A lipoic acid-loaded polylactic acid complex nanoparticle and a preparation method and application thereof

Abstract

The application discloses a kind of alpha-lipoic acid loaded polylactic acid composite nanoparticles and preparation method and application thereof.The nanoparticles are formed with polylactic acid-glycolic acid copolymer to form a hydrophobic inner core, with distearoyl phosphatidyl ethanolamine-polyethylene glycol- triphenylphosphine to form a surface functional layer, and alpha-lipoic acid is loaded. The system has the advantages of mild preparation conditions, uniform particles, high drug loading efficiency, slow release in gastric acid environment, sustained release in neutral or weak acid environment, good cell compatibility, and enhanced mitochondrial regional positioning. The nanoparticles can reduce hypoxia-induced reactive oxygen species and Ca²⁺ elevation, improve ATP deficiency, and reduce myocardial and lung tissue damage, and are suitable for preparing pharmaceutical preparations for alleviating hypoxia-induced mitochondrial dysfunction and related organ damage.

Description

Technical Field

[0001] This invention belongs to the fields of pharmaceutical formulation, nanobiomaterials and hypoxia injury protection, specifically relating to a polylactic acid composite (PLGA / DSPE-PEG-TPP) nanoparticle loaded with α-lipoic acid, its preparation method, pharmaceutical composition and its application in the preparation of drugs to alleviate hypoxia-induced mitochondrial dysfunction. Background Technology

[0002] Alpha-lipoic acid (ALA) has antioxidant properties and improves mitochondrial energy metabolism, but free ALA suffers from instability and insufficient target site delivery efficiency. PLGA possesses degradable and drug-loadable properties, but there is still room for improvement in the encapsulation efficiency, early release control, and organelle localization capabilities of PLGA nanoparticles for ALA.

[0003] Existing PLGA delivery systems still face technical challenges in simultaneously addressing particle stability, drug encapsulation, controlled release, and target site delivery. To address these challenges, this invention combines PLGA, DSPE-PEG-TPP, and α-lipoic acid through nanoprecipitation and self-assembly to form composite nanoparticles. This achieves higher encapsulation efficiency, suitable particle size, sustained release, good cell compatibility, and mitochondrial localization ability, thereby enhancing the effective utilization of α-lipoic acid in hypoxia injury intervention. Summary of the Invention

[0004] The present invention aims to provide an α-lipoic acid nanodelivery system that is easy to prepare, has high encapsulation efficiency, controllable release, and has the potential for mitochondrial regional localization, so as to improve the problems of insufficient stability and delivery efficiency of free α-lipoic acid.

[0005] (1) This invention provides PLGA / DSPE-PEG-TPP nanoparticles loaded with α-lipoic acid. The nanoparticles use PLGA as the main framework material, forming a hydrophobic core in an aqueous environment to encapsulate the hydrophobic drug α-lipoic acid. DSPE-PEG-TPP, as a functional amphiphilic molecule, participates in the self-assembly of the nanoparticles. The hydrophobic fatty acid segments of DSPE can be embedded into the PLGA core or anchored to the surface of the nanoparticles through hydrophobic interactions, while the PEG segments extend outwards into the aqueous phase, improving the dispersibility and colloidal stability of the nanoparticles and reducing particle aggregation. The TPP groups are located on the outer layer of the nanoparticles, endowing them with certain mitochondrial localization potential. α-Lipoic acid is mainly encapsulated in the hydrophobic core of PLGA and its adjacent region, thereby improving its dispersibility in the aqueous system and achieving a certain degree of slow release.

[0006] (2) In the nanoparticles of the present invention, the mass ratio of PLGA, DSPE-PEG-TPP, and α-lipoic acid is 100:2-5:10-20. By adjusting the ratio of the three components, nanoparticles with relatively uniform particle size distribution, good stability, and superior drug loading performance can be obtained. Preferably, the mass ratio of PLGA, DSPE-PEG-TPP, and α-lipoic acid is 100:2.5:10. At this ratio, the nanoparticles can simultaneously possess good granulation properties, drug loading capacity, and dispersion stability, which is beneficial for subsequent formulation preparation and in vitro and in vivo applications.

