A platycodin d double-targeting nanoparticle and a preparation method and application thereof

Abstract

The application belongs to the technical field of nano drug carrier, and particularly relates to a double-targeted nanoparticle for loading platycodin D as well as a preparation method and application thereof. The platycodin D is prepared into PD-NPs through an enzymatic reaction, and then a mitochondrion-targeting molecule (5-carboxy-pentyl) triphenylphosphonium bromide (TPP) and a macrophage-targeting molecule hyaluronic acid (HA) are sequentially connected to obtain PT-HNPs. The nanoparticle has a multi-stage targeting capability and can deliver drugs to inflammatory sites and mitochondria. Experiments show that the PT-HNPs can significantly improve the weight loss of ulcerative colitis model mice, reduce the disease activity index, relieve colon shortening and hematochezia, protect the integrity of the colon structure, and improve mitochondrial dysfunction. The combination of the platycodin D, the TPP and the HA achieves a synergistic effect of '1+1+1>3', and provides an efficient targeted nano delivery system for the treatment of acute ulcerative colitis.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine carrier technology, specifically relating to a dual-targeting nanoparticle loaded with platycodon saponin D, its preparation method, and its application. Background Technology

[0002] Ulcerative colitis (UC) is a chronic inflammatory bowel disease affecting the colon and rectum. Its pathogenesis is complex and influenced by various factors such as dietary patterns, unhealthy lifestyle habits (e.g., lack of exercise, excessive stress, insufficient sleep), and environmental pollution. Clinical manifestations include persistent or recurrent abdominal pain, diarrhea, bloody and mucous stools, tenesmus, and varying degrees of systemic symptoms. In recent years, the incidence of UC has shown a year-on-year upward trend worldwide, and UC is often accompanied by mental disorders such as depression and anxiety. Despite extensive research, the etiology of UC remains unclear, and a complete cure is currently lacking.

[0003] Currently, there is no effective treatment to completely cure ulcerative colitis (UC). Clinical drugs such as glucocorticoids and immunosuppressants are commonly used to alleviate UC symptoms, but they have drawbacks such as high relapse rates, significant side effects, and short-lasting treatment effects. Therefore, there is an urgent need to design well-developed and universal drug delivery systems for targeted therapy of UC, in order to develop more effective treatment methods.

[0004] Platycodin D (PD), molecular formula: C 57 H 92 O 28 With a molecular weight of 1225.324, PD is a triterpenoid monomeric compound isolated and extracted from Platycodon grandiflorus. It is the main active ingredient of Platycodon grandiflorus and has been found to possess pharmacological activities such as anti-inflammatory, analgesic, antitumor, hepatoprotective, and immunomodulatory effects as a natural product. Existing studies have shown that PD has clear anti-inflammatory properties by participating in the regulation of the NF-κB signaling pathway and reducing the levels of NO, TNF-α, and iNOS in lipopolysaccharide (LPS)-stimulated macrophages. Simultaneously, some in vivo studies have also confirmed the anti-inflammatory properties of PD; PD can attenuate DSS-induced colitis and reduce the inflammatory response, and its mechanism is related to alleviating intestinal inflammation through the TLR4 / NOD signaling pathway. However, PD suffers from low bioavailability and high hemolysis rate, which greatly limits its clinical application.

[0005] Existing technologies have been reported to use hyaluronic acid (HA) to target inflamed tissues in ulcerative colitis (UC) or to combine HA with TPP for dual-target delivery, but none have addressed specific protocols for using PD as an active ingredient to improve mitochondrial dysfunction in the treatment of UC. Therefore, constructing a novel multifunctional nanomaterial capable of targeted delivery of PD to inflamed sites is of significant scientific importance and application value for improving the efficacy and safety of UC treatment. Summary of the Invention

[0006] The primary objective of this invention is to overcome existing deficiencies and provide a multi-level targeted nanomedicine delivery system (PT-HNPs) that targets inflammatory cells and mitochondria.

