Nanoparticles for atherosclerosis diagnosis and treatment and application thereof
By combining a lipid-soluble photoacoustic imaging agent, SS-31 peptide, and oxidized dextran onto a human serum albumin carrier to form nanoparticles, the problem of insufficient targeting of photosensitive therapeutic agents in existing technologies is solved, enabling precise imaging and treatment of atherosclerotic plaques and improving treatment efficacy.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-27
- Publication Date
- 2026-04-14
AI Technical Summary
Existing photosensitive therapeutic agents lack active targeting capabilities and are difficult to effectively accumulate at atherosclerotic plaque sites, resulting in poor treatment outcomes. Furthermore, existing albumin nanodelivery systems have limited targeted therapeutic capabilities, making it difficult to meet the multiple needs of precise imaging, active targeting, and treatment of complex atherosclerotic plaques.
Using human serum albumin as a carrier, combined with lipid-soluble photoacoustic imaging agent, SS-31 peptide and oxidized dextran modification, nanoparticles with cascade targeting capabilities are formed to achieve stepwise delivery from arterial plaques to mitochondria, integrating photoacoustic imaging and drug therapy functions.
It improves the targeting of nanodelivery systems, exhibits excellent photoacoustic imaging performance and free radical scavenging ability, enhances bioaffinity for macrophages and anti-lipid deposition performance, and enables precise imaging and treatment of atherosclerotic plaques.
Smart Images
Figure CN121846302A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of novel nanomedicine preparation technology, specifically relating to a nanoparticle for the diagnosis and treatment of atherosclerosis and its application. Background Technology
[0002] Atherosclerosis (AS) is the core pathological basis of cardiovascular disease, causing approximately 17.9 million deaths globally each year and posing a serious public health challenge. AS is essentially a pathological remodeling of blood vessels driven by chronic inflammation, with complex molecular mechanisms involving pathological processes such as oxidative stress, lipid metabolism disorders, and endothelial dysfunction. Current clinically relied-upon statins and stent interventions face limitations such as insufficient targeting and significant side effects, making precise pathological intervention difficult. Therefore, developing novel targeted therapies has become a key direction for overcoming the bottlenecks in AS prevention and treatment.
[0003] Photoacoustic technology (PA) is a platform technology that mediates energy conversion through photoexcitation. It exhibits excellent scalability, deep tissue action capability, and high-precision intervention potential, offering the possibility of targeted therapy for atherosclerotic plaques. However, traditional photosensitizing agents generally lack active targeting capabilities and are difficult to effectively accumulate at plaque sites due to intracellular metabolic influences, severely limiting their therapeutic effects. Human serum albumin, as the main natural protein in serum, possesses abundant functional groups and an amphiphilic structure, enabling it to bind to various organic molecules and drugs, showing great potential in drug delivery applications. Nevertheless, existing albumin nanodelivery systems still have relatively limited targeted therapeutic capabilities, often exhibiting singular functions and failing to simultaneously meet the multiple needs of precise imaging, active targeting, and treatment of complex atherosclerotic plaques. Therefore, there is an urgent need to improve the functionality of albumin nanodelivery systems and develop a highly efficient integrated product for the diagnosis and treatment of atherosclerosis. Summary of the Invention
[0004] To address the aforementioned problems, this invention provides nanoparticles for the diagnosis and treatment of atherosclerosis and their applications. These nanoparticles enable stepwise delivery of arterial plaques to mitochondria, improving the targeting accuracy of the leukorrhea nanoparticle delivery system. They hold significant promise for applications in the preparation of rapid early detection reagents and therapeutic drugs for atherosclerotic plaques.
[0005] To achieve the above objectives, the specific technical solution of the present invention is as follows: The first aspect of the present invention provides nanoparticles for the diagnosis and treatment of atherosclerosis. The nanoparticles for the diagnosis and treatment of atherosclerosis are formed by using human serum albumin as a carrier, combining a lipid-soluble photoacoustic imaging agent and a drug with lipid-lowering and autophagy-regulating effects, and then modifying the carrier with SS-31 peptide with antioxidant and mitochondrial-targeting effects and oxidized dextran with antioxidant and macrophage-targeting effects. Furthermore, the nanoparticles used for the diagnosis and treatment of atherosclerosis have a particle size of 180nm~240nm.
