CN115487314A - Bis-5HT-Glu-NH 2 Modified paris polyphylla saponin II nano-micelle and preparation method and application thereof - Google Patents
Bis-5HT-Glu-NH 2 Modified paris polyphylla saponin II nano-micelle and preparation method and application thereof Download PDFInfo
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- CN115487314A CN115487314A CN202211389998.XA CN202211389998A CN115487314A CN 115487314 A CN115487314 A CN 115487314A CN 202211389998 A CN202211389998 A CN 202211389998A CN 115487314 A CN115487314 A CN 115487314A
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- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to bis-5HT-Glu-NH 2 Modified polyphyllin II photosensitive nano-micelle (bis-5 HT-Glu-NH) 2 -Ce 6-PSII-PLGA) and a preparation method and application thereof, wherein the photosensitive nano-micelle can identify Myeloperoxidase (MPO) by the surface specificity of targeted neutrophils; photodynamic therapy with photosensitizing properties combined with the ability to enhance neutrophil aggregation: (Photosensitizer Ce 6) can be used as a new strategy for treating AR, and an alternative platform is provided for in vitro or in vivo targeted treatment of AR.
Description
Technical Field
The invention relates to the technical field of pharmacy, in particular to bis-5HT-Glu-NH 2 Modified paris polyphylla saponin II nano-micelle and preparation method and application thereof.
Background
Allergic Rhinitis (AR) is an inflammatory disease of the nasal mucosa caused by IgE-mediated inflammatory reactions, characterized by nasal congestion, runny nose, sneezing and itching. The impact of AR on the perceived health of the patient is enormous. In addition, AR has been shown to be a significant risk factor for a variety of diseases. It is now clear that allergic inflammation and Th in AR 2 Is related to lymphocytes. Dendritic Cells (DCs) can activate the selective differentiation of naive T cells into Th according to the external environment 1 Or Th 2 A subgroup which is believed to play an important role in the occurrence and maintenance of AR. Current drugs for treatment of AR are mainly oral and nasal antihistamines, acute AR treatment requires short-term use of alpha-mimetics and intranasal glucocorticoids. Although these have some effect on AR treatment, in part, possibly by modulating DC function to exert its therapeutic effect, the specific mechanism is not clear. The allergic rhinitis infiltrated by the neutrophil is a special type of the allergic rhinitis, and the inflammatory reaction of the allergic rhinitisIs heavier than the common AR, has poor traditional treatment effect at present, and brings heavy burden to social medical treatment. At present, allergic rhinitis drugs targeting neutrophil infiltration are extremely lacking in clinic.
Paridis Saponin (PS) is a natural compound separated from rhizoma Paridis, and has antiinflammatory, antitumor, immunity regulating, and antibacterial effects. However, the clinical utility of PSII is limited by the bioactive properties of poor solubility, short retention time and low bioavailability. Thus, the present invention recognizes: in order to better exert the anti-inflammatory effect of PSII on allergic diseases and improve the bioavailability and safety of PSII, a high-efficiency PSII delivery system is urgently needed.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides bis-5HT-Glu-NH 2 Modified polyphyllin II photosensitive nano-micelle (bis-5 HT-Glu-NH) 2 -Ce 6-PSII-PLGA) and a preparation method and application thereof, wherein the photosensitive nano-micelle can identify Myeloperoxidase (MPO) by the surface specificity of targeted neutrophils; the photosensitive property of the compound can be combined with photodynamic therapy (photosensitizer Ce 6) capable of enhancing neutrophil aggregation, can be used as a new strategy for treating AR, and provides an alternative platform for in vitro or in vivo targeted therapy of AR.
In order to achieve the above object, the present invention includes the following technical contents:
in a first aspect of the invention, bis-5HT-Glu-NH is provided 2 Modified paris polyphylla saponin II (PSII) photosensitive nano micelle (bis-5 HT-Glu-NH) 2 -Ce 6-PSII-PLGA) comprising: bis-5HT-Glu-NH 2 And PSII and Ce6 (Ce 6-PSII-PLGA), wherein the bis-5HT-Glu-NH 2 The chemical structural formula of (A) is shown in formula 1, and the CAS No.50773-42-7 of PSII is shown in the specification.
Further, the bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA is in a core-shell structure, wherein PSII and Ce6 are inner cores and are wrapped in shells bis-5HT-Glu-NH 2 -PLGA.
