Drug-loaded nanoparticles for targeted removal of senescent cells and preparation method and application thereof

Abstract

This invention relates to the field of biomedical materials, specifically disclosing a drug-loaded nanoparticle for targeted senescent cell elimination, its preparation method, and its applications. The drug-loaded nanoparticle is composed of dopamine, a β-galactosidase substrate, and a senescent cell scavenger. This invention encapsulates the senescent cell scavenger; when the drug-carrying nanoparticle targets and enters senescent cells, it releases the senescent cell scavenger intracellularly in response, inducing apoptosis and thus eliminating senescent cells. Compared to free drugs, the apoptosis-inducing effect is significantly enhanced, indicating that the drug-loaded nanoparticle significantly improves the senescent cell elimination effect. The drug-loaded nanoparticles of this invention, which target and eliminate senescent cells, can be applied to eliminate natural aging and treat induced cellular senescence (including radiotherapy and chemotherapy), showing good potential application value in anti-aging and enhancing tumor radiotherapy and chemotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials, specifically to a drug-loaded nanoparticle for targeted removal of senescent cells, its preparation method, and its application. Background Technology

[0002] Cellular senescence is a crucial protective mechanism for the body in response to stress responses such as DNA damage and oxidative stress, preventing the replication of senescent, damaged, or transformed cells. However, the abnormal accumulation of senescent cells (SnCs) has been proven to be closely related to atherosclerosis, osteoarthritis, fibrotic diseases, and age-related degenerative diseases. These cells disrupt the homeostasis of the microenvironment by secreting pro-inflammatory cytokines (SASPs). Evidence from genetic models suggests that eliminating senescent cells can prolong lifespan, improve health, and benefit various diseases. Therefore, clearing senescent cells has become a new direction in anti-aging therapy, and improving the efficiency of this clearance has become a key issue in anti-aging treatment.

[0003] Several "senolytics," drugs that selectively kill senescent cells, have been discovered, including sartinib and quercetin, long peppermint, FOXO4 interfering peptide, and Bcl-2 family inhibitors. Among these, Bcl-2 family inhibitors can eliminate a range of senescent cells in the body and replicate the effects observed in transgenic mice undergoing senescence ablation, making them the gold standard for existing anti-aging drugs. However, in the clinical application of existing senolytics, ABT-263 (Navitoclax), as a Bcl-2 family protein inhibitor, while effectively inducing apoptosis in senescent cells, inhibits platelets and B-cell lymphoma-extra large (Bcl-XL) proteins in normal tissues, leading to dose-limiting toxicity (DLT) such as thrombocytopenia. Furthermore, its oral bioavailability is less than 15%, and excessive systemic exposure can cause hepatotoxicity and nephrotoxicity. In addition, ABT-263 exhibits off-target effects, lacks targeting of senescent cells, and has a narrow therapeutic window (therapeutic index TI < 3). Therefore, it is necessary to improve the specificity of senescent cell scavengers in targeting senescent cells, thereby improving drug bioavailability and reducing cytotoxicity.

[0004] Another strategy for targeting aging is to leverage the properties that distinguish senescent cells from normal cells. High expression of aging-associated β-galactosidase (SA-β-gal) is one of the more conserved and decisive characteristics of senescent cells. While galactoside modification has improved the pharmacokinetic properties or delivery of existing drugs, current technologies have failed to effectively utilize this biomarker. Conventional chemical conjugation methods (such as maleimide linkages) result in the masking of the substrate's active site, leading to targeting efficiencies of less than 40%. Therefore, there is a need to focus on improving the senescent cell targeting specificity of existing drugs.

[0005] In summary, the key technical problem this invention addresses is how to design a universal drug delivery system that targets senescent cells and possesses good targeting, high efficiency in eliminating senescent cells, and good safety for delivering targeted drugs for eliminating aging in vivo. Summary of the Invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to address the shortcomings of the prior art by providing a drug-loaded nanoparticle for targeted removal of senescent cells, its preparation method, and its application.

