Self-assembled polypeptide for loading multiple traditional Chinese medicine components and magnolia flower multi-component delivery system prepared from self-assembled polypeptide

Abstract

The invention discloses a self-assembled polypeptide for loading multiple traditional Chinese medicine components, a magnolia flower multi-component delivery system prepared from the self-assembled polypeptide and application of the magnolia flower multi-component delivery system in treatment of allergic rhinitis. The self-assembled polypeptide is synthesized firstly, and then the magnolia flower multi-component delivery system is prepared through stirring in combination with a probe ultrasonic method. According to the self-assembled polypeptide IIFFSSKKGE-Dopa disclosed by the invention, through construction of a nasal cavity delivery system FCSP and NFs, co-loading of traditional Chinese medicine components such as desmogenin and eudesmol can be realized, and the nasal cavity residence time of volatile oil components such as eudesmol and indissolvable components such as desmogenin is effectively prolonged. Meanwhile, the FCSP (at) NFs overcomes a mucous cilia removal barrier, a physical protection net is formed in the nasal cavity, allergens are effectively intercepted, nasal mucosa damage is relieved, and the treatment effect on allergic rhinitis is improved.

Description

Technical Field

[0001] The present invention belongs to the polypeptide delivery drug system, and particularly relates to a multi-component delivery system of Magnolia biondii and its application in the treatment of allergic rhinitis. Background Art

[0002] Magnolia biondii is the dried flower bud of Magnolia biondii Pamp. or Magnolia denudata Desr. of the Magnoliaceae family. It is pungent and warm in nature, with the effects of aromaticity, running through the meridians, lightness and upward movement. As a traditional important medicine for the treatment of allergic rhinitis (AR), Magnolia biondii has the therapeutic advantages of multiple components and multiple targets. Among them, the volatile component eucalyptol is a strong inhibitor of inflammatory cytokines IL-1β and IL-6, and the non-volatile component magnolignan can not only inhibit mast cell degranulation, but also inhibit the enhancement of anti-allergic capillary permeability. Although the main active ingredients of Magnolia biondii have been analyzed, due to the volatility of eucalyptol and the poor solubility of magnolignan, the preparations of Magnolia biondii are still mainly traditional nasal drops, sprays, etc. At the same time, the mucociliary clearance mechanism existing in the nasal cavity results in a half-life of the drug of only about 20 minutes, making it difficult to maintain the long-term effect of the drug. Only by increasing the dosing frequency can the symptoms be relieved, and the compliance is poor. Therefore, more long-acting nasal drug delivery preparations are needed for the treatment of AR. In addition to drug treatment, wearing a mask can effectively reduce the incidence of respiratory diseases such as AR through physical interception, but the mask will increase the breathing resistance and needs to be worn for a long time, which is extremely unfriendly to AR patients. Therefore, designing a convenient nasal physical protection net has become an effective way to improve the treatment of AR. Summary of the Invention

[0003] The purpose of the present invention is to solve the problem of drug delivery for existing allergic rhinitis (AR). The present invention proposes to construct a multi-component delivery system of Magnolia biondii by designing self-assembled polypeptides, overcome the physiological barriers of nasal drug delivery, explore the formation of a protection net in the nasal cavity, and finally realize a long-term treatment strategy that combines physical protection and drug treatment for AR.

[0004] In this invention, a self-assembling polypeptide (IIFFSSKKGE-Dopa, Pep) was synthesized by the solid-phase synthesis method, and its physicochemical properties, assembly behavior, drug-loading function, safety, etc. were systematically investigated. The results showed that Pep had appropriate amphiphilicity (oil-water partition coefficient was -1.135), and its isoelectric point was about 6.58, showing near neutrality, which was in line with the physiological environment of nasal administration. Moreover, Pep could spontaneously aggregate at low concentrations (critical aggregation concentration was 0.20 μM) to form nanofibers with a diameter of 58.13 ± 12.59 nm, indicating strong self-assembly ability. Subsequently, a multi-component delivery system of Magnolia officinalis Sieb. et Zucc. FCSP@NFs was prepared by stirring combined with probe sonication method. The drug-loading amount of magnolol in it reached 3.50%, which was 8.75 times its solubility in the aqueous environment; the drug-loading amount of eucalyptol reached 13.09%, and its room-temperature stability time (drug content > 90%) was greater than 48 h, which was more than 16 times that of free eucalyptol, indicating that FCSP@NFs could improve the solubility and stability of the loaded drugs. Infrared spectroscopy and differential scanning calorimetry results showed that magnolol existed in an amorphous state inside the nanofibers of FCSP@NFs, and at the same time, the volatilization rate of the stably loaded eucalyptol slowed down. TEM results showed that after FCSP@NFs loaded magnolol and eucalyptol into the hydrophobic cavity of the self-assembling polypeptide, it still presented a cross-linked fibrous structure. In addition, the results of nasal mucosa irritation and organ safety experiments proved that FCSP@NFs had good safety for nasal administration.

[0005] To explore the dual functions and mechanisms of physical protection and drug treatment of FCSP@NFs, within the safe concentration range of cell administration, the present invention investigated experimental indicators such as its assembled morphology on the cell surface, TEER, FITC-Dextran permeation amount, and intercellular tight junctions. SEM morphology confirmed that FCSP@NFs formed an expected physical protection network structure on the surface of HNEPC cells. Subsequently, in the HDM-induced barrier damage model, FCSP@NFs could maintain normal TEER values and effectively protect the integrity of the epithelial barrier. Although the TEER value of the Pep group (single fiber protection network) reached 1.31 times that of the HDM group, showing a certain effect of reducing barrier damage, its TEER value was significantly lower than that of the FCSP@NFs group, which revealed the importance of the dual combination of the physical protection network and the loaded drug. In the permeability experiment with FITC leakage as the detection index, the FCSP@NFs group and the Pep component reduced the permeability of FITC-Dextran by 33.55% and 24.99% respectively compared with the HDM group. In particular, the permeation amount of the FCSP@NFs group had no significant difference from that of the Control group, which further confirmed the dual efficacy of FCSP@NFs in maintaining the permeability of the epithelial barrier. In addition, the results of RT-qPCR, WB, and IF experiments showed that FCSP@NFs could promote the expression of Occludin and ZO-1 to return to normal levels, and the intercellular tight junctions were intact and continuous. However, the tight junctions of the Pep group had intermittent small breaks, and the mRNA expression of Occludin and ZO-1 was significantly lower than that of the FCSP@NFs group, which again indicated that compared with the single fiber protection network (Pep), FCSP@NFs with dual effects had stronger ability to protect the epithelial barrier.

[0006] Based on the excellent dual-effect ability of FCSP@NFs to protect the epithelial barrier, in order to further explore its retention and release behavior in the nasal cavity, experiments such as SEM, in vivo imaging, laser confocal microscopy, and intranasal release were carried out. The SEM results showed that FCSP@NFs formed a nanofiber network structure by in-situ crosslinking at the root of rat nasal cilia, still conforming to the expected morphology of the physical protection network. In vivo imaging of rats showed that Pep-CY7.5 extended the nasal retention time of CY7.5 to 10 h (with 40% fluorescence intensity) by virtue of its crosslinked fiber network, which was 5 times longer than that of free CY7.5, indicating that the crosslinked fiber network could achieve long nasal retention. The confocal results confirmed that the mucosal penetration depth of Pep-FITC reached 75 μm after 1 h in the nasal cavity, which was about 7 times that of free FITC, and its nasal retention time was as long as 9 h, indicating that the crosslinked fiber network could penetrate the nasal mucosa and extend the retention time. The GC analysis results showed that the retention amount of eucalyptol in FCSP@NFs in the nasal cavity at 0.5 h was 2 times that of free eucalyptol, and its nasal retention time was as long as 9 h (the retention amount at 9 h in the nasal cavity was 2.09 ± 0.39 μg / g). At the same time, the HPLC data showed that the retention amount of magnolol in FCSP@NFs in the nasal cavity at 0.5 h was increased by 4.04 times compared with the free group, and its nasal retention time was as long as 24 h (the retention amount at 24 h in the nasal cavity was 2.31 ± 0.58 μg / g), which confirmed that FCSP@NFs could overcome the nasal clearance effect and achieve the sustained release of magnolol and eucalyptus oil in the nasal cavity.

[0007] To investigate the therapeutic effect of FCSP@NFs on AR, an AR rat model was successfully constructed by the methods of basic sensitization and OVA nasal drip atomization challenge in rats, and systematic evaluations were carried out from aspects such as behavioral evaluation, inflammation inhibition, immune regulation, and barrier repair. The results of biological behavior evaluation showed that after continuous administration for 7 days, the total score of typical AR symptoms (nose scratching, sneezing, runny nose) in the rats of the FCSP@NFs treatment group decreased significantly from 6.0 in the Model group to 2.0, and the curative effect was better than that of other treatment groups. The RT-qPCR and WB results showed that compared with the Model group, FCSP@NFs could significantly inhibit the expression of pro-inflammatory factors (IL-6, TNF-α) and Th2-type cytokines (IL-4, IL-13), down-regulate the activation of immune signaling pathways such as STAT6 and GATA3, and up-regulate the expression levels of tight junction proteins (ZO-1, Occludin) in the nasal mucosa. The histopathological results further confirmed that the nasal mucosa structure in the FCSP@NFs group was similar to that in the normal group, with almost no infiltration of eosinophilic granulocytes and degranulation of mast cells, and the nasal mucosa epithelial layer showed a continuous and complete tight junction structure, confirming that FCSP@NFs had its best anti-inflammatory, anti-allergic and barrier damage repair abilities.

[0008] Technical solution: In order to achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A self-assembling polypeptide for loading multiple components of traditional Chinese medicine, characterized in that the self-assembling polypeptide is IIFFSSKKGE.

[0010] The self-assembling polypeptide described in the present invention can improve the solubility of poorly soluble components and enhance the stability of volatile oil components.

[0011] As a preferred embodiment, the poorly soluble components loaded by the above self-assembling polypeptide include magnolol and geraniol, and the volatile oil components include eucalyptol; it can improve the solubility of magnolol and geraniol and reduce the volatility of eucalyptol.

[0012] The self-assembling polypeptide loaded with multiple components of traditional Chinese medicine can achieve long-term nasal adhesion and extend the release time of magnolol, geraniol and eucalyptol in the nasal cavity.

[0013] The self-assembling polypeptide can assemble into a fibrous structure, and by performing Dopa modification on the polypeptide sequence, wet adhesion can be achieved.

[0014] The structural characteristics of this type of self-assembling polypeptide provided by the present invention are:

[0015] For volatile oils, intermolecular forces such as aromatic interactions (π-π stacking) and hydrophobic interactions of aromatic and aliphatic amino acid residues can be used for effective loading; for flavonoids and terpenoid components, intermolecular forces such as aromatic interactions, hydrogen bonds and electrostatics in aromatic and acid / base amino acid residues can be used for loading. The specific amino acid types include: aromatic amino acid phenylalanine (Phe, F), aliphatic hydrophobic amino acid isoleucine (Ile, I) and serine rich in hydroxyl groups (Ser, S).

[0016] A multi-component delivery system for magnolia, which is prepared by the following method:

[0017] (1) Synthesis of self-assembling polypeptide;

[0018] (2) Preparation of a multi-component delivery system for magnolia by stirring combined with probe sonication.

[0019] As a preferred embodiment, for the multi-component delivery system for magnolia described above, the synthesis of the self-assembling polypeptide in step (1) includes the following steps:

[0020] (1) Resin swelling: Weigh accurately Rink-Amide-MBHA resin, place it in a centrifuge tube, add DMF, shake it in an ice bath to swell, transfer it to a polypeptide synthesis tube, and wash it successively with DMF (N,N-dimethylformamide) and DCM (dichloromethane);

[0021] (2) Deprotection: Add piperidine and shake in an ice bath to remove the Fmoc protecting group on the resin. After repeating the deprotection once, wash with DMF and DCM respectively; Take a small amount of resin for ninhydrin color reaction. After verifying that the deprotection is complete, start the condensation reaction;

[0022] (3) Condensation reaction: Add the Fmoc-DOPA(acetonide)-OH amino acid solution to the polypeptide synthesis tube, add HBTU (O-benzotriazole-tetramethylurea hexafluorophosphate) and DIEA (N,N-diisopropylethylamine) solutions respectively, shake and react in an ice bath, and wash with DMF and DCM successively; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the first amino acid is completed;

[0023] (4) Cleavage of resin: After the condensation and deprotection of the last amino acid are completed, transfer the resin to a centrifuge tube, add the cleavage solution for cleavage and lysis reaction;

[0024] (5) Ether precipitation: Add the polypeptide solution to ice-cold ether, centrifuge, discard the supernatant, add ice-cold ether, mix and precipitate, then centrifuge again. Repeat this process, and place it in a vacuum drying oven to remove the solvent to obtain the crude polypeptide Pep.

