Nanometer vesicle with tissue targeting and mitochondrial positioning functions as well as preparation and application of nanometer vesicle
By preparing nanovesicles with tissue targeting and mitochondrial localization, and loading them with the small molecule mitochondrial membrane stabilizer BAI1, the problem of insufficient intervention in mitochondrial membrane stability in existing technologies has been solved, achieving effective intervention in mitochondrial function stabilization and cell senescence, especially in diseases such as intervertebral disc degeneration.
Patent Information
- Application Number
- CN202511004349.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-21
- Publication Date
- 2025-11-11
AI Technical Summary
Existing technologies lack tissue-specific and mitochondrial-targeting drug delivery systems, resulting in limited means of intervening in mitochondrial membrane stability. This makes it impossible to effectively block mtDNA release and the production of SASP factors, and consequently, to effectively intervene in cellular senescence and related degenerative diseases.
Nanovesicles with tissue targeting and mitochondrial localization were prepared. By modifying the nucleus pulposus cell membrane with triphenylphosphine cations and loading small molecule mitochondrial membrane stabilizers, such as the Bax pathway inhibitor BAI1, targeted delivery to mitochondria and intervention on membrane stability were achieved.
It significantly inhibits mtDNA leakage and SASP factor expression, improves mitochondrial function, delays cell senescence and related degenerative diseases, especially intervertebral disc degeneration, and has broad clinical application prospects.
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Figure CN120919073A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of medical device technology, and relates to a mitochondrial delivery vesicle system, specifically to a nanovesicle with tissue targeting and mitochondrial localization, and its preparation and application. Background Technology
[0002] Cellular senescence is a widespread cellular state that accompanies aging, characterized by permanent cell cycle arrest, mitochondrial dysfunction, and the secretion of senescence-associated secretory phenotype (SASP) factors. Recent studies have shown that increased mitochondrial outer membrane permeability is a key event inducing cellular senescence and tissue degeneration. Particularly in age-related diseases such as intervertebral disc degeneration (IVDD), the mitochondrial membrane structure of nucleus pulposus cells (NPCs) in the center of the intervertebral disc is unstable, leading to leakage of mitochondrial DNA (mtDNA) into the cytoplasm. This activates the cGAS-STING signaling pathway, inducing an inflammatory response and promoting the release of SASP factors, accelerating local tissue degeneration.
[0003] Current technologies for treating diseases caused by cellular senescence mainly focus on inhibiting the expression of downstream inflammatory factors or clearing senescent cells. Some methods involve injecting stem cells to promote cell regeneration and matrix synthesis. However, interventions targeting upstream mitochondrial membrane stability remain very limited.
[0004] The development of biomimetic nanotechnology has provided new ideas for achieving precise intracellular drug delivery. Biomimetic nanovesicles derived from natural cell membranes have become a promising drug delivery platform due to their excellent biocompatibility, immune evasion capabilities, and cell-specific targeting. Currently, mitochondrial-targeted drugs lack tissue-specific delivery methods, resulting in unstable efficacy and high side effects in practical applications. Therefore, there is an urgent need to develop a drug delivery system that possesses both tissue-targeting and mitochondrial localization capabilities. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention proposes a nanovesicle with tissue targeting and mitochondrial localization, as well as its preparation and application. By delivering mitochondrial membrane stabilizers to mitochondria, it inhibits the increase in mitochondrial outer membrane permeability at the source, thereby blocking mtDNA release and SASP factor production, achieving anti-aging, anti-inflammatory, and tissue degeneration intervention, effectively delaying the progression of cell aging and related degenerative diseases, especially degenerative diseases related to mitochondrial dysfunction.
[0006] A type of nanovesicle with tissue targeting and mitochondrial localization is formed by encapsulating a small molecule mitochondrial membrane stabilizer on the nucleus pulposus cell membrane, wherein the surface of the nucleus pulposus cell membrane is modified with triphenylphosphine cations (TPP). + ).
[0007] Preferably, the small molecule mitochondrial membrane stabilizer is a Bax pathway inhibitor (BAI1).
[0008] As a preferred method, triphenylphosphine cations are modified on the surface of the nucleus pulposus cell membrane via a bioorthogonal reaction.
[0009] Preferably, the bioorthogonal reaction is a copper ion-free click chemistry.
[0010] A method for preparing tissue-targeting and mitochondrial-localized nanovesicles involves extracting nucleus pulposus cell membranes, modifying the membrane surface with triphenylphosphine cations, and then obtaining uniformly sized nucleus pulposus cell membrane vesicles via membrane extrusion. Finally, the vesicles are loaded with a small-molecule mitochondrial membrane stabilizer through co-incubation to obtain the tissue-targeting and mitochondrial-localized nanovesicles.