[0007] Inventive Concept: Regarding material design, recent studies have attempted to improve drug targeting using anti-β-gal antibody conjugation strategies, but this strategy is not adopted due to the high cost and potential for immunogenic reactions. In contrast, nanoparticles demonstrate promising potential for targeted drug delivery. Various types of nanoparticles, including liposomes, cationic polymers, inorganic nanoparticles, and nanocapsules, have been proven to successfully deliver drugs to target sites. Polydopamine (PDA) nanocarriers are biomimetic materials inspired by mussel adhesion proteins, formed by the self-oxidative polymerization of dopamine under alkaline conditions. PDA exhibits excellent surface adhesion, forming a uniform coating on almost all material surfaces. Its surface is rich in active groups such as amino and phenolic hydroxyl groups, facilitating chemical modification and functionalization. Furthermore, PDA possesses good biocompatibility and biodegradability, with its degradation products being naturally occurring dopamine monomers, ensuring high safety. In drug delivery, PDA achieves efficient drug loading through various intermolecular forces such as π-π stacking, hydrogen bonding, and electrostatic adsorption, achieving drug loading capacities exceeding 20% ​​(w / w). PDA nanocarriers possess pH-responsive properties, enabling controlled drug release. To enhance targeting, PDA nanocarriers can be actively targeted by surface modification with antibodies, ligands, or enzyme-responsive molecules.

[0008] Based on the high expression of SA-β-gal unique to senescent cells, this invention creatively employs the β-galactosidase-specific substrate X-Gal as a targeting molecule. A senescent cell scavenger is loaded using polydopamine surface grafting technology, achieving enzymatic targeting and microenvironment-responsive release. The galactosidic groups on the surface of the drug-loaded nanoparticles prepared according to this invention are hydrolyzed by SA-β-gal, inducing the nanoparticles to accumulate on the surface of senescent cells, thus significantly enhancing their targeting ability. Furthermore, PDA undergoes charge reversal in the slightly acidic environment of senescent cells, improving drug release efficiency and ultimately enhancing the clearance efficiency of senescent cells, thus showing broad application prospects in anti-aging therapy.

[0009] To solve the above-mentioned technical problems, the present invention discloses the following technical solution:

[0010] In a first aspect, the present invention discloses a drug-loaded nanoparticle for targeted removal of senescent cells and a method for preparing the same.

[0011] The preparation method includes the use of dopamine, β-galactosidase substrate and senescent cell scavenger.

[0012] The β-galactosidase substrate is 5-bromo-4-chloro-3-indole-β-D-galactosidase X-Gal.

[0013] The manufacturing process includes:

[0014] (1) Disperse dopamine hydrochloride in water to obtain a polydopamine prepolymer solution; mix the polydopamine prepolymer solution with an organic solution of an aging cell scavenger to obtain a mixed solution;

[0015] (2) An alkaline substance was added to the resulting mixed solution to carry out a polymerization reaction, resulting in a polydopamine dispersion loaded with a senescent cell scavenger;

[0016] (3) The resulting dispersion was reacted with β-galactosidase substrate to obtain a reaction solution containing drug-loaded nanoparticles that target and eliminate senescent cells.

[0017] In step (1), the senescent cell scavenger is any one or a combination of several of Bcl-2 family inhibitors, flavonoid fisetin and tyrosine kinase inhibitors; the Bcl-2 family inhibitors include ABT-263.

[0018] In step (1), the mass-to-volume ratio of dopamine hydrochloride to water is 1.7-2.7 mg / mL, such as 2.2 mg / mL.

[0019] In step (1), the concentration of the organic solution of the senescent cell scavenger is 0.1-110 mg / mL, such as 90, 100, or 110 mg / mL; the solvent of the solution is an organic solvent, such as DMSO.

[0020] In step (1), the mixing is vortex mixing, such as stirring at a speed of 200-400 rpm for 2-4 hours.

[0021] In step (2), the alkaline substance includes any one or a combination of sodium hydroxide, ammonia, and Tris; the concentration of the sodium hydroxide is 0.05-0.2 M, such as 0.1 M. The volume ratio of the alkaline substance to the polydopamine prepolymer solution is 1:8-10.

