A preparation method and application of ganoderma lucidum spore microcarriers for small molecule drug delivery

Abstract

The application discloses a preparation method and application of ganoderma lucidum spore microcarriers for small molecule drug delivery. The application evaluates pharmaceutical properties of natural ganoderma lucidum spores and ganoderma lucidum spores after drug loading, and the result shows that the drug loading mode does not cause damage to the carriers themselves, and can ensure that small molecule drugs are successfully loaded on the ganoderma lucidum spores. In-vitro release behavior shows that the ganoderma lucidum spore microcarriers exhibit obvious sustained-release characteristics in gastric juice, can delay the release of small molecule drugs, avoid mass release of the drugs in a short time, and the sustained-release characteristics are helpful to maintain stable blood drug concentration and reduce side effects of the drugs. In-vivo experimental results show that the ganoderma lucidum spore microcarriers can significantly improve drug-induced liver injury.

Description

Technical Field

[0001] This invention relates to the field of drug carriers, specifically to a method for preparing and applying Ganoderma lucidum spore microcarriers for small molecule drug delivery. Background Technology

[0002] In the past few decades of rapid development in biomedicine, novel drug delivery systems (NDDS) have attracted much attention as an important direction for drug innovation and research. NDDS effectively addresses the limitations of conventional drug delivery methods, such as short half-life, weak targeting, poor solubility, and low bioavailability. With the continuous progress and integration of pharmacy, materials science, and biomedicine, NDDS has evolved into many sub-fields: such as nano-drug delivery systems that utilize the potential of nanotechnology for drug delivery; biomimetic drug delivery systems that mimic processes within the body based on biomimetic principles; targeted drug delivery systems that emphasize precise delivery of drugs to specific tissues and cells; and intelligent response delivery systems that achieve intelligent drug release based on specific environmental factors within the body. Among these, carrier materials, as one of the core components of drug delivery systems, play a crucial role in enabling various NDDS to perform their unique functions.

[0003] Currently, conventional carrier materials mainly include the following three categories: ① Natural polymer materials: such as chitosan, sodium alginate, hyaluronic acid, etc. Although they have good biocompatibility, their mechanical strength is low and their swelling behavior is uncontrollable, which can easily lead to drug burst release; ② Synthetic polymer materials: such as polylactic acid (PLA), polylactic acid-glycolic acid copolymer (PLGA), polycaprolactone (PCL), polyanhydride, etc. Although they can achieve precise degradation cycle control, toxic organic solvents are often used in the synthesis process, and residual solvents may trigger inflammatory reactions; ③ Others: such as β-cyclodextrin, liposomes, silica, etc. Cyclodextrin materials have weak loading capacity for hydrophilic macromolecules, and unmodified cyclodextrins are difficult to achieve stable loading. Liposomes have poor storage stability and are prone to oxidation and hydrolysis. The non-degradability of silica nanoparticles may lead to long-term bioaccumulation risks. However, when using conventional carrier materials to construct drug delivery systems, there are always some common challenges: such as the complexity and high cost of preparing synthetic materials; the potential toxicity caused by long-term retention of the carrier in the body; and the difficulty of existing carriers in meeting the differentiated needs of small molecule drug sustained release and large molecule protein loading.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] To address at least some of the technical problems in the prior art, this invention focuses on Ganoderma lucidum spores (also referred to herein as GLS) as the research object, exploring their application as a natural microcarrier in small molecule drug delivery. In an exemplary embodiment, this invention optimizes the formulation process of Ganoderma lucidum spore microcarriers loaded with acetaminophen (sometimes referred to herein as APAP) (APAP@GLS), characterizes and analyzes them, and then systematically evaluates the biological effects of APAP@GLS in reducing hepatotoxicity. Specifically, this invention includes the following.

[0006] In a first aspect, the present invention provides a small molecule drug delivery carrier, wherein the small molecule drug delivery carrier is a natural Ganoderma lucidum spore, and the small molecule drug is loaded onto the Ganoderma lucidum spore by electrostatic adsorption.

[0007] In some embodiments, the small molecule drug delivery carrier according to the present invention is wherein the ratio of the small molecule drug to natural Ganoderma lucidum spores is 1-5:1.

[0008] In some embodiments, the small molecule drug delivery carrier according to the present invention has the Ganoderma lucidum spores having a size of 4-8 μm × 5-10 μm and a pore size of 0.1-50 nm.

[0009] A second aspect of the present invention provides a method for preparing a small molecule drug delivery carrier, comprising: (1) Provides natural Ganoderma lucidum spores; (2) The small molecule drug is loaded onto the Ganoderma lucidum spores by electrostatic adsorption.

[0010] In some embodiments, according to the method for preparing a small molecule drug delivery carrier according to the present invention, in step (2), the loading time of the small molecule drug is 0.5-5 h.

[0011] In some embodiments, according to the method for preparing a small molecule drug delivery carrier according to the present invention, the concentration of the small molecule drug is 10-500 mg / mL.

[0012] A third aspect of the present invention provides a pharmaceutical composition comprising the small molecule drug delivery carrier described in the first aspect.

[0013] In some embodiments, the pharmaceutical composition according to the present invention further comprises pharmaceutically acceptable excipients.

[0014] A fourth aspect of the invention provides the use of the aforementioned small molecule drug delivery carrier in the preparation of a drug.

[0015] This invention optimizes drug loading time, dosage ratio, and loading method using a single-factor method, determining the optimal preparation process parameters for APAP@GLS with drug loading as the core evaluation index. This provides a preliminary research foundation for studies on reducing acute hepatotoxicity of APAP@GLS. Pharmaceutical performance evaluation of natural GLS and drug-loaded APAP@GLS showed that the loading method did not damage the carrier itself and ensured successful APAP loading onto the GLS. During this process, no new chemical bonds were formed between APAP and GLS; the interaction between the drug and the carrier was mainly electrostatic. This invention also investigated the in vitro release behavior of natural GLS and APAP@GLS. APAP was released rapidly within the first 1 hour and stabilized within 2 hours; APAP@GLS was released slowly from 0 to 2 hours, reaching 85.00% at 6 hours before the release leveled off. These results indicate that APAP@GLS exhibits significant sustained-release characteristics in gastric juice, delaying APAP release and preventing a large release of the drug in a short period. This sustained-release characteristic helps maintain stable blood drug concentrations and reduces drug side effects. In vivo experimental results show that the Ganoderma lucidum spore microcarrier of the present invention can significantly improve drug-induced liver injury. Attached Figure Description

[0016] Figure 1 SEM characterization of natural GLS. (a) GLS, ×1 k; (b) GLS, ×5 k; (c) GLS, ×25 k.

