Biodegradable particles for drug- or gene delivery

Abstract

Biodegradable particle comprising at least one cationic polymeric compound, the cationic polymeric compound being independently selected from the list comprising PEI, PLL, PLA and at least one drug or at least one genetic material, the genetic material being independently selected from the list comprising genes, siRNA, antisense oligonucleotides, DNA-sequences, RNA-sequences, long non- coding RNA, whereat the ratio of the sum of the negative charges of the phosphate groups in the genetic material or the sum of the negative charges of the at least one drug is equivalent to the sum of the positive charges in the at least one cationic polymer material.

Description

[0001] Patent Application

[0002] Biodegradable particles for drug- or gene delivery

[0003] The invention relates to the production of biodegradable particles for the drug delivery and / or gene delivery into immune cells and their use for the delivery of drugs and / or genes into immune cells. The biodegradable particles are either composed of one or more biodegradable polymers forming polymeric biodegradable capsules (core-shell structured particles, containing the drug / gene within a core which is free of drug or gene) or are complexes of polymers with nucleic acids, being called polyplexes.

[0004] Description

[0005] Ex-vivo CAR-T / NK technologies face difficulties in transducing DNA or RNA-based receptors or activating molecules efficiently by standard transfection and non-viral methods. Polymer-based complexes represent safe methods to deliver nucleic acids into immune cells. An advantage of polymers over other gene delivery systems is their potential capability for decoration with ligands, proteins, other biological substances for cell-specific targeting ex vivo and in vivo. We invent here a fabrication method for polymeric multilayer capsules to achieve high internalization rate by human immune cells. Subsequently, we invent a method for loading polymeric capsules with DNA, siRNA or mRNA. The invention further comprises delivering the loaded (manufactured) capsules into immune cells. We also invent a composition of polymers poly-L-arginine (and / or poly-L-lysine) and dextran sulfate, capable of binding nucleic acids (DNA, siRNA, mRNA) in solution and internalizing into human immune cells, subsequently delivering and expressing DNA vectors, siRNA and mRNA in the cells. Our data represent a potentially powerful method to utilize polymeric capsules and polyplexes for a broad spectrum of immune cell gene editing applications. Field of the invention

[0006] The invention is located in the field of drug delivery, resp. gene delivery, especially the manufacturing of drug / gene delivery systems. Polymer-based systems are provided, capable to deliver nucleic acids (DNA, siRNA, mRNA) into immune cells.

[0007] Background of the technology

[0008] Genetic manipulation of blood cells is key to understanding and developing treatments for solid and haematological cancers and potentially for a broad range of infectious diseases. The delivery of nucleic acids to blood and immune cells has the potential to improve therapeutic outcomes through the development of cellbased therapies. However, transfection of immune cells and circulating blood cells is generally very difficult. T lymphocytes and NK cells, which are particularly interesting because of their therapeutic promise due to cytotoxic activity, have proven to be refractory to delivery of DNA and RNA by standard methods, including commercial liposome- and polymer-based reagents. Transduction with lenti- / adenoviruses is now the most widely used and effective approach to introducing genes into immune cells. However, the use of live viruses is limited by low loading capacity, high cost; time-consuming and unscalable with potential mutagenicity and immunogenicity that drives research towards alternative solutions. Physical methods, including electroporation, show high transfection efficiency yet suffer from some level of toxicity (about 20-30%), variable transfection efficiency in different tissues and lack of cell-specific targeting, which hinders their potential for further in vivo applicability. To this end, biodegradable polymers are highly potent substances for fabricating complexes capable of binding and delivering nucleic acids into cells in vitro, ex vivo and in vivo. Considering their biocompatibility, good stability in the bloodstream, high loading capacity, facile and cost-effective fabrication potential, large-scale production and capability for decoration with ligands for cell-specific targeting polymer-based complexes might represent great potential in the delivery of gene cargos into hard-to-transfect immune cells in vitro, ex vivo and in vivo. An advantage of polymeric capsules is that they can be safely used in vivo and potentially can house pharmaceutical drug and nucleic acids simultaneously. The versatility of polymer chemical and physical structures provides a wide range of properties that can be optimized. Many attempts have been made to improve ex vivo transfection of immune cells with mRNA and DNA, including hyaluronan nanocapsules, poly(s-caprolactone) nanocapsules, alginate-azo / chitosan nanocapsules. A search for new combinations of polymers to achieve high transfection efficiency accompanied by low cell toxicity, the capability to decorate with ligands, and use in vivo is highly awaited. Our previous research reported a methodology of biodegradable polymeric multilayer capsules (PMC) for drug delivery. The key of the methodology is that poly-L-arginine (PLA) and dextran sulfate (DS) alternately adsorb on a ~500 nm vaterite (p-CaCOs) sacrificial template followed by removal of the inorganic core to obtain hollow capsules for consequent loading with drug molecules. These PMC show high internalization rate into tumor cells and macrophages in vitro and in vivo. In the current disclosure, we used different types of polymers for capsule fabrication and found a most potent combination of poly-L-lysine (PLL) with DS achieving up to 100% of capsule uptake by human immune cells. These capsules can be used for pre-loading of immune cells ex vivo with drugs boosting their cytotoxic activity. We subsequently tested PLL / DS capsules to be used for loading with nucleic acids (DNA, siRNA, mRNA). We found that at this current stage, PMC are not suitable to deliver DNA into immune cells (but it works for tumor cells), however our methodology can be used to deliver siRNA and / or mRNA into immune cells. In general, further optimization of the methodology for packing siRNA and / or mRNA in capsules is a preferable approach for potential application in vivo.

