Plantain herb acidic polysaccharide PLP-II preparation as well as preparation method and application thereof

Abstract

The invention relates to a plantain herb acidic polysaccharide PLP-II preparation as well as a preparation method and application thereof. The preparation comprises PLP-II and a lipidosome nano-carrier encapsulating the PLP-II, the lipidosome nano-carrier comprises lipidosome and a gellan derivative connected to the surface of the lipidosome, and the preparation has the effects of specifically targeting dendritic cells in a tumor microenvironment, reducing the off-target effect, enhancing the bioavailability of the PLP-II and improving the drug effect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical preparations, specifically relating to a plantain acidic polysaccharide PLP-II preparation, its preparation method, and its application. Background Technology

[0002] Plantago asiatica L. acidic polysaccharide (PLP-II) is a purified acidic polysaccharide isolated from the aqueous extract of the whole plant of Plantago asiatica, and is known to have a variety of beneficial pharmacological activities. PLP-II (Mw = 12726.85 Da) is mainly composed of HG-type domains, RG-I pectin domains, and a small amount of AGX-type domains. Its side chains are composed of type I and type II arabinogalactan (AG-I and AG-II), containing →3)-β-D-Galp-(1→ and →3,6)-β-D-Galp-(1→). The glycoside moiety is mainly composed of arabinose, rhamnose, galactose, and galacturonic acid. Preliminary screening experiments in our laboratory on the antitumor immunomodulatory activity of the crude extract of Plantago asiatica polysaccharides PLP and its purified products PLP-I and PLP-II showed that PLP-II had the best antitumor immunomodulatory properties. For details on the extraction and purification of PLP-II and its antitumor effects, please refer to the reference: Jiatong Zhang et al., Purified polysaccharides from Plantago asiatica L.: Preparation, characterization and immune-activating effects, International Journal of Biological Macromolecules, Volume 318, Part 1, July 2025, 144771. However, there are few reports on how to improve its bioavailability and reduce its off-target toxicity.

[0003] In view of this, the present invention is hereby proposed. Summary of the Invention

[0004] This invention provides a plantain acidic polysaccharide PLP-II formulation, a method for preparing the formulation, and the application of the formulation in the preparation of antitumor drugs. The invention aims to specifically target dendritic cells in the tumor microenvironment, reduce off-target effects, enhance the bioavailability of PLP-II, and improve efficacy.

[0005] In a first aspect, the present invention provides a psyllium acid polysaccharide PLP-II formulation, the formulation comprising PLP-II and a liposome nanocarrier encapsulating the PLP-II, the liposome nanocarrier comprising liposomes and a gelatin derivative attached to the surface of the liposomes.

[0006] Plantain acidic polysaccharide PLP-II is a high-molecular-weight polymer. In vivo, due to hepatic metabolism and the first-pass effect, the amount of the original drug entering systemic circulation is significantly reduced, greatly decreasing its bioavailability and ultimately weakening its clinical therapeutic effect. Furthermore, once the drug enters the body without any modification, it cannot actively target the tumor site or accumulate in tumor tissue. Instead, it is distributed to various organs and tissues via the bloodstream, damaging normal cells.

[0007] To address this technical problem, this invention encapsulates plantain acidic polysaccharide PLP-II using liposome nanocarriers. This protects PLP-II from external environmental influences, prevents degradation, and improves its bioavailability. The gelatin derivative on the surface of the liposome nanocarriers enables the liposomes to exhibit pH sensitivity, releasing PLP-II in acidic environments. The gelatin derivative acts as a target, targeting the Dectin-1 receptor on the surface of dendritic cells (DCs) to deliver the nanoliposome carriers directly to the tumor microenvironment, activating DCs and achieving PLP-II accumulation in tumor tissue and immune cell activation. Therefore, the liposome nanocarriers described in this invention improve the bioavailability of PLP-II, enabling it to accurately reach the tumor site and continuously act on immune cells in the tumor microenvironment, significantly enhancing anti-tumor efficacy and reducing off-target toxicity.

[0008] In some specific embodiments, the liposome nanocarrier has a core-shell structure.

[0009] In some specific embodiments, the liposomes are composed of phospholipids (preferably soybean lecithin) and cholesterol.

