Determining specificity of microcapsules for specific cell types

Abstract

It is an object of the present invention to provide transport microcapsules that are ingested by a target cell type and permanently or temporarily alter the target cell without thereby causing a toxic effect on the cell. The solution according to the invention consists in the use of a monodisperse core to thereby produce polyelectrolyte nanocapsules having cell-specific dimensions, in which the dimensions for hematopoietic cells range from 20 to 80 nm, preferably from 40 to 60 nm. The size of the particles must be in a very narrow range to prevent the occurrence of toxic effects. In order to maintain the low toxicity of nanomicrocapsules, it is also important to remove nanoparticles (cores) surrounding which create microcapsules prior to use. The method for this purpose is known in the prior art (e.g., using EDTA removal). Another object of the invention is the stabilization of transport microcapsules. The solution according to the invention consists in modifying microcapsules, layers and / or carriers to be packaged by functional groups, allowing stabilization and thus long-term storage at room temperature. The third purpose of the invention is targeted introduction of transport microcapsules. The solution according to the invention is functionalization of the layer by chemical modification and / or supplementation of the layer with antibodies, proteins or peptides.

Description

[0001] This divisional application is a divisional application based on the PCT invention patent application with application number 202080088128.1, which was filed on December 17, 2020 and entered the Chinese national phase, and the invention name is "Determining the specificity of microcapsules to specific cell types".

[0002] In WO 002019020665 A1, polyelectrolyte nanocapsules as transport media for biomolecules and "small molecules" in cells are described. Such nanocapsules can be constructed around a solid or liquid core using the so-called layer-by-layer technique. The size of the nanocapsule depends on the size of the core used. For the selection and efficiency of specific cell uptake, in addition to the surface structure, the size of the nanoparticles is also important. In WO 002019020665 A1, it is described that the microcapsules are formed around a core consisting of calcium carbonate, wherein this core is formed by a precipitation reaction of calcium nitrate and sodium carbonate solution. Various additives affect the precipitation and are related to the different sizes of the precipitated calcium carbonate core.

[0003] The problem is that the size distribution is very wide, i.e., when producing 60nm particles, particles over 100nm will also be produced, and vice versa. However, for the successful application of these nanocapsules in certain cell types, it is necessary to be able to accurately size the particles to about 10 to a maximum of 20nm, so that the uptake of unwanted cells can be reduced. Because hematopoietic cells are mainly transfected with nanocapsules of 20-60nm size, tumor cells prefer a size of 100-200nm. This means that the nanocapsules must be of the correct size to avoid nonspecific effects caused by nonspecific uptake.

[0004] Another important problem in the prior art is the toxicity of particles larger than 100 nm to cells of hematopoietic origin, such as CD34+ hematopoietic progenitor cells and T cells. Such cells can only be transduced or electroporated using viruses, with all the problems associated therewith.

[0005] Another important problem in the prior art is the relatively low stability of microcapsules filled with cargo. Sometimes only microcapsules loaded with siRNA have proven to be extremely stable; other molecules, especially mRNA and DNA, are unstable in microcapsules and prevent long-term storage, resulting in degradation of genomic cargo and / or aggregation of microcapsules. In addition, long-term storage can lead to diffusion effects of small molecules and chemotherapeutic drugs, which currently do not allow the effective use of these classes of substances. By chemically modifying the microcapsule layer and / or RNA or DNA, the stability can be significantly improved. In addition, cell or organ-specific introduction can be achieved through these modifications.

[0006] The problem with particles produced so far in the bottom-up process is that they have a wide size distribution and the production process is very variable. Therefore, this method is not suitable for the technical production of nanoparticles of the same particle size. On the other hand, particles produced in the top-down process are easily available and inexpensive, but have the problem of non-uniform shape.

[0007] Therefore, a first object of the present invention is to provide a microcapsule which is taken up by a target cell type and permanently or temporarily modifies the target cell without thereby causing any toxic effects on said specific target cell type. A second task is to stabilize the microcapsules for long term storage by means of the chemical modification described above.

[0008] The microcapsule according to the present invention is to use monodisperse nanoparticles to produce polyelectrolyte nanocapsules with cell-specific size and chemically modified as needed, and its size for hematopoietic cells is in the range of 20-80nm, preferably in the range of 40-60nm, and for epithelial cells and non-hematopoietic cells, it is 60-280nm, as described in WO0020199020665. The size of the particles must be in a very narrow range here to prevent toxic effects. Biodegradable polymers used for microcapsule construction, such as dextran and poly-L-arginine, can be chemically modified as needed (by connecting functional groups, such as functional groups containing hydrocarbons, oxygen, nitrogen, sulfur or phosphate groups; methods for this are known in the prior art) to prevent spontaneous aggregation and spontaneous carrier diffusion of microcapsules and improve the stability of microcapsules. In order to maintain the low toxicity of nanocapsules, it is also advantageous in some cases to remove the nanoparticles (so-called cores) around which the microcapsules are constructed before use. The method for this is known in the prior art (e.g., dissolving with EDTA).