[0025] (6) Polypeptide purification

[0026] Dissolve the crude polypeptide Pep in a mixed solvent of acetonitrile and water, centrifuge, take the supernatant, purify it by preparative liquid chromatography, and obtain the pure polypeptide Pep after freeze-drying the receiving solution.

[0027] As a preferred scheme, for the magnolia multi-component delivery system described above, the synthesis of the self-assembled polypeptide in step (1) includes the following steps:

[0028] (1) Resin swelling: Weigh accurately Rink-Amide-MBHA resin, place it in a centrifuge tube, add DMF, shake in an ice bath for 15 - 30 min to swell, transfer it to the polypeptide synthesis tube, and wash 3 - 4 times with DMF and DCM successively;

[0029] (2) Deprotection: Add piperidine and shake in an ice bath to remove the Fmoc protecting group on the resin. After repeating the deprotection once, wash 3 - 4 times with DMF and DCM respectively; Take a small amount of resin for ninhydrin color reaction. After verifying that the deprotection is complete, start the condensation reaction;

[0030] (3) Condensation reaction: Add the Fmoc-DOPA(acetonide)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with shaking in an ice bath for 20 - 30 min, wash 3 - 4 times with DMF and DCM respectively; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the first amino acid.

[0031] (4) Cleavage of resin: After the condensation and deprotection of the last amino acid are completed, transfer the resin to a centrifuge tube, add the cleavage solution and carry out a cleavage and lysis reaction for 2 - 3 h;

[0032] (5) Precipitation with ether: Add the peptide solution to ice-cold ether, centrifuge, discard the supernatant, add ice-cold ether, mix well and precipitate, then centrifuge again. Repeat this process, place it in a vacuum drying oven to remove the solvent, and obtain the crude polypeptide Pep.

[0033] (6) Polypeptide purification

[0034] Dissolve the crude polypeptide Pep in a mixed solvent of water and acetonitrile with a volume ratio of 3:2, centrifuge, take the supernatant, purify it by preparative liquid chromatography, and obtain the pure polypeptide Pep after freeze-drying the received solution.

[0035] As a preferred scheme, for the multi-component delivery system of magnolia officinalis described above, the conditions for preparative liquid chromatography purification in step (6) are: Chromatographic column: C18 reversed-phase chromatographic column, with a specification of 50×250 mm, packing 10 μm, 100 AA; Mobile phase: Acetonitrile containing 0.1% TFA is phase A - water containing 0.1% TFA is phase B; Elution program: 0 - 10 min, 5% - 15% A; 10 - 50 min, 15 - 31% A; Flow rate: 20 mL / min, Detection wavelength: 220 nm; Injection volume: 1 mL; Column temperature: 30 °C.

[0036] As a preferred scheme, the condensation reaction in step (3) above includes the following steps:

[0037] The polypeptide sequence is IIFFSSKKGE-Dopa from the N-terminus to the C-terminus. In the synthesis process, an amino resin is used. Therefore, the order of synthesizing amino acids is Dopa, E, G, K, K, S, S, F, F, I, I in sequence;

[0038] ① Dopa: Add the Fmoc-DOPA(acetonide)-OH dopa solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with shaking in an ice bath for 20 min, wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the dopa-modified part;

[0039] ② Glutamic acid (E): Add the Fmoc-Glu(OtBu)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 20 min, and wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the first amino acid;

[0040] ③ Glycine (G): Add the Fmoc-Gly-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 20 min, and wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the second amino acid;

[0041] ④ Lysine (K): Add the Fmoc-Lys(Boc)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 20 min, and wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the third amino acid; Continue to repeat this step to synthesize the fourth amino acid K;

[0042] ⑤ Serine (S): Add the Fmoc-Ser-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 20 min, and wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the fifth amino acid; Continue to repeat this step to synthesize the sixth amino acid S;

[0043] ⑥ Phenylalanine (F): First, add the first portion of the Fmoc-Phe-Phe-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 30 min, and wash successively with DMF and DCM; Then add the second portion of the Fmoc-Phe-Phe-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 30 min, and wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the seventh and eighth amino acids F;

[0044] ⑦Isoleucine (I): First, add the first portion of the Fmoc-Ile-OH amino acid solution into the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath oscillation for 30 min, and wash successively with DMF and DCM; then add the second portion of the Fmoc-Ile-OH amino acid solution into the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath oscillation, and wash successively with DMF and DCM; take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the ninth amino acid; continue to repeat this step to synthesize the tenth amino acid (I).

[0045] As a preferred embodiment, for the multi-component delivery system of Magnolia officinalis described above, the steps of preparing the multi-component delivery system of Magnolia officinalis by stirring combined with probe sonication include:

[0046] Weigh the polypeptide and dissolve it in ultrapure water, adjust the pH to neutral with NaOH and HCl solutions, and prepare a polypeptide solution with a certain concentration; then weigh magnolol, add anhydrous ethanol and sonicate to dissolve it, and then add eucalyptol and mix evenly to obtain a drug mixed solution; stir the polypeptide solution, dropwise add the drug mixed solution into the polypeptide solution, continue to stir, after treatment with probe sonication, place the sample at low temperature for a certain period of time to stabilize, centrifuge to remove free magnolol, and the obtained supernatant is the FCSP@NFs solution of the multi-component delivery system of Magnolia officinalis.

[0047] As a more preferred embodiment, for the multi-component delivery system of Magnolia officinalis described above, the steps of preparing the multi-component delivery system of Magnolia officinalis by stirring combined with probe sonication include:

[0048] Weigh the polypeptide and dissolve it in ultrapure water, adjust the pH to neutral with 1 - 2 M NaOH and HCl solutions, and prepare a polypeptide solution with a concentration of 10 - 20 mg / mL; then weigh 5.00 - 10 mg of magnolol, add 1 - 2 mL of anhydrous ethanol and sonicate to dissolve it, and then add 40 - 80 μL of eucalyptol and mix evenly to obtain a drug mixed solution; place the polypeptide solution at 40 - 45 °C and stir at a speed of 3000 - 5000 rpm / min, dropwise add 200 - 400 μL of the drug mixed solution into the polypeptide solution, continue to stir, then, after treatment with probe sonication at a frequency of 60 Hz for 5 - 10 min, place the sample at 4 °C for 24 - 48 h to stabilize, and centrifuge at a speed of 2000 - 4000 rpm for 10 - 20 min to remove free magnolol, and the obtained supernatant is the FCSP@NFs solution.

[0049] Beneficial effects:

[0050] In this invention, the multi-component delivery system of magnolia officinalis, FCSP@NFs, was prepared by stirring combined with probe ultrasound method, and its drug loading capacity, room temperature stability, TEM morphology, DSC spectrum, etc. were investigated. The results of drug loading capacity and stability showed that the construction of FCSP@NFs increased the solubility of magnolol in the aqueous environment by 8.75 times, and the stable time of cineole loaded by FCSP@NFs at room temperature was about 16 times longer than that of free cineole, indicating that the construction of FCSP@NFs was beneficial to improve the solubility and stability of the loaded substances and was expected to achieve the long-term effect of drugs. FTIR and DSC were used to prove that magnolol and cineole stably existed inside the FCSP@NFs delivery system, and magnolol was in an amorphous state, which also indicated that the construction of FCSP@NFs improved the thermal stability of cineole. The TEM results showed that

[0051] After FCSP@NFs loaded magnolol and cineole into the hydrophobic cavity of the self-assembled polypeptide, it still presented a cross-linked fiber network structure. Finally, the nasal mucosa irritation and organ safety experiments showed that FCSP@NFs had good safety for nasal administration.

[0052] Based on the verification of the drug loading performance, physical protection and long retention function of the FCSP@NFs delivery system, in order to evaluate its therapeutic effect on AR, first, an AR rat model was successfully constructed by the method of OVA systemic sensitization and local challenge. The results showed that the behavioral score of the FCSP@NFs treatment group decreased from 6.0 to 2.0, significantly inhibiting the expression of pro-inflammatory factors IL-6, TNF-α and Th2-type cytokines IL-4, IL-13, which decreased by 72.59%, 87.11%, 57.69% and 63.63% respectively compared with the Model group. At the same time, the protein expression of the STAT6 / GATA3 pathway was down-regulated and the protein expression of ZO-1 and Occludin was up-regulated to normal levels. Pathological analysis showed that in the nasal mucosa tissue of AR rats treated with FCSP@NFs, there was no obvious eosinophil infiltration and mast cell degranulation, and the tight junctions of the epithelial barrier were intact and continuous, proving that FCSP@NFs had the best effects of inflammation inhibition, immune regulation and barrier repair.

[0053] Therefore, in this invention, the self-assembled polypeptide IIFFSSKKGE-Dopa was synthesized, and through the construction of the nasal delivery system FCSP@NFs, the co-loading of magnolol and cineole was achieved, effectively prolonging the nasal retention time of the volatile oil component cineole and the poorly soluble component magnolol. At the same time, FCSP@NFs overcame the mucociliary clearance barrier, formed a physical protection network in the nasal cavity, effectively intercepted allergens, reduced nasal mucosa damage, and improved the therapeutic effect of AR. Description of the Drawings

[0054] Figure 1 Mass spectrum of self-assembled polypeptide (IIFFSSKKGE-Dopa, Pep).

[0055] Figure 2 High performance liquid chromatography of Pep.

[0056] Figure 3 Potential diagram of Pep at different pH values.

[0057] Figure 4 Pyrene fluorescence spectra of Pep solutions with different concentrations (A) and critical aggregation concentration of Pep (B).

[0058] Figure 5 TEM morphology of Pep.

[0059] Figure 6 Specificity diagram of magnolin.

[0060] Figure 7 Specificity diagram of eucalyptol.

[0061] Figure 8 Stability of eucalyptol in FCSP@NFs.

[0062] Figure 9 Infrared spectrum analysis of FCSP@NFs.

[0063] Figure 10 Nasal mucosa irritation of Pep and FCSP@NFs.

[0064] Figure 11 Main organ safety of Pep and FCSP@NFs.

[0065] Figure 12 Cell viability of Pep (A) and FCSP@NFs (B) acting on HNEPC cells (compared with the group with polypeptide concentration of 0 μg / mL, * P < 0.05, ** P < 0.01, *** P < 0.001).

[0066] Figure 13 Change in resistance of HNEPC cell barrier (compared with Control group, *** P < 0.001; compared with HDM group, ### P < 0.001; compared with FCSP@NFs group, &&& P < 0.001).

[0067] Figure 14 Permeation amount of FITC-Dextran beside HNEPC cells (compared with Control group, **P < 0.01, *** P < 0.001; Compared with the HDM group, ### P < 0.001; Compared with the FCSP@NFs group, & P < 0.05).

[0068] Figure 15 For the mRNA expression of ZO-1 (A) and Occludin (B) in HNEPC cells (compared with the Control group, ** P < 0.01, *** P < 0.001; Compared with the HDM group, ## P < 0.01, ### P < 0.001; Compared with the FCSP@NFs group, & P < 0.05).

[0069] Figure 16 For the immunoblotting image (A) and the quantitative results of ZO-1 (B) and Occludin (C) (*P < 0.05, **P < 0.01, ***P < 0.001 compared with the Control group; ##P < 0.01 compared with the HDM group).

[0070] Figure 17 Behavioral scores of rats after nasal administration treatment (compared with the Model, *** P < 0.001; compared with the FCSP@NFs, # P < 0.05, ## P < 0.01, ### P < 0.001).

[0071] Figure 18 mRNA expression levels of TNF-α (A) and IL-6 (B) in nasal mucosa tissues (compared with the Control, * P <

[0072] 0.05, ** P < 0.01, *** P < 0.001; compared with the Model, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with the FCSP@NFs, &P < 0.05, &&P < 0.01, &&&P < 0.001).

[0073] Figure 19 Immunoblotting images (A) and quantitative results (B) of TNF-α and IL-6 in nasal mucosa tissues (*P < 0.05, **P < 0.01 compared with the Control; #P < 0.05, ##P < 0.01 compared with the Model).

