EP4076409A2 - Détermination de la spécificité capsulaire de types cellulaires spécifiques - Google Patents

Détermination de la spécificité capsulaire de types cellulaires spécifiques

Info

Publication number
EP4076409A2
EP4076409A2 EP20859677.5A EP20859677A EP4076409A2 EP 4076409 A2 EP4076409 A2 EP 4076409A2 EP 20859677 A EP20859677 A EP 20859677A EP 4076409 A2 EP4076409 A2 EP 4076409A2
Authority
EP
European Patent Office
Prior art keywords
cells
nanocapsules
layers
capsules
size
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP20859677.5A
Other languages
German (de)
English (en)
Inventor
Irina Nazarenko
Holger Klapproth
Arnold Martin
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Capco Bio GmbH
Albert Ludwigs Universitaet Freiburg
Original Assignee
Capco Bio GmbH
Albert Ludwigs Universitaet Freiburg
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Capco Bio GmbH, Albert Ludwigs Universitaet Freiburg filed Critical Capco Bio GmbH
Publication of EP4076409A2 publication Critical patent/EP4076409A2/fr
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5169—Proteins, e.g. albumin, gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes

Definitions

  • WO 002019020665 A1 Polyelectrolyte nanocapsules as a transport medium for biomolecules and “small molecular compounds” in cells are described in WO 002019020665 A1. These can be built up around solid or liquid cores using the so-called layer-by-layer technique. The size of the nanocapsules depends on the size In addition to the surface design, the size of the nanoparticles is also important for the selection and the efficiency of uptake in specific cells. In WO 002019020665 A1 it is described that the capsules are formed around cores made of calcium carbonate, these cores being formed by a Precipitation reaction can be formed from a solution of calcium nitrate and sodium carbonate. Various solvents influence the precipitation and ensure different sizes of the precipitated calcium carbonate cores.
  • the size of the particles can be precisely set to approx. 10 to a maximum of 20 nm, so that uptake in undesired cells can be reduced.
  • hematopoietic cells can be transfected primarily with nanocapsules with a size of 20-60 nm, and sizes of 100-200 nm are preferred for tumor cells. That is, in order to avoid unspecific effects due to unspecific intake, the nanocapsules must be the right size.
  • Another significant problem in the art is the toxicity of particles above 100 nm to cells of hematopoietic origin such as CD34 + hematopoietic progenitor cells and T cells. So far, such cells can only be transduced or electroporated through the use of viruses. With all the problems that come with it.
  • CONFIRMATION COPY Stability can be improved significantly.
  • a cell- or organ-specific introduction can be achieved via these modifications.
  • the particles produced so far in the bottom-up process have the problem that they have a broad size distribution and the production process is very variable.
  • the process is therefore not suitable for the technical production of nanoparticles of the same size.
  • particles produced in the top-down process are easy and inexpensive to obtain, but have the problem of non-uniform shapes.
  • the first object of the invention is thus to provide a capsule which is taken up by the target cell type and which permanently or transiently modifies the target cell without exerting toxic effects on the specific target cell type.
  • the second task is to stabilize long-term storage through the chemical modifications mentioned above.
  • the capsules according to the invention consist in the use of monodisperse nanoparticles in order to produce polyelectrolyte nanocapsules with cell-specific sizes and, if required, chemically modified, the sizes for hematopoietic cells being in a range of 20-80 nm, preferably in a range of 40-60 nm, for epithelial cells and non-hematopoietic cells of 60-280 nm as described in W00020199020665.
  • the sizes of the particles must be in a very narrow range in order to prevent toxic effects from occurring.
  • Biodegradable polymers such as dextran and poly-L-arginine, which are used for capsule construction, can be chemically modified as required (by attaching functional groups such as hydrocarbon, oxygen, nitrogen, sulfur or phosphate-containing functional groups; Methods for this are known from the prior art) in order to prevent the spontaneous aggregation and the spontaneous cargo diffusion of the capsules and to improve the stability of the capsule.
  • functional groups such as hydrocarbon, oxygen, nitrogen, sulfur or phosphate-containing functional groups
  • nanoparticles and nanocapsules with a narrow, defined size distribution of ⁇ 15 nm or optionally ⁇ 25% around the mean value, preferably ⁇ 10 nm (or ⁇ 20% around the mean value) are referred to as monodisperse.
  • the problem of the size of the cores cannot be solved by using mechanically produced nanoparticles sorted by size. For example, by grinding calcium carbonate in ball mills, nanoparticles with a size of 15-60 nm can be generated and then by fractionation (by sedimentation or centrifugation) uniform fractions ( ⁇ 10 nm) can be generated.
  • These particles can then be coated with polymers in a layer-by-layer process.
  • the non-uniform shape of these particles is, however, disruptive because it disrupts the sorting of the particles. Only the further purification of these particles, for example by cross-flow filtration after the cores have been dissolved, enables a sufficiently precise size selection.
  • Nanocapsules can be produced using the well-known layer-by-layer method.
  • Molecules such as chemotherapeutic drugs, small molecules and macromolecules such as nucleic acids and proteins can be inserted between the layers, which can then be released in the cell.
  • chimeric T-cell receptors can be generated by introducing DNA-loaded nanocapsules into T-cells.