[0007] One of the objectives of this invention is to provide a method for preparing dual-targeting nanoparticles loaded with platycodon saponin D. The prepared PT-HNPs have multi-level targeting capabilities for inflammatory cells and mitochondria, which can efficiently deliver platycodon saponin D to the lesion site of ulcerative colitis, thereby improving the therapeutic effect and drug safety.

[0008] To achieve the above objectives, the present invention employs the following technical solution: a method for preparing platycodon saponin D-loaded dual-targeting nanoparticles, comprising the following steps: Step S1: Platycodin D (PD), horseradish peroxidase (HRP), polyethylene glycol 2000 (PEG-2000), and 1,4-dioxane were added to phosphate buffer (PBS) and stirred. During stirring, 5% hydrogen peroxide (H2O2) solution was added in portions at intervals. The reaction was continued in the dark and stirred. The reaction solution was dialyzed and freeze-dried to obtain nanoparticles loaded with platycodin D, denoted as PD-NPs. Step S2: Take (5-carboxypentyl)triphenylphosphine bromide (TPP), 4-dimethylaminopyridine (DMAP), and N,N'-dicyclohexylcarbodiimide (DCC), add them to dimethyl sulfoxide (DMSO), and stir in the dark to activate the carboxyl group in TPP; then add the PD-NPs obtained in step S1, continue stirring in the dark, dialyze the reaction solution, and freeze-dry to obtain mitochondrial-targeting nanoparticles loaded with platycodon saponin D, denoted as PT-NPs; Step S3: Hyaluronic acid (HA) was dispersed in phosphate buffer (PBS), and 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added. The mixture was stirred in the dark to activate the carboxyl groups in HA. Then, PT-NPs prepared in step S2 were added, and the reaction was stirred in the dark. The reaction solution was dialyzed and freeze-dried to obtain platycodon saponin D dual-targeting nanoparticles, denoted as PT-HNPs.

[0009] Further improvements to the preparation method of platycodon saponin D dual-targeting nanoparticles: Preferably, the mass-to-volume ratio of platycodon saponin D, horseradish peroxidase, PEG-2000, 1,4-dioxane, phosphate buffer, and 5% H2O2 in step S1 is 50 mg:10 mg:50 mg:50 mL:72.5 mL:12.5 mL.

[0010] Preferably, the mass-to-volume ratio of (5-carboxypentyl)triphenylphosphine bromide to 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide, dimethyl sulfoxide, and PD-NPs in step S2 is 25 mg:7.5 mg:12.5 mg:25 mL:25 mg.

[0011] Preferably, the volume-to-mass ratio of hyaluronic acid, phosphate buffer, 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and PT-NPs in step S3 is 50 mg:50 mL:25 mg:25 mg:50 mg.

[0012] Preferably, the time for HA activation by stirring in the dark in step S3 is 1-2 h.

[0013] Preferably, the time for continued stirring in the dark during steps S1, S2, and S3 is 20-24 hours, and the reaction temperature is room temperature.

[0014] Preferably, in steps S1, S2, and S3, dialysis uses dialysis bags with a molecular weight cutoff of 500 Da. The dialysis bags are activated before use, the dialysate is pure water, and dialysis is performed in the dark for 48-52 hours. During dialysis, the water is changed at intervals of 2-4 hours, 4-6 hours, and 10-12 hours.

[0015] Preferably, the freeze-drying conditions in steps S1, S2, and S3 are as follows: the sample is pre-cooled in a -80°C freezer for 10-12 hours, and then freeze-dried in a pre-cooled freeze dryer for 40-48 hours.

[0016] The second objective of this invention is to provide a method for preparing platycodon D-loaded dual-targeting nanoparticles as described in any one of the above-mentioned methods, wherein the chemical structural formula of the platycodon D-loaded dual-targeting nanoparticles is as follows: .

