Microcarriers for delivering active mitochondria and methods of making and using the same
By encapsulating engineered mitochondria in porous hydrogel microspheres and coating them with a polydopamine coating, the problem of exogenous mitochondria being easily inactivated was solved, and efficient mitochondrial function recovery and wound healing were achieved.
Patent Information
- Application Number
- CN202610494937.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-04-15
- Publication Date
- 2026-07-03
AI Technical Summary
In existing technologies, exogenous mitochondria are easily inactivated in vitro and are difficult for damaged cells to effectively take up, thus failing to effectively restore their function and limiting their application in treating refractory wounds.
Engineered mitochondria were encapsulated in porous hydrogel microspheres and coated with a polydopamine coating. Microfluidic technology and UV curing were used to prepare microcarriers for delivering active mitochondria, which protected mitochondrial activity and promoted uptake by target cells.
It significantly prolonged the survival time and functional activity of mitochondria, improved the uptake efficiency of target cells, promoted cell migration and proliferation, and significantly accelerated wound healing.
Smart Images
Figure CN122320897A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of biomedical materials and tissue engineering, and more specifically, to a microcarrier capable of delivering active mitochondria, a method for preparing the same, and the application of the microcarrier in the preparation of medicaments for treating skin wounds (especially photodamaged skin wounds). Background Technology
[0002] Skin wounds, especially refractory wounds caused by photodamage such as ultraviolet (UV) radiation, pose a significant challenge to clinical treatment. Photodamage leads to the production of excessive reactive oxygen species within cells, triggering a severe oxidative stress response that severely impairs the structure and function of mitochondria, including mitochondrial DNA damage and electron transport chain dysfunction. Mitochondrial dysfunction results in cellular energy metabolism disorders, prolonged inflammation, decreased cell proliferation and migration capacity, ultimately hindering tissue remodeling and wound healing.
[0003] Current treatment strategies largely focus on the molecular level, such as exogenous supplementation of antioxidants (e.g., vitamins C and E), minerals, or amino acids, in an attempt to repair mitochondrial function. However, these methods have limited effectiveness in fully restoring damaged mitochondrial function and achieving efficient cellular energy replenishment. In recent years, inspired by the natural transfer of mitochondria between cells, exogenous supplementation of active mitochondria has emerged as a new therapeutic approach. However, isolated mitochondria are extremely fragile in vitro, their biological activity is difficult to maintain, and they are difficult to be efficiently taken up by target cells, which greatly limits their translational prospects for direct application. Therefore, developing a delivery system that can effectively protect mitochondrial activity and promote its uptake by target cells is of great significance for treating refractory wounds such as photodamage. Summary of the Invention
[0004] This invention aims to solve the technical problem in the prior art that exogenous mitochondria are easily inactivated in vitro and are difficult to be effectively taken up by damaged cells to restore their function. It provides a microcarrier that can protect mitochondrial activity, promote cell uptake and efficiently repair the function of damaged cell mitochondria, as well as its preparation method and application.
[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:
[0006] In a first aspect, the present invention provides a microcarrier for delivering active mitochondria.
[0007] The microcarrier includes: porous hydrogel microspheres; engineered mitochondria encapsulated inside the porous hydrogel microspheres; and a polydopamine coating covering the surface of the porous hydrogel microspheres.
[0008] The engineered mitochondria are obtained by modifying the surface of active mitochondria with phenylboronic acid-modified polyamide-amine dendritic polymer (PPBA).
[0009] Secondly, the present invention provides a method for preparing the above-mentioned delivery active mitochondrial microcarrier, comprising the following steps:
[0010] S1. Preparation of engineered mitochondria Mito PPBA : Active mitochondria are mixed with phenylboronic acid-modified polyamide-amine dendritic polymer (PPBA), and surface modification is carried out through electrostatic or coordination interactions to obtain engineered mitochondria;
[0011] S2. Preparation of water-in-oil emulsion: The engineered mitochondria obtained in step S1 are used to prepare the water-in-oil emulsion. PPBA It is mixed with a hydrogel prepolymer solution as the inner phase of a microfluidic device; an oil phase containing surfactants is used as the outer phase; the inner phase is dispersed in the outer phase using microfluidic technology to form water-in-oil monodisperse droplets.