  • RNA molecules can also be introduced into cells in order to produce only a transient modification of the cells.
  • all of the named molecules and macromolecules can be introduced either individually or in any imaginable combination. This property enables, for example, the transfection of CRISPR / Cas complexes.
  • the chemical modifications mentioned can be used both for the capsule polymers and for the cargo in order to regulate the stability and consequently the release in the target cells.
  • Primary cells are cells that have been taken from the body and have not lost their tissue-specific properties through cultivation. Primary cells are, for example, but not exclusively, stem cells, germ cells, hematopoietic cells, tumor cells, or else mesenchymal stem cells, tissue cells in vivo and ex vivo.
  • stem cells that can differentiate into different cell types or tissues are generally referred to as stem cells. Depending on the type of stem cell and how it is influenced, they have the potential to develop in any tissue (embryonic stem cells) or in certain specified types of tissue (adult stem cells / precursors).
  • Polycations and polyanions which alternately form the layers of the nanocapsules, are suitable polymers for building up layers of the nanocapsules. Due to the opposing charge, the polymers can form the layers through self-organization. After the formation of each layer, the unused portion of the polymers has to be separated from the nanocapsules.
  • Polymers within the meaning of the invention also include classical polymers and copolymers as well
  • Biopolymers such as macromolecules with an ordered structure of amino acids, nucleic acids and poly- or oligosaccharides.
  • polycations are: polyethylenimine (PEI), chitosan (CS), poly (L-lysine) (PLL),
  • PAA Poly (amido amines) s
  • polyanions examples include poly (methacrylic acid) (PMA) poly (N-vinylpyrrolidone) (PVP) tannic acid (TA) poly (2-n-propyl-2-oxazoline) s (PnPropOx)
  • Substances that can be used to stabilize the nucleic acids are substances that are applied together with the nucleic acids in the capsules or substances that diffuse into the capsules and into the lumen of the capsules through incubation with the finished capsules and there until the capsules enter the Cells linger at least partially. Examples of such substances are:
  • nanoparticles that are suitable as cores for the production of nanocapsules:
  • Iron nanoparticles including magnetites
  • the surface of the nanoparticles can be modified by further groups in order to improve the penetration of the target cells.
  • groups are e.g. folic acid groups, COOH groups, NH3 groups. These groups are also suitable for bioconjugation in order to bind specific binding molecules such as ligands and receptors including antibodies to them.
  • Calcium carbonate nanoparticles were purchased from SkySpring Nanomaterials. The average particle size was 15-40 nm. A 10 mg / ml suspension in PBS was created with the particles. Ultrasound was used for 5 minutes to separate the particles. These were then purified by size by fractionated centrifugation in an Eppendorf centrifuge. 2 ml of suspension were centrifuged at 500 RCF, 1000 RCF, 2000 RCF, 5000 RFC, 10000 RCF, 15000 RFC and 20000 RCF. It started with the lowest RCF, removed the supernatant and centrifuged with the next higher RCF. The centrifugation pellet was then measured. The 500 RCF fraction was discarded (particles were aggregated or too large).
  • dextran sulfate as sodium salt
  • poly-L-arginine hydrochloride as described in WO 002019020665 A1.
  • nucleic acids plus additional biomolecules
  • the nucleic acids adhere to the capsule wall by electrostatic bonding, so that it is sufficient to incubate the capsules with the biomolecules. The procedure is described in W0002019020665A1.
  • Example 3 Purification of the Finished Nanocapsules by Tangential Flow Filtration Finished nanocapsules were then purified using a KrosFlo Research 11 / system with a 50 nm filter module to remove any larger nanocapsules that might be present. Particles larger than 50 nm in size were retained. For this purpose, 50 ml of a 10 mg / ml suspension of the nanocapsules in PBS were produced. This was filtered according to the manufacturer's instructions.
  • Finished nanocapsules were then cleaned up with an Eclipse AF4 from Wyatt Technology to remove any larger nanocapsules that might be present. Particles were separated according to charge and size. For this purpose, 50 ml of a 10 mg / ml suspension of the nanocapsules in PBS were produced. This was separated according to the manufacturer's instructions.
  • nanocapsules with a size of 40-80 nm are significantly more advantageous.
  • Protein, DNA, mRNA, miRNA and siRNA were used as cargo for nanocapsules between 50 nm and 80 nm in size.
  • CD 34+ hematopoietic stem cells, CD4 + and CD8 + T cells were incubated with the capsules for 48 hours. 10 capsules / cell were used. Successful introduction was checked by means of confocal microscopy in the case of the fluorescence-labeled capsules and PCR.
  • iPS cells For embryonic stem cells as well as for induced pluripotent stem cells (iPS cells) nanocapsules with a size of 50-120 nm were produced. Protein, DNA, mRNA, miRNA and siRNA were used as cargo for nanocapsules 50 to 120 nm in size. Embryonic stem cells and iPS cells were incubated with the capsules for 48 hours. 20 capsules / cell were used. Successful introduction was checked by means of confocal microscopy in the case of the fluorescence-labeled capsules and PCR.
  • Example 7 Stabilization and storage at room temperature (RT) of capsules.
  • Example 9 targeted introduction: with the help of AK, peptides, proteins
  • Example 10 Stabilization of capsules with small molecules as cargo
  • the small molecules or chemotherapeutic drugs were immobilized in the capsules using click chemistry, thus preventing diffusion effects.