In a second aspect of the invention, a bis-5HT-Glu-NH is provided 2 Modified PSII photosensitive nanomicelle (bis-5 HT-Glu-NH) 2 -Ce 6-PSII-PLGA) comprising the steps of:
(1) Preparation of PLGA nanoparticles loaded with PSII and Ce6
Weighing PLGA-PEG-COOH in a centrifuge tube, adding into an organic solvent, dissolving by ultrasonic, and adding into methanol or chloroform solution containing PSII and Ce6 to obtain an oil phase; adding the oil phase into the external water phase PVA water solution to obtain a mixed emulsion; the emulsion was transferred to a beaker and stirred overnight; after the reaction is finished, purified water is used for ultrafiltration washing and heavy suspension, and PLGA-PSII-Ce6 nanoparticles are obtained;
(2) PSII and Ce6 loaded PLGA nanoparticle coupling bis-5HT-Glu-NH 2
Taking the PLGA-PSII-Ce6 nanoparticles prepared in the step (1), and performing ultrafiltration washing once by using MES; vortex addition of a solution containing bis-5HT-Glu-NH 2 And EDC aqueous solution, reacting for 8-12 h; after the reaction is finished, collecting the supernatant through ultrafiltration, detecting the polypeptide coupling rate, and washing to obtain bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA。
Further, the mass-to-volume ratio of PSII, ce6 to the organic solvent in step (1) is 16; the organic solvent is methanol or chloroform.
Further, the mass-to-volume ratio of PLGA-PEG-COOH to chloroform or dichloromethane in step (1) is 32 to 48, preferably 40.
Further, the volume ratio of the methanol or chloroform solution of PSII and Ce6 to the chloroform or dichloromethane solution of PLGA-PEG-COOH in the step (1) is 1 to 1, preferably 1.5.
Further, the concentration of the PVA aqueous solution in the step (1) is 1%; the volume ratio of the PVA aqueous solution to the PLGA-PEG-COOH organic solvent solution is 1.
Further, the mode of adding the oil phase into the external water phase PVA aqueous solution in the step (1) is selected from vortex, ultrasonic, water bath heating and the like.
Further, the vortex condition for adding the oil phase into the external water phase by vortex in the step (1) is 40% power (192 w) probe ultrasonic for 5 min.
Further, the stirring process described in step (1) is to add a magnetic stirrer to stir at 100 rpm overnight.
Further, bis-5HT-Glu-NH in the aqueous solution in step (2) 2 And the concentration of EDC is 10 mg/mL; the PLGA-PSII-Ce6 nanoparticle and bis-5HT-Glu-NH 2 And the mass ratio of EDC is (1); preferably 10.
Further, the reaction temperature in the step (2) was 37 ℃.
Further, said bis-5HT-Glu-NH 2 The specific preparation method of the-Ce 6-PSII-PLGA comprises the following steps:
(1) Preparation of PLGA nanoparticles loaded with PSII and Ce6
A. Preparing a medicament: 10 Dissolving PSII and 4.5 mg Ce6 in 1 mL methanol by ultrasonic wave;
B. oil phase: weighing 100 mg PLGA-PEG-COOH in a 15 mL centrifuge tube, adding 2.5 mL chloroform for ultrasonic dissolution, and adding 1 mL methanol solution containing 10 mg PSII and 4.5 mg Ce6;
C. external water phase: 10 mL 1% of an aqueous PVA solution;
D. adding the oil phase into the external water phase by vortex, and carrying out ultrasonic treatment for 5 min by a 40% power (192 w) probe;
E. the emulsion was transferred to a 50 mL beaker and stirred overnight at 100 rpm with the addition of a magnetic stirrer;
F. after the reaction is finished, transferring the solution into a 100 KD ultrafiltration tube, performing ultrafiltration washing for 5 times by using sterilized purified water, and then performing heavy suspension by using the purified water to obtain the PLGA-PSII-Ce6 nanoparticles.
(2) PSII and Ce6 loaded PLGA nanoparticle coupling bis-5HT-Glu-NH 2
A. Taking 100 mg PLGA-PSII-Ce6 nanoparticles, carrying out ultrafiltration washing once by adopting 15 mM MES, and adding water to a constant volume of 8 mL;
C. after the reaction is finished, collecting the supernatant by ultrafiltration through a 100 KD ultrafiltration tube, and washing for 3 times by purified water to obtain bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA。
In a third aspect of the invention, there is provided a bis-5HT-Glu-NH 2 The modified PSII photosensitive nano-micelle is applied to the fields of medicine, biology and the like, in particular to the application in preparing medicines for treating Allergic Rhinitis (AR).
Compared with the prior art, the invention has the following beneficial effects:
first, the present invention employs polymeric micelles to enhance PSII to target neutrophils, thereby improving the treatment of AR. On this basis, the research strategy of the present invention is to increase accumulation of PSII in neutrophil-infiltrated tissues by increasing the permeability and residence time of PSII in vivo. However, nanoparticles are easily cleared by some phagocytic cells, including monocytes and macrophages in the liver, spleen, lymph Nodes (LN), kidney, and blood, which are key to drug-controlled delivery. In order to develop a synthetic structure for enhancing dendritic cell targeting and a unique molecular marker over-expressed in inflammatory diseases, the invention promotes the recruitment of neutrophils by simultaneously carrying a photosensitizer Ce6, thereby providing treatment for allergic rhinitis.