[0009] Due to limitation of PMC in DNA delivery, we generated a protocol to combine two polymers - Poly-L-arginine and dextran sulfate to bind nucleic acids in a solution (not in capsules), forming polyplexes for transfection in vitro and ex vivo. Subsequently, these polyplexes / DNA / RNA complexes are internalized by immune cells as high as 100% and release nucleic acids. The efficiency of expression is in a range of 4-80% for plasmid DNA, 12-18% for siRNA and 30-50% for mRNA. We conclude that poly-L-arginine / dextran sulfate polyplexes show high potential for nucleic acid delivery ex vivo into human immune cells with toxicity rate about 20- 30% depending on cell type. Further optimization of polyplexes is required to reduce cell toxicity. An advantage of polyplexes is that they are potentially suitable for tuning with biomolecules through electrostatic interactions or covalent binding and relatively easy and fast to prepare. There is no need to establish S2 facilities and techniques for viral transduction if using polyplexes for genetic modification. This is an important technical effect (advantage) of polyplexes.

[0010] There are some strategies known in the state of the art to provide cell-specific delivery of therapeutics (API’s, genes) in order to enhance the therapeutic efficacy of the drug / gene. But the known ex-vivo CAR-T / NK technologies face difficulties in transducing DNA / RNA-based receptors or activating molecules efficiently by non- viral methods. Viral methods for gene delivery also have several disadvantages, including immunogenicity, genotoxicity, low loading capacity, high cost and timeconsumption.

[0011] Ex-vivo CAR-T / NK technologies face difficulties in transducing DNA-based receptors or activating molecules efficiently by non-viral methods. Polymer-based complexes represent safe methods to deliver nucleic acids into immune cells. We invent a composition of at least one of two polymers capable of delivering DNA vectors, siRNA and / or mRNA into hard-to-transfect human immune cells. Our data represent a potential to utilize polyplexes for a broad spectrum of applications for immune cell editing. The at least two polymers being used for the polymer-based complexes can be applied together in combination or each one solely. The scope of the invention not only comprises usage of PLL, PLA as cationic polymers, but also other cationic polymers having comparable properties of degradability.

[0012] Content of the invention

[0013] An objective of the present invention is to provide biodegradable particles which can be used for the effective and cell specific delivery of drugs / API’s and / or genes specifically into immune cells. Drugs, genes, API’s are herein all considered to be therapeutics.

[0014] An additional objective of the present invention is to provide substances (resp. chemical compositions, resp. particles) that can be used for transfection of human immune cells with DNA, siRNA and mRNA (acute myeloid leukemia, Jurkat, NK-92, primary NK-cells, primary T-cells) with acceptable rate of cellular toxicity. One aspect of the subject matter of the present invention is a protocol, resp. method, to load PLA (and / or PLL) / DS capsules with DNA, siRNA and mRNA and the polymer-based complexes resulting from this method.

[0015] Another aspect of the subject matter of the present invention is a new method of cell transfection by forming complexes between nucleic acids (DNA, siRNA or mRNA) and a combination of poly-L-arginine and dextran sulfate in 150 mM NaCI solution to deliver them into immune cells in serum-free media, and the nanoparticle delivery systems provided by this method.

[0016] Both aspects (PLA (and / or PLL) / DS capsules and complexes between nucleic acids and polymers, i.e. polyplexes) are embodiments of the biodegradable particles according to the invention.

[0017] The Polymer-based complexes and nanoparticle delivery systems according to the invention represent safe methods for drug and / or gene delivery which have the following advantages over other non-viral methods like electroporation or lipid nanoparticles, which are already known in the state of the art:

[0018] - Higher intracellular uptake

[0019] - Higher stability in the blood stream, allowing lower dosing in manufacturing and / or application

[0020] - Capability to house drugs / API’s / bioactive molecules and / or DNA / RNA simultaneously

[0021] - Capability to be decorated with ligands for cell-specific interaction, thus allowing cell-specific targeting

[0022] - Cost-effective manufacturing

[0023] The biodegradable particles according to the invention are of nanoparticular size, i.e. the average size (measured by SEM) of the biodegradable particles ranges from 1 nm until 1000 nm.