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  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Biomedical Technology (AREA)
  • Nanotechnology (AREA)
  • Optics & Photonics (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Physics & Mathematics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Inorganic Chemistry (AREA)
  • Medicinal Preparation (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention vise à fournir une des capsules de transfert qui sont absorbées par le type de cellules cibles et modifient de manière permanente ou transitoire la cellule cible, sans exercer pour autant d'effets toxiques sur la cellule. A cet effet, la solution selon l'invention consiste à utiliser des noyaux monodispersés, de manière à en dériver des nanocapsules à polyélectrolyte ayant des dimensions spécifiques de cellules, les dimensions de cellules hématopoïétiques se situant dans une plage comprise entre 20 et 80 nm, de préférence dans une plage comprise entre 40 et 60 nm. Les dimensions des particules doivent de fait se situer dans une plage très étroite de manière à empêcher l'émergence d'effets toxiques. Pour maintenir la toxicité des nanocapsules la plus faible possible, il est important d'éliminer les nanoparticules formées (noyaux) autour des capsules avant l'utilisation. Des procédés appropriés sont connus de l'état de la technique (par ex. dissolution par acide éthylène-diamine-tétracétique). L'invention a pour autre objectif de stabiliser les capsules de transfert. Selon l'invention, la solution consiste à modifier les capsules, les couches et/ou le cargo à emballer par des groupes fonctionnels, ce qui a pour effet de permettre une stabilisation et par conséquent un stockage de longue durée à température ambiante. L'invention a pour autre objectif encore d'introduire les capsules de transfert de manière ciblée. La solution selon l'invention consiste à fonctionnaliser les couches par des modifications chimiques et/ou à compléter les couches par des anticorps, des protéines ou de peptides.
EP20859677.5A 2019-12-19 2020-12-17 Détermination de la spécificité capsulaire de types cellulaires spécifiques Pending EP4076409A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP19000577 2019-12-19
PCT/EP2020/000212 WO2021121646A2 (fr) 2019-12-19 2020-12-17 Détermination de la spécificité capsulaire de types cellulaires spécifiques

Publications (1)

Publication Number Publication Date
EP4076409A2 true EP4076409A2 (fr) 2022-10-26

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ID=69167559

Family Applications (1)

Application Number Title Priority Date Filing Date
EP20859677.5A Pending EP4076409A2 (fr) 2019-12-19 2020-12-17 Détermination de la spécificité capsulaire de types cellulaires spécifiques

Country Status (5)

Country Link
US (1) US20230014648A1 (fr)
EP (1) EP4076409A2 (fr)
JP (1) JP2023507218A (fr)
CN (2) CN114828835A (fr)
WO (1) WO2021121646A2 (fr)

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP3954522B2 (ja) * 2003-04-18 2007-08-08 日清紡績株式会社 生物学的活性物質を固定化した素子
KR101021788B1 (ko) * 2008-08-21 2011-03-17 한국지질자원연구원 비즈 밀링을 이용한 탄산칼슘 나노입자의 제조방법
US20120061608A1 (en) * 2010-09-10 2012-03-15 Hitachi Maxell, Ltd. Functional particle with rough-surfaced polymer coating
US10465189B2 (en) * 2013-12-13 2019-11-05 Nanyang Technological University Multilayered nanoparticle and methods of manufacturing and using the same
JP6588039B2 (ja) * 2014-05-28 2019-10-09 エボニック レーム ゲゼルシャフト ミット ベシュレンクテル ハフツングＥｖｏｎｉｋ Ｒｏｅｈｍ ＧｍｂＨ ナノ粒子
US11253482B2 (en) * 2017-07-26 2022-02-22 Albert-Ludwigs-Universitaet Freiburg Biodegradable multilayer nanocapsules for the delivery of biologically active agents in target cells

Also Published As

Publication number Publication date
WO2021121646A3 (fr) 2021-08-12
WO2021121646A2 (fr) 2021-06-24
CN114828835A (zh) 2022-07-29
CN119770457A (zh) 2025-04-08
JP2023507218A (ja) 2023-02-21
US20230014648A1 (en) 2023-01-19

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