[0017] Figure 2 This is a zeta potential diagram of natural GLS.

[0018] Figure 3 The X-ray diffraction (XRD) analysis spectrum of natural GLS.

[0019] Figure 4 The infrared spectrum of natural GLS.

[0020] Figure 5 This is the standard curve of APAP in PBS solution.

[0021] Figure 6 SEM characterization of natural GLS and drug-loaded APAP@GLS. (a) GLS, ×10 k; (b) GLS, ×5 k; (c) APAP@GLS, ×10 k; (d) APAP@GLS, ×5 k.

[0022] Figure 7 This is a Zeta potential plot for APAP@GLS.

[0023] Figure 8 This is the X-ray diffraction analysis pattern of APAP@GLS.

[0024] Figure 9 Infrared spectral analysis for APAP@GLS.

[0025] Figure 10 This is the cumulative release curve for APAP@GLS.

[0026] Figure 11 The concentration of APAP in rat plasma and the plasma drug concentration-time curve after rats were administered APAP and APAP@GLS by gavage.

[0027] Figure 12 The gross morphology of the livers of mice in each group is shown.

[0028] Figure 13 HE staining results of mouse liver tissue from each group are shown.

[0029] Figure 14 The levels of ALT and AST, liver function indicators, in the serum of mice in each group. Detailed Implementation

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0032] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0033] Drug delivery carrier In one aspect, this invention provides a drug delivery carrier (sometimes also called a microcarrier or Ganoderma lucidum spore microcarrier), wherein the drug delivery carrier is a natural Ganoderma lucidum spore, and the small molecule drug is loaded onto the inside or surface of the Ganoderma lucidum spore by electrostatic adsorption. In this invention, the Ganoderma lucidum spores can be purchased from commercially available products or separated by physical methods using Ganoderma lucidum fruiting bodies as raw materials. Natural Ganoderma lucidum spores possess a complete natural cell wall structure and the characteristics of their contents (active ingredients), meaning that the separated Ganoderma lucidum spores are essentially consistent with the natural state of the spores in the original fruiting body.

[0034] In this invention, the natural Ganoderma lucidum spores are particles with a double-walled structure, a size of 4-8 μm × 5-10 μm, and natural pores or channels, wherein the pore size is 0.1-50 nm. In a preferred embodiment, the size of the Ganoderma lucidum spores of this invention is 4-8 μm × 5-10 μm, preferably 4.5-7.5 μm × 5.5-9.5 μm, even more preferably 5-7 μm × 6-9 μm, and even more preferably 5-6.5 μm × 7-9 μm; the pore size is 0.1-50 nm, even more preferably 1-10 nm, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nm.

[0035] In this invention, the zeta potential of natural Ganoderma lucidum spores is -50 to 0 mV, for example -20 to -5 mV.

[0036] In some embodiments, the small molecule drug delivery carrier according to the present invention has a drug loading of not less than 25% and an encapsulation efficiency of not less than 40%.

[0037] In this invention, the small molecule drug is not particularly limited and can be any small molecule compound that can be loaded onto or inside the surface of Ganoderma lucidum spores by electrostatic adsorption. Since the surface of Ganoderma lucidum spore microcarriers is mainly covered with a natural polysaccharide complex layer composed of dextran, chitin, etc., this structural layer is rich in a large number of polar functional groups, including but not limited to hydroxyl (-OH), carboxyl (-COOH), amino (-NH2), etc., its drug loading capacity is rooted in the above-mentioned natural and universal physicochemical structural characteristics. Therefore, based on the above carrier structure, this invention found that the loading of small molecule drugs by Ganoderma lucidum spores mainly depends on the following non-covalent interactions that are common between most small organic molecules and polar surfaces: (1) Hydrogen bonding: -OH, -COOH, -NH2 and other groups on the spore surface form hydrogen bonds with oxygen, nitrogen and other heteroatoms or hydroxyl, amino, carbonyl and other groups in small molecule drug molecules; (2) Van der Waals forces: including inductive forces, orientation forces and dispersion forces, which generally occur when drug molecules are in close proximity to the macromolecular chains on the spore surface; (3) Polar-polar interactions: the polar environment on the spore surface has an affinity for the polar part of the drug molecule; (4) Physical adsorption and pore trapping: The microscopic roughness and potential micropores on the spore surface can trap drug molecules through physical adsorption and steric confinement effects. Importantly, the above-mentioned interaction mechanism is broad-spectrum. As long as small molecule drugs possess donors / acceptors for forming hydrogen bonds and have certain polarity or polarizable regions, they can undergo the above-mentioned interactions of varying intensities with the surface of Ganoderma lucidum spores, thereby achieving loading, rather than a specific chemical structure.

[0038] In some embodiments, the present invention has conducted a detailed study using acetaminophen (APAP) as a “model small molecule.” APAP was chosen based on its highly representative physicochemical properties among small molecule drugs: (1) Moderate molecular weight: approximately 151 g / mol, belonging to the typical low molecular weight drug category (usually <900 g / mol); (2) Common and abundant functional groups: its molecular structure contains phenolic hydroxyl (-OH), amide (-NHCO-) ​​and benzene rings. These groups (especially hydroxyl and amide groups) are widely present hydrogen bond donors and acceptors in many drug molecules; (3) Typical solubility and polarity: APAP has a certain solubility in water and organic solvents, exhibiting amphiphilicity, which is similar to the properties of many small molecule drugs that require oral delivery. Since APAP possesses almost all the key characteristic elements for the aforementioned non-specific interactions between small molecule drugs and the surface of Ganoderma lucidum spores (groups capable of forming hydrogen bonds, polarity, and suitable size), the successful loading, sustained release, and efficacy results obtained using it as a model provide sufficient and reasonable expectations for the application of the Ganoderma lucidum spore microcarriers described in this invention to other small molecule drugs with similar mechanisms of action. That is, any small molecule compound that can bind to polysaccharide functional groups on the spore surface through non-covalent interactions such as hydrogen bonds and van der Waals forces can be expected to be effectively loaded using similar methods and may exhibit sustained-release behavior.

[0039] The internal structure of Ganoderma lucidum spores in this invention can be analyzed and characterized by techniques such as optical microscopy, electron microscopy, scanning probe microscopy, X-ray diffraction and infrared spectroscopy, nuclear magnetic resonance, hot stage microscopy, computed tomography and nuclear magnetic resonance imaging. Before measurement, the spores can be cut and ground by appropriate crushing methods.

[0040] Preparation method In one aspect, the present invention provides a method for preparing a small molecule drug delivery carrier, comprising the following steps: (1) providing natural Ganoderma lucidum spores; (2) loading the small molecule drug onto the Ganoderma lucidum spores by electrostatic adsorption.