[0022] In step (2), the polymerization reaction is carried out under the conditions of avoiding light, 20-30℃, and vortex stirring at 200-500 rpm for 3-10 h, such as 7 h, 8 h or 9 h.

[0023] In step (3), the mass of the β-galactosidase substrate is 0.3-3 times that of dopamine hydrochloride, such as 1 or 2 times.

[0024] In step (3), the reaction is carried out at 20-30℃ and 150-300 rpm for 1-3 hours.

[0025] In step (3), after the reaction is completed, the reaction solution is dialyzed through a dialysis bag to remove unreacted impurities.

[0026] In a second aspect, the present invention discloses the application of the drug-loaded nanoparticles described in the first aspect above in the preparation of drugs for clearing senescent cells and / or anti-tumor drugs.

[0027] Mechanism of action: Polydopamine-loaded nanoparticles modified with the galactosidase substrate X-Gal on their surface are designed and synthesized, and loaded with senescence-clearing targeted drugs (such as ABT-263, a small molecule drug targeting the anti-apoptotic protein BCL2). When senescent cells engulf the nanoparticles, the high galactosidase in their lysosomes degrades X-Gal, leading to the degradation of the polydopamine nanospheres. The loaded ABT-263 is then released, causing apoptosis in the senescent cells.

[0028] Beneficial effects:

[0029] (1) This invention provides a method for preparing drug-loaded nanoparticles that target and clear senescent cells. The nanoparticles are prepared from polydopamine, β-galactosidase substrate (X-Gal) and senescent cell clearing agent. The targeting of senescent cells is significantly enhanced, and the nanoparticles can accumulate in large quantities in senescent cells, thus solving the problem of insufficient targeting of senescent cell clearing agent.

[0030] (2) This invention utilizes a senescent cell scavenger encapsulated in nanoparticles. Upon targeted entry into senescent cells, the drug-loaded nanoparticles responsively release the senescent cell scavenger, inducing apoptosis and thus clearing the senescent cells. Compared to free drugs, the apoptosis-inducing effect is significantly enhanced, indicating that the drug-loaded nanoparticles exhibit a significantly improved senescent cell clearing effect. The drug-loaded nanoparticles of this invention, which target and clear senescent cells, can be applied to clearing naturally aging cells and treating (including radiotherapy and chemotherapy) induced cell senescence. They have promising potential applications in anti-aging and enhancing tumor radiotherapy and chemotherapy. Attached Figure Description

[0031] 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.

[0032] Figure 1 This is a TEM image of the dispersion of galactoside-modified polydopamine drug-loaded nanoparticles (ABT@Gal-PDA) prepared in Example 1 of the present invention.

[0033] Figure 2 This is a schematic diagram showing the stability of drug-loaded nanoparticles modified with galactoside loaded with ABT-263 under different conditions.

[0034] Figure 3 The cumulative release curves of FITC from galactoside-modified polydopamine-loaded nanoparticles loaded with FITC in different pH values ​​(left) and in solutions with and without β-galactosidase (right).

[0035] Figure 4 CLSM plot showing the uptake of drug-loaded nanoparticles by non-senescent / senescent cells.

[0036] Figure 5 This figure shows the effect of drug-loaded nanoparticles on cell apoptosis.

[0037] Figure 6 Survival curves (A) of mice in the control group (no treatment), TMZ combined with ABT-263 monotherapy group, TMZ combined with ABT-263@PDA treatment group, and TMZ combined with ABT-263@Gal-PDA treatment group, and tumor solid images and immunohistochemical staining of aging and apoptosis indicators (B). Detailed Implementation

[0038] The present invention can be better understood from the following embodiments. However, those skilled in the art will readily understand that the descriptions in the embodiments are for illustrative purposes only and should not, and will not, limit the invention as detailed in the claims.

