Centipeda minima outer vesicle nose drop as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to centipeda minima extracellular vesicle nose drops and a preparation method and application thereof.The centipeda minima extracellular vesicle nose drops comprise active ingredients, plant extracellular vesicles (Cm-EVs) derived from centipeda minima, the concentration of the plant extracellular vesicles (Cm-EVs) is 1 * 10 < 10 >-5 * 10 < 11 > particles / ml; the key active substances are as follows: the content of the Cm-EVs naturally encapsulated sesquiterpene lactone compound centipederin is more than or equal to 50 micrograms per milliliter (detected by HPLC-MS); the pharmaceutical auxiliary materials comprise 0.05 to 0.2 percent (w / v) of sodium hyaluronate, 0.8 to 1.0 percent (w / v) of NaCl and 0.005 to 0.02 percent (v / v) of polysorbate 80; the method has the beneficial effects that the targeted enrichment capability is realized: mucous membrane adhesion is enhanced through surface pectin protein, and the nasal cavity residence time is prolonged to 6 hours (the free medicine is less than 1 hour); the nano-scale particle size (80-200nm) can efficiently penetrate through the mucous layer, and the nasal mucosa drug concentration reaches 20 times of the plasma concentration (rat pharmacokinetic data); the long-acting slow-release property is as follows: the active ingredients are protected by the lipid bilayer, the cumulative release rate of the centipederin within 72 hours reaches 82%, and the free medicine can be completely released within 24 hours.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a nasal drop solution containing *Centipeda minima* vesicles, its preparation method, and its application. Background Technology

[0002] Rhinitis is a prevalent disease worldwide (prevalence rate 10%-30%), and the prevalence rate of allergic rhinitis in my country has reached 17.6%. Current treatments have significant limitations: antihistamines (such as loratadine) are ineffective for nasal congestion and have a resistance rate >30%; nasal corticosteroids (such as budesonide) can cause mucosal atrophy with long-term use (incidence rate 12%); decongestants (pseudoephedrine) have a rebound congestion risk >40%; immunotherapy requires a treatment course of 3-5 years and is very costly.

[0003] The core problem is that the first-pass effect of systemic administration results in a bioavailability of <20%, making it impossible to target and accumulate in the nasal mucosa.

[0004] Centipederin, the core anti-inflammatory component of Centipeda minima, has the following effects: inhibiting mast cell degranulation (reducing histamine release by 50% in the RBL-2H3 cell model); downregulating the NF-κB pathway (downregulating IL-4 / IL-5 expression by 70%); and enhancing the epithelial barrier (increasing occludin protein expression by 2 times). However, its direct application is limited: the hydrophobicity of centipederin results in a nasal retention time of less than 1 hour, and it is easily deactivated by enzymes.

[0005] Therefore, we propose a nasal drop solution containing *Centipeda minima* vesicles, its preparation method, and its application to solve the above problems. Summary of the Invention

[0006] In view of the problems existing in the prior art, this invention discloses a Centipedea herb exovesicular nasal drop, its preparation method, and its application. This formulation utilizes a nanoscale carrier (80-200 nm) to naturally load the anti-inflammatory component centipedederin and immunomodulatory miRNAs, achieving targeted delivery to the nasal mucosa. It is used to treat allergic rhinitis (AR), hormone-resistant chronic rhinitis, and other diseases, solving the problems of low bioavailability, systemic toxicity, and mucosal damage associated with traditional drugs. The technical solution adopted includes: Active ingredient: plant extracellular vesicles (Cm-EVs) derived from Centipeda minima, at a concentration of 1×10⁻⁶. 10 –5×10 11 Particles / ml; Key active ingredient: The content of the naturally encapsulated sesquiterpene lactone compound centipederin in the Cm-EVs is ≥50 μg / mL (detected by HPLC-MS). Pharmaceutical excipients: 0.05–0.2% (w / v) sodium hyaluronate, 0.8–1.0% (w / v) NaCl, 0.005–0.02% (v / v) polysorbate 80.

[0007] As a preferred embodiment of the present invention, the physical and biological properties of the Cm-EVs include: Particle size range 80–200 nm (dynamic light scattering detection); Surface features: Transmission electron microscopy (TEM) confirmed an intact lipid bilayer structure carrying pectin proteins; As a preferred embodiment of the present invention, the Cm-EVs further comprise at least one of the following active ingredients: Arnicolide D (a sesquiterpene lactone), quercetin-3-O-glucoside (a flavonoid), and miR-159a (an immunomodulatory miRNA).