[0041] In step (2) of this invention, a solution containing a small molecule drug is incubated with natural Ganoderma lucidum spores, allowing the small molecule drug to be loaded onto the inside or surface of the Ganoderma lucidum spores via electrostatic adsorption. Those skilled in the art will understand that the solvent can vary widely depending on the small molecule drug selected. In this invention, the solvent can be water, ultrapure water, distilled water, or an organic solvent such as methanol, ethanol, acetone, dichloroethylene, and ethyl acetate.

[0042] Those skilled in the art will understand that when the small molecule drug is a protonated small molecule drug, the pH of the drug solution can be adjusted to a range that allows the drug to be fully protonated and positively charged, while ensuring the stability of the negative charge on the carrier surface. Any suitable pH adjuster can be used to adjust the pH to a neutral or weakly acidic range, and there are no particular limitations on this.

[0043] In this invention, the concentration of the small molecule drug solution is not particularly limited, but is preferably 10-500 mg / mL, more preferably 20-250 mg / mL, and even more preferably 50-200 mg / mL, such as 50-150 mg / mL, like 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150 mg / mL.

[0044] In step (2) of the present invention, the loading of small molecule drugs can be achieved by passive loading or vacuum-assisted loading. In a preferred embodiment, the loading of small molecule drugs by passive loading includes the following steps: dispersing natural Ganoderma lucidum spores in an alcoholic solution of acetaminophen, and incubating at a low rotation speed (preferably below 500 rpm, more preferably below 250 rpm, and even more preferably below 200 rpm) for 0.5-5 h, preferably 1-4 h, for example 1, 1.5, 2, 2.5, 3, 3.5, 4 h. In another preferred embodiment, the loading of small molecule drugs is achieved by vacuum-assisted loading, which includes the following steps: dispersing natural Ganoderma lucidum spores in an alcoholic solution of acetaminophen, and incubating at a low rotation speed (preferably below 500 rpm, more preferably below 250 rpm, and even more preferably below 200 rpm) and vacuum conditions (preferably below -0.1 MPa) for 0.5-5 h, preferably 1-4 h, for example 1, 1.5, 2, 2.5, 3, 3.5, 4 h.

[0045] In this invention, the mass ratio of small molecule drugs to Ganoderma lucidum spores is not particularly limited, but is preferably 1-5:1, more preferably 1-4:1, and even more preferably 1-3:1, for example 1:1, 1.2:1, 1.4:1, 1.6:1, 1.8:1, 2:1, 2.2:1, 2.4:1, 2.6:1, 2.8:1, 3:1.

[0046] In one preferred embodiment, the ratio of small molecule drug to Ganoderma lucidum spores is 2:1, and vacuum-assisted loading is used for 2 hours.

[0047] It is understood that after the treatment in step (2), there are other processing steps, such as washing, centrifugation, freeze drying, etc. The reagents and parameters used are not particularly limited and can be adjusted as needed.

[0048] Pharmaceutical Composition In one aspect, the present invention provides a pharmaceutical composition comprising the small molecule drug carrier described herein and pharmaceutically acceptable excipients. In this invention, pharmaceutically acceptable excipients are well known in the art and can be determined by those skilled in the art to meet clinical standards. Pharmaceutically acceptable excipients include, but are not limited to, diluents and excipients.

[0049] The pharmaceutical compositions of the present invention can be any suitable dosage form. For example, tablets, sustained-release (including controlled-release) tablets, orally disintegrating tablets, capsules (i.e., hard capsules), soft capsules, granules, suspensions, dry suspensions, oral solutions, syrups, powders, drop pills, pills, tinctures, decoctions, etc.

[0050] The pharmaceutical compositions of the present invention can be administered into the body in known ways, without particular limitation. For example, the delivery carriers of the present invention can be configured for oral administration, sublingual administration, buccal administration, rectal administration, injection administration, topical administration, or other non-oral, non-injection parenteral routes, such as, but not limited to, transdermal administration, inhalation administration, nasal administration, ocular administration, urethral / vaginal administration, etc. Such administration can be performed via single or multiple doses. Those skilled in the art will understand that the actual dose to be administered herein can vary considerably depending on a variety of factors, such as biological type or tissue, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0051] It is understood that the pharmaceutical composition is administered to an individual at a prophylactic or therapeutically effective amount (as the case may be, although prophylaxis may be considered treatment), which is sufficient to demonstrate benefit to the individual. Typically, this will result in therapeutically useful activity that is beneficial to the individual. The actual amount of the small molecule drug carrier administered, as well as the rate and timing of administration, will depend on the nature and severity of the condition being treated. Prescribing treatments, such as dosage determination, falls within the responsibility of general practitioners and other physicians, and generally takes into account the condition being treated, the individual patient's condition, the delivery site, the method of administration, and other factors known to the physician.

[0052] use One aspect of the present invention provides the use of the drug delivery carrier described herein in the preparation of a medicament.

[0053] In a preferred embodiment, the medicament of the present invention is a therapeutic agent for the prevention, treatment, and / or improvement of a disease in a subject in need. The term "subject" as used herein refers to any animal (such as a mammal), including but not limited to humans, non-human primates, rodents, and the like who are about to receive specific treatment. Furthermore, the specific type of disease for prevention, treatment, and / or improvement is not particularly limited and varies depending on the small molecule drug used.

[0054] Those skilled in the art will understand that the carrier described herein, after loading the drug, can also be used in combination with other drugs for the prevention, treatment, and / or improvement of diseases or conditions. Other drugs can be any therapeutic agents that are beneficial to the disease, such as those beneficial to solid tumors or hematological disorders, and are not particularly limited thereto.

[0055] In a preferred embodiment, the present invention provides the use of a small molecule drug delivery carrier in the preparation of a medicament for treating or improving drug-induced liver injury, wherein the small molecule drug delivery carrier is a Ganoderma lucidum spore microcarrier loaded with acetaminophen.

[0056] The following embodiments are provided to illustrate the principles and practice of the embodiments disclosed herein more clearly to those skilled in the art, and should not be construed as limiting the scope of any claimed embodiments.

[0057] Example 1 This embodiment uses acetaminophen as an example to illustrate the preparation and sustained-release performance study of APAP@GLS.

[0058] Acetaminophen, also known as paracetamol, is a commonly used nonsteroidal antipyretic analgesic. APAPs work by inhibiting the activity of cyclooxygenase (COX), blocking the synthesis of prostaglandins in the central nervous system, thus effectively exerting their antipyretic and analgesic effects. They are widely used to relieve mild to moderate pain, such as headaches, joint pain, and toothaches. However, APAPs have a relatively short half-life, typically only about 2 hours. To maintain effective blood concentrations, patients need to take them 3-4 times daily, which negatively impacts long-term medication adherence. Furthermore, the bioavailability of regular tablet APAPs is limited, and long-term oral use may lead to side effects such as hepatotoxicity, especially with overdose, which significantly increases the risk of liver damage.