[0017] The third objective of this invention is to provide the above-mentioned platycodin D-loaded dual-targeting nanoparticles as a nanomedicine delivery system for the preparation of drugs for treating acute ulcerative colitis.

[0018] The advantages of this invention compared to the prior art are as follows: 1. This invention provides a method for preparing dual-targeting nanoparticles loaded with platycodon saponin D. Platycodon saponin D is reacted into PD-NPs through an enzymatic reaction. Then, TPP is coupled by an ester bond reaction to obtain PT-NPs with mitochondrial targeting. Finally, HA is encapsulated on PT-NPs to obtain PT-HNPs with macrophage targeting.

[0019] 2. The dual-targeting nanoparticles loaded with platycodon saponin D prepared in this invention not only significantly improve the bioavailability of platycodon saponin D and ensure its full therapeutic efficacy, but also exert a dual-targeting effect on the cell membrane and mitochondria of macrophages through TPP and HA. Platycodon saponin D exhibits good pharmacological activities in anti-inflammatory, analgesic, antitumor, hepatoprotective, and immunomodulatory aspects. Hyaluronic acid (HA) can specifically bind to CD44 receptors and target macrophages, while (5-carboxypentyl)triphenylphosphine bromide (TPP), as a delocalized lipophilic cation, can selectively accumulate in the negatively charged mitochondrial membrane and target mitochondria. The combination of these three substances achieves multi-stage intervention in UC and achieves a synergistic effect of "1+1+1>3".

[0020] 3. Experimental results show that the platycodon saponin D-loaded dual-targeting nanoparticles of this invention improve weight loss caused by acute ulcerative colitis, reduce the disease activity index, alleviate colonic shortening and rectal bleeding, protect the structural integrity of the colon, and improve mitochondrial dysfunction. This invention is the first to use PD as an active ingredient and combines TPP and HA to improve mitochondrial dysfunction for the treatment of UC. Experimental results show that PT-HNPs are significantly superior to PD compounds, PD-NPs, and PT-NPs. The PT-HNPs of this invention are significantly superior to the unmodified HA control in terms of colonic targeting enrichment ability and have good biocompatibility (no hemolysis, low cytotoxicity), overcoming the shortcomings of low bioavailability and high toxicity of free PD. Attached Figure Description

[0021] Figure 1 This is a flowchart illustrating the preparation process of platycodon saponin D prepared in Example 1 of the present invention.

[0022] Figure 2 Transmission electron microscopy (TEM) images of the PD-NPs, PT-NPs, and PT-HNPs nanoparticles prepared in Examples 1-3 of this invention.

[0023] Figure 3 The particle size diagrams are of the PD-NPs, PT-NPs, and PT-HNPs nanoparticles prepared in Examples 1-3 of this invention.

[0024] Figure 4 The results of zeta potential detection are for the PD-NPs, PT-NPs, and PT-HNPs nanoparticles prepared in Examples 1-3 of this invention.

[0025] Figure 5 The infrared spectra (FT-IR) of PD-NPs, PT-NPs, PT-HNPs, PD, TPP, and HA prepared in Examples 1-3 of this invention are shown.

[0026] Figure 6 The effects of PD-NPs, PT-NPs, and PT-HNPs prepared in Examples 1-3 of this invention on NO release in LPS-induced RAW264.7 cells (Control group was the blank control group, and LPS was the model group).

[0027] Figure 7 The effects of PD-NPs, PT-NPs, and PT-HNPs prepared in Examples 1-3 of this invention on LPS-induced mitochondrial membrane potential (JC-1) in RAW264.7 cells.

[0028] Figure 8 The effects of PD-NPs, PT-NPs, and PT-HNPs prepared in Examples 1-3 of this invention on the level of reactive oxygen species (ROS) in LPS-induced RAW264.7 cells.