Secondly, PSII can participate in the treatment of AR by modulating the function of nasal mucosal DCs, overcoming the limitations of PSII delivery and therapeutic efficacy, and bis-5HT-Glu-NH 2 Coupled Ce6-PSII-PLGA micelles can significantly increase the penetration of PSII in neutrophil-infiltrated allergic rhinitis local tissues. Since PSII allows modulation of inflammatory responses in AR by inhibiting maturation of DC cells in lymph nodes and migration of mature DCs to the nasal mucosa, bis-5HT-Glu-NH 2 Coupled Ce6-PSII-PLGA micelles can enhance the limited penetration of PSII in the local tissue of allergic rhinitis by targeting MPO on the surface of neutrophilsAnd inhibit maturation and migration of DCs, induce tolerance immune responses and reduce allergic activity.
In addition, the present invention demonstrates: (1) bis-5HT-Glu-NH 2 The modification can enhance the infiltration of the PSII nano-micelle in local tissues of allergic rhinitis. (2) bis-5HT-Glu-NH 2 The modification may enhance the AR inhibitory effect of PSII in vitro. (3) The photosensitizer Ce6 can recruit neutrophils, so that PSII can efficiently and intensively inhibit the inflammatory reaction of AR. (4) bis-5HT-Glu-NH 2 The modified PSII nano micelle can enhance the anti-allergic activity of OVA sensitized mice, enhance the inhibition effect of PSII on lymph node DC maturation, and generate the inhibition effect of PSII on AR through a metabolic pathway. (5) bis-5HT-Glu-NH 2 The modification effectively improves the permeability and in vivo residence time of PSII, thereby achieving the purpose of increasing the accumulation of PSII in allergic rhinitis tissues.
Drawings
FIG. 1 preparation of bis-5HT-Glu-NH according to the invention 2 Schematic representation of the principle of Ce 6-PSII-PLGA.
FIG. 2 shows hydrodynamic size detection of PLGA-PSII-Ce6 nanoparticles prepared by the present invention.
FIG. 3 shows the hydrodynamic size detection of PLGA-PSII-Ce6-bis nanoparticles prepared by the present invention.
FIG. 4 shows Zeta potential detection of PLGA-PSII-Ce6 nanoparticles prepared by the present invention.
FIG. 5 shows Zeta potential detection of PLGA-PSII-Ce6-bis nanoparticles prepared by the present invention.
FIG. 6 shows the ultraviolet full-wavelength absorption spectrum of a Ce6 (0.08 mg/mL) methanol solution prepared by the invention.
FIG. 7 shows the ultraviolet full-wavelength absorption spectrum of PLGA-PSII-Ce6 prepared by the present invention after demulsification.
FIG. 8 is a PSII standard curve prepared according to the present invention.
FIG. 9 bis-5HT-Glu-NH prepared according to the invention 2 A standard curve.
FIG. 10 shows TEM results of PSII and Ce6 loaded PLGA nanoparticles prepared by the present invention.
FIG. 11. Bis-5HT-Glu-NH prepared according to the invention 2 The Ce6-PSII-PLGA is small for allergic rhinitisEffect of neutrophils in peripheral blood of mice.
FIG. 12 bis-5HT-Glu-NH prepared according to the invention 2 Effect of Ce6-PSII-PLGA on DC cells in mice with allergic rhinitis.
FIG. 13 bis-5HT-Glu-NH prepared according to the invention 2 Th of-Ce 6-PSII-PLGA for allergic rhinitis mice 2 The effect of the cells.
FIG. 14 bis-5HT-Glu-NH prepared according to the invention 2 Effect of-Ce 6-PSII-PLGA on Th17 cells in allergic rhinitis mice.
FIG. 15 bis-5HT-Glu-NH prepared according to the invention 2 -effect of Ce6-PSII-PLGA on neutrophil expression in mouse tissue of allergic rhinitis.
FIG. 16. Bis-5HT-Glu-NH prepared according to the invention 2 -effect of Ce6-PSII-PLGA on the expression of inflammatory factors in allergic rhinitis mice.
FIG. 17 bis-5HT-Glu-NH prepared according to the invention 2 -Ce6-PSII-PLGA inhibits the inflammatory response of AR via metabolic pathways.
Detailed Description
The present application is described in further detail below by way of examples to enable those skilled in the art to practice the present application. It is to be understood that other embodiments may be utilized and that changes may be made without departing from the spirit or scope of the present application. To avoid detail not necessary to enable those skilled in the art to practice the application, the description may omit certain information known to those skilled in the art. The following detailed description is, therefore, not to be taken in a limiting sense, and the scope of the present invention is defined only by the appended claims. The following examples facilitate a better understanding of the present application and are not intended to limit the scope of the present application.
In the following examples, paris saponin II (PSII) (purity > 99%) was purchased from Kyormant Biotechnology Ltd (Chengdu, china).
