Biodegradable multilayer nanocapsules for the delivery of biologically active agents in target cells

Abstract

The present invention relates to a biodegradable multilayer nanocapsule for the delivery of at least one biologically active agent into at least one target cell consisting of at least two layers of at least two biodegradable polymers which are laid one onto the other and whereby the biologically active agent is layered onto a layer of a biodegradable polymer and covered with a further layer of a biodegradable polymer, whereby one biologically active agent is a nucleic acid.

Claims

1. A biodegradable multilayer nanocapsule for the delivery of at least one biologically active agent into at least one target cell or organ, said nanocapsule comprised of a void center coated with at least a first and second layer, each of which is fabricated of a biodegradable polymer, whereby said at least one biologically active agent is layered onto said first layer of a biodegradable polymer and then covered with said second layer of a biodegradable polymer, further wherein said at least one biologically active agent is a nucleic acid and said nanocapsule has a diameter of 60 nm to 150 nm, wherein the void center of the nanocapsule is free of active agent.

2. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the biodegradable polymer of said first layer is dextran sulfate sodium salt.

3. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the biodegradable polymer of said second layer is poly-L-arginine-hydrochloride.

4. The biodegradable multilayer nanocapsule according to claim 1, further comprising a second biologically active agent that is a protein.

5. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the nucleic acid is an RNA molecule selected from among a non coding RNA, small non coding RNA, miRNA, mRNA, and long non coding RNA.

6. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the nucleic acid is selected from among an RNA, a synthetic analogue of RNA and a hybrid RNA/DNA molecule.

7. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the nucleic acid is an RNA selected from among siRNA, gRNA, and combinations thereof.

8. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the nucleic acid is mRNA.

9. The biodegradable multilayer nanocapsule according to claim 1, characterized in that the nucleic acid is selected from among DNA, synthetic DNA analogues, and hybrid DNA/RNA molecules comprising linear fragments of DNA, circle DNA, plasmids, single- and double stranded DNA, RNA/DNA hybrids and synthetic nucleic acids that are able to bind intracellular DNA or RNA fragments.

10. The biodegradable multilayer nanocapsule according to claim 1, wherein said nanocapsule further includes specific compounds that increase the efficiency of uptake of a biologically active agent into said target cell or organ.

11. A process for the preparation of a biodegradable multilayer nanocapsule according to claim 1, said process comprising the following steps: a) preparing a core consisting of CaCO.sub.3; b) coating the core particles with said first layer of a biodegradable polymer; c) optionally coating with at least one further layer of biodegradable polymer whereby the polymer is different from the polymer as used in step b); d) coating the core which has already been coated with biodegradable polymer with said at least one biologically active agent; e) coating the product obtained from step d) with said second layer of biodegradable polymer; f) removing the core.

12. The process according to claim 11, wherein said process further includes the performance of washing and centrifugation steps after one or more of steps a) to f).

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a characterization of the capsules by scanning electron microscopy (SEM). FIG. 1A shows SEM image of a vaterite CaCO.sub.3 particle; FIG. 1B shows an SEM image of core-shell capsules showing spheroid structures of slightly different sizes. The size of capsules is directly dependent of the size of CaCO.sub.3 valerites, which is determined by concentration of the reagents, salts solubility, reaction time, and rotation speed during mixing.

(2) FIG. 2 shows the preparation of nanocapsules loaded with a fluorescent dye and siRNA. FIG. 2A shows a schematic representation of capsule loading with siRNA: first, two layers of DS and PARG are coated on the CaCO.sub.3 core; then siRNA layer can be coated directly on a PARG and covered with an additional PARG layer. As the last step, core can be removed with EDTA; FIG. 2B shows images of capsules loaded with encapsulated Rhodamin B isocyanate (RdnB) and control siRNA, labeled with Alexa Fluor 488 (ctrsiRNA-488); RdnB/(DS/PARG)2/siRNA/PARG). In the red channel, Rhodamin B is visible, showing capsules loaded with RdnB; in the green channel Alexa Fluor 488 can be visualized. Overlay of both images shows a clear merge of both colors giving the color yellow, indicating successful encapsulation of siRNA into the capsules.

(3) FIG. 3 shows the efficient uptake and intracellular localization of polyelectrolyte nanocapsules in HT1080 cells. FIG. 3A shows that for determination of optimal capsule concentration 10, 20 and 50 capsules/cell were applied for treatment of HT1080 cells; 24 h post-treatment cells were fixed; stained with phalloidin-Alexa488 and DAPI and subjected to confocal microscopy. Only few capsules could be visualized by application of 10 capsules/cell; concentration of 20 capsules/cell was considered as optimal; whereas 50 capsules/cell exhibited toxic effect, causing nuclei deformation (white arrow heads) and access of not internalized capsules in the solution (white arrows). FIG. 3B shows a comparison of intracellular localization of nanocapsules (left image, showing uptake of capsules loaded with siRNA-Alexa488) and vesicles (right image, showing uptake of vesicles loaded with red dye PKH26). Very similar localization in the perinuclear region suggests that nanocapsules are able to deliver their payloads, e.g. RNA to physiological intracellular sites mimicking extracellular vesicles in their function. Scale bar 50 μm.