[0006] Furthermore, the nanoparticles used for the diagnosis and treatment of atherosclerosis are prepared by the following steps: Drugs with lipid-lowering and autophagy-regulating effects are mixed with lipid-soluble photoacoustic imaging agents and human serum albumin to form albumin-co-loaded drug imaging nanoparticles. Albumin-co-loaded drug imaging nanoparticles were mixed with SS-31 peptide, which has antioxidant and mitochondrial targeting effects, so that the albumin-co-loaded drug imaging nanoparticles and SS-31 peptide were chemically cross-linked to form mitochondrial-targeting autophagy-regulating nanoparticles. Mitochondrial-targeted autophagy-regulating nanoparticles were mixed with oxyglucan, which has antioxidant and macrophage-targeting effects, and the nanoparticles and oxyglucan were linked by Schiff base bonds to obtain nanoparticles for the diagnosis and treatment of atherosclerosis.
[0007] Furthermore, the lipid-soluble photoacoustic imaging agent is a thiophene dye or IR-1061.
[0008] Furthermore, the drug is a statin, rapamycin, or a SIRT5 activator.
[0009] Furthermore, the SIRT5 activator is MC3138.
[0010] Furthermore, the molar ratio of human serum albumin, MC-3138, and IR-1061 is 1:4~6:1~3. Within this range, the particle size of the nanoparticles used for the diagnosis and treatment of atherosclerosis is 180nm~240nm, avoiding the problems of drug metabolism being too fast due to excessively small size or difficulty in entering cells due to excessively large size. The mass ratio of the albumin co-loaded drug imaging nanoparticles to SS-31 peptide is 5:0.5~2. The mass ratio of the mitochondrial-targeted autophagy-regulating nanoparticles to oxidized dextran is 5:0.5~2.
[0011] Furthermore, the mass ratio of the albumin-co-loaded drug-imaging nanoparticles to the SS-31 peptide is 5:1.
[0012] Furthermore, the mass ratio of the mitochondrial-targeted autophagy-regulating nanoparticles to oxidized dextran is 5:1.
[0013] Furthermore, the mixing of the drug with lipid-lowering and autophagy-regulating effects with the lipid-soluble photoacoustic imaging agent and human serum albumin, the mixing of albumin-co-loaded drug imaging nanoparticles with SS-31 peptide, and the mixing of mitochondrial-targeted autophagy-regulating nanoparticles with oxidized dextran were all carried out under light-protected conditions, and the mixing time was 6h~24h.
[0014] A second aspect of the present invention provides the application of the nanoparticles described above for the diagnosis and treatment of atherosclerosis in the preparation of a reagent for detecting atherosclerotic plaques.
[0015] A third aspect of the present invention provides the application of the nanoparticles described above for the diagnosis and treatment of atherosclerosis in the preparation of a medicament for treating atherosclerosis.
[0016] A fourth aspect of the present invention provides a medicament for treating atherosclerosis, the medicament comprising the nanoparticles for the diagnosis and treatment of atherosclerosis, and pharmaceutically acceptable excipients or carriers.
[0017] Furthermore, the drug uses the nanoparticles used for the diagnosis and treatment of atherosclerosis as its sole effective active ingredient.
