure, i.e., with the free surface S of the structure. Guiding cells to the vicinity of a structure means guiding the cells, or at least a portion of the cells, to reach the structure at a distance of 100 μm or less, for example, at a distance of 50 μm, 20 μm, 10 μm, 5 μm, 3 μm, 2 μm, 1 μm, 0.5 μm, or 0.1 μm. This means that the (shortest) distance between the cells, or at least a portion of the cells, and the free surface S of the structure is less than 100 μm, for example, 50 μm, 20 μm, 10 μm, 5 μm, 3 μm, 2 μm, 1 μm, 0.5 μm, or 0.1 μm.

[0028] Furthermore, an advantage of the method of the present invention is that no or substantially no material of the particles present in the structure is released. Substantially no material is released means that less than 1%, preferably less than 0.5%, less than 0.1%, or less than 0.05% of the total particle mass of the particles present in the structure is released.

[0029] For the purposes of this invention, the term “volume” (V) of a structure is also referred to as the internal volume of the structure, which is defined as the total space occupied by the structure, that is, the total space occupied by the material and particles of the structure.

[0030] The term "free surface" (S) of a structure is defined as the total outer surface of the structure that surrounds its (internal) volume, i.e., the total surface of the structure that is in direct contact with the environment. When the structure is immersed in a fluid, such as a liquid (e.g., a medium containing (biological) cells, or a gas, such as air), and the structure contains a material impermeable to that fluid, the free surface of the structure can also be defined as the total surface of the structure's material that is in contact with or may be in contact with the fluid.

[0031] Preferably, the ratio of the free region surface S of the structure to the volume of the structure, i.e., the ratio S / V, is 10. -2 Up to 10 2 μm -1 For example, 10 -1 Up to 50μm -1 or 1 to 10 μm -1 .

[0032] The shortest distance L from a particle to the free region surface S of a structure is defined as the shortest distance measured from the outer surface of the particle to the free region surface S of the structure.

[0033] The average equivalent sphere diameter *d* of a particle (e.g., spherical, elongated, or irregularly shaped particles) is defined as the average diameter of a sphere having the same volume as the particle. The average equivalent sphere diameter *d* can also be called the average equivalent volume diameter of the particle. In the case where the particle contains spherical particles, the average equivalent sphere diameter obviously corresponds to the average diameter of those particles.

[0034] As described above, the concentration of particles capable of absorbing electromagnetic radiation in the structure of the present invention is from 0.001 vol% to 20 vol%. More preferably, the concentration of particles capable of absorbing electromagnetic radiation in the structure of the present invention is from 0.01 vol% to 10 vol%, or from 0.01 vol% to 5 vol%, and for example, 0.05 vol%, 0.1 vol%, 0.2 vol%, 0.5 vol%, 1 vol%, 2 vol%, or 5 vol%.

[0035] The particles present in the structure of the present invention are preferably embedded in the material such that at least 60% of the particles present in the structure have a minimum distance L of 1 nm to 500 nm from the free region surface S, for example, 2 nm to 500 nm, or 5 nm to 500 nm. More preferably, the particles are embedded in the material such that at least 70%, at least 80%, or at least 90% of the particles present in the structure have a minimum distance L of 1 nm to 500 nm from the free region surface S of the structure, for example, 2 nm to 500 nm, or 5 nm to 500 nm.

[0036] In a preferred embodiment, the embedding of particles in the material is such that at least 60% of the particles present in the structure have a minimum distance L of 1 nm to 250 nm from the free region surface S, for example, 2 nm to 250 nm or 5 nm to 250 nm. More preferably, the embedding of particles in the material is such that at least 70%, at least 80%, and at least 90% of the particles present in the structure have a minimum distance L of 1 nm to 250 nm from the free region surface S of the structure, for example, 2 nm to 250 nm or 5 nm to 250 nm.

[0037] In other embodiments, the embedding of particles in the material is such that at least 60% of the particles present in the structure have a minimum distance L of 1 nm to 100 nm from the free region surface S, for example, 2 nm to 100 nm or 5 nm to 100 nm. More preferably, the embedding of particles in the material is such that at least 70%, at least 80%, and at least 90% of the particles present in the structure have a minimum distance L of 1 nm to 100 nm from the free region surface S of the structure, for example, 2 nm to 100 nm or 5 nm to 100 nm.

[0038] Preferably, the surface density of particles with a minimum distance L from the surface S of the free region of the structure to be 1 nm to 500 nm is 10. -4 μm -2 Up to 1 / d 2 (d is the average equivalent sphere diameter of the particle, expressed in μm), for example, 2 × 10⁻⁶. -4 μm -2 up to 2μm -2 Or 2×10 -3 μm -2 Up to 0.2μm -2The surface density of particles is thus defined as the percentage P of particles N present in the structure, multiplied by the percentage of particles P at the shortest distance L from the surface of the free region S (L is 1 nm to 500 nm, for example, 5 nm to 500 nm), and then divided by the surface area of ​​the free region of the structure. The surface density of particles at distances of 1 nm to 500 nm, for example, 5 nm to 500 nm, from L can be calculated using the formula NP / S.

[0039] In a preferred embodiment, the surface density of particles with a minimum distance L from the surface S of the free region of the structure being 1 nm to 250 nm is 10. -4 μm -2 Up to 1 / d 2 For example, 2×10 -4 μm -2 up to 2μm -2 Or 2×10 -3 μm -2 Up to 0.2μm -2 .

[0040] In other preferred embodiments, the surface density of particles with a minimum distance L from the surface S of the free region of the structure of 1 nm to 100 nm is 10. -4 μm -2 Up to 1 / d 2 For example, 2×10 -4 μm -2 up to 2μm -2 Or 2×10 -3 μm -2 Up to 0.2μm -2 .

[0041] The surface density range of iron oxide particles with an average equivalent sphere diameter of 160 μm is, for example, 2 × 10⁻⁶. -4 μm -2 up to 2μm -2 1×10 -3 μm -2 Up to 0.4μm -2 Or 2×10 -3 μm -2 Up to 0.2μm -2 .

[0042] In a preferred embodiment of the structure of the present invention, all or substantially all particles are completely embedded in the material of the structure. This means that all or substantially all particles of the structure are completely surrounded by the material of the structure. Therefore, no particles or substantially no particles are exposed on the free surface of the structure.

[0043] For the purposes of this invention, the term "particle exposed to the surface of the free region" refers to all particles whose outer surface is exposed from the surface of the free region of the structure and in contact with the external environment surrounding the structure.

[0044] For the purposes of this invention, "substantially all particles" means at least 95% of the particles, preferably at least 99% of the particles, for example at least 99.9% of the particles.

[0045] Similarly, for the purposes of this invention, "substantially no particles" means less than 5% particles, preferably less than 1% particles, for example less than 0.1% particles.

[0046] The particles embedded in the structure of the present invention may include any particles capable of absorbing electromagnetic radiation and suitable for generating a photothermal effect when irradiated with electromagnetic radiation.

[0047] Particles may include microparticles, nanoparticles, or a combination of microparticles and nanoparticles.

[0048] The term "microparticle" refers to particles with an equivalent sphere diameter of 1 μm to 100 μm. The term "nanoparticle" refers to particles with an equivalent sphere diameter of 1 nm to 1000 nm.

[0049] Particles can have any shape. For example, they can be spherical, ellipsoidal, rod-shaped, pyramidal, branched, or they can have irregular shapes.

[0050] The particles can be solid particles and can have a shell structure or a core-shell structure containing one or more materials.

[0051] Preferred particles include metal particles, metal oxide particles, carbon or carbon-based particles, particles containing one or more light-absorbing compounds, or particles loaded with or functionalized with one or more light-absorbing compounds.