[0074] Figure 20 The expression levels of IL-4 (A) and IL-13 (B) mRNA in nasal mucosa tissue (compared with Control, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with Model, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with FCSP@NFs, & P < 0.05, && P < 0.01, &&& P < 0.001).

[0075] Figure 21 The immunoblot images (A) and quantitative results (B) of STAT6 and GATA3 in nasal mucosa tissue (compared with Control, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with Model, # P < 0.05, ## P < 0.01, ### P <

[0076] 0.001).

[0077] Figure 22 The expression levels of ZO-1 (A) and Occludin (B) mRNA in nasal mucosa tissue (compared with Control, * P < 0.05, ** P < 0.01, *** P < 0.001; compared with Model, # P < 0.05, ## P < 0.01, ### P < 0.001; compared with FCSP@NFs, & P < 0.05, && P < 0.01, &&& P < 0.001). Detailed implementation manners

[0078] The present invention will be further illustrated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. After reading the present invention, various equivalent modifications made by those skilled in the art to the present invention all fall within the scope defined by the appended claims of this application.

[0079] Example 1 Synthesis of self-assembled polypeptide

[0080] 1. Reagent Preparation

[0081] 0.6M HBTU: Weigh 22.7448 g of HBTU with a ten-thousandth balance and add it to 100 mL of anhydrous DMF, then mix well.

[0082] 1.2M DIEA: Pipette 19.82 mL of DIEA, add 100 mL of anhydrous DMF, and mix well.

[0083] 20% Piperidine: Measure 100 mL of piperidine, add 400 mL of anhydrous DMF, and mix well.

[0084] Cutting Solution: Carefully pipette 9.50 mL of trifluoroacetic acid (TFA) into a 15 mL centrifuge tube in a fume hood. Then, use a pipettor to respectively pipette 0.25 mL of Tis and 0.25 mL of ultrapure water, mix well, and use it immediately after preparation.

[0085] 1% Ninhydrin Solution: Weigh 0.3000 g of ninhydrin with a ten-thousandth balance, dissolve it in 30 mL of anhydrous DMF, mix well, and store it in the dark for later use.

[0086] Ice Ether: Measure 30 mL of anhydrous ether with a 50 mL graduated cylinder and place it in six 50 mL centrifuge tubes, a total of 180 mL. Seal it and pre-freeze it in a -80°C refrigerator for 3 h, then seal it for later use.

[0087] Amino Acid Solution: Prepare according to Table 1. Weigh the corresponding mass of amino acid and place it in a 15 mL centrifuge tube, then add 3.3 mL of anhydrous DMF and mix well.

[0088] 1M HCl Solution: Take concentrated hydrochloric acid with a percentage concentration of 38% and a density of 1.194 g / cm 3 Add 88 μL of concentrated hydrochloric acid to 912 μL of ultrapure water and mix well.

[0089] 1M NaOH Solution: Weigh 40.01 mg of NaOH with a ten-thousandth balance, add 1 mL of ultrapure water, and mix well.

[0090] Table 1 Amino Acids Used in the Preparation of Amino Acid Solution

[0091]

[0092] 2. Synthesis Steps

[0093] 2.1 Resin Swelling: Weigh accurately 1.0028 g of Rink-Amide-MBHA resin (substitution degree 0.655 mmol / g) and place it in a 50 mL centrifuge tube. Add 15 mL of DMF, shake in an ice bath for 15 min for swelling, then transfer it to a polypeptide synthesis tube, and wash it 3 times with 10 mL of DMF and 10 mL of DCM respectively.

[0094] Deprotection: Add 15 mL of 20% piperidine and shake in an ice bath for 15 min to remove the Fmoc protecting group on the resin. After repeating the deprotection once, wash it 3 times with 10 mL of DMF and 10 mL of DCM respectively. Take a small amount of resin for ninhydrin color reaction. After verifying that the deprotection is complete, start the condensation reaction.

[0095] 2.2 Condensation Reaction:

[0096] The polypeptide sequence is IIFFSSKKGE-Dopa from the N-terminus to the C-terminus. Since an amino resin is used in the synthesis, the order of synthesizing amino acids is Dopa, E, G, K, K, S, S, F, F, I, I in turn;

[0097] ① Dopa: Add the Fmoc-DOPA(acetonide)-OH dopa solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the dopa-modified part is completed;

[0098] ② Glutamic acid (E): Add the Fmoc-Glu(OtBu)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the first amino acid is completed;

[0099] ③ Glycine (G): Add the Fmoc-Gly-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the second amino acid is completed;

[0100] ④ Lysine (K): Add the Fmoc-Lys(Boc)-OH amino acid solution to the peptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 20 min, wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the third amino acid; Continue to repeat this step to synthesize the fourth amino acid K;

[0101] ⑤ Serine (S): Add the Fmoc-Ser-OH amino acid solution to the peptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 20 min, wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the fifth amino acid; Continue to repeat this step to synthesize the sixth amino acid S;

[0102] ⑥ Phenylalanine (F): First, add the first portion of Fmoc-Phe-Phe-OH amino acid solution to the peptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 30 min, wash successively with DMF and DCM; Then add the second portion of Fmoc-Phe-Phe-OH amino acid solution to the peptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 30 min, wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the seventh and eighth amino acids F;

[0103] ⑦ Isoleucine (I): First, add the first portion of Fmoc-Ile-OH amino acid solution to the peptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking for 30 min, wash successively with DMF and DCM; Then add the second portion of Fmoc-Ile-OH amino acid solution to the peptide synthesis tube, add HBTU and DIEA solutions respectively, react with ice bath shaking, wash successively with DMF and DCM; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates the end of the synthesis of the ninth amino acid; Continue to repeat this step to synthesize the tenth amino acid (I).

[0104] 2.3. Cleavage of resin:

[0105] After the condensation and deprotection of the last amino acid are completed, transfer the resin to a 15 mL centrifuge tube, add 10 mL of cleavage solution for a 2 h cleavage and lysis reaction.

[0106] Precipitation with ether: Add 5 mL of the peptide solution to 30 mL of ice-cold ether, centrifuge at 8000 rpm for 5 min at 4 °C, discard the supernatant, add 30 mL of ice-cold ether, mix the precipitate and centrifuge again. Repeat this 3 times, and place it in a vacuum drying oven to remove the solvent to obtain the crude product Pep.

[0107] 2.4, Polypeptide Purification

[0108] Dissolve the crude Pep in 10 mL of a mixed solvent (water:acetonitrile = 6:4), centrifuge at 12000 rpm for 10 min, take the supernatant and set aside to obtain the preparative liquid chromatography sample.

[0109] Preparative liquid chromatography purification conditions: Chromatographic column: C18 reversed-phase chromatographic column (50×250 mm, packing 10 μm, 100 Å); Mobile phase: acetonitrile (0.1% TFA) (A) - water (0.1% TFA) (B); Elution program: 0 - 10 - 50 min (5% - 15% - 31% A); Flow rate: 20 mL / min, Detection wavelength: 220 nm; Injection volume: 1 mL; Column temperature: 30 °C. The receiving solution is lyophilized to obtain pure Pep.

[0110] 2.5, Purity Detection

[0111] Weigh 1 mg of pure Pep and dissolve it in 1 mL of a mixed solvent (water:acetonitrile = 7:3), centrifuge at 12000 rpm for 10 min, take 100 μL of the supernatant and transfer it into a liquid phase vial for standby to obtain the analytical liquid chromatography sample.

[0112] Analytical liquid chromatography conditions: Chromatographic column: Kromasil C18-5 (4.6×250 mm); Mobile phase: acetonitrile (A) - water (0.1% TFA) (B); Elution program: 0 - 22 min (5% - 50%); Flow rate: 1 mL / min, Detection wavelength: 220 nm; Injection volume: 15 μL; Column temperature: 30 °C.

[0113] 5. Identification of Self-Assembled Polypeptides

[0114] Weigh 1.00 mg of the purified product Pep with a balance, dissolve it in 1 mL of ultrapure water, and then dilute it to 500 ng / mL. After centrifuging at 12000 rpm for 5 min, take 200 μL of the supernatant and transfer it into a liquid phase vial. Use electrospray ionization mass spectrometry (ESI-MS) to identify the molecular weight of the pure polypeptide. The mass spectrometry conditions are: Probe Bias: +5.0 kV; Nebulizer Gas Flow: 0.2 mL / min; Detector: 1.5 kV; CDL: -20.0 V; CDL Temp.: 250 °C.

[0115] 6. Physicochemical Property Characterization of Self-Assembled Polypeptides

[0116] 6.1 Determination of Isoelectric Point

[0117] Prepare 9 portions of 2 mL of 1 mM polypeptide solution in parallel. Adjust the pH value of the polypeptide solution to 4.0, 4.5, 5.5, 6.5, 7.0, 8.0, 9.0 with 1 mol / L HCl or NaOH for standby. Use Nano ZS 90 to measure the Zeta potential of the polypeptide solution at different pH values, measure it in parallel 3 times, take the average value of the measurement results, plot the Zeta-pH curve of Pep, calculate the intersection point with the horizontal axis, and the obtained pH value is the isoelectric point.

[0118] 6.2 Determination of oil-water partition coefficient

[0119] Precisely weigh 3.23 mg of Pep polypeptide sample, dissolve it in 10.0 mL of n-octanol saturated aqueous solution to prepare a 240.00 μM polypeptide n-octanol saturated aqueous solution, and serially dilute it to 160, 80.00, 40.00, 20.00, 10.00, 5.00 μM. Measure its absorbance at 220 nm and plot the Pep standard curve. Prepare n-octanol saturated aqueous solution and water-saturated n-octanol solution. Weigh 2.00 mg of Pep sample and add it to 15.0 mL of equal-volume n-octanol saturated aqueous solution and water-saturated n-octanol solution to obtain a 100 μM Pep solution. Shake it overnight and let it stand for 2 h, then separate the upper and lower layer solutions. Measure the absorbance of the Pep solution in different layers at 220 nm, and calculate the oil-water partition coefficient of Pep according to the formula: P = Co / Cw. The larger the Log P value, the more lipophilic Pep is; conversely, the more hydrophilic and the better the water solubility.

[0120] 7 Study on the self-assembly behavior of polypeptides

[0121] 7.1 Determination of critical aggregation concentration

[0122] Prepare a 1×10 -2 mol / L methanol solution of pyrene and serially dilute it to 1×10 -4 mol / L for standby. Take 40 μL of the methanol solution of pyrene in a centrifuge tube and volatilize it in the dark. Add 4 mL of different concentrations of Pep solution (0.005, 0.05, 0.5, 1, 5, 50 μM) to each centrifuge tube, sonicate for 5 min to fully disperse pyrene. Use a fluorescence spectrophotometer with an excitation wavelength of 334 nm, an excitation slit of 5.0 nm, an emission slit of 1.5 nm, a scanning speed of Medium, and a scanning range of 350 - 450 nm. Plot the ratio of the peak intensities of the first vibration band and the third vibration band (I1 / I3) against the logarithm of the polypeptide concentration.

[0123] 7.2 Investigation of assembly morphology

[0124] The self-assembled morphology of the polypeptide was observed using TEM. A 1 mM Pep solution was prepared. 10 μL of the solution was dropped onto a 200-mesh carbon organic film copper grid. After waiting for 30 s, the excess solution was aspirated, and it was left to dry naturally. Then, 10 μL of saturated uranyl acetate staining agent was added for negative staining. After 4 min, the excess staining solution was blotted dry with filter paper and left to dry naturally. Then, it was placed under the TEM for observation and photography.

[0125] 8 Experimental results

[0126] 8.1 Confirmation of the molecular weight and purity analysis of the self-assembled polypeptide

[0127] The ESIMS detection results of the pure Pep product are as Figure 1 shown. 668.15 is the trivalent peak of Pep ([M + 3H] 3+ ), and 445.85 is the divalent peak ([M + 2H] 2+ ), which is consistent with the theoretical molecular weight (1334.75). The HPLC results ( Figure 2 ) showed that the retention time of Pep was 9.330 min and the purity was 96.64%, meeting the requirements of subsequent experiments.

[0128] 8.2 Physicochemical properties of the self-assembled polypeptide

[0129] 8.2.1 Isoelectric point

[0130] To avoid the adsorption and retention of Pep by negatively charged mucin when penetrating mucus, 2 lysines (Lys, K) were selected in the Pep sequence to adjust the self-assembled polypeptide to be uncharged or weakly charged. As Figure 3 shown, the Zeta potential of Pep at different pH values was measured by Zetasizer Nano ZS90, and the isoelectric point of Pep was calculated to be approximately 6.58, showing near neutrality, which is in line with the physiological environment of nasal drug delivery (pH 5.5 - 6.5).