[0010] In some specific embodiments, the gelatin derivative includes MGlu-Curd-C 10 .

[0011] In some specific embodiments, the liposome nanocarriers have a particle size of 150–160 nm. Liposome nanoparticles with suitable particle size exhibit prolonged blood circulation, increase the likelihood of their effective payload interacting with tumor tissue, and enhance tumor penetration, thereby improving bioavailability and antitumor efficacy.

[0012] In a second aspect, the present invention also provides a method for preparing the formulation described in the first aspect of the present invention, the method comprising the following steps: (1) encapsulating PLP-II in liposomes; (2) attaching a gelatin derivative to the surface of the liposomes to form a liposome nanocarrier encapsulating PLP-II.

[0013] In some specific embodiments, step (1) includes: forming liposomes encapsulating PLP-II by dispersing PLP-II with components for forming liposomes using a thin-film dispersion method.

[0014] In some specific embodiments, step (2) includes: attaching the gelatin derivative to the surface of the liposome by co-incubation.

[0015] In a third aspect, this aspect also provides the use of the formulation described in the first aspect of the invention in the preparation of antitumor drugs.

[0016] In some specific embodiments, the tumor is a breast tumor. Attached Figure Description

[0017] Figure 1 Electron micrograph of MGlu-Curd-Lips.

[0018] Figure 2 This is a particle size distribution diagram of MGlu-Curd-Lips.

[0019] Figure 3 Electron micrograph of PLP-II / Liposomes.

[0020] Figure 4 This is a particle size distribution diagram of PLP-II / Liposomes.

[0021] Figure 5 This is a schematic diagram of the preparation of PLP-II / MGlu-Curd-Lips.

[0022] Figure 6 Electron micrograph of PLP-II / MGlu-Curd-Lips.

[0023] Figure 7 PLP-II / MGlu-Curd-Lips particle size distribution diagram.

[0024] Figure 8 The graph shows the in vitro acid-responsive release curve of PLP-II / MGlu-Curd-Lip.

[0025] Figure 9 This is a flow cytometry result of MGlu-Curd-Lip targeting dendritic cells (Dectin-1) in vitro.

[0026] Figure 10 The average fluorescence intensity of Dectin-1 in dendritic cells targeted in vitro by MGlu-Curd-Lip is shown.

[0027] Figure 11 Non-invasive in vivo optical images of mice with breast tumors 24 h after injection of PBS, free DiR, Liposomes, and MGlu-Curd-Lip.

[0028] Figure 12 Optical images of tumors outside the body.

[0029] Figure 13 Images of ex vivo tumors from animal experiments using PLP-II / MGlu-Curd-Lip anti-4T1 breast tumors.

[0030] Figure 14 The quality of ex vivo tumors from each group in the animal experiment of PLP-II / MGlu-Curd-Lip against 4T1 breast tumors.

[0031] Figure 15 The daily tumor volume of each group in the animal experiment of PLP-II / MGlu-Curd-Lip against 4T1 breast tumors. Detailed Implementation

[0032] To better understand the present invention, specific embodiments will be given to further illustrate the invention. However, it should be understood that the described embodiments are exemplary embodiments, and the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0033] Example 1

[0034] 120 mg of soybean lecithin and 20 mg of cholesterol were weighed and dissolved in 5 mL of anhydrous ethanol to form a mixture. The mixture was homogenized in an ice-water bath for 10 min using an ultrasonic cleaner until no obvious solids were observed. The organic solvent was removed by rotary evaporation under reduced pressure in a 100 mL flask at 45 °C to form a colloidal film. 10 mL of PBS buffer was added to the flask, and the mixture was rotated at 50 rpm for 10 min at 45 °C under normal pressure, and then allowed to stand at room temperature for 20 min to allow the film to swell and hydrate. The resulting liposome suspension was homogenized in an ice-water bath for 10 min (5 s on, 5 s off) using a 30% ultrasonic probe to form small single-chamber liposomes. The liposome suspension was placed in cryovials and frozen in liquid nitrogen for 6 h. After thawing, the freeze-thaw cycle was repeated four times. The liposome suspension was sterilized by passing it through 0.45 μm and 0.22 μm filters, and then stored in centrifuge tubes at 4 °C to obtain the drug delivery carriers, Liposomes.