(4) FIG. 4 shows an analysis of degradation kinetic of polyelectrolyte nanocapsules. FIG. 4A shows that to examine degradation of capsules within the cells, HT1080 cells were treated with capsules loaded with RdnB and ctrsiRNA-488. Then 4, 24 and 48 h after treatment the cells were fixed, stained with phalloidin and DAPI and subjected to confocal microscopy (scale bar 30 μm). Yellow color on the images indicates merged RdnB and ctrsiRNA-488 signals (mostly visible after 4 h) and intact capsules. Appearance of red and green dots after 24 h indicates capsule degradation. FIG. 4B shows a quantitative analysis of capsule degradation. A black line (□) indicates intensity of yellow signal, showing that majority of capsules is intact; grey lines show intensity of yellow color after 24 (○) and 48 (Δ) h respectively, showing decrease of a number of intact capsules. FIG. 4C shows a diagram, showing decrease of a portion of the intact capsules from 100% after 4 hours to only 19% after 48 h. This reflects the efficient uptake of the nanocapsules.

(5) FIG. 5 shows the efficient GFP knockdown by transfer of the GFPsiRNA in biodegradable nanocapsules in HT1080-GFP cells. FIG. 5A shows a confocal microscopy of the HT1080 cells treated with capsules loaded with GFP-specific siRNA or transfected with Lipofectamine 2000 using the same amount of siRNA/cell. As a control, unspecific ctrsiRNA-488 was used. Images were taken 48 h post treatment. FIG. 5B shows a plot diagram showing quantitative analysis of the knockdown efficiency extrapolated from the reduction of the GFP signal intensity. Image freeware was used for the analysis. FIG. 5C shows a diagram showing decrease of green fluorescence indicating 80% knockdown efficiency by capsules and only 21% by Lipofectamine application.

(6) FIG. 6 shows an efficient apoptosis induction by transfer of AllStar Cell Death siRNA in biodegradable nanocapsules in HT1080 cells. FIG. 6A shows confocal images of HT-1080 cells treated with capsules loaded with AllStar Cell Death Control siRNA inducing apoptosis (apoptsiRNA) or with a control siRNA. Images were taken 24 and 48 hours of incubation with capsules. In samples treated with the apoptosis-inducing RNA, nuclei deformation and condensation typical for apoptotic cells were observed (white arrows). FIG. 6B shows a diagram showing results of WST-1 viability assay for quantification of apoptosis induced by capsules loaded with apoptsiRNA and either maintaining their core or core-free. Treatment of cells with Tween-20 was used as a positive control of cell death.

(7) FIG. 7 shows an application of nanocapsules for transfer of the pro-apoptotic AllStar Death Control siRNA in mesenchymal stem cell (MSCs). FIG. 7A shows confocal images of MSCs treated either with capsules loaded with apoptsiRNA and ctrsiRNA, or transfected with Lipofectamine 2000 using the same siRNAs. To control morphology of intact cells, images of untreated MSCs were taken. All cells were cultured for 48 h, fixed as stained with phalloidin-Alexa488 and DAPI prior microscopy. To control capsule stability, MSCs were treated with capsules stored for 1 year by 4° C. FIG. 7B shows that to quantify apoptosis, WST-1 assay was performed. Highly significant reduction of cell number was measured after application of fresh capsules and capsules stored for 1 year capsules after loaded with AllStar Cell Death siRNA, no significant reduction of cell viability could be detected upon transfection of corresponding amounts of siRNA with Lipofectamine 2000.

(8) FIG. 8 shows extracellular vesicles (EV) isolated from cell culture supernatants of HT1080 cells. FIG. 8A shows that to control vesicle integrity, transmission electron microscopy was performed. Typical for exosomes and other extracellular vesicles, structures of 60-150 nm diameter exhibiting so-called “cap-like” shape which membrane vesicles may acquired during drying procedures, are detectable on the grid, indicating that intact vesicles were isolated from the cell culture supernatants. FIG. 8B shows that to calculate EV number and size distribution, NTA analysis was performed. 2.25×10.sup.9 particle/ml was detected in the preparation.