[0029] Figure 9 The in vivo fluorescence imaging (a) and the quantitative analysis of average fluorescence intensity (b) of Cy5.5-labeled PD, PD-NPs, PT-NPs, and PT-HNPs prepared in Examples 1-3 of this invention at different time points (3, 6, 9, 12 h) in mice with ulcerative colitis model are shown.

[0030] Figure 10 The effects of PT-HNPs prepared for Examples 1-3 of this invention on the disease activity index DAI (a) and body weight change (b) of DSS-induced UC mice.

[0031] Figure 11 The results show the effects of PD-NPs, PT-NPs, PT-HNPs, PD, and 5-ASA prepared in Examples 1-3 of this invention on the colon length of DSS-induced UC mice.

[0032] Figure 12 HE staining images of the effects of PD-NPs, PT-NPs, PT-HNPs, and PD and 5-ASA on the histopathological effects of DSS-induced UC mice prepared in Examples 1-3 of this invention.

[0033] Figure 13 Transmission electron microscopy images of PD-NPs, PT-NPs, PT-HNPs prepared in Examples 1-3 of this invention, and the effect of PD on the ultrastructure of mitochondria in the colon tissue of DSS-induced UC mice.

[0034] Figure 14 The results of hemolysis experiments of PD-NPs, PT-NPs, PT-HNPs and PD prepared in Examples 1-3 of this invention are shown.

[0035] Figure 15 The PD-NPs, PT-NPs, PT-HNPs prepared in Examples 1-3 of this invention, and the effect of PD on the survival rate of RAW264.7 cells and Caco-2 cells (MTT method). Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] Example 1

[0038] This embodiment provides a method for preparing platycodon saponin D nanoparticles (PD-NPs), the specific steps of which are as follows: Step 1: Dissolve 10 mg of HRP (horseradish peroxidase) in 10 mL of PBS, mix well, and prepare solution A. Step 2: Dissolve 50 mg of PD (platycoside D) in 6.5 mL of PBS, and dissolve 50 mg of PEG-2000 (polyethylene glycol-2000) in 6 mL of PBS. Then mix the PEG-2000 and PD solutions to prepare solution B. Step 3: Mix 50 mL of 1,4-dioxane with 50 mL of PBS to prepare solution C; Step 4: Add solution C to the beaker, then add solutions B and A in sequence, and stir until homogeneous. Stir the reaction under light-protected conditions for 24 hours. After the reaction begins, add 625 μL of 5% H2O2 solution every 30 minutes, for a total of 12.5 mL, until the solution is completely added.

[0039] Step 5: The product obtained after 24 hours of reaction was placed in an activated dialysis bag (500 Da) and dialyzed in the dark (dialysis solution was water) for 48 hours. Finally, the sample was dried to powder by freeze drying to obtain platycodon saponin D nanoparticles, denoted as PD-NPs.

[0040] Example 2

[0041] A method for preparing mitochondrial-targeting nanoparticles loaded with platycodon saponin D (PT-NPs) includes the following steps: Step 1: 25 mg of TPP ((5-carboxypentyl)triphenylphosphine bromide) was dispersed in 25 mL of DMSO (dimethyl sulfoxide), 12.5 mg of DCC (N,N'-dicyclohexylcarbodiimide) and 7.5 mg of DMAP (4-dimethylaminopyridine) were added, and the mixture was stirred in the dark for 1 h to activate the carboxyl groups in the TPP. Then, 25 mg of PD-NPs obtained in Example 1 were added, and the mixture was stirred in the dark for 24 h to allow for a complete reaction.

[0042] Step 2: The product obtained after 24 hours of reaction was placed in an activated dialysis bag (500 Da) and dialyzed in the dark (dialysis solution was water) for 48 hours. Finally, the sample was dried to powder by freeze-drying to obtain mitochondrial-targeting nanoparticles loaded with platycodon saponin D, denoted as PT-NPs.

