Intracellular delivery by 2d cell flattening (ID2D)

Abstract

The present invention relates to a method for the introduction of macromolecules and / or particles into living cells comprising (a) flattening the living cells between two surfaces such that the cells become amenable for the uptake of the macromolecules and / or particles, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and (b) incubating the fattened cells, wherein the macromolecules and / or particles become introduced into the cells.

Description

[0001] Intracellular Delivery by 2D Cell Flattening (ID2D)

[0002] The present invention relates to a method for the introduction of macromolecules into living cells comprising (a) flattening the living cells between two surfaces such that the cells become amenable for the uptake of the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and (b) incubating the fattened cells, wherein the macromolecules become introduced into the cells.

[0003] In this specification, a number of documents are cited. The disclosure of these documents, while not considered relevant for the patentability of this invention is herewith incorporated by reference in its entirety. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

[0004] Intracellular delivery as described herein refers to delivering of external substances inside a living cell. Such delivery has a wide range of application potential in cell-based therapy, gene editing, regenerative medicine and fundamental biology. Particles, molecules or macromolecules could be subjected of the delivery depending on the desired application or study. This task is mostly achieved in the prior art either by carrier-mediated delivery [1] or membrane- disruption-based systems [2], Carrier-based approaches use the biochemical assemblies, in the nano-scale range, that can enter inside the cell by endocytosis. Membrane-disruption is a mechanical approach in which the shock to the cell membrane (plasma membrane) increases the permeability of it in order to introduce a wide range of material in-vitro or ex-vivo. In the case of membrane-disruption, the plasma membrane of the cell is perturbed in a physical manner by applying mechanical or thermal shocks, electromagnetic radiation and electric fields. Nevertheless, the permeability can be caused by adding chemical agents such as peptides, detergents and pore-forming toxins [3],

[0005] A comprehensive overview of intracellular delivery methods can be found in the prior art [4,5], The ID2D method fits in the category of intracellular delivery by applying mechanical shocks. The most relevant available method is the cell squeezing approach [6], It was proven that squeezing a cell inside a microfluidic channel result in sufficient membrane disruption to achieve intracellular delivery for a wide range of macromolecules [7,8], Another similar approach is the cell poking method that applies mechanical shock to the cell by using nanoneedle arrays to disrupt the membrane [9], These methods are capable of introducing vector free particles inside the cell with claimed efficiency of up to 80 %.

[0006] The main problem of the existing microfluidic method, that is the most similar approach to ID2D method, is the operation complexity. In other words, the cell squeezing requires optimization of the operation parameters of the microfluidic setup in order to have sufficient cell squeezing. The squeezing degree depends on the flow rate and the dimensions of the microfluidic constriction channel in relation to cell size, shape and rigidity. Therefore, it requires designing steps based on the physical characteristics of the cell, high precision microfluidics and pumping system and tools. These factors make the method only applicable by well-equipped laboratories and it cannot be used without professional microfluidics skills.

[0007] Hence, the present invention aims at providing a novel intracellular delivery method that is less complex and, thus, easy to be carried out.

[0008] Accordingly, the present invention relates to a method for the introduction of macromolecules into living cells comprising (a) flattening the living cells between two surfaces such that the cells become amenable for the uptake of the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and (b) incubating the fattened cells, wherein the macromolecules become introduced into the cells.

[0009] The term ..macromolecule" as used herein designates a high molecular compound (preferably in excess of 1 ,000 atoms). The macromolecule can be an organic macromolecule (such as DNA, RNA, protein etc.) or an inorganic macromolecule. An inorganic macromolecule is a macromolecule not found in living matter, such as organisms and cells (e.g. a macromolecule made of petroleum-based monomers). The macromolecule is preferably an organic macromolecule and preferred examples thereof will be described herein below.

[0010] The nature and material of the surfaces that face the cells is not particularly limited as long as the surfaces can be functionalized by an antifouling coating that comprises biocompatible polymeric brushes together with theses brushed results in the flattening of the cell. Preferred examples of the nature and material of the surfaces will be described herein below. The surface is generally a surface of solid material with a considerable degree of flatness to achieve a minimum distance in between two surfaces. The minimum distance assures a sufficient flattening of the cell that results in an adequate membrane disruption of the flattened cell.

[0011] The cells to be used in the method of the invention are not particularly limited. Any live cell is flattened when it is confined in between the two functionalized surfaces. In this connection it is preferred that the cells have a plasma membrane but are not covered with a rigid cell wall (such as in plant cells) that might resist the flattening of the cells. The cell may be a prokaryotic cell such as an Escherichia coli cell, but is preferably a eukaryotic cell such as a yeast cell, fungal cell, more preferably an animal cell, even more preferred a vertebrate cell and most preferred a mammalian cell such as a human cell. The cell may be single cells or part of a cell line.

[0012] Living (or “live”) cells are characterized by an intact plasma membrane barrier function. In addition, living cells are preferably characterized by one or more abilities to grow, divide, respond to their environment, and carry out metabolic activities.