[0039] Unless otherwise specified, the experimental methods described in the following examples are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0040] Cellular senescence is an intrinsic cause of many diseases. Cellular senescence leads to the decline of tissue and organ function, thereby inducing various age-related diseases, including: core mechanisms such as decreased cell proliferation capacity and senescence-associated secretory phenotype (SASP) triggering chronic inflammation. This results in neurodegenerative diseases such as Alzheimer's and Parkinson's diseases; cardiovascular diseases such as atherosclerosis; metabolic diseases such as type 2 diabetes; eye diseases such as age-related macular degeneration (AMD) and cataracts; skin diseases such as photoaging, the formation of age spots and senile lentigines; and tumor drug resistance, metastasis, and recurrence caused by radiotherapy and chemotherapy. Therefore, this approach has broad application scenarios. In the future, this approach is expected to be transformed into related medical drugs, using precise targeted therapies and local injection to intervene in the early and mid-stages of the above-mentioned diseases. The following examples use the postoperative adjuvant therapy of glioblastoma as an example to illustrate that senescent cell-targeted drug-loaded nanoparticles can be used in the postoperative adjuvant therapy of tumors.

[0041] Example 1: Preparation of drug-loaded nanoparticles for targeted elimination of senescent cells

[0042] 40 mg of dopamine hydrochloride was dispersed in 18 mL of water to obtain a polydopamine prepolymer solution; 10 mg of ABT-263 was dissolved in 100 μL of DMSO to obtain an ABT-263 solution; the ABT-263 solution was added dropwise to the polydopamine prepolymer solution, vortexed at 300 rpm, and stirred for 3 h to obtain a mixed solution of polydopamine prepolymer solution and ABT-263; 2 mL of sodium hydroxide solution (0.1 M) was added dropwise to the mixed solution, and the polymerization reaction was carried out in air at room temperature and in the dark, vortexed at 300 rpm, and the reaction time was 8 h to obtain a polydopamine dispersion loaded with ABT-263; then 40 mg of X-Gal was added to the dispersion and vortexed at 300 rpm for 3 h. h, after dialysis with a dialysis bag (1000Da) to remove unreacted impurities, a dispersion of ABT-263-loaded galactoside-modified polydopamine drug-loaded nanoparticles (ABT@Gal-PDA) was obtained.

[0043] Morphology and size characteristics were analyzed using TEM.

[0044] The dispersion of polydopamine-loaded nanoparticles modified with galactoside loaded with ABT-263 was diluted and the morphology of the samples was observed using transmission electron microscopy. The results are shown in the figure. Figure 1 As shown in the figure. The results indicate that this method can be used to prepare nanoparticles with uniform size.

[0045] Example 2: Stability testing of drug-loaded nanoparticles

[0046] The stability of the prepared drug-loaded nanoparticles was investigated at different temperatures (4°C, 37°C). The specific implementation scheme is as follows: A dispersion of ABT-263-loaded galactoside-modified polydopamine drug-loaded nanoparticles was dispersed in water and placed at 4°C or 37°C. The particle size distribution of the nanoparticles at different time points was measured using a dynamic light scattering instrument. The results are shown in [Figure number missing]. Figure 2 As shown in the figure. The results indicate that the prepared ABT-263-loaded galactoside-modified polydopamine drug-loaded nanoparticles have an average particle size of approximately 150 nm and maintain good dimensional stability for up to 14 days at both 4°C and 37°C.

[0047] Example 3: Detection of drug release from drug-loaded nanoparticles

[0048] To assess the in vitro drug release performance of galactoside-modified polydopamine-loaded nanoparticles, galactoside-modified polydopamine-loaded nanoparticles (FITC@Gal-PDA) were prepared using fluorescent isothiocyanate (FITC) as a model drug. The drug release performance of the drug-loaded nanoparticles in this invention was evaluated by detecting the release of FITC. The specific implementation scheme is as follows:

[0049] (1) 40 mg of dopamine hydrochloride was dispersed in 18 mL of water to obtain a polydopamine prepolymer solution. Then, 10 mg of FITC was dissolved in 100 μL DMSO to obtain a FITC solution. The FITC solution was added dropwise to the polydopamine prepolymer solution, vortexed at 300 rpm, and stirred for 3 h to obtain a mixed solution of polydopamine prepolymer solution and FITC. 2 mL of sodium hydroxide solution (0.1 M) was added dropwise to the mixed solution, and the polymerization reaction was carried out in air under the conditions of room temperature and protection from light. The mixture was vortexed in air at 300 rpm for 8 h to obtain a polydopamine dispersion loaded with FITC. Then, 40 mg of X-Gal was added to the dispersion and vortexed at 300 rpm for 3 h. After removing unreacted impurities by dialysis with a dialysis bag (1000 Da), a dispersion of FITC-loaded galactoside-modified polydopamine drug-loaded nanoparticles was obtained.

[0050] (2) Phosphate-buffered saline (PBS) solutions at pH 5.5 and pH 7.4 were used to simulate the physiological environment in vivo. 5 mL of the prepared FITC-loaded galactoside-modified polydopamine drug-loaded nanoparticle dispersion was placed in a dialysis bag (1000 Da), sealed, and immersed in 50 mL of 0.2 M PBS solutions at different pH values ​​(5.5 / 7.4). Dialysis was performed at 37°C for 7 days. During this period, 2 mL of dialysate was taken each time and the UV absorbance of the dialysate was monitored at 495 nm (the UV characteristic absorption peak of FITC) using a UV spectrophotometer. The cumulative release concentration was obtained from the UV absorption-concentration standard curve of FITC, and the cumulative release rate of FITC was calculated as shown in the following formula:

[0051] .

[0052] (3) Using phosphate buffer solution (PBS) (50 nM, pH 6.5) containing (but not containing) β-galactosidase to simulate the physiological environment in vivo, 5 mL of the prepared FITC-loaded galactoside-modified polydopamine drug-loaded nanoparticle dispersion was placed in a dialysis bag (1000 Da) and tied. The bag was then immersed in 50 mL of phosphate buffer solution (0.2 M, β-galactosidase concentrations (0, 2 U / mL)) and dialyzed at 37°C for 7 days. During this period, the dialysate was taken and the UV absorbance value of the dialysate was monitored at 495 nm (the UV characteristic absorption peak of FITC) using a UV spectrophotometer. The amount of FITC loaded on the drug-loaded nanoparticles at different times was recorded using the method described in step (2), and the cumulative dissolution release rate of FITC was calculated.

[0053] Implementation results are shown Figure 3 The results showed that after 7 days of dialysis, the UV absorbance of the dialysate remained stable at pH 7.4, while at pH 5.5, the absorbance of the dialysate increased significantly with increasing dialysis time. In the presence of β-galactosidase, the absorbance of the dialysate increased significantly with increasing dialysis time compared to the control group. These results indicate that the material showed no significant release at pH 7.4, but significant drug release at pH 5.5. Figure 3 (Left), in the presence of β-galactosidase, significant release ( Figure 3 (Right) This shows that the galactosyl-modified polydopamine drug-loaded nanoparticles prepared in this invention exhibit different drug release behaviors under different pH conditions and with or without β-galactosidase, thus demonstrating significant drug controlled release performance.

[0054] Example 4: Targeting detection of galactose-modified nanoparticles on senescent cells

[0055] To detect the targeting ability of galactoside-modified polydopamine-loaded nanoparticles to senescent cells, fluorescent isothiocyanate (FITC) was used as the fluorescent dye to label the nanoparticles. Gal-PDA (FITC) nanoparticles were prepared using FITC, and the cellular uptake of galactoside-modified nanoparticles by senescent cells was investigated. The specific implementation scheme is as follows:

[0056] (1) 40 mg of dopamine hydrochloride was dispersed in 18 mL of water to obtain a polydopamine prepolymer solution. 2 mL of sodium hydroxide solution was added dropwise to the mixed solution, and polymerization was carried out in the dark in air. The reaction conditions were room temperature, dark, and vortexed in air at 300 rpm for 8 h. Then 40 mg of X-Gal was added and vortexed again at 300 rpm for 3 h to obtain galactosyl-modified polydopamine nanoparticles (Gal-PDA). Unmodified polydopamine nanoparticles (PDA) were prepared as a control.