[0012] S3. Curing and Post-Modification: The water-in-oil droplets obtained in step S2 were photocured, followed by washing to remove the oil phase, yielding porous hydrogel microspheres (Mito) encapsulating engineered mitochondria. PPBA @HMs; Finally, the microspheres were mixed with a dopamine solution, and a polydopamine coating was formed on the surface of the microspheres through self-polymerization, yielding the final microcarrier Mito. PPBA @DHMs.
[0013] In a preferred embodiment, in step S1, the PPBA is prepared by reacting 4-bromomethylphenylboronic acid with second to fifth generation polyamide-amine resins at a molar ratio of (50~150):1, preferably 128:1, in an organic solvent (preferably methanol solution) at 60-80 °C for 20-28 hours, and then purifying the mixture.
[0014] As a preferred embodiment, in step S1, the preparation conditions of the engineered mitochondria are as follows: the mass ratio of the phenylboronic acid-modified polyamide-amine dendritic polymer (PPBA) to the active mitochondria is (1:1) to (3:1), preferably 2:1; the standing time after mixing is 15-60 minutes, preferably 30 minutes.
[0015] In a preferred embodiment, in step S2, the hydrogel prepolymer solution is a PBS solution of methacrylamide gelatin (GelMA) or methacrylamide hyaluronic acid (HAMA) with a mass fraction of 5%-10%; the internal phase also contains a photoinitiator, which is 2-hydroxy-2-methyl-1-phenyl-1-propanone with a volume concentration of 0.1%-0.5%.
[0016] In a preferred embodiment, in step S2, the concentration of engineered mitochondria in the inner phase is 10.8 -10 10 per milliliter.
[0017] In a preferred embodiment, in step S2, the external phase is a mixture of liquid paraffin and surfactant Span-80 in a volume ratio of (90-98):(2-10), preferably 95:5.
[0018] In a preferred embodiment, in step S2, the parameters of the microfluidic device are set as follows: the internal phase flow rate is 2-10 μL / min, and the external phase flow rate is 40-60 μL / min, preferably 50 μL / min.
[0019] In a preferred embodiment, in step S3, the photocuring is performed using ultraviolet light (e.g., 365 nm wavelength) for 30-120 seconds, preferably 60 seconds.
[0020] As a preferred embodiment, in step S3, the conditions for forming the polydopamine coating are as follows: immersing the hydrogel microspheres in an aqueous dopamine solution (concentration of 1-5 mg / mL, pH 8.0-9.0) and stirring at room temperature for 2-12 hours.
[0021] Thirdly, the present invention also provides the use of the above-mentioned microcarriers in the preparation of medicaments for treating skin wounds.
[0022] As a preferred application, the skin wound is a skin wound caused by photodamage (especially UVB photodamage). The microcarrier delivers active mitochondria to the wound cells, achieving one or more of the following functions: repairing mitochondrial function of damaged cells, reducing intracellular reactive oxygen species levels, resisting oxidative stress, promoting vascular endothelial cell tube formation, promoting cell migration, and promoting cell proliferation.
[0023] Beneficial effects:
[0024] Compared with the prior art, the present invention has the following beneficial effects:
[0025] (1) Significantly prolonging mitochondrial activity and improving uptake efficiency: This invention first uses phenylboronic acid-modified dendritic polymers (PPBA) to engineer mitochondria. This modification not only provides a protective layer for fragile mitochondria, significantly prolonging their survival time and functional activity (such as complex I and complex V enzyme activities) in vitro, but more importantly, PPBA modification promotes the endocytosis of mitochondria by target cells (such as HUVECs), efficiently providing high-quality functional mitochondria to damaged cells.