[0013] Flattening is a measure of the compression of a circle or sphere along a diameter to form an ellipse or an ellipsoid of revolution (spheroid), respectively. The preferred notation for flattening is f = a - b / a, wherein a is the larger dimension (e.g. semimajor axis), and b is the smaller (semiminor axis) of the circle or sphere. In this connection it is of note that cells are generally spheroids. In this connection f is in the range of 8 to 10, depending of the cell type. For instance, an adherent cell like Dictyostelium discoideum with an average diameter of 10 pm (the values for a and b), was shown to display an average “a” value around 80 pm and a negligible “b” value (around 0.5 pm). Therefore, giving an f value around 8.

[0014] Fouling (i.e., unwanted adhesion) is a complex and undesirable process where material from the environment, such as macromolecules, microorganisms, or suspended particles, adhere reversibly or irreversibly to a surface. The term “antifouling coating” in accordance with the present invention refers to a coating that repels proteins and cell membranes and thus block their attachment to surfaces, in the present case to the two surfaces. In particular, the “biocompatible polymeric brushes” of the antifouling coating repels proteins and cell membranes and thus blocks their attachment to surfaces, in the present case preferably to the surfaces of microscopy slides. The polymeric brushes are biocompatible which means that they do not or essentially do not interfere with the “live" status of the cells.

[0015] In accordance with the invention, a “polymeric brush” is defined as a surface coating consisting of polymers tethered to a surface or, in other terms, as polymers being attached by one end to an interface at relatively high coverage that stretch away from the interface to avoid overlapping (Milner (1991), Science, 251 (4996):905-914). A polymer brush coating used for adhesion is typically <100 nm in thickness resulting in extremely low material consumption, low risk of chemical leaching, and high precision in the dimensions of the joint. Polymer brushes achieve precise control of surface chemistry, enhance material compatibility, and allow the introduction of reactive functional groups to enable strong chemical bonding. The brush may be either in a solvated state, where the tethered polymer layer consists of polymer and solvent, or in a melt state, where the tethered chains completely fill up the space available. These polymer layers can be tethered to flat substrates such as silicon wafers, or highly curved substrates such as nanoparticles. Also, polymers can be tethered in high density to another single polymer chain, noting that this arrangement has been referred to as “bottlebrush”.

[0016] Means for the functionalization of surfaces with an antifouling coating are known in the art and comprise (1) physical bonding via entanglement and (2) chemical bonding (see, for example, Buhl et al. (2020), 12(7): 1475). In more detail, functionalization can be obtained either via physical interactions where the polymer brushes unify with the bulk polymer material through interpenetration and entanglement, or via chemical crosslinks where reactive groups in the polymer brush react and form covalent bonds to the bulk polymer melt.

[0017] As is illustrated by the appended examples, for the delivery of macromolecules into cells, the method of the invention uses the logic of cell confinement between two fictionalized surfaces. It was found that such cell flattening in between two surfaces disrupts the membrane to an extent that allows delivery of macromolecules inside livings cells without or essentially without impairing the “live” status of the cells. This method is also called ID2D method herein, standing for intracellular delivery by 2D cell flattening. For the proof of concept, two classes of macromolecules namely as vector DNA (plasmid) and protein structures with high molecular weight were subjected to delivery into cells in the appended examples. The ID2D method is shown to deliver the macromolecules with above 95% efficiency and with the cell vitality rate over 90%. To the best knowledge of the inventors this constitutes an improvement as compared to the delivery efficiency of up to 80% as achieved by the prior art as discussed herein above.

[0018] The advantages of the method of the present invention in comparison to available cell membrane disruption methods and specially the squeezing approach may be summarized as follows:

[0019] • Remarkably short time required for conducting the delivery process (in the range of few minutes only). • Application of delivery to large number of the cells in a single flattening step. There is no limitation for the size of the surfaces. As the result, large number of cells can be subjected to delivery in between bigger surface in a single process. This offers great advantage in saving the time and material when the delivery is intended for large number of the cells.

[0020] • Considerably low costs of the setup, which only requires two functionalized surfaces such as microscope glass slides. The coating material and the surfaces are basic consumables in many laboratories.

[0021] • Applicability of the method to any type of cell containing an exposed plasma membrane, regardless of the size and shape of the cell.

[0022] • The anti-fouling coating of the glass cover slips does not show any attachment of cells or organic macromolecules. Therefore, a waste of macromolecules, by binding of macromolecules to the surface, is totally avoided.

[0023] • The method is considerably easy to apply and does not require profound professional skills.

[0024] • Remarkably high delivery efficiency in vector-based and vector free deliveries, around 95 % with a cell viability rate over 90 %.

[0025] The procedure for the delivery of macromolecules into flattened cells is shown in Figure 2.

[0026] In a preferred embodiment, the method further comprises prior to (a) (a’) adding the macromolecules to the living cells.

[0027] This step (a’) can be carried out at a desired timing in order to control the delivery of macromolecules to the living cells. For instance, and in the case of growing cells, the cell cycle of the cells can be synchronized, so that the delivery of the macromolecules can be initiated at a desired time point during the cell cycle (e.g. G1 , S or G2 phase).

[0028] In another preferred embodiment, the method further comprises (c) separating the two surfaces from each other and optionally collecting the cells.