[0131] 8.2.2 Determination of the oil-water partition coefficient

[0132] A standard curve was plotted with the solution concentration as the abscissa and the absorbance as the ordinate, and linear fitting was performed on this. The regression equation of Pep was y = 0.01288x - 0.05249, and R 2 = 0.9997, indicating good linearity in the range of 5 - 240 μM. The absorbance of Pep in the oil solution and aqueous solution was measured separately by an ultraviolet spectrophotometer. According to the standard curve of Pep, it was calculated and analyzed that the Log P value was -1.135 (Table 2), showing appropriate amphiphilicity, which is beneficial to the stable assembly of the polypeptide in water.

[0133] Table 2 Oil-water partition coefficient of Pep

[0134] Polypeptide Oil phase Co (μM) Aqueous phase Cw (μM) Oil-water partition coefficient P Log P Pep 16.36±1.05 223.64±1.05 0.0732±0.005 -1.135±0.03

[0135] 8.3 Self-assembly behavior of polypeptides

[0136] 8.3.1 Determination of critical aggregation concentration

[0137] Pyrene, as a hydrophobic fluorescent probe, has a fluorescence spectrum with a five-claw peak structure. In aqueous solution, the fluorescence intensity ratio of the first characteristic peak I1 to the third characteristic peak I3 is about 1.8. The fluorescence spectrum of pyrene in aqueous solutions of different concentrations of Pep was measured by a fluorescence spectrophotometer to find the inflection point of the I1 / I3 vs. concentration curve. The results are as Figure 4 shown. The critical aggregation concentration of Pep is 0.2036 μM, indicating that Pep aggregates at low concentrations and has good assembly performance.

[0138] 3.3.2 Investigation of assembly morphology

[0139] As Figure 5 shown, Pep can self-assemble into nanofiber structures with a diameter of 58.13 ± 12.59 nm in aqueous solution. Its morphology conforms to the expected design and is expected to meet the requirements of a nasal protective net.

[0140] In this invention, Pep was synthesized by the solid-phase synthesis method, and the product was confirmed to be Pep by mass spectrometry with a purity of 96.64%. The isoelectric point was determined by zeta potential to be about 6.58, showing near neutrality, which is in line with the physiological environment of nasal drug delivery. The oil-water partition coefficient Log P value is -1.1435, indicating appropriate amphiphilicity. The critical aggregation concentration of Pep was determined to be 0.2036 μM by the pyrene fluorescence probe spectroscopy method. Combining with the TEM morphology of Pep, it shows that Pep can aggregate to form nanofibers with a diameter of 58.13 ± 12.59 nm at low concentrations and has strong self-assembly ability.

[0141] Example 2 Construction and characterization of a multi-component delivery system of Magnolia officinalis

[0142] 1 Experimental animals

[0143] Male Sprague-Dawley rats, weighing 180 - 220 g, were purchased from the Experimental Animal Center of Hangzhou Medical College, license number: SCXK(Zhe)2019 - 0002.

[0144] 2 Experimental methods

[0145] 2.1 Establishment of HPLC methodology for magnolignan

[0146] 2.1.1 HPLC conditions

[0147] Chromatographic column: Kromasil 100 - 5 - C18(4.6×250 mm);

[0148] Mobile phase: 0.1% trifluoroacetic acid in water (A) - acetonitrile (B);

[0149] Elution gradient: 1 - 20 min, 10 - 95% B; 20 - 22 min, 95 - 10% B; 22 - 25 min, 10 - 10% B;

[0150] Flow rate: 1 mL / min; Detection wavelength: 220 nm / 278 nm; Column temperature: 25 °C

[0151] 2.1.2 Investigation of linear relationship

[0152] Accurately weigh 5.00 mg of magnolialide in a 5 mL volumetric flask, add an appropriate amount of methanol until magnolialide is dissolved, dilute to the mark with methanol, shake well, and prepare a magnolialide standard solution with a concentration of 1000 μg / mL. Then, serially dilute it to prepare standard solutions with concentrations of 500, 250, 125, 62.5, 31.25, and 1.95 μg / mL. Take 10 μL of the standard solution for HPLC detection, record its peak area, perform linear regression with the peak area against the concentration, and plot the standard curve of magnolialide.

[0153] 2.1.3 Investigation of specificity

[0154] Prepare a 1 mg / mL Pep methanol solution and a 1 mg / mL magnolialide methanol solution, perform HPLC detection, record the chromatogram, and investigate the method specificity.

[0155] 2.1.4 Investigation of precision

[0156] Take magnolialide standard solutions with concentrations of 1.95, 62.5, and 500 μg / mL, inject 5 consecutive injections, and calculate the relative standard deviation RSD%.

[0157] 2.1.5 Investigation of repeatability

[0158] Take magnolialide standard solutions with concentrations of 1.95, 62.5, and 500 μg / mL, make 5 parallel samples for each, perform HPLC detection, record their peak areas and calculate the relative standard deviation RSD%.

[0159] 2.1.6 Investigation of stability

[0160] Take magnolialide standard solutions with concentrations of 1.95, 62.5, and 500 μg / mL, perform HPLC detection at 0, 4, 8, 12, 24, 48, and 72 h respectively, record their peak areas and calculate the relative standard deviation RSD%, and investigate the stability within 72 h.

[0161] 2.2 Establishment of eucalyptol GC methodology

[0162] 2.2.1 GC conditions

[0163] Chromatographic column: HP-5 (30 m × 250 μm × 0.25 μm);

[0164] Chromatographic conditions: injection port temperature 280 °C;

[0165] Programmed temperature rise: initial temperature 40 °C, maintained for 3 min, then raised to 100 °C at a rate of 15 °C / min, and then raised to 250 °C at a rate of 25 °C / min, maintained for 3 min;

[0166] Carrier gas: He, 40 mL / min, splitless.

[0167] 2.2.2 Investigation of linear relationship

[0168] Use a pipette to aspirate 10 μL of eucalyptol and place it in a 15 mL centrifuge tube. Add 8.99 mL of ethyl acetate until the eucalyptol is dissolved, mix well to prepare a 1000 μg / mL eucalyptol standard solution, and serially dilute it to obtain standard solutions with concentrations of 500, 250, 125, 62.5, 31.25, 7.81, 3.91, 1.95 μg / mL. Take 10 μL of the standard solution for GC detection, record its peak area, perform linear regression with the peak area against the concentration, and plot the standard curve.

[0169] 2.2.3 Investigation of specificity

[0170] Prepare a 125 μg / mL eucalyptol solution for GC detection, record the chromatogram, and investigate the method specificity.

[0171] 2.2.4 Investigation of precision

[0172] Take 1.95, 62.5, 500 μg / mL eucalyptol standard solutions, inject continuously for 5 times, and calculate the relative standard deviation RSD%.

[0173] 2.2.5 Investigation of accuracy

[0174] Take 1.95, 62.5, 500 μg / mL eucalyptol standard solutions, with three parallel samples for each concentration, perform GC detection, record their peak areas and calculate the relative standard deviation RSD%.

[0175] 2.2.6 Investigation of stability

[0176] Take 1.95, 62.5, 500 μg / mL eucalyptol standard solutions, place them in an environment at 4 °C, perform GC detection at 0, 4, 8, 12, 24 h respectively, record their peak areas and calculate the relative standard deviation RSD%, and investigate the stability within 24 h.

[0177] 2.3 Preparation of FCSP@NFs and investigation of drug loading

[0178] Using stirring combined with probe ultrasound method [2] Preparation of FCSP@NFs: Weigh 20.00 mg of the polypeptide and dissolve it in 2 mL of ultrapure water. Use 1 M NaOH and HCl solutions to adjust the pH to neutral, and prepare a polypeptide solution with a concentration of 10 mg / mL. Then weigh 5.00 mg of magnolol, add 1 mL of absolute ethanol, and ultrasonically dissolve it. Then add 40 μL of eucalyptol and mix well to obtain a mixed solution of the drug. Place the polypeptide solution at 45 °C and stir it at a speed of 3600 rpm / min. Gradually add 200 μL of the drug mixed solution dropwise to the polypeptide solution and continue stirring for 2.5 h. Then, use probe ultrasound to treat it at a frequency of 60 Hz for 5 min. After that, place the sample at 4 °C for 24 h and then centrifuge it at a speed of 2000 rpm for 10 min to remove free magnolol. The obtained supernatant is the FCSP@NFs solution. Prepare 3 parallel samples of the sample.

[0179] Investigation of the drug loading of magnolol: Use a pipette to transfer 0.5 mL of the prepared 3 FCSP@NFs solutions to 5 mL centrifuge tubes, add 2 mL of methanol, and ultrasonically treat it for 15 min to completely destroy the structure. After centrifuging twice at 12000 rpm, take 200 μL of the supernatant and place it in a liquid phase vial, and use an HPLC instrument to detect the content of magnolol in FCSP@NFs.

[0180] Investigation of the drug loading of eucalyptol: Use a pipette to transfer 0.5 mL of 3 FCSP@NFs solutions to 5 mL centrifuge tubes, add 2 mL of ethyl acetate, and perform ultrasonic treatment for 15 min in the same way to destroy the structure. After centrifuging twice at 12000 rpm, take 200 μL of the supernatant and place it in a liquid phase vial, and use GC to detect the content of eucalyptol. According to the detection results, calculate the drug loading of FCSP@NFs by taking the average value.

[0181] 2.4 Investigation of the stability of FCSP@NFs

[0182] Parallelly prepare 3 4 mL FCSP@NFs solutions according to the preparation method of FCSP@NFs under item 2.3. The Control group is an aqueous solution of free eucalyptol with the same eucalyptol concentration as FCSP@NFs. Then, place the 3 FCSP@NFs solutions and 3 Control solutions in the dark at room temperature. At 0, 6, 24, 48, and 72 h after placement, use a pipette to transfer 500 μL of the sample solution, treat it according to the treatment method in item 2.3, and use GC to detect the concentration of eucalyptol.

[0183] 2.5 FTIR detection of FCSP@NFs

[0184] The loading status of fargesin and cineole in FCSP@NFs was investigated by FTIR. The volatile oil was prepared into a sample by the potassium bromide smear method, while fargesin, Pep, and FCSP@NFs were prepared by the potassium bromide tablet pressing method. Infrared spectrum scanning was performed in the wavenumber range of 600 - 4000 cm -1 The wavenumber range.

[0185] 2.6 DSC detection of FCSP@NFs

[0186] DSC was used to study the loading status and thermal stability of the drugs in FCSP@NFs. Under the condition of nitrogen flow, with an empty aluminum crucible as the reference and a heating rate of 10 °C / min, DSC curves of fargesin, cineole, Pep, and FCSP@NFs in the range of 30 - 200 °C were obtained.

[0187] 2.7 Investigation of the assembled morphology of FCSP@NFs

[0188] 10 μL of the FCSP@NFs solution prepared by the above method 2.3 was taken and dropped onto a 200-mesh carbon organic film copper grid. After 90 s, the excess solution was blotted with a filter paper and air-dried naturally. Then, 10 μL of saturated uranyl acetate staining agent was added for negative staining. After 4 min, the excess staining solution was blotted with a filter paper and air-dried naturally. After that, it was placed under a TEM for observation and photography.

[0189] 2.8 Investigation of nasal mucosa irritation

[0190] Eighteen SD rats were randomly divided into 3 groups: Control group, Pep group, and FCSP@NFs group. Each rat was instilled with 200 μL per day. The Control group was instilled with normal saline. After continuous administration for 7 days, 3 SD rats were randomly selected from each group. After anesthesia and sacrifice, their nasal mucosa tissues were dissected and fixed in 4% paraformaldehyde for standby. The remaining 3 SD rats in each group were continuously administered for another 7 days, then anesthetized, sacrificed, and their nasal mucosa tissues were dissected and fixed in 4% paraformaldehyde for standby. After paraffin embedding, tissue sectioning, and H&E staining of all tissue samples in sequence, the morphological structure of the nasal mucosa was observed under a microscope to evaluate the nasal mucosa irritation of Pep and FCSP@NFs in SD rats.