[0043] Example 3

[0044] A method for preparing platycodon saponin D-loaded dual-targeting nanoparticles (PT-HNPs) includes the following steps: Step 1: First, activate the carboxyl groups in HA (hyaluronic acid). Disperse 50 mg of HA in 50 mL of PBS, add 25 mg of EDC (3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride) and 25 mg of NHS (N-hydroxysuccinimide), and stir in the dark for 2 h to activate the carboxyl groups in HA. Then, add 50 mg of PT-NPs obtained in Example 2 to the above compound, and stir in the dark for 24 h to allow for complete reaction.

[0045] Step 2: The product obtained after 24 hours of reaction was placed in an activated dialysis bag (500 Da) and dialyzed in the dark (dialysis solution was water) for 48 hours. Finally, the sample was dried to powder by freeze drying to obtain platycodon saponin D dual-targeting nanoparticles, denoted as PT-HNPs.

[0046] To analyze and test the functionality of the prepared PT-HNPs nanoparticles, the following methods are provided for analysis and verification: (1) Material characterization Figure 1The flowchart for the preparation of platycodon saponin D prepared in Example 1 of the present invention is as follows: First, platycodon saponin D is reacted into PD-NPs by enzymatic reaction. Then, TPP is coupled by DCC and DMAP ester bond reaction to obtain PT-NPs. Finally, HA is encapsulated onto PT-NPs by EDC and NHS to obtain PT-HNPs.

[0047] The morphology of PD-NPs, PT-NPs, and PT-HNPs prepared in Examples 1-3 was observed using transmission electron microscopy. Figure 2 As shown, all three groups of nanoparticles are well-dispersed and nearly spherical. With the increase of hierarchical modification, the particle size gradually increases, indicating that stable nanostructures have been successfully formed.

[0048] The PD-NPs, PT-NPs, and PT-HNPs prepared in Examples 1-3 were further characterized, and the results are as follows: Figure 3 , 4 As shown in Figure 5, including particle size ( Figure 3 ), potential ( Figure 4 ) and infrared ( Figure 5 ).like Figure 3 The particle sizes of PD-NPs, PT-NPs, and PT-HNPs increase sequentially. Furthermore, their zeta potentials all change after tiered loading. Figure 4 Infrared results () Figure 5 This also indicates the formation of dual-targeted nanoparticles loaded with platycodon saponin D, and both PT-NPs and PT-HNPs exhibit infrared peaks for PD, TPP, and HA. The above characterization results confirm that the obtained product meets the basic characteristics and requirements of nanoparticles.

[0049] (2) Functional verification

[0050] To verify the application of PT-HNP nanoparticles in anti-inflammatory, mitochondrial targeting, and treatment of acute ulcerative colitis, the PD-NPs, PT-NPs, and PT-HNPs nanoparticles prepared in Examples 1-3 were used in RAW264.7 inflammatory cells and mice with DSS-induced acute ulcerative colitis. Firstly, nitric oxide (NO) level detection: In 24-well cell culture plates, 1 × 10⁻⁶ ppm was applied to each well. 5RAW264.7 cells were seeded at a density of 500 μL of cell culture solution per well. After seeding, the cells were incubated in a cell culture incubator for 24 h. Cell growth was then observed using an inverted microscope. Once cells showed good growth and adherence, the supernatant was discarded. Experimental groups were prepared with 1 μg / mL PD-NPs, PT-NPs, and PT-HNPs nanosolutions. Control and model groups (LPS) were also established. Except for the control group, all other groups received 1 μg / mL lipopolysaccharide (LPS). Each group had three replicates, with 500 μL of the relevant solution added to each well. After sample loading, the 24-well plates were returned to the cell culture incubator for another 24 h. The supernatant was then collected, and NO content was measured according to the kit instructions. The results are shown below. Figure 6 .