[0018] Furthermore, the excipients include any one or more of fillers, stabilizers, diluents, and adjuvants.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses a nanoparticle for the diagnosis and treatment of atherosclerosis and its applications. For the first time, this invention assembles a photoacoustic imaging molecule IR-1061, a SIRT5 activator MC3138 with lipid-lowering and autophagy-regulating properties, an SS-31 peptide with antioxidant and mitochondrial-targeting effects, and an oxidized dextran with antioxidant and macrophage-targeting effects into a nanoparticle for the diagnosis and treatment of atherosclerosis using human serum albumin. The nanoparticle for the diagnosis and treatment of atherosclerosis provided by this invention improves the targeting ability of conventional albumin nanodelivery systems. On the one hand, it exhibits photoacoustic imaging performance above 850 nm and excellent free radical scavenging ability; on the other hand, it shows good biocompatibility, anti-lipid deposition, and anti-inflammatory properties on macrophages. The nanoparticle for the diagnosis and treatment of atherosclerosis is a cascaded targeted-multimodal imaging nanoparticle constructed based on human serum albumin carrier, integrating photoacoustic imaging, specific drugs, and targeted modification, providing a new solution for reducing the incidence of cardiovascular and cerebrovascular events. Furthermore, the preparation method of nanoparticles for the diagnosis and treatment of atherosclerosis provided by this invention is simple and easy to control, meets the needs of actual production, and is conducive to large-scale promotion. Attached Figure Description
[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0021] Figure 1 The hydrodynamic particle size distribution, morphology, light absorption and photoacoustic imaging performance of nanoparticles (ATMC-PA nanoparticles) used for the diagnosis and treatment of atherosclerosis were characterized. Figure 1 In the image, 'a' is a transmission electron microscope (TEM) image of the nanoparticle ATMC-PA. Figure 1 In the diagram, b represents the hydrodynamic particle size distribution of the ATMC-PA nanoparticles. Figure 1 In the diagram, c represents the optical absorption spectrum of the ATMC-PA nanoparticles. Figure 1 In the image, d represents the photoacoustic imaging image of the ATMC-PA nanoparticles. Figure 1 In this context, 'e' represents the photoacoustic imaging signal intensity of the ATMC-PA nanoparticles at different wavelengths.
[0022] Figure 2 The results were obtained to determine the in vitro free radical scavenging ability of ATMC-PA nanoparticles. Figure 2 In the figure, 'a' represents the effect of different concentrations of ATMC-PA nanoparticles on superoxide radicals (O2). ·- The ability to clear ( ); Figure 2 In the figure, b represents the scavenging ability of ATMC-PA nanoparticles at different concentrations for hydroxyl radicals (·OH); Figure 2 In the figure, c represents the ability of ATMC-PA nanoparticles of different concentrations to scavenge reactive oxygen species, as determined by the ABTS method. Figure 2 In the figure, d represents the ability of ATMC-PA nanoparticles of different concentrations to scavenge reactive oxygen species using the DPPH method.
[0023] Figure 3 The results of the test on the protective performance of different concentrations of ATMC-PA nanoparticles against Hcy-stimulated macrophages are shown in **. p <0.01, *** indicates p <0.001.
[0024] Figure 4 After Hcy combined with ATMC-PA intervention in macrophages SIRT5 mRNA expression status, ** indicates p <0.01, **** indicates p <0.0001.
[0025] Figure 5 Oil Red O staining was used to assess lipid deposition in macrophages after Hcy combined with ATMC-PA intervention.
[0026] Figure 6 To evaluate the antioxidant performance of macrophages after Hcy combined with ATMC-PA intervention using flow cytometry, Figure 6 In the table, a, b, and c represent the flow cytometry analysis results of the Control group, the Hcy stimulation group, and the Hcy+ATMC-PA group, respectively.Figure 6 In this context, d represents the statistical result, and **** indicates... p <0.0001.
[0027] Figure 7 To assess the reactive oxygen species status of macrophages after Hcy combined with ATMC-PA intervention using laser confocal microscopy, Figure 7 In the image, 'a' represents a confocal microscope image, scale bar: 50μm; Figure 7 In this context, 'b' represents the statistical result, and **** indicates... p <0.0001. Detailed Implementation
[0028] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.
[0029] This invention uses human serum albumin as a carrier, combining the lipid-soluble photoacoustic imaging agent IR-1061 with the SIRT5 activator. After modifying the carrier with SS-31 peptide and oxidized dextran, a nanoparticle for the diagnosis and treatment of atherosclerosis is obtained. This nanoparticle for atherosclerosis diagnosis and treatment possesses cascaded targeted multimodal imaging capabilities, enabling stepwise delivery from arterial plaques to mitochondria, and shows great promise for rapid early detection and treatment of atherosclerotic plaques.
[0030] Example 1: A nanoparticle for the diagnosis and treatment of atherosclerosis is prepared by the following steps: 1) Dissolve 66 mg of human serum albumin in water to obtain an aqueous solution of human serum albumin; dissolve 2.2 mg of MC-3138 in dimethyl sulfoxide solution to obtain an MC-3138 solution; dissolve 1.5 mg of IR-1061 in dimethyl sulfoxide solution to obtain an IR-1061 solution.
