A self-activated NO nanopump, its construction method and its application

Abstract

This invention discloses a self-activated NO nanopump, its construction method, and its application. The self-activated NO nanopump comprises a chemiluminescent substrate CPPO and a NO donor BNN6 encapsulated in DSPE-PEG-Mal. The DSPE-PEG-Mal surface is conjugated with a LepR antibody. The self-activated NO nanopump of this invention can specifically target LepR... + It accumulates in cells and releases intracellular NO, thereby effectively reversing cellular aging and differentiation fate. It rescues bone aging by upregulating the glycolysis pathway. It possesses strong H2O2 scavenging capacity, NO production capacity, and anti-aging ability. In vivo administration significantly reverses osteoporosis in OVX mice. The self-activated NO nanopump provided by this invention offers a novel strategy for the treatment of bone aging.

Description

Technical Field

[0001] This invention relates to a self-activated NO nanopump, its construction method, and its application, belonging to the field of biotechnology. Background Technology

[0002] In vivo fate mapping of stromal cells expressing LepR-Cre shows that they are present in the bone marrow at birth and are a major source of osteoblasts and adipocytes in the bone marrow during development. Increasing evidence suggests that LepR-Cre expression increases with age. + Cells age and differentiate into more fat cells, while osteoblasts decrease, ultimately leading to osteoporosis. Maintaining bone balance relies on a healthy LepR cell line. + Cells, thus causing aging LepR + Cellular revitalization is a crucial strategy for treating osteoporosis. Nitric oxide (NO) is an endogenous gaseous neurotransmitter that has been found to participate in various metabolic processes in the body. Studies have shown that age-related decreases in nitric oxide production are associated with an increased incidence of age-related diseases such as osteoporosis and cardiovascular events. Because it is difficult to directly obtain gaseous NO in the body, exogenous NO donor therapy has been used for the prevention and treatment of osteoporosis. Wang et al. treated osteoporosis induced by oophorectomy (OVX) by direct subcutaneous injection of diethylenetriamine diphenyl phthalate (NONOate, NO donor). However, the rapid clearance rate of NONOate reduced its effectiveness. Therefore, the short duration of sustained NO release is a major obstacle to the delivery of exogenous NO donors. To overcome these challenges, Sung and his team synthesized an injectable microparticle system to encapsulate NONOate, achieving active capture of NO bubbles and prolonging the half-life of NO. However, this therapy relies on frequent subcutaneous injections and has poor bone tissue targeting, requiring further improvement. As an attractive alternative, Wang et al. constructed a bone-targeted photosensitive NO-generating nanoplatform (UCPA) doped with lanthanides (Ln). 3+ The nanoplatform can convert irradiated near-infrared light into ultraviolet / blue light, causing the NO donor BNN6 to release NO, thereby reversing osteoporosis. However, the poor penetration and low efficiency of excitation light (even near-infrared light) severely affect its effect on deep bone tissue. Summary of the Invention

[0003] Objectives of the Invention: The first objective of this invention is to provide a self-activated NO nanopump. The second objective of this invention is to provide a method for preparing the aforementioned self-activated NO nanopump. The third objective of this invention is to provide applications of the aforementioned self-activated NO nanopump.

[0004] Technical solution: The self-activated NO nanopump of the present invention comprises a chemiluminescent substrate bis(oxalate) CPPO and a NO donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine BNN6 encapsulated in phospholipid polyethylene glycol maleimide (DSPE-PEG-Mal). The nanopump is prepared by adding BNN6, soybean oil, CPPO, and DSPE-PEG-Mal to THF, mixing them thoroughly under nitrogen protection, removing the solvent, drying, adding Milli-Q water, and shaking vigorously.

[0005] Furthermore, the DSPE-PEG-Mal surface conjugated LepR antibody.

[0006] This invention encapsulates the chemiluminescent substrate CPPO and the NO donor BNN6 in DSPE-PEG-Mal (CB-NPs) using a nanoprecipitation method. Then, through the reaction of the antibody's thiol groups with the Mal groups of the nanoparticles, a LepR-targeting antibody is conjugated on the surface of the CB-NPs, yielding a LepR-targeting antibody. + Cellular CB-LepR. After entering the bone aging microenvironment, CB-LepR can effectively remove harmful H2O2 through the spontaneous reaction of CPPO and H2O2. At the same time, DOD, a byproduct of CPPO oxidation, is a high-energy intermediate whose energy can be directly transferred to the NO donor, leading to the in-situ release of NO.