[0051] Secondly, mitochondrial membrane potential detection (JC-1): RAW264.7 cells were harvested when their confluence reached 80% for subsequent studies. The mitochondrial membrane potential changes induced by lipopolysaccharide (LPS) in each experimental group were detected using the mitochondrial membrane potential detection kit (JC-1). The ratio of JC-1 monomer (green fluorescence) to JC-1 polymer (red fluorescence) in each group was analyzed using an inverted fluorescence microscope to determine the changes in mitochondrial membrane potential. The detection results are as follows: Figure 7 Displayed. By Figure 7 The test results show that PT-HNPs significantly restored mitochondrial membrane potential levels.

[0052] Third, intracellular ROS scavenging capacity: A reactive oxygen species (ROS) detection kit was used to detect changes in intracellular ROS levels in lipopolysaccharide (LPS)-induced RAW264.7 cells under different experimental treatment groups. RAW264.7 cells (1.5 × 10⁻⁶) were... 5 Cells were cultured in 24-well plates (each cell well) for 24 hours, then fresh culture medium containing nanoparticles was added and the cells were cultured together for another 24 hours. Next, the cells were washed with phosphate-buffered saline (PBS) and stained with 10 μM DCFH-DA in the dark for 30 minutes. After staining, the DCFH-DA was removed, and the cells were washed with PBS and the fluorescence was observed using an inverted microscope. Figure 8 The test results show that ROS levels decreased significantly after PT-HNPs treatment.

[0053] Fourth, targeting ability observation: Cy5.5 was used as a fluorescent probe and loaded into the nanoparticle coating formulations. Cy5.5@PD, cy5.5@PD-NPs, cy5.5@PT-NPs, and cy5.5@PT-HNPs were prepared using the same Cy5.5 concentration and preparation process. Whole-body fluorescence images of mice were obtained at predetermined time points (3, 6, 9, 12 h) using the IVIS Lumina LT imaging system, as shown... Figure 9 As shown in (a). The results of semi-quantitative analysis using an in vivo imaging system are shown in [reference needed]. Figure 9 As shown in (b). From Figure 9 The test results in (a) and (b) show that PT-HNPs can target the colon and have a good accumulation capacity.

[0054] Fifth, in vivo pharmacodynamic evaluation

[0055] 1. Establishment and grouping of a mouse model of acute ulcerative colitis induced by sodium dextran sulfate (DSS).

[0056] C57BL / 6 mice were randomly divided into six groups: Control group, DSS group, DSS+5-ASA group (5-aminosalicylic acid, positive control group), DSS+PD group, DSS+PD-NPs group, DSS+PT-NPs group, and DSS+PT-HNPs group, with six mice in each group. After one week of acclimatization, an acute colitis mouse model was induced by free drinking of 3% (w / v) DSS for 6 days. The acute colitis mice were then randomly divided into six groups and administered PBS orally via gavage to the DSS+5-ASA group, DSS+PD group, DSS+PD-NPs group, DSS+PT-NPs group, and DSS+PT-HNPs group. Healthy mice not treated with DSS were given PBS orally as a normal control. During the DSS treatment period, the drugs in each group were administered orally daily from day 2 to day 7.

[0057] 2. Animal sample collection and experimental records

[0058] (1) The weight, blood in stool and characteristics of feces of the mice were recorded daily in the experiment.

[0059] (2) After anesthesia, the mice were euthanized, fixed in a supine position, and the abdomen was disinfected with alcohol. Using sterile surgical scissors, the abdominal skin and peritoneum were cut open, and the colon tissue was quickly removed at the pubic symphysis and cecum. The length of the entire colon was measured and images and samples were kept for subsequent experiments.

[0060] (3) After the experimental mice were euthanized, the colon, liver and kidney were collected and packaged. Some tissue pieces were fixed in a pre-prepared formaldehyde reagent for histopathological observation; other tissue pieces were quick-frozen in liquid nitrogen, transported to the laboratory with dry ice, and stored in a -80℃ refrigerator for subsequent physiological and biochemical index analysis.

