CN109692327B

CN109692327B - Application of nano-particles for carrying melittin

Info

Publication number: CN109692327B
Application number: CN201710995177.3A
Authority: CN
Inventors: 骆清铭; 张智红; 刘征; 范展
Original assignee: Huazhong University of Science and Technology; Ezhou Institute of Industrial Technology Huazhong University of Science and Technology
Current assignee: Huazhong University of Science and Technology; Ezhou Institute of Industrial Technology Huazhong University of Science and Technology
Priority date: 2017-10-23
Filing date: 2017-10-23
Publication date: 2022-04-08
Anticipated expiration: 2037-10-23
Also published as: CN109692327A

Abstract

The invention discloses an application of melittin, relating to the technical field of bioscience and drug carriers. The invention also discloses an application of the nano-particles for carrying melittin, the nano-particles for carrying melittin consist of polypeptide, phospholipid and cholesterol ester for carrying melittin, and the nano-particles for carrying melittin can be used for preparing a medicament for treating allergic skin diseases, can be accumulated in sensitized skin drainage lymph nodes through a skin lymphatic system, can play the effects of inhibiting antigen specific T cell proliferation and reducing excessive immune reaction in the sensitized skin drainage lymph nodes, and has low toxic and side effects.

Description

Application of nano-particles for carrying melittin

Technical Field

The invention relates to the technical field of bioscience and drug carriers, in particular to application of melittin and application of nano-particles for carrying melittin.

Background

Allergic dermatitis is the most common immunological skin disease. At present, hormone medicines are mainly used for treating allergic dermatitis, however, a series of side effects of osteoporosis, hyperglycemia, skin atrophy and the like are induced in the treatment process of hormones, and the hormonal dermatitis can be induced after long-term use. The development of a new medicine for treating allergic dermatitis has great application prospect.

The natural bee venom has wide application in traditional medicine, and one of the natural bee venom has the immunosuppressive effect of the natural bee venom. Bee-needle therapy has been effective for suppressing immune diseases such as multiple sclerosis, allergic rhinitis and arthritis. Allergic dermatitis is also an immune response mediated dermatological disorder. Therefore, the application of the traditional medicine, namely bee venom, to the treatment of allergic dermatitis has great potential. However, there is insufficient medical evidence for the use of all components of bee venom to treat immune diseases. Particularly, bee venom has complex components, and is difficult to directly study and apply to clinical characteristics as a mixture extracted from the body of bees in a natural state. Melittin is an amphiphilic polypeptide with alpha helical structure, is the main active component in bee venom, and accounts for 40-60% of dry weight. Melittin is very destructive to cell membrane structures, including the membrane structure of the plasma membrane and some organelles within the cell. The action mechanism is mainly that the membrane is embedded into a phospholipid membrane structure through an amphiphilic structure, and then the membrane is perforated, so that the membrane structure is broken, and the change of the internal and external environments of the membrane finally causes the death of cells; apoptosis can also be induced by affecting key proteins in multiple signaling pathways that mediate tumor proliferation within cells. Meanwhile, the melittin also has broad-spectrum antibacterial function and anti-AIDS function.

Meanwhile, due to the toxic characteristics of melittin, it is difficult to directly apply it to the treatment of diseases of living bodies. Since melittin rapidly binds to erythrocytes after injection into blood vessels, and destroys cell membranes, so that a strong hemolytic reaction occurs. And the melittin is directly applied to living body treatment, which can damage normal cells of an organism and bring about great toxic and side effects. In addition, melittin has a short half-life in the body and a high metabolic rate, and is not suitable for clinical treatment of diseases.

Therefore, a technical scheme for melittin to treat allergic skin diseases and explore the regulation mechanism of melittin in the immune system is urgently needed, and a new direction is provided for the therapeutic agent for allergic skin diseases.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of melittin, which is used for preparing the medicament for treating the allergic skin diseases and can effectively inhibit the proliferation of antigen-specific T cells and inhibit the release of cell inflammatory factors.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: use of melittin for the preparation of a medicament for the treatment of an allergic skin disease.

Compared with the prior art, the invention has the advantages that:

the melittin is used for preparing a medicament for treating allergic skin diseases, can effectively inhibit inflammation and swelling caused by allergy, has similar effect to that of dexamethasone which is a clinical common hormone medicament, and can effectively inhibit antigen-specific T cell proliferation and inhibit release of cell inflammatory factors.

The invention also aims to provide application of the melittin-carrying nanoparticles in preparation of a medicament for treating allergic skin diseases, and the melittin-carrying nanoparticles have good treatment effect and low toxic and side effects.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows: the melittin-carrying nanoparticles consist of melittin-carrying polypeptides and phospholipids, cholesterol esters; the polypeptide carrying melittin is formed by connecting alpha helical polypeptide, a connecting sequence and melittin in series in a covalent bond mode; the melittin-carrying nanoparticles are used for the preparation of a medicament for the treatment of allergic skin diseases.

Based on the scheme, the medicament can inhibit antigen-specific T cell proliferation and inhibit the release of cell inflammatory factors in lymph nodes.

On the basis of the scheme, the melittin-carrying nanoparticle is also used for preparing a drug carrier, and the drug carrier is used for loading dye molecules or drug molecules for disease diagnosis.

On the basis of the above scheme, the phospholipid is dimyristoyl phosphatidylcholine (DMPC).