[0052] 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.

[0053] Examples of metal oxide particles include iron oxide, titanium oxide, zirconium oxide, cerium oxide, zinc oxide, and magnesium oxide.

[0054] Examples of carbon or carbon-based particles include graphene quantum dots, (reduced) graphene oxide, and carbon nanotubes.

[0055] Examples of particles containing one or more light-absorbing compounds, or particles loaded with or functionalized with one or more light-absorbing compounds, include particles containing, loaded with, or functionalized with synthetic organic or inorganic absorbents, and particles containing, loaded with, or functionalized with naturally occurring absorbents or derivatives thereof. Specific examples include liposomes, solid lipid nanoparticles, and polymer particles containing, loaded with, or functionalized with light-absorbing dye molecules (e.g., indocyanine green), inorganic quantum dots (with low fluorescence quantum yield), naturally occurring light absorbers such as pigments (e.g., melanin, rhodopsin, photoopin, or rhodopsin), and synthetic analogs (e.g., polydopamine), or photosensitizers used in photodynamic therapy.

[0056] The particles preferably include biocompatible particles. More preferably, the particles include or consist of clinically approved particles.

[0057] Particles may include a single particle or a combination or cluster of two or more particles located close to each other.

[0058] The structure of the present invention may include one type of particle or a combination of different particles, such as particles with different sizes, different compositions and / or different shapes.

[0059] The size of the particles, such as their width, height, or diameter, can be determined using transmission electron microscopy (TEM), scanning electron microscopy (SEM), or atomic force microscopy (AFM).

[0060] The size of the particles is preferably defined by the equivalent sphere diameter d (also known as the equivalent volume diameter).

[0061] Structural materials embedded with particles capable of absorbing electromagnetic radiation include, for example, inorganic materials or inorganic-based materials (e.g., silica or silica-based materials, ceramics or ceramic-based materials), organic materials or organic-based materials (e.g., carbon materials or carbon-based materials, polymers or polymer-based materials). Structural materials may also include composite materials comprising at least one of the above materials, such as composite materials comprising organic and inorganic materials.

[0062] Preferred structural materials include or are based on polystyrene, polycaprolactone, ethyl cellulose, 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.

[0063] In a preferred embodiment, the structure includes a surface-modifying material, such as a surface-modified polymer material. Surface modification includes, for example, applying a coating (e.g., collagen) to enhance cell adhesion to the structure material.

[0064] Structures can be continuous or discontinuous.

[0065] The structure may include porous or non-porous structures. Porous structures are preferred because they have the advantage of a large free-area surface, thus providing a large surface area that can be used to expose cells guided onto or near the structure according to the method of the invention. Preferably, the pore size of the porous structure allows cells guided onto or near the structure to partially or completely permeate into the pores. Preferably, the pore size of the porous structure does not restrict the arrival of molecules present in the cell culture medium into the cells.

[0066] The porosity of a structure is defined as the ratio of the volume of its pores or voids to the total volume occupied by the structure, where the total volume is the sum of the structure's volume V (the volume of the material and the particles embedded in the material) and the volume of its pores or voids. Porosity can range from 0% to 100%. When the structure comprises 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%.

[0067] The structure can be flat or planar, or it can be non-planar, such as tubular. The surface of the structure irradiated by electromagnetic radiation can be flat or non-planar.

[0068] The structure can have smooth or non-smooth surfaces. Non-smooth surfaces include, for example, surfaces with protrusions.

[0069] The thickness of the structure is preferably from 0.1 μm to 1000 μm, for example from 0.1 μm to 100 μm or from 1 μm to 10 μm.

[0070] The thickness of a structure is defined as the distance traversed through the structural material along its shortest dimension. For example, for a flat or planar structure, the thickness corresponds to the distance measured along a direction perpendicular to the horizontal plane. For a long tubular structure, the thickness corresponds to the radial diameter of the tubular structure.

[0071] The first set of embodiments includes a non-porous structure comprising a material and particles embedded in the material capable of absorbing electromagnetic radiation. Examples include polymer sheets or polymer foils containing particles embedded in the polymer sheets or foils capable of absorbing electromagnetic radiation.

[0072] Particularly preferred embodiments include polymer sheets comprising or based on polystyrene, polycaprolactone, ethyl cellulose, 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.

[0073] The polymer sheet contains, for example, iron oxide particles and / or carbon particles embedded in the polymer sheet.

[0074] The structure of the first group has, for example, a thickness t, a length A, and a width A'. The thickness t is preferably from 0.1 μm to 100 μm, for example from 0.1 μm to 10 μm.

[0075] The volume V of the structure corresponds to tAA'.

[0076] Since the length and width of the first group of structures are typically significantly greater than the thickness t of the structure, the free region surface of the first group of structures can be estimated to be equal to 2.A.A'. The irradiated free region surface of the structure is a surface of the structure (e.g., the top or bottom surface), and therefore can be estimated to be equal to A.A'.

[0077] Therefore, the ratio of the free region surface S of the structure to the volume V of the structure, i.e., S / V, is 1 / t.

[0078] The second set of embodiments includes a porous structure comprising a material and particles embedded in the material capable of absorbing electromagnetic radiation. Examples include porous polymer structures in which particles are embedded.

[0079] Examples of porous structures include: structures comprising fibers (e.g., polymer fibers), structures comprising particles (e.g., polymer particles), structures comprising a combination of fibers and particles (e.g., a combination of polymer fibers and / or polymer particles), and structures comprising foams (e.g., polymer foams). The fibers and / or particles may be interconnected or not interconnected. The particles, such as polymer particles, may include spherical particles as well as irregularly shaped particles. Particles capable of absorbing electromagnetic radiation are preferably embedded in the fibers, particles, or foams, preferably such that the particles are not (partially) exposed on the free surface of the structure.

[0080] A first example of the second group of structures is a structure comprising (polymer) fibers. The (polymer) fibers have a diameter of, for example, 0.1 μm to 10 μm, such as 0.5 μm or 1 μm. The (polymer) fibers may or may not be interconnected. The (polymer) fibers can be obtained by any technique known in the art. A preferred technique for manufacturing (polymer) fibers is electrospinning. Alternative techniques include wet spinning, melt spinning, extrusion spinning, dry-spray wet spinning, emulsion spinning, and suspension spinning. Preferred examples of polymers include polystyrene fibers, polycaprolactone fibers, ethyl cellulose fibers, cellulose acetate phthalate fibers, polylactic acid fibers, and polylactic acid-glycolic acid copolymer-based fibers. The (polymer) fibers may undergo surface modification.

[0081] Since (polymer) fibers can be considered as long cylinders, their diameter corresponds to the fiber diameter d. 纤维 The length corresponds to the fiber length L. 纤维 Therefore, the volume V of the fiber 纤维 yes And the free region surface S of the fiber 纤维 It is πd 纤维 L 纤维 Therefore, the free region surface S 纤维 With volume V 纤维 The ratio is 4 / d 纤维 .

[0082] A second example of the second group of structures is a structure comprising polymer particles, such as polymer (micro)spheres. The particle diameter is, for example, from 0.1 μm to 10 μm, such as 0.5 μm or 1 μm. The polymer particles may or may not be interconnected. The (polymer) particles can be obtained by any technique known in the art. Preferred examples of the particles include polystyrene, polycaprolactone, ethyl cellulose, cellulose acetate phthalate, polylactic acid, and polylactic acid-glycolic acid copolymer-based fibers. The polymer particles may undergo surface modification.

[0083] If the particulate matter has a diameter of d ms If the microsphere is a sphere, then the volume V of the microsphere is... ms yes And the free region area S of the microsphere ms yes Therefore, the free region surface S of the microsphere ms With volume V ms The ratio is 6 / d ms .