[0007] (3) The nanoparticles of this invention are prepared by a nanoprecipitation-self-assembly method. Specifically, PLGA and α-lipoic acid are dissolved in an organic solvent to form an organic phase, and DSPE-PEG-TPP is dispersed or dissolved in an aqueous phase. Under stirring conditions at room temperature, the organic phase is added dropwise to the aqueous phase, allowing PLGA, DSPE-PEG-TPP, and α-lipoic acid to form nanoparticles through hydrophobic interactions, molecular self-assembly, and solvent diffusion. This method is simple to operate, has mild reaction conditions, does not require high temperature, high pressure, strong acid or strong base conditions, and does not require complex chemical modification or additional coupling reactions of α-lipoic acid, which is beneficial for maintaining the stability of the drug structure and facilitates scale-up preparation.

[0008] (4) The nanoparticles of the present invention can be further prepared into various pharmaceutical formulations, including but not limited to oral suspensions, oral solutions, capsules, tablets, granules, or lyophilized formulations. Depending on the specific application requirements, suitable pharmaceutically acceptable excipients, such as stabilizers, lyophilization protectants, suspending agents, fillers, disintegrants, binders, or lubricants, can be added to improve formulation stability, storage convenience, and dosing suitability. The nanoparticles or their pharmaceutical formulations can be used to alleviate hypoxia-induced mitochondrial functional damage and further reduce damage to related organs such as the heart, lungs, and brain caused by hypoxia.

[0009] The beneficial effects of this invention are:

[0010] (1) The preparation process is simple. Composite nanoparticles can be formed by nanoprecipitation-self-assembly, which involves few steps, mild conditions, and commercially available materials, making it easy to standardize and scale up.

[0011] (2) High drug loading efficiency. The α-lipoic acid encapsulation efficiency of the preferred formulation is 81.78%, and the drug loading efficiency is 8.77%; under the same drug dosage ratio, both the encapsulation efficiency and the drug loading efficiency are higher than those of α-LA@PLGA.

[0012] (3) The particle properties are suitable for delivery. Preferably, the nanoparticles have an average particle size of 125.6 nm, a Zeta potential of -20.8 mV, and are spherical or near-spherical with clear particle boundaries and relatively uniform distribution.

[0013] (4) Release characteristics are favorable for oral application. Nanoparticles release slowly at pH 1.2, which can reduce rapid drug release in strong acid environments; release is more complete at pH 6.5 and pH 7.4, and have sustained release characteristics.

[0014] (5) It has good compatibility and mitochondrial regional localization potential. The blank vector did not show obvious toxicity to H9C2, A549 and SH-SY5Y cells after treatment with 200 μg / mL for 24 h; the overlap between TPP functionalized particles and mitochondrial fluorescence signals was higher than that of unmodified PLGA particles.

[0015] (6) It has a protective effect against hypoxia-induced damage. The nanoparticles can reduce the levels of ROS and Ca²⁺ in hypoxic cells, improve ATP deficiency, and reduce myocardial and lung tissue damage and serum CK and LDH elevation after oral administration. Attached Figure Description

[0016] Figure 1 This is a schematic diagram illustrating the preparation process and structure of PLGA / DSPE-PEG-TPP nanoparticles.

[0017] Figure 2 The images show transmission electron microscopy (TEM) images, particle size distribution, and zeta potential results for the nanoparticles.

[0018] Figure 3 The in vitro release curves of α-LA@PLGA and α-LA@PLGA / DSPE-PEG-TPP are shown, as well as the release curve of the latter under different pH conditions.

[0019] Figure 4 The results show the colocalization of Rh123@PLGA and Rh123@PLGA / DSPE-PEG-TPP with mitochondria and Golgi apparatus in three cell types.

[0020] Figure 5 To investigate the cell compatibility of PLGA / DSPE-PEG-TPP and the effect of α-LA@PLGA / DSPE-PEG-TPP on representative mitochondrial functional indicators in hypoxic cells.

[0021] Figure 6 The effects of α-LA@PLGA / DSPE-PEG-TPP on myocardial and lung tissue damage and serum CK and LDH in hypoxic mice. Detailed Implementation

[0022] The technical solution of the present invention will be described in detail below with reference to specific embodiments. The following embodiments are only used to better explain the principles and features of the present invention and are not intended to limit the scope of the invention. Unless otherwise specified, specific conditions in the embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments used, unless otherwise specified, are all commercially available products. Quantitative experiments in the present invention are performed in triplicate unless otherwise specified, and the results are averaged.