(9) FIG. 9 shows that the HT1080 cells were stably transfected with the GFP-expressing plasmid and sorted for GFP expression. FIG. 9A shows prior experiments with capsules, GFP expression was controlled by FACS analysis. 98.9% of the HT1080-GFP cells exhibited GFP expression, detected in the FL1 channel as a green fluorescence (red line). The parental HT1080 cells served as a negative control (black line). FIG. 9B shows that additionally, GFP expression was controlled by Western Blot analysis. GFP signal was detected in the HT1080-GFP cells, but not in the HT1080 cells. GAPDH served as a loading control.

(10) FIG. 10 shows the knockdown of Tspan8 and E-Cadherin using nanocapules loaded simultaneously with corresponding siRNAs. The MDA-MB-361 breast cancer cells and breast cancer stem cells (BCSC), which are excessively characterized were treated with capsules containing a mixture of the siRNAs targeting E-Cadherin and siRNAs targeting Tspan8, which were loaded as a mixture between the PARG layers analogous to the application of a single siRNA. As a control, untreated cells and scrambled oligonucleotides were applied. Images were taken 48 hours post treatment. Strong diminishment of the Tspan8- and E-Cadherin-specific staining was observed in both type of cells, supporting that the nanocapsules are universally applicable for both, cell lines and primary cells, e.g. breast cancer stem-like cells. These data demonstrate a way of manipulation of tumors in vivo, including cancer stem cells, also referred as cancer-initiating cells, inaccessible until now with conventional non-viral methods of gene transfer.

(11) FIG. 11 demonstrates the transfer of the full length functional GFP mRNA in breast cancer stem-like cells. To test if nanocapsules can be used for transfer of functional mRNA, GFP mRNA supplemented with RNase Inhibitor was incorporated between the PARG layers. Images were taken 48 hours post-treatment. Green fluorescence was obtained in BCSC cells treated with nanocapsules, whereas the untreated cells did not exhibit green fluorescence.

(12) FIG. 12 shows the transfer of genetic material to primary T cells and CD34+ hematopoietic progenitor cells. To adapt nanocapsules-based gene transfer for application on primary immune cells and hematopoietic progenitor cells, protocol for capsule preparation was slightly changed in order to produce capsules of a smaller size to ensure efficient uptake and low toxicity as described in Example 8.

(13) FIG. 12 shows capsules for transfer of genetic material to tumor and primary cells differ in their size.

(14) A) Scanning electron microscopy of capsules used for siRNA and mRNA transfer in tumor cell lines and primary tumor cells, showing CaCO.sub.3 core (left panel) and the capsules after loading (right panel)

(15) B) For transfer of genetic material to primary immune- and hematopoietic cells smaller capsules were produced using a slightly modified protocol. CaCO.sub.3 core (left panel) was coated with several layers of polymers (right panel).

(16) C) Nanoparticle tracking analysis showing nanocapsules with a size distribution between 50 and 280 nm

(17) FIG. 13 shows the uptake efficiency and viability of CD34 and T cells treated with Rhodamine-labeled capsules. The details of the experiment are disclosed in Example 9.

(18) FIG. 14 shows the activity of CRISPR/Cas9 nuclease upon capsule-mediated delivery in primary T cells assessed by T7E1 assay at the “HEK site 4” locus. Cells were either left untreated (UT), or treated with capsules (cap) containing Cas9 mRNA+gRNA, or nucleofected (nuc) with 5 μg of mRNA encoding Cas9 and 75 pmol gRNA.

DETAILED DESCRIPTION OF THE PRESENT INVENTION

(19) Particularly preferred embodiments of the present invention are shown in the following Examples:

Example 1

(20) First, a new protocol allowing production of small biodegradable capsules with efficient encapsulation of high RNA amounts was established. The capsules are shown in FIG. 1. For this purpose, preparation of a CaCO.sub.3 core was done using a recently developed protocol (FIG. 1A). Salt concentration and duration of stirring conditions were adjusted to produce particles 100-600 nm diameter (FIG. 1B). Then two biodegradable polymers, dextran sulfate sodium salt (DS) and poly-L-arginine hydrochloride (PARG), were assembled using LbL technique to produce biodegradable capsules.

(21) The following materials were used: Anhydrous sodium carbonate, sodium chloride, ethylene glycol, calcium chloride, dextran sulfate sodium salt (DS, MW>70 000), poly-L-arginine hydrochloride (PARG, MW>70 000), Rhodamine B isothiocyanate (MW 536.08), phosphate buffered saline (PBS, 0.01M), calcium chloride dihydrate, ethylenediaminetetraacetic acid disodium salt (EDTA), dimethyl sulfoxide (DMSO), were all obtained from Sigma-Aldrich. RPMI-1640 medium, fetal bovine serum (FBS), was purchased from Thermo-Fischer Scientific. Control siRNA labeled with Alexa 488 (ctrsiRNA-488) and AllStar Death Control siRNA (apoptsiRNA) were purchased from Qiagen.