PLGA-PEG-COOH was obtained from Shanghai \33411, ce6 was obtained from Nanjing Kangman chemical industries, inc., EDC was obtained from Sigma, bis-5HT-Glu-NH 2 Biochemical family of peptides from Shanghai ChuTechnical Co., ltd.
RPMI-1640 medium was purchased from HyClone (USA); fetal Bovine Serum (FBS) was purchased from Gibco (australia). Ammonium chloride solutions were purchased from Shanghai Yunwu Biotech, inc.
Recombinant murine IL-4 and granulocyte macrophage colony stimulating factor (GM-CSF) were purchased from PeproTech (USA).
Ovalbumin (OVA, grade V), aluminum hydroxide, lipopolysaccharide (LPS) and 4' -6-diamidino-2-phenylindole (DAPI) were purchased from Sigma-Aldrich (St. Louis, USA).
Percp/cy 5.5-conjugated anti-mouse interferon-gamma, PE coupled anti-mouse IL-4, alexa Fluor 647 coupled anti-mouse IL-17 alpha, APC coupled anti-mouse CD11c, FITC coupled anti-mouse CD80, PE coupled anti-mouse CD86 were purchased from BioLegend (USA).
The Mouse IL-4 ELISA Kit, mouse TNF-. Alpha.ELISA Kit, mouse CCL17 ELISA Kit, and Mouse IgE ELISA Kit were purchased from Multisciences (China), mouse Histamine ELISA Kit (DRG International Inc., germany).
Other chemicals are reagent grade and can adopt commercial products.
Example 1 bis-5HT-Glu-NH 2 Preparation of-Ce 6-PSII-PLGA
1. Preparation of PSII and Ce6 loaded PLGA nanoparticles
(1) Preparing a medicament: 10 Dissolving PSII and 4.5 mg Ce6 in 1 mL methanol by ultrasonic wave;
(2) Oil phase: weighing 100 mg PLGA-PEG-COOH in a 15 mL centrifuge tube, adding 2.5 mL chloroform for ultrasonic dissolution, and adding 1 mL methanol solution containing 10 mg PSII and 4.5 mg Ce6;
(3) External water phase: 10 mL 1% of an aqueous PVA solution;
(4) Adding the oil phase into the external water phase by vortex, and carrying out ultrasonic treatment for 5 min by a 40% power (192 w) probe;
(5) The emulsion was transferred to a 50 mL beaker and stirred overnight at 100 rpm with the addition of a magnetic stirrer;
(6) After the reaction is finished, transferring the solution into a 100 KD ultrafiltration tube, carrying out ultrafiltration washing for 5 times by using sterilized purified water, and then carrying out heavy suspension by using the purified water to obtain the PLGA-PSII-Ce6 nanoparticles.
2. PSII and Ce6 loaded PLGA nanoparticle coupling bis-5HT-Glu-NH 2
(1) Taking 100 mg of PLGA-PSII-Ce6 nanoparticles prepared in the step 1, carrying out ultrafiltration washing once by adopting 15 mM MES, and diluting to 8 mL by using water;
(2) Vortex to add 1 mL of solution containing bis-5HT-Glu-NH 2 And EDC, wherein bis-5HT-Glu-NH is present in the aqueous solution 2 EDC concentration is 10 mg/mL, and the reaction is carried out overnight at 37 ℃;
(3) After the reaction is finished, collecting the supernatant by ultrafiltration through a 100 KD ultrafiltration tube, washing the supernatant for 3 times by purified water, and then adding water to a constant volume of 10 mL to obtain bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA。
Example 2 product characterization and detection:
characterization of bis-5HT-Glu-NH Using zeta-sizer Nano ZS90 (Malvern Instrument, UK) 2 And particle size and zeta potential of PLGA-PSII-Ce6, the results are shown in FIGS. 4 and 5.
(ii) pairing said bis-5HT-Glu-NH using Transmission Electron Microscopy (TEM) 2 The observation of-Ce 6-PSII-PLGA, the results of which are shown in FIG. 10, shows that the bis-5HT-Glu-NH was prepared 2 Crystal analysis of-Ce 6-PSII-PLGA (bis-5 HT-Glu-NH) 2 And Ce 6-PSII-PLGA) were measured using an X-ray diffractometer (XRD, bruker D8 forward), at a voltage of 40kV and at a current of 40mA. The XRD patterns were obtained at a scan rate of 3 deg./min from 5 deg. to 50 deg.. Before analysis, each sample was diluted with distilled water and shaken well to obtain the appropriate concentration.