[0031] 2) Mix the human serum albumin aqueous solution, MC-3138 solution and IR-1061 solution from step 1), stir in the dark for 12 hours, collect the supernatant by centrifugation, dialyze the supernatant for 24 hours (MW 14kDa), freeze-dry the dialysate to obtain albumin co-loaded drug imaging nanoparticles AT-PA, labeled as AT-PA.
[0032] 3) Disperse 10 mg AT-PA in deionized water, add 2 mg SS-31 peptide, stir in the dark for 12 hours, and then dialyze in water for 24 hours (MW 3kDa) to obtain dialysate containing mitochondrial-targeted autophagy-regulating nanoparticles ATM-PA. Label the mitochondrial-targeted autophagy-regulating nanoparticles as ATM-PA.
[0033] 4) Add 2 mg of oxidized dextran to the dialysis solution (containing 10 mg ATM-PA) obtained in step 3), stir in the dark for 12 hours, then dialyze in deionized water for 24 hours (MW 15 kDa), and freeze-dry to obtain solid nanoparticles for the diagnosis and treatment of atherosclerosis, and label them as ATMC-PA.
[0034] Example 2: A nanoparticle for the diagnosis and treatment of atherosclerosis is prepared by the following steps: 1) Dissolve 66 mg of human serum albumin in water to obtain an aqueous solution of human serum albumin; dissolve 1.98 mg of MC-3138 in dimethyl sulfoxide solution to obtain an MC-3138 solution; dissolve 0.606 mg of IR-1061 in dimethyl sulfoxide solution to obtain an IR-1061 solution.
[0035] 2) Mix the human serum albumin aqueous solution, MC-3138 solution and IR-1061 solution from step 1), stir in the dark for 24 hours, collect the supernatant by centrifugation, dialyze the supernatant for 24 hours (MW 14kDa), and freeze-dry the dialysate to obtain AT-PA.
[0036] 3) Disperse 10 mg AT-PA in deionized water, add 1 mg SS-31 peptide, stir in the dark for 24 hours, and then dialyze in water for 24 hours (MW 3kDa) to obtain dialysate containing ATM-PA.
[0037] 4) Add 4 mg of oxidized dextran to the dialysis solution (containing 10 mg ATM-PA) obtained in step 3), stir for 24 hours in the dark, then dialyze in deionized water for 24 hours (MW 15 kDa), and freeze dry to obtain ATMC-PA.
[0038] Example 3: A nanoparticle for the diagnosis and treatment of atherosclerosis is prepared by the following steps: 1) Dissolve 66 mg of human serum albumin in water to obtain an aqueous solution of human serum albumin; dissolve 2.99 mg of MC-3138 in dimethyl sulfoxide solution to obtain an MC-3138 solution; dissolve 1.99 mg of IR-1061 in dimethyl sulfoxide solution to obtain an IR-1061 solution.
[0039] 2) Mix the human serum albumin aqueous solution, MC-3138 solution and IR-1061 solution from step 1), stir in the dark for 6 hours, collect the supernatant by centrifugation, dialyze the supernatant for 24 hours (MW 14kDa), and freeze-dry the dialysate to obtain AT-PA.
[0040] 3) Disperse 10 mg AT-PA in deionized water, add 4 mg SS-31 peptide, stir in the dark for 6 hours, and then dialyze in water for 24 hours (MW 3kDa) to obtain dialysate containing ATM-PA.
[0041] 4) Add 1 mg of oxidized dextran to the dialysate (containing 10 mg ATM-PA) obtained in step 3), stir for 6 hours in the dark, then dialyze in deionized water for 24 hours (MW 15 kDa), and freeze dry to obtain ATMC-PA.
[0042] Examples 1-3 have similar effects. For ease of subsequent discussion and reference, the experimental results of Example 1 will be used as an example. The following are the experimental results of Example 1.
[0043] Experimental Example 1: Characterization of hydrodynamic particle size distribution, morphology, light absorption and photoacoustic imaging performance of ATMC-PA nanoparticles The hydrodynamic particle size distribution of ATMC-PA nanoparticles at a concentration of 0.1 mg / mL was determined using a dynamic light scattering instrument (Litesizer DLS 501, AntonPaar); the morphology of the ATMC-PA nanoparticles was analyzed using field emission transmission electron microscopy (Talos F200s, FEI); the light absorption performance of the ATMC-PA nanoparticles was tested using a UV-Vis spectrophotometer (T2602, Shanghai Youke); and the photoacoustic imaging performance of the TMC-PA nanoparticles was tested using a photoacoustic integrated imaging platform (Marsonics PIIP-FC, Beijing Guangsheng Biotechnology).