[0023] Example 1

[0024] Preparation of polylactic acid composite nanoparticles

[0025] The main materials used in this embodiment are sourced from the following sources: carboxyl-terminated PLGA (lactic acid / glycolic acid ratio: 50:50; molecular weight: 40-50kDa); DSPE-PEG-TPP (Chongqing Yusi Pharmaceutical Technology Co., Ltd.; PEG molecular weight: 2kDa); α-lipoic acid (Shanghai Maclean Biochemical Technology Co., Ltd.); dimethyl sulfoxide (Beijing Solarbio Technology Co., Ltd.); acetone (Sinopharm Chemical Reagent Co., Ltd.).

[0026] This embodiment uses a nanoprecipitation-self-assembly method to prepare α-LA@PLGA / DSPE-PEG-TPP nanoparticles, specifically including the following steps:

[0027] (1) Preparation of aqueous phase: Accurately weigh 0.25 mg DSPE-PEG-TPP, add 6.25 μL of dimethyl sulfoxide to dissolve it completely, and then add 4 mL of deionized water to dilute it. Mix thoroughly with a shaker to obtain a clear or uniformly dispersed DSPE-PEG-TPP aqueous phase.

[0028] (2) Preparation of organic phase: Accurately weigh 10 mg of carboxyl-terminated PLGA and 1 mg of α-lipoic acid, add 1 mL of acetone, and gently shake or vortex until PLGA and α-lipoic acid are fully dissolved to obtain a homogeneous organic phase. The mass ratio of PLGA, DSPE-PEG-TPP and α-lipoic acid in this formulation is 100:2.5:10.

[0029] (3) Nanoprecipitation and self-assembly: Under room temperature and magnetic stirring, the above organic phase was slowly and dropwise added to the aqueous phase. As acetone diffused into the aqueous phase, PLGA gradually precipitated and formed a hydrophobic core; the hydrophobic double chains of DSPE were embedded in the surface or adjacent region of the PLGA particles, the PEG segments extended outward, the terminal TPP groups were distributed on the outer layer of the particles, and α-lipoic acid was encapsulated in the PLGA core and its adjacent region. After the dropwise addition was completed, stirring was continued for 30 min to allow the system to fully self-assemble, obtaining an α-LA@PLGA / DSPE-PEG-TPP nanoparticle dispersion. The obtained dispersion can be directly used for particle size, potential, and release detection, or stored at 4 ℃.

[0030] (4) Preparation of control samples: When preparing blank PLGA / DSPE-PEG-TPP carriers, α-lipoic acid was not added to the organic phase, and other conditions remained unchanged; when preparing α-LA@PLGA control particles, DSPE-PEG-TPP was not added to the aqueous phase, and other feed amounts and operating conditions remained consistent. By setting up the above controls, the effects of the DSPE-PEG-TPP functional layer on particle properties, drug loading, and release behavior can be compared.

[0031] This method does not require high temperature, high pressure, or additional chemical coupling reactions. It involves few steps, is mild, and can form a uniform nano-dispersion system in a short time. The preparation process and particle structure are described below. Figure 1 .

[0032] Example 2

[0033] Properties and drug loading characteristics of polylactic acid composite nanoparticles

[0034] The nanoparticles obtained in Example 1 were characterized using transmission electron microscopy (TEM), dynamic light scattering (DLS), and Zeta potential detection. For TEM, the sample was appropriately diluted with deionized water or ethanol and ultrasonically dispersed. One to two drops were added to a copper grid with a supporting film, and the particles were observed after natural drying. For DLS, the sample was uniformly dispersed in deionized water, and an appropriate amount of the dispersion was added to a clean sample cell, avoiding interference from air bubbles and visible impurities. Each sample was measured three times, and the average particle size and particle size distribution were recorded. Zeta potential was measured using a corresponding potential cell to evaluate the particle surface charge and the stability of the underlying colloidal structure.

[0035] Test results are shown Figure 2See Table 1. The average particle size of the blank PLGA particles was 146.1 nm, and the Zeta potential was -26.7 mV. After introducing DSPE-PEG-TPP, the average particle size of the blank PLGA / DSPE-PEG-TPP particles was 169.9 nm, and the Zeta potential was -28.0 mV. After loading 10% α-lipoic acid, the average particle size of α-LA@PLGA / DSPE-PEG-TPP was 125.6 nm, and the Zeta potential was -20.8 mV. Under transmission electron microscopy, the particles were spherical or near-spherical with relatively clear boundaries, and no large-area irregular agglomeration was observed. The above results indicate that the present invention can obtain drug-loaded particles with nanometer-sized particles, relatively complete morphology, and certain surface charge, which are suitable for forming aqueous nano-dispersion systems.