[0008] As a preferred embodiment of the present invention, a method for preparing a Centipeda minima nasal droplet is characterized by comprising the following steps: a) Extraction of external vesicles from *Centipeda minima* by polyethylene glycol precipitation: ①Pretreatment: Take fresh *Centipeda minima* tissue, wash with pre-cooled phosphate-buffered saline (PBS), grind into a homogenate, add PBS to suspend, and obtain crude extract; ② Centrifugation to remove impurities: Centrifuge the crude extract at 3,000×g and 4℃ for 15 minutes, and collect the supernatant; then centrifuge at 10,000×g and 4℃ for 20 minutes, and collect the supernatant. ③ Polyethylene glycol precipitation: Add polyethylene glycol 6000 (final concentration 10% w / v) and NaCl (final concentration 0.15 M) to the supernatant obtained in step b, mix gently at 4°C and let stand for 3 hours. ④ Centrifugal purification: Centrifuge the mixture obtained in step c at 12,000×g and 4℃ for 30 minutes, discard the supernatant, resuspend the precipitate in PBS, and centrifuge again at 12,000×g and 4℃ for 30 minutes. Finally, resuspend the precipitate in an appropriate amount of PBS to obtain the exovesicles from Centipeda minima. ⑤ Purity verification: Particle size analysis (NTA): Main peak 80-150nm, PDI=0.18; b. Preparation of excipient solution: ① Dissolve sodium hyaluronate: Take 80 mL of pre-cooled PBS (4℃), slowly add 0.1 g of sodium hyaluronate, and stir magnetically (500 rpm, 2 h) until completely dissolved; let stand at 4℃ overnight to defoam (to avoid clogging of subsequent filters); if the dissolution is incomplete, it can be assisted by ultrasound (40 kHz, 30 s pulse). ② Add other excipients: Add 0.9g NaCl, 10μL polysorbate 80, and 0.005g benzalkonium chloride sequentially. Stir until completely dissolved (about 30 minutes); bring PBS to a final volume of 100mL and adjust the pH to 6.8 (fine-tune with 0.1M HCl / NaOH); ③ Filtration and sterilization: Filter sequentially through 0.45μm and 0.22μm filter membranes (aseptic operation required); sample and test pH (6.5-7.0) and osmotic pressure (290±10 mOsm / kg); c. Final preparation of Centipeda minima nasal drops: ①External vesicle addition: Add purified external vesicle suspension (concentration ≥ 1×10⁻⁶) 10 Add the excipient solution at a ratio of 1:10 (granules / ml); ② Filling and storage: Fill into brown nose dropper bottles (5mL / bottle) under nitrogen purging conditions; store at 4℃ (3 months), freeze at -80℃ (12 months); As a preferred embodiment of the present invention, the pH in the polyethylene glycol precipitation step is strictly controlled at 4.75 ± 0.02 (pH meter calibrated). The particle size distribution of the final product was confirmed by NanoSight NTA analysis, and the endotoxin content was <0.1 EU / mL (Limulus amebocyte lysate (LAL) method).

[0009] As a preferred technical solution of the present invention, it includes: ①The Centipeda minima nasal drops according to any one of claims 1-3; ② Synergistic drug: mometasone furoate, with a concentration of 10±2 micrograms / mL; ③ The mass ratio of Cm-EVs to mometasone furoate in the composition is 100:1.

[0010] As a preferred embodiment of the present invention, the nasal drops are used as a medication for treating the following diseases: ①IgE-mediated allergic rhinitis (serum IgE > 100 IU / mL); ② Hormone-resistant chronic rhinitis (requiring treatment for ≥14 days) (defined as ineffective use of glucocorticoids for >4 weeks and nasal congestion score ≥ 5 / 10); ③ Non-allergic rhinitis with damage to the nasal mucosal epithelial barrier (downregulation of tight junction protein ZO-1 expression ≥50%).

[0011] As a preferred embodiment of the present invention, the administration regimen satisfies the following: ① Single nasal administration volume: 10±2μL, containing 1×10 Cm-EVs 10 Particles; ② Frequency of administration per day: 1-2 times; ③ Treatment course: 7-28 days (hormone-resistant type requires ≥ 14 days).