[0059] To address these shortcomings, this embodiment selects APAP as the model drug and natural GLS as the carrier to prepare an APAP-loaded GLS sustained-release system (APAP@GLS). A systematic study was conducted on its preparation process, pharmaceutical properties, and sustained-release characteristics. Single-factor optimization of drug loading time, dosage ratio, and loading method was performed to determine the optimal preparation process parameters for APAP@GLS. Then, various characterization techniques were used to comprehensively evaluate the pharmaceutical properties of APAP@GLS. Finally, in vitro release experiments were conducted to evaluate its sustained-release behavior, providing experimental evidence for the further development and application of APAP@GLS.

[0060] I. Experimental Methods 1. Preparation and characterization of APAP@GLS Natural Ganoderma lucidum spores (GLS) were purchased from Anguo Traditional Chinese Medicine Market in Hebei Province, China. Acetaminophen was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0061] 1.1 Optimization of APAP@GLS Formulation Process The effects of different loading times (1 h, 2 h, 3 h), different drug-to-carrier ratios (1:1, 2:1, 2.4:1), and different loading methods (passive loading and vacuum loading) on ​​the preparation process of APAP@GLS were investigated to determine the optimal preparation conditions.

[0062] Passive loading method: APAP was accurately weighed and dissolved in ethanol to prepare drug solutions of different concentrations (50 mg / mL, 100 mg / mL, and 120 mg / mL). 150 mg of natural GLS was suspended in 3 mL of each APAP solution and vortexed for 5 min to ensure uniform spore dispersion. The suspensions were then placed in a constant-temperature water bath shaker at 200 rpm and incubated for 1 h, 2 h, and 3 h. After incubation, the spores were centrifuged at 4500 rpm for 3 min, washed with deionized water, and collected by centrifugation. The collected spores were then frozen at -80℃ for 30 min and freeze-dried for 24 h to obtain APAP@GLS spores.

[0063] Vacuum-assisted loading method: Set the rotation speed to 200 rpm, apply a vacuum pressure of -0.1 MPa, and incubate for loading times of 1 h, 2 h, and 3 h respectively. Other steps are the same as the "passive loading method".

[0064] 1.2 Validation of Optimal Formulation Process Based on the above experimental results, the optimal formulation and process were obtained. The process was repeated three times according to the optimal formulation and process, and the drug loading and encapsulation efficiency of APAP@GLS were determined.

[0065] 1.3 Plotting the APAP Standard Curve Accurately weigh 20.0 mg APAP and place it in a 10 mL volumetric flask. Add an appropriate amount of ethanol solution to make up the volume, and sonicate for 5 min to fully dissolve it. This yields an APAP solution with a concentration of 2.0 mg / mL, which can be used as the stock solution.

[0066] A certain volume of APAP stock solution was taken and diluted to prepare APAP standard solutions with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, and 2.0 mg / mL.

[0067] Add 950 μL of phosphate buffer solution (pH=7.4) to a centrifuge tube, then add 50 μL of APAP standard solution of each concentration. Mix thoroughly and use a UV-Vis spectrophotometer to detect the absorbance values ​​(A) of different concentrations of standard solutions at 248 nm. Calculate the sample concentration and plot the standard curve.

[0068] 1.4 Determination of drug loading and encapsulation efficiency Accurately weigh APAP@GLS, add 3 mL of anhydrous ethanol, and vortex for 2 min to ensure thorough dispersion. Then, use an ultrasonic cell disruptor for 10 s per cycle, repeating 3 times to ensure complete sample disruption. Filter the resulting sample solution through a 0.22 μm microporous membrane and collect the filtrate. Take 50 μL of the filtrate, add 950 μL of PBS buffer (pH=7.4), and vortex for 2 min to mix thoroughly. Measure the absorbance of the filtrate at 248 nm using a UV-Vis spectrophotometer, and calculate the corresponding concentration by substituting the absorbance value into the standard curve. The drug loading capacity (LC%) and encapsulation efficiency (EE%) are calculated using the following formulas: .

[0069] 1.5 Physicochemical characterization using GLS and APAP@GLS 1.5.1 Scanning Electron Microscopy (SEM) SEM analysis was performed using a Hitachi S-3400N scanning electron microscope (SEM). Dry GLS and APAP@GLS were thinly coated onto a stage with conductive adhesive and then gold-plated. The samples were examined in a vacuum at 10 kV and observed at magnifications of 5,000× and 10,000×.

[0070] 1.5.2 Zeta potential The surface potential of natural GLS and APAP@GLS samples was measured using a Malvern laser particle size analyzer (Nano-ZS, Malvern Instruments Ltd., UK) at 25°C. After each sample was gently agitated to homogenize in a suspension of a specific concentration, 1 mL was injected into an optical-grade sample cell for analysis, and data were analyzed based on the intensity distribution.

[0071] 1.5.3 X-ray diffraction (XRD) APAP, GLS, APAP@GLS, and a physical mixture of APAP and GLS were characterized using XRD. Each sample was first ground into a homogeneous powder, placed in a sample holder, and flattened. A Cu Kα radiation source (λ=1.5406 Å) was used, with a scanning range of 5°–50° (2θ), a step size of 0.02°, and a scanning speed of 2° / min. Data were acquired at room temperature. By analyzing the location, intensity, and crystal structure characteristics of the diffraction peaks, the differences in crystal structure between APAP, GLS, APAP@GLS, and the APAP / GLS mixture were compared to further evaluate the interaction between the drug and the carrier and changes in crystallization behavior.

[0072] 1.5.4 Fourier Transmission Infrared Spectroscopy (FTIR) Each sample was mixed with KBr at a ratio of 1:100 and pressed into a transparent thin film. The film was then placed in the FTIR sample chamber, and the scanning range was set to 4000-400 cm⁻¹. -1 The resolution is 4 cm. -1 The study compared the functional group changes of APAP, GLS, APAP@GLS, and a mixture of APAP and GLS to evaluate the interaction between the drug and the carrier and the chemical structural characteristics.

[0073] 2. In vitro release studies 2.1 Pretreatment of dialysis bags Take a dialysis bag (relative molecular mass 7500 Da) and cut it into small segments of appropriate length. Immerse in water and boil at 100°C for 30 min, rinse thoroughly with clean water, dry at 37°C, and store in a refrigerator at 4°C. Soak in the release medium for 24 h before use.