[0191] 2.9 Investigation of the safety of major organs

[0192] Nine SD rats were randomly divided into three groups: Control group, Pep group, and FCSP@NFs group. Each rat was instilled with 200 μL per day. The rats in the Control group were instilled with normal saline. After 7 consecutive days of administration, the rats were anesthetized and sacrificed, and their hearts, livers, spleens, lungs, and kidneys were dissected and fixed in 4% paraformaldehyde for more than 48 h. After paraffin embedding, tissue sectioning, and H&E staining of the fixed tissues, the tissue morphological structure was observed under a microscope to evaluate the safety of Pep and FCSP@NFs on the major organs of SD rats.

[0193] 3 Experimental results

[0194] 3.1 HPLC methodology for magnolin

[0195] 3.1.1 Specificity

[0196] The HPLC results ( Figure 6 ) showed that the retention times of magnolin and polypeptide were 18.143 and 8.632 min, respectively, and the peak positions did not interfere with each other, proving that the specificity of this method meets the requirements of quantitative detection.

[0197] 3.1.2 Standard curve

[0198] A standard curve was plotted with the magnolin concentration as the abscissa and the peak area as the ordinate. The linear regression equation was: y = 18007x + 26372, R 2 = 0.9998, showing a good linear relationship within the range of 1.95 - 500 μg / mL.

[0199] 3.1.3 Accuracy, precision, and stability

[0200] The results of method precision, repeatability, and stability are shown in Table 3. The RSDs of precision and repeatability for detecting magnolin at the concentrations of 1.95, 62.5, and 500 μg / mL were all less than 2%, and the RSDs of stability were all less than 5%, indicating that the detection method meets the requirements of quantitative detection.

[0201] Table 3 Precision, accuracy, and stability of the in vitro analysis method for magnolin

[0202]

[0203] 3.2 GC methodology for eucalyptol

[0204] 3.2.1 Specificity

[0205] The GC results were as Figure 7 shown. The retention time of eucalyptol was 8.561 min, and the elution time of the ethyl acetate solvent peak was 3.311 min. The peak positions did not interfere with each other, proving that the specificity of this method meets the requirements of quantitative detection.

[0206] 3.2.2 Standard curve

[0207] A standard curve was plotted with the eucalyptol concentration on the abscissa and the peak area on the ordinate. The linear regression equation was: y = 14.54x + 33.41, R 2 = 0.999, and there was a good linear relationship within the range of 1.95 - 500 μg / mL.

[0208] The results of method precision, repeatability, and stability are shown in Table 4. The RSDs of precision and repeatability for magnolol detection at the concentrations of 1.95, 62.5, and 500 μg / mL were all less than 2%, and the RSDs of stability were all less than 5%, indicating that the detection method met the requirements of quantitative detection.

[0209] Table 4 Precision, repeatability, and stability of the in vitro analysis method of eucalyptol

[0210]

[0211]

[0212] 3.3 Preparation and drug loading of FCSP@NFs

[0213] The experimental results are shown in Table 5. The drug loadings of magnolol and eucalyptol were 3.5% and 13.09% respectively, achieving the co - loading of magnolol and eucalyptol. In addition, for the poorly soluble magnolol (solubility of 0.048 mg / mL), the construction of FCSP@NFs increased its solubility in the aqueous environment by 8.75 times.

[0214] Table 5 Drug loading of FCSP@NFs

[0215] Component Drug concentration (mg / mL) Drug loading capacity (%) Eucalyptol 1.57±0.04 13.09±0.33 Magnolol 0.42±0.02 3.5±0.16

[0216] 3.4 Stability of FCSP@NFs

[0217] Stability is an important prerequisite for ensuring the biological activity of volatile oils. Therefore, a stability experiment was carried out to evaluate the stability of eucalyptol. The results are as Figure 8 shown. At room temperature, the stable time of eucalyptol (drug content greater than 90%) was greater than 48 h, and the stable time of eucalyptol after loading was about 16 times more than that of free eucalyptol, indicating that the polypeptide drug - loading improved the stability of eucalyptol.

[0218] 3.5 FTIR analysis of FCSP@NFs

[0219] As Figure 9 shown, after loading, the aromatic ether bond representing magnolol (1027 cm -1) and the absorption peaks of the cyclic ether in eucalyptol (984 cm -1 ) are shifted to 1016 cm and 950 cm respectively in the infrared spectrum of FCSP@NFs -1 . Compared with Pep, the characteristic absorption peaks at 1202 cm and 1140 cm that should originally belong to Pep in the infrared spectrum of CSP@NFs -1 almost disappear, confirming that FCSP@NFs has successfully achieved the co-loading of the two drugs through the intermolecular interaction between Pep and the drug (magnolin and eucalyptol) molecules. 3.6 DSC analysis of FCSP@NFs -1 -1 -1 . The DSC results show that the characteristic crystal form transformation peak (137 °C) of magnolin and the endothermic peak of eucalyptol volatilization (114 °C) do not appear in the DSC of FCSP@NFs, indicating that FSCP@NFs can change the crystal form structure of magnolin and reduce the volatilization of eucalyptol. And compared with Pep, the thermodynamic behavior of FSCP@NFs is similar and stable in the temperature range of 30 - 200 °C, indicating that FSCP@NFs has achieved the stable loading of the two drugs.

[0220] 3.7 Assembly morphology of FCSP@NFs

[0221] The assembly morphology of FCSP@NFs is similar to that of Pep, still presenting a cross-linked fiber network structure, indicating that its morphology remains unchanged after loading magnolin and eucalyptol into the hydrophobic cavity of the self-assembled polypeptide.

[0222]

[0222]

[0223] 3.8 Nasal mucosa irritation

[0224] As Figure 10 shown, after continuous nasal drops for 7 d and 14 d, the nasal mucosa epithelial structure of the rats in the FCSP@NFs group is intact, the cilia are continuous and without shedding, and no obvious inflammatory cell infiltration, bleeding, edema or necrosis are seen, which is similar to the normal nasal mucosa, indicating that FCSP@NFs has no obvious irritation to the nasal mucosa.

[0225] 3.9 Safety of major organs

[0226] Rats were administered for one week to evaluate the safety of FCSP@NFs on the major organs of rats( Figure 11 ). Compared with the normal group, the heart tissue of the rats in the FCSP@NFs group shows a regular myocardial fiber structure, the hepatic lobule structure is intact, and no obvious inflammatory damage, edema and other pathological changes are seen in the spleen, lung, kidney, etc., indicating that FCSP@NFs has good biosafety.

[0227] In this invention, a multi-component delivery system of magnolia officinalis, FCSP@NFs, was prepared by stirring combined with probe sonication method, and its drug loading capacity, room temperature stability, TEM morphology, DSC spectrum, etc. were investigated. The results of drug loading capacity and stability showed that the construction of FCSP@NFs increased the solubility of magnolol in aqueous environment by 8.75 times, and the stable time of eucalyptol after being loaded by FCSP@NFs at room temperature was about 16 times longer than that of free eucalyptol, indicating that the construction of FCSP@NFs was beneficial to improve the solubility and stability of the loaded substances and was expected to achieve the long-term effect of drugs. FTIR and DSC were used to prove that magnolol and eucalyptol stably existed inside the FCSP@NFs delivery system, in which magnolol was in an amorphous state, and at the same time, it was shown that the construction of FCSP@NFs improved the thermal stability of eucalyptol. TEM results showed that after loading magnolol and eucalyptol into the hydrophobic cavity of self-assembled polypeptide, FCSP@NFs still presented a cross-linked fiber network structure. Finally, nasal mucosa irritation and organ safety experiments showed that FCSP@NFs had good safety for nasal administration.

[0228] Example 3 Study on the physical protection function of the multi-component delivery system of magnolia officinalis

[0229] 1 Experimental method

[0230] 1.1 Cell culture

[0231] Preparation of MEM complete medium: Add 50 mL fetal bovine serum and 5 mL double antibody solution to 445 mL MEM single medium, and mix well.

[0232] Cell resuscitation: Take out the cryopreserved HNEPC cell cryopreservation tube from liquid nitrogen, quickly put it into a 37°C water bath and shake it quickly to melt. Then transfer the cell suspension to a 10 mL centrifuge tube pre-added with 6 mL complete medium in a laminar flow hood, centrifuge at 1000 rpm for 5 min, carefully discard the supernatant, add 5 mL complete medium to resuspend the cells, transfer them to a cell culture flask, and culture them in an incubator at 37°C and 5% CO2.

[0233] Cell passage: When the cells grow to 80%-90%, discard the old medium, wash the cells 3 times with PBS buffer, and then add 1 mL of 0.25% trypsin. Observe the cells under a microscope. When the cell morphology changes and the gap increases, quickly add 2 mL complete medium to terminate digestion, gently pipette the cell layer to blow it off and disperse it, transfer it to a centrifuge tube, centrifuge at 1000 rpm for 5 min, discard the supernatant, add MEM complete medium to resuspend the cells, and transfer the cell suspension to a new cell culture flask at a ratio of 1:2, and culture it in an incubator at 37°C and 5% CO2.

[0234] 1.2 Cytotoxicity

[0235] Take HNEPC cells in the logarithmic growth phase and seed 1×10 4 cells per well in a 96-well plate. Culture the cells in an incubator at 37°C and 5% CO2 for 24 h. For each drug administration group, calculate the amount of Pep as the calculated dose, use MEM single medium as the solvent, and prepare FCSP@NFs solutions and Pep solutions with polypeptide concentrations of 0.75, 3.75, 7.5, 37.5, 75, and 375 μg / mL. After the cells adhere to the wall, aspirate the culture medium, wash the cells 3 times with PBS, add 100 μL of FCSP@NFs solutions and Pep solutions with different concentrations to each well, repeat 6 wells for each concentration, and incubate at 37°C and 5% CO2 for 24 h. After the drug administration is completed, aspirate the supernatant, wash 3 times with PBS, then add 100 μL of 10% CCK-8 solution prepared with MEM single medium to each well, and incubate at 37°C in the dark for 2 h. Measure the absorbance value at a wavelength of 490 nm with an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the survival rate of HNEPC cells.

[0236] Cell survival rate = (ODexperiment - ODblank) / (ODcontrol - ODblank) × 100%

[0237] 1.3 Investigation of the assembly morphology of FCSP@NFs in HNEPC cells

[0238] Take HNEPC cells in the logarithmic growth phase and seed 2×104 cells per well in a 48-well plate pre-placed with cell slides. Culture the cells in an incubator at 37°C and 5% CO2. After the cells adhere to the wall, discard the old culture medium, wash 3 times with PBS, add 200 μL of MEM single medium and FCSP@NFs solution (with a polypeptide concentration of 0.375 mg / mL) respectively, and incubate in an environment of 37°C and 5% CO2 for 24 h. After the drug administration is completed, aspirate the supernatant and wash 3 times with PBS. Then, take out the cell slides from the well plate and fix them in 2.5% glutaraldehyde electron microscopy solution for more than 48 h, and perform SEM photography on the HNEPC cell samples using a SU8010 scanning electron microscope.

[0239] 1.4 Measurement of transepithelial electrical resistance

[0240] HNEPC cells in the logarithmic growth phase were seeded at a density of 2×104 cells / well in the upper chamber of a transwell in a 24-well plate. 200 μL of cell suspension was added to the upper chamber of the transwell, and 600 μL of complete MEM medium was added to the lower chamber. The cells were cultured in an incubator at 37°C and 5% CO2. There were a total of 4 groups, namely the Control group, the HDM group, the FCSP@NFs group, and the Pep group. After the cells had fused to a monolayer, they were washed 3 times with PBS. In the upper chamber of the transwell in the Control group and the HDM group, MEM single medium was added. In the upper chamber of the FCSP@NFs group and the Pep group, 200 μL of FCSP@NFs and Pep solution (with a polypeptide concentration of 0.375 mg / mL) was added, and 600 μL of MEM single medium was added to the lower chamber of all groups. The cells were incubated in an incubator at 37°C and 5% CO2. After 24 hours of drug administration, the cells were washed 3 times with PBS. In the upper chamber of the Control group, 200 μL of MEM single medium was added. In the upper chamber of the other three groups, 200 μL of 10 μg / mL HDM solution was added, and 600 μL of MEM single medium was added to the lower chamber of all groups. The cells were placed in an incubator at 37°C and 5% CO2. After 24 hours of allergen stimulation, the solutions in the upper and lower chambers were aspirated, and the cells were washed 3 times with PBS. MEM single medium (300 μL in the upper chamber and 800 μL in the lower chamber of the transwell) was added. The epithelial cell resistance value was measured using a Millipore millicell ESR-2 according to the instructions.