[0044] The results are as follows Figure 1 As shown, Figure 1 Figures a and b show that the size of the ATMC-PA nanoparticles is approximately 200 nm. Figure 1 The 'c' in the figure indicates that the ATMC-PA nanoparticles exhibit significant light absorption performance in the range of 700nm to 1000nm, and show the strongest light absorption ability at 800nm. Figure 1 The d-values in the image show that the ATMC-PA nanoparticles exhibit clear photoacoustic imaging. Figure 1 The 'e' in the figure indicates that the ATMC-PA nanoparticles exhibit strong photoacoustic signals in the 800 nm to 900 nm range.
[0045] Experimental Example 2: Determination of the In Vitro Free Radical Scavenging Ability of ATMC-PA Nanoparticles 1. The superoxide radical scavenging ability was detected using a superoxide dismutase (SOD) kit (WST-8 method). Following the kit instructions, the microplate reader wavelength was adjusted to 450 nm. Reagents from the kit and aqueous solutions of ATMC-PA nanoparticles at different concentrations (10 μg / mL to 150 μg / mL) were added sequentially, and the absorbance at 450 nm was measured.
[0046] Suppression percentage = [(A 空白管1 -A 空白管2 )-(A 样本管 -A 样本对照管* )] / (A 空白管1 -A 空白管2 )×100%.
[0047] 2. The hydroxyl radical scavenging ability was tested using the following method: Equimolar amounts (1 mM) of ferrous sulfate and salicylic acid were mixed, and 2 mM of H2O2 was added. After incubation for 15 minutes, the working solution was obtained. 100 μL of the working solution was added to a 96-well plate, followed by an equal volume of aqueous solutions containing different concentrations (10 μg / mL~150 μg / mL) of ATMC-PA nanoparticles. The plate was incubated in the dark for 15 minutes, and then the absorbance at 510 nm was measured using a microplate reader.
[0048] Suppression percentage = [(A 空白管1 -A 空白管2 )-(A 样本管 -A 样本对照管* )] / (A 空白管1 -A 空白管2 )×100%.
[0049] 3. ABTS scavenging ability was tested using the following method: ABTS free radical stock solution was generated using ABTS solution and potassium persulfate, and then diluted to prepare working solution (5-fold dilution). 100 μL of working solution was added to a 96-well plate, followed by an equal volume of aqueous solution containing different concentrations (10 μg / mL~150 μg / mL) of ATMC-PA nanoparticles. The plate was incubated in the dark for 10 minutes, and then the absorbance at 734 nm was measured using a microplate reader.
[0050] Suppression percentage = [(A 空白管1 -A 空白管2 )-(A 样本管 -A 样本对照管* )] / (A 空白管1 -A 空白管2 )×100%.
[0051] 4. The DPPH scavenging ability was tested using the following method: 100 μL of aqueous solution containing different concentrations (10 μg / mL~150 μg / mL) of ATMC-PA nanoparticles was added to a 96-well plate, followed by the addition of DPPH solution. The plate was incubated at 37°C for 15 minutes, and then the absorbance at 517 nm was measured using a microplate reader.
[0052] Suppression percentage = [(A 空白管1 -A 空白管2 )-(A 样本管 -A 样本对照管* )] / (A 空白管1 -A 空白管2 )×100%.
[0053] MC3138 and oxidized dextran in ATMC-PA nanoparticles not only serve as targeted modifications, but also possess antioxidant properties themselves. Figure 2 a in Figure 1 and b in Figure 2 show the effect of ATMC-PA nanoparticles on O2. ·- Both ·OH and ·OH exhibited concentration-dependent scavenging performance; when the added ATMC-PA concentration was 150 µg / mL, it significantly reduced O2 scavenging activity. ·- The scavenging rates of ·OH and ·OH can reach over 80% and 90%, respectively. The overall antioxidant capacity of the ATMC-PA nanoparticles is shown in the following results. Figure 2 As shown in c and d, the ATMC-PA nanoparticles also exhibit concentration-dependent scavenging performance.