[0084] For example, cells can be directed to or near a structure by applying a suspension containing cells to or near the structure. Cells can be directed to or near the structure continuously or discontinuously.

[0085] The preferred cell concentration in the suspension is 1-10. 6 Cells / mL.

[0086] In a preferred method, a suspension, i.e., cells are cultured on or near a structure for a specific period of time.

[0087] In an alternative approach, the cells are treated as follows: the structure is activated by electromagnetic radiation immediately or shortly after the cells are guided onto or near the structure.

[0088] The structure, and particularly the particles embedded within it, is preferably irradiated with a pulsed radiation source, but irradiation with a continuous wave radiation source is also acceptable. The structure may be irradiated with one or more pulses.

[0089] When using a pulsed radiation source, the pulse duration is preferably 1 fs to 1 ms, for example 1 fs to 100 μs, 10 fs to 10 μs, 10 fs to 1 μs, or 10 fs to 10 ns.

[0090] The flux (electromagnetic energy transferred per unit area) of each pulse from the radiation source is preferably from 0.001 to 1000 J / cm². 2 For example, 0.001 to 100 J / cm 2 0.01 to 10 J / cm 2 For example, 0.1 J / cm 2 Up to 1J / cm 2 .

[0091] The wavelength range of the radiation source can range from the ultraviolet to the infrared region. In a preferred method, the radiation wavelength range used is from visible light to the near-infrared region.

[0092] Compared to methods known in the art, such as those described in EP2272945, the method of the present invention exhibits improved efficiency, such as improved transfection efficiency. While the applicant does not wish to be bound by any theory, the applicant believes that this improved efficiency is a direct result of increased contact between the cell and the structure. Due to the increased contact between the cell and the structure, a larger surface area of ​​the cell membrane becomes permeable, resulting in more and / or larger molecules being able to enter the cell. In the case where the structure comprises a porous structure, the efficiency can be further improved because the free region surface is larger and the cell can reach the free region surface of the structure from different sides.

[0093] To date, it has been believed that delivering large macromolecules into cells requires high-intensity laser pulses to generate vapor nanobubbles and induce localized pressure waves, which could make cell membranes (e.g., the cytoplasmic membrane) permeable. Surprisingly, the method of this invention allows the use of single laser pulses with much lower intensity (e.g., a flux of 0.001 J / cm²). 2 Up to 1J / cm 2 0.01 J / cm 2 Up to 0.5 J / cm 2 More preferably 0.05 J / cm 2 Up to 0.2 J / cm 2A single laser pulse makes the cell membrane (e.g., the cytoplasmic membrane) permeable. Furthermore, even when using low laser intensity, the method of the present invention allows the induction of relatively large pores and does indeed allow the delivery of relatively large macromolecules, such as macromolecules with a nominal size of 500 kDa, into the cell.

[0094] Another advantage of the method of the present invention is that it avoids the breakage or release of particles embedded in the structure. ICP-MS analysis showed no release of detectable amounts of particulate material. On the one hand, this means that cells are not exposed to the potential toxic substances of the particles; on the other hand, it means that the particles remain intact and functional after irradiation. In known photoperforation techniques, irradiated (nanoparticles) often break down after a single laser pulse. Therefore, in techniques known in the art, (nanoparticles) are often only usable once. In the method of the present invention, the structure can be used for repeated irradiation.

[0095] Another advantage of the method of the present invention is that the structure is easy to manufacture.

[0096] According to a second aspect of the invention, a structure is provided that is suitable for permeating the plasma membrane of cells, particularly cells, guided to or near the structure during a photothermal process. The structure comprises a material and particles embedded in the material capable of absorbing electromagnetic radiation. The particles have an average equivalent sphere diameter d. The structure defines a volume V and a free region surface S. The concentration of the particles present in the structure is from 0.001 vol% to 20 vol% (particle volume / structure volume), for example, from 0.01 vol% to 10 vol% or 0.01 vol% to 10 vol%. At least P% of the particles present in the structure are located at the shortest distance L from the free region surface S of the structure, where L is from 1 nm to 500 nm; P is at least 60.

[0097] This structure can include any of the above types of structures.

[0098] The structure of this invention is particularly suitable for drug screening, cell therapy, immunotherapy, gene therapy, cell labeling, and the production of engineered cells.

[0099] This structure is particularly suitable for intracellular delivery of nucleic acids, including oligonucleotides, siRNA, mRNA, or pDNA.

[0100] This structure is also suitable for intracellular delivery of nucleoproteins, including ribonucleoproteins such as Cas9 / gRNA.

[0101] Furthermore, this structure is suitable for intracellular delivery of peptides and proteins, such as nanobodies or antibodies.

[0102] Furthermore, this structure is suitable for intracellular delivery of contrast agents, such as fluorescently labeled polymers, quantum dots, iron oxide nanoparticles, and gadolinium chelates.

[0103] This structure is also suitable for intracellular delivery of plasmonic nanoparticles, for example for sensing and characterization purposes, such as LSPR sensors (localized surface plasmon resonance) or for SERS (surface enhanced Raman spectroscopy).

[0104] The structure of this invention is suitable for in vitro and ex vivo applications. It is also suitable for in vivo applications.

[0105] According to a third aspect of the invention, uses of the structure of the invention are provided, particularly in drug screening, cell therapy, immunotherapy, gene therapy, cell labeling, production of engineered cells, and protein interference research. The structure can be used for in vitro and ex vivo applications. The structure can also be used for in vivo applications.

[0106] In a preferred application, the structure is used in a method for increasing cell permeability as described above. Attached Figure Description

[0107] The invention will now be discussed in more detail with reference to the accompanying drawings, in which:

[0108] Figure 1 The method for increasing cell membrane permeability according to the present invention is illustrated schematically;

[0109] Figure 2 Confocal images are shown, which demonstrate intracellular delivery of calcein AM activity staining and red fluorescently labeled 10 kDa dextran (RD10) under a single 7 ns laser pulse with increasing flux.

[0110] Figure 3 The delivery efficiency and cell viability (calcein-positive cells) of red fluorescently labeled 10 kDa dextran (RD10) are shown when using different laser pulse doses and iron oxide nanoparticle (IONP) concentrations.

[0111] Figure 4 The confocal image shows the result of repeated optical perforation, first with red fluorescent 10 kDa dextran (RD10) and then with green fluorescent FITC dextran (FD10).

[0112] Figure 5 The flow cytometry data for repeated photoperforation with RD10 and FD10 are shown, indicating that 90% of the cells contain both RD10 and FD10.

[0113] Figure 6The delivery efficiency of FD10 in HELA cells under continuous photoperforation was shown, with its concentration doubled between each photoperforation step (N=1 to 4);

[0114] Figure 7 The mean relative average fluorescence intensity (rMFI) per cell is shown as the number of photoperforation steps increases when HELA cells are subjected to continuous photoperforation and FD10 is used.

[0115] Figure 8 The delivery efficiency of FITC-glucan molecules with different molecular weights (10, 40, 70, 150 and 500 kD) is shown with increasing number of photoperforations (N = 1, 2, 4).

[0116] Figure 9 The relative mean fluorescence intensity (rMFI) per cell for different FITC-glucan molecules is shown as the number of photoperforations increases;

[0117] Figure 10 The efficiency and viability of FD10 delivery in Jurkat cells were shown for structures containing fibers with different concentrations of IONP and irradiated with laser pulses of different doses.

[0118] Figure 11 The iron concentrations, as measured by ICP-MS, are shown in untreated cells (negative control), cells incubated with IONP (positive control), and cells treated with light-perforated structures composed of fibers containing different concentrations of IONP.

