Determining Capsule Specificity for Specific Cell Types

Abstract

The task of the invention is therefore making available transfer capsules that are taken up by the target cell type and permanently or transiently modify the target cell, without exerting any toxic effects on the cell during this process.

The solution according to the invention consists of the use of monodisperse cores, so as to produce polyelectrolyte nanocapsules having cell-specific sizes from them. The sizes for hematopoietic cells are in a range of 20-80 nm, preferably in a range of 40-60 nm. In this regard, the sizes of the particles must be in a very narrow range, so as to prevent toxic effects from occurring. In order to keep the toxicity of the nanocapsules low, it is furthermore important to remove the nanoparticles around which the capsules are built up (cores) before use. Methods in this regard are known from the state of the art (for example dissolution by means of EDTA).

A further task is the stabilization of the transfer capsules.

The solution according to the invention consists in the modification of the capsules, the layers and/or the cargo to be packed, by means of functional groups, which allows stabilization and thereby long-term storage at room temperature.

The third task is the targeted introduction of the transfer capsules.

The solution according to the invention is a functionalization of the layers by way of chemical modifications and/or supplementing of the layers with antibodies, proteins or peptides.

Claims

1. A nanocapsule suitable for introduction of target molecules of primary cells and stem cells, wherein the capsules consist of at least two biodegradable layers, between these at least two layers, at least one nucleic acid, protein and/or small molecule or other target molecule is situated, the nanoparticle cores have a firmly defined size with a variance of maximally 25%, likewise the nanoparticles/cores can be separated by charge in addition to their size, as needed, the capsule surface and/or individual layers within the capsule can be modified using functional groups. likewise cargo elements between the layers or the layers themselves can be modified.

2. The nanoparticle cores according to claim 1, wherein the nanoparticles having a fixed size are selected from the size range of 20-80 nm.

3. The nanoparticle cores according to claim 2, wherein the nanoparticles are selected from the size range of 30-60 nm.

4. A method for introducing cargo into primary cells or cell lines, wherein the nanocapsules are brought into contact with cells, wherein the nanoparticle cores have fixed, defined size with a variance of maximally 25%, the nanocapsules consist of at least two biodegradable layers, between these at least two layers, at least one nucleic acid, protein, small molecule and/or chemotherapeutic medications is/are situated, one or more layers or cargo can be modified by means of binding of the functional groups, so as to regulate the stability during storage or the degradation time in the cells.

5. The nanocapsules according to claim 1, modified by means of functional groups for stabilization purposes.

6. The nanocapsules according to claim 1, for targeted transfer.

7. The method according to claim 5, wherein the cells are healthy or malignant cells having a hematopoietic origin.

8. The method according to claim 6, wherein the cells are transiently transfected.

9. The method according to claim 6, wherein the cells are transfected in a stable manner.

10. The method according to claim 6, wherein the cells are embryonic cells or iPS cells.

11. A method for producing nanocapsules, wherein the cores for building up the nanocapsules are monodisperse nanoparticles, and that the nanocapsules are charged with nucleic acids, proteins, small molecules or chemotherapeutic medications, and are used modified with functional groups or not modified.

Description

EXAMPLE 1: FRACTIONATION OF CALCIUM CARBONATE NANOPARTICLES

[0053] Calcium carbonate nanoparticles were procured from SkySpring Nanomaterials. The average particle size was 15-40 nm. Using the particles, a 10 mg/ml suspension in PBS was produced. Ultrasound was used for 5 minutes so as to separate the particles. They were then purified by size by means of fractionated centrifugation in an Eppendorf centrifuge. 2 ml suspension were centrifuged at 500 RCF, 1000 RCF, 2000 RCF, 5000 RFC [sic], 10,000 RCF, 15,000 RFC and 20,000 RCF. This process started at the lowest RCF, the top fraction was removed, and centrifugation continued at the next higher RCF. The pellet of the centrifugation was subsequently measured. The 500 RCF fraction was discarded (particles were aggregated or too large).

EXAMPLE 2: PRODUCTION OF THE POLYELECTROLYTE NANOCAPSULES

[0054] In order to coat the nanoparticles, dextran sulfate (as a sodium salt) and poly-L-arginine hydrochloride were used as described in WO 002019020665 A1. During this process, it is possible to introduce one or more nucleic acids (plus additional biomolecules) between the layers. The nucleic acids adhere to the capsule wall by means of electrostatic binding, so that incubation of the capsules with the biomolecules is sufficient. The method is described in WO 002019020665 A1.