(22) The capsules were prepared as follows: One ml of 0.33M Na.sub.2CO.sub.3 and 1 ml of 0.33M CaCl.sub.2 were dissolved in 10 mL ethylene glycol (EG) and rapidly mixed under magnetic stirring for 3 h. The final size of the vaterite particles depends strongly on the concentration of the reagents, the solubility of the salts, the reaction time, and the rotation during mixing. The size of CaCO.sub.3 was obtained in the range of 100-600 nm. After 3 h of stirring, the particles were sedimented by centrifugation, resuspended in 1 ml of ddH.sub.2O and stored at 4° C. until further use.

Example 2

(23) Loading of Core Particles

(24) To demonstrate their applicability for regulation of gene expression and their efficiency, the transfer of siRNA molecules into the cells a test model was used. For quantitative analysis of RNA incorporation and release, RNA labeled with AlexaFluor488 dye (ctrsiRNA-488) was applied. Additionally, an external cationic dye, Rhodamine B isothiocyanate (RdnB) conjugated with polymer PARG, was chosen for visualization of capsules by confocal microscopy. To allow simultaneous loading of different payloads and a controlled consecutive siRNA release, a new encapsulation method was developed. The RdnB dye served as a first layer if labeling of capsules was desired, followed by 4 alternating DS and PARG layers.

(25) Employing electrostatic interactions between positively charged PARG and negatively charged oligonucleotides; siRNA was positioned between 2 PARG layers as shown in FIG. 2A. As the last step of capsule preparation, CaCO.sub.3 core was removed with EDTA (FIG. 2A). Imaging of siRNA-containing capsules labeled with RdnB showed a complete overlay between siRNA-Alexa488 (green channel) and RdnB (red channel), indicating highly efficient incorporation of siRNA into the capsules (FIG. 2B). Measurement of RNA concentration revealed 1.25 pmol siRNA/1×10.sup.6 capsules.

(26) RNA and dye encapsulation. Encapsulation of a dye was developed using the layer-by-layer (LbL) technique. The LbL technique is based on the sequential adsorption of oppositely charged molecules, such as polyelectrolytes, onto a charged sacrificial template. For the layers biocompatible polyelectrolytes Dextran Sulfate (DS) 1 mg/ml (2 ml) and Poly-L-arginine hydrochloride (PARG) 1 mg/ml (1 ml) were applied. For preparation of labeled capsules, rhodamine isocyanate (concentration 1 mg/ml) was added as a first layer to CaCO.sub.3 particles diluted in 2 mL of ddH.sub.2O which was conjugates with polymer PARG. Then 2 layers of DS and PARG were coated consequently.

(27) For encapsulation of siRNA a new method was developed. First, 50 μl of the 20 pmol siRNA solution was diluted in 1 ml RNAse-free, DNase-free ddH.sub.2O. Next, the siRNA layers were coated on the PARG layer and covered again with a PARG layer. It is important that the last layer has a positive charge. Next, the core was removed with Ethylenediaminetetraacetic acid (EDTA); capsules were resuspended in 1 ml ddH.sub.2O and final concentration 8×10.sup.8/ml.

(28) The key advantages of the method of the present invention include:

(29) 1) highly efficient incorporation of oligonucleotides from the starting solution into the final formulation with more than 90% of the capsules covered with RNA;

(30) 2) quantitative loading of oligonucleotides/capsule;

(31) 3) a unique possibility to simultaneously use several payloads under controlled conditions, e.g. a drug, filling a CaCO.sub.3 core using a conventional technique, different nucleic acid layers positioned between PARG layers, and functional groups positioned on the top layer, e.g. peptides, ligands, polysaccharides or nanoparticles.

Example 3

(32) Uptake Efficiency and Delivery to Physiological Intercellular Sites

(33) The uptake efficiency of capsules using HT1080 fibrosarcoma cells as a model was assessed. For estimation of a potential impact of capsules on cell viability, confocal microscopy was employed, monitoring nuclear morphology by staining with DAPI and building actin stress fibers by staining the actin filaments with phalloidin. The uptake efficiency was tested using different capsule concentrations: 10 capsules/cell, 20 capsules/cell and 50 capsules/cell. Capsules labeled with RdnB were used for their intracellular visualization; confocal images were taken 18 h after capsules were added to the cell culture medium. By application of 10 and 20 capsules/cell, no residual capsules were detected in the cell culture medium, indicating high uptake efficiency. No evidence of stress fibers or deformation of nuclei indicating toxic effects were observed (FIG. 3A left and middle panels). In contrast, application of 50 capsules/cell resulted in deformation of cell and nuclear shapes (FIG. 3A, right panel, arrow heads), indicating toxic effects as described elsewhere; furthermore, a number of capsules remained in the cell culture medium (FIG. 3A, right panel, arrows).