[0024] The biodegradable particles according to the invention can deliver pharmaceuticals (API’s, drugs) and / or nucleic acids (RNA, DNA) into any types of cells of interest in vitro, in vivo or ex vivo. The delivery of drugs / API’s is - according to current experimental status - much more efficient with the capsules than with the polyplexes.

[0025] The biodegradable particles according to the invention are polymer-based, i.e. are comprising at least one biodegradable polymer. They are either multilayer- structured (exerting at least a core-shell structure) or are solidly structured. If they are solidly structured, the API / drug / gene is dissolved, resp. complexed, within the solid polymer phase, thus forming - in case of genes being included - polyplexes. If they are multilayer-structured, they are also called polymeric multilayer capsules (PMC).

[0026] The biodegradable particles are able to efficiently internalize into epithelial cells and / or macrophages in the lungs and liver in vitro as well as in vivo.

[0027] The experimental data of pre-treatment of macrophages with biodegradable particles according to the invention, comprising clodronate or chitin or ralimetinib, shows that the tumor-promoting functions of TAMs are blocked (ex vivo experiments).

[0028] According to the invention, the surface of the biodegradable particles can be modified chemically by attaching molecules upon the surface (chemical grafting) which are interacting with one or more membrane proteins of a certain desired type of cells in order to direct them to these cells of interest. The biodegradable particles modified this way show a high internalization rate into human immune cells (ex-vivo experiments, e.g. macrophages, dendritic cells, T- and NK-cells, human leukemia cells). Inventive biodegradable particles of this type can thus be used for pre-loading immune cells with activating drugs prior to adoptive cell transfer.

[0029] The invention also comprises a special composition of biodegradable particle of polyplex-type, which is able to deliver DNA, mRNA and siRNA into human immune cells. Summarizing the subject matter of the invention, it is characterized by the following features:

[0030] PLL (or PLA) / DS capsules show high uptake by immune cells. Loading capsules with DNA is possible with high expression level in HEK293T cells, but not in immune cells. Capsules can be used for drug delivery ex vivo and in vivo settings. Optimized protocol to load siRNA in PLA / DS capsules is provided with tested knock-down effect is about 90% in NK92 cells and PBMC-derived human macrophages. The first version of the protocol loading mRNA into PLL / DS capsules is developed with an efficiency of 2.94-9.13 % in human macrophages, NK92 and MOLM13 cells. Further optimization and improvement to load capsules with mRNA is possible, up to at least 15, 20, 25, 30 or even 50 % efficacy. In general, capsules are not toxic and suitable to use ex vivo and in vivo.

[0031] The subject matter of the invention provides a method to fabricate complexes between polymers poly-L-arginine and dextran sulfate and nucleic acids in solution provide relatively efficient delivery of DNA, siRNA and mRNA into human immune cells with - for example - 20-30% of cellular toxicity. It is well known that toxicity varies upon cell type (e.g. regarding macrophage-like human cells THP1 , there is no toxicity at all observed by polyplexes, according to current experiments). So, toxicity varies depending on cell type in a range, from about 0% to 30%. Additional optimization is possible to reduce toxicity.

[0032] In summary, the biodegradable particle according to the invention comprises at least one cationic polymeric compound and at least one drug and / or at least one genetic material. Genetic material, as is understood here, comprises - for example - the following materials, whereat this list is not considered to be confining the scope of the invention: genes, siRNA, antisense oligonucleotides, long non-coding RNA (abbreviation: IncRNA) etc. In fact, all of the exemplarily listed genetic materials may be of arbitrary structure / sequence, for the ratio of cationic polymeric material (i. e. cationic polymer) to genetic material can be individually adjusted according to individual structure / sequence of the genetic material. Therefore, generally arbitrary DNA-sequences and / or RNA-sequences can be applied without leaving the scope of the invention. The ratio of the sum of the negative charges of the phosphate groups in the genetic material (e.g. gene) and / or the sum of the negative charges of the at least one drug on one hand is equivalent to the sum of the positive charges of the polymer material. Thus, the at least one cationic polymeric compound (material) and the genetic material resp. the drug (or a combination of both) are admixed in equivalent ratio with respect to each backbone-charge, i.e. the charge of the organic structure, the corresponding counter ions being out of consideration.