[0007] The method for constructing the self-activated NO nanopump of the present invention includes the following steps: adding BNN6, soybean oil, CPPO and DSPE-PEG-Mal to THF, mixing evenly under nitrogen protection, removing the solvent, drying, adding Milli-Q water and shaking vigorously to obtain CB-NPs.

[0008] Furthermore, it also includes a step of combining LepR antibody, specifically: mixing LepR antibody with Traut's reagent, reacting at room temperature, removing excess Traut's reagent after the reaction, adding the resulting liquid to CB-NPs and incubating overnight in the dark, adding Milli-Q water and shaking vigorously to obtain CB-LepR.

[0009] Furthermore, the mass ratio of BNN6:CPPO:DSPE-PEG-Mal is 0.75:1:5.

[0010] The application of the self-activated NO nanopump of the present invention in the preparation of drugs to reverse bone aging.

[0011] The application of the self-activated NO nanopump of the present invention in the preparation of H2O2 scavenging drugs.

[0012] The application of the self-activated NO nanopump of the present invention in the preparation of drugs that promote NO generation.

[0013] The application of the self-activated NO nanopump of the present invention in the preparation of anti-aging drugs.

[0014] Furthermore, the concentration of the self-activated NO nanopump is 50 μg / mL.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following outstanding advantages: The self-activated NO nanopump of the present invention can specifically activate in LepR + It accumulates in cells and releases intracellular NO, thereby effectively reversing cellular aging and differentiation fate. It rescues bone aging by upregulating the glycolysis pathway. It possesses strong H2O2 scavenging capacity, NO production capacity, and anti-aging ability. In vivo administration significantly reverses osteoporosis in OVX mice. The self-activated NO nanopump provided by this invention offers a novel strategy for the treatment of bone aging. Attached Figure Description

[0016] Figure 1 A schematic diagram illustrating the construction route of CB-LepR in an embodiment of the present invention and its application mechanism in the treatment of bone aging;

[0017] Figure 2 The figure shows the characterization results of the self-activated NO nanopump according to an embodiment of the present invention.

[0018] Figure 3 Bone-targeting performance of the self-activated NO nanopump in an embodiment of the present invention;

[0019] Figure 4 The H2O2 scavenging ability of the self-activated NO nanopump in an embodiment of the present invention;

[0020] Figure 5 The NO generation capability of the self-activated NO nanopump in an embodiment of the present invention;

[0021] Figure 6 The anti-aging ability of the self-activated NO nanopump in an embodiment of the present invention;

[0022] Figure 7 The self-activated NO nanopump's effect on reversing osteoporosis in OVX mice, as an embodiment of the present invention;

[0023] Figure 8 Analysis of the anti-aging mechanism of the self-activated NO nanopump in embodiments of the present invention. Detailed Implementation

[0024] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0025] Example 1: Fabrication of a nanopump

[0026] S1: Synthesis of self-activated CB nanoparticles CB-NPs

[0027] BNN6 (0.75 mg), soybean oil (2 μL), CPPO (1 mg), and DSPE-PEG-Mal (5 mg) were added to 1 mL of THF and mixed thoroughly for 12 hours under nitrogen protection. The resulting mixture was then subjected to a rotary evaporator to remove the solvent and dried in a vacuum drying oven. 1 mL of Milli-Q water was added to the dried mixture and the mixture was shaken vigorously to prepare CB-NPs.

[0028] S2: Binding LepR antibody

[0029] Mix LepR antibody (2 μM) with Traut's reagent (20 μM) and react at room temperature for 1 h. Separate the excess Traut's reagent using a desalting column. Add the resulting liquid to 1 mL of CB-NPs from step S1 and incubate overnight in the dark. Add Milli-Q water and shake vigorously to obtain CB-LepR.

[0030] The CB-NPs and CB-LepR prepared above were characterized. First, the highest occupied molecular orbital (HOMO) energy level of BNN6 and the lowest unoccupied molecular orbital (LUMO) energy level of DOD were calculated to be -6.22 eV and -3.33 eV, respectively, using Gaussian 09 calculations. Figure 2 B). An energy gap of 2.89 eV is sufficient for CPPO to directly excite BNN6, thereby generating NO( Figure 2 C). The content of CB-NPs was then determined by HPLC. The co-localization of characteristic peaks of CPPO, BNN6, and CB-NPs in HPLC indicated the successful synthesis of CB-NPs. Figure 2 D). Both CB-NPs and CB-LepR exhibit dispersed spherical shapes ( Figure 2 The average diameters of the nanoparticles (EF) were 118.3 ± 10.5 nm and 125.9 ± 0.25 nm, respectively. Both nanoparticles maintained a constant size for 3 days in 10% fetal bovine serum and exhibited satisfactory stability. Figure 2 G).