(34) Cells culture and viability assay. HT-1080 and HT1080-GFP cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS). To measure cytotoxicity and viability, WST-1 assay was performed according to the manufacturers recommendations. Briefly, cells were seeded in a 96-well plate and incubated overnight. After administration of capsules loaded with corresponding siRNAs or treated with Tween-20 used as a positive control for cell death, cells were cultured for 24, 48 or 72 h, respectively as required. WST1 reagent was added to each well and maintained for 4 h. Optical density was measured using TECAN Elisa Reader.

(35) siRNA Transfer by Capsules and Transfection

(36) One day before transfection or treatment with capsules, HT1080 and HT1080-GFP cells were seeded in 4-well or 8-well chamber slides (Ibidi) using 2×10.sup.4 cells in 300 μL of cell culture medium per well and grown overnight to the expected cell density of 60-70%. Capsules were added at concentration 20 capsules/cell and incubated for the desired duration. For transfection Lipofectamine 2000 was used; transfection was performed according to the recommendation of the supplier. Cells were transfected with an amount of siRNA, corresponding to the amount of siRNA loaded in the capsules. Thus, 1.6×10.sup.6 capsules and 20 pmol siRNA were used for 8×10.sup.4 cells for treatment or transfection respectively.

(37) In pursuing the main goal of developing a carrier system mimicking natural extracellular vesicles containing functional RNA, e.g. exosomes or microvesicles, it is assumed that if capsules resembled them, the same intracellular traffic routes to deliver encapsulated RNA to the corresponding physiological intracellular commitment sites are used. To prove this, extracellular vesicles from cancer cell supernatants were isolated by conventional ultracentrifugation, allowing enrichment of exosomes as follows:

(38) Isolation of Cell Culture Derived Extracellular Vesicles

(39) Fibrosarcoma HT1080 cells were cultured in RPMI+10% FBS at 37° C. and 5% CO.sub.2. 36 h prior to harvesting the vesicles produced, medium was changed to serum-free RPMI. Harvested medium was centrifuged for 15 min at 2,000×g, followed by 45 min at 5,000×g and 30 min at 12,000×g. The supernatant was filtered with a 0.2 μm membrane and concentrated in a concentration chamber to achieve a volume of 30 to 50 ml. This concentrated supernatant was centrifuged at 120,000×g for 1.5 h. The resulting supernatant was discarded and the exosome pellet washed with 11.5 ml Sodium Chloride, followed by a second centrifugation step at 120,000×g for 2 h. The supernatant was discarded and the exosome pellet resuspended with sodium chloride with a final volume of 200 μl per cell line.

(40) Transmission Electron Microscopy

(41) The quality of exosomes was controlled by Transmission electron microscopy (TEM). 10 μl of the vesicle preparation were loaded on a 300-mesh copper grid and fixed with 1% glutaraldehyde. Next, they were washed with double distilled water and negatively stained with 10 μl drop of 1% uranyl acetate and washed. Images were taken by the electron microscope (LEO 906 E, Zeiss, Oberkochen, Germany) using SIS software (Olympus, Hamburg, Germany)

(42) Nanoparticle Tracking Analysis

(43) Exosome and Capsule concentration and size distribution were analyzed by nanoparticle tracking analysis (NTA) using the ZetaView system PMX110 (Particle Metrix, Meerbusch, Germany) according to the manufacturer's instructions. Briefly, samples were diluted in filter-sterilized HEPES buffers; using ration 1:500 for exosomes and 1:100 for capsules. Images were recorded at 11 positions and 5 cycles with camera sensitivity 95%; shutter position 70; temperature was monitored manually, ranged from 21 to 22° C.

(44) Staining of Vesicles with PKH26

(45) Staining of vesicles with PKH26 (Sigma-Aldrich) was performed. Briefly, vesicle pellets after ultracentrifugation were resuspend in 200 μl PBS. Then 500 μl of Diluent C provided by the supplier was added to the solution and mixed with 1 μl of PKH dye diluted in 500 μl diluent C. Vesicles in a final volume of 1.2 ml were incubated for 5 min, washed. Remaining dye was removed by centrifugation in 100 kDa centrifugal filter unit (Amicon, Sigma-Aldrich).