[0011] As a preferred method, tetraacetylated N-acetylaminus is added during the culture of nucleus pulposus cells to express sialic acid derivatives with azide groups (–N3) on the surface of the nucleus pulposus cell membrane, thereby modifying the surface of the nucleus pulposus cell membrane with triphenylphosphine cations through a bioorthogonal reaction.
[0012] Applications of nanovesicles with tissue-targeting and mitochondrial localization in the preparation of mitochondrial-targeted anti-aging drugs, or in the preparation of drugs for the intervention of tissue degenerative diseases related to mitochondrial membrane damage, or in the preparation of drugs for the regulation of mtDNA leakage and SASP release in cellular aging models.
[0013] Preferably, the tissue degenerative disease associated with mitochondrial membrane damage is intervertebral disc degeneration.
[0014] The present invention has the following beneficial effects: This study provides a strategy to intervene in cellular senescence at its source, overcoming the limitations of traditional anti-inflammatory and anti-aging therapies such as dispersed targets and low drug delivery efficiency. By stabilizing the mitochondrial membrane structure, it significantly improves the release of cellular senescence-related secretory phenotypes and mitochondrial function, and has broad application prospects and important clinical translational value. Attached Figure Description
[0015] Figure 1 The particle size determination results of the nanovesicles BNPMT in Example 1; Figure 2 The results of the zeta potential measurement of BNPMT nanovesicles in Example 1; Figure 3 This is a cryo-electron microscope image of the BNPMT nanovesicles in Example 1; Figure 4 The encapsulation efficiency of the small molecule drug by the cell membrane in Example 1; Figure 5 The results of fluorescence staining in Example 1 are shown in the figure; the scale bar is 10 μm. Figure 6 The results of fluorescence co-localization in Example 1; Figure 7 The results of fluorescence intensity changes in Example 2; Figure 8 The results of mtDNA content detection in Example 2; Figure 9 The results of inflammatory factor concentration detection in Example 2; Figure 10 The results of the cell senescence ratio detection in Example 2; Figure 11 The results of inflammatory factor concentration detection in Comparative Example 1; Figure 12 The results of the apoptosis rate detection in Comparative Example 1; Figure 13 The results show the protein expression level detection in Comparative Example 1. Detailed Implementation
[0016] The present invention will be further explained below with reference to the accompanying drawings; Example 1 This embodiment provides a method for preparing nanovesicles with tissue targeting and mitochondrial localization. The nanovesicles are small molecule mitochondrial membrane stabilizers encapsulated in the nucleus pulposus cell membrane. The specific steps are as follows: Step 1: Culture and metabolic labeling of nucleus pulposus cells Primary rat nucleus pulposus cells (NP cells) were selected, seeded in high-glucose DMEM medium containing 10% fetal bovine serum and 1% penicillin antibiotics, and cultured in a 37°C, 5% CO2 incubator.
[0017] Once the cells adhered to the culture medium and grew to approximately 70% confluence, 50 μM tetraacetylated N-acetylammonomannose (Ac4ManNAz) was added, and the cells were cultured for another 48 hours. This allowed the nucleus pulposus cells to express sialic acid derivatives with an azide group (–N3) on the cell membrane surface through the endogenous sialic acid metabolic pathway.
[0018] Step 2: Extraction of nucleus pulposus cell membrane Nucleus pulposus cells enriched with azide were washed twice with cold PBS, and the cells were collected and resuspended in ultrapure water. Cells were lysed by freezing at -80°C for 5 minutes and then thawing at 37°C for 5 minutes, repeated three times. Subsequently, the cells were centrifuged sequentially at 1000 g and 10000 g to remove the nucleus and other organelles, collecting the cell membrane fraction from the supernatant. The cell membrane fraction was further collected by centrifugation at 12000 g to obtain the azide-enriched nucleus pulposus cell membrane (NPm-Azide).
[0019] Step 3: DBCO-TPP modification The obtained NPm-Azide was resuspended in PBS buffer, and DBCO-TPP (diphenylcyclooctynyl modified triphenyl phosphate) was added at a molar ratio of 1:5 to a final concentration of 100 μM. The mixture was mixed and reacted for 2 hours. Copper ion free click chemical reaction was carried out under room temperature and dark conditions. After the reaction was completed, unreacted DBCO-TPP was removed by ultrafiltration or centrifugation.
[0020] By TPP + Covalent modification of the NPm-Azide surface yielded mitochondrial-targeted nucleus pulposus cell membrane (TPP-NPm).
[0021] Step 4: Construction and drug loading of nanovesicles TPP-NPm was repeatedly extruded five times through a polycarbonate membrane with a pore size of 200 nm to obtain vesicles with uniform particle size. Subsequently, the small molecule drug BAI1 was co-incubated with the vesicles in PBS solution and sonicated under ice bath conditions, with a 10 s working time, 5 s rest time, and 10 cycles to promote drug loading.