[0031] Example 2: Performance Verification of CB-NPs and CB-LepR

[0032] The CB-NPs prepared in Example 1 were mixed with CY5.5-SH at a mass ratio of 10:1 and reacted overnight in the dark. Then, unreacted Cy5.5-SH and CB-NPs were removed by dialysis with deionized water to obtain CB. CY5.5 -NPs.

[0033] LepR FITCAntibody (2 μM) was mixed with Traut's reagent (20 μM) and reacted at room temperature for 1 h. Excess Traut's reagent was separated using a desalting column. Then, it was reacted with CB... CY5.5 -NPs were incubated overnight in the dark, and unreacted Cy5.5-SH and CB-NPs were removed by dialysis with deionized water to obtain CB. CY5.5 -LepR FITC .

[0034] LepR antibody (2 μM) was mixed with Traut's reagent (20 μM) and reacted at room temperature for 1 h. Excess Traut's reagent was separated using a desalting column. Then, it was reacted with CB... CY5.5 -NPs were incubated overnight in the dark, and unreacted Cy5.5-SH and CB-NPs were removed by dialysis with deionized water to obtain CB. CY5.5 -LepR.

[0035] (1) Validation of bone-targeting performance

[0036] In vitro experiments involved separately applying CB CY5.5 -NPs, CB CY5.5 -LepR FITC , with LepR + The cells were cultured together for 72 hours. CB was found. CY5.5 -LepR FITC Enter LepR + The number of cells increased significantly ( Figure 3 B). Control nanoparticles (CB) CY5.5 Nonspecific binding of -NPs is less ( Figure 3 A).

[0037] In vivo experiments involved injecting 20 μL of CB into 6-week-old C57 / BL6 mice from the Comparative Medicine Center of Yangzhou University. CY5.5 -NPs or CB CY5.5 -LepR (concentration 50 μg / mL) was used to collect mouse femurs on days 0, 3 and 7 to prepare sections.

[0038] Fluorescence microscopy revealed that during the evaluation period, the non-targeted CB... CY5.5 Compared to -NPs, CB CY5.5 -LepR of LepR + Stronger cell targeting ability Figure 3 These data confirm that LepR antibody modification significantly enhances the nanoparticles' response to LepR. + Cell targeting.

[0039] (2) Verification of H2O2 scavenging ability

[0040] 20 μL of CB-NPs and CB-LepR (concentration 50 μg / mL) prepared in Example 1 were injected into the femoral medullary cavity of 6-week-old C57 / BL6 OVX mice from the Comparative Medicine Center of Yangzhou University. Six weeks later, 50 μL of an H2O2 probe (Beyotime Biotechnology Co., Ltd., catalog number: ST010) was injected. The H2O2 content was detected by in vivo imaging 20 minutes later. In vivo imaging showed a large accumulation of H2O2 in the medullary cavity of OVX mice. This accumulation was cleared after treatment with CB-NPs or CB-LepR. Figure 4 The above data indicate that CB-LepR can effectively remove malignant H2O2 from the bone aging microenvironment.

[0041] (3) Verification of NO generation capacity

[0042] CB CY5.5 -LepR (50 μg / mL) and LepR + After co-culturing the cells for 24 hours, the medium was replaced with fresh medium containing hydrogen peroxide (200 μM). 50 μL of the NO probe (Beyotime Biotechnology, catalog number: S0019) was quickly added, and the cells were placed in a live cell workstation to capture LepR. + Cellular absorption of CB CY5.5 The behavior after -LepR was observed. Results showed that red fluorescence in cells gradually decreased, while green fluorescence gradually increased. Figure 5 A). These results provide evidence that CB-LepR releases NO via chemically activated BNN6 after exposure to H2O2.

[0043] (4) Verification of anti-aging ability

[0044] Mix PBS, H2O2 (200uM), CB-NPs (50ug / mL), H2O2+CB-NPs (50ug / mL) or H2O2+CB-LepR (50ug / mL) with LepR + After 7 days of cell incubation, cell senescence was observed using SA-β-Gal staining. The results showed that H2O2+CB-LepR exhibited better anti-senescence activity compared to other treatment groups. Figure 6 AB).