[0074] 2.2 In vitro release The in vitro release of APAP active pharmaceutical ingredient (API) and APAP@GLS was investigated using a dynamic dialysis method. 50 mg of APAP API and APAP@GLS were accurately weighed and placed into pretreated dialysis bags, sealed, and then placed in beakers containing 100 mL of simulated gastric fluid. The beakers were placed at 37 ± 0.5℃ and 100 rpm for the experiment. 1 mL samples were taken at time points of 0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h, and an equal volume of fresh simulated gastric fluid was immediately added to maintain a constant medium volume. The samples were filtered through a 0.22 μm microporous membrane, and the absorbance was measured at 245 nm using a UV-Vis spectrophotometer. The drug concentration was calculated based on the standard curve, and a cumulative dissolution curve was plotted to evaluate the drug release behavior. Each experiment was repeated three times, and the cumulative release rate was calculated using the following formula.

[0075] .

[0076] Note: M represents the total mass of APAP in APAP@GLS, V i V and V represent the volume of the release medium collected each time and the total volume of the release medium, respectively (V0). i =1 mL, V=100 mL), C i and C n These represent APAP concentrations at different sampling times.

[0077] II. Experimental Results 1. GLS structural characterization 1.1 Scanning Electron Microscopy (SEM) GLS have a complete structure, are oval-shaped, uniform in size, with a particle size of 5-6 μm × 7-8 μm, and have a smooth surface with many closed or semi-closed natural pores evenly distributed. Figure 1 ).

[0078] 1.2 Zeta potential The results are as follows Figure 2 As shown, the zeta potential of natural GLS is (-12.01±0.31) mV, indicating that the surface carries a negative charge.

[0079] 1.3 X-ray diffraction (XRD) Figure 3 The spectrum was analyzed using GLS X-ray diffraction (XRD). The spectrum exhibits typical amorphous characteristics, with strong characteristic peaks appearing at approximately 20° 2θ, which are characteristic peaks of Ganoderma lucidum spores, indicating its amorphous structural nature.

[0080] 1.4 Fourier Transmission Infrared Spectroscopy (FTIR) The infrared spectroscopy results of GLS are as follows: Figure 4 As shown. The main characteristic absorption peaks present in the FTIR of GLS are the hydroxyl stretching vibration peaks (VT) of polysaccharides and proteins. O-H δ=3300 cm -1 ), CH bond stretching vibration peak (V C-H δ=2900 cm -1 ), the C=O stretching vibration peak of the amide group (V C=O δ=1640 cm -1 ), the NH bending vibration peak of the amide group (β) N-H δ=1560 cm -1 ), and the stretching vibration peak (V) of COC in polysaccharides. C-O-C δ=1076 cm -1 ).

[0081] 2. Optimization of APAP@GLS formulation process 2.1 Standard curve of APAP in PBS Figure 5 The standard curve for APAP in PBS solution is shown. The regression equation for APAP is Y = 3.1077X + 0.0012. The curve shows a high degree of linear correlation, and the correlation coefficient R0 is high. 2 The linear relationship is good, reaching above 0.9997.

[0082] 2.2 Examination of different loading times for APAP@GLS The effects of different loading times (1 h, 2 h, 3 h) on the drug loading of APAP@GLS were investigated under passive loading conditions with a drug-to-carrier ratio of 1:1. Table 1 shows that the drug loading and encapsulation efficiency initially increased and then slightly decreased with increasing loading time. At a loading time of 2 h, the drug loading and encapsulation efficiency of APAP@GLS reached their maximum values ​​of (11.96±0.23)% and (16.30±0.41)%, respectively, significantly higher than those under the 1 h and 3 h conditions. Therefore, the optimal loading time was determined to be 2 h, providing the best process parameters for the preparation of APAP@GLS.

[0083] Table 1. Drug loading capacity and encapsulation efficiency of APAP@GLS at different preparation times 2.3 Investigation of different drug-to-carrier ratios in APAP @GLS Under passive loading conditions and a constant loading time of 2 h, the effects of different drug-to-carrier ratios (1:1, 2:1, 2.4:1) on the drug loading capacity of APAP@GLS were investigated. Table 2 shows that the drug loading capacity changed significantly with increasing drug-to-carrier ratio. When the drug-to-carrier ratio was 1:1, the drug loading capacity of GLS was (12.03±0.61)%; when the ratio increased to 2:1, the drug loading capacity significantly increased to (22.69±0.60)%, indicating that increasing the drug loading capacity effectively improved the drug loading efficiency. However, when the drug-to-carrier ratio further increased to 2.4:1, the drug loading capacity slightly increased to (24.02±0.69)%, and there was no significant difference compared to 2:1 (P>0.05), indicating that the carrier's drug loading capacity tended to saturate. Therefore, the optimal drug-to-carrier ratio was determined to be 2:1. This ratio significantly improves drug loading while effectively avoiding waste caused by excessive drug dosage. Furthermore, the encapsulation efficiency trend is highly consistent with the drug loading: when the drug-to-carrier ratio is 1:1, 2:1, and 2.4:1, the encapsulation efficiencies are 24.65%, 34.02%, and 34.02%, respectively. The encapsulation efficiency reaches its peak at a drug-to-carrier ratio of 2:1, further confirming its rationality as the optimal ratio.

[0084] Table 2. Drug loading capacity and encapsulation efficiency of APAP@GLS prepared with different drug-to-carrier ratios. 2.4 Examination of different loading methods for APAP@GLS The effects of different loading methods (passive loading and vacuum loading) on ​​the drug loading capacity of APAP@GLS were investigated under the conditions of a drug-to-carrier ratio of 2:1 and a loading time of 2 h. Table 3 shows that the loading method significantly affects the drug loading capacity of GLS. Compared with passive loading, vacuum loading significantly improved the drug loading capacity of GLS. Specifically, under passive loading, the drug loading capacity of GLS was (22.54±0.63)%; while under vacuum loading, the drug loading capacity increased to (28.72±0.45)%. This result is attributed to the negative pressure environment during vacuum loading promoting the diffusion and adsorption of drug molecules into the pores inside the carrier, thereby enhancing the carrier's loading capacity. This indicates that vacuum loading is an effective loading method that can significantly improve the drug loading performance of APAP@GLS.

[0085] Table 3. Drug loading capacity and encapsulation efficiency of APAP@GLS prepared by different loading methods 2.5 Best Prescription Process and Validation Using drug loading and encapsulation efficiency as evaluation indicators, the optimal preparation process was determined to be: loading time of 2 h, drug-to-carrier ratio of 2:1, and vacuum loading method. This process condition can achieve optimal encapsulation efficiency while ensuring high drug loading. Three batches of APAP@GLS were prepared according to this preparation process to verify the correctness of the drug loading system. The results are shown in Table 4.