1. Preparation of BM-DCs:
preparation of BM-DCs: c57BL/6 mice were sacrificed by cervical dislocation. The femur and tibia were separated under sterile conditions. The tissue was removed with sterile gauze and the bone marrow was flushed into petri dishes with a syringe. The cells were harvested, centrifuged, 5 mL of erythrocyte lysate was preheated at 37 deg.C, 5 mL of RPMI-1640 was added to stop lysis, centrifuged 2 times, counted and complete medium (containing 10 ng/mL recombinant murine IL-4, 20 ng/mL recombinant GM-CSF, 10% inactivated FBS and 1% diabody) was used. Inoculating the cells at 37 deg.C with 5% CO 2 In 6-well plate, 3 h later, the supernatant was discarded, and the same factor was addedThe culture was continued in complete medium at a concentration, and the medium was changed at 48 to 96 hours. After 8 days, cells were adjusted to 2X 105 cells/well and 10. Mu.g/mL LPS was added. LPS stimulated maturation of DCs.
2. And (3) detecting mature cells of BM-DCs:
evaluation of bis-5HT-Glu-NH by flow cytometry 2 Inhibition of BM-DC by-Ce 6-PSII-PLGA for 24 h. Briefly, cells were plated at 1X 10 per well 4 Individual cells were seeded at a density in 3 mL DMEM medium and pretreated with different types of PSII for 10 minutes and incubated with or without LPS (1 mg/mL) for 24 h. Then, the cells were washed twice with PBS and left for 30 min at 4 ℃ with Fluorescein Isothiocyanate (FITC) -conjugated anti-CD 80, (Percp/cy 5.5) -conjugated anti-IA/IE, phycoerythrin (PE) -conjugated anti-CD 86 and (APC) -conjugated anti-CD 11 c. Cells were washed and suspended in 0.5 mL Phosphate Buffered Saline (PBS) and cytometric analysis was performed on a FACSCalibur (BD Biosciences).
3. Sensitization and treatment of AR mice:
an allergic rhinosinusitis mouse model is established to detect the anti-inflammatory action of NGR-PSII-PM in vivo. Male C57BL/6J mice in the experimental group (6-8 weeks, 20 ± 2 g) were sensitized with i.p. Mu.g OVA and 2 mg aluminum hydroxide were injected on days 0 and 8, followed by intranasal instillation of 200. Mu.g solution in 20. Mu.l PBS daily from day 15 to day 22. Thereafter, experimental mice were injected 3 times a week with OVA at a concentration of 6% by subsequent nasal exposure for 30 days. Control mice were sensitized with PBS instead of OVA. The experimental mice were then randomly divided into 6 groups of 5 mice each, and the treatment was as follows: (1) group A (negative control group) PBS 100. Mu.l; (2) For group B (WT + bis-5 HT-Glu-NH) 2 -Ce6-PSII-PLGA group); (3) for group C (AR group); (4) group D (AR + PSII); (5) For group F (AR + bis-5 HT-Glu-NH) 2 -Ce6-PSII-PLGA group); (6) Group E (AR + bis-5 HT-Glu-NH) 2 -Ce6-PSII-PLGA + light group) all mice received 3 treatments per week for 4 weeks. Two hours after the last nasal instillation of OVA, the nasal scraping frequency was estimated to be 10 min.
Secondary C57BL/6J mice (6-8 weeks, 20. + -.2 g) were obtained from Shandong university without mouse-specific pathogens and respiratory diseases. The experimental mice will be quarantined for one week prior to treatment. All animal care and experimental procedures were performed according to institutional animal care and use guidelines.
4. Hematoxylin and eosin staining:
the mice were sacrificed and the heads of the mice were dissected, and the resulting samples were excised from the second alveolar ridge onto the first upper molars and fixed in PFA at a mass concentration of 4% for 2 days, followed by decalcification in ethylenediaminetetraacetic acid (EDTA) at a mass concentration of 10% for 1 week. The samples were then embedded in paraffin and cut into sections 4 μm thick. Inflammation and histology of the nasal mucosa are characterized by hematoxylin and eosin staining, with eosinophils being sirius red staining. Lymph node and spleen staining were performed in the same way.
5. Enzyme-linked immunosorbent assay:
after the sensitized mice were sacrificed, nasal lavage fluid and peripheral blood were collected and inflammatory cytokines were detected. We used the Mouse IL-17 ELISA Kit and the Mouse IgE ELISA Kit to detect nasal lavage and serum, as well as neutrophil counts in blood routine.
6. Detection of Th2, th17 and mature DCs in nasal lavage, lymph nodes and spleen:
flow cytometry (FCM, FACSCalibur, BDBiosciences, USA) was used to examine the relative abundance of Th1, th2 and DCs cells in lymph nodes. Lymphocytes were first stained on the cell surface for CD4 and CD8, then fixed, permeabilized and stained with the intracellular antigens IL-4, IL-17 and CD80/86 of DC according to the manufacturer's instructions. MHCII was used for dc maturity testing. The percentage of CD80/86+ cells, MHCII + cells, th2 cells and Th17 cells was evaluated. All analyses were processed using flowjo10.0 software.