[0026] (2) Excellent microenvironment protection and tissue adhesion: Engineered mitochondria are encapsulated in porous hydrogel microspheres such as GelMA or HAMA using microfluidic technology, providing mitochondria with a 3D microenvironment that mimics the extracellular matrix in vivo, further maintaining their activity. At the same time, the surface-modified polydopamine coating endows the microcarrier with excellent wet tissue adhesion ability, enabling it to be stably fixed at the wound site and continuously release and deliver mitochondria.
[0027] (3) Highly effective healing promotion effect: In vitro cell experiments have confirmed that the Mito prepared in this invention has a high efficiency in promoting healing. PPBA @DHMs can significantly promote the formation and migration of vascular endothelial cells. In vivo animal experiments show that this microcarrier can effectively repair the mitochondrial function of damaged cells in photo-damaged skin wounds, resist oxidative stress, and promote cell proliferation, thereby significantly accelerating wound healing and improving healing quality.
[0028] (4) Controllable and scalable preparation method: This invention uses microfluidic technology combined with ultraviolet curing process to prepare porous hydrogel microspheres with uniform size (good monodispersity) and controllable structure in batches. The method is simple, easy to operate, and has mild conditions, with little impact on mitochondrial activity, and has good scalability and batch stability. Attached Figure Description
[0029] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, and the advantages of the present invention in the above and / or other aspects will become clearer.
[0030] Figure 1 This is a schematic diagram illustrating the preparation process and structure of the delivery active mitochondrial microcarrier provided in an embodiment of the present invention.
[0031] Figure 2 The mitochondrial engineering modification before (Mito) and after (Mito) provided in the embodiments of the present invention PPBA Transmission electron microscope (TEM) image of ).
[0032] Figure 3 The engineered mitochondria (Mito) provided in the embodiments of the present invention PPBA A comparative analysis of enzyme activities of mitochondrial complex I and complex V from unengineered mitochondria (Mito).
[0033] Figure 4 The engineered mitochondria (Mito) provided in the embodiments of the present invention PPBA Figure 1 shows the validation results of promoting tube formation and migration of human umbilical vein endothelial cells (HUVECs); where a is the cell scratch assay, b is the cell tube formation assay, and c is the Transwell cell migration assay.
[0034] Figure 5 The figure shows the therapeutic evaluation results of the microcarrier loaded with active mitochondria provided in the embodiments of the present invention in the treatment of mouse skin wounds. Detailed Implementation
[0035] The present invention can be better understood from the following embodiments.
[0036] like Figure 1 As shown, the preparation process of the microcarrier for delivering active mitochondria in this invention is as follows:
[0037] S1. Active mitochondria are mixed with phenylboronic acid-modified polyamide-amine dendritic polymer for surface modification to obtain engineered mitochondria;
[0038] S2. The engineered mitochondria are mixed with a hydrogel prepolymer solution as the inner phase, and an oil phase containing surfactants is used as the outer phase. Water-in-oil monodisperse droplets are prepared by microfluidic technology.
[0039] S3. The water-in-oil droplets are photocured and washed to obtain porous hydrogel microspheres encapsulating engineered mitochondria; then the porous hydrogel microspheres are mixed with dopamine solution to form a polydopamine coating on the surface of the microspheres, thus obtaining the microcarrier.
[0040] Example 1: Engineered Mitochondria PPBA Preparation and characterization of )
[0041] Mitochondrial extraction: Active mitochondria were isolated and extracted from umbilical cord mesenchymal stem cells (MSCs) using a commercially available mitochondrial extraction kit (such as Thermo Fisher's Mitochondrial Isolation Kit). The extracted mitochondria were then stored in a special mitochondrial preservative and kept on ice for later use.
[0042] Synthesis of phenylboronic acid-modified polyamide-amine dendritic polymer (PPBA): 4-bromomethylphenylboronic acid (128 mmol) and a fifth-generation polyamide-amine dendritic polymer (PAMAM G5, 1 mmol) were dissolved in methanol. The mixture was refluxed at 70 °C for 24 hours. After the reaction, the reaction solution was transferred to a dialysis bag (molecular weight cutoff: 3500 Da), and dialyzed against methanol for 24 hours, followed by dialyzed against deionized water for 24 hours to remove unreacted small molecules. Finally, the product in the dialysis bag was freeze-dried to obtain purified PPBA powder.