[0029] The cell are preferably collected by washing off the cells from the surface. For instance, once the two surfaces are separated the cells can be washed off by pipetting the cell medium on the surfaces, thereby resulting in the collection of the cells (e.g. in a petri dish). This is shown in Figure 2.

[0030] In a more preferred embodiment, the method further comprises (d) washing off the cells from the surfaces, and / or (e) concentrating the cells, preferably by centrifugation. The washing off the cells from the flattening surfaces is used in order to collect the cells and / or to further concentrate the cells. In this respect steps (e) can be repeated, if necessary.

[0031] For the washing preferably a physiological buffer is used.

[0032] In a preferred embodiment, the two surfaces are made of glass, quartz, silicon wafers, polyolefin (preferably cyclic olefin copolymer (COC) or cyclo-olefin polymers (COP)), plasma- activated polymers, and / or metal oxides (preferably Ta2Os, indium tin oxide (ITO), TiO2 or ZrO2).

[0033] The above materials for the two surfaces are preferred because these materials have a considerably flat surface with minimum roughness and can be functionalized by an antifouling coating that comprises biocompatible polymeric brushes.

[0034] In another preferred embodiment, the two surfaces are microscopy slides, preferably glass microscopy slides.

[0035] The microscopy slides are used in the examples. Microscopy slides at various sizes and of various thickness are commercially available. Microscopy slides are usually made of optical quality glass, such as soda lime glass or borosilicate glass, but specialty plastics are also used.

[0036] In a more preferred embodiment, for example, in the case of fluorescently-labelled macromolecules, the method further comprises after step (b) (1) microscopic imaging of the flattened cells, and (2) optionally storing the images on a storage medium.

[0037] Microscopic imaging refers to the visual inspection of the flattened cells by taking images of the cells.

[0038] In a more preferred embodiment, the microscopic imaging is imaging by fluorescence microscopy, confocal microscopy, multiphoton microscopy, total internal reflection microscopy, FRET microscopy, lifetime imaging microscopy, super-resolution microscopy, transmitted light microscopy, brightfield microscopy, oil immersion microscopy, phase contrast microscopy, Kohler illumination microscopy, darkfield microscopy, and combinations thereof.

[0039] All these microscopy methods are well known in the art. Images can be taken, for example, from cellular compartments, extracellular vesicles being released from the cells, or the cytoskeleton of the cells. It is also possible to observe and localize liposomes, synthetically formed nano-size vesicle, which have wide range of application in gene therapy and precise drug delivery.

[0040] Thus, in connection with the present invention in particular the uptake of the macromolecules into the living cells can be visualized.

[0041] In a preferred embodiment, the two surfaces have a size of 24 mm ± 50% x 50 mm ± 50%, a thickness of 0.170 mm ± 50%, and / or a weight of 0.605 ± 50% gram.

[0042] Surfaces in the form of microscopy slides with the above size and thickness are illustrated by the examples. Together with the brush-like structures they were found to be suitable to flatten various eukaryotic cell types, in particular neural stem cells, Dictyostelium discoideum cells, fibroblasts, HeLa cells, cardiomyocytes and epithelial cells. They impose a pressure on the cells that flattens the cells without destroying the cells.

[0043] In another preferred embodiment, the cells are within a medium.

[0044] As discussed above, the method of the invention is applied to living cells and the medium is helpful to keep the cells alive. Accordingly, the medium is preferably a cell maintenance medium and preferably a growth medium. In a maintenance medium the cells stay alive and in a growth medium the cells can survive and divide.

[0045] In a further preferred embodiment, the cells are suspension cells.

[0046] Both adherent and suspension cells can be used in the method of the invention. The method is particularly beneficial for suspension cells, because they generally tend to not adhere to surfaces as contrasted to adherent cells. Therefore, they tend to cluster inside the cell medium as floating spheroids that is not convenient in the case of methods introduced in literature [7,8], The ID2D method is capable of flattening of cell spheroids as a cluster of multiple cells. Flattening does not require or result in adhering of the cells, because the anti-fouling coating avoids the adhesion.

[0047] In a still further preferred embodiment, the biocompatible polymeric brushes of the antifouling coating are (a) linear brushes comprising polyethylene of glycol (PEG), lubricin, polyoxazolines, polyglycerol, dendrons, polyvinylpyrrolidone (PVP), polysaccharides, polypeptoids, polyacrylamide and / or zwitterionic polymers; and / or

[0048] (b) bottlebrushes comprising polyvinylpyrrolidone (PVP), poly[(oligoethylene glycol) methyl ether methacrylate] (POEGMA), polyoxazoline family, poly(2-methyl-2-oxazoline) (PMOXA), and / or thiol end-capped poly(methacrylic acid)-g-poly(2-methyl-2-oxazoline) (PMAA-g- PMOXA).

[0049] The above preferred biocompatible polymeric brushes are reviewed, for example, in Maan et al. (2020), Advanced functional materials, 30(32), 2000936.

[0050] There are many alternative coatings and any one of the above preferred antifouling coatings that are all biocompatible can be used. Another aspect to be considered is the hydrophilicity of the coating that is necessary for spreading of the cell solution in between two glasses. The flattening happens due to high hydrophilicity of the coated surfaces that result in strong capillary flow. Also, in this respect the above biocompatible polymeric brushes are expected to be particularly useful.