[0022] After loading, the nanovesicles (BNPMT) with tissue targeting and mitochondrial localization were purified by centrifugation at 12000 g.
[0023] The particle size and zeta potential of the obtained nanovesicles were measured using a Zetasizer Nano ZS particle size analyzer, such as Figure 1 , 2 As shown, the average particle size is 170 nm and the Zeta potential is -16.7 mV.
[0024] The nanovesicles were observed using cryo-electron microscopy, such as Figure 3 As shown, the vesicle structure is intact. The encapsulation efficiency of the small molecule drug BAI1 in the mitochondrial-targeted nucleus pulposus cell membrane was analyzed by high-performance liquid chromatography (HPLC), as shown... Figure 4 As shown, the encapsulation rate is 17.8%.
[0025] Fluorescent staining was performed on the cell nucleus, BNPMT, and mitochondria using blue, green, and red dyes, respectively. Figure 5 As shown in the figure. The degree of co-localization of BNPMT with mitochondria was observed by fluorescence confocal microscopy, and the fluorescence intensity was counted. The results are as follows. Figure 6 As shown, the green fluorescence of BNPMT highly overlaps with the red fluorescence of mitochondria, indicating that BNPMT can target mitochondria and exert its function.
[0026] Example 2 This embodiment applies the tissue-targeting and mitochondrial-localized nanovesicles (BNPMTs) prepared in Example 1 to the preparation of mitochondrial-targeted anti-aging drugs and verifies their in vitro intervention effects on mitochondrial membrane stability and cell senescence. The specific steps are as follows: Step 1: After culturing primary rat nucleus pulposus cells to passage 4-5 under standard conditions, treat them with 100 nM doxorubicin (Doxo) for 2 hours to induce early senescence. After treatment, wash twice with PBS and replace with normal culture medium without Doxo for another 48 hours.
[0027] Step 2: Select a portion of Doxo-induced nucleus pulposus cells and co-incubate them with BNPMT prepared in Example 1, as the BNPMT treatment group. Select a portion of Doxo-induced nucleus pulposus cells without any intervention, as the senescence group (Doxo). Then select nucleus pulposus cells that have not been induced by Doxo, as the blank control group (Control).
[0028] Step 3: After 24 hours, incubate each group of cells with TMR working solution for 30 minutes, wash with PBS, and observe the changes in TMR fluorescence intensity using a fluorescence microscope. Figure 7 As shown, the fluorescence intensity of the BNPMT-treated group was significantly higher than that of the senescent group, indicating that the mitochondrial membrane potential was restored and membrane integrity was improved.
[0029] Step 4: Differential centrifugation was performed on each group of cells to separate the cytoplasmic components. Total cytoplasmic DNA was extracted, and the mtDNA content was detected using qPCR. Results are as follows: Figure 8 As shown, the cytoplasmic mtDNA level in the BNPMT-treated group was significantly lower than that in the senescent group, indicating that BNPMT successfully inhibited mtDNA leakage.
[0030] Step 5: Detect the concentration of SASP-related inflammatory cytokine IL-6 in the supernatant using an enzyme-linked immunosorbent assay (ELISA). Figure 9 As shown, the concentrations of inflammatory factors in the BNPMT-treated group were significantly lower than those in the senescent group. The proportion of senescent cells was analyzed using SA-β-gal staining, as shown... Figure 10 As shown, the proportion of positively stained cells in the BNPMT-treated group was significantly lower than that in the senescent group.
[0031] The results of the above in vitro experiments show that the tissue-targeting and mitochondrial-localized nanovesicles BNPMT proposed in this application can stabilize the mitochondrial membrane, inhibit the release of mtDNA into the cytoplasm, and thus significantly inhibit the expression of SASP factor and the senescence process of NP cells, demonstrating strong mitochondrial protection and anti-aging efficacy, and can be applied to the preparation of anti-aging drugs.
[0032] Comparative Example 1 Based on Example 1, this comparative example uses mitochondrial-targeted nucleus pulposus cell membrane (TPP-NPm) to encapsulate the traditional anti-inflammatory drug curcumin, preparing biomimetic anti-inflammatory drug nanovesicles (CNPMT), and comparing its differences with BNPMT prepared in Example 1 in intervening in the senescence and apoptosis process of nucleus pulposus cells: Step 1: After culturing primary rat nucleus pulposus cells to passage 4-5 under standard conditions, treat them with 100 nM doxorubicin (Doxo) for 2 hours to induce early senescence. After treatment, wash twice with PBS and replace with normal culture medium without Doxo for another 48 hours.