[0033] Biodegradable polymeric nanoparticles for drug and / or gene delivery (delivery of genetic material of arbitrary type) exert many advantages:

[0034] Developing targeting strategies to enable cell-specific delivery of therapeutics holds promise for enhancing a drug’s therapeutic efficacy. The main challenge in ex vivo CAR-T / NK technologies is the efficient delivery of nucleic acids by non-viral transfection methods. T lymphocytes and NK cells have proven to be refractory to the delivery of dsDNA and RNAs by standard methods. The present invention relates to producing biodegradable polymeric particles to deliver nucleic acids (DNA, mRNA, siRNA) or drugs into primary human immune cells and potentially to any other hard-to-transfect cells. These nanoparticles are either biodegradable polymeric capsules containing nucleic acids or drugs within a core or are spontaneous complexes of polymers with only nucleic acids, being called polyplexes. An advantage of polymer nanocapsules over other gene delivery systems is their capability to simultaneously house drugs, different types of nucleic acids and other biological substances. The new technology has the following advantages over other nucleic acids delivery methods:

[0035] Capsules

[0036] • Up to 100% uptake by all cell types

[0037] • High efficiency to deliver and express into cells nucleic acids such as dsDNA, mRNA, siRNA and potentially any other type of RNAs (for example IncRNAs) and antisense oligonucleotides targeting RNAs

[0038] • Transfection of primary naive and activated T and NK cells

[0039] • Suitable for in vivo use

[0040] • Non-toxic ex vivo and in vivo • Potential capability to stably load with active pharmaceutical ingredients / bioactive molecules and / or DNA / RNA simultaneously

[0041] • High loading capacity - up to 200 000 bp per one capsule

[0042] • High stability in the bloodstream, allowing lower drug dosing in manufacturing and / or application

[0043] • Slow drug release

[0044] • Potential capability to be decorated with ligands for cell-specific targeting

[0045] • Cost- and time-effective manufacturing

[0046] Polyplexes

[0047] • High and selective uptake by cells ex vivo, preferably by T and NK cells, less by macrophages or dendritic cells

[0048] • High efficiency to deliver and express into cells nucleic acids such as dsDNA, mRNA, siRNA and potentially any other type of RNAs (e.g. IncRNAs) and antisense oligonucleotides targeting RNA

[0049] • Transfection of primary naive and activated T and NK cells

[0050] • High loading capacity - up to 25 000 bp per one particle

[0051] • Low acceptable levels of toxicity depending on cell type

[0052] • Capable to deliver several different types of RNAs or DNAs simultaneously

[0053] • Potential capability to be decorated with ligands for cell-specific targeting

[0054] • Cost- and time-effective manufacturing

[0055] • Not tested yet in vivo

[0056] One of the challenges in gene editing of immune cells is that viruses can target only activated T and NK cells. Polymeric nanoparticles can deliver nucleic acids in both - naive and activated immune cells.

[0057] Overall:

[0058] • Transfection of human cells, including primary naive or activated T- and NK- cells, with dsDNA, mRNA, siRNA and potentially with other types of RNA (e.g. IncRNA) or antisense oligonucleotides with an acceptable rate of cellular toxicity

[0059] • Effective delivery of drugs / genes to immune cells

[0060] Detailed embodiments of the invention

[0061] First exemplary preliminary example, variant 1 :

[0062] First, an aqueous solution of a cationic polymer (e.g. PEI) is prepared (solution 1 ) and an aqueous solution of the gene is prepared (solution 2). Then, solution 1 and solution 2 are admixed by use of a microfluidic system in such a way that the ratio of the negative charges of the phosphate groups in the genetic material is suitable (i.e. equivalent) to the sum of the positive charges in the polymer material (N / P-ratio, cationic-excess) in order to form stable particles.

[0063] Further, more detailed / alternative elaborations:

[0064] Materials

[0065] • Cell culture plastic (plates, tubes and tips) - Sarstedt, Germany

[0066] • CaCI_2 H2O, ChemSolut 2641 .0500, Germany. Stock solution: 1 M

[0067] • Na2COs anhydrous, Riedel de Haen 31432, Germany. Stock solution: 1 M

[0068] • Glycerol, Sigma G7893-500ml, Germany

[0069] • Water (sterile), Braun Melsungen, Germany

[0070] • Sodium chloride - CarlRoth 3957.1 , Germany. Stock solution: 3M

[0071] • EDTA Dihydrat, Carl Roth 8043.2, Germany. Stock solution: 0,2M pH=7,2.

[0072] • 5xsiRNA buffer (300 mM KCI, 30 mM HEPES-pH 7.5, 1.0 mM MgCI2), Sigma, Germany. Prepare Ixsolution by adding sterile water.

[0073] • Poly-L-arginine Mw>70kDa, Sigma P3892-100 mg, Germany

[0074] • Poly-L-lysine Mw 30-70kDa, Sigma P2636-25mg, Germany

[0075] • TRITC - Sigma 87918-10 mg Germany

[0076] • Dextran sulfate Mw=40000, Sigma 42867-5g

[0077] • FAM-labelled siRNA, ThermoFischer AM4620

[0078] • mRNA of EGFP with ARCA cap and polyA tail was synthetised with New England Biolabs kit, according the manufacture's protocol, NEB E2060S

[0079] • OptiMEM, Gibco 31985070, USA

[0080] • RNAse Inhibitor, recombinant, Sigma R1158, Germany

[0081] The two variants of the particles according to the invention can be manufactured as is descibed below, sections I) and II). These detailed descriptions are not to be understood as confining the scope of the invention. I) Manufacturing of PLA / DS capsules loaded with DNA, siRNA or mRNA

[0082] Synthesis of sacrificial -CaCOs vaterite template for LbL assembling of capsules

[0083] 1 . Weight 4,0 g of glycerol into glass beaker with lid and magnetic bar. Put it on the magnetic stirrer and start to mix 500 rpm.