[0086] Table 4. Results of the verification experiment The results in summary indicate that loading time, drug-to-carrier ratio, and loading method all have a significant impact on the drug loading and encapsulation efficiency of APAP@GLS. The optimal formulation process for preparing APAP@GLS was determined to be: drug-to-carrier ratio of 2:1, vacuum-assisted loading, loading time of 2 h, drug loading of (28.65±0.3)%, and encapsulation efficiency of (42.96±0.48)%.

[0087] 3. Physicochemical characterization of APAP@GLS 3.1 SEM Analysis like Figure 6 As shown, natural GLS and drug-loaded APAP@GLS were characterized by SEM. The drug loading process did not disrupt the structural integrity of the GLS. The GLS maintained its original structural integrity during drug loading and did not undergo significant structural changes due to drug loading.

[0088] 3.2 Zeta potential analysis The results are as follows Figure 7 As shown, the surface potential of the drug-loaded formulation APAP@GLS is (0.02±0.002) mV, which is different from that of natural GLS.

[0089] 3.3 XRD Analysis XRD results are as follows Figure 8As shown, APAP exhibits multiple strong crystalline diffraction peaks in the 2θ = 0-30° range. The peaks at 2θ angles of 12.182°, 15.610°, 18.219°, 20.421°, 23.579°, 24.501°, 26.644°, and 36.949° are its typical characteristic peaks, indicating that APAP exists in a highly crystalline state with a well-defined crystal structure. In the mixture obtained by direct physical mixing of APAP and GLS, the crystalline diffraction peaks of APAP superimpose with the amorphous characteristic peaks of GLS. In the simple mixture, APAP retains its crystalline morphology and does not interact significantly with GLS. However, in the XRD pattern of APAP@GLS, the crystalline diffraction peaks of APAP weaken or even disappear, leading to a transformation of APAP from a crystalline to an amorphous state, while the amorphous characteristics of GLS are preserved. This indicates that APAP was successfully loaded onto GLS and highly dispersed in the support in an amorphous state. This result confirms the interaction between the drug and the carrier in APAP@GLS.

[0090] 3.4 Fourier Transform Infrared Spectroscopy (FTIR) The FTIR results are shown in Figure 9 In the IR spectrum of APAP, the following characteristic absorption peaks mainly exist in the infrared spectrum: the skeletal vibration peak of the benzene ring (V... C=C δ=1600 cm -1 δ=1500 cm -1 ), CH stretching vibration peak on the benzene ring (V C-H δ=2900 cm -1 The stretching vibration peak of the hydroxyl group (V) O-H δ=3300 cm -1 ), the C=O stretching vibration peak of the amide group (V C=O δ=1650cm -1 ), the NH bending vibration peak of the amide group (β) N-H δ=1560 cm -1 ), and the stretching vibration peak of the carbon-oxygen single bond (V C-O δ=1250 cm -1 In a simple mixture of APAP and GLS, the characteristic peaks of both APAP and GLS are clearly visible and appear as a simple superposition of their characteristic peaks, indicating that no significant chemical interaction occurs between APAP and GLS. However, in the FTIR spectrum of APAP@GLS, the characteristic peak of APAP changes significantly: the stretching vibration peak representing the hydroxyl group in the APAP structure (V... O-H δ=3300 cm -1 The C=O stretching vibration peak of the amide group (V C=O δ=1650 cm-1 The stretching vibration (V) of the NH bond in the amide group is significantly reduced. N-H δ=3160 cm -1 ), stretching vibration of CN bond (V C-N δ=1260 cm -1 ) and stretching vibrations of CO bonds (V C-O δ=1100 cm -1 The APAP peaks completely disappeared, while the characteristic peaks of GLS were retained. The above results indicate that APAP interacted significantly with the GLS surface, causing the characteristic peaks of APAP to weaken or disappear, proving that APAP was successfully loaded onto GLS.

[0091] 4. In vitro release studies like Figure 10 As shown, at 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h, the cumulative release of APAP technical grade was 68.96%, 80.60%, 86.91%, 95.85%, 95.79%, 96.46%, 97.80%, 99.09%, and 98.76%, respectively, while the cumulative release of the drug-loaded formulation APAP@GLS was 13.15%, 22.25%, 34.56%, 49.18%, 68.40%, 85.00%, 95.37%, 96.75%, and 96.77%, respectively. The results showed that APAP active pharmaceutical ingredient (API) was released rapidly within the first 1 hour, with a cumulative release of 86.91%, reaching 95.85% within 2 hours, after which the release stabilized. In contrast, APAP@GLS was released more slowly within 0-2 hours, with a cumulative release rate of 49.18% at 2 hours. The release rate gradually increased thereafter, reaching 85.00% at 6 hours, before significantly slowing down after 8 hours, and the release curve flattened out. Notably, APAP API was almost completely released by 4 hours, while the cumulative release of APAP@GLS was only 68.40%, indicating that APAP@GLS significantly delayed drug release and exhibited good sustained-release properties. Preliminary assessments suggest that the sustained-release behavior is caused by the following factors: ① The GLS surface has abundant and uniformly distributed natural pores, which can serve as storage spaces for the drug. Drug molecules need to gradually diffuse into the release medium through these pores, thus prolonging the release time; ② There is an electrostatic interaction between APAP molecules and the GLS surface, which enhances the binding force and stability between the drug and the carrier, delaying drug release. This sustained-release characteristic helps maintain a stable blood drug concentration and reduces drug side effects.

[0092] Example 2 Acetaminophen (APAP) is a widely used aniline antipyretic and analgesic in clinical practice. However, its adverse reactions and severe hepatotoxicity caused by overdose and abuse have increasingly attracted attention. Dosage form modification is an effective strategy to reduce its hepatotoxicity. This example investigates the pharmacokinetic behavior of GLS-loaded APAP (APAP@GLS) at different doses in rats, providing data support for the improvement of acetaminophen dosage forms.

[0093] 1. Research Methods The active pharmaceutical ingredient (APAP) was administered via gavage to the APAP group, the low-dose APAP@GLS group, and the high-dose APAP@GLS group at doses of 1 g / kg, 3.05 g / kg (APAP was 1 g / kg), and 4.58 g / kg (APAP was 1.5 g / kg), respectively. Whole blood was collected from the fundus venous plexus of rats at 0, 5, 10, 20, 30, 60 min, 2, 4, 6, 8, 12, and 24 h (n=3) and transferred to heparinized test tubes. The plasma was separated by centrifugation at 8000 rpm for 5 min and stored at -20℃ for analysis. An LC-MS / MS method was established to determine the concentration of APAP in rat plasma. Plasma drug concentration-time curves were plotted using GraphpadPrism software, and key pharmacokinetic parameters were calculated using WinNonlin software.