7. Metabonomic analysis:
nasal lavage solutions were taken from 4 groups of mice and snap frozen in liquid nitrogen. After all samples are collected, slowly thawing at 4 ℃, adding a proper amount of samples into a precooled methanol/acetonitrile/water solution (2. Separating the sample by adopting an Agilent 1290 Infinity LC ultra-high performance liquid chromatography system (UHPLC) HILIC chromatographic column; the column temperature is 25 ℃; the flow rate is 0.5 mL/min; the sample size is 2 mu L; mobile phase composition A: water +25 mM ammonium acetate +25 mM ammonia, B: acetonitrile; the gradient elution procedure was as follows: 0-0.5 min,95% B; 0.5-7 min, B varies linearly from 95% to 65%; 7-8 min, B varies linearly from 65% to 40%; 8-9 min, B is maintained at 40%;9- -9.1 min, B varies linearly from 40% to 95%; 9.1-12 min, B is maintained at 95%; samples were placed in a 4 ℃ autosampler throughout the analysis. In order to avoid the influence caused by the fluctuation of the detection signal of the instrument, the continuous analysis of the samples is carried out by adopting a random sequence. QC samples are inserted into the sample queue and used for monitoring and evaluating the stability of the system and the reliability of experimental data.
8. Statistical analysis:
data are shown as mean ± SD from at least three independently performed experiments. Parameter comparisons between the two groups were analyzed using unpaired student's t-test. Statistical significance was analyzed by two-tailed paired t-test using GraphPad Prism Software 6.0 (GraphPad Software, la Jolla, calif.). Apvalue <0.05 is considered statistically significant. Each data point is expressed as a mean (standard deviation).
And (3) detection results:
1.bis-5HT-Glu-NH 2 preparation and characterization of-Ce 6-PSII-PLGA (refer to fig. 2 to 5):
referring to the chemical formula, in bis-5HT-Glu-NH 2 In the preparation process of-Ce 6-PSII-PLGA, carboxyl in a PSII chemical formula and amino in a formula 1 are covalently bonded to form an amido bond target. As can be seen from table 1, the loading of Ce6 in 1 mg PLGA nanoparticles was 0.0599 mg.
TABLE 1.1 Ce6 loading in mg PLGA nanoparticles
Referring to fig. 2 and 3, the hydrodynamic size of plga-PSII-Ce6 nanoparticles is 150 nm;loaded bis-5HT-Glu-NH 2 Thereafter, the hydrodynamic size of the PLGA nanoparticles increased to 210.8 nm.
Referring to FIG. 3, PSII-PM and bis-5HT-Glu-NH 2 The peak sizes in the particle size detection were similar, indicating bis-5HT-Glu-NH 2 The coupling to the PSII-PM does not change the physical properties of the PSII-PM.
Referring to FIG. 4 and FIG. 5, the Zeta potential of PLGA-PSII-Ce6 nanoparticles is-23.39 mV; loading bis-5HT-Glu-NH 2 Then, the Zeta potential of the PLGA nano-particle is changed to-12.29 mV.
Referring to FIG. 6, the Ce6 methanol solution has a plurality of absorption peaks in the range of 190 nm to 800 nm, and the absorption peak at 500 nm is selected as the detection peak in the report.
3 mg PLGA microspheres (227 mu L) are added with acetonitrile (HPLC purity) to 1 mL, after full ultrasonic dissolution, 0.5 mL methanol is added for ultrasonic dissolution, 14000 g is centrifuged to take supernatant, and HPLC is detected.
TABLE 2 HPLC detection result after demulsification of PLGA nanoparticles loaded with PSII and Ce6
Note: the solid content of nanoparticles prepared from 100 mg PLGA was 50.14 mg, so that 3 mg PLGA nanoparticles contained 0.598 mg (about 0.6 mg) of PSII.
From the results in table 2, it is understood that the encapsulation efficiency of PSII was 70% and the drug loading was 14%.
PLGA nanoparticle coupling bis-5HT-Glu-NH 2 Thereafter, BCA detection was performed by diluting the supernatant 2-fold with 100 KD ultrafiltration.
TABLE 3 bis-5HT-Glu-NH 2 Dilution of the supernatant after coupling for 2-fold BCA detection results
M-Abs | Concentration mg/mL | Concentration mg/mL before dilution | Volume mL | Xiuqing polypeptide (mg) | Polypeptide batch (mg) | Amount of polypeptide coupled (mg) | The coupling rate of the polypeptide% |
1.261333 | 0.19091398 | 0.381828 | 10 | 3.81828 | 10 | 6.182 | 61.8 |
As can be seen from Table 3, bis-5HT-Glu-NH was attached to 100 mg PLGA nanoparticles 2 The coupling amount of (3) was 6.182 mg, and the coupling rate was 61.8%.