[0084] 2. Add 400 pl 0,25M CaCI_2 in sterile water, mix at least 5 minutes. Then drop by drop, slowly add 400 pl 0,25M Na2COs. Close the lid and mix at 3000 rpm for 75 minutes. After this time, centrifuge the mixture at 17000 g for 5 minutes in several Eppendorf tubes per 2 ml.

[0085] 3. Pour out supernatant and wash three times in 96% ethanol. Centrifugation regime: 10000g 2 min. In the last steps with ethanol, combine all vaterite in one tube.

[0086] 4. Wash once in sterile water. Centrifugation regime: 14000g 2 min.

[0087] Protocol for assembling capsules loaded plasmid DNA

[0088] 1 . To precipitate of vaterite add 1 ml of sterile water. Make sure that there is no visible aggregation before LbL assembly! It should be homogeneous, without any clamps. For this purpose, it is comfortable to use an ultrasound bath (but not in the mixtures with nuclec acids!); also it's preferable to check the sample at a microscope with 40-1 OOx magnification for the presence of cubic calcite.

[0089] 2. Perform LbL assembly.To the suspension of particles in 1 ml sterile water, add 500 pl 1 mg / ml PLA-TRITC solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 14000g 2 min RT and wash precipitate two times with sterile water.

[0090] 3. To the suspension of particles in 1 ml sterile water 1 ml 1 mg / ml dextran sulfate solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 14000g 2 min RT and wash precipitate two times with sterile water.

[0091] 4. Repeat steps 2-3.

[0092] 5. Perform dissolving of vaterite core with EDTA. After the last washing step, add 1 ml of sterile water and up to 200 mM EDTA pH=7,2. Incubate on rotor shaker two minutes, centrifuge 17000g 5 min RT. Resuspend in 1 ml of sterile water and repeat treatment with EDTA.

[0093] 6. Wash three times with sterile water by centrifuging at 17000g 5 min RT.

[0094] 7. To the suspension of particles in 1 ml sterile water add 500 pl 1 mg / ml PLL solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 17000g 5 min RT and wash precipitate two times with sterile water.

[0095] 8. Add 1 ml of sterile water with 20 mkg plasmid DNA to the suspension of particles. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 17000g 5 min RT and wash precipitate 2 times with sterile water.

[0096] 9. Repeat step 6.

[0097] 10. Store capsules in dark place at +4°C.

[0098] Protocol for assembling capsules loaded siRNA

[0099] 1 . Add 1 ml of sterile water to the precipitate of vaterite. Make sure that there is no visible aggregation before LbL assembly! It should be homogeneous, without any clamps. For this purpose, it is comfortable to use an ultrasound bath (but not in the mixtures with nucleic acids!); also, it's preferable to check the sample at a microscope with 40-1 OOx magnification for the presence of cubic calcite. Add 1 pl of RNAse inhibitor, vortex briefly and add 5 pg of target siRNA, vortex briefly and incubate at RT on rotor shaker for 10 minutes. Add 1 ml of 96% ethanol, vortex briefly, incubate on ice for 5 minutes. Centrifuge 10000g 2 min, pour out supernatant and resuspend precipitate in 1 ml of sterile water.

[0100] 2. Perform LbL assembly.

[0101] To the suspension of vaterite in 1 ml sterile water, add 500 pl 1 mg / ml PLA- TRITC solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 14000g 2 min RT and wash precipitate two times with sterile water.

[0102] 3. To the suspension of vaterite in 1 ml sterile water 1 ml 1 mg / ml dextran sulfate solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 14000g 2 min RT and wash precipitate two times with sterile water.

[0103] 4. Repeat steps 2a-b.

[0104] 5. Dissolve vaterite core with EDTA. After last washing step, add 1 ml of sterile water and add up to 50 mM EDTA pH=7,2. Incubate on rotor shaker two minutes, centrifuge 17000g 5 min RT. Resuspend in 1 ml of sterile water and repeat treatment with EDTA.