[0043] Engineered mitochondria Mito PPBAPreparation: The synthesized PPBA was dissolved in PBS buffer to prepare a 1 mg / mL solution. An appropriate amount of mitochondrial suspension (protein concentration approximately 0.5 mg / mL) was taken, and the PPBA solution was slowly added to the mitochondrial suspension at a PPBA to mitochondrial mass ratio of 2:1, and gently mixed by pipetting. The mixture was allowed to stand at room temperature for 30 minutes to allow PPBA to fully bind to the glycoproteins on the mitochondrial membrane surface through the interaction of phenylboronic acid. Subsequently, the precipitate was collected by centrifugation (e.g., 4°C, 10000 g, 10 min), and washed twice with PBS to remove unbound PPBA, yielding engineered mitochondria (Mito). PPBA Resuspend in PBS for later use.
[0044] Characterization: A small amount of natural mitochondria (Mito) and prepared Mito were taken. PPBA The samples were observed using transmission electron microscopy (TEM). The results are as follows: Figure 2 As shown, compared to unmodified mitochondria, Mito PPBA A hazy halo appeared on the surface, indicating that PPBA was successfully modified on the mitochondrial surface. Equal amounts of Mito and Mito were measured using mitochondrial complex I and complex V activity assay kits, respectively. PPBA The enzyme activity. Results are as follows: Figure 3 As shown, Mito PPBA The Complex I and Complex V activities of the modified mitochondria were significantly higher than those of the unmodified mitochondria (p < 0.05), indicating that the engineered modification strategy of the present invention not only did not damage mitochondrial function, but may have enhanced its enzyme activity by stabilizing the membrane structure.
[0045] Example 2: Engineered Mitochondria PPBA In vitro functional assessment
[0046] This embodiment uses human umbilical vein endothelial cells (HUVECs) to evaluate Mito PPBA Biological activity, results are shown in Figure 4 .
[0047] Cell scratch assay: HUVECs were seeded in 6-well plates. After the cells grew into a monolayer, straight scratches were made using a 200 μL sterile pipette tip. After washing with PBS, the cells were divided into three groups: (1) blank control group (basal medium containing 2% serum); (2) Mito group (addition of an equal amount of natural mitochondria, approximately 5 × 10⁻⁶ mcg). 5 (3) Mito PPBA Group (with added equal amounts of engineered mitochondria, approximately 5 × 10⁻⁶) 5 (Number / well). Photos were taken at 0 h, 12 h, and 24 h. Results are as follows: Figure 4As shown in a, Mito PPBA The cells in the Mito group showed almost complete closure of the scratches after 24 hours, while the blank control group and the Mito group still showed obvious scratches, indicating that Mito... PPBA It can more effectively promote endothelial cell migration.
[0048] Cell tube formation assay: In a 96-well plate pre-coated with Matrigel, seed approximately 2 × 10⁶ cells per well. 4 HUVECs were cultured. Cell grouping and treatment were the same as in the scratch assay. Tubular structure formation was observed after 6 hours of culture. Results are as follows: Figure 4 As shown in b, Mito PPBA The group formed a more complete and dense luminal network, with significantly better tube length and number of branch points than the Mito group and the blank control group, proving that Mito... PPBA It has a stronger ability to promote angiogenesis.
[0049] Transwell cell migration assay: Transwell chambers with a pore size of 4 μm were used. 2 × 10⁶ cells were seeded in the upper chamber. 4 HUVECs were cultured in the lower chamber with medium containing 10% FBS as a chemotactic agent. Grouping and treatment were the same as above. After 16 hours of culture, cells that migrated to the lower chamber were fixed and stained. Results are as follows. Figure 4 As shown in c, Mito PPBA The number of cells that migrated to the lower chamber in group A was significantly greater than in the other two groups, further confirming its excellent ability to promote cell migration.