2.bis-5HT-Glu-NH 2 Effect of Ce6-PSII-PLGA on peripheral blood neutrophils in allergic rhinitis mice
Further, to investigate bis-5HT-Glu-NH 2 Effect of PSII-PM on AR, mice were sensitized by intraperitoneal injection of OVA and aluminium hydroxide adjuvant on days 1, 5 and 14 of this experiment. OVA solution (50. Mu.g in 20. Mu.L PBS)OVA) was instilled intranasally once daily from day 15-22 1 hour prior to intranasal OVA challenge, as shown in the schematic (fig. 11A). The results are shown in FIG. 11, and FIG. 11A is a pattern diagram of OVA-sensitized mice. FIG. 11B is the peripheral blood neutrophil count of OVA-sensitized mice. Bis-5HT-Glu-NH compared to other PSII treatment groups 2 The group Ce6-PSII-PLGA showed a lower inflammatory cell infiltration, each value representing the mean. + -. SD of three independent experiments. Statistical significance was shown using student test analysis; * P is<0.05;**P<0.01;***P<0.001。
The results show that: after 4 weeks of nasal OVA exposure, the morphological observation of the nasal mucosa of mice shows that the mucosal lesions of the AR group are raised, eosinophilic granulocyte infiltration of the nasal mucosa tissues is carried out, and no obvious inflammatory cells are found in the PBS control group. PSII and post-phototherapy (free PSII, bis-5 HT-Glu-NH) 2 -Ce6-PSII-PLGA、bis-5HT-Glu-NH 2 Ce6-PSII-PLGA + light group) significantly reduced inflammatory cell infiltration and mucosal lesions compared to the untreated AR group. Furthermore, bis-5HT-Glu-NH as compared to PSII alone treatment 2 The Ce6-PSII-PLGA group had fewer inflammatory cells and inflammatory factors.
Subsequently, this assay also evaluated bis-5HT-Glu-NH 2 -effect of Ce6-PSII-PLGA on allergic symptoms in AR mice. In the OVA-induced AR mouse model, the frequency of nasal scratching was estimated to be 10 min. Symptoms of nasal scratching frequency and sneezing were significantly increased and neutrophil infiltration was significantly increased in AR mice compared to PBS-treated mice (figure 11B).
3. bis-5HT-Glu-NH 2 -effect of Ce6-PSII-PLGA on OVA-sensitized mouse chemokines:
in addition, as shown in FIG. 16, the levels of IL-17 and IgE in the nasal lavage fluid and peripheral blood serum were also measured. AR mice increased significantly. However, they are in bis-5HT-Glu-NH 2 The Ce6-PSII-PLGA group was significantly lower than the PSII-only treatment group. The above results indicate that PSII can alleviate the symptoms of allergic rhinitis in AR mice, bis-5HT-Glu-NH 2 The modification (2) can obviously enhance the effect of PSII on allergic rhinitis.
4.bis-5HT-Glu-NH 2 The modification can enhance the inhibition of PSII on lymph node DC maturationThe function is as follows:
to study bis-5HT-Glu-NH 2 Effect of Ce6-PSII-PLGA on maturation of DCs cells in lymph nodes of AR mice, the lymph nodes of AR mice were harvested and the proportion of CD80, CD86 positive cells was examined by flow cytometry. The results are shown in FIG. 12 for CD80+ CD86+ cells in nasal lavage, lymph nodes and spleen, respectively. Each value represents the mean ± SD of three independent experiments. Statistical significance was shown using student test analysis; * P<0.05;**P<0.01;***P<0.001。
The results show that the cell ratio of CD80+ CD86 marker in the lymph node of AR mice is obviously increased, and PSII can obviously reduce the cell ratio of CD80+ CD86+ cells. Bis-5HT-Glu-NH in contrast to Free-PSII 2 The expression of CD80+ CD86+ in DCs is most obviously inhibited by-Ce 6-PSII-PLGA. bis-5HT-Glu-NH 2 The modification of-Ce 6-PSII-PLGA obviously enhances the inhibition effect of PSII on the maturation of lymph node DCs cells.
5.bis-5HT-Glu-NH 2 The modification can enhance the inhibition of the Th2 inflammatory response in lymph nodes of OVA-sensitized mice by PSII:
given that maturation of DCs plays an irreplaceable role in the differentiation of naive T cells to Th1 or Th2 subsets, the present invention considers that: bis-5HT-Glu-NH 2 The inhibitory effect of Ce6-PSII-PLGA on cell maturation of DCs may affect the differentiation of the AR mouse naive T cells. Meanwhile, because the number of Th17 and Th2 cells in the nasal cavity lavage fluid is low, the analysis is not facilitated, and the proportion of the Th17 cells to the Th2 cells in the lymph nodes of the mice is further detected. Nasal lavage fluid, peripheral lymph nodes and spleen tissues of OVA mice were collected in this experiment, and it was found that lymph nodes of AR mice were significantly enlarged as compared with non-sensitized control mice. PSII has certain anti-inflammatory effect on lymph nodes of AR mice, and is bis-5HT-Glu-NH 2 Lymph node recovery was most evident in AR mice treated with-Ce 6-PSII-PLGA. In subsequent flow cytometry, IL-17 and IL-4 were used to label Th17 and Th2 cell subsets, respectively, in CD4 cells. The results showed that the proportion of Th2 cell subsets in the lymph nodes of AR mice was significantly increased, consistent with the change in the proportion of Th17 cell subsets. PSII inhibits differentiation of Th2 cell subsets bis-5HT-Glu-NH 2 The modification significantly enhances PSIIThis inhibitory effect. These data indicate that the percentage of Th17 and Th2 cells in lymph nodes of AR mice are affected by bis-5HT-Glu-NH 2 By regulating the maturation of DCs cells.