[0054] Experimental Example 3: Performance determination of ATMC-PA nanoparticles in inhibiting macrophage lipid deposition and inflammatory response The lipid deposition-inhibiting and anti-inflammatory properties of the ATMC-PA nanoparticles were evaluated using mouse mononuclear macrophages (RAW 264.7). RAW 264.7 cells were cultured in high-glucose medium (DMEM) containing 10% (v / v) fetal bovine serum.
[0055] 1. CCK-8 cytotoxicity assay The cytotoxicity of ATMC-PA nanoparticles was investigated using a CCK-8 assay. 5 × 10⁶ nanoparticles were used per well. 3 RAW 264.7 cells were seeded in 96-well plates and incubated for 24 h. Then, the medium was replaced with 200 μL of fresh medium containing 0.1 mM Hcy and different concentrations (2.5 µg / mL to 80 µg / mL) of ATMC-PA and cultured for another 24 h. Finally, 20 μL of CCK-8 solution was added and the cells were co-cultured for 2 h. The absorbance at 450 nm was measured using a microplate reader, and in vitro cytotoxicity was calculated. The Control group and the Hcy group were treated with equal volumes of medium and medium containing 0.1 mM Hcy, respectively, with all other steps remaining the same.
[0056] The results are as follows Figure 3 As shown, compared with the Control group, the cell survival rate of the Hcy stimulation group alone was significantly reduced; however, as the ATMC-PA concentration increased (2.5 µg / mL~80 µg / mL), the cell survival rate rebounded in a concentration-dependent manner. The cell survival rate of the 80 µg / mL ATMC-PA group recovered to a level close to that of the Control group, confirming that ATMC-PA can effectively antagonize Hcy-induced macrophage damage without significant cytotoxicity.
[0057] 2. SIRT5 mRNA expression detection SIRT5 expression was detected using qRT-PCR: Total RNA was extracted from macrophages using an RNA extraction kit; first-strand cDNA was synthesized from the total RNA using a Revert Aid first-strand cDNA synthesis kit (MBI, Vilnius, Lithuania); and qRT-PCR was performed using Talent qPCR premix (SYBR Green) on a QuantStudio 5 real-time PCR system (ThermoFisher Scientific, USA).
[0058] The results are as follows Figure 4 As shown, the expression level of SIRT5 mRNA in the Hcy stimulation group was significantly downregulated compared with that in the Control group; while the expression of SIRT5 in the Hcy+ATMC-PA combined intervention group was significantly restored, suggesting that ATMC-PA can reverse the inhibition of SIRT5 expression caused by Hcy and restore its anti-inflammatory regulatory function.
[0059] 3. Oil Red O staining to assess lipid deposition ATMC-PA was used to assess lipid deposition in macrophages using Oil Red O staining: RAW 264.7 cells were seeded in 12-well plates and cultured for 24 h in medium containing Hcy, followed by 24 h in medium containing ox-LDL and ATMC-PA nanoparticles. The medium was discarded, and the cells were washed three times with PBS. Then, 0.3% Oil Red O working solution was added for staining for 60 min, followed by rinsing with 60% isopropanol for 5 s. Cell lipid uptake was observed under an inverted microscope. The control group received an equal volume of medium, while the Hcy group received only ox-LDL after Hcy stimulation, with other parameters unchanged.
[0060] like Figure 5 Microscopic observation showed that no obvious red lipid droplets were observed in the cells of the Control group; a large number of red lipid droplets appeared in the cytoplasm of the Hcy-stimulated group, and lipid deposition was significantly enhanced; while the number of red lipid droplets in the Hcy+ATMC-PA group was significantly reduced and the staining intensity was weakened.
[0061] 4. ROS testing Flow cytometry used a reactive oxygen species (ROS) detection kit (DHE) to stain for ROS; laser confocal microscopy used a ROS detection kit (CM-H2DCFDA) to stain for ROS. RAW 264.7 cells were seeded in 6-well plates and cultured for 24 hours. After discarding the original culture medium, the cells were cultured for another 24 hours in medium containing 0.1 mM Hcy. Cells were then processed according to the instructions for the DHE and CM-H2DCFDA kits. Finally, flow cytometry analysis and imaging analysis were performed on a CytoFLEX SRT instrument using a 711-6 laser confocal imaging system. Statistical data were analyzed using GraphPad Prism 8.0 and One-way analysis of variance software.