[0057] (2) Take 10 mL of Gal-PDA and PDA, add 1 mL of FITC (DMSO, 1 mg / mL) to each, and continue to vortex to mix. The rotation speed is 200 rpm and the reaction time is 8 h to obtain FITC-labeled galactoside modified nanoparticles Gal-PDA (FITC) and FITC-labeled polydopamine drug-loaded nanoparticles PDA (FITC).

[0058] (3) Seed cells in 6-well plates (5 × 10⁻⁶ cells / well). 4 Cells were incubated in medium (cells / well) for 24 hours, and then incubated for another 12 hours in different formulations of medium (FITC, PDA (FITC), Gal-PDA (FITC)) at the same concentration. Cell uptake was observed by confocal microscopy.

[0059] Implementation results are shown Figure 4 The results showed that only galactoside-modified nanoparticles, specifically Gal-PDA (FITC), accumulated significantly in senescent cells, while no accumulation of Gal-PDA (FITC) was observed in non-senescent cells. Furthermore, only small amounts of free FITC and PDA (FITC) were taken up in senescent and normal cells, indicating that the nanoparticles modified with galactoside exhibited significant senescent cell specificity.

[0060] Example 5: Detection of the effect of drug-loaded nanoparticles ABT@Gal-PDA on promoting apoptosis in chemotherapy-induced senescent glioma cells.

[0061] The purpose of this study is to evaluate whether ABT@Gal-PDA inhibits tumor cell proliferation by inducing apoptosis in senescent cells. We used flow cytometry to quantitatively detect the level of apoptosis, and the specific implementation plan is as follows:

[0062] (1) ABT@Gal-PDA galactoside-modified polydopamine-loaded nanoparticles loaded with ABT were prepared according to the steps in Example 1, and free drug ABT-263 was used as a control.

[0063] (2) To detect cell apoptosis under different treatment conditions, LN18 cells were cultured at a density of 1×10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in six-well plates and cultured for 24 hours. For each cell type, there were three control groups: a non-senescent cell control group (Control), a senescent cell control group (TMZ), senescent cell experimental group 1 (single drug, TMZ+ABT-263), and senescent cell experimental group 2 (treated with galactoside-modified polydopamine-loaded nanoparticles, TMZ+ABT@Gal-PDA). The non-senescent cell control group was cultured for 5 days without additional treatment; the senescent cell control group was treated for 5 days with complete medium containing 400 μM TMZ; senescent cell experimental group 1 was treated for 5 days with complete medium containing 400 μM TMZ combined with 10 μM ABT-263; and senescent cell experimental group 2 was treated for 5 days with complete medium containing 400 μM TMZ combined with 40 μL ABT@Gal-PDA.

[0064] (3) After collecting cells, they were stained with Annexin V-FITC; the apoptosis rate was determined using a BD FACSCalibur flow cytometer and FlowJo flow cytometry software.

[0065] Implementation results are shown Figure 5 Compared to the blank control group and the temozolomide-induced cell senescence group, the TMZ+ABT@Gal-PDA combined administration regimen induced a significantly stronger apoptosis effect, primarily in early-stage apoptotic cells. Compared to the control group, the TMZ+ABT@Gal-PDA combined administration regimen significantly increased the apoptosis rate of TMZ-induced senescent cells (82%), significantly higher than that treated with free drug ABT-263 (56%). This indicates that the galactoside-modified polydopamine-loaded nanoparticles ABT@Gal-PDA can effectively induce apoptosis in senescent cells, demonstrating excellent anti-aging properties and significantly enhancing the chemotherapeutic efficacy of TMZ.