6.bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA inhibits the allergic rhinitis inflammatory response through a lipid metabolic pathway.
As shown in FIG. 17, all metabolites identified by the present invention (metabolites identified by combining positive and negative ions) were classified and counted according to the attribution information of their Chemical classification (Chemical Taxonomy), and the ratio of the number of each type of metabolites is shown in the figure. The different colored patches in the graph represent different chemical taxonomy assignment entries, and the percentage represents the number of metabolites in the chemical taxonomy assignment entry as a percentage of the number of all identified metabolites. Metabolites without chemical taxonomic assignment were defined as undefined.
The above description is only illustrative of several embodiments of the present invention, and should not be understood as limiting the scope of the present invention. It should be noted that other persons skilled in the art can make modifications, substitutions, improvements and the like without departing from the spirit and scope of the present invention, and all of them belong to the protection scope of the present invention.
Claims (9)
1. Bis-5HT-Glu-NH 2 The modified PSII photosensitive nano-micelle is characterized in that the chemical formula of the micelle is bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA comprising: bis-5HT-Glu-NH 2 The PLGA nanoparticle loaded with PSII and Ce6, wherein the bis-5HT-Glu-NH is provided 2 The chemical structural formula of (2) is shown as formula 1:
formula 1.
2. The photosensitive nanomicelle according to claim 1, wherein the bis-5HT-Glu-NH is 2 -Ce6-PSII-PLGA is core-shell structure, wherein the PSII and Ce6 is an inner core, which is wrapped in a shell bis-5HT-Glu-NH 2 -PLGA.
3. Bis-5HT-Glu-NH 2 The preparation method of the-Ce 6-PSII-PLGA is characterized by comprising the following steps:
(1) Preparation of paris polyphylla saponin II and Ce6 loaded PLGA nano particle
Adding PLGA-PEG-COOH into an organic solvent to dissolve to obtain a solution 1, and adding an organic solution 2 containing PSII and Ce6 to obtain an oil phase; adding the oil phase into a PVA aqueous solution to obtain a mixed emulsion; stirring the emulsion for 8-12 h; ultra-filtering, washing and re-suspending to obtain PLGA-PSII-Ce6 nanoparticles;
(2) PSII and Ce6 loaded PLGA nanoparticle coupling bis-5HT-Glu-NH 2
Taking the PLGA-PSII-Ce6 nanoparticles prepared in the step (1), and performing MES ultrafiltration washing; vortex addition of a solution containing bis-5HT-Glu-NH 2 And EDC aqueous solution, reacting for 8-12 h; after the reaction is finished, collecting the supernatant through ultrafiltration, and washing to obtain bis-5HT-Glu-NH 2 -Ce6-PSII-PLGA。
4. The preparation method according to claim 3, wherein the mass-to-volume ratio of PSII, ce6 and the organic solution of PSII and Ce6 in the step (1) is (16 to 24) mg: (7 to 11) mg:2 mL; wherein the organic solution is methanol or chloroform.
5. The preparation method according to claim 3, wherein the PLGA-PEG-COOH is added into an organic solvent in the step (1), wherein the mass volume ratio of the PLGA-PEG-COOH to the organic solvent is 32 mg to 48 mg; the organic solvent is chloroform or dichloromethane.
6. The preparation method according to claim 3, wherein the volume ratio of the solution 2 to the solution 1 in the step (1) is 1 to 2-1.
7. The method according to claim 3, wherein the concentration of the aqueous PVA solution in the step (1) is 1%; the volume ratio of the PVA aqueous solution to the solution 1 is 1.
8. The process according to claim 3, wherein bis-5HT-Glu-NH is present in the aqueous solution in step (2) 2 And the concentration of EDC is 10 mg/mL; the PLGA-PSII-Ce6 nanoparticle and bis-5HT-Glu-NH 2 And the mass ratio of EDC is 8.
9. Use of the photosensitive nanomicelle according to claim 1 or 2 or the photosensitive nanomicelle prepared by the preparation method according to any one of claims 3 to 9, wherein the photosensitive nanomicelle is used for the preparation of a medicament for the treatment of allergic rhinitis.
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