[0050] Example 3: Delivery of active mitochondrial microcarriers (Mito PPBA Preparation of @DHMs)
[0051] Internal phase preparation: Methacrylamide gelatin (GelMA) was dissolved in sterile PBS to prepare a 5% (w / w) prepolymer solution. The photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone (HMPP) was added to this solution to a final concentration of 0.1% (v / v). Finally, Mito prepared in Example 1 was added. PPBA To make its final concentration 10 9 Mix each sample at a concentration of 1 / mL gently to form the internal phase for microfluidics.
[0052] External phase preparation: Liquid paraffin and surfactant Span-80 were mixed at a volume ratio of 95:5 and stirred thoroughly to form the external phase for microfluidics.
[0053] Microfluidic preparation: The internal phase and external phase are injected into the corresponding inlets of a microfluidic chip (e.g., a chip with a flow focusing structure). The internal phase flow rate is set to 5 μL / min, and the external phase flow rate is set to 50 μL / min. Under the combined action of shear force and surface tension of the chip, the internal phase is cut into uniformly sized water-in-oil (W / O) monodisperse droplets, which are collected in centrifuge tubes.
[0054] Curing and washing: The collected emulsion was irradiated under a 365 nm UV lamp for 60 seconds to crosslink and cure the GelMA. Then, a large amount of sterile PBS was added, the mixture was vigorously shaken and centrifuged (1000 rpm, 3 minutes). The supernatant was discarded, and the emulsion was washed three times to remove paraffin oil and surfactants, yielding encapsulated Mito. PPBA Porous hydrogel microspheres, denoted as Mito PPBA @HMs.
[0055] Polydopamine surface modification: Prepare a 2 mg / mL aqueous solution of dopamine hydrochloride and adjust the pH to 8.5 with Tris-HCl buffer. Then, apply the cleaned Mito... PPBA @HMs were immersed in a dopamine solution and reacted with stirring at room temperature in the dark for 4 hours. After the reaction, the microspheres were repeatedly washed with sterile PBS to remove unreacted dopamine monomers, yielding the final microcarriers coated with polydopamine, denoted as Mito. PPBA @DHMs. A schematic diagram of the entire preparation process of this microcarrier is shown below. Figure 1 As shown.
[0056] Example 4: Delivery of active mitochondrial microcarriers (Mito PPBA In vivo evaluation of the treatment of photodamaged skin wounds by @DHMs
[0057] Animal model establishment: Thirty male BALB / c mice aged 6-8 weeks were selected. The backs of 24 mice were shaved and dehaired, while the remaining skin was covered with lead plates. The exposed back skin was irradiated with a UVB light source (irradiance: 100 mJ / cm²) for three consecutive days to establish a photodamage model. On the third day after irradiation, two symmetrical full-thickness skin wounds were created on the backs of all mice (including 6 unirradiated normal controls) using a 10 mm diameter skin biopsy puncturer.
[0058] Grouping and Treatment: Twenty-four photodamage model mice were randomly divided into four groups of six each: (1) PBS control group (PBS was applied to the wound); (2) DHMs group (microcarrier suspension without mitochondrial encapsulation was applied to the wound); (3) Mito@DHMs group (microcarriers without PPBA modification encapsulating mitochondria were applied to the wound); (4) Mito PPBA@DHMs group (microcarriers prepared in Example 3 of this invention were applied to the wound). Another 6 unirradiated normal mice served as (5) normal wound control group (PBS was applied to the wound). All treatment groups were given the same mitochondrial equivalent of the preparation. Dressings were changed and the medication was re-administered every 2 days.
[0059] Healing assessment: The wound was photographed on days 0, 2, 4, 6, 8, and 10 post-treatment, and the wound area was calculated. On day 10, mice were sacrificed, and wound tissue was collected for H&E staining and Masson staining to assess re-epithelialization and collagen deposition.