[0241] TEER = (TEER cell -TEER blank ) / effective filter membrane area

[0242] (where TEERcell is the TEER reading of the seeded cells; TEERblank is the TEER reading of the blank insert)

[0243] 1.5 Measurement of paracellular pathway

[0244] After 24 hours of allergen stimulation, the solutions in the upper and lower chambers were aspirated, and the cells were washed 3 times with PBS. 200 μL of 0.5 mg / mL FITC-Dextran solution prepared with phenol red-free MEM single medium was added to the upper chamber of the transwell, and 800 μL of phenol red-free MEM single medium was added to the lower chamber. The cells were incubated in an incubator at 37°C and 5% CO2 for 2 hours. 100 μL of the solution was aspirated from the lower chamber and added to a black microplate. The absorbance value was measured using a microplate reader (excitation at 495 nm and emission at 520 nm), and the FITC-Dextran permeability was calculated.

[0245] Relative permeability = OD value of sample well / OD value of normal well × 100%

[0246] 1. Detection of the expression level of 1.6HNEPC epithelial barrier protein

[0247] 1.6.1 Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0248] (1) Stimulation with allergens: HNEPC cells in the logarithmic growth phase were seeded at 5×104 cells / well in a 24-well plate and cultured in an incubator at 37°C and 5% CO2. There were a total of 4 groups, namely the Control group, the HDM group, the FCSP@NFs group, and the Pep group. After the cells reached confluence as a monolayer, they were washed 3 times with PBS. The Control group and the HDM group were added with MEM single medium, and the FCSP@NFs group and the Pep group were added with 500 μL of FCSP@NFs and Pep solution (where the polypeptide concentration was 0.375 mg / mL). After 24 hours of drug administration, they were washed 3 times with PBS. The Control group was added with 500 μL of MEM single medium, and the other three groups were added with 500 μL of HDM solution (10 μg / mL) per well. After 24 hours of allergen stimulation, they were washed 3 times with PBS, digested with trypsin, centrifuged at 1000 rpm for 5 minutes, and the precipitate was collected for RT-qPCR detection.

[0249] (2) RNA extraction: 100 μL of Dilution Buffer was added to the above lysis solution, vortexed until the solution was completely mixed, allowed to stand at room temperature for 5 minutes, then centrifuged at 11200 rpm at room temperature for 15 minutes. The centrifuge tube was taken out, and 500 μL of the supernatant was aspirated into a new centrifuge tube. An equal volume of isopropanol was added, and the centrifuge tube was inverted several times to mix evenly, allowed to stand at room temperature for 10 minutes, then centrifuged at 11200 rpm at room temperature for 10 minutes, and the supernatant was discarded, retaining the precipitate. 1 mL of 75% ethanol (prepared with RNase-free ddH2O) was added, and the precipitate was gently pipetted with a pipette until it was suspended, then inverted several times, centrifuged at 9100 rpm at room temperature for 3 minutes, the supernatant was discarded, retaining the precipitate. The above operation was repeated, and 75% ethanol was added again to remove impurities. After centrifugation, all the supernatant was discarded. It was left to dry at room temperature, and 20 μL of RNase-free ddH2O was added to dissolve the precipitate, and vortexed at room temperature for 3 minutes to dissolve the RNA precipitate.

[0250] (3) RNA reverse transcription:

[0251] Component Volume Total RNA 1 μg 4×gDNA wiper mix 4 μL <![CDATA[RNase-free ddH2O]]> To 16 μL 5×HiScriptII qRT SuperMixII 4 μL

[0252] Reaction temperature and time: 42°C, 2 minutes, 50°C, 15 minutes, 85°C, 5 seconds.

[0253] (4) Real-time fluorescence quantitative PCR

[0254] ① Prepare the following mixed solution in the RT-qPCR tube

[0255] Reagent Volume 2×ChamQ Universal SYBR qPCR Master Mix 10.0 μL Primer1 (10 μM) 0.4 μL Primer2 (10 μM) 0.4 μL Template DNA / cDNA 1.0 μL <![CDATA[ddH2O]]> To 20.0 μL

[0256] ②Perform RT-qPCR reactions according to the following conditions

[0257]

[0258] 1.6.2 Immunoblotting (WB)

[0259] (1) Allergen stimulation: Perform experimental operations according to the method in 1.6.1.

[0260] (2) Extraction of cellular proteins: Add an appropriate amount of RAPI cell lysis buffer (in a ratio of 100:1 with protease inhibitor) to the cells and lyse them on ice for 30 min. Use a cell scraper to scrape the cells in different directions and collect them into a centrifuge tube. Centrifuge at 13000 rpm for 20 min to remove the precipitate.

[0261] (3) Determine the protein concentration by the BCA method: Prepare an appropriate amount of BCA working solution according to the product manual. Add a certain amount of BSA, 1×PBS, and BCA working solution to the 96-well plate in sequence to draw a standard curve. Take 20 μL of the sample and add 200 μL of BCA working solution to the 96-well plate, and mix well. Incubate at 37 °C for 30 min, and measure the OD value (A = 562 nm) with an enzyme-linked immunosorbent assay (ELISA) reader. Calculate the cellular protein concentration according to the drawn standard curve.

[0262] (4) Protein denaturation: Add the corresponding amount of 5× loading buffer to the protein solution according to a volume ratio of protein to loading buffer of 4:1, and perform metal bath at 100 °C for 5 min to denature the protein.

[0263] (5) Electrophoresis conditions: Constant voltage of 80 V for the stacking gel for 30 min; constant voltage of 120 mV for the separating gel, and electrophorese until the bromophenol blue reaches the end of the electrophoresis tank.

[0264] (6) Blotting conditions: Constant current of 300 mA, and the blotting times for ZO-1 and Occludin proteins are 1 h and 35 min respectively.

[0265] (7) Blocking: Add protein-free serum blocking solution and block on a shaker for 2 h.

[0266] (8) Primary antibody incubation: After washing the membrane 3 times, add ZO-1 (1:10000) and Occludin (1:100000) antibodies and incubate overnight at 4 °C.

[0267] (9) Secondary antibody incubation: After washing the membrane 3 times, add the secondary antibody (1:10000) and incubate at room temperature for 2 h.

[0268] (10) Protein band imaging: In the dark box, add ECL developing solutions A and B in a volume ratio of 1:1, mix well, add the PVDF membrane, and after 30 s, place the PVDF membrane into the chemiluminescence imaging system for development.

[0269] 1.7 Investigation of the integrity of tight junctions of the cell epithelial barrier

[0270] Perform the allergen stimulation experiment according to the method in 2.6.1, but replace the 24-well plate with a confocal dish for the immunofluorescence experiment. After 24 h of allergen stimulation, add 500 μL of ice-cold methanol (pre-cooled at -20 °C for 2 h) to each confocal dish, fix at 4 °C for 15 min, wash 3 times with PBS, 1 min for each wash. Add the blocking solution, block at room temperature for 30 min, then add 200 μL of the primary antibody to each well and incubate overnight at 4 °C (the dilution ratios of Occludin and ZO-1 antibodies are 1:100). Aspirate the primary antibody, wash 3 times with PBS, add 200 μL of the fluorescent secondary antibody to each well, and incubate at room temperature in the dark for 1 h. Aspirate the secondary antibody, wash 3 times with PBS, add a mounting medium containing DAPI to the confocal dish, and perform immunofluorescence imaging using a Nikon AX laser confocal microscope.

[0271] 2 Experimental results

[0272] 2.1 Cytotoxicity

[0273] As Figure 12 shown, calculated based on the polypeptide concentration, after administering HNEPC cells with Pep and FCSP@NFs within the polypeptide concentration range of 0.75 - 375 μg / mL, the cell survival rate was above 90%. This indicates that Pep as a carrier material and FCSP@NFs as a novel delivery system have no obvious toxicity to cells and good biocompatibility.

[0274] 2.2 Assembly morphology of FCSP@NFs in HNEPC cells

[0275] To explore whether FCSP@NFs can form the expected physical protection network structure on the cell surface, we examined its morphology on human nasal mucosa epithelial cells by SEM. The morphology of HNEPC cells in the FCSP@NFs group was similar to that of HNEPC cells in the normal group, with natural extension of microvilli. At the same time, FCSP@NFs could form a network covering on the cell surface, meeting the expected morphology.

[0276] 2.3 Trans-epithelial electrical resistance value

[0277] As Figure 13As shown, there was no significant difference in TEER between the FCSP@NFs group and the Control group, indicating that the tight junctions were intact. For the HDM group, however, the TEER decreased significantly, which was the result of allergen-induced damage to the tight junctions and subsequent disruption of epithelial barrier integrity. Compared with the HDM group, the TEER value of the Pep group was 1.31 times that of the HDM group, suggesting that the single fiber protection network could intercept allergens to a certain extent and reduce the damage of allergens to the tight junctions of the epithelial barrier. Compared with the FCSP@NFs group, the TEER value of the Pep group was significantly decreased, indicating that the loaded drug could contribute to the repair of tight junctions.

[0278] 2.4 Paracellular pathway leakage of FITC-Dextran

[0279] The effect of FCSP@NFs on the permeability of the nasal mucosa epithelial barrier was investigated using FITC-Dextran leakage as the detection index, and the results are as Figure 14 shown. Compared with the Control group, the permeability of FITC-Dextran in the HDM group increased significantly, indicating that HDM damaged the epithelial tight junctions, enhanced permeability, and promoted more FITC-Dextran to penetrate the epithelial barrier (upper layer), resulting in the highest leakage detected in the lower layer. Compared with the HDM group, after treating cells with Pep and FCSP@NFs, the FITC-Dextran penetration amounts decreased significantly by 24.99% and 33.55% of the HDM group, respectively. In addition, there was no significant difference in the penetration amount between the FCSP@NFs group and the Control group, indicating that FCSP@NFs could maintain the normal permeability of the epithelial barrier.

[0280] 2.5 Expression levels of epithelial barrier tight junction proteins in HNEPC cells

[0281] 2.5.1 Detection of ZO-1 and Occludin mRNA expression levels by RT-qPCR

[0282] The results showed ( Figure 15 ) that compared with the Control group, the invasion of HDM led to downregulation of the mRNA expressions of Occludin and ZO-1, which was not conducive to epithelial tight junctions. After administration of FCSP@NFs, the expression levels of ZO-1 and Occludin in HNEPC cells were 3.15 times and 3.23 times that of the HDM group, respectively, and FCSP@NFs could promote the expression levels of Occludin and ZO-1 to return to normal levels, indicating that FCSP@NFs could protect the nasal mucosa from the effects of HDM. The expression level in the Pep treatment group was significantly lower than that in the FCSP@NFs group, indicating that the barrier protection ability of Pep was weaker than that of FCSP@NFs.

[0283] 2.5.2 WB detection of the expression levels of ZO-1 and Occludin proteins

[0284] HDM is a widely recognized common allergen that triggers AR. Its allergenic protein, Der p 1, has cysteine protease activity and can cleave the extracellular domains of proteins such as Occludin and ZO-1, reducing the expression of tight junction proteins, disrupting the tight junctions between adjacent cells, increasing barrier permeability, and destroying the integrity of the epithelial barrier, thereby inducing AR. As Figure 16 shown, due to the stimulation of HDM, the expressions of tight junction proteins Occludin and ZO-1 were also significantly decreased compared with the Control group. Compared with the HDM group, the expressions of Occludin and ZO-1 in the Pep group and the FCSP@NFs group were significantly up-regulated. In particular, there was no significant difference in the expression levels of the tight junction proteins Occludin and ZO-1 in the FCSP@NFs group and the protein expression levels in the Control group, indicating that FCSP@NFs can defend against the stimulation of house dust mites and protect the nasal mucosal epithelial barrier.

[0285] 2.6 Integrity of tight junctions of the epithelial barrier in HNEPC cells

[0286] Immunofluorescence staining of ZO-1 and Occludin was used to visually demonstrate that FCSP@NFs can effectively maintain the tight junctions between cells. The tight junctions of FCSP@NFs were complete and continuous, consistent with the normal group, while the tight junctions in the HDM group were extensively disrupted. Although the tight junctions of cells in the Pep group were relatively complete, there were intermittent small disruptions. This indicates that FCSP@NFs protects the tight junctions between cells and maintains the integrity of the epithelial barrier by maintaining the normal expression of Occludin and ZO-1.