[0036] Table 1. Particle size and zeta potential of representative formulations sample Particle size / nm Zeta potential / mV PLGA 146.1 -26.7 PLGA / DSPE-PEG-TPP (2.5%) 169.9 -28.0 α-LA@PLGA / DSPE-PEG-TPP (10% dosage) 125.6 -20.8

[0037] The encapsulation efficiency and drug loading rate of α-lipoic acid were determined by ultraviolet spectrophotometry. First, a series of α-lipoic acid standard solutions of known concentrations were prepared, and the absorbance was measured at 334 nm. A standard curve was established with α-lipoic acid concentration as the x-axis and absorbance as the y-axis. The prepared drug-loaded nanoparticle dispersion was centrifuged at 12,000 rpm for 15 min, and the supernatant was collected. The free α-lipoic acid content in the supernatant was calculated based on the standard curve. The encapsulation efficiency was calculated as "(drug dosage - free drug dosage) / drug dosage × 100%". The absorption of the blank carrier at 334 nm was used as background correction to determine the α-lipoic acid content in the drug-loaded nanoparticles, and the drug loading rate was calculated as "drug mass in nanoparticles / total mass of drug-loaded nanoparticles × 100%".

[0038] The results are shown in Table 2. At the same 10% drug dosage ratio, the encapsulation efficiency and drug loading rate of α-LA@PLGA were 47.89% and 3.62%, respectively, while those of α-LA@PLGA / DSPE-PEG-TPP reached 81.78% and 8.77%, respectively. When the α-lipoic acid dosage ratio was increased to 20%, the encapsulation efficiency of the nanoparticles of this invention reached 67.00%, and the drug loading rate increased to 13.16%. The results indicate that, under the same drug dosage conditions, the composite nanostructure of this invention can reduce the proportion of free drug and increase the effective encapsulation capacity of α-lipoic acid. Furthermore, by adjusting the drug dosage ratio, a choice can be made between higher encapsulation efficiency and higher drug loading rate, facilitating the formulation of appropriate formulations according to different formulation requirements.

[0039] Table 2 Encapsulation efficiency and drug loading rate of α-lipoic acid sample Encapsulation rate / % Drug loading rate / % α-LA@PLGA (10% dosage) 47.89 3.62 α-LA@PLGA / DSPE-PEG-TPP (10% dosage) 81.78 8.77 α-LA@PLGA / DSPE-PEG-TPP (20% dosage) 67.00 13.16

[0040] Example 3

[0041] In vitro drug release characteristics of polylactic acid composite nanoparticle drug delivery system

[0042] This embodiment evaluates the in vitro release behavior of α-LA@PLGA / DSPE-PEG-TPP, using α-LA@PLGA as a comparison sample. 5 mL of the nanoparticle dispersion was added to 25 mL of sterilized release medium and incubated at 37°C with constant temperature shaking at 150 rpm. At preset time points, 1 mL of sample was taken and centrifuged at 4°C and 14,000 rpm for 20 min, collecting the supernatant. A blank nanocarrier supernatant treated under the same conditions was used as a background control. The absorbance was measured at 334 nm, and the amount of drug released and the cumulative release percentage at each time point were calculated based on the α-lipoic acid standard curve.

[0043] First, the release processes of the two drug-loaded particles were compared in a neutral release medium. The results are shown below. Figure 3 The cumulative release of α-LA@PLGA / DSPE-PEG-TPP on days 1 and 2 was approximately 33% and 55%, respectively, which was lower than that of α-LA@PLGA (approximately 47% and 68%), indicating that the DSPE-PEG-TPP functional layer can reduce the rapid release of the formulation in the early stages. Subsequently, the drug was released continuously, with the cumulative release reaching nearly 95% by day 7, demonstrating that the system can achieve sufficient subsequent release while reducing the initial burst release.

[0044] The release differences were further investigated using media at pH 1.2, pH 6.5, and pH 7.4. The results showed that the nanoparticles were released more slowly at pH 1.2, and more fully at pH 6.5 and pH 7.4. These pH-related release characteristics help reduce the rapid leakage of α-lipoic acid in strongly acidic environments and enable sustained drug release under conditions close to the intestinal or physiological environment, thus demonstrating the technical advantages of this invention in oral delivery and prolonged drug release time.