(46) After conventional quality controls using electron microscopy and nanoparticle tracking analysis (FIG. 8), the vesicles were labeled with PKH26 membrane dye for their visualization within the cells. Tumor cells were treated with the vesicles and capsules for 8 h. Next, the cells were fixed and stained with DAPI and phalloidin for samples treated with vesicles. Analysis of images by confocal microscopy revealed remarkably similar intracellular localization of capsules and vesicles (FIG. 3B). Both were visualized in the perinuclear regions and endocytic compartments supporting our rationale that nanosized capsules will be delivered to the same intracellular compartments as the vesicles, possibly employing their intracellular routes. Based on current knowledge, one can speculate that in contrast to microcapsules, which have been frequently characterized in previous works and are reported as being internalized mostly by a cholesterol-, and caveolin-dependent pathway and as being located in the cytoplasm, nanocapsules, can, due their smaller size, be encapsulated via clathrin-mediated endocytosis, which is described as one of the main pathways for the internalization of exosomes. However, since not only particle size but also charge, types of recipient cell and perhaps other as yet undefined parameters play a role in determination of the internalization pathways, more efforts will be required to characterize the intracellular traffic routes of nanocapsules in different cell types.

Example 4

(47) Analysis of the Kinetic of siRNA Release

(48) Next, kinetic of RNA release was addressed. As is shown in FIG. 4A, 4 h after treatment, the Rdn-ctrsiRNA-488 capsules were already detected within the cells. RdnB (red color) was fully merged with the siRNA-488 (green color), indicating that the capsules were intact and that the RNA was still entrapped within the capsules (FIG. 4A, left panel, arrows). The fluorescence signal was increased after 24 h, suggesting that more capsules have internalized. Only a portion of RdnB signal was merged with the siRNA-488 signal, indicating capsule degradation and release of the siRNA-488 from the capsules (FIG. 4A, middle panel, arrows).

(49) Electron microscopy. Capsule morphologies were provided by scanning electron microscopy (SEM MIRA II LMU (TESCAN). Capsule suspension was dropped to the silicon surface, dried, coated with gold. SEM observation was carried out using an accelerating voltage of 10 kV. To visualize cells uptake and evaluate capsules, a confocal laser microscopy system was used.

(50) Immunofluorescence. Two days before experiment, 1.5×104 cells/well were seeded in ibidi 8-well μ-slide chamber. On the day of staining, cells were fixed with 4% paraformaldehyde for 5 min at 37° C., washed and permeabilized with 0.1% Triton-X 100. For staining, cytoskeleton phalloidin conjugated with either Alexa488 or Alexa594 fluorophores was applied for 1 h and washed. Next, the nuclei were stained with DAPI for 20 min at room temperature, washed and incubated with Prolong Diamond anti-fade mountant, allowed to heal overnight at room temperature. Images were taken using a Leica confocal microscope (Leica TCS SP2 AOBS) equipped with a HCX PL APO 63× NA 1.4 oil immersion objective. Images for the different fluorophores were scanned sequentially. Further image processing was carried out using image J software.

(51) Image analysis. For quantitative evaluation of green signal intensity, Image J freeware was used. The experiment was done in biological triplicates. For statistical analysis, five images of each delivery method were taken. For each image, intensity distribution graph of the green signal over the area was plotted. Area statistics was calculated for the complete image and the average intensity value was calculated.

(52) A decrease of the fluorescent signal was observed after 48 h of incubation, suggesting that majority of capsules had degraded. Only few large red spots, which could represent agglomerated dye, were detected in the cytoplasm partly colocalized with actin filaments or residual RNA, both stained in this image in green (FIG. 4A right panel, arrows). Quantitative analysis revealed that about 43% of the capsules had degraded after 24 h and about 81% after 48 h (FIG. 4B, C). This indicates that due to the intracellular proteolytic activity, the majority of the capsules had degraded between 24 and 48 h, allowing consecutive release of active RNA molecules within the cells and supporting their sustainable effect and high efficiency, which we addressed in our next experiments.

Example 5

(53) Efficient GFP Knockdown in Cancer Cells

(54) To show the universality of our approach, conventional method of knocking down GFP was employed, stably overexpressed at a high level in HT1080 cells. This is shown in FIG. 9. For this purpose, capsules loaded with a control siRNA (AllStars negative control siRNA labeled with Alexa488 dye, ctrsiRNA-488), and capsules loaded with siRNA specific for GFP (GFPsiRNA) were produced; 20 capsules/cell were used for treatment of the HT1080-GFP cells (FIG. 5A upper panel). Additionally, cells were transfected with Lipofectamine 2000 using the same amount of siRNA, corresponding to 2.5×10.sup.−4 pmol siRNA/cell (FIG. 5A, bottom panel). No residual GFP signal could be detected in the cells treated for 48 h with the capsules containing GFPsiRNA, in contrast to the cells transfected with GFPsiRNA using Lipofectamine 2000. Quantitative analysis based on calculation of the intensity of the green fluorescence signal (FIG. 5B) revealed 80% reduction of green fluorescence in the cells treated with nanocapsules filled with GFPsiRNA and 21% reduction of GFP signal in the cells transfected with Lipofectamine 2000; no unspecific effect was observed by the application of capsules loaded with the control siRNA (FIGS. 5B, C).