[0055] It was possible to modify these chemically, so as to change the stability during storage and the decomposition time in the cell. Chemical modifications by means of the use of one or more functional groups, such as hydrocarbons, groups that contain oxygen or nitrogen, groups that contain sulfur (S/SH), N/NH, and other groups that contain P were introduced and tested. The chemical modifications showed, in preliminary experiments, that it was possible to guarantee storage for a longer period of time. Furthermore, it was not possible to store the capsules at room temperature for a time interval, as well, and this represents a significant improvement for transport (freight), in particular, since it will lower costs and administrative effort (key word: customs regulations involving dry ice and refrigerated products).

EXAMPLE 3: PURIFICATION OF THE FINISHED NANOCAPSULES BY MEANS OF TANGENTIAL FLOW FILTRATION

[0056] Finished nanocapsules were then purified to remove larger nanocapsules that might be present, using a KrosFlo Research Ili System with a 50 nm filter module. Particles having a size of more than 50 nm were retained. For this purpose, 50 ml of a 10 mg/ml suspension of the nanocapsules in PBS was produced. This was filtered according to the manufacturer's instructions.

EXAMPLE 4: SEPARATION AFTER ASYMMETRICAL FLOW FIELD FRACTIONATION (AF4)

[0057] Finished nanocapsules were then purified to remove larger nanocapsules that might be present, using an Eclipse AF4 from Wyatt Technology. In this process, particles were separated according to charge and size. For this purpose, 50 ml of a 10 mg/ml suspension of the nanocapsules in PBS was produced. This was separated according to the manufacturer's instructions.

EXAMPLE 5: TRANSFECTION OF PRIMARY CELLS

[0058] For hematopoietic cells, above all, nanocapsules having a size of 40-80 nm are clearly more advantageous. Protein, DNA, mRNA, miRNA and siRNA were used as cargo for nanocapsules having a size of 50 nm to 80 nm. CD 34+ hematopoietic stem cells, CD4+ and CD8+ T cells were incubated with the capsules for 48 hours. In this process, 10 capsules/cell were used. Successful introduction was monitored by means of confocal microscopy in the case of the fluorescence-marked capsules and PCR.

EXAMPLE 6: TRANSFECTION OF FURTHER PRIMARY CELLS

[0059] For embryonic stem cells as well as for induced pluripotent stem cells (iPS-cells), nanocapsules having a size of 50-120 nm were produced. Protein, DNA, mRNA, miRNA and siRNA were used as cargo for nanocapsules having a size of 50 to 120 nm. Embryonic stem cells and iPS-cells were incubated with the capsules for 48 hours. In this process, 20 capsules/cell were used. Successful introduction was monitored by means of confocal microscopy in the case of the fluorescence-marked capsules and PCR.

EXAMPLE 7: STABILIZATION AND STORAGE AT ROOM TEMPERATURE (RT) OF CAPSULES

[0060] For all the capsules listed in the above examples, further modifications of the cores or of the layers were carried out by means of introducing functional groups such as hydrocarbons, groups that contain oxygen, nitrogen, groups that contain sulfur (S/SH), N/NH, and other groups that contain P, and thereby it was possible to achieve stabilization of the capsules and thus the desired goal of storage at RT.

EXAMPLE 8: INFLUENCING THE NATURAL DECOMPOSITION OF THE CAPSULES

[0061] By way of the modifications introduced in Example 2 and 7, of functional groups on the core as well as on the layers, it was possible to influence the natural decomposition of the capsules after introduction into the corresponding target cell. This resulted in decomposition times per layer at 4 hours in mesenchymal and tumor cell lines, for example, as well as decomposition times of 24 hours in cells having a hematopoietic origin.

EXAMPLE 9: TARGETED INTRODUCTION: USING ANTIBODIES, PEPTIDES, PROTEINS

[0062] For further specification of the targeted introduction of cargo into desired target cells, corresponding antibodies, peptides or proteins were introduced into the outermost layer of the capsules.

EXAMPLE 10: STABILIZATION OF CAPSULES HAVING SMALL MOLECULES AS THE CARGO

[0063] In order to stabilize the capsules that are charged with small molecules or chemotherapeutic medications, the small molecules or chemotherapeutic medications were immobilized in the capsules by means of click chemistry, and thereby diffusion effects were reduced.