[0012] The beneficial effects of this invention are: Targeted enrichment capability: Enhanced mucosal adhesion through surface pectin protein, extending nasal retention time to 6 hours (compared to less than 1 hour for free drug); Nanoscale particles (80-200nm) efficiently penetrate the mucus layer, achieving 20 times the plasma concentration in the nasal mucosa (rat pharmacokinetic data); Long-lasting sustained-release properties: Lipid bilayer protects the active ingredient, with a cumulative release rate of 82% for centipederin within 72 hours, while free drug is completely released within 24 hours; Synergistic effects of core active ingredients: Centipederin inhibits mast cell degranulation and reduces histamine release by 50% (verified in vitro using the RBL-2H3 cell model); miR-159a targets and inhibits the TLR4 / MyD88 pathway, reducing NF-κB activity by 70% (verified using dual-luciferase reporter gene assay); quercetin-3-O-glucoside upregulates the expression of tight junction protein ZO-1 by 3-fold (immunofluorescence detection), repairing the nasal mucosal barrier; Multicenter trial validation (n=300): 7-day nasal congestion relief rate was 85%, superior to budesonide (68%) and azelastine hydrochloride (45%); serum IL-13 level decreased by 76%, significantly higher than traditional drugs (budesonide group 52%); effective rate for hormone-resistant chronic rhinitis was 78% (budesonide group only 35%); onset of action: symptom relief time was shortened to within 30 minutes (traditional drugs require 1-2 hours); Zero mucosal damage: No epithelial cell apoptosis was observed after 28 days of continuous administration (TUNEL test negative); No systemic toxicity: Plasma centipederin concentration remained below 0.5 μg / mL (LC-MS / MS detection); Long-term stability: With the addition of 5% trehalose as a cryoprotectant, the active ingredient retention rate was >95% after 3 months of storage at 4°C. Attached Figure Description

[0013] Figure 1 This is a particle size distribution diagram of the NTA in the outer vesicles of *Gnaphalium affine* of the present invention.

[0014] Figure 2 This is a TEM image of the outer vesicles of the present invention. Detailed Implementation

[0015] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0016] This invention discloses a Centipeda minima nasal drop solution, its preparation method, and its application. The technical solution adopted includes: Active ingredient: plant extracellular vesicles (Cm-EVs) derived from Centipeda minima, at a concentration of 1×10⁻⁶. 10 –5×10 11 Particles / ml; Key active ingredient: The content of the naturally encapsulated sesquiterpene lactone compound centipederin in the Cm-EVs is ≥50 μg / mL (detected by HPLC-MS). Pharmaceutical excipients: 0.05–0.2% (w / v) sodium hyaluronate, 0.8–1.0% (w / v) NaCl, 0.005–0.02% (v / v) polysorbate 80.

[0017] As a preferred embodiment of the present invention, the physical and biological properties of the Cm-EVs include: Particle size range 80–200 nm (dynamic light scattering detection); Surface features: Transmission electron microscopy (TEM) confirmed an intact lipid bilayer structure carrying pectin proteins; Marker proteins: positive for tumor susceptibility gene 101 protein (TSG101) and heat shock protein 70 (HSP70) (Western blot verification).

[0018] As a preferred embodiment of the present invention, the Cm-EVs further comprise at least one of the following active ingredients: Arnicolide D (a sesquiterpene lactone), quercetin-3-O-glucoside (a flavonoid), and miR-159a (an immunomodulatory miRNA).

[0019] As a preferred embodiment of the present invention, a method for preparing a Centipeda minima nasal droplet is characterized by comprising the following steps: a) Extraction of external vesicles from *Centipeda minima* by polyethylene glycol precipitation: ①Pretreatment: Take fresh *Centipeda minima* tissue, wash with pre-cooled phosphate-buffered saline (PBS), grind into a homogenate, add PBS to suspend, and obtain crude extract; ② Centrifugation to remove impurities: Centrifuge the crude extract at 3,000×g and 4℃ for 15 minutes, and collect the supernatant; then centrifuge at 10,000×g and 4℃ for 20 minutes, and collect the supernatant. ③ Polyethylene glycol precipitation: Add polyethylene glycol 6000 (final concentration 10% w / v) and NaCl (final concentration 0.15 M) to the supernatant obtained in step b, mix gently at 4°C and let stand for 3 hours. ④ Centrifugal purification: Centrifuge the mixture obtained in step c at 12,000×g and 4℃ for 30 minutes, discard the supernatant, resuspend the precipitate in PBS, and centrifuge again at 12,000×g and 4℃ for 30 minutes. Finally, resuspend the precipitate in an appropriate amount of PBS to obtain the exovesicles from Centipeda minima. ⑤ Purity verification: Particle size analysis (NTA): Main peak 80-150nm, PDI=0.18; b. Preparation of excipient solution: ① Dissolve sodium hyaluronate: Take 80 mL of pre-cooled PBS (4℃), slowly add 0.1 g of sodium hyaluronate, and stir magnetically (500 rpm, 2 h) until completely dissolved; let stand at 4℃ overnight to defoam (to avoid clogging of subsequent filters); if the dissolution is incomplete, it can be assisted by ultrasound (40 kHz, 30 s pulse). ② Add other excipients: Add 0.9g NaCl, 10μL polysorbate 80, and 0.005g benzalkonium chloride sequentially. Stir until completely dissolved (about 30 minutes); bring PBS to a final volume of 100mL and adjust the pH to 6.8 (fine-tune with 0.1M HCl / NaOH); ③ Filtration and sterilization: Filter sequentially through 0.45μm and 0.22μm filter membranes (aseptic operation required); sample and test pH (6.5-7.0) and osmotic pressure (290±10 mOsm / kg).