(55) These results suggest that biodegradable nanocapsules possess utmost high transfer efficiency of RNA molecules with no toxic effect. Interestingly, by application of comparable biodegradable microcapsules 1-3 μm in diameter considerably higher loading capacity using conventional loading technique into the CaCO.sub.3 core was recently reported. However, to enrich a comparable knockdown efficiency of 80%, 500 pmol/10 capsules/cell siRNA were required as described previously, which is 2×10.sup.6 fold more than used in the current work applying 2.5×10.sup.−4 pmol siRNA/20 capsules/cell. This comparison argues strongly that microcapsules definitely possess a higher loading capacity and may be favorable for delivery of substances to target cells if a high amount of a payload is the primary goal. However, for transfer of small amounts of regulatory molecules such as RNA or DNA, application of biodegradable nanocapsules allowing highly efficient transfer of payloads to target cells may be favorable.

Example 6

(56) High Efficiency of Capsule-mediated Cancer Cell Death by Transfer of Pro-apoptotic siRNAs

(57) Increased resistance to apoptosis, enabling survival under abnormal growth stimulation and various forms of cellular stress, such as DNA damage, hypoxia, or nutrient deprivation are among the hallmarks of cancer cells. Consequently, strategies for a specific targeting of cancer cells and apoptosis induction may provide a rational basis for development of new therapeutic tools, for example, by transfer of apoptosis-inducing agents into tumor cells. Therefore, we tested whether transfer of corresponding siRNAs by capsules may be sufficient to induce apoptosis in cancer cells. For this purpose, capsules were loaded with AllStars Cell Death Control siRNA (Qiagen) containing highly potent validated siRNAs targeting ubiquitous cell survival genes. To allow quantification of transfer efficiency and functionality, cell phenotype was controlled after 24 and 48 h of incubation with capsules by staining of the cells with a tubulin-specific antibody for cytoskeleton and DAPI for nuclei. As it is shown on the FIG. 6A, after 24 h of treatment both viable cells and fragmented nuclei characteristic for apoptosis could be detected (FIG. 6A, upper panel, white arrows), whereas only few cells with fragmented nuclei characteristic for apoptosis could be detected after 48 h (FIG. 6A, bottom panel, white arrows). This result shows that transfer of siRNA by capsules is sufficient to induce apoptosis in cancer cells and is consistent with the observation of capsules degradation. This shows that the majority of the capsules degrade between 24 and 48 h, suggesting an enhancement of siRNA effect within this time frame.

(58) A quantitative analysis using a WST-1 viability assay was performed. Additionally, efficiency of capsules still containing a CaCO.sub.3 core and core-free capsules was compared (FIG. 6B). The data demonstrate that treatment of cells with capsules containing a control siRNA exhibited no significant impact on cell viability. Application of capsules with a core led to 45% reduction of cells viability, whereas application of core-free capsules loaded with apoptotic siRNA resulted in 73% reduction of cell viability, which is comparable with the effect of Tween-20 detergent, disrupting cell membranes and used standard wise as a positive control for cell death, and showing 83% efficiency if measured by WST1 assay (FIG. 6B). These results further support efficiency and usability of biodegradable core-free nanocapsules for transfer of functional RNA molecules. By application in a ratio of 20 capsules/cells corresponding to 2.5×10.sup.−4 pmol siRNA/cell, 80% functional efficiency can be reached, as demonstrated by GFP knockdown and apoptosis induction, showing no unspecific or toxic effects, which, based on the current state of technology, is one of the most efficient tools for targeted delivery of regulatory RNA.

Example 7

(59) Efficient Apoptosis Induction by Transfer of Pro-apoptotic siRNA to the Mesenchymal Stem Cells and Stability Test of siRNA in the Capsules

(60) The delivery system of the present invention was tested for RNA delivery to mesenchymal stem cells (MSCs) due to the therapeutic applications envisaged for these cells. Consequently, development of an efficient, easily accessible technique allowing MCSs manipulation that is compatible with GMP (good manufacturing practice) is exceptionally relevant. Therefore, polyelectrolyte nanocapsules were tested for RNA delivery into MSCs using AllStars Cell Death Control and AllStars negative control siRNAs as described above.

(61) Forty eight hours after treatment, MSCs were stained with phalloidin and DAPI for visualization of treatment effect using confocal microscopy. Application of apoptosis-inducing siRNA resulted in a strong reduction of cell number; remaining cells exhibited reduction of cytoplasma volume and nuclei fragmentation (FIG. 7A, upper panel, white arrows). Additionally, conventional transfection of siRNA using Lipofectamine 2000 with equal amount of siRNA/cells was performed as a control. A considerable change in cell number was not observed (FIG. 7A, middle panel).