[0066] Example 6: In vivo efficacy and brain tissue staining of drug-loaded nanoparticles ABT@Gal-PDA

[0067] 6-8 week old mice (20 g) of the C57BL / 6J strain were injected with 1×10⁻⁶ mice on day 0. 5 One GL261-Luc cell was injected into the right frontal lobe of a mouse (2.5 mm lateral to the frontal lobe, 1.0 mm inferior to the frontal lobe, depth 2.5 mm). Tumor growth was observed using a small animal in vivo imaging system. When the tumor volume approached 50-100 mm², [the tumor was monitored]. 3 Mice were randomly divided into groups. After surgical removal of GBM tumors in the brains of mice, complete culture media containing 400 μM TMZ, 400 μM TMZ + 10 μM ABT-263, 400 μM TMZ + 40 μL ABT@PDA, and 400 μM TMZ + 40 μL ABT@Gal-PDA were injected in situ into the surgical cavity, and the wound was sutured. Mice were sacrificed on day 21 and brain tissue was collected. Fresh brain tissue was embedded in OCT, frozen sectioned, and β-Gal stained. Fresh brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin for 24 h, sectioned, and stained with H&E and TUNEL.

[0068] Implementation results are shown Figure 6 .like Figure 6 Results A show that the combination therapy of TMZ and drug-loaded nanoparticles (TMZ+ABT@Gal-PDA) significantly prolonged the survival of mice. Figure 6 As shown in the brain tissue photographs, H&E, and TUNEL staining results (B), the control group, the TMZ+ABT-263 group, and the TMZ+ABT@PDA group all showed significant tumor tissue and severe damage to normal brain tissue; the TMZ+ABT@Gal-PDA group showed the least impact of tumor damage on brain tissue and the most significant treatment effect. Figure 6 The β-gal staining results in B showed that significant cellular senescence was observed in the control group; the TMZ+ABT-263 and TMZ+ABT@PDA groups showed a reduction in β-gal-positive senescent cells due to the senescent cell clearance effect of ABT-263; the TMZ+ABT@Gal-PDA group had the fewest β-gal-positive senescent cells and the most significant senescent cell elimination effect compared with other groups.

[0069] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for preparing drug-loaded nanoparticles that target and eliminate senescent cells, characterized in that, It is made from dopamine, β-galactosidase substrate, and senescent cell scavenger.

2. The preparation method according to claim 1, characterized in that, The β-galactosidase substrate is 5-bromo-4-chloro-3-indole-β-D-galactoside.

3. The preparation method according to claim 1, characterized in that, The manufacturing process includes: (1) Disperse dopamine hydrochloride in water to obtain a polydopamine prepolymer solution; mix the polydopamine prepolymer solution with an organic solution of an aging cell scavenger to obtain a mixed solution; (2) An alkaline substance was added to the resulting mixed solution to carry out a polymerization reaction, resulting in a polydopamine dispersion loaded with a senescent cell scavenger; (3) The resulting dispersion was reacted with β-galactosidase substrate to obtain a reaction solution containing drug-loaded nanoparticles that target and eliminate senescent cells.

4. The preparation method according to claim 4, characterized in that, In step (1), the senescent cell scavenger is any one or a combination of several of the following: Bcl-2 family inhibitors, flavonoid fisetin, and tyrosine kinase inhibitors.

5. The preparation method according to claim 4, characterized in that, In step (1), the mass-to-volume ratio of dopamine hydrochloride to water is 1.7-2.7 mg / mL, and the concentration of the organic solution of the senescent cell scavenger is 0.1-110 mg / mL.

6. The preparation method according to claim 4, characterized in that, In step (2), the alkaline substance includes any one or a combination of sodium hydroxide, ammonia and Tris; the polymerization reaction is carried out for 3-10 h under the conditions of avoiding light, 20-30℃ and vortex stirring at 200-500 rpm.

7. The preparation method according to claim 4, characterized in that, In step (3), the mass of the β-galactosidase substrate is 0.3-3 times that of dopamine hydrochloride.

8. The preparation method according to claim 4, characterized in that, In step (3), the reaction is carried out at 20-30℃ and 150-300 rpm for 1-3 h.

9. A drug-loaded nanoparticle for targeting and clearing senescent cells, prepared by the method of any one of claims 1-8.

10. The use of the drug-loaded nanoparticles according to claim 9 in the preparation of drugs for clearing senescent cells and / or antitumor drugs.

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