KR-20260062949-A - Microparticles loaded with enzymes and nanozymes

Abstract

The present invention relates to a microcapsule composition and method for encapsulating gas-regulating enzymes or nanozymes with unprecedented concentrations and stability, thereby enabling the control of local gas concentrations. This system can replace expensive incubators/chambers and is the first system capable of generating spatiotemporal gas gradients within a limited volume. The microcapsules can be widely applied in tissue culture, bioprinting, in vivo applications, and industrial uses.

Inventors

• 킬리언 크리스토퍼 앨런
• 몰리 토마스

Assignees

• 뉴사우스 이노베이션스 피티와이 리미티드

Dates

Publication Date

20260507

Application Date

20230901

Claims (20)

1. As a group of microcapsules, the microcapsules are It comprises gelatin coated with a polydopamine nanoscale layer, and The size is 1 µm to 10 mm, and A group that encapsulates a payload.
2. In claim 1, the group of microcapsules having an average pore size of about 1 nm.
3. A group in which the microcapsules exclude proteases, in paragraph 1 or 2.
4. A group according to any one of claims 1 to 3, wherein the group is capable of withstanding a shear stress value of at least 10 kPa.
5. A group in which, in any one of claims 1 to 4, the payload is at least one enzyme.
6. In claim 5, the group comprising at least one enzyme including glucose oxidase and catalase.
7. A group in which the payload is nanoceria, in any one of claims 1 to 4.
8. A hydrogel comprising a group according to any one of claims 1 to 7.
9. A pharmaceutical composition comprising a group according to any one of claims 1 to 7.
10. A solid or semi-solid matrix comprising a group according to any one of claims 1 to 7.
11. As a method for manufacturing a group of microcapsules, i) a step of adding gelatin and a payload to an oil bath to form an emulsion containing loaded microcapsules; ii) a step of washing the loaded microcapsules; iii) a step of forming a polydopamine coating on the loaded microcapsules by adding dopamine hydrochloride; iv) A method comprising the step of washing the coated, loaded microcapsules.
12. A method according to claim 11, further comprising shaking or stirring in step i).
13. A method according to claim 11 or 12, further comprising shaking or stirring in step iii).
14. A bioprinting method comprising printing a group according to any one of claims 1 to 7.
15. In paragraph 14, the method in which the above group is in a jammed state.
16. A method in which the above group is combined with a microgel in claim 14 or 15.
17. A method for reducing the oxygen concentration in an environment, comprising adding a group according to any one of claims 1 to 6 to the environment.
18. In claim 17, the above environment is a cell culture medium in a test tube.
19. In paragraph 17, the method wherein the environment is an in vitro tissue sample or an organoid.
20. In paragraph 17, the method wherein the above environment is localized within the body of the subject.

Description

Microparticles loaded with enzymes and nanozymes Statement on Federally Funded Research This study was conducted with the support of the National Cancer Institute (#R01CA251443) of the National Institutes of Health (NIH) and the Australian Research Council (#DP210103654). The rights to this invention belong to the government. Technology field The present invention relates to microcapsules loaded with active substances such as enzymes or nanozymes, which allow small molecules to diffuse into and out of the microcapsules. The microcapsule layer protects the payload, preventing the ingress of proteases and other degrading agents. This system is inexpensive, easy to manufacture, and possesses unprecedented loading capacity, and can be used in a number of forms, including deposition in hydrogels, granular solids for 3D bioprinting, and injectable biomaterials. Oxygen is one of the most important molecules sustaining life on Earth. For humans and other mammals, oxygen tension plays a critical role not only in tissue development and the maintenance of normal tissue function but also in pathological processes such as tumor progression. A state of low oxygen concentration within tissues is called hypoxia, and hypoxic conditions mediate the transcriptional activity of hypoxia-inducible factor (HIF). This factor regulates gene expression associated with numerous functional activities, including angiogenesis, cell migration, differentiation, metabolism, and apoptosis. These HIF proteins are sensitive to local oxygen tensions, which vary significantly within and between various tissues in the body. While oxygen saturation in air is approximately 20%, circulating blood concentration is about 5%, and can drop as low as 1% in various tissues such as bone marrow and cartilage. These figures stand in stark contrast to the 18.6% oxygen level in cell culture incubators, which induces cellular hyperoxia in vitro. For decades, finding a way to easily and effectively fine-tune oxygen concentration in in vitro cultures has remained a difficult challenge. Currently, the standard method for mimicking in vitro hypoxic environments is the use of hypoxic incubators and hypoxic chambers. These devices limit the amount of diffusible oxygen within the cell culture environment by controlling the atmospheric oxygen concentration. However, these tools are difficult to control precisely. They are expensive, making them inaccessible to many researchers, and the oxygen concentration resets when the device is opened. To overcome these issues, researchers have been studying methods to create hypoxic environments using chemical and enzymatic approaches. While existing systems allow for more precise control of local oxygen concentration, their specialized methodologies may limit their application to local hydrogel networks. To broaden the utility of this concept, we sought an approach applicable to any hydrogel system while leveraging the benefits provided by enzymatic oxygen control. This enables long-term control of high oxygen concentrations in any 2D or 3D cell model without compromising cell-matrix interactions. To achieve this, we aimed to encapsulate enzymes within microcapsules. Since the substrates of glucose oxidase and catalase are small molecules, a polymer membrane is required that is large enough to allow for the diffusion of these small molecules but too large to accommodate the diffusion of the enzymes. The capsules must be processed gently to contain a large amount of active enzymes, and their presence must not interfere with the overall network characteristics of the hydrogel or the resident cells. Currently, there are no microencapsulation methods that satisfy these criteria. A controlled gas environment is typically provided through a chamber connected to a gas tank. Alternatively, the gas concentration can be attenuated by dispersing enzymes or nanozymes in a solution, thereby allowing control of the gas concentration without a chamber. However, as described above, none of these approaches can precisely control the gas spatially or temporally. A new approach to gas concentration control that can conveniently mimic the biological environment is needed. Fig. 1. Schematic diagram of an exemplary capsule synthesis procedure. First, gelatin at 37°C is loaded into a warm oil bath with stirring. After cooling and adding acetone, the particles are washed with hexane and air-dried. Subsequently, the particles are placed in Tris-HCl buffer (pH 8.5, 50 mM) containing 10 mg/mL of dopamine, shaken at 300 rpm for 24 hours, and then washed four times with PBS. Fig. 2. Schematic diagram of a hypoxic capsule within a microgel. A) Schematic diagram of the reaction of glucose oxidase and catalase. B) Schematic representation of glucose oxidase within the capsule. The enzyme is trapped within the capsule wall, but the pore size of the wall is small enough to allow for the exchange of enzyme substrates (glucose and oxygen). (Right) Schematic repres