[0061] 3. Disease Activity Index (DAI) score measurement

[0062] Observe and record the general condition of the mice daily (weight, bleeding and fecal characteristics), calculate the disease activity index (DAI) as (body weight index + fecal characteristics + bleeding) / 3, and the scoring details are shown in Table 1.

[0063]

[0064] The results are as follows Figure 10 As shown, compared with the Normal group, the mice in the DSS group showed a significant decrease in body weight and a significant increase in DAI score. Compared with the DSS group, the DSS+5-ASA group and the DSS+PT-HNPs group could effectively alleviate the weight loss caused by DSS and the increase in DAI score was smaller.

[0065] Measurement of colon length in mice: The colon is significantly shortened in ulcerative colitis (UC) patients, and the severity of the disease is positively correlated with the degree of colon shortening. In animal experiments with UC, the degree of inflammation in mice with ulcerative colitis can also be assessed by observing the length of the colon. Results are as follows... Figure 11 As shown in (a), (b) is a quantitative graph of the measurement results. The results showed that compared with the Normal group, the colon of mice in the DSS group was significantly shortened, and the treatment with 5-ASA and each drug administration group could inhibit the shortening of the colon length in mice, among which the intervention effect of the PT-HNPs group was the best.

[0066] Observation of histopathological sections of mouse colon: HE staining images of colon sections of DSS-induced UC mice in each drug administration group are shown below. Figure 12 As shown in the HE staining results, the colonic structure of the DSS group was severely damaged, mainly manifested as crypt destruction, gland disappearance, and extensive inflammatory cell infiltration. Treatment with PD, PD-NPs, PT-NPs, PT-HNPs, and 5-ASA significantly improved the pathological damage to the intestinal barrier structure induced by DSS. These results indicate that each treatment group could alleviate the colonic injury symptoms induced by DSS in UC mice, with PT-HNPs showing the best intervention effect.

[0067] Observation of mouse mitochondrial morphology: Transmission electron microscopy (TEM) was used to examine the ultrastructure of mitochondria in DSS-induced mouse colon tissue. The results are as follows: Figure 13 As shown, electron microscopy revealed morphological changes in the ultrastructure of mitochondria, characterized by an increase in the number and density of cristae per mitochondria in all drug-treated groups. This indicates that the nanogroups have a protective effect on mitochondrial health.

[0068] Hemolysis test: Blood was collected in tubes containing ethylenediaminetetraacetic acid (EDTA), and red blood cells were separated by centrifugation. The red blood cell suspension was incubated with different samples at 37°C for 2 hours. After centrifugation, the absorbance of the supernatant was measured at 540 nm. Figure 14 The results of the multi-level targeted nanoparticles provided by this invention on DSS-induced hemolysis in UC mice are presented in the experimental results. Figure 14 The test results showed that a hemolysis rate exceeding 5% was observed at a concentration of 25 ug / mL of free PD. However, PT-HNPs did not cause significant hemolysis even at a concentration of 400 ug / mL. In conclusion, PT-HNPs not only effectively treat UC but also prevent hemolysis associated with intravenous administration of free PD.

[0069] In the cell viability assay, RAW264.7 and Caco-2 cells were cultured at 1.5... 10 3 Cells were seeded at a density of 0.5 g / well in 96-well plates and cultured for 24 hours in DMEM medium containing 10% fetal bovine serum (FBS) (37°C, 5% CO2). Then, PD-NPs, PT-NPs, and PT-HNPs nanoparticle solutions at concentrations of 0.25, 0.5, 1, and 2 μg / mL were added, and incubation continued for another 24 hours. For MTT assay, 200 μL of MTT reagent was added to each well, and after incubation in the dark for 3 hours, the absorbance was measured at 590 nm using a microplate reader. The results are shown below. Figure 15 Displayed. By Figure 15 The test results show that PT-HNPs have good cell compatibility.