[0119] Figure 12 The iron concentrations, measured by ICP-MS, are shown in distilled water (negative control), fibers digested with aqua regia at different concentrations of IONP (positive control) (the amount of fiber digested was comparable to that in culture wells with different concentrations of IONP), and distilled water collected from the structure after photoperforation.

[0120] Figure 13 The MFI, knockdown efficiency, and cell viability of H1299 cells that stably expressed GFP and grew on a fiber substrate after N repeated light perforations (N=14) were shown. The fiber substrate contained IONP and siRNA of different concentrations of C (C=0.5-50 μM).

[0121] Figure 14 The study demonstrates intracellular delivery of FD10 in human T cells following photoperforation using different IONP concentrations and laser doses.

[0122] Figure 15Intracellular delivery of FD10 in human T cells was demonstrated under repeated light perforation (N=1 to 4);

[0123] Figure 16 The study demonstrated siRNA delivery performance in stimulated human T cells after electroporation (EP), photoporation (PEN) of the present invention, and sensitized photoporation (PP) using gold nanoparticles, exhibiting viability (…). Figure 16 a) and transfection yield ( Figure 16 b) Transfection yield is the percentage of live transfected cells obtained by multiplying the percentage of positive cells by the percentage of live cells.

[0124] Figure 17 An exemplary histogram of PD1 expression in CD3+ T cells is shown;

[0125] Figure 18 The levels of PD1 knockdown in human CD3 T cells were shown within 72 hours following delivery via electroporation (EP), photoporation (PEN) of the present invention, and gold nanoparticle-sensitized photoporation (PP).

[0126] Figure 19 The application of a structure containing nanofibers derived from polycaprolactone (PCL) and 1% IONP in knocking out the Cas-9 gene in H1299 is shown.

[0127] Figure 20 The application of a structure containing nanofibers derived from polycaprolactone (PCL) and 1% IONP in delivering macromolecules in H9 human embryonic stem cells is shown.

[0128] Figure 21a and Figure 21b Another example of a structure containing a polymer sheet that increases the permeability of the cell membrane is shown;

[0129] Figure 22 The results show the FD500 positive cells, viability, and relative mean fluorescence intensity after photoperforation of HeLa cells using polymer sheets with different concentrations of IONP.

[0130] Figure 23 The results show the percentage of FD500 positive cells, viable cells, and relative average fluorescence intensity after photoperforation of HeLa cells using polymer sheets with specific concentrations of IONP and different laser doses. Detailed Implementation

[0131] The invention will be described with reference to specific embodiments and certain accompanying drawings, but is not limited thereto; it is limited only by the claims. The drawings are illustrative only and are not restrictive. For illustrative purposes, the dimensions of some elements in the drawings may be exaggerated and not drawn to scale. Dimensions and relative dimensions do not represent actual reductions in practice of the invention.

[0132] When referring to the endpoints of a range, the endpoint values ​​of the range are included.

[0133] When describing this invention, unless otherwise stated, the terms used are to be interpreted according to the following definitions.

[0134] The terms “first,” “second,” etc., used in the specification and claims are used to distinguish similar elements and do not necessarily describe an order in time, space, sequence, or any other way. It should be understood that the terms thus used are interchangeable where appropriate, and the embodiments of the invention described herein can operate in orders other than those described or illustrated herein.

[0135] When more than two items are listed, the term "and / or" means that any one of the listed items can be used alone, or any combination of more than two listed items can be used.

[0136] The term "cell" refers to all types of biological cells, including eukaryotic cells and prokaryotic cells.

[0137] The terms “increase permeability,” “permeable,” “become permeable,” and “permeable” refer to any method that alters the permeability of a membrane or barrier (e.g., the plasma membrane of a cell) at least partially or locally. After permeability is achieved, the alteration of the membrane or barrier (e.g., the plasma membrane of a cell) makes it more permeable to one or more types of compounds (e.g., molecules, macromolecules, particles, or nanoparticles).

[0138] The terms “perforation,” “pore opening,” or “perforation action” refer to any method of providing one or more openings, pores, or holes in a membrane or barrier, such as the plasma membrane of a cell. By perforating a membrane or barrier, creating openings in the membrane or barrier, such as the plasma membrane of a cell, compounds (such as molecules, macromolecules, particles, or nanoparticles) are allowed to pass through the membrane or barrier, for example, through the plasma membrane of a cell.

[0139] For the purposes of this invention, the terms “increased permeability,” “permeable,” “becomes permeable,” and “permeable,” as well as the terms “perforation,” “opening,” or “perforation effect,” are used interchangeably.

[0140] Similarly, for the purposes of this invention, the terms “opening,” “hole,” or “cavity” may be used interchangeably.

[0141] Example 1 comprises a porous structure containing nanofiber meshes and particles embedded in the nanofibers.

[0142] A second embodiment of the structure of the present invention includes a porous structure comprising nanofibers and particles embedded within the nanofibers capable of absorbing electromagnetic radiation. The examples described below include polycaprolactone as the structural material and iron oxide nanoparticles as the particles capable of absorbing electromagnetic radiation. Clearly, other materials and other particles are also contemplated.

[0143] 1.a Synthesis and Characterization of Photothermal Electrospun Nanofibers

[0144] The following materials are used to synthesize nanofiber webs:

[0145] • Polycaprolactone (PCL, Mw≈70,000 g / mol);

[0146] ·N,N-Dimethylformamide (DMF);

[0147] Tetrahydrofuran (THF);

[0148] • Iron oxide (Fe3O4) nanopowder (IONP) (#MKBW3262, Sigma-Aldrich, Belgium);

[0149] • Poly(allylamine hydrochloride) (PAH, Mw = 17,560 g / mol, #MKBZ2824V, Sigma-Aldrich, Belgium);

[0150] • Concentrated sulfuric acid solution (96%) (Sigma-Aldrich);

[0151] • Collagen I rat protein (Thermo Fisher Scientific, #A1048301, Gibco) TM ,Belgium).

[0152] IONP was redispersed in a 1:1 DMF / THF solution containing varying concentrations of PCL, ranging from 0% to 1.15% by volume.

[0153] Nanofibers were fabricated using the resulting mixture via electrospinning. The nanofibers were collected on a microscope slide (#1000912, Marienfeld, Germany) mounted on a grounded rotating collector.

[0154] During electrospinning, unless otherwise specified, the applied voltage, flow rate, and electrospinning distance are fixed at 10 kV, 0.3 ml / h, and 20 cm, respectively. The rotation speed of the grounded rotating collector is set to 500 rpm. After 30 minutes (or a specially specified time), the electrospinning process is stopped, the glass slide with the nanofiber mesh is separated from the rotating collector, and sterilized by ultraviolet irradiation in a laminar flow cabinet for 45 minutes.

[0155] The size and diameter of the nanofibers were determined using scanning electron microscopy. The average diameter of the fibers without IONP was 300 nm. When up to 1.15 vol% IONP was included, the average diameter did not change significantly.

[0156] The thickness of the structure was studied using confocal microscopy. The structure gradually thickened with increasing electrospinning duration, reaching 4 μm after 1 hour. Since the web did not change significantly after 30 minutes, a 30-minute electrospinning time was chosen.

[0157] When an increased amount of IONP was used on the nanofibers, the thickness of the nanofiber web did not change significantly. This clearly demonstrates that the thickness of the nanofiber web is independent of the IONP content within the range used.