[0062] Flow cytometry results ( Figure 6 The results showed that the DHE fluorescence signal (proportion of ROS-positive cells) in the Hcy-stimulated group was significantly higher than that in the Control group; while the fluorescence peak in the Hcy+ATMC-PA group shifted to the left and the proportion of ROS-positive cells decreased significantly, indicating that ATMC-PA can significantly reduce Hcy-induced ROS production and enhance the antioxidant capacity of cells.
[0063] Confocal microscopy imaging ( Figure 7 The results showed that cells in the Control group exhibited weak green fluorescence; the green fluorescence intensity of cells in the Hcy-stimulated group was significantly enhanced, indicating the large-scale generation of ROS; the fluorescence intensity of the Hcy+ATMC-PA group was significantly reduced, approaching the level of the Control group, indicating that ATMC-PA effectively scavenged Hcy-induced reactive oxygen species, consistent with the flow cytometry results.
[0064] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of the invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of this invention.
[0065] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A nanoparticle for the diagnosis and treatment of atherosclerosis, characterized in that, The nanoparticles are prepared by the following steps: Drugs with lipid-lowering and autophagy-regulating effects are mixed with lipid-soluble photoacoustic imaging agents and human serum albumin to form albumin-co-loaded drug imaging nanoparticles. Albumin-co-loaded drug imaging nanoparticles were mixed with SS-31 peptides, and the albumin-co-loaded drug imaging nanoparticles and SS-31 peptides were chemically cross-linked to form mitochondrial-targeted autophagy-regulating nanoparticles. Mitochondrial-targeted autophagy-regulating nanoparticles were mixed with oxidized dextran, and the nanoparticles and oxidized dextran were linked by Schiff base bonds to obtain nanoparticles for the diagnosis and treatment of atherosclerosis.
2. The nanoparticles for the diagnosis and treatment of atherosclerosis according to claim 1, characterized in that, The lipid-soluble photoacoustic imaging agent is an ester-soluble thiophene dye or IR-1061.
3. The nanoparticles for the diagnosis and treatment of atherosclerosis according to claim 2, characterized in that, The drug is a statin, rapamycin, or a SIRT5 activator.
4. The nanoparticles for the diagnosis and treatment of atherosclerosis according to claim 3, characterized in that, The SIRT5 activator is MC3138.
5. The nanoparticles for the diagnosis and treatment of atherosclerosis according to claim 4, characterized in that, The molar ratio of human serum albumin, MC-3138, and IR-1061 is 1:4~6:1~3; the mass ratio of albumin-co-loaded drug imaging nanoparticles to SS-31 peptide is 5:0.5~2; and the mass ratio of mitochondrial-targeted autophagy-regulating nanoparticles to oxidized dextran is 5:0.5~2.
6. The nanoparticles for the diagnosis and treatment of atherosclerosis according to claim 5, characterized in that, The mixing of the drug with lipid-lowering and autophagy-regulating effects with a lipid-soluble photoacoustic imaging agent and human serum albumin, the mixing of albumin-co-loaded drug imaging nanoparticles with SS-31 peptide, and the mixing of mitochondrial-targeted autophagy-regulating nanoparticles with oxidized dextran were all carried out under light-protected conditions, with mixing times ranging from 6 to 24 hours.
7. The nanoparticles for the diagnosis and treatment of atherosclerosis according to claim 6, characterized in that, The nanoparticles used for the diagnosis and treatment of atherosclerosis have a particle size of 180nm~240nm.
8. The use of the nanoparticles for the diagnosis and treatment of atherosclerosis as described in claim 1 in the preparation of a reagent for detecting atherosclerotic plaques.
9. The use of the nanoparticles of claim 1 for the diagnosis and treatment of atherosclerosis in the preparation of a medicament for treating atherosclerosis.
10. A drug for treating atherosclerosis, characterized in that, It includes the nanoparticles for the diagnosis and treatment of atherosclerosis as described in claim 1, and pharmaceutically acceptable excipients or carriers.