[0045] Example 4

[0046] Mitochondrial regional localization and cell compatibility of polylactic acid composite nanoparticle drug delivery system

[0047] The main materials used in this embodiment are from the following sources: MitoTracker Red (Thermo Fisher Scientific, USA); Golgi-Tracker (Thermo Fisher Scientific, USA); Hoechst 33342 (Yisheng Biotechnology (Shanghai) Co., Ltd.); CCK-8 reagent kit (Yisheng Biotechnology (Shanghai) Co., Ltd.).

[0048] To evaluate the intracellular localization characteristics of the nanocarriers of this invention, Rhodamine 123 (Rh123) was used as a fluorescent tracer. Rh123@PLGA / DSPE-PEG-TPP was prepared according to the nanoprecipitation-self-assembly method of Example 1, and Rh123@PLGA without DSPE-PEG-TPP was prepared as a control. Specifically, Rh123 and PLGA were thoroughly mixed in acetone to make the concentration of Rh123 relative to the organic phase 0.1 wt%, and then the corresponding aqueous phase was added to complete the particle preparation. The obtained samples were stored at 4 °C in the dark.

[0049] H9C2, A549, and SH-SY5Y cells in good growth condition were seeded into confocal culture dishes. After the cells were fully adhered, fresh medium containing Rh123-labeled nanoparticles was added and the cells were incubated for 24 h. After incubation, the medium was discarded, and the cells were gently washed three times with PBS. MitoTracker Red or Golgi-Tracker working solution was added and incubated for 30 min, followed by three washes with PBS. Hoechst 33342 staining solution was then added and incubated for 15 min to label the cell nuclei. After staining, the cells were washed with PBS, and fresh medium was added to cover the cells. Laser confocal imaging was performed under the same excitation, emission, and acquisition parameters.

[0050] See results Figure 4 In the three cell types, the green fluorescence of Rh123@PLGA / DSPE-PEG-TPP showed greater overlap with the red fluorescence of mitochondria than that of Rh123@PLGA, while the overlap with the Golgi apparatus signal was relatively weaker. This result indicates that DSPE-PEG-TPP modification can enhance the localization tendency of nanoparticles in the mitochondrial region, providing a structural basis for the delivery of α-lipoic acid to mitochondrial damage-related regions. Given that this experiment is a fluorescence co-localization evaluation, this paper describes the effect of this technique as having the potential to enhance mitochondrial regional localization, rather than equating it with direct proof of intact intracellular transport processes.

[0051] The cell compatibility of the blank PLGA / DSPE-PEG-TPP carrier was evaluated using the CCK-8 assay. H9C2, A549, and SH-SY5Y cells in logarithmic growth phase were digested and prepared into a homogeneous cell suspension, seeded in 96-well plates, and 100 μL of culture medium was added to each well. After cell attachment, different concentrations of the blank nanocarrier were added, and the cells were cultured for another 24 h. For assay, working solutions were prepared at a CCK-8 reagent to culture medium ratio of 1:9. The original culture medium was discarded, and the solutions were added to each well. The cells were incubated in the dark for 30–60 min, and the absorbance was measured at 450 nm. Relative cell viability was calculated based on the results of the blank wells and the normal control wells.

[0052] See results Figure 5A. Within the detection concentration range, none of the three cell types showed a significant concentration-dependent decrease in viability after treatment with blank PLGA / DSPE-PEG-TPP; even when the concentration was increased to 200 μg / mL and treated for 24 h, the overall cell viability remained at a high level. This indicates that the blank nanocarrier of the present invention has good basic cell compatibility under the above-mentioned cell and detection conditions, and can meet the needs of subsequent loading of α-lipoic acid and evaluation of hypoxia injury intervention.

[0053] Example 5

[0054] Cellular hypoxia protection effect of polylactic acid-based alpha-lipoic acid nanocomposite drug delivery system

[0055] The main materials used in this embodiment were sourced from the following sources: cobalt chloride (Shanghai Myrui Biochemical Technology Co., Ltd.); Fluo-3 calcium ion fluorescent probe (Yisheng Biotechnology (Shanghai) Co., Ltd.); DCFH-DA reactive oxygen species detection reagent (Shanghai Beyotime Biotechnology Co., Ltd.); ATP detection kit and ATP standards (Shanghai Beyotime Biotechnology Co., Ltd.). The H9C2, A549, and SH-SY5Y cells used were sourced from the same sources as in Example 4.