[0045] (5) Verification of the reversal effect on osteoporosis in OVX mice

[0046] In this embodiment, an osteoporosis model was established by removing both ovaries of mice. Then, 20 μL of the complex (concentration 50 μg / mL) was locally injected into the femoral medullary cavity. Mice were sacrificed 6 weeks after treatment. Finally, the bone quality of the mouse femur was observed to determine the anti-osteoporosis effect of the self-activated NO nanopump. Results are shown in the appendix. Figure 7(Control group: mice without ovarian removal). The integrity of the femur in each group was observed using micro-computed tomography (micro-CT) and 3D reconstruction. Figure 7 AB). The results showed that, compared with the OVX group, the OVX+CB-NPs group and the OVX+CB-LepR group had significantly increased trabecular bone volume and significantly decreased intertrabecular spacing in the femur of mice. Figure 7 These changes reflect the release of NO to promote bone formation when CB-NPs and CB-LepR are exposed to H2O2 in the bone aging microenvironment. Compared with other groups, CB-LepR significantly reduced the release of NO to promote bone formation. + The cells showed greater targeting ability, thus maximally reducing bone loss in the OVX+CB-LepR group. These results suggest that the self-activated NO nanopump holds promise as a precise targeting strategy for rescuing osteoporosis.

[0047] (6) Verification of anti-aging mechanisms

[0048] To further investigate the effect of NO release from nanopumps on LepR + The mechanism of cell senescence was discussed in this example, focusing on LepR cells after different treatments. + Cells were stained with SA-β-Gal (specific conditions as in (4)). The results showed that cells in the H2O2 group exhibited significant senescence, while cells in the H2O2+CB-NPs group and the H2O2+CB-LepR group showed significantly reduced senescence. Figure 8 (AB). Notably, after the addition of 2-DG (a glycolysis inhibitor) to the H2O2+CB-LepR group, cellular senescence recurred. Figure 8 (AB), which means that NO nanopumps may regulate LepR through the glycolysis pathway. + Cellular senescence. Furthermore, evidence suggests that metabolic processes are closely related to cellular senescence. Therefore, the extracellular acidification rate (ECAR, used to measure glycolysis) was quantified in each group. The results showed that H2O2 addition led to a decrease in both basal and glycolytic capacity in LepR+ cells. Notably, the addition of H2O2+CB-NPs or H2O2+CB-LepR partially restored basal and glycolytic levels and capacity. Figure 8 These data suggest that the NO-mediated glycolysis pathway is crucial for mitigating cellular senescence.

[0049] In summary, this embodiment successfully constructed a self-activated NO nanopump for anti-bone aging, and the binding of the LepR antibody enhanced the LepR... + Cellular targeting. CB-LepR exhibits strong H2O2 scavenging, NO production, and anti-aging capabilities. In vivo administration significantly reversed osteoporosis in OVX mice. Our study proposes a novel strategy to combat bone aging.

Claims

1. A self-activated NO nanopump, characterized in that, The nanopump comprises a chemiluminescent substrate bis(oxalate) CPPO and a NO donor N,N'-di-sec-butyl-N,N'-dinitroso-1,4-phenylenediamine BNN6 encapsulated in phospholipid polyethylene glycol maleimide (DSPE-PEG-Mal). The nanopump is prepared by adding BNN6, soybean oil, CPPO, and DSPE-PEG-Mal to THF, mixing thoroughly under nitrogen protection, removing the solvent, drying, adding Milli-Q water, and shaking vigorously. The DSPE-PEG-Mal surface conjugated LepR antibody.

2. A method for constructing the self-activated NO nanopump according to claim 1, characterized in that, Includes the following steps: The preparation method involves adding BNN6, soybean oil, CPPO, and DSPE-PEG-Mal to THF, mixing them evenly under nitrogen protection, removing the solvent, drying, adding Milli-Q water, and shaking vigorously to obtain CB-NPs.

3. The method for constructing a self-activated NO nanopump according to claim 2, characterized in that, It also includes the step of combining LepR antibody, specifically: mixing LepR antibody with Traut's reagent, reacting at room temperature, removing excess Traut's reagent after the reaction, adding the resulting liquid to CB-NPs and incubating overnight in the dark, adding Milli-Q water and shaking vigorously to obtain CB-LepR.

4. The method for constructing a self-activated NO nanopump according to claim 2, characterized in that, The mass ratio of BNN6:CPPO:DSPE-PEG-Mal is 0.75:1:

5.

5. The use of the self-activated NO nanopump according to claim 1 in the preparation of a drug for reversing bone aging.

6. The application of the self-activated NO nanopump according to claim 1 in the preparation of anti-aging drugs.

7. The application according to any one of claims 5 to 6, characterized in that, The concentration of the self-activated NO nanopump is 50 μg / mL.

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