[0009] Using cores obtained by precipitation does not allow for sufficiently narrow size selection, since the size distribution is very broad. In the context of the present invention, nanoparticles and nanocapsules with a narrow size distribution of ±15 nm or alternatively ±25%, preferably ±10 nm (or ±20% of the mean) are referred to as monodisperse.

[0010] Surprisingly, the problem of core size is not solved solely by using mechanically produced nanoparticles that are size-sorted. By grinding calcium carbonate in a ball mill, nanoparticles with a size of 15-60 nm can be produced, which can then be graded (by sedimentation or centrifugation) to produce uniform fractions (± 10 nm). These particles can then be coated with polymers in a layer-by-layer process. However, the inhomogeneous shape of these particles is disturbing because it disrupts the sorting of the particles. Only further purification of these particles, for example by cross-flow filtration after core dissolution, can achieve sufficiently accurate size selection.

[0011] Nanocapsules can be produced using the well-known layer-by-layer method. Molecules such as chemotherapeutics, small molecules and macromolecules such as nucleic acids and proteins can be introduced between the layers and then released in the cells. For example, chimeric T cell receptors can be produced by introducing DNA-loaded nanocapsules into T cells. RNA molecules can also be introduced into cells to produce only temporary modifications of cells. In addition, all the molecules and macromolecules mentioned can be introduced alone or in any conceivable combination. This property can, for example, transfect CRISPR / Cas complexes. The chemical modifications mentioned can be used for microcapsule polymers and carriers to adjust stability and thus regulate release in target cells.

[0012] Primary cells are cells that have been obtained from the body and have not lost their tissue-specific properties through culture. Primary cells are, for example, but not limited to, stem cells, germ cells, hematopoietic cells, tumor cells, or also mesenchymal stem cells, in vivo and ex vivo tissue cells.

[0013] Stem cells are generally referred to as somatic cells that can differentiate into different cell types or tissues. Depending on the type of stem cell and its influence, they have the potential to develop into any tissue (embryonic stem cells) or certain specific tissue types (adult stem cells / progenitor cells).

[0014] Suitable polymers for building nano-microcapsule layer are polycations and polyanions, which alternately form nano-microcapsule layer. Due to opposite electric charge, polymer can form layer by self-organization. After each layer is formed, the unused part of polymer must be separated from nano-microcapsule. In the meaning of the present invention, polymer also comprises classical polymers and copolymers and also biopolymers, for example macromolecules with the ordered structure of amino acid, nucleic acid and polysaccharide or oligosaccharide.

[0015] Examples of polycations are:

[0016] - Polyethyleneimine (PEI),

[0017] -Chitosan (CS),

[0018] - poly(L-lysine) (PLL),

[0019] - poly(amidoamine) type (PAA),

[0020] -Oligolipid Lysine

[0021] -Poly-(I)-ornithine (PLO)

[0022] -Poly(diallyldimethylammonium chloride) (PDAC)

[0023] - Histidine- and lysine-rich peptides and proteins, such as Cys-Gly-Ala-Gly-

[0024] Pro-(Lys)3-Arg-Lys-Val-Cys

[0025] Examples of polyanions are:

[0026] -Poly(methacrylic acid) (PMA)

[0027] -Poly(N-vinylpyrrolidone) (PVP)

[0028] -Tannic acid (TA)

[0029] -Poly (2-n-propyl-2-oxazoline) (PnPropOx)

[0030] -Poly(1-vinylimidazole)

[0031] -Poly(lactic-co-glycolic acid) (PLGA)

[0032] - Dextran sulfate,

[0033] - polyethylene glycol,

[0034] - Bovine serum albumin,

[0035] -Human serum albumin

[0036] -Protamine sulfate

[0037] -1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)

[0038] Substances that stabilize nucleic acids can be used, either substances that are applied together with the nucleic acids in the microcapsules or substances that diffuse into the microcapsules and enter the lumen of the microcapsules by incubation with the finished microcapsules and remain there at least partially until the microcapsules enter the cells. Examples of such substances are:

[0039] -Protamine sulfate

[0040] -Cell Penetrating Peptides

[0041] -Glutathione

[0042] -TAT protein

[0043] - RNase inhibitors

[0044] -DNAase inhibitors

[0045] Examples of nanoparticles suitable as cores for producing nanocapsules are:

[0046] -Iron nanoparticles (including magnetite)

[0047] -Silica nanoparticles

[0048] - Commercially available CaCO3 nanoparticles

[0049] - Other carbonate nanoparticles, such as MgCO3

[0050] -Vesicles

[0051] -Gold nanoparticles

[0052] -Latex Nanoparticles

[0053] The surface of the nanoparticle can be modified with additional groups to improve the permeability of the target cell. Such groups are, for example, folic acid groups, COOH groups, NH3 groups. These groups are also suitable for biocoupling to bind specific binding molecules, such as ligands and receptors, including antibodies.

[0054] Example 1: Classification of calcium carbonate nanoparticles

[0055] Calcium carbonate nanoparticles were purchased from SkySpring Nanomaterials. The average particle size was 15-40 nm. A 10 mg / ml suspension in PBS was prepared with the particles. Ultrasonic waves were used for 5 minutes to disperse the particles. They were then purified by size fractionation centrifugation in an Eppendorf centrifuge. 2 ml suspension was centrifuged at 500 RCF, 1000 RCF, 2000 RCF, 5000 RCF, 10000 RCF, 15000 RCF and 20000 RCF. Starting at the lowest RCF, the supernatant was removed and centrifuged at the next higher RCF. The precipitation spots from the centrifugation were then measured. The 500 RCF fraction was discarded (the particles were aggregated or too large).

[0056] Example 2: Preparation of polyelectrolyte nanocapsules

[0057] As described in WO 002019020665 A1, dextran sulfate (as sodium salt) and poly-L-arginine hydrochloride are used to coat the nanoparticles. One or more nucleic acids (plus additional biomolecules) can be introduced between the layers. The nucleic acids adhere to the microcapsule wall by electrostatic binding, so it is sufficient to incubate the microcapsules with the biomolecules. The method is described in WO002019020665 A1.

[0058] These can be chemically modified to change the storage stability and the degradation time in the cell. Chemical modifications were introduced and tested by applying one or different functional groups such as hydrocarbons, oxygen-containing, nitrogen-containing groups, sulfur (S / SH), N / NH and other P-containing groups. The chemical modifications have shown in first tests that longer storage times can be guaranteed. In addition, the microcapsules can now also be stored for a period of time at room temperature, which represents a great convenience especially for transport (freight), as it will reduce costs and administrative work (keywords: customs regulations for dry ice and refrigerated products).

[0059] Example 3: Purification of finished nanocapsules by tangential flow filtration

[0060] The finished nanocapsules were then cleaned using a KrosFlo Research II system with a 50nm filter module to remove any larger nanocapsules that may be present. Particles larger than 50nm were retained. To this end, 50ml of a 10mg / ml nanocapsule suspension in PBS was prepared. This was filtered according to the manufacturer's instructions.

[0061] Example 4 Separation after asymmetric flow field fractionation (AF4):

[0062] The finished nanocapsules were then cleaned using an Eclipse AF4 from Wyatt Technology to remove any larger nanocapsules that might be present. The particles were separated based on charge and size. For this purpose, 50 ml of a 10 mg / ml nanocapsule suspension in PBS was prepared. This was separated according to the manufacturer's instructions.

[0063] Example 5: Transfection of primary cells

[0064] Especially for hematopoietic cells, nanocapsules of 40-80nm size are obviously more advantageous. Proteins, DNA, mRNA, miRNA and siRNA are used as carriers of nanocapsules with sizes ranging from 50nm to 80nm. CD34+ hematopoietic stem cells, CD4+ and CD8+ T cells were incubated with microcapsules for 48 hours. 10 microcapsules / cell were used. In the case of fluorescently labeled microcapsules and PCR, successful introduction was checked with the help of confocal microscopy.

[0065] Example 6: Transfection of other primary cells

[0066] Nanocapsules of 50-120 nm in size were prepared for embryonic stem cells and induced pluripotent stem cells (iPS cells). Proteins, DNA, mRNA, miRNA and siRNA were used as carriers of nanocapsules of 50 to 120 nm in size. Embryonic stem cells and iPS cells were incubated with microcapsules for 48 hours. 20 microcapsules / cell were used. In the case of fluorescently labeled microcapsules and PCR, successful introduction was checked by confocal microscopy.