[0158] IONPs are embedded in nanofibers. This can be clearly seen using SEM with a voltage of 20 kV. SEM images show that IONPs can exist as individual particles or as clusters of two or more individual particles. For simplicity, the embedded IONPs are referred to as “IONP clusters” or “clusters”, and it should be understood that the term “IONP cluster” or “cluster” includes both individual particles and clustered particles. SEM allows for quantitative analysis of each 1000 μm in SEM images. 2 Apparent density of IONP clusters across the entire network over area. As the IONP content increases from 0.0046 vol% to 1.15 vol%, the density increases from 1.7 clusters / 1000 μm. 2 The number of clusters increased linearly to 192 per 1000 μm. 2 .

[0159] 1.b Preparation of nanofiber mesh as a cell culture substrate

[0160] In the laminar flow cabinet, use the 8-hole Secure-Seal TM Double-sided adhesive pads (#S24737, Invitrogen) were sterilized by UV irradiation for 45 minutes. After removing the protective sealing layer from one side of the adhesive pads, they were gently adhered to the nanofiber mesh. Next, the samples were immersed in distilled water for 3 minutes to allow for easy removal of the mesh from the slide (with the adhesive pads on top). The mesh was manually cut into small pieces, each with 1 or 4 adhesive wells (in which cells can grow) and stored in PBS buffer.

[0161] Next, these cell culture substrates were further modified with collagen to obtain optimal cell adhesion. The cell culture substrates were immersed in 32% sulfuric acid solution (3 ml per well in a 6-well plate) for 3 minutes. After washing with distilled water, they were immersed in an aqueous solution of polyelectrolyte PAH (2 mg / ml, 0.5 M NaCl) for 15 minutes, and then rinsed three times with distilled water. PAH physically adsorbed onto the nanofiber surface, giving the nanofibers a positive charge. Next, the PAH-coated nanofibers were immersed in an aqueous solution of 0.5 mg / ml collagen I rat tail protein for 15 minutes, and then rinsed with PBS solution. Finally, the modified substrates were stored in PBS for further use.

[0162] 1.c. Culture or collect cells in a cell culture substrate for photoperforation treatment.

[0163] HeLa cells (#CCL-2) and Jurkat clone E6.1 (#TIB-152) were obtained from ATCC (American Type Culture Collection) and used as models for transfecting adherent and suspension cells, respectively, via light perforation. Human lung epithelial cells (H1299) stably expressing enhanced green fluorescent protein (eGFP) were used to validate siRNA knockdown experiments. HeLa cell culture medium was prepared using DMEM / F-12 containing 2 mM glutamine, 100 U / mL penicillin / streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). H1299 and Jurkat cell culture media consisted of RPMI 1640 containing 2 mM glutamine, 100 U / mL penicillin / streptomycin, and 10% FBS.

[0164] To promote adherent cell growth, place the cell culture medium in a 6-well titration plate (#10062-892, VWR) and add HeLa or H1299 (approximately 1×10⁻⁶ per 2 ml of cell culture medium). 6 (cells). Cells were allowed to attach and grow over 24 hours in a cell culture incubator at 37°C under a humidified atmosphere of 5% CO2. Target molecules to be delivered into the cells were added to the cell culture medium just before the photoperforation treatment.

[0165] Jurkat cells at 75 cm 2 Or 175cm 2 Flasks (#734-2313, #734-2315) ) in 1×10 5 Up to 1×10 6 Cells were cultured at a cell density of approximately 2 × 10⁶ cells / ml. For photoperforation, the target molecule was added to the cell culture medium at a concentration of approximately 2 × 10⁶ cells / ml. 5Cells are transferred to the cell substrate at individual cells / wells. Cells are allowed to settle on the fiber mesh for 5 minutes before starting the photoperforation laser scan.

[0166] The final experiment was conducted using human T cells obtained from Ghent University Hospital. Erythrocyte sedimentation rate (ESR) and tannin levels were obtained from healthy donors. Peripheral blood mononuclear cells (PBMCs) were separated by density centrifugation using Lymphoprep (Alere Technologies, Oslo, Norway). Next, the PBMCs were incubated in IMDM (Gibco, Invitrogen, Merelbeke, Belgium) supplemented with 10% fetal bovine serum (FCS, Bovogen), 100 U / ml penicillin (Gibco, Invitrogen), 100 μg / ml streptomycin (Gibco, Invitrogen), 2 mM glutamine, and 5 ng / ml IL-2 (Roche, Vilvoorde, Belgium), and stimulated with CD23 / CD28 beads (Stemcell Technologies, Vancouver, Canada) at a 1:1 bead-cell ratio. Seven days later, cells were harvested and re-incubated in complete IMDM supplemented with 1 μg / ml phytohemagglutinin (Remel Europe, KENT, UK) along with X-ray irradiated (40 Gy) (SARRP) PBMCs (1:2 ratio) and X-ray irradiated (50 Gy) JY cells (5:1 ratio). After another 14 days, CD3+ cells were harvested for further illustrative experiments. Feeder cells were irradiated using a small animal radiation research platform (Xstrahl, Surrey, UK). For photoperforation treatment, T cells were introduced at approximately 8 × 10⁸ cells per minute, provided transfection molecules were already present. 5 Transfer cells at a density of 1 cell / well to the culture medium substrate. Allow the cells to settle on the fiber mesh for 5 minutes before starting laser treatment.

[0167] 1. Light perforation of adherent cells

[0168] Figure 1 The method of the present invention is illustrated schematically. First, a structure comprising a material and particles capable of absorbing electromagnetic radiation is provided. Figure 1 a.). This structure is synthesized, for example, as described above. Subsequently, cells are grown on the structure, for example, as described above. Figure 1b). Photoporation of cells using a previously reported custom optical device with some minor modifications (RHXiong et al., Comparison of Gold Nanoparticle Mediated Photoporation: Vapor Nanobubbles Outperform Direct Heating for Delivering Macromolecules in Live Cells, Acs Nano, 8(2014)6288-6296) Figure 1 c.) In short, a pulsed laser with a pulse duration of 7 ns was tuned to a wavelength of 647 nm (Opolette™ HE 355LD, OPOTEK Inc, CA) and used to irradiate the structure containing nanofibers and IONPs. The collimated pulsed laser beam was guided by a 1° light-shaping diffuser (Physical Optics Corporation, Torrance, CA), which, in combination with an achromatic lens in front of the microscope entrance and a 10X objective lens (Plan Fluor, Nikon), produced a laser beam diameter of approximately 250 μm at the sample. The laser pulse energy was monitored by an energy meter (J-25MB-HE&LE, Coherent) synchronized with the pulsed laser. To scan all cells (approximately 9 mm in diameter) on the structure containing nanofibers and IONPs of the present invention, the sample was scanned using a motorized microscope stage with a fixed laser beam. Since the laser repetition frequency was 20 Hz, the scanning speed was set to 3 mm / s, and the distance between subsequent lines was 0.15 mm. This ensured that all cells received at least one, and at most four, laser pulses in the overlapping areas between adjacent irradiated regions. In some experiments using Jurkat or human T cells, the cells are scanned multiple times, as shown in the text. In this case, the cells are resuspended in the wells and allowed to settle again between scans, allowing the cells to randomly attach to the nanofibers at new locations. Transfected cells are shown... Figure 1 As shown in d.

[0169] 1.e. Delivery of molecules into cells via photoporation

[0170] To evaluate intracellular delivery via photoperforation using the structure of the present invention, 10 kDa (RD10) red fluorescently labeled dextran was added to HeLa cells cultured in a structure containing nanofibers and 0.23 vol% IONP. The cells were scanned using a 7 ns pulsed laser beam (λ = 647 nm) as described above. After laser treatment, the cells were washed and calcein AM activity staining agent was added to the cells. Figure 2 Exemplary confocal images are shown using different laser flux amounts. Figure 2Image a shows a confocal image of green fluorescence from calcein AM activity staining, indicating cytotoxicity at only 0.12 J / cm². 2 This becomes more apparent at the highest laser flux. Figure 2 b shows a confocal image of red fluorescence from RD10, indicating that intracellular delivery of RD10 increases with increasing laser flux.