[0051] In a preferred embodiment, the antifouling coating that comprises biocompatible polymeric brushes is a co-polymer having a poly(L-lysine) backbone and poly (ethylene glycol) brushes.

[0052] This co-polymer is also known as PLL-g-PEG. PLL-g-PEG is a random graft co-polymer with a poly(L-lysine) backbone and poly(ethylene glycol) side-chains. The PLL backbone interacts electrostatically with the substrate, while the sidechains extend from the surface to form a densely packed polymeric brush.

[0053] A backbone polymer is generally used when the main polymer does not covalently bind to the surface. For instance, in PLL-g-PEG, as the glass surface is negatively charged the poly-(L- Lysine) is used which has positive surface charge. This results in covalent binding. In all the bottlebrushes wherever the name indicates -g-, it refers to grafted polymers. The first polymer is the backbone and the second one is the brush. For example, in the case of PMMA-g- PMOXA, PMMA is the backbone polymer.

[0054] PLL-g-PEG can adhere to surfaces, such as microscopy slides by electrostatic adsorption from aqueous buffers. The PLL-g-PEG coating has a brush-like structure well characterized by protein absorption and friction coefficient 9 measurements. PLL-g-PEG is preferably applied as a coating solution. The coating solution preferably has a final PLL-g-PEG concentration 0.1 mg / ml ±50% in 10 mM ±50% HEPES (pH 7.4, at room temperature). The coating solution is preferably applied as a drop on a parafilm layer and the coating surfaces are put on the drop. Therefore, the drop in between the parafilm layer and the coating surface expands and covers whole surface which is normally incubated for 30 min ±50%, preferably in a ventilated hood. Remaining liquid may be removed and slides can be dried by air flow.

[0055] In a more preferred embodiment, the poly(L-lysine) forming the backbone has a molecular weight of 20kDa ± 50% and / or the poly (ethylene glycol) brushes have a molecular weight in the range of 2kDa ± 50% and 5kDa ± 50%.

[0056] The poly(L-lysine) having a molecular weight of 20kDa ± 50% has with increasing preference a molecular weight of 20kDa ± 40%, 20kDa ± 30%, 20kDa ± 20%, 20kDa ± 10%, and 20kDa ± 5%.

[0057] Similarly, the poly (ethylene glycol) brushes having a molecular weight in the range of 2kDa ± 50% and 5kDa ± 50% have with increasing preference a molecular weight in the range of 2kDa ± 40% and 5kDa ± 40%, 2kDa ± 30% and 5kDa ± 30%, 2kDa ± 20% and 5kDa ± 20%, 2kDa ± 10% and 5kDa ± 10%, and 2kDa ± 5% and 5kDa ± 5%.

[0058] In another more preferred embodiment, the copolymer comprises or has the formula

[0059] ((H .

[0060] (. ii PL L backbone i ■ tn i

[0061] MH. In the above formula m refers to the PEG molecular weight, 5 kDa j refers to the grafting ratio, ~3. and k refers to the PLL molecular weight, 20 kDa.

[0062] In a preferred embodiment, the method comprises prior to (a):

[0063] (1) activating the two surfaces in oxygen plasma, preferably for 3 min ± 50% at 0.5 mbar ± 50%;

[0064] (2) coating the activated two surfaces with the antifouling agent, preferably the co-polymer of poly(L-lysine) and poly (ethylene glycol), wherein the co-polymer preferably is used at a concentration 0.1 mg / ml± 50% in a buffer, wherein the buffer is preferably 10 mM ± 50% HEPES; and

[0065] (3) optionally drying the coated two surfaces, preferably in ventilated hood.

[0066] Surface contaminations, in particular the residues remaining after mechanical and wetchemical cleaning, are often organic. In many cases, these are residues of oils, greases, release agents, and silicones which many solvents cannot remove completely. If these substances remain on the surface, they impair all downstream processing steps and have a particularly negative effect on all types of bonding and coating. These substances can be removed residue-free in an oxygen plasma, often also an air plasma. Excited oxygen molecules and radicals are highly reactive and form very stable bonds.

[0067] The UV radiation of the radiation splits the polymer chains of the organic residues. Oxygen radicals immediately occupy the released bonds and prevent the polymer fragments from reuniting. In this way, short-chain volatile substances which can be sucked off by the vacuum pump are increasingly formed from the macromolecules.

[0068] In the above preferred embodiment the ± 50% are with increasing preference ± 40%, ± 30%, ± 20%, ± 10% and ± 5%.

[0069] In a more preferred embodiment, the method further comprises prior to (1) the sterilization of the two surfaces, preferably by UV light.

[0070] UV light can optionally be combined with ethanol and preferably only UV light is used.

[0071] Alternatively, or in addition the surfaces, such as microscopy slides can be sterilized or cleaned by one or more and preferably all of (i) washing in 100% ethanol (e.g. for 10min ±50%) and rinsing in deionized water,

[0072] (ii) sonication in acetone (e.g. for 30 min ±50%) optionally followed by incubated in 96% ethanol (e.g. for 10 min ±50%), and

[0073] (iii) incubation in a Hellmanex III solution (Hellma Analytics) (e.g. 2% ±50% for 2h ±50%).