[0070] Those skilled in the art should understand that the above descriptions are merely several specific embodiments of the present invention, and not all embodiments. It should be noted that many modifications and improvements can be made by those skilled in the art, and all modifications or improvements not exceeding the scope of the claims should be considered within the protection scope of the present invention.

Claims

1. A method for preparing platycodon saponin D-loaded dual-targeting nanoparticles, characterized in that, Includes the following steps: Step S1: Take platycodon saponin D, horseradish peroxidase, polyethylene glycol 2000, and 1,4-dioxane, add them to phosphate buffer and stir to mix. During the stirring process, add 5% hydrogen peroxide solution in batches at intervals, continue stirring in the dark, dialyze the reaction solution and freeze dry to obtain platycodon saponin D-loaded nanoparticles, denoted as PD-NPs. Step S2: Take (5-carboxypentyl)triphenylphosphine bromide, 4-dimethylaminopyridine, and N,N'-dicyclohexylcarbodiimide, add them to dimethyl sulfoxide, and stir in the dark to activate the carboxyl group in TPP; then add the PD-NPs obtained in step S1, continue stirring in the dark, dialyze the reaction solution, and freeze-dry to obtain mitochondrial-targeting nanoparticles loaded with platycodon saponin D, denoted as PT-NPs; Step S3: Disperse hyaluronic acid in phosphate buffer, add 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, stir in the dark to activate the carboxyl group in HA; then add PT-NPs obtained in step S2, continue stirring in the dark, dialyze and freeze dry the reaction solution to obtain platycodon saponin D dual-targeting nanoparticles, denoted as PT-HNPs.

2. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, characterized in that, The mass-to-volume ratio of platycodon saponin D, horseradish peroxidase, PEG-2000, 1,4-dioxane, phosphate buffer, and 5% H2O2 in step S1 is 50 mg:10 mg:50 mg:50 mL:72.5 mL:12.5 mL.

3. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, characterized in that, The mass-to-volume ratio of (5-carboxypentyl)triphenylphosphine bromide to 4-dimethylaminopyridine, N,N'-dicyclohexylcarbodiimide, dimethyl sulfoxide, and PD-NPs in step S2 is 25 mg:7.5 mg:12.5 mg:25 mL:25 mg.

4. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, 2, or 3, characterized in that, The volume-to-mass ratio of hyaluronic acid, phosphate buffer, 3-(3-dimethylaminopropyl)-1-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and PT-NPs in step S3 is 50 mg:50 mL:25 mg:25 mg:50 mg.

5. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, characterized in that, In step S3, the HA is activated by stirring in the dark for 1-2 hours.

6. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, characterized in that, The reaction time for continued stirring in the dark as described in steps S1, S2, and S3 is 20-24 h, and the reaction temperature is room temperature.

7. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, characterized in that, In steps S1, S2, and S3, dialysis bags with a molecular weight cutoff of 500 Da are used. The dialysis bags are activated before use. The dialysate is pure water. Dialysis is performed in the dark for 48-52 hours. During dialysis, the water is changed at intervals of 2-4 hours, 4-6 hours, and 10-12 hours.

8. The method for preparing platycodon saponin D-loaded dual-targeting nanoparticles according to claim 1, characterized in that, The freeze-drying conditions described in steps S1, S2, and S3 are as follows: the sample is pre-cooled in a -80°C freezer for 10-12 hours, and then freeze-dried in a pre-cooled freeze dryer for 40-48 hours.

9. The method for preparing the platycodin D-loaded dual-targeting nanoparticles according to any one of claims 1-8, wherein the chemical structural formula of the platycodin D-loaded dual-targeting nanoparticles is as follows: 。 10. The use of the platycodin D-loaded dual-targeting nanoparticles as described in claim 9 in the preparation of a drug for treating acute ulcerative colitis.

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