[0171] For structures prepared with different laser injection doses and different IONP concentrations, intracellular delivery and cell viability of RD10 were systematically evaluated using confocal microscopy. Figure 3 Delivery efficiency was quantified as the percentage of RD10-positive cells, while viability was expressed as the percentage of calcein-positive cells. As expected, in the absence of laser irradiation (0 J / cm²), delivery efficiency was [data missing]. 2 In the case of laser irradiation, no significant RD10 uptake occurred in HeLa. RD10 was successfully delivered into cells after laser irradiation, to varying degrees depending on the applied laser flux and IONP concentration. Increasing the laser flux or IONP concentration generally resulted in greater intracellular delivery, but cytotoxicity also gradually increased. Interestingly, several combinations of laser flux and IONP concentration were found to achieve optimal delivery efficiency. For example, for the structure with the lowest IONP concentration of 0.023% (corresponding to 3.6 IONP / cell), 0.56 J / cm² was optimal. 2 The laser dose produced >85% positive cells with a cell viability of approximately 87%. Results were almost identical using a structure containing 0.23% IONP (43 IONP / cell), but with a laser dose of 0.08 J / cm². 2 It was almost 7 times lower.

[0172] 1.f Repeated activation of the structures used for transfecting cells

[0173] Known nanoparticle-sensitized photoperforation methods in the art utilize nanoparticles, such as gold nanoparticles, which can only be activated once because they tend to fragment after the first laser pulse, resulting in the loss of their photothermal function. However, to further improve delivery efficiency, multiple irradiation cycles of the structure of the present invention were evaluated.

[0174] Cells on the structure comprising nanofibers and IONP of the present invention were irradiated twice. RD10 was delivered in the first round, as previously described. The cells were then washed. Figure 4 Image a shows the confocal image after the first round of irradiation. Cells of the same structure were then irradiated a second time in the presence of 10 kDa green fluorescent FITC-dextran macromolecules (FD10). The confocal image after the second round is shown below. Figure 4 As shown in b. Figure 4 a and Figure 4The superposition of b is displayed Figure 4 In c, it is shown that many cells simultaneously exhibit green and red fluorescence.

[0175] Figure 5 The quantitative analysis performed by flow cytometry presented in the paper confirmed that 90% of the cells were positive for RD10 and FD10.

[0176] To provide further evidence for repeated photoperfusion using the same structure, HELA cells were photoperfused up to four times with FD10. The FD10 concentration was doubled between each round of photoperfusion (from 0.2 mg / ml to 1.6 mg / ml) to more easily observe the increase in intracellular delivery (this is diffusion-driven and therefore requires a concentration gradient). The percentage of positive cells after each photoperfusion was [not specified in the original text]. Figure 6 The relative average fluorescence intensity per cell after each photoperforation is given in [the figure]. Figure 7 The percentage of positive cells increased from approximately 70% to approximately 90%. Figure 6 The increased delivery is quite evident from the almost linear increase in relative average fluorescence (rMFI) per cell with each additional round of light perforation. Figure 8 ).

[0177] 1.g macromolecules delivered into cells via photoporation

[0178] To evaluate the intracellular delivery of larger macromolecules (i.e., molecules with the molecular weight of proteins or mRNA), FITC-glucan molecules of 40 kDa, 70 kDa, 150 kDa, and 500 kDa (FD40, FD70, FD150, and FD500) were delivered to HeLa cells via 1x, 2x, and 4x light perforation. Uptake was determined by flow cytometry and expressed as the percentage of positive cells. Figure 8 ) and rMFI( Figure 9 ).

[0179] like Figure 8 and Figure 9 As shown, delivery efficiency gradually decreases with increasing molecular weight, due to a combination of the molecules becoming larger relative to the pore size and slower molecular diffusion. Repeated photoporation procedures typically produce slightly more positive cells, but they do not improve the average delivery rate per cell.

[0180] from Figure 8 and Figure 9 It can be concluded that the method of the present invention successfully transfected cells with compounds up to at least 500 kDa, and the percentage of transfected cells was 65% to 90%, depending on the molecular size.

[0181] 1.h Transfection of suspension cells via light perforation

[0182] To investigate the success of the method of the present invention in transfecting suspension cells, Jurkat cells (an immortalized cell line of human T lymphocytes, a widely used model of difficult-to-transfect primary human T cells) were used. Before adding the cells to the structure containing nanofibers and IONPs, 2 mg / ml FD10 was added to the Jurkat cell suspension. The cells were allowed to settle for 5 minutes, sufficient to collect them on top of the fiber web. They were then photoperforated by laser beam scanning in exactly the same manner as adherent cells. The number of available IONP clusters per cell was quantified by multiplying the Jurkat cell area by the IONP density, which in this case ranged from 7.7 to 28.4 IONPs / cell.

[0183] Next, the transfection efficiency as a function of laser flux and IONP content was investigated. For example... Figure 10 a, Figure 10 As shown in b and 10c, delivery efficiency increases with increasing laser flux, but at the cost of cell viability, as measured by calcein red-orange AM viability staining. Similarly, for a given laser flux, delivery efficiency generally increases with increasing IONP content. A minimum 80% viability threshold was set using a structure containing nanofibers and 0.46 volume % IONP (approximately 12 IONP / cell) and 0.16 J / cm². 2 The optimal laser injection rate yielded the best transfection efficiency (approximately 75% positive cells). Finally, repeated photoperfusion was tested. Figure 10 d) It was found again that repeating this process could increase the percentage of positive cells, with almost no effect on cell viability. Note that for this experiment, a structure containing nanofibers and 0.46 vol% IONP was used, along with 0.08 J / cm². 2 A suboptimal laser flux is used to better visualize the gradual increase. Between subsequent laser scans, the cells are gently resuspended and re-sedimented, allowing them to randomly attach to nanofibers in new locations.

[0184] 1.i. ICP-MS measurements were performed to detect potential leakage of IONP from structures containing nanofibers and IONP under laser irradiation.

[0185] To assess whether there is direct contact between cells and particles embedded in structural materials that can absorb electromagnetic radiation, the iron content of cells after photoperforation was measured by ICP-MS (inductively coupled plasma mass spectrometry).

[0186] Structures containing nanofibers and IONPs were irradiated with and without cells on the fibers. In the absence of cells, distilled water was added to the structures containing nanofibers and particles. The distilled water was collected again after laser treatment for ICP-MS analysis. Samples with cells were prepared as described above. After laser irradiation, cells were collected by washing with PBS in the case of suspended cells, and by trypsin treatment in the case of adherent cells. Finally, 100 μl of aqua regia (a 3:1 mixture of hydrochloric acid and nitric acid) was added to the sample to digest any cells or other organic matter that might be present. Next, the iron content was measured by ICP-MS (Agilent 8800, Santa Clara, CA, USA). Specifically, the sample solution was diluted 100-fold with 2% HNO3 to a final volume of 10 mL in a metal-free tube, and Y was added as an internal standard (final concentration 1 μg / L). -1 This is to correct for instrument instability and / or signal drift. External calibration standards (0, 0.5, 1, 2.5, 5, and 10 μg / L) are used. -1 Fe+1μgL -1 Y) from 1,000 mg / L -1 The Fe standard stock solution was prepared by diluting an appropriate amount with a slightly acidic solution (2% HNO3) to simulate the matrix of the sample solution. Throughout the sample preparation process, the solution was thoroughly mixed using a vortex mixer.