[0033] Step 2: Select a portion of Doxo-induced nucleus pulposus cells and co-incubate them with BNPMT prepared in Example 1 (BNPMT treatment group). Add CNPMT to these cells and co-incubate them (CNPMT treatment group). Select a portion of Doxo-induced nucleus pulposus cells without any intervention (senescence group, Doxo). Select un-Doxo-induced nucleus pulposus cells as the blank control group (Control).
[0034] Step 3: Detect the mRNA expression of typical inflammatory factors IL-6 and TNF-α in cells using qPCR, such as... Figure 11 As shown, the BNPMT treatment group had lower levels of inflammatory factors, demonstrating a significant inhibitory effect, and its anti-inflammatory ability was superior to that of traditional anti-inflammatory drugs.
[0035] Step 4: Detect the apoptosis rate using live / dead cell staining and flow cytometry. The results are as follows: Figure 12 As shown, the apoptosis rate of NP cells in the BNPMT-treated group was significantly lower than that in the CNPMT-treated group and the senescent group, suggesting that it has a stronger cell protection ability.
[0036] Step 5: Detect the upregulated expression of type II collagen (COL2) and matrix-degrading enzyme MMP13 using qPCR. Figure 13 As shown, the BNPMT treatment group had a stronger ability to upregulate the expression level of type II collagen (COL2) and a more significant inhibitory effect on the expression of matrix degrading enzyme MMP13, which helps to maintain the matrix metabolic balance of NP cells.
[0037] The results of the comparative experiments above show that BNPMT is superior to traditional anti-inflammatory drug-loaded biomimetic vesicle CNPMT in terms of anti-inflammation, anti-apoptosis and maintenance of ECM homeostasis. It demonstrates its multidimensional advantages in the intervention of nucleus pulposus cell degeneration and can be applied to the preparation of drugs for the intervention of tissue degenerative diseases related to mitochondrial membrane damage, especially intervertebral disc degenerative disease (IVDD).
Claims
1. A nanovesicle with tissue targeting and mitochondrial localization, characterized in that: The nanovesicles are nucleus pulposus cell membranes encapsulating small molecule mitochondrial membrane stabilizers, and the surface of the nucleus pulposus cell membranes is modified with triphenylphosphine cations.
2. The nanovesicle with tissue targeting and mitochondrial localization as described in claim 1, characterized in that: The small molecule mitochondrial membrane stabilizer is a Bax pathway inhibitor.
3. The nanovesicle with tissue targeting and mitochondrial localization as described in claim 1, characterized in that: Triphenylphosphine cations were modified on the surface of nucleus pulposus cell membranes via bioorthogonal reactions.
4. A method for preparing nanovesicles with tissue targeting and mitochondrial localization as described in any one of claims 1 to 3, characterized in that: Triphenylphosphine cations were modified on the surface of nucleus pulposus cell membranes, and then nucleus pulposus cell membrane vesicles were prepared by membrane extrusion. Finally, the nucleus pulposus cell membrane vesicles were loaded with small molecule mitochondrial membrane stabilizers by co-incubation to obtain the nanovesicles with tissue targeting and mitochondrial localization.
5. The method for preparing nanovesicles with tissue targeting and mitochondrial localization as described in claim 4, characterized in that: Adding tetraacetylated N-acetylaminus to the nucleus pulposus cell culture process allows the expression of sialic acid derivatives with azide groups on the cell membrane surface, thereby modifying the cell membrane surface with triphenylphosphine cations through a bioorthogonal reaction.
6. The method for preparing nanovesicles with tissue targeting and mitochondrial localization as described in claim 5, characterized in that: Triphenylphosphine cations were modified on the surface of nucleus pulposus cell membranes via copper ion-free click chemistry.
7. The method for preparing nanovesicles with tissue targeting and mitochondrial localization as described in claim 6, characterized in that: Nucleus pulposus cell membranes with surface-modified triphenylphosphine cations were resuspended in PBS buffer, and diphenylcyclooctynyl modified triphenyl phosphate at a molar ratio of 1:5 was added to a final concentration of 100 μM. The mixture was reacted for 2 hours, and copper ion free click chemistry was carried out at room temperature in the dark.
8. The application of a nanovesicle with tissue targeting and mitochondrial localization as described in any one of claims 1 to 3, characterized in that: The nanovesicles with tissue targeting and mitochondrial localization are used to stabilize the mitochondrial membrane structure.
9. The application of a nanovesicle with tissue targeting and mitochondrial localization as described in claim 8, characterized in that: The nanovesicles with tissue targeting and mitochondrial localization can be used to prepare drugs for the treatment of tissue degenerative diseases related to mitochondrial membrane damage, or to prepare drugs for the regulation of mtDNA leakage and SASP release in senescent cells.
10. The application of a nanovesicle with tissue targeting and mitochondrial localization as described in claim 9, characterized in that: The tissue degenerative disease associated with mitochondrial membrane damage is intervertebral disc degeneration.