[0074] The Hellmanex is generally a step which is also beneficial for better imaging through the surfaces, such as microscopy slides. In this connection it is to be understood that although Hellmanex step improves the optical properties of the glass cover slips it is mostly used in the appended examples as a cleaning agent. While the optical improvement is beneficial the main purpose of the is the optional Hellmanex step is cleaning. According to the company description of the product “Hellmanex® III significantly reduces the surface tension of water. The Hellmanex aqueous solution helps with the removal of dirt particles, and its high emulsifying and dispersing capabilities prevent the redeposition of the loosened particles. Special surface-active substances facilitate the residue-free rinsing of the optical components once they have been cleaned. (see https: / / www.hellma.com / en / laboratory- supplies / cuvettes / hellmanex-cleaning-concentrate / )”

[0075] In a preferred embodiment, the macromolecules are proteins, peptides, nucleic acids molecules, carbohydrates, lipids, nanogels, macrocycles or a combination thereof.

[0076] The term “protein” as used herein interchangeably with the term “polypeptide” describes linear molecular chains of amino acids, including single chain proteins or their fragments, containing at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting of up to 49 amino acids, whereas the term “polypeptide” (also referred to as "protein") as used herein describes a group of molecules consisting of at least 50 amino acids. The term “peptide” as used herein describes a group of molecules consisting with increased preference of at least 15 amino acids, at least 20 amino acids at least 25 amino acids, and at least 40 amino acids. The group of peptides and polypeptides are referred to together by using the term "(poly)peptide". (Poly)peptides may further form oligomers consisting of at least two identical or different molecules. The corresponding higher order structures of such multimers are, correspondingly, termed homo- or heterodimers, homo- or heterotrimers etc. Furthermore, peptidomimetics of such proteins / (poly)peptides where amino acid(s) and / or peptide bond(s) have been replaced by functional analogues are also encompassed by the invention. Such functional analogues include all known amino acids other than the 20 gene-encoded amino acids, such as selenocysteine. The terms “(poly)peptide” and “protein” also refer to naturally modified (poly)peptides and proteins where the modification is effected e.g. by glycosylation, acetylation, phosphorylation and similar modifications which are well known in the art. The term “nucleic acid molecule” in accordance with the present invention includes DNA, such as cDNA or double or single stranded genomic DNA and RNA. In this regard, "DNA" (deoxyribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and thymine (T), called nucleotide bases, that are linked together on a deoxyribose sugar backbone. DNA can have one strand of nucleotide bases, or two complimentary strands which may form a double helix structure. "RNA" (ribonucleic acid) means any chain or sequence of the chemical building blocks adenine (A), guanine (G), cytosine (C) and uracil (II), called nucleotide bases, that are linked together on a ribose sugar backbone. RNA typically has one strand of nucleotide bases, such as mRNA. Included are also single- and double-stranded hybrids molecules, i.e., DNA-DNA, DNA-RNA and RNA- RNA. The nucleic acid molecule may also be modified by many means known in the art. Nonlimiting examples of such modifications include methylation, "caps", substitution of one or more of the naturally occurring nucleotides with an analog, and internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) and with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.). Nucleic acid molecules, in the following also referred as polynucleotides, may contain one or more additional covalently linked moieties, such as, for example, proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalators (e.g., acridine, psoralen, etc.), chelators (e.g., metals, radioactive metals, iron, oxidative metals, etc.), and alkylators. The polynucleotides may be derivatized by formation of a methyl or ethyl phosphotriester or an alkyl phosphoramidate linkage. Further included are nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of DNA or RNA and mixed polymers. Such nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include phosphorothioate nucleic acid, phosphoramidate nucleic acid, 2’-O-methoxyethyl ribonucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA) (see Braasch and Corey, Chem Biol 2001 , 8: 1). LNA is an RNA derivative in which the ribose ring is constrained by a methylene linkage between the 2’-oxygen and the 4’-carbon. Also included are nucleic acids containing modified bases, for example thio-uracil, thio-guanine and fluorouracil. A nucleic acid molecule typically carries genetic information, including the information used by cellular machinery to make proteins and / or polypeptides. The nucleic acid molecule of the invention may additionally comprise promoters, enhancers, response elements, signal sequences, polyadenylation sequences, introns, 5'- and 3'- non-coding regions, and the like.

[0077] A carbohydrate refers to any of the group of organic compounds consisting of carbon, hydrogen, and oxygen, usually in the ratio of 1 :2:1 , hence the general formula: Cn(H2O)n. Carbohydrates are the most abundant among the major classes of biomolecules. Lipids are a broad group of organic compounds which include fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), monoglycerides, diglycerides, phospholipids, and others. Lipids be in the form of exosomes. Exosomes are lipid packages which can carry a variety of different molecules including RNA (Alexander et al. (2015), Nat Commun; 6:7321).