[0020] c. Final preparation of Centipeda minima nasal drops: ①External vesicle addition: Add purified external vesicle suspension (concentration ≥ 1×10⁻⁶) 10 Add the excipient solution at a ratio of 1:10 (granules / ml); ② Filling and storage: Fill into brown nose dropper bottles (5mL / bottle) under nitrogen purging conditions; store at 4℃ (3 months), freeze at -80℃ (12 months).

[0021] As a preferred embodiment of the present invention, the pH in the polyethylene glycol precipitation step is strictly controlled at 4.75 ± 0.02 (pH meter calibrated). The particle size distribution of the final product was confirmed by NanoSight NTA analysis, and the endotoxin content was <0.1 EU / mL (Limulus amebocyte lysate (LAL) method).

[0022] As a preferred technical solution of the present invention, it includes: ①The Centipeda minima nasal drops according to any one of claims 1-3; ② Synergistic drug: mometasone furoate, with a concentration of 10±2 micrograms / mL; ③ The mass ratio of Cm-EVs to mometasone furoate in the composition is 100:1.

[0023] As a preferred embodiment of the present invention, the nasal drops are used as a medication for treating the following diseases: ①IgE-mediated allergic rhinitis (serum IgE > 100 IU / mL); ② Hormone-resistant chronic rhinitis (requiring treatment for ≥14 days) (defined as ineffective use of glucocorticoids for >4 weeks and nasal congestion score ≥ 5 / 10); ③ Non-allergic rhinitis with damage to the nasal mucosal epithelial barrier (downregulation of tight junction protein ZO-1 expression ≥50%).

[0024] As a preferred embodiment of the present invention, the administration regimen satisfies the following: ① Single nasal administration volume: 10±2μL, containing 1×10 Cm-EVs 10 Particles; ② Frequency of administration per day: 1-2 times; ③ Treatment course: 7-28 days (hormone-resistant type requires ≥ 14 days).

[0025] Clinical efficacy observation Case 1 (Seasonal Allergic Rhinitis): The patient was a 32-year-old female with a 5-year history of pollen allergy. Her nasal itching and sneezing scores were 8 / 10. She was treated with 10 μL of nasal drops (containing 1×10^9 exovesicles) in each nasal cavity daily for 14 days. The results showed that the nasal itching was relieved within 24 hours, and the sneezing frequency decreased by 75% within 7 days. Her serum IgE decreased from 350 IU / mL to 120 IU / mL (ELISA test).

[0026] Case 2 (steroid-resistant chronic rhinitis): The patient was a 45-year-old male who had been using glucocorticoids for a long time without effect and had a nasal congestion score of 7 / 10. He used nasal drops twice a day in combination with low-dose mometasone furoate (synergistic group). After 14 days, his nasal ventilation improved by 60% (nasal resistance meter test). Histological biopsy showed that IL-4 expression was downregulated by 70% (immunohistochemistry).

[0027] Comparative Examples (with Traditional Drugs)

[0028] Components not described in detail in this article are existing technologies.

[0029] While the specific embodiments of the present invention have been described in detail above, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention, and modifications or variations without creative effort are still within the protection scope of the present invention.

Claims

1. A type of Centipeda minima vesicle nasal drops, characterized in that, include: Active ingredient: extracellular vesicles (Cm-EVs) derived from Centipeda minima, at a concentration of 1×10⁻⁶. 10 –5×10 11 Particles / ml; Key active ingredient: The content of the naturally encapsulated sesquiterpene lactone compound centipederin in the Cm-EVs is ≥50 μg / mL (detected by HPLC-MS); Pharmaceutical excipients: 0.05–0.2% (w / v) sodium hyaluronate, 0.8–1.0% (w / v) NaCl, 0.005–0.02% (v / v) polysorbate 80.

2. The Centipeda minima nasal drops according to claim 1, characterized in that, The physical and biological properties of the Cm-EVs include: Particle size range 80–200 nm (dynamic light scattering detection); Surface features: Transmission electron microscopy (TEM) confirmed an intact lipid bilayer structure carrying pectin.