[0187] Internal standard correction is performed according to the following equation:

[0188]

[0189] Where R Fe，corr It has been corrected. 56 Fe(NH3)2 + Signal response, R Fe It is a measurement 56 Fe(NH3)2 + Signal response, R Y yes 89 Y(NH3)6 + Signal response. For all calculation steps (internal normalization and external calibration), the relative standard deviation is calculated via error propagation. The background equivalent concentration (BEC) is calculated instead of the limit of detection / limit of quantitation (LOD / LOQ) because BEC is a more representative measure of analytical performance, while the background concentration of Fe is typically slightly elevated.

[0190] As described above, HeLa and Jurkat cells were photoperforated using structures containing nanofibers and 0.23 vol% or 0.46 vol% IONP, respectively. As a positive control, cells were also incubated at 37°C for 4 hours with 500 μg / ml of 30 nm IONP coated with polyethylene glycol. Figure 11 As shown, for both cell types, the positive control did indeed have a significantly higher iron concentration compared to the negative control (untreated cells). However, it is important that the laser flux (0.08 to 0.16 J / cm²) was constant for any test. 2 The number of laser scans (maximum N=4) showed no significant difference in iron content between the photoperforated cells and the untreated cells. While this demonstrates that there was no measurable increase in iron content in the cells, it also suggests that the endogenous iron content in the cells was already quite high, making it difficult to detect small increases. Therefore, the potential iron release from the structure of the present invention was evaluated when immersed in pure distilled water and irradiated with a laser (in the absence of any cells). Figure 12 The results showed that after laser activation of the structure containing nanofibers and IONP, the iron content in distilled water did not increase significantly and remained below the instrument's detection sensitivity of 0.082 mg / L. This was true not only for the structure containing nanofibers and 0.23 vol% IONP, but also for the structure with the highest IONP content of 1.15 vol%, even after multiple laser activation cycles (maximum N=4) using a flux of up to 0.16 J / cm². As a positive control, a similar amount of fiber present in the nanofiber-containing structure was digested with aqua regia, which should have released all the IONP. In this case, ICP-MS did indeed detect a very high iron concentration proportional to the embedded IONP content (0.23 vol%, 0.46 vol%, or 1.15 vol%). It can be concluded that the structure of the present invention achieves the desired goal of efficient cell transfection after laser activation while avoiding any direct exposure of cells to potentially toxic sensitized nanoparticles or their components.

[0191] 1.j Efficient gene silencing in adherent cells via light perforation

[0192] To evaluate the intracellular delivery of siRNA as a functional macromolecule, anti-eGFP siRNA was delivered to adherent H1299 cells stably expressing green fluorescent protein (GFP). Cells were grown at 37°C for 24 h on a collagen-coated nanofiber mesh with 0.23% IONP, and then photoperforated (0.08 J / cm²) under both control and anti-GFP siRNA conditions. 2The cells were allowed to continue growing for 24 hours before GFP expression was measured. Confocal microscopy of the experimental experiments with 5 μM siRNA showed that GFP was significantly downregulated when treated with anti-GFP siRNA instead of control siRNA. Flow cytometry confirmed these results: 77% of GFP-positive cells were treated with control siRNA, which decreased to 28% after treatment with functional siRNA. Knockdown efficiency and cytotoxicity as a function of siRNA concentration (0.5, 1, 2, and 5 μM) were systematically evaluated. Higher siRNA concentrations resulted in decreased eGFP expression, with 75% of cells showing significant gene silencing at 5 μM siRNA. Figure 13 a, 13b). The question of whether repeated photoperforation also facilitated siRNA gene silencing was assessed. Indeed, with up to four repeated laser scans, eGFP expression gradually decreased with each scan, reaching a maximum knockdown efficiency of 75% after four repeated laser irradiations. Cell viability, as measured by the cell titer-Glo luminescence assay, remained very good (>75%) for all conditions. Figure 13 b).

[0193] 12k performs efficient gene silencing in primary human T cells via light perforation.

[0194] The photoperforation of human patient-derived CD3+ T cells on the nanofiber- and IONP-containing structures of the present invention was evaluated. Structures with IONP contents of 0.23 vol%, 1.15 vol%, and 2.3 vol% were prepared and photoperforated using 0.16 J / cm². 2 Fixed laser pulse transfection of T cells was performed (as this is optimal for Jurkats). Optimal transfection efficiency (approximately 30% positive cells) was obtained using 1.15% (v / v) IONP. Figure 14 Next, we optimized the laser flux and confirmed that the transfection efficiency was 0.16 J / cm². 2 The timing is optimal. Interestingly, increasing the laser flux to 0.32 J / cm² is also effective. 2 It did not further increase transfection efficiency as predicted by our theoretical simulations. Similar to Jurkats, repeated light perforation did increase the percentage of FD10-positive cells ( Figure 15 For example, with three light pores, a transfection efficiency of 53% and cell viability of >60% were achieved. Based on these results, we selected I = 0.16 J / cm². 2 Further experiments were conducted on human T cells using 1.15% by volume of IONP neutral nanofibers and N=3.

[0195] The siRNA delivery performance via photoporation in stimulated human T cells was tested using fluorescently labeled model siRNA (without biological function). Direct comparisons were made with two other established physical transfection techniques: electroporation and conventional gold nanoparticle-sensitized photoporation. Figure 16 In this study, electroporation is referred to as EP, photoporation is referred to as PEN, and gold nanoparticle-sensitized photoporation is referred to as PP. As is often observed in electroporation, only a small percentage of cells survive after treatment (14.2%). Figure 16 b), however, almost all of them were siRNA positive (94.2%). Figure 16 a). The product of these two measurements is the so-called transfection yield, which is the percentage of viable transfected cells, only 13.5% for electroporation. Gold nanoparticle-sensitized photoporation and photoporation using the structure of the present invention are much gentler on cells, with cell viability >60% and 40-50% positive cells. This results in a transfection yield of 35% for photoporation using the structure of the present invention, compared to 30% for gold nanoparticle-sensitized photoporation. Figure 16 (b) Therefore, it can be concluded that the transfection yield of photoperfusion according to the present invention is more than 2.5 times that of electroporation, while being similar to that of conventional photoperfusion. This is a remarkable achievement because, according to the present invention, this is achieved without direct contact between particles and cells.

[0196] To assess gene silencing with functional siRNA, the PD-1 receptor was targeted. On day 1, T cells were collected from donors and subjected to the first stimulation. After 7 days, cells were collected for siRNA transfection and subjected to a second stimulation. Cells were transfected with 1 μM siPD1, and PD1 expression was quantified by flow cytometry after PD-1 antibody staining at 24, 48, and 72 hours. Transfection was again compared between electroporation, photoporation, and gold nanoparticle-sensitized photoporation. Exemplary flow cytometry histograms are shown. Figure 17 In the study, k was used on cells 48 hours after photoperforation under control siRNA and siPD1, showing decreased PD1 expression in the latter case. The reduction in PD-1 antibody staining across the entire viable cell population quantified the knockdown efficiency over time. Figure 18 All three transfection methods achieved similar levels of PD-1 gene silencing, reaching up to approximately 40% knockdown after 48 hours. Note that photoporation yielded 2.5 times higher transfection productivity than electroporation due to reduced toxicity, confirming it as a very promising and efficient transfection method for producing engineered T cells for adoptive T-cell therapy.

[0197] Figure 19The application of a structure containing nanofibers derived from polycaprolactone (PCL) and 1% IONP in Cas-9 gene knockout in H1299 is shown.

[0198] Figure 19 Image a shows a confocal image displaying green fluorescence in H1299 cells stably expressing GFP before (left) and after (right) photoperforation with 4 μM Cas-9 ribonucleoprotein (designed to knock out GFP expression). Using 0.08 J / cm² 2 The laser flux is used to scan the sample once.