[0078] Nanogels are three-dimensional hydrogel materials in the nanoscale size range formed by crosslinked swellable polymer networks with a high capacity to hold water, without actually dissolving into the aqueous medium. Nanogels can be composed of a variety of naturally occurring polymers, synthetic polymers or a combination thereof. Their characteristics such as size, charge, porosity, amphiphilicity, softness, and degradability can be fine-tuned by varying the chemical composition of the nanogels. They are mostly spherical particles but the current advancement in synthetic strategies allow for the fabrication of nanogels of different shapes. They can be also designed to have either a core-shell or a core-shell-corona structure, with at least one of the layers crosslinked for structural integrity. Being mostly hydrophilic in nature, nanogels are highly biocompatible with a high loading capacity for guest molecules and their unique physical properties offer them distinct advantages over other types of nanomaterials for biomedical applications.

[0079] Macrocycles are molecules or ions containing a ring of seven or more, preferably twelve or more atoms. Classical examples include the crown ethers, calixarenes, porphyrins, and cyclodextrins.

[0080] In a preferred embodiment, the macromolecules are particles or are in the form of particles.

[0081] The particles are generally microparticles. Microparticles are particles having a site of 100 pm or below. Commercially available microparticles are available in a wide variety of materials, including ceramics, glass, polymers, and metals. Microparticles encountered in daily life include pollen, sand, dust, flour, and powdered sugar. The microparticles are preferably microspheres or microbeads. The surface of the particles can be functionalized with the abovedescribed macromolecules.

[0082] As regards the embodiments characterized in this specification, in particular in the claims, it is intended that each embodiment mentioned in a dependent claim is combined with each embodiment of each claim (independent or dependent) said dependent claim depends from. For example, in case of an independent claim 1 reciting 3 alternatives A, B and C, a dependent claim 2 reciting 3 alternatives D, E and F and a claim 3 depending from claims 1 and 2 and reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0083] Similarly, and also in those cases where independent and / or dependent claims do not recite alternatives, it is understood that if dependent claims refer back to a plurality of preceding claims, any combination of subject-matter covered thereby is considered to be explicitly disclosed. For example, in case of an independent claim 1 , a dependent claim 2 referring back to claim 1 , and a dependent claim 3 referring back to both claims 2 and 1 , it follows that the combination of the subject-matter of claims 3 and 1 is clearly and unambiguously disclosed as is the combination of the subject-matter of claims 3, 2 and 1. In case a further dependent claim 4 is present which refers to any one of claims 1 to 3, it follows that the combination of the subject-matter of claims 4 and 1 , of claims 4, 2 and 1 , of claims 4, 3 and 1 , as well as of claims 4, 3, 2 and 1 is clearly and unambiguously disclosed.

[0084] The figures show:

[0085] Figure 1 : Examples of the experiments for delivery of high molecular weight vector free proteins inside HeLa and Dictyostelium discoideum cells. a,b) Two examples of the transfected HeLa cells with high and low concentration of proteins, respectively. Low and high concentration of the proteins were subjected to determine the presence and location of them inside the HeLa cells. The experiments show no free proteins around the cell and a successful delivery of them inside the cells, c) show the example of protein transfection inside Dictyostelium discoideum cells.

[0086] Figure 2: The experimental method of applying the ID2D method, a) Schematics of the experiment to introduce external material inside the cell. The coated glass covers, the PLL-g- PEG coating is shown as a brush like structure which provides the antifouling property of the substrate (I). A droplet of the cell mixture with external material intended for delivery is pipetted on the coated glass cover, seen as a sessile droplet (II). A second glass cover was dropped on the droplet with the coated surface facing the droplet, indicated by the arrows (III). The washing of cell from the glass covers after few minutes of incubation time. The cells were washed off by pipetting the cell medium on the coated surface of the glasses used for flattening and the cells were collected inside a petri dish (IV). b,c) show the logic behind the intracellular delivery by the ID2D method. The flattening mechanism causes the membrane disruption that allows delivery of the external material inside the cell.

[0087] The examples illustrate the invention.

[0088] Example 1 : Delivery of vector free protein complexes inside HeLa and Dictyostelium discoideum cells

[0089] The vector free delivery of labelled globular proteins was tried, namely, phosphorus-histone H3, Alexa Fluor 555 and E-cadherin, Alexa Fluor 488. These proteins are large protein structures with around 150 kDa molecular weight plus the Alexa Flour molecules, adding extra weight to them.

[0090] The mixture of the cells in buffer plus the mentioned proteins was prepared prior to application of cell flattening. A drop of the mixture was introduced on the first glass cover with PLL-g-PEG coating. Afterwards, the second coated glass cover was dropped on the first glass cover, such that the two coated surfaces face each other. In both cases of the cells, two minutes of incubation was applied for the flattening process. After the incubation, the glass covers were separated and the cells were washed out from the glass covers with pipetting the cell buffer. The collected cell solution was centrifuged to acquire cell concentration and was subjected to the 2DLCM method to visualize the cells. The results of fluorescent microscopy images depict the presence of protein structures inside the cells (Figure 1).