[0105] 6. Wash three times with sterile water by centrifuging at 17000g 5 min RT. After last washing step resuspend capsules in 1 ml IxsiRNA buffer, add 0,5 RNAse inhibitor. Store capsules in dark place at +4°C

[0106] Protocol for assembling capsules loaded with mRNA

[0107] 1 . To precipitate of vaterite add 1 ml of sterile water. Make sure that there is no visible aggregation before LbL assembly! It should be homogeneous, without any clamps. For this purpose, it is comfortable to use an ultrasound bath (but not in the mixtures with nuclec acids!); also it's preferable to check the sample at a microscope with 40-1 OOx magnification for the presence of cubic calcite.

[0108] 2. After last centrifugation in water resuspend vaterite in 100 ul of water, add 1 pl of RNAse Inhibitor (5 units), pipet up and down, then add 1 pg of mRNA, pipet up and down again and incubate at RT on rotor shaker for 10 min. Then add 100 pl CHCh, briefly vortex and incubate at RT on rotor shaker for 10 min.

[0109] 3. Centrifuge 10000g 2 min, suck out supernatant and resuspend precipitate in 1 ml of sterile water as fast as you can. Immediately add 500 pl 1 mg / ml PLA- TRITC solution. Briefly vortex and incubate at RT on rotor shaker for 15 min. Centrifuge 14000g 2 min RT and wash precipitate two times with DEPC- treated water.

[0110] 4. To the suspension of particles in 1 ml sterile water add 1 ml 1 mg / ml dextran sulfate solution. Briefly vortex and incubate at RT on rotor shaker for 15 min. Centrifuge 14000g 2 min RT and wash precipitate two times with DEPC- treated water. 500 pl 1 mg / ml PLL solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 14000g 2 min RT and wash precipitate two times with DEPC-treated water.

[0111] 5. Resuspend particles in 1 ml DEPC-treated water. Add 500 pl 1 mg / ml PLL, vortex briefly and incubate at RT on rotor shaker for 15 min. Centrifuge 14000g 2 min RT and wash precipitate two times with DEPC-treated water.

[0112] 6. To the suspension of particles in 1 ml DEPC-treated water add 1 ml 1 mg / ml dextran sulfate solution. Briefly vortex and incubate at RT on rotor shaker for 15 min. Centrifuge 14000g 2 min RT and wash precipitate two times with DEPC-treated water. 500 pl 1 mg / ml PLL solution. Briefly vortex and incubate at RT on rotor shaker for 15 minutes. Centrifuge 14000g 2 min RT and wash precipitate two times with DEPC-treated water.

[0113] 7. Perform dissolving of vaterite core with EDTA. After the last washing step, add 1 ml of sterile water and up to 50 mM EDTA pH=7,2. Incubate on rotor shaker two minutes, centrifuge 17000g 5 min RT. Resuspend in 1 ml of sterile water and repeat treatment with EDTA. Repeat this treatment 3-4 times.

[0114] 8. Wash three times with sterile water by centrifuging at 17000g 5 min RT.

[0115] Store capsules in dark place at +4°C.

[0116] Protocol for transfection of cells with capsules loaded with DNA and / or RNA

[0117] 1. Seed immune cells (primary human NK cells, primary human T cells, Jurkat cells, NK92, MOLM13 human AML) at density 5*105cells / well into 12-well plate in in 0,5 ml respective growth media.

[0118] 2. Add prepared capsules (1 :50 celkcapsule ratio) into Eppendorf tube with 0,5 ml respective growth media. Resuspend well by pipetting up and down. Add suspension to the cells drop by drop and pipet again up and down gently.

[0119] 3. Incubate at least 24h in an incubator at +37°C 5% CO2.

[0120] 4. Perform flow cytometry to detect GFP-expression or SDS-PAGE for siRNA knockdown efficacy after 48-96h. Exemplary experimental results (the methods for performing the following measurements being well known within the state of the art):

[0121] Uptake of PLA / DS capsules

[0122] Jurkat - 98%

[0123] Human primary NK cells - 100%

[0124] Human primary T cells 100%

[0125] Human primary macrophages - 98%

[0126] Human primary dendritic cells - 98%

[0127] PLL / DS and PLA / DS capsules are non-toxic.

[0128] DNA delivery in capsules

[0129] Jurkat - 2%

[0130] Human primary NK cells - 0.5%

[0131] Human primary macrophages - 1 .7%

[0132] Human primary dendritic cells - 0.2%

[0133] HEK293 - 70% siRNA delivery in capsules

[0134] NK-92 - about 90% (tested by Western blot)

[0135] Human macrophages - about 90% (tested by Western blot) mRNA delivery in capsules human macrophages - 9.14%

[0136] NK92 - 3.49%

[0137] MOLM13 - 2.94% II) Method for forming complexes between nucleic acids (DNA, and / or siRNA and / or mRNA) and polymers

[0138] Protocol for preparation of polyplexes.

[0139] Each cell type and each type of nucleic acid has its own optimal proportions of reagents. Examples of concentrations of pDNA, siRNA and polymers for respective transfections are presented in Tables 1-3.