[0067] Example 7: Stabilization and storage of microcapsules at room temperature (RT).

[0068] For all the microcapsules listed in the previous examples, further modifications on the core or layer by introducing functional groups such as hydrocarbons, oxygen-containing, nitrogen-containing groups, sulfur (S / SH), N / NH and other phosphorus-containing groups can achieve stabilization of the microcapsules and thus achieve the desired goal of storage at room temperature.

[0069] Example 8: Effect of natural degradation of microcapsules

[0070] The natural degradation of the microcapsules after introduction in the corresponding target cells can be influenced by the functional group modification on the core and the layers as described in Examples 2 and 7. It was found that, for example, the degradation time per layer was 4 hours in mesenchymal cell lines and tumor cell lines, while the degradation time in cells of hematopoietic origin was 24 hours.

[0071] Example 9: Targeted introduction: with the help of AK, peptides, and proteins

[0072] In order to further specify the targeting of the cargo into the desired target cells, the corresponding antibodies, peptides or proteins are introduced into the outer layer of the microcapsule.

[0073] Example 10: Stabilization of microcapsules with small molecule cargo

[0074] In order to stabilize the microcapsules loaded with small molecules or chemotherapeutic drugs, click chemistry is used to immobilize the small molecules or chemotherapeutic drugs in the microcapsules and thereby prevent the diffusion effect.

[0075] In summary, this application provides the following technical solutions:

[0076] 1. Nanocapsules suitable for introducing target molecules from primary cells and stem cells, characterized in that

[0077] - The microcapsule consists of at least two biodegradable layers

[0078] - at least one nucleic acid, protein and / or small molecule or other target molecule is located between the at least two layers

[0079] -Nanoparticles - cores have well-defined dimensions with a maximum variance of 25%

[0080] -Nanoparticles-cores can be separated based on charge in addition to size

[0081] - If desired, the microcapsule surface and / or the various layers within the microcapsule can be modified with functional groups.

[0082] - It is also possible to modify the carrier elements between the layers or the layers themselves.

[0083] 2. The nanoparticle-core according to technical solution 1 is characterized in that the fixed size of the nanoparticle is selected from the size range of 20-80nm.

[0084] 3. The nanoparticle-core according to technical solution 2 is characterized in that the nanoparticles are selected from the size range of 30-60nm.

[0085] 4. A method for introducing a cargo into a primary cell or a cell line, characterized in that the nanocapsule is brought into contact with the cell, wherein

[0086] -Nanoparticles - cores have well-defined dimensions with a maximum variance of 25%

[0087] -Nanocapsules consist of at least two biodegradable layers

[0088] - at least one nucleic acid, protein, small molecule and / or chemotherapeutic drug is located between the at least two layers

[0089] - One or more layers or cargoes can be modified by attaching functional groups to modulate stability during storage or degradation in cells.

[0090] 5. The nanocapsule according to technical solution 1, which is modified with functional groups for stabilization purposes.

[0091] 6. The nanocapsule according to technical solution 1 is used for targeted transport.

[0092] 7. The method according to Technical Solution 5, wherein the cells are healthy or malignant cells of hematopoietic origin.

[0093] 8. A method according to technical solution 6, wherein the cells are temporarily transfected.

[0094] 9. The method according to technical solution 6, wherein the cells are stably transfected.

[0095] 10. The method according to technical solution 6, wherein the cells are embryonic cells or iPS cells.

[0096] 11. A method for preparing nanocapsules, characterized in that the core of the nanocapsules is monodispersed nanoparticles, and the nanocapsules are loaded with nucleic acids, proteins, small molecules or chemotherapeutic drugs, and are used with or without functional group modification.

Claims

1. A method for preparing nanocapsules, wherein nanoparticles with a size of 15-60 nm are produced by grinding calcium carbonate in a ball mill, and then uniform nanoparticle fractions are produced by fractionating the nanoparticles, each of which has a size distribution of ±10 nm of the mean value, wherein these nanoparticles are coated with a polymer in a layer-by-layer process, wherein molecules are introduced between the layers thus obtained, which molecules can be released into target cells.

2. The method according to claim 1, characterized in that: The nanoparticles around which the nanocapsule is constructed are removed before the nanocapsule is used.

3. The method according to claim 2, characterized in that The nanoparticles around which the nanocapsules are built are removed by dissolution and thereafter the nanocapsules are purified for size selection.

4. The method according to claim 3, characterized in that: The nanocapsules were purified by cross-flow filtration for size selection.

5. Nanocapsules prepared according to the method according to any one of claims 1 to 4.