[0091] Example 2: Delivery of DNA vectors inside Dictyostelium discoideum cells

[0092] Although the vector free intracellular delivery generally is more challenging as compared to vector-based gene delivery, for a proof of concept the experiment of delivering myosin vectors (plasmid) inside Dictyostelium discoideum cells was conducted. Linearised DNA myosin GFP and cycled pDM502 Cathrin-GFP was subjected to the delivery following the same procedure as described in Example 1. After 24 hours from the delivery process, antibodies were used to screen for transfected cells with intended vectors. After a week of incubation in medium cells were observed to be attached and replicated which proved the successful delivery of the plasmid.

[0093] Example 3: Studying the cell vitality and delivery efficiency In two separate experiments the vitality and delivery efficiency of the method was investigated. First, the flattening procedure was applied without adding macromolecules to study the vitality rate of the cells. Various cells such a HeLa, fibroblasts, cardiomyocytes and pancreas cells were studied. The vitality rate was measured by a standard selection method and cell vitality was to be over 90 %. Second, after application of the ID2D method, the transfection efficiency was measured by counting the number of transfected cells and the total cell population.

[0094] Example 4: Studying the shape changes of the flattened cells

[0095] The ID2D method, as it flattens the cell mechanically, is a membrane-disruption method. To confirm this effect, the cell sizes before and after flattening were measured. The flattened cells showed a 15-20 % larger size. This confirms the expansion of the plasma membrane without damaging the biological functions of the cell and allow delivery of the external substances.

[0096] Example 5: Further experiments

[0097] In order to prove and amplify the application range of the ID2D method, the following further experiments can be conducted:

[0098] • Delivery of mRNA to T cells to be applied as messenger RNA (mRNA)-based therapy.

[0099] • Intracellular delivery of multiple mixtures of macromolecules and nano-particles.

[0100] • Study of the release of plasmid from the cells subjected to the ID2D method, and to investigate whether the released plasmid can be delivered to the neighboring cells.

[0101] Example 6: Cell flattening and delivery

[0102] Figure 2 shows the procedure for the ID2D method. The experiment was done under an air circulated hood to avoid contamination. The coated glass covers were introduced inside the hood with UV light, for 30 mins prior to conducting the experiment, in order to sterilize them.

[0103] Example 7: Material and methods

[0104] Preparation of cover glass slides:

[0105] Microscopy slides (Zeiss company, 24*50 mm, thickness of 0.170 mm ± 0.005 mm and weight of 0.605 ± 0.001 gr) were cleaned by washing with 100% ethanol for 10min and rinsing in deionized water. They were further sonicated in acetone for 30 min, followed by incubation in 96% ethanol for 10 min. The last step was a 2h incubation in a 2% Hellmanex III solution (Hellma Analytics). Finally, slides were extensively washed in deionized water and dried with a filtered airflow. All cleaning procedures were performed at room temperature. The cleaned slides were immediately activated in oxygen plasma (FEMTO, Diener Electronics, Germany) for 3 min at 0.5 mbar. The coating solution with a final concentration 0.1 mg / ml of the Poly(L- lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) (SuSoS AG, Switzerland) in 10 mM HEPES (pH 7.4, at room temperature) was prepared. The coating solution was applied as drops on a clean surface and the activated glass slides were put on it and incubated for 30 min in a ventilated hood. Remaining liquid was removed and slides were dried by air flow.

[0106] Microscopy and Image Analysis:

[0107] Image acquisition was performed using an inverted fluorescence microscope Olympus IX-71 with a 60x-oil objective (Olympus, Japan), with various binning and window sizes depending on the experiment. For excitation, a Lumen 200 metal arc lamp (Prior Scientific Instruments, U.S.A.) was applied. The images were recorded with a CCD camera (CoolSnap HQ2, Photometries). The frames were acquired with at 1 Hz. Image J software was used for the analysis of the acquired images.

[0108] References

[0109] [1] Martin P Stewart, Anna Lorenz, James Dahlman, and Gaurav Sahay. Challenges in carrier-mediated intracellular delivery: moving beyond endosomal barriers. Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology, 8(3):465-478, 2016.

[0110] [2] Martin P Stewart, Robert Langer, and Klavs F Jensen. Intracellular delivery by membrane disruption: mechanisms, strategies, and concepts. Chemical reviews, 118 (16):7409-7531 , 2018.

[0111] [3] Andrey A Gurtovenko, Jamshed Anwar, and llpo Vattulainen. Defect-mediated trafficking across cell membranes: insights from in silico modeling. Chemical reviews, 110 (10):6077- 6103, 2010.

[0112] [4] Martin P Stewart, Armon Sharei, Xiaoyun Ding, Gaurav Sahay, Robert Langer, and Klavs F Jensen. In vitro and ex vivo strategies for intracellular delivery. Nature, 538 (7624): 183-192, 2016.

[0113] [5] Dorsa Morshedi Rad, Maryam Alsadat Rad, Sajad Razavi Bazaz, Navid Kashaninejad, Dayong Jin, and Majid Ebrahimi Warkiani. A comprehensive review on intracellular delivery. Advanced Materials, 33(13):2005363, 2021.