[0056] This embodiment was used to verify the protective effect of the drug-loaded nanoparticles of the present invention on chemically simulated hypoxic cells. H9C2 rat cardiomyocytes, A549 human lung epithelial cells, and SH-SY5Y human neuron-like cells were selected as cardiac, lung, and nerve-related cell models, respectively. Based on the concentrations near IC25 determined in preliminary experiments, H9C2 cells were treated with 350 μM CoCl2, A549 cells with 400 μM CoCl2, and SH-SY5Y cells with 100 μM CoCl2 for 24 h each time, to induce hypoxia-related changes such as ROS accumulation, Ca²⁺ homeostasis disturbance, and decreased ATP production.

[0057] The experiment included a normal control group, a CoCl2-hypoxia group, a free α-lipoic acid group, an α-LA@PLGA group, a blank PLGA / DSPE-PEG-TPP group, and an α-LA@PLGA / DSPE-PEG-TPP group. All groups underwent the corresponding treatments under the same cell culture conditions. Intracellular Ca²⁺ levels were detected using the Fluo-3 fluorescent probe, and ROS levels were detected using the DCFH-DA probe; ATP content was determined using luciferase immunoassay. All assays were performed at least three times independently.

[0058] See results Figure 5B. CoCl2 treatment increased Ca²⁺ and ROS levels and decreased ATP content in all three cell types, indicating damage to cellular calcium homeostasis, redox homeostasis, and energy metabolism. Treatment with α-LA@PLGA / DSPE-PEG-TPP reduced elevated Ca²⁺ and ROS levels to varying degrees in all three cell types, and improved ATP deficiency. Among multiple indicators, the drug-loaded nanoparticle group showed a more significant recovery trend than the free α-lipoic acid or ordinary α-LA@PLGA group. Combined with the limited improvement of the above abnormal indicators by the blank carrier itself, this indicates that the present invention mainly improves the utilization efficiency of α-lipoic acid in hypoxia-related mitochondrial dysfunction by enhancing its encapsulation, sustained release, and effective intracellular delivery.

[0059] Example 6

[0060] In vivo hypoxia protection effect of polylactic acid-based alpha-lipoic acid nanocomposite drug delivery system

[0061] The main materials used in this embodiment were sourced from the following sources: experimental mice (strain: BALB / c; Shanghai Jinuo Biotechnology Co., Ltd.); CK detection kit (Nanjing Jiancheng Bioengineering Institute); LDH detection kit (Nanjing Jiancheng Bioengineering Institute); the sources of α-lipoic acid, PLGA and DSPE-PEG-TPP used were the same as in Example 1.

[0062] This embodiment was used to verify the in vivo protective effect of the nanoparticles of the present invention after oral administration by gavage. Five-week-old mice were randomly divided into a normal control group, a hypoxia model group, a hypoxia + free α-lipoic acid group, a hypoxia + blank PLGA / DSPE-PEG-TPP group, a hypoxia + α-LA@PLGA / DSPE-PEG-TPP group, and a blank carrier-only administration group, with six mice in each group. Except for the normal control group and the blank carrier-only administration group, the other groups were administered CoCl2 40 mg / kg by gavage daily for two consecutive weeks to establish a chemically simulated hypoxia injury model. After model establishment, the normal control group and the hypoxia model group were administered an equal volume of physiological saline by gavage daily; the free α-lipoic acid group and the drug-loaded nanoparticle group of the present invention were administered α-lipoic acid 20 mg / kg / day by gavage; and the corresponding blank carrier groups were administered PLGA / DSPE-PEG-TPP 150 mg / kg / day by gavage, with intervention lasting for two consecutive weeks.

[0063] During the drug administration period, the activity status, fur, respiratory function, and weight changes of mice were observed and recorded. After drug administration, serum was collected, and creatine kinase (CK) and lactate dehydrogenase (LDH) activities were measured according to the kit method. Heart and lung tissues were isolated, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Histological structure was observed by HE staining, and collagen fiber deposition was observed by Masson staining. All groups used the same sampling, staining, and imaging conditions to ensure comparability of results.