[0035] Example 2

[0036] Take the flask from Example 1, which was used to remove the organic solvent by rotary evaporation under reduced pressure to form a colloidal film, and weigh the synthesized 3-methylglutaric acid gel polysaccharide decylamine (MGlu-Curd-C). 10 20 mg was dissolved in 10 mL of phosphate buffered saline (PBS, pH 7.4), vortexed to mix, and added to a flask. A rotary evaporator was installed, and the flask containing the liposome suspension was rotated at 50 rpm for 60 min in a 45°C water bath under normal pressure. The mixture was then allowed to stand at room temperature for 20 min to allow the membrane to swell and hydrate. MGlu-Curd-C was then incubated using a co-incubation method. 10 The liposomes were attached to the lipid membrane surface. The resulting liposome suspension was homogenized in an ice-water bath for 10 min (5 s on, 5 s off) using a 30% ultrasonic probe to form small single-compartment liposomes. The liposome suspension was placed in cryovials and stored in liquid nitrogen for 6 h, then thawed. This freeze-thaw cycle was repeated four times. After sterilization by passing the liposome suspension through 0.45 μm and 0.22 μm filters, it was stored in centrifuge tubes at 4°C to obtain the drug delivery carrier MGlu-Curd-Lips.

[0037] MGlu-Curd-C 10For details on the preparation method, please refer to the literature: Eiji Yuba et al., Bioactive polysaccharide-based pH-sensitive polymers for cytoplasmic delivery of antigen and activation of antigen-specific immunity, Biomaterials, Volume 120, March 2017, Pages 32-45.

[0038] Example 3

[0039] The MGlu-Curd-Lips prepared in Example 2 were observed under a low-power electron microscope. The drug delivery carrier was spherical and horseshoe-shaped (e.g., ...). Figure 1 (As shown).

[0040] Example 4

[0041] The particle size and zeta potential of the MGlu-Curd-Lips prepared in Example 2 were measured using dynamic light scattering (DLS) technology: Intensity mean data were selected as the reference particle size. The MGlu-Curd-Lips prepared in Example 2 were placed in a PBS environment, and their average particle size was approximately 152.7 nm (e.g., ...). Figure 2 (As shown), corresponding to TEM data; PDI is 0.152, and the Zeta value for measuring the particle surface charge is -10.5 mV.

[0042] Example 5

[0043] The drug delivery system PLP-II / Liposomes was prepared using a thin-film dispersion method. PLP-II was dissolved in phosphate-buffered saline (PBS, pH 7.4). Using the flask from Example 1 (which under reduced pressure was used to remove organic solvents and form a colloidal film), 10 mL of PBS buffer containing the drug (1 mg / mL) was added to the flask. The flask was rotated in a water bath at normal pressure for 10 min, then allowed to stand at room temperature for 20 min to allow the film to swell and hydrate. The resulting liposome suspension was homogenized in an ice-water bath for 10 min (5 s on, 5 s off) using an ultrasonic probe to form small single-compartment liposomes. These liposomes were then frozen in liquid nitrogen for 6 h, thawed, and subjected to repeated freeze-thaw cycles four times to improve encapsulation efficiency. The liposome suspension was sterilized by passing it through 0.45 μm and 0.22 μm filters, and then stored in centrifuge tubes at 4°C to obtain the final drug delivery system PLP-II / Liposomes.

[0044] Example 6

[0045] The PLP-II / Liposomes prepared in Example 5 were observed under a low-power electron microscope. The drug delivery carrier was spherical and horseshoe-shaped (e.g., ...). Figure 3 (As shown).

[0046] Example 7

[0047] The particle size and zeta potential of the PLP-II / Liposomes prepared in Example 5 were measured using dynamic light scattering (DLS) technology: Intensity mean data were selected as the reference particle size. The PLP-II / Liposomes prepared in Example 5 were placed in a PBS environment, and their average particle size was approximately 188.8 nm (e.g., ...). Figure 4 (As shown), corresponding to TEM data; PDI is 0.262, and the Zeta value for measuring particle surface charge is -7.2 mV.