[0199] Figure 19 b shows the corresponding cell count histogram, illustrating how eGFP expression was distributed in the cell population before and after photoperforation, with 90.5% and 33.5% of the cells being eGFP-positive, respectively. Figure 19 c and Figure 19 d shows the mean fluorescence intensity (MFI) and knockdown efficiency (the percentage of eGFP-negative cells) of H1299 cells photoperforated with increasing concentrations of Cas-9 ribonucleoprotein (0.5, 1, 2, 4 μM) and with multiple treatments at a concentration of 0.5 μM (N = 2, 3, 4).

[0200] Figure 20 The application of a structure containing nanofibers derived from polycaprolactone (PCL) and 1% IONP in delivering macromolecules in H9 human embryonic stem cells is shown.

[0201] Figure 20 Image a shows confocal images demonstrating successful delivery of fluorescently labeled 10 kDa dextran (RD10) before light perforation (top row), after one light perforation cycle (second row), and after two light perforation cycles (bottom row). Live cells were stained with calcein AM, while dead cells were identified by a positive propidium iodide (PI) signal. 0.08 J / cm² was used. 2 Laser injection is used for optical perforation.

[0202] Figure 20 b shows the laser flux (I = 0.08, 0.12, and 0.24 J / cm²). 2 Cell viability and percentage of RD-positive cells were quantified by imaging processing using functions of multiple photoperforation cycles (N=2, 3, 4) and multiple photoperforation cycles (N=2, 3, 4).

[0203] Example 2: Non-porous structure comprising polymer materials and nanoparticles

[0204] Figure 21a A schematic diagram showing an embodiment of the structure 1 of the present invention is displayed. Figure 21b Showing Figure 21a The structure 1 shown is a cross-section along line A-A'. Structure 1 includes a polymer sheet containing a polymer material 2 and particles 3 capable of absorbing electromagnetic radiation. Particles 3 include, for example, carbon particles, iron oxide particles, or a combination of carbon particles and iron oxide particles. Particles 3 are embedded in the material 2 and have, for example, an average equivalent sphere diameter d of 1000 nm.

[0205] The structure has a thickness t ranging from 0.1 μm to 100 μm, for example, 1 μm, 2 μm or 5 μm.

[0206] The ratio of the surface area S of the free region of the structure to the volume V of the structure, i.e., the ratio S / V, is 1 / t.

[0207] The polymer sheet preferably comprises a polymer that includes or is based on polystyrene, polycaprolactone, ethyl cellulose, cellulose acetate phthalate or polylactic acid-glycolic acid copolymer, cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginate, hyaluronic acid, dextran, starch, polycarbonate or polyacrylate.

[0208] The concentration of particles in the material ranges from 0.001 vol% to 20 vol% (particle volume / structure volume), for example, 1 vol%, 2 vol%, or 5 vol%.

[0209] Preferably, all or substantially all of the particles are completely embedded in the material of the structure, which means that all or substantially all of the particles of the structure are completely surrounded by the material of the structure and no particles or substantially no particles are exposed on the free surface of the structure.

[0210] At least 60% of the particles present in the structure are embedded in the material such that the shortest distance L between the particle and the free region surface S of the structure is 1 nm to 100 nm.

[0211] Figure 22 The results show the percentage of FD 500 (FICT-500kDa dextran) positive cells, cell viability, and relative mean fluorescence intensity as measured by CellTiter Glo metabolic assay for HeLa cells using photoperforation with PLA membranes containing no IONP (control), 0.005% IONP, 0.01% IONP, and 0.1% IONP.

[0212] Furthermore, for the use of PLA films with 0.025% IONP (2% PLA) and different laser fluxes (0.3 J / cm², respectively)... 2 (=E1), 0.5J / cm 2 (=E2), 0.84J / cm2 (=E3), 1.26J / cm 2 (=E4) and 1.6J / cm 2 Photoperforation (one photoperforation cycle) of HeLa cells (=E5) determined the percentage, viability, and relative mean fluorescence intensity (rMFI) of FD500 positive cells. Results showed... Figure 23 middle.

Claims

1. A method for increasing the permeability of the cell membrane to facilitate intracellular delivery of cell-impermeable substances, the method comprising the steps of: A structure is provided comprising a material and including particles embedded in the material capable of absorbing electromagnetic radiation, the particles having an average equivalent sphere diameter d, the structure defining a volume V and a free region surface S, the concentration of the particles present in the structure being from 0.01 vol% to 10 vol% in terms of particle volume / structure volume, at least P% of the particles present in the structure being embedded in the material such that the shortest distance L between the P% of particles and the free region surface S of the structure is from 1 nm to 500 nm; P is at least 60%, wherein at least 95% of the particles are not exposed on the free region surface of the structure. Guide at least one cell to the structure or to a distance of less than 100 μm from the structure; The structure is irradiated with electromagnetic radiation.

2. The method as described in claim 1, wherein, The concentration of the particles in the structure is from 0.05% to 5% by volume.

3. The method as described in claim 1 or 2, wherein, The surface density of particles with a minimum distance L from the surface S of the free region of the structure of 5 nm to 500 nm is 10. -4 μm -2 Up to 1 / d 2 The surface density of particles is defined as: the number of particles N present in the structure multiplied by the percentage P of the particles located at the shortest distance L from the surface of the free region, and then divided by the surface area of ​​the free region of the structure (NP / S).

4. The method as described in claim 1 or 2, wherein, At least 99% of the particles are not exposed on the free area surface of the structure.

5. The method as described in claim 1 or 2, 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 loaded with or functionalized with one or more light-absorbing compounds.

6. The method as described in claim 1 or 2, wherein, The materials include: inorganic materials or inorganic-based materials, ceramics or ceramic-based materials, organic materials or organic-based materials, or composite materials containing at least one of these materials.

7. The method as described in claim 1 or 2, wherein, The material includes materials selected from or surface-modified with materials selected from the following: polystyrene, polycaprolactone, ethyl cellulose, cellulose acetate phthalate, polylactic acid, polylactic acid-glycolic acid copolymer, cellulose, polyvinyl alcohol, polyethylene glycol, gelatin, collagen, silk, alginate, hyaluronic acid, dextran, starch, polycarbonate, and polyacrylate.

8. The method as described in claim 1 or 2, wherein, The structure includes a porous structure or a non-porous structure.

9. The method as described in claim 1 or 2, wherein, The structure includes a porous structure with a porosity of at least 50%.

10. The method of claim 9, wherein, The porous structure includes fibers, particles, a combination of fibers and particles, or foam, wherein the particles are embedded in the fibers, the particles, or the foam.

11. The method as described in claim 1 or 2, wherein, The irradiation includes irradiation with a pulsed radiation source, the pulse duration of which is from 1 fs to 1 μs and / or the flux of each pulse is from 0.001 to 10 J / cm. 2 .

12. A structure adapted to permeate cells guided to or near the structure during a photothermal process for intracellular delivery of cell-impermeable substances, the structure comprising a material and particles embedded in the material capable of absorbing electromagnetic radiation, the particles having an average equivalent sphere diameter d, the structure defining a volume V and a free region surface S, the concentration of the particles present in the structure being from 0.01 vol% to 10 vol% in a particle volume / structure volume ratio, at least P% of the particles present in the structure being embedded in the material such that the shortest distance L between the P% of particles and the free region surface S of the structure is from 1 nm to 500 nm; P is at least 60%; in, At least 95% of the particles are not exposed on the free area surface of the structure.

13. Use of the structure of claim 12 in drug screening, cell labeling, production of engineered cells, and protein interference research.

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