[0140] Table 1. Reagents composition for plasmid DNA transfection

[0141] Table 2. Reagents composition for siRNA transfection

[0142] Table 3. Reagents composition for mRNA transfection Add 50 l 150 mM NaCI in water and 500 ng of plasmid vector (pEGFP-N1 , ClonTech, USA) or 100 ng FITC-labelled scrambled siRNA into 0,5 ml Eppendorf tube (Sarstedt, Germany) or 500 ng mRNA, mix well by vortexing. In a second 0,5 ml Eppendorf tube, add 50 pl 150mM NaCI in water and 6 pg of poly-arginine-TRITC. Mix well by vortexing, and incubate at room temperature for at least 3 minutes. In the third 0,5 ml Eppendorf tube, add 50 pl 150mM NaCI in water and 1 pg of dextran sulfate. Mix well by vortexing, and incubate at room temperature for at least 3 minutes. After these steps, start mixing. Add prepared polyarginine solution to solution with vector. Pipet up and down well intensively; also vortexing. Incubate this mixture for 15 min. Add a prepared solution of dextran sulfate into the mix of vector with polyarginine. Pipet up and down well intensively; also vortexing. Incubate this mixture for 15 min. Seed immune cells (primary human NK cells, primary human T cells, Jurkat cells, NK92, MOLM13 human AML) at density 5*105cells / well into 12-well plate in 1 ml OptiMEM media. Add prepared polyplexes from (point 5) to the cells. Don't pipet! Mix by shaking\swirling of a plate. Incubate for at least six h in an incubator at +37°C 5% CO2. Then change media to suitable for transfected cells FBS-containing media. Perform flow cytometry to detect GFP-expression and siRNA delivery after 48h. Exemplary experimental results (the methods for performing the following measurements being well known within the state of the art):

[0143] Cellular uptake of polyplexes

[0144] KG1a * 18%

[0145] NB4 * 16%

[0146] MOLM * 80%

[0147] Jurkat « 98%

[0148] Primary human macrophages - 19%

[0149] Primary NK cells - 79%

[0150] Primary T cells - 80%

[0151] Primary DC cells - 0.2%

[0152] Toxicity of polyplexes

[0153] Jurkat - 8%

[0154] Primary macrophages - 3%

[0155] NK cells - 25%

[0156] MOLM13 - 30%

[0157] NK-92 - 20%

[0158] Mouse T-cells - 19%

[0159] THP1 cells - 1%

[0160] DNA delivery (GFP) by polyplexes

[0161] MOLM13 - 80%

[0162] Jurkat - 20%

[0163] Human primary NK cells - 40%

[0164] Human primary macrophages - 4%

[0165] Human primary T cells 4%

[0166] THP1 cells - 25% siRNA delivery by polyplexes

[0167] First example of measurement: Jurkat - 19%

[0168] Second example of measurement:

[0169] Jurkat - 18%

[0170] NK92 - 12% mRNA delivery by polyplexes

[0171] Jurkat - 18%

[0172] MOLM13 - 51 %

[0173] NK92 - 45%

[0174] Measurement of the particle size was done by use of a NanoFCM machine, the way how to perform such measurements being well known to the person skilled in the art. Alternatively particle size may be measured via DLS (dynamic light scattering, a method also being well known to the person skilled in the art. The size of the particles is within 1-1000 nm, preferred within 10 - 800 nm, more preferred within 50-500 nm, most preferred within about 80-250 nm.

[0175] Description of the drawings

[0176] Fig. 1 : Examples of capsule uptake by different cell types. Flow cytometry analysis of TRITC-positive immune cells. DC - dendritic cells, NK - nature killer cells, Jurkat - human T-cell like cell line, h - human, m - mouse.

[0177] Fig. 2: Examples of Plasmid DNA delivery by capsules and expression of EGFP. Flow cytometry analysis of cells expressing GFP. First graph: Uptake of PMC with PLA or PLL in the second polymer bilayer by HEK293T cells. Flow cytometry analysis of TRITC- positive cells. Third graph: Flow cytometry analysis of immune cells expressing GFP delivered as a plasmid encapsulated in PMC with PLL in the second polymer bilayer.

[0178] Fig. 2.1 : Examples of Intracellular localization of TRITC-labelled PMC in primary human macrophages (cytoplasm) and in primary human NK cells (cytoplasm and nucleus). Nuclei exemplarily denoted with refence mark 10, capsules (PMC) exemplarily denoted with reference mark 20. Nuclei and capsules are also denoted in same grey scale as legend on vertical axis.

[0179] Fig. 3: Examples of siRNA delivery by PMC into human NK-92 cells. Western Blot analysis of human p38a-MAPK14 protein and a-Tubulin as a loading control in NK-92 cells transfected with capsules loaded with siRNA targeting human MAPK14.