[0114] [6] Armon Sharei, Janet Zoldan, Andrea Adamo, Woo Young Sim, Nahyun Cho, Emily Jackson, Shirley Mao, Sabine Schneider, Min-Joon Han, Abigail Lytton-Jean, et al. A vector- free microfluidic platform for intracellular delivery. Proceedings of the National Academy of Sciences, 110(6):2082-2087, 2013. [7] Alina Kollmannsperger, Armon Sharei, Anika Raulf, Mike Heilemann, Robert Langer, Klavs F Jensen, Ralph Wieneke, and Robert TampALe. Live-cell protein labelling with nanometre precision by cell squeezing. Nature communications, 7(1):10372, 2016. [8] Tia DiTommaso, Julie M Cole, Luke Cassereau, Joshua A BuggALe, Jacquelyn L Sikora Hanson, Devin T Bridgen, Brittany D Stokes, Scott M Loughhead, Bruce A Beutel, Jonathan B Gilbert, et al. Cell engineering with microfluidic squeezing preserves functionality of primary immune cells in vivo. Proceedings of the National Academy of Sciences, 115(46): E10907- E10914, 2018.

[0115] [9] Ying Wang, Yang Yang, Li Yan, So Ying Kwok, Wei Li, Zhigang Wang, Xiaoyue Zhu, Guangyu Zhu, Wenjun Zhang, Xianfeng Chen, et al. Poking cells for efficient vector-free intracellular delivery. Nature communications, 5(1):4466, 2014.

Claims

CLAIMS1 . A method for the introduction of macromolecules into living cells comprising(a) flattening the living cells between two surfaces such that the cells become amenable for the uptake of the macromolecules, wherein the surfaces that face the cells are functionalized by an antifouling coating that comprises biocompatible polymeric brushes, and(b) incubating the fattened cells, wherein the macromolecules become introduced into the cells.

2. The method of claim 1 , further comprising prior to (a) (a’) adding the macromolecules to the living cells.

3. The method of claim 1 or 2, further comprising(c) separating the two surfaces from each other and optionally collecting the cells.

4. The method of claim 3, further comprising(d) washing the separated cells, and / or(e) concentrating the cells, preferably by centrifugation.

5. The method of any one of claims 1 to 4, wherein the two surfaces are made of glass, quartz, silicon wafers, polyolefin (preferably cyclic olefin copolymer (COC) or cycloolefin polymers (COP)), plasma-activated polymers, and / or metal oxides (preferably Ta2Os, indium tin oxide (ITO), TiO2, ZrO2).

6. The method of any one of claims 1 to 5, wherein the two surfaces are microscopy slides, preferably glass microscopy slides.

7. The method of claim 5 or 6, wherein the method further comprises after step (b)(1) microscopic imaging of the flattened cells, and(2) optionally storing the images on a storage medium.

8. The method of any one of claims 1 to 7, wherein the two surfaces have a size of 24 mm ± 50% x 50 mm ± 50%, a thickness of 0.170 mm ± 50%, and / or a weight of 0.605 ± 50% gram.

10. The method of any one of claims 1 to 9, wherein the cells are within a medium.

11. The method of any one of claims 1 to 10, wherein the cells are suspension cells.

12. The method of any one of claims 1 to 11, wherein the biocompatible polymeric brushes of the antifouling coating are(a) linear brushes comprising polyethylene of glycol (PEG), lubricin, polyoxazolines, polyglycerol, dendrons, polyvinylpyrrolidone (PVP), polysaccharides, polypeptoids, polyacrylamide and / or zwitterionic polymers; and / or(b) bottlebrushes comprising polyvinylpyrrolidone (PVP), poly[(oligoethylene glycol) methyl ether methacrylate] (POEGMA), polyoxazoline family, poly(2-methyl-2- oxazoline) (PMOXA), and / or thiol end-capped poly(methacrylic acid)-g-poly(2-methyl- 2-oxazoline) (PMAA-g-PMOXA).

13. The method of any one of claims 1 to 12, wherein the antifouling coating that comprises biocompatible polymeric brushes is a co-polymer having a poly(L-lysine) backbone and poly (ethylene glycol) brushes.

14. The method of claim 13, wherein the poly(L-lysine) forming the backbone has a molecular weight of 20kDa ± 50% and / or the poly (ethylene glycol) brushes have a molecular weight in the range of 2kDa ± 50% and 5kDa ± 50%.

15. The method of claim 14, wherein the copolymer comprises or has the formula( ii„ PI 1 backbone nr iNH16. The method of any one of claims 1 to 15, further comprising prior to (a):(1) activating the two surfaces in oxygen plasma, preferably for 3 min ± 50% at 0.5 mbar ± 50%;(2) coating the activated two surfaces with the antifouling agent, preferably the copolymer of poly(L-lysine) and poly (ethylene glycol), wherein co-polymer preferably is used at a concentration 0.1 mg / ml± 50% in a buffer, wherein the buffer is preferably 10 mM ± 50% HEPES; and(3) optionally drying the coated two surfaces, preferably in ventilated hood.

17. The method of claim 16, further comprising prior to (1) the sterilization of the two surfaces, preferably by UV light.

18. The method of any one of claims 1 to 17, wherein the macromolecules are proteins, peptides, nucleic acids molecules, carbohydrates, lipids, nanogels, macrocycles or a combination thereof.

19. The method of any one of claims 1 to 18, wherein the macromolecules are particles.

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