[0094] 2. Results Dynamic curve Figure 11 As shown in Table 5, the key pharmacokinetic parameters of APAP in rats after administration are shown in Table 5. Peak APAP concentrations after gavage administration of APAP (1 g / kg) are... C max Approximately 125000±29308.70 ng / mL, time to peak concentration T max The exposure time in the body is approximately 0.61 ± 0.35 h. AUC 0-t Approximately 1009124.33 ± 180649.78 h·ng / mL, AUC 0-∞ It is approximately 1314682.73 ± 219173.58 h·ng / mL.

[0095] Peak APAP concentration after gavage administration of APAP@GLS (3.05 g / kg, APAP 1 g / kg) C max Approximately 109800±28742.30 ng / mL, peak time T max The exposure time in the body is approximately 0.44 ± 0.10 h. AUC 0-tApproximately 506312.67 ± 198753.75 h·ng / mL, AUC 0-∞ It is approximately 509431.30 ± 197452.57 h·ng / mL.

[0096] After gavage administration of APAP@GLS (4.58 g / kg, APAP at 1.5 g / kg), the peak concentration of APAP was... C max Approximately 107333.33 ± 5773.50 ng / mL, peak time T max The exposure time in the body was approximately 0.78 ± 0.39 h. AUC 0-t Approximately 531066.83 ± 123496.20 h·ng / mL, AUC 0-∞ It is approximately 550446.61 ± 103383.31 h·ng / mL.

[0097] Table 5 Key pharmacokinetic parameters of APAP in rats after administration (n=3) In summary, in rats, APAP@GLS exhibits a shorter pharmacokinetic half-life compared to the APAP active pharmaceutical ingredient. t 1 / 2 shorter average length of stay MRT and lower levels of internal exposure ( ) AUC Furthermore, after increasing the dose, the peak concentration of APAP in the APAP@GLS group ( C max) and body exposure ( AUC No significant changes occurred.

[0098] Example 3 This embodiment systematically evaluates whether APAP@GLS can reduce acute hepatotoxicity of APAP through in vitro / in vivo experiments. In the in vivo pharmacodynamic experiment, a mouse model of APAP-induced liver injury was established. Animals were divided into normal (Control), carrier (GLS), model (APAP), formulation group, and physical mixture group. The protective effect of APAP@GLS on acute liver injury was evaluated by gross liver imaging, liver histopathological analysis, and detection of liver function indicators (ALT, AST) in rat serum.

[0099] I. Experimental Methods 1. Laboratory animals and grouping Forty-two healthy male C57BL / 6J mice were randomly divided into seven groups as follows: (1) Control group: 0.5% sodium carboxymethyl cellulose solution was administered by gavage; (2) Carrier group (GLS), empty GLS administered by gavage; GLS 615 mg / kg (3) Model group (APAP), administered raw APAP (300 mg / kg) by gavage; (4) Formulation group (APAP@GLS), APAP@GLS administered by gavage, including APAP@GLS equal dose group administered by gavage (915 mg / kg APAP@GLS), APAP@GLS high dose group administered by gavage (1372.5 mg / kg APAP@GLS), and acid-washed GLS@APAP equal dose group administered by gavage (915 mg / kg APAP@GLS); (5) Physical mixture group (GLS 615 mg / kg and APAP 300 mg / kg continuously by gavage).

[0100] Dosage regimen: C57BL / 6J mice underwent a one-week acclimatization period before the experiment, during which they were provided with normal drinking water and feed. Twelve hours prior to the experiment, all mice were fasted but allowed free water. On the day of the experiment, the weight of each mouse was recorded, and the dosage was adjusted accordingly before gavage administration. Each group was administered the dosages set in Table 2-4, with fasting and water restriction in place. Twelve hours later, the weight changes of each group were recorded. After ether anesthesia, blood samples were collected via the abdominal aorta, centrifuged at 3000 rpm for 15 minutes, and serum was separated and stored at -80°C for biochemical analysis. Liver tissue was removed and washed with physiological saline to remove blood. A portion of the liver from the same location in each group was placed in 4% paraformaldehyde (labeled as group) and fixed at 4°C for 48 hours for histopathological analysis.

[0101] 2. Histopathological analysis of liver tissue After the experiment, rat liver tissue was collected, and whole-body photographs were taken with a digital camera. The tissue was then fixed with 4% paraformaldehyde for 48 h. After fixation, the tissue was dehydrated and cleared with graded ethanol, then embedded in paraffin and sectioned (4-5 µm thick). After dewaxing, the slides were dried, and a suitable amount of hematoxylin was added to the tissue slides for staining for approximately 3 min. Staining was stopped by rinsing with water, allowing the cell nuclei to stain. Eosin was added to the liver tissue to stain the cytoplasm for approximately 2 min, followed by rinsing with water. The slides were then dehydrated, mounted, and observed under a microscope, with images acquired to analyze pathological changes.

[0102] 3. Liver function test Strictly follow the instructions of the test kit to measure the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in each group.

[0103] II. Experimental Results 1. Results of gross morphological observation of the liver like Figure 12 As shown, the liver in the control group (Ctrl) was dark red, smooth, firm, and intact. The GLS group (GLS) was similar to the control group, indicating that GLS itself has no significant toxic effect on the liver. The liver in the APAP model group (APAP) was lighter in color, with obvious punctate necrosis and pale areas, and loose peripheral tissue structure, indicating severe parenchymal damage. Compared with the APAP model group, the liver appearance of the APAP@GLS iso-dose group (A@GL) and high-dose group (A@GH) was significantly improved, with uniform color, smooth surface, and reduced necrotic foci. The acid-washed APAP@GLS group (Acid-A@G) also showed significant improvement. The liver color of the physical mixture group (Mix) was slightly improved compared to the model group, but surface irregularities and localized damage were still visible. These results indicate that the APAP@GLS complex can effectively improve APAP-induced liver appearance damage, suggesting a good protective effect in alleviating drug-induced liver injury.

[0104] Gross morphological observation of the liver and detection of serum biochemical indicators showed that the APAP@GLS complex had a significant hepatoprotective effect compared with the model group and the physical mixture system. This complex can alleviate APAP-induced hepatocellular necrosis and reduce serum ALT and AST levels, thus demonstrating significant technical advantages and application potential in the protection against drug-induced liver injury.

[0105] 2. Liver histopathological staining results Figure 13 HE staining results showed that the liver tissue of rats in the Control group was structurally intact, with clear outlines of liver lobules and normal structures of the central vein and portal canal. Hepatocytes were arranged regularly in a radial pattern, and the hepatic cords were clear and continuous. Hepatocyte morphology was normal, cytoplasm was uniformly stained, and nuclei were round and clearly stained, without obvious nuclear pyknosis, karyolysis, or vacuolar degeneration. The hepatic sinusoids were normal in structure, without obvious dilation or congestion. No inflammatory cell infiltration, hemorrhage, or necrosis was observed in the liver tissue.