[0180] Fig. 3.1 : Examples of Plasmid DNA (pDNA) delivery by capsules and expression of EGFP. Percentage of HEK293T cells expressing EGFP delivered as a plasmid encapsulated in PMC with PLA or PLL in the second bilayer. Flow cytometry analysis of EGFP-positive cells (left); immunofluorence images (right).

[0181] Fig. 4: Examples of Cellular uptake of polyplexes. Flow cytometry analysis of TRITC-positive cells 48 hrs after transfection.

[0182] Fig. 5: Examples of Cellular toxicity of polyplexes. Flow cytometry analysis of propidium iodide-positive cells 48 hrs after transfection.

[0183] Fig. 5.1 : Examples of siRNA delivery into human macrophages. Western Blot analysis of human p38a-MAPK14 protein and a-Tubulin as a loading control in PBMC-derived human macrophages transfected with capsules loaded with siRNA targeting human MAPK14.

[0184] Fig. 5.2: Examples of GFP-mRNA delivery by PMC into human cells. A: Flow cytometry analysis of GFP-positive human macrophages, human NK-92 cells and MOLM13 cells. Capsules are labelled with TRITC, Zombie NIR used to estimate a percentage of dead cells. B: Percentage of GFP- positive human cells, based on flow cytometry data at Figure 6A.

[0185] Fig. 6: Examples of DNA delivery by polyplexes and expression of EGFP in cells. A: Flow cytometry analysis of cells 48 hrs after transfection by polyplexes with a plasmid coding EGFP. B: Experiment 1 with many cell types. B: Representative flow cytometry data of cells transfected by polyplexes with mRNA of EGFP gene. C: Examples of Transfection efficiency of plasmid DNA by commercially available reagents as a referral. Flow cytometry analysis of cells 48 hrs after transfection by of a plasmid coding EGFP by Lipofectamine 3000 or Mirus IT2020 according to the manufacturer instructions, polyplexes with a plasmid coding EGFP.

[0186] Fig. 7: Examples of siRNA delivery by polyplexes into Jurkat cells. Flow cytometry analysis of Jurkat cells 48 hrs after transfection by polyplexes with scrambled siRNA labelled with FAM (green). Uptake of TRITC- labelled polyplexes is in purple 7-AAD (red) was used to estimate level of cytotoxicity.

[0187] Fig. 8: Examples of mRNA delivery by polyplexes into human immune cells. Flow cytometry analysis of Jurkat, MOLM13 and NK92 cells 48 hrs after transfection by polyplexes with mRNA of EGFP gene (degree of expression indicated by horizontal bar). Cytotoxicity measurement was performed with propidium iodide (degree of toxicity indicated by horizontal bar).

[0188] Fig. 9: Examples of plasmid DNA loading efficiency into PMC. The concentration of the plasmid was measured before and after loading into the capsules, suggesting an efficiency of approximately 50%.

[0189] Fig. 10: Comparison of different methods for gene delivery into immune cells, showing the advantages of the inventive method over the state of the art.

Claims

Claims Biodegradable particle comprising- at least one cationic polymeric compound, the cationic polymeric compound being independently selected from the list comprising PEI, PLL, PLA and- at least one drug or at least one genetic material, the genetic material being independently selected from the list comprising genes, siRNA, antisense oligonucleotides, DNA-sequences, RNA-sequences, long non-coding RNA, whereat the ratio of the sum of the negative charges of the phosphate groups in the genetic material or the sum of the negative charges of the at least one drug is equivalent to the sum of the positive charges in the at least one cationic polymer material. Biodegradable particle according to claim 1 comprising- at least one polymeric compound- at least one drug or at least one genetic material, whereat the surface of the biodegradable particle is chemically modified. Biodegradable particle according to claim 1 or claim 2, characterised in that the average size of the biodegradable particle, measured by dynamic light scattering, is between 1 nm and 1000 nm. Biodegradable particle according to claim 1 or claim 2 or claim 3, characterized in that it comprises siRNA and / or mRNA and / or DNA. Biodegradable particle according to claim 1 or claim 2 or claim 3 or claim 4, characterized in that it comprises chitin or ralimetinib.

6. A product for use in a medical treatment, characterised in that it comprises a biodegradable particle according to any one of the proceeding claims.

7. Usage of a biodegradable particle according to claim 1 or claim 2 or claim 3 or claim 4 or claim 5 for immune cell editing.

8. Manufacturing of a biodegradable particle according to claim 1 , characterised in that- first, an aqueous solution of a cationic polymer is prepared (solution 1 ) and an aqueous solution of the gene is prepared (solution 2),- then solution 1 and solution 2 are admixed in such a way that the ratio of the negative charges of the phosphate groups in the genetic material is suitable to the sum of the positive charges in the polymer material in order to form stable particles.

9. Method of manufacturing according to claim 8, characterized in that mixing of solutions 1 and 2 is achieved by vortexing.

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