[0048] Example 8

[0049] Preparation of drug delivery system PLP-II / MGlu-Curd-Lips by thin film dispersion method (illustration shown in figure) Figure 5 (As shown): PLP-II was dissolved in phosphate buffer (PBS, pH 7.4). 5 mL of PBS buffer containing the drug (2 mg / mL) was added to the flask containing the colloidal film from Example 1. The flask was rotated in a water bath at normal pressure for 10 min, then allowed to stand at room temperature for 20 min. 5 mL of PBS buffer containing MGlu-Curd-C was then added. 10 Add 4 mg / mL PBS buffer to the flask, rotate at 50 rpm for 60 min in a water bath at normal pressure, and let stand at room temperature for 20 min to allow the membrane to swell and hydrate. Then, incubate the MGlu-Curd-C membrane using a co-incubation method. 10 The liposomes were attached to the lipid membrane surface. The resulting liposome suspension was homogenized in an ice-water bath for 10 min (5 s on, 5 s off) using an ultrasonic probe to form small single-compartment liposomes. These were then frozen in liquid nitrogen for 6 h, thawed, and subjected to repeated freeze-thaw cycles four times to improve encapsulation efficiency. The liposome suspension was sterilized by passing it through 0.45 μm and 0.22 μm filters, and then stored in centrifuge tubes at 4 °C to obtain the drug delivery system PLP-II / MGlu-Curd-Lips.

[0050] Example 9

[0051] The encapsulation efficiency of PLP-II in the drug delivery system prepared in Example 5, determined by the phenol-sulfuric acid method, was 79.66%. Low-magnification electron microscopy revealed that the drug delivery system was spherical (e.g., ...). Figure 6 (As shown).

[0052] Example 10

[0053] The PLP-II / MGlu-Curd-Lips prepared in Example 5 were subjected to DLS analysis, and their average diameter was measured to be 156.2 nm (e.g., Figure 7 (As shown).

[0054] Example 11

[0055] In vitro drug release was analyzed using dialysis, with PBS (pH 5.5 and pH 7.4) selected as the release medium. Liposomes were prepared under optimal conditions. 4 mL of the PLP-II / MGlu-Curd-Lips sample was added to a dialysis bag (8kDa~14kD), and the bags were sealed at both ends with clamps. The bags were then placed in 40 mL of release medium at different pH values ​​to simulate a body fluid environment. At regular intervals (1, 2, 4, 8, 24, 48, 72 h), 2 mL of release medium was removed each time, and an equal amount of release medium was added to an Erlenmeyer flask. The polysaccharide content in the release medium was determined using the phenol-sulfuric acid method, and cumulative drug release curves were plotted. The results showed that PLP-II / MGlu-Curd-Lips exhibited acid-responsive drug release (e.g., ... Figure 8 (As shown).

[0056] Example 12

[0057] To evaluate the potential of the Dectin-1 receptor in mediating the targeting of MGlu-Curd-Lips, mouse bone marrow-derived dendritic cells (DCs) were pretreated with different concentrations of Curdlan (1, 2, and 4 mg / mL). The DCs were then incubated at 37°C with MGlu-Curd-Lips loaded with Cou-6 (0.6 mol%) for 1 h. Cells were collected, washed three times with PBS containing 1% FBS, labeled with PE-antimouse-CD11c antibody, washed with cold PBS, and analyzed by flow cytometry (BD Fortessa, BD Biosciences, USA). Figure 9-10 (As shown). The results showed that, compared with the control group without inhibitors, gelatin at concentrations of 2 and 4 mg / mL significantly reduced the cell affinity of Cou-6-loaded MGlu-Curd-Lips (P < 0.001), confirming the Dectin-1 receptor-mediated targeting of DCs by MGlu-Curd-Lips.