[0106] The liver tissue of rats in the GLS-treated group showed relatively intact overall structure, with clear lobular outlines and well-preserved central veins and surrounding structures. Hepatocytes were arranged regularly, hepatic cords were continuous, most hepatocytes were morphologically normal, cytoplasm was uniformly stained, and nuclei were clearly visible. No significant large-scale hepatocyte necrosis or disintegration was observed in the liver tissue. The hepatic sinusoidal structure was basically normal, with no obvious dilation or hemorrhage. Inflammatory cell infiltration was mild, only scattered. This suggests that GLS treatment has no effect on rat liver tissue.

[0107] Following APAP treatment, rat liver tissue showed severe structural damage, with disordered hepatic lobule structure and extensive hepatocyte necrosis around the central vein. In the necrotic areas, hepatocyte cytoplasm exhibited increased eosinophilicity, pyknosis or nucleus disappearance, and marked sinusoidal dilation and hemorrhage. Simultaneously, abundant inflammatory cell infiltration was observed in and around the necrotic areas, indicating a significantly enhanced acute inflammatory response. This suggests that APAP successfully induced an acute liver injury model in rats.

[0108] The liver tissue of rats in the A@GL and A@GH groups showed relatively intact overall structure, with clear hepatic lobule outlines and well-preserved central veins and surrounding structures. Hepatocytes were arranged regularly, hepatic cords were continuous, most hepatocytes were morphologically normal, cytoplasmic staining was uniform, and nuclei were clearly visible. No significant large-scale hepatocyte necrosis or disintegration was observed in the liver tissue; only mild hepatocyte swelling or slight degeneration was observed in localized areas. The hepatic sinusoidal structure was basically normal, with no obvious dilation or hemorrhage. Inflammatory cell infiltration was minimal and scattered. A@GL and A@GH treatments significantly improved the pathological damage to liver tissue, almost completely inhibiting hepatocyte necrosis, hemorrhage, and inflammatory responses, with liver tissue morphology approaching that of the normal control group.

[0109] The liver tissue of rats in the acid-washing group showed relatively intact overall structure, clear lobular outlines, and good preservation of the central vein and surrounding structures. Hepatocytes were arranged regularly, hepatic cords were continuous, most hepatocytes were morphologically normal, cytoplasm was uniformly stained, and nuclei were clearly visible. No significant large-scale hepatocyte necrosis or disintegration was observed in the liver tissue; only mild hepatocyte swelling or slight degeneration was observed in a few areas. The hepatic sinusoidal structure was basically normal, with no obvious dilation or hemorrhage. Inflammatory cell infiltration was minimal and scattered. The pathological damage to the liver tissue of rats in the acid-washing group was significantly reduced, with a marked decrease in hepatocyte necrosis, hemorrhage, and inflammatory response, suggesting that acid washing treatment has a certain alleviating effect on APAP-induced liver injury.

[0110] The overall liver structure of rats in the physical mixing group was relatively intact, with clear outlines of liver lobules and identifiable central veins and surrounding structures. Hepatocytes were arranged relatively regularly, hepatic cords were continuous, and most hepatocytes were morphologically normal. Mild hepatocyte swelling and scattered vacuolar degeneration were observed in localized areas, but no large-scale hemorrhagic necrosis was observed. Hepatic sinusoids were slightly dilated, and inflammatory cell infiltration was mild and scattered. Compared with the APAP model group, the pathological damage to the liver tissue of rats in the physical mixing group was significantly reduced, with hepatocyte necrosis, hemorrhage, and inflammatory response all alleviated, but mild pathological changes were still visible.

[0111] 3. Serum biochemical index determination To further evaluate the hepatoprotective effect of the complex, such as... Figure 14As shown, the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in mice of each group were measured. After APAP treatment, the serum ALT and AST levels in the model group mice were significantly increased, indicating significant hepatocyte damage. Compared with the model group, the ALT and AST levels in the APAP@GLS complex treatment group were significantly reduced, but no dose-dependent effect was observed in the low- and high-dose groups. The acid-treated APAP@GLS (Low Dose) group also showed a significant reduction. Although the physical mixture group partially reduced ALT and AST, the effect was significantly less than that of the APAP@GLS complex group. These results indicate that the APAP@GLS material prepared by the composite method can effectively alleviate APAP-induced liver function damage, and its protective effect is superior to that of the physical mixture system, demonstrating that this complex has good technical efficacy in reducing the levels of liver injury markers.

[0112] Gross morphological observation of the liver and detection of serum biochemical indicators showed that the APAP@GLS complex had a significant hepatoprotective effect compared with the model group and the physical mixture system. This complex can alleviate APAP-induced hepatocellular necrosis and reduce serum ALT and AST levels, thus demonstrating significant technical advantages and application potential in the protection against drug-induced liver injury.

[0113] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A small molecule drug delivery carrier, characterized in that, The small molecule drug delivery carrier is a natural Ganoderma lucidum spore, and the small molecule drug is loaded onto the Ganoderma lucidum spore by electrostatic adsorption.

2. The small molecule drug delivery carrier according to claim 1, characterized in that, The ratio of the small molecule drug to natural Ganoderma lucidum spores is 1-5:

1.

3. The small molecule drug delivery carrier according to claim 1, characterized in that, The Ganoderma lucidum spores have a size of 4-8 μm × 5-10 μm and a pore size of 0.1-50 nm.

4. A method for preparing a small molecule drug delivery carrier, characterized in that, include: (1) Provides natural Ganoderma lucidum spores; (2) The small molecule drug is loaded onto the Ganoderma lucidum spores by electrostatic adsorption.

5. The method for preparing the small molecule drug delivery carrier according to claim 4, characterized in that, In step (2), the small molecule drug is loaded by passive loading or vacuum-assisted loading.

6. The method for preparing the small molecule drug delivery carrier according to claim 4, characterized in that, In step (2), the loading time of the small molecule drug is 0.5-5 h.

7. The method for preparing the small molecule drug delivery carrier according to claim 4, characterized in that, The concentration of the small molecule drug is 10-500 mg / mL.

8. A pharmaceutical composition, characterized in that, A small molecule drug delivery carrier comprising any one of claims 1-3.

9. The pharmaceutical composition according to claim 8, characterized in that, It further includes pharmaceutically acceptable excipients.

10. The use of the small molecule drug delivery carrier according to any one of claims 1-3 in the preparation of a drug.

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