[0287] The present invention demonstrates the function and mechanism of the FCSP@NFs delivery system in physically protecting the nasal epithelial barrier through systematic in vitro experiments. The SEM results show that FCSP@NFs can successfully form the expected physical protection network on the surface of HNEPC cells. The results of transepithelial electrical resistance measurement show that the TEER value of the FCSP@NFs group is not different from that of the Control group, indicating that it can protect the integrity of the epithelial barrier. The TEER value of the Pep group without loaded drug is higher than that of the HDM group but lower than that of the FCSP@NFs group, suggesting that a single fiber network needs to be combined with drug treatment to effectively defend against epithelial barrier damage. The results of the paracellular pathway permeability experiment show that the FCSP@NFs group and the Pep group can reduce the leakage of FITC-Dextran, and the FITC-Dextran permeability of the FCSP@NFs group has no significant difference from that of the normal group, indicating that FCSP@NFs can inhibit the enhanced permeability of the epithelial barrier by HDM due to its dual effects. It is demonstrated by RT-qPCR and WB experiments that FCSP@NFs can maintain the normal expression of tight junction proteins Occludin and ZO-1 and protect the tight junctions between cells. Further immunofluorescence imaging confirms that FCSP@NFs can protect HNEPC cells from allergen stimulation, thereby maintaining the continuity and integrity of tight junctions between cells.

[0288] Pharmacodynamic Study of the Magnolia Multicomponent Delivery System in Example 4

[0289] 1. Experimental Animals

[0290] Male SD rats, weighing 180 - 220 g, were purchased from Qinglongshan Biotechnology Co., Ltd., Jiangsu Province, with the license number: SCXK(Su)2024 - 0001.

[0291] 2. Experimental Methods

[0292] 2.1 Establishment of the Allergic Rhinitis Model

[0293] According to the preparation specification of the allergic rhinitis animal model [1] and adding the OVA aerosol challenge method, an AR rat model was constructed. In the basic sensitization stage, each rat was intraperitoneally injected with 1 mL of physiological saline solution containing 0.3 mg of OVA and 30 mg of aluminum hydroxide, once every other day, for a total of 7 times. On the 15th day of model establishment, the rats were challenged. After 50 μL of 3% OVA physiological saline was instilled into each nasal cavity of the rats, they were aerosolized with 5 mg / mL OVA solution for 30 min, once a day, for 7 consecutive days. After the last challenge, the mice were observed behaviorally for 30 min, and the symptom scores were evaluated according to the biological behaviors such as the frequency of nose scratching, the number of sneezes, and nasal discharge. A total score > 5 points indicates successful model establishment.

[0294] Table 6 Scoring Criteria for the Biological Behaviors of AR Rats

[0295] Integration / minute Nose scratching (times) Sneezing (times) Runny nose condition 1 1-5 1-3 Runny nose to the anterior naris 2 6-15 4-10 Runny nose out of the anterior naris 3 ＞15 ＞11 Runny nose covering the face

[0296] 2.2 Administration and Behavioral Evaluation

[0297] Table 7 Nasal Administration Dosage of AR Rats

[0298]

[0299]

[0300] The 36 successfully constructed AR model rats were randomly divided into groups of 6 each. The specific experimental grouping and administration dosage are shown in Table 6. Each group was given the corresponding drug solution (200 μL / rat) via nasal cavity 1 h before OVA challenge every day for 7 consecutive days. 1 h after the last administration, OVA challenge was performed, and the number of allergic behaviors such as nose scratching, sneezing, and rhinorrhea in rats within 30 min was recorded. After the behavioral recording was completed, the animals were anesthetized and sacrificed, and the nasal mucosa tissues were taken out. Part of them was quickly frozen in liquid nitrogen and stored at -80 °C for later use, and part of them was fixed in 4% paraformaldehyde for more than 48 h, and after paraffin embedding and tissue section processing, they were reserved for use.

[0301] 2.3 Detection of the Expression Levels of Inflammatory Factors TNF-α and IL-6 in Nasal Mucosa of Rats

[0302] The experimental procedures of RT-qRCR and WB were the same as those in Example 3. The WB membrane transfer conditions for TNF-α and IL-6 were both constant current of 200 mA for 30 min, and the dilution ratios were both 1:1000.

[0303] 2.4 Detection of the Expression Levels of IL-4, IL-13 mRNA and GATA 3, STAT 6 Pathway Proteins in Nasal Mucosa of Rats

[0304] The experimental procedures of RT-qRCR and WB were the same as those in Example 4. The WB membrane transfer conditions for GATA3 and STAT6 were both constant current of 300 mA for 40 min, and the dilution ratios were both 1:10000.

[0305] 2.5 Detection of the Expression Levels of Tight Junction Proteins Occludin and ZO-1 in Nasal Mucosa of Rats

[0306] The experimental procedures of RT-qRCR and WB were the same as those in Example 3.

[0307] 2.6 Pathological Examination of Nasal Mucosa of Rats

[0308] 2.6.1 H&E Staining

[0309] 2.6.2 Toluidine Blue Staining

[0310] The paraffin sections were successively placed in xylene Ⅰ for 20 min → xylene Ⅱ for 20 min → absolute ethanol Ⅰ for 5 min → absolute ethanol Ⅱ for 5 min → 75% alcohol for 5 min → rinsed with water for 2 min → toluidine blue staining solution for 5 min → rinsed with water until clear → differentiated with 1% glacial acetic acid → cleared with xylene for 5 min → sealed with neutral gum → examined under a microscope, and the images were collected and analyzed.

[0311] 2.6.3 Immunofluorescence

[0312] The paraffin sections were successively dewaxed with xylene Ⅰ and Ⅱ for 15 min each, hydrated with gradient ethanol (100% - 75%) and then rinsed with distilled water; subsequently, EDTA microwave antigen repair was performed (medium fire for 8 min → stop fire for 8 min → medium-low fire for 7 min), and after natural cooling, it was washed three times with PBS (5 min each time); after the tissue area was circled with a histochemical pen, autofluorescence quenching (5 min), 5% BSA blocking (30 min), and primary antibody incubation overnight at 4℃ in a wet box were carried out in sequence; the next day, after washing with PBS, secondary antibody was added dropwise and incubated in the dark for 50 min, and the nucleus was stained with DAPI for 10 min; finally, after washing with PBS, it was sealed with an anti-fluorescence quenching agent and stored at 4℃ in the dark.

[0313] 3 Experimental methods

[0314] 3.1 Biological behavior evaluation

[0315] After continuous administration for 7 days, the symptom scores of each group were recorded and statistically analyzed ( Figure 17 ). Compared with the Model group, FCSP@NFs could improve the nose-rubbing, sneezing and rhinorrhea conditions of AR rats, and the total score decreased from 6.0 to 2.0. The scores of the other treatment groups were all greater than 3.0, indicating that FCSP@NFs had the most obvious improvement on allergic rhinitis.

[0316] 3.2 Expression levels of inflammatory factors TNF-α and IL-6 in rat nasal mucosa

[0317] 3.2.1 Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0318] The RT-qPCR results are shown in the figure ( Figure 18), compared with the Model group, after 7 days of continuous nasal administration, the inflammation levels of each treatment group were significantly reduced, among which the FCSP@NFs treatment group had the largest reduction in inflammatory factor levels, with its TNF-α and IL-6 mRNA expression levels significantly reduced by 87.11% and 72.59% of the model group, respectively. In particular, its TNF-a mRNA expression was not significantly different from that of the normal group, which strongly proves that FCSP@NFs can significantly inhibit AR inflammation. Further comparison with the Model, both the FC group and the Pep group were able to reduce TNF-a and IL-6 mRNA expression, respectively verifying the effectiveness of drug treatment and physical interception in improving AR inflammation. However, compared with the Model group, although the FCSP-Blend treatment group reduced the expression of inflammatory factors in the nasal mucosa of AR rats, its expression level was significantly higher than that of the FCSP@NFs treatment group, which further emphasizes the long-term advantage of FCSP@NFs in the nose.

[0319] 3.2.2 Western blotting (WB)

[0320] WB analysis results ( Figure 19 Consistent with RT-qPCR results, compared to the model group, the expression levels of inflammatory proteins TNF-α and IL-6 decreased in the FC, Pep, and FCSP-Blend treatment groups, but remained higher than those in the FCSP@NFs group. FCSP@NFs, through its unique dual-action mechanism, demonstrated optimal inhibitory effects on inflammatory protein expression, reducing protein expression of the inflammatory factors TNF-α and IL-6 by 52.34% and 38.02%, respectively, compared to the model group. These levels of inflammatory expression remained similar to normal levels, demonstrating that the dual-action effect of FCSP@NFs contributes to the effective treatment of AR inflammation.

[0321] 3.3 Expression levels of IL-4, IL-13 mRNA and GATA 3, STAT 6 pathway proteins in rat nasal mucosa

[0322] 3.3.1 Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0323] The RT-qPCR results are shown in the figure ( Figure 20) Compared with the Model group, the expression levels of IL-4 and IL-13 were decreased in each treatment group. Among them, the FCSP@NFs group had the largest downregulation, which were decreased by 57.69% and 63.63% respectively compared with the Model group, and there was no difference from the normal group, fully demonstrating the excellent AR allergy treatment effect of FCSP@NFs. In particular, the FC, Pep, and FCSP-Blend treatment groups had comparable effects to the positive drug in downregulating the expression levels of IL-4 and IL-13, indicating that the fiber network structure composed of drugs and polypeptides had comparable treatment effects on AR immune response to clinically commonly used drugs, and both could effectively improve the allergic reaction of AR. However, the expression levels of IL-4 and IL-13 in these three treatment groups were significantly higher than those in the FCSP@NFs group, and this result was consistent with the results of FCSP@NFs in inflammatory treatment, further providing strong evidence for the dual effect of FCSP@NFs in the highly effective treatment of AR.

[0324] 3.3.2 Western Blot (WB)

[0325] The results of WB are shown in Figure ( Figure 21 ) and showed that the expression levels of STAT6 and GATA3 in the nasal mucosa of the FCSP@NFs group were the lowest,

[0326] which were decreased by 43.48% and 48.19% of the Model group, and 38.69% and 24.50% of the Positive Drug group respectively, and both were restored to normal levels, indicating that FCSP@NFs could effectively inhibit the allergic response. The FC, Pep, and FCSP-Blend treatment groups had comparable effects to the Positive Drug group in downregulating the expression level of STAT6, and this result was consistent with the RT-qPCR results. This indicated that the dual effect of FCSP@NFs inhibited the activation of STAT6, downregulated the expression of GATA3, and thus reduced the secretion of Th2 cytokines IL-4 and IL-13, significantly improving the allergic immune response of AR.

[0327] 3.4 Expression levels of tight junction proteins Occludin and ZO-1 in rat nasal mucosa

[0328] 3.4.1 Real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-qPCR)

[0329] The results of RT-qPCR are shown in Figure ( Figure 22) As shown, except for the FCSP@NFs and Pep treatment groups, there were no significant differences in the expression levels of ZO-1 and Occludin mRNA in the remaining treatment groups compared with the model group. This indicates that the fibrous structure of the polypeptide can significantly repair the nasal mucosal barrier damage, emphasizing the effectiveness of physical interception of allergens as an AR protection strategy. However, compared with the Model group, although both the FCSP@NFs and Pep treatment groups could significantly increase the mRNA expression levels of the tight junction proteins ZO-1 and Occludin, only the expression level of the scaffolding protein ZO-1 mRNA in the FCSP@NFs treatment group was close to the normal physiological level. This shows that only on the basis of the Pep fibrous structure providing physical interception of allergens and combined with the slow release of magnolol and eucalyptol can the significant repair of epithelial barrier damage be achieved to the normal level.

[0330] 3.4.2 Immunoblotting (WB)

[0331] As shown by the WB results, compared with the Model group, the protein expression levels of ZO-1 and Occludin in FCSP@NFs were the highest (P < 0.01), being 4.20 times and 2.56 times that of the model group respectively, and there was no difference from the normal level. Combining with the ability of FCSP@NFs to physically intercept allergens demonstrated in in vitro experiments, it fully proved that FCSP@NFs could effectively maintain the structure and function of the nasal mucosal epithelial barrier.

[0332] 3.5 Histopathological analysis of rat nasal mucosa

[0333] 3.5.1 H&E staining

[0334] By analyzing the inflammatory pathology of nasal mucosal tissues through H&E staining, the nasal mucosal tissue structure of FCSP@NFs was complete, similar to the normal group, and almost no EOS infiltration was observed. While in the model group, cilia were disordered and shed, and a large number of inflammatory cells infiltrated, indicating that FCSP@NFs could effectively inhibit the infiltration of inflammatory cells.