[0064] Representative results are shown in Figure 6 In the hypoxia model group, myocardial fibers in mice showed swelling, disordered arrangement, widened interstitial spaces, and localized rupture. Masson staining revealed increased collagen deposition in the myocardial interstitium. Lung tissue showed thickening of alveolar septa, narrowing of alveolar cavities, interstitial widening, and increased collagen deposition. Simultaneously, serum CK and LDH levels were elevated in the hypoxia model group. These changes were alleviated after intervention with free α-lipoic acid. After gavage intervention with α-LA@PLGA / DSPE-PEG-TPP, myocardial arrangement and alveolar structure were further improved, tissue collagen deposition decreased, and serum CK and LDH levels showed a decreasing trend. The blank carrier group did not show a comparable improvement effect to the drug-loaded nanoparticles. These results indicate that the nanoparticles of this invention can still exert the protective effect against hypoxia-induced damage from α-lipoic acid under gavage administration conditions, and demonstrate mutually reinforcing technical effects at multiple levels, including myocardial tissue, lung tissue, and serum enzyme indicators.

[0065] The PLGA, DSPE-PEG-TPP, and α-lipoic acid used in this invention are all commercially available. The preparation process mainly employs nanoprecipitation and self-assembly, with mild conditions and few operational steps. The resulting nanoparticles exhibit high encapsulation, suitable particle size, sustained release, good cell compatibility, and mitochondrial regional localization potential, and can be used to develop oral nanoformulations of α-lipoic acid or other mitochondrial protective drugs.

[0066] The above embodiments are for illustrative purposes only. Any conventional adjustments or equivalent substitutions made by those skilled in the art to the molecular weight of materials, the feeding ratio, the amount of solvent, the stirring conditions, and the dosage form without departing from the concept of this invention should fall within the protection scope of this invention.

Claims

1. A polylactic acid composite nanoparticle loaded with α-lipoic acid, characterized in that, It includes α-lipoic acid, polylactic acid-glycolic acid copolymer, and distearylphosphatidylethanolamine-polyethylene glycol-triphenylphosphine (PLGA / DSPE-PEG-TPP); the polylactic acid-glycolic acid copolymer forms a hydrophobic core, the hydrophobic end of the distearylphosphatidylethanolamine is embedded in the core or its surface, the polyethylene glycol segments extend into the aqueous phase, and the triphenylphosphine groups are located on the outer layer of the nanoparticles.

2. The nanoparticles according to claim 1, characterized in that, The mass ratio of the polylactic acid-hydroxyacetic acid copolymer, distearate phosphatidylethanolamine-polyethylene glycol-triphenylphosphine, and α-lipoic acid is 100:2-5:10-20, preferably 100:2.5:

10.

3. The nanoparticles according to claim 1 or 2, characterized in that, The nanoparticles are spherical or near-spherical in shape, with an average particle size of 100–250 nm and a zeta potential of -15–-30 mV.

4. The nanoparticles according to any one of claims 1 to 3, characterized in that, The encapsulation efficiency of α-lipoic acid is 60%–90%, and the drug loading rate is 8%–14%.

5. A method for preparing the nanoparticles according to any one of claims 1 to 4, characterized in that, include: Distearate phosphatidylethanolamine-polyethylene glycol-triphenylphosphine was dispersed in an aqueous phase; Polylactic acid-glycolic acid copolymer and α-lipoic acid are dissolved in an organic solvent to form an organic phase; The organic phase is added to the aqueous phase under stirring conditions, so that the components are precipitated and self-assembled to form nanoparticles.

6. The preparation method according to claim 5, characterized in that, Each 10 mg of polylactic acid-glycolic acid copolymer is prepared with 0.20–0.50 mg of distearylphosphatidylethanolamine-polyethylene glycol-triphenylphosphine and 1–2 mg of α-lipoic acid; the organic solvent is acetone, and the self-assembly is carried out at 20–30 °C.

7. A pharmaceutical composition, characterized in that, It contains the nanoparticles as described in any one of claims 1 to 4 and pharmaceutically acceptable excipients.

8. The pharmaceutical composition according to claim 7, characterized in that, The pharmaceutical composition is an oral suspension, oral solution, capsule, tablet, or lyophilized preparation.

9. The use of the nanoparticles according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 7 or 8 in the preparation of a drug for alleviating hypoxia-induced mitochondrial dysfunction.

10. The application according to claim 9, characterized in that, The hypoxia-induced injury includes myocardial tissue injury, lung tissue injury, or multi-organ hypoxia injury.

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