[0060] Result: As Figure 5 As shown, compared with the PBS control group, DHMs group, and Mito@DHMs group, Mito PPBA The @DHMs treatment group achieved the fastest wound closure, with almost complete healing by day 10. Histological analysis showed that Mito PPBA The @DHM group exhibited more intact new epidermis and more ordered, denser collagen fiber deposition. These results demonstrate that the microcarriers prepared in this invention, by delivering highly active engineered mitochondria, can effectively resist UVB-induced oxidative stress, repair mitochondrial function, and thus significantly accelerate the healing of photodamaged skin wounds.
[0061] This invention provides a microcarrier for delivering active mitochondria, its preparation method, and its application. Many methods and approaches exist for implementing this technical solution; the above description is merely a preferred embodiment of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications should also be considered within the scope of protection of this invention. All components not explicitly stated in this embodiment can be implemented using existing technologies.
Claims
1. A microcarrier for delivering active mitochondria, characterized in that, include: Porous hydrogel microspheres; Engineered mitochondria encapsulated inside the porous hydrogel microspheres; as well as A polydopamine coating covering the surface of the porous hydrogel microspheres; The engineered mitochondria are obtained by surface modification of active mitochondria with a polyamide-amine dendritic polymer modified with phenylboronic acid.
2. The microcarrier according to claim 1, characterized in that, The porous hydrogel microspheres are made of methacrylated gelatin or methacrylated hyaluronic acid. The active mitochondria are derived from mesenchymal stem cells, including but not limited to umbilical cord mesenchymal stem cells and adipose-derived mesenchymal stem cells.
3. A method for preparing the microcarrier as described in claim 1 or 2, characterized in that, Includes the following steps: S1. Active mitochondria are mixed with phenylboronic acid-modified polyamide-amine dendritic polymers for surface modification to obtain engineered mitochondria; S2. The engineered mitochondria are mixed with a hydrogel prepolymer solution as the inner phase, and an oil phase containing surfactants is used as the outer phase. Water-in-oil monodisperse droplets are prepared by microfluidic technology. S3. The water-in-oil droplets are photocured and washed to obtain porous hydrogel microspheres encapsulating engineered mitochondria; then the porous hydrogel microspheres are mixed with dopamine solution to form a polydopamine coating on the surface of the microspheres, thus obtaining the microcarrier.
4. The preparation method according to claim 3, characterized in that, In step S1, the phenylboronic acid-modified polyamide-amine dendritic polymer is prepared by reacting 4-bromomethylphenylboronic acid with second to fifth generation polyamide-amine dendritic polymers in an organic solvent at a molar ratio of (50~150):
1.
5. The preparation method according to claim 3, characterized in that, In step S1, the mass ratio of the phenylboronic acid-modified polyamide-amine dendritic polymer to the active mitochondria is 1:1 to 3:1; the mixing and standing time is 15-60 minutes.
6. The preparation method according to claim 3, characterized in that, In step S2, the hydrogel prepolymer solution is a PBS solution of methacrylamide gelatin or methacrylamide hyaluronic acid, with a mass fraction of 5%-10%; the inner phase also contains the photoinitiator 2-hydroxy-2-methyl-1-phenyl-1-propanone, with a volume concentration of 0.1%-0.5%; the concentration of engineered mitochondria in the inner phase is 10. 8 -10 10 per ml.
7. The preparation method according to claim 3, characterized in that, In step S2, the external phase is a mixture of liquid paraffin and Span-80 with a volume ratio of (90-98):(2-10); the microfluidic parameters are: internal phase flow rate 2-10 μL / min, external phase flow rate 40-60 μL / min.
8. The preparation method according to claim 3, characterized in that, In step S3, the photocuring is performed by irradiating with ultraviolet light for 30-120 seconds; the polydopamine coating is formed by immersing hydrogel microspheres in a dopamine aqueous solution with a concentration of 1-5 mg / mL and reacting for 2-12 hours.
9. The use of the microcarrier according to claim 1 or 2 in the preparation of a medicament for treating skin wounds.
10. The application according to claim 9, characterized in that, The skin wound is caused by photodamage.