[0058] Example 13

[0059] The concentration of the mouse mammary tumor cell line 4T1 cell suspension was adjusted to 1×10⁻⁶. 7 Cells / mL were subcutaneously injected into the right abdominal mammary region of 12 female Balb / c mice, with each mouse receiving 100 μL (containing 1×10⁶ cells / mL). 6After establishing the tumor-bearing mouse model, the mice were weighed and the tumor size was measured. Twelve mice were randomly divided into four groups (control group: 150 μL PBS, free DiR group: 150 μL DiR, Liposomes group: 150 μL DiR / Liposomes, MGlu-Curd-Lips group: 150 μL DiR / MGlu-Curd-Lips, where the dosage of DiR was 2 mg / kg), with three mice in each group. The mice were administered the drugs via tail vein injection. After administration, the mice were scanned using the IVIS® spectral imaging system at 24 h, and the tumors were removed and their fluorescence intensity was measured. The results showed that MGlu-Curd-Lips had superior aggregation and retention capabilities at the tumor site (e.g., ...). Figure 11-12 (As shown).

[0060] Example 14

[0061] The concentration of the mouse mammary tumor cell line 4T1 cell suspension was adjusted to 1×10⁻⁶. 7 Cells / mL were subcutaneously injected into the right abdominal mammary region of 36 female Balb / c mice, with each mouse receiving 100 μL (containing 1×10⁶ cells / mL). 6 After establishing the tumor-bearing mouse model, the mice were weighed and the tumor size was measured. The tumor-bearing mice were divided into 6 groups (G1-PBS control group; G2-Liposomes group; G3-PLP-II group; G4-PLP-II / Liposomes group; G5-PLP-II / MGlu-Curd-Lips peritumoral injection group; and G6-PLP-II / MGlu-Curd-Lips intravenous injection group), with six mice in each group. The G3-PLP-II group received PLP-II (10 mg / kg) via peritumoral administration. The G1-PBS control group, G2-Liposomes group, G4-PLP-II / Liposomes group, and G6-PLP-II / MGlu-Curd-Lips intravenous injection group received the drug via intravenous injection at a dose of 150 μL. The G5-MGlu-Curd-Lips peritumoral injection group received 150 μL. The dosage of PLP-II in the G4-PLP-II / Liposomes group, the G5-PLP-II / MGlu-Curd-Lips peritumoral injection group, and the G6-PLP-II / MGlu-Curd-Lips intravenous injection group was 10 mg / kg. The administration cycle was injections on days 5, 7, 9, 11, and 13 after tumor cell inoculation. Mice were euthanized on day 15, and tumor tissue was harvested for analysis. The results are as follows: Figure 13-15As shown in the figure. The results indicated that, compared with the PBS control group, the peritumoral injection group and the intravenous injection group of PLP-II / MGlu-Curd-Lips significantly reduced tumor mass and volume.

[0062] The above descriptions are merely specific embodiments of the present invention. These examples are not intended to limit the present invention to the precise form disclosed. Various choices and improvements made by those skilled in the art based on the present invention are all within the protection scope of the present invention.

Claims

1. A plantain acidic polysaccharide PLP-II formulation, characterized in that, The formulation includes PLP-II and a liposome nanocarrier encapsulating the PLP-II, wherein the liposome nanocarrier includes liposomes and a gelatin derivative attached to the surface of the liposomes.

2. The formulation according to claim 1, characterized in that, The liposome nanocarrier has a core-shell structure.

3. The formulation according to claim 1 or 2, characterized in that, The components of the liposomes include phospholipids and cholesterol.

4. The formulation according to claim 1 or 2, characterized in that, The gelatin derivative includes MGlu-Curd-C 10 .

5. The formulation according to claim 1 or 2, characterized in that, The liposome nanocarrier has a particle size of 150~160nm.

6. A method for preparing the plantain acidic polysaccharide PLP-II formulation according to any one of claims 1-5, characterized in that, The method includes the following steps: (1) encapsulating PLP-II in liposomes; (2) attaching a gelatin derivative to the surface of the liposomes to form a liposome nanocarrier encapsulating PLP-II.

7. The method according to claim 6, characterized in that, Step (1) includes: forming liposomes encapsulating PLP-II by dispersing PLP-II with components for forming liposomes using a thin film dispersion method.

8. The method according to claim 6, characterized in that, Step (2) includes: attaching the gelatin derivative to the surface of the liposome by co-incubation.

9. The use of the plantain acidic polysaccharide PLP-II preparation according to any one of claims 1-5 in the preparation of antitumor drugs.

10. The application according to claim 9, characterized in that, The tumor is a breast tumor.

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