3. The Centipeda minima nasal drops according to claim 1 or 2, characterized in that, The Cm-EVs also contain at least one of the following active ingredients: Arnicolide D (a sesquiterpene lactone), quercetin-3-O-glucoside (a flavonoid), and miR-159a (an immunomodulatory miRNA).

4. The method for preparing the Centipeda minima nasal drops according to any one of claims 1-3, characterized in that, Includes the following steps: a) Extraction of external vesicles from *Centipeda minima* by polyethylene glycol precipitation: ①Pretreatment: Take fresh *Centipeda minima* tissue, wash with pre-cooled phosphate-buffered saline (PBS), grind into a homogenate, add PBS to suspend, and obtain crude extract; ② Centrifugation to remove impurities: Centrifuge the crude extract at 3,000×g and 4℃ for 15 minutes, and collect the supernatant; then centrifuge at 10,000×g and 4℃ for 20 minutes, and collect the supernatant. ③ Polyethylene glycol precipitation: Add polyethylene glycol 6000 (final concentration 10% w / v) and NaCl (final concentration 0.15 M) to the supernatant obtained in step b, mix gently at 4°C and let stand for 3 hours. ④ Centrifugal purification: Centrifuge the mixture obtained in step c at 12,000×g and 4℃ for 30 minutes, discard the supernatant, resuspend the precipitate in PBS, and centrifuge again at 12,000×g and 4℃ for 30 minutes. Finally, resuspend the precipitate in an appropriate amount of PBS to obtain the exovesicles from Centipeda minima. ⑤ Purity verification: Particle size analysis (NTA): Main peak 80-150nm, PDI=0.18; b. Preparation of excipient solution: ① Dissolve sodium hyaluronate: Take 80 mL of pre-cooled PBS (4℃), slowly add 0.1 g of sodium hyaluronate, and stir magnetically (500 rpm, 2 h) until completely dissolved; let stand at 4℃ overnight to defoam (to avoid clogging of subsequent filters); if the dissolution is incomplete, it can be assisted by ultrasound (40 kHz, 30 s pulse). ② Add other excipients: Add 0.9g NaCl, 10μL polysorbate 80, and 0.005g benzalkonium chloride sequentially. Stir until completely dissolved (about 30 minutes); bring PBS to a final volume of 100mL and adjust the pH to 6.8 (fine-tune with 0.1M HCl / NaOH); ③ Filtration and sterilization: Filter sequentially through 0.45μm and 0.22μm filter membranes (aseptic operation required); sample and test pH (6.5-7.0) and osmotic pressure (290±10 mOsm / kg); c. Final preparation of Centipeda minima nasal drops: ①External vesicle addition: Add purified external vesicle suspension (concentration ≥ 1×10⁻⁶) 10 Add the excipient solution at a ratio of 1:10 (granules / ml); ② Filling and storage: Fill into brown nose dropper bottles (5mL / bottle) under nitrogen purging conditions; store at 4℃ (3 months), freeze at -80℃ (12 months).

5. The method for preparing the Centipeda minima nasal drops according to claim 4, characterized in that: During the polyethylene glycol precipitation step, the pH was strictly controlled at 4.75 ± 0.02 (pH meter calibrated). The particle size distribution of the final product was confirmed by NanoSight NTA analysis, and the endotoxin content was < 0.1 EU / mL (Limulus amebocyte lysate (LAL) method).

6. A pharmaceutical composition for treating refractory rhinitis, characterized in that, Include: ①The Centipeda minima nasal drops according to any one of claims 1-3; ② Synergistic drug: mometasone furoate, at a concentration of 10±2 μg / mL; ③ The mass ratio of Cm-EVs to mometasone furoate in the composition is 100:

1.

7. The Centipeda minima nasal drops according to any one of claims 1-3, characterized in that, The nasal drops are used in medications to treat the following conditions: ①IgE-mediated allergic rhinitis (serum IgE > 100 IU / mL); ② Steroid-resistant chronic rhinitis (requiring treatment for ≥14 days) (defined as ineffective use of glucocorticoids for >4 weeks and nasal congestion score ≥5 / 10); ③ Non-allergic rhinitis with damage to the nasal mucosal epithelial barrier (downregulation of tight junction protein ZO-1 expression ≥ 50%).

8. The Centipeda minima nasal drops according to claim 7, characterized in that, The dosing regimen meets the following requirements: ① Single nasal administration volume: 10±2μL, containing 1×10 Cm-EVs 10 Particles; ② Frequency of administration per day: 1-2 times; ③ Treatment course: 7-28 days (hormone-resistant type requires ≥ 14 days).

Images