[0335] 3.5.2 Toluidine blue staining

[0336] By analyzing the changes of mast cells in nasal mucosal tissues through toluidine blue staining, like the normal group, FCSP@NFs had fewer mast cells and almost no degranulation was observed. However, the number of mast cells in the remaining treatment groups increased. The model group had the largest number of mast cells and obvious degranulation, indicating that FCSP@NFs could inhibit mast cell degranulation and the effect was comparable to that of the normal group.

[0337] 3.5.3 Immunofluorescence

[0338] The effect of FCSP@NFs on the tight junctions of the nasal mucosal epithelial barrier in rats was further investigated by immunofluorescence experiments. As shown in the figure, the tight junctions in the model group were the most severely damaged, which would exacerbate the inflammation and allergic response of AR. After administration of FCSP@NFs, the nasal mucosal epithelial layer showed continuous and intact tight junctions, similar to the normal group, while small-scale breaks occurred in the other treatment groups, indicating that FCSP@NFs could repair the nasal mucosal barrier damage and maintain the integrity of tight junctions.

[0339] In this example, the therapeutic effect of the FCSP@NFs delivery system on AR was comprehensively explored from multiple dimensions. First, behavioral assessment showed that the total scores of nose scratching, sneezing and rhinorrhea in the AR rats in the FCSP@NFs treatment group were significantly reduced from 6.0 in the Model group to 2.0, which was significantly better than the other treatment groups. Next, the AR inflammation treatment study found that FCSP@NFs could significantly down-regulate the expression of TNF-α and IL-6 inflammatory factors, reducing by 87.11% and 72.59% of the Model group respectively, and the expression levels of inflammatory factors were significantly lower than those in the FC, Pep, and FCSP-Blend groups. H&E staining further confirmed that it could effectively inhibit the infiltration of inflammatory cells, indicating that while physically defending against the continuous stimulation of allergens, FCSP@NFs slowly released magnolol and eucalyptol with anti-inflammatory activity to treat AR, significantly reducing the secretion of inflammatory factors and showing the best anti-inflammatory ability. In terms of regulating Th2-type allergic reactions, the FCSP@NFs had the strongest allergy inhibitory effect (the expression levels of IL-4, IL-13, STAT6, and GATA3 decreased by 57.69%, 63.63%, 43.48%, and 48.19% respectively compared with the Model group), and there was no significant difference from the normal group. Toluidine blue staining verified that its effect on inhibiting mast cell degranulation was equivalent to that of the normal group, indicating that the dual effect of FCSP@NFs regulated the balance of Th1 and Th2 cells by inhibiting the activation of STAT6 and down-regulating the expression of GATA3, thereby reducing the secretion of allergic inflammatory mediators IL-4 and IL-13 and significantly improving the allergic immune response of AR. Finally, the role of FCSP@NFs in nasal epithelial barrier repair was also explored in this chapter. By up-regulating the expression of tight junction proteins ZO-1 and Occludin (the protein expression levels were 4.20 and 2.56 times that of the Model group respectively), FCSP@NFs successfully repaired the tight junction structure of the nasal mucosal epithelial layer. The results of immunofluorescence experiments also observed that after administration of FCSP@NFs, the damaged nasal mucosal epithelial barrier recovered to a continuous tight junction structure, indicating that FCSP@NFs had the best ability to repair nasal mucosal damage and could accelerate the recovery of the nasal epithelial barrier to normal.

Claims

1. A self-assembling polypeptide for loading multiple components of traditional Chinese medicine, characterized in that, The self-assembling polypeptide is IIFFSSKKGE-Dopa.

2. The application of the self-assembling polypeptide according to claim 1 in loading multi-components of traditional Chinese medicine, so as to improve the solubility of poorly soluble components and enhance the stability of volatile oil components.

3. The application according to claim 2, wherein The poorly soluble components loaded by the self-assembling polypeptide include magnolin and geraniin, and the volatile oil components include eucalyptol; it improves the solubility of magnolin and geraniin and reduces the volatility of eucalyptol.

4. According to the application described in claim 2, the self-assembling polypeptide loaded with multi-components of traditional Chinese medicine realizes long-term nasal adhesion and prolongs the release time of magnolin, geraniin and eucalyptol in the nasal cavity.

5. The self-assembling polypeptide for loading multiple components of traditional Chinese medicine according to claim 1, characterized in that, The self-assembling polypeptide can assemble into a fibrous structure, and wet adhesion can be achieved by modifying Dopa on the polypeptide sequence.

6. A multi-component delivery system of Magnolia officinalis, characterized in that, It is prepared by the following method: (1) Synthesis of the self-assembling polypeptide; (2) Preparation of a multi-component delivery system for magnolia by stirring combined with probe sonication.

7. The magnolia multifunctional delivery system according to claim 6, characterized in that, Step (1) The synthesis of the self-assembling polypeptide includes the following steps: (1) Resin swelling: Weigh accurately Rink-Amide-MBHA resin, place it in a centrifuge tube, add DMF, shake it in an ice bath to swell, transfer it to a polypeptide synthesis tube, and wash it successively with DMF and DCM; (2) Deprotection: Add piperidine and shake it in an ice bath to remove the Fmoc protecting group on the resin. After repeating the deprotection once, wash it with DMF and DCM respectively; Take a small amount of resin for ninhydrin color reaction. After verifying that the deprotection is complete, start the condensation reaction; (3) Condensation reaction: Add the Fmoc-DOPA(acetonide)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake it in an ice bath for reaction for 20 - 30 min, and wash it 3 - 4 times with DMF and DCM respectively; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the first amino acid is completed; (4) Cutting the resin: After the condensation and deprotection of the last amino acid are completed, transfer the resin to a centrifuge tube, add the cutting solution for cutting and cleavage reaction; (5) Ether precipitation: Add the polypeptide solution to ice-cold ether, centrifuge, discard the supernatant, add ice-cold ether, mix and precipitate, then centrifuge again. Repeat this process, and place it in a vacuum drying oven to remove the solvent to obtain the crude polypeptide Pep; (6) Polypeptide purification Dissolve the crude polypeptide Pep in an acetonitrile-water mixed solvent, centrifuge, take the supernatant, purify it by preparative liquid chromatography, and freeze-dry the received solution to obtain the pure polypeptide Pep.

8. The multi-component delivery system of Magnolia officinalis described in claim 7, characterized in that, Step (1) The synthesis of the self-assembling polypeptide includes the following steps: (1) Resin swelling: Weigh accurately Rink-Amide-MBHA resin, place it in a centrifuge tube, add DMF, shake it in an ice bath for 15 - 30 min to swell, transfer it to a polypeptide synthesis tube, and wash it 3 - 4 times successively with DMF and DCM; (2) Deprotection: Add piperidine and shake in an ice bath to remove the Fmoc protecting group on the resin. After repeating the deprotection once, wash with DMF and DCM 3 - 4 times each; Take a small amount of resin for ninhydrin color reaction. After verifying that the deprotection is complete, start the condensation reaction; (3) Condensation reaction: The polypeptide sequence is IIFFSSKKGE - Dopa from the N - terminal to the C - terminal. In the synthesis process, amino resin is used. Therefore, the order of synthesizing amino acids is Dopa, E, G, K, K, S, S, F, F, I, I in turn; ① Dopa: Add the Fmoc - DOPA(acetonide)-OH dopa solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake and react in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the dopa - modified part is completed; ② Glutamic acid (E): Add the Fmoc - Glu(OtBu)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake and react in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the first amino acid is completed; ③ Glycine (G): Add the Fmoc - Gly - OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake and react in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the second amino acid is completed; ④ Lysine (K): Add the Fmoc - Lys(Boc)-OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake and react in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the third amino acid is completed; Continue to repeat this step to synthesize the fourth amino acid K; ⑤ Serine (S): Add the Fmoc - Ser - OH amino acid solution to the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, shake and react in an ice bath for 20 min, and wash with DMF and DCM in turn; Take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the fifth amino acid is completed; Continue to repeat this step to synthesize the sixth amino acid S; ⑥Phenylalanine (F): First, add the first portion of the Fmoc-Phe-Phe-OH amino acid solution into the polypeptide synthesis tube, then add HBTU and DIEA solutions respectively, and react with ice bath shaking for 30 min. Wash with DMF and DCM successively; then add the second portion of the Fmoc-Phe-Phe-OH amino acid solution into the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, and react with ice bath shaking for 30 min. Wash with DMF and DCM successively; take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the seventh and eighth amino acids F is completed; ⑦Isoleucine (I): First, add the first portion of the Fmoc-Ile-OH amino acid solution into the polypeptide synthesis tube, then add HBTU and DIEA solutions respectively, and react with ice bath shaking for 30 min. Wash with DMF and DCM successively; then add the second portion of the Fmoc-Ile-OH amino acid solution into the polypeptide synthesis tube, add HBTU and DIEA solutions respectively, and react with ice bath shaking, and wash with DMF and DCM successively; take a small amount of resin for ninhydrin color reaction. After verifying that the condensation reaction is complete, it indicates that the synthesis of the ninth amino acid is completed; continue to repeat this step to synthesize the tenth amino acid (I); (4) Cleavage of resin: After the condensation and deprotection of the last amino acid are completed, transfer the resin into a centrifuge tube, add the cleavage solution and carry out a cleavage and lysis reaction for 2 - 3 h; (5) Ether precipitation: Add the peptide solution into ice-cold ether, centrifuge, discard the supernatant, add ice-cold ether, mix and precipitate, then centrifuge again. Repeat this process, and place it in a vacuum drying oven to remove the solvent to obtain the crude polypeptide Pep; (6) Polypeptide purification Dissolve the crude polypeptide Pep in a mixed solvent of water and acetonitrile with a volume ratio of 3:2, centrifuge, take the supernatant, purify it by preparative liquid chromatography, and freeze-dry the received solution to obtain the pure polypeptide Pep.

9. The magnolia multi-component delivery system according to claim 7 or 8, characterized in that, The conditions for preparative liquid chromatography purification in step (6) are: Chromatographic column: C18 reversed-phase chromatographic column, specifications 50×250 mm, packing 10 μm, 100 AA; Mobile phase: Acetonitrile containing 0.1% TFA is phase A - water containing 0.1% TFA is phase B; Elution program: 0 - 10 min, 5% - 15% A 10 - 50 min, 15 - 31% A; Flow rate: 20 mL / min, Detection wavelength: 220 nm; Injection volume: 1 mL; Column temperature: 30 °C.

10. The magnolia multi-component delivery system according to claim 6, characterized in that, The steps for preparing the multi-component delivery system of magnolia officinalis by the method of stirring combined with probe sonication include: Weigh the polypeptide and dissolve it in ultrapure water, adjust the pH to neutral with NaOH and HCl solutions to prepare a polypeptide solution with a certain concentration; then weigh magnolignan, add absolute ethanol and sonicate to dissolve it, and then add eucalyptol and mix evenly to obtain a drug mixed solution; stir the polypeptide solution, dropwise add the drug mixed solution into the polypeptide solution, continue to stir, after treatment with probe sonication, place the sample at low temperature for a certain period of time to stabilize, centrifuge to remove the free magnolignan, and the obtained supernatant is the multi-component delivery system FCSP@NFs solution of magnolia officinalis.

11. The magnolia multi-component delivery system according to claim 10, wherein, The steps for preparing the multi-component delivery system of magnolia flower by stirring combined with probe ultrasound method include: Weigh the polypeptide and dissolve it in ultrapure water. Use 1-2M NaOH and HCl solutions to adjust the pH to neutral, and prepare a polypeptide solution with a concentration of 10-20mg / mL. Then weigh 5.00-10mg of magnolol, add 1-2mL of absolute ethanol and sonicate to dissolve it, and then add 40-80μL of eucalyptol and mix evenly to obtain a drug mixed solution. Place the polypeptide solution at 40-45°C and stir at a speed of 3000-5000rpm / min. Dropwise add 200-400μL of the drug mixed solution into the polypeptide solution and continue stirring. Then, use probe ultrasound to treat at a frequency of 60Hz for 5-10min. After that, place the sample at 4°C for 24-48h and then centrifuge at a speed of 2000-4000rpm for 10-20min to remove the free magnolol. The obtained supernatant is the FCSP@NFs solution.

12. Use of the multi-component delivery system of magnolia flower according to any one of claims 1-6 in the preparation of a drug for treating rhinitis.

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