(62) Because stability counts as one of the important parameters for choice of delivery method, functionality of capsules stored for 1 year at +4° C. was additionally tested. As is shown in FIG. 7A, MSCs treated with the capsules and stored for 1 year, exhibited similar phenotype as MSCs, treated with freshly prepared capsules (FIG. 7A, bottom and upper panels, respectively). Quantitative analysis of cell viability revealed non toxicity of capsules loaded with a control siRNA, and over 80% reduction of MSC viability through treatment with capsules loaded with AllStars Cell Death Control siRNA. Both were freshly prepared after 1 year of storage, which indicates that along with such advantages as cost-, and functional efficiency, the capsules offer excellent payload stability and stable exploitation.

Example 8

(63) Capsules Preparations for Transfer of RNA in Primary Cells

(64) CaCO.sub.3 nanoparticles were prepared as described with some modification. Firstly, gelatin (3 g) was dissolved in ddH.sub.2O (50 ml) and heated to 90° C. After that, gelatin solution was rapidly mixed upon magnetic stirring with 99% Glycerol (50 ml). Taking into account that the size of valerit crystals is strictly dependent on salt concentration, 0.1M Na.sub.2CO.sub.3 (10 ml) and 0.1M CaCl.sub.2 (10 ml) were mixed and stirred for 24 h. The fabricated particles were sedimented by ultracentrifugation at 40 000×g and washed with hot water (70° C.). Coating of particles with layers with and without RNA was undertaken as described previously.

Example 9

(65) Transfer of PBMCs with Nanocapsules Comprising gRNA and Cas Protein

(66) Peripheral blood mononuclear cells (PBMCs) were isolated using phase separation and then frozen in liquid nitrogen until used. PBMCs were thawed 4 days prior to use and let to recover for 24 hours to deplete the monocytes in RPMI complete medium [RPMI 1640 medium supplemented with 10% fetal calf serum, penicillin (100 U/ml), streptomycin (100 mg/L) and HEPES (10 mM). Then, T cells or CD34+ cells were harvested from the supernatant; T-cells were activated using anti-CD2/CD3/CD2 antibodies and cultured with RPMI complete medium supplemented with 100 U/ml of IL-2, 25 U/ml of IL-7 and 50 U/ml of IL-15 for 3 days before treatment. At day 3, 1×10.sup.6 activated T cells and CD34+ cells were treated with nanocapsules loaded with Rhodamine, in order to define concentration of capsules allowing maximal uptake by minimal toxicity. For T cells 10 capsules/cells and for CD34 cells 5 capsules/cells were considered as an optimal concentration.

(67) To test applicability of nanocapsules to manipulate primary T cells, the cells were isolated as described above; the nanocapsules were loaded with Cas9 mRNA and a guide RNA (gRNA) targeting the “HEK site 4” genomic locus (PMID: 25513782). As a positive control, 1×10.sup.6 activated T cells were nucleofected with 5 μg of mRNA encoding Cas9 and 75 pmol of gRNA targeting the “HEK site 4” locus using the 4D nucleofector according to the manufacturer recommendation (P3 kit, EO-115 program). After transfer of capsules or nucleofection, respectively, T cells were recovered in 96-well plates for 4 days before assessing the nuclease cleavage activity at the target locus.

(68) The activity of the nuclease was assessed by measuring the extent of non-homologous end joining (NHEJ)-mediated mutagenic repair at the target site using the mismatch-sensitive T7 endonuclease 1 (T7E1) assay. At day 4 post-transfection, cells were harvested and genomic DNA was extracted using direct lysis buffer mixed with proteinase K (20 mg/ml). An amplicon encompassing the nuclease target site in the “HEK site 4” locus was generated by PCR using the primer pair (5′-AGGCAGAGAGGGGTTAAGGT-3′ (SEQ ID NO:1) and 5′-GGGTCAGACGTCCAAAACCA-3′) (SEQ ID NO:2). Afterwards, amplicons were purified using QlAquick PCR Purification Kit and subjected to digestion with T7E1 as previously described (PMID: 21813459). Cleaved fragments are an indication for the activity of the nuclease at the intended target site compared to the un-transfected (UT) sample were no cleaved fragments can be observed. As shown in FIG. 14, distinct bands can be detected in the sample treated with capsules (cap) containing the CRISPR/Cas9 RNAs, thus proving evidence for efficient capsule-mediated RNA delivery into primary T cells. A similar pattern can also be detected in the positive control samples, where cells were subjected to nucleofection (nuc).