Based on the above protocol, the alpha-helical polypeptide has the amino acid sequence of DWFKAFYDKVAEKFKEAF.

On the basis of the above scheme, the amino acid sequence of melittin is GIGAVLKVLTTGLPALISWIKRKRQQ.

Based on the above scheme, the amino acid sequence of the linker sequence is GSG.

Compared with the prior art, the method has the following advantages:

(1) has good therapeutic effect

The medicament for treating allergic skin diseases, which is prepared by the nano-particles carrying the melittin, can be accumulated in sensitized skin drainage lymph nodes through a skin lymphatic system, and plays the effects of inhibiting antigen-specific T cell proliferation and reducing excessive immune response in the sensitized skin drainage lymph nodes, thereby achieving good anti-allergic treatment effect.

(2) Has low adverse side effect

The medicament for treating allergic skin diseases, which is prepared by the nano-particles carrying the melittin, has the advantages that the melittin is buried in the single-layer phospholipid layer due to the increase of the interaction between the melittin and the phospholipid, so that the direct contact between the melittin and blood cells and normal cells is avoided, and the toxic and side effects of intradermal administration are low.

(3) Function expandable

The nano-particles carrying melittin can also be used for preparing a drug carrier, and the drug carrier can be loaded with drug molecules for treating diseases, such as steroid hormones and the like, at the core of the drug carrier, and can also be loaded with other types of targeting polypeptides or therapeutic polypeptides at the same time, so that the effect of cooperative targeting or cooperative treatment of diseases is realized.

Drawings

FIG. 1 is a graph of the evaluation of various dosages of melittin for the treatment of contact allergic dermatitis;

FIG. 2 is an evaluation of melittin treatment for chronic contact allergic dermatitis;

FIG. 3 is a graph of HE staining of ear sections for treatment of chronic allergic skin disease in C57BL/6 mice by caudal intradermal injection of nanoparticles of melittin;

FIG. 4 is a pictorial representation of a comparison of mouse skin lesions following intradermal injection of melittin-carrying nanoparticles and free melittin, respectively, in the abdomen of a living subject;

FIG. 5 is a graph comparing swelling experiments by intracutaneous injection of melittin-carrying nanoparticles and free melittin in vivo ears;

FIG. 6 is a time-swelling thickness plot of melittin-carrying nanoparticles and free melittin tail root intradermal injection in a model of chronic allergic skin disease in C57BL/6 mice;

FIG. 7 is a graph of HE staining of ear sections of melittin-carrying nanoparticles with free melittin and PBS tail injected intradermally into the root for treatment of chronic allergic skin disease in C57BL/6 mice;

FIG. 8 is an overall fluorescence imaging diagram of in vivo detection of distribution and infiltration of nanoparticles in skin draining lymph nodes and tissues and organs of a melittin-carrying nanoparticle core loaded with a fluorescent dye DiR-BOA, C57BL/6 mouse;

FIG. 9 is a graph showing the results of a cell proliferation assay in which melittin-carrying nanoparticles are incubated with hapten-stimulated lymphocytes in vitro to co-culture;

FIG. 10 is a statistical chart of the results of in vitro incubation co-culture assays for detecting cell proliferation of melittin-carrying nanoparticles with hapten-stimulated lymphocytes;

FIG. 11 shows that the nanoparticles carrying melittin inhibit IFN- γ expression in co-incubation culture with allergen-stimulated lymphocytes;

FIG. 12 is a graph showing that melittin-carrying nanoparticles are effective in reducing the expression levels of the pro-inflammatory cytokines IFN-. gamma.and IL-4 in serum.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

Example 1: application of melittin

The embodiment of the invention discloses application of melittin, and the melittin is used for preparing a medicament for treating allergic skin diseases.

Example 2: use of nanoparticles carrying melittin

The embodiment of the invention discloses application of melittin-carrying nanoparticles in preparation of a medicament for treating allergic skin diseases. The preparation can inhibit antigen specific T cell proliferation and cell inflammatory factor release in lymph node. The nanoparticles carrying melittin are also used for the preparation of drug carriers for loading dye molecules or drug molecules for disease diagnosis.

Wherein the melittin-carrying nanoparticles are composed of melittin-carrying polypeptides and phospholipids, cholesterol esters. The phospholipid is dimyristoyl phosphatidylcholine (DMPC, 1, 2-dimyristoyl-sn-glycerol-3-phosphorylcholine). The polypeptide carrying melittin is formed by connecting alpha helical polypeptide, a connecting sequence and melittin in series in a covalent bond mode. The amino acid sequence of the alpha-helical polypeptide is DWFKAFYDKVAEKFKEAF. The amino acid sequence of the alpha helix polypeptide is shown as SEQ ID NO.1 in the sequence table. The amino acid sequence of melittin is GIGAVLKVLTTGLPALISWIKRKRQQ, and the amino acid sequence is shown as SEQ ID NO.2 in the sequence table. The amino acid sequence of the linker sequence is GSG.

EXAMPLE 3 study of the use of free melittin in vivo for the treatment of chronic allergic skin diseases

The melittin of example 1 was used in living body to treat allergic skin diseases and chronic allergic skin diseases, and the study is shown in fig. 1, fig. 2, and fig. 3. In figure 1, it is shown that free melittin dermal injection can effectively inhibit the inflammation and swelling degree caused by allergy in a single hapten-induced allergic skin disease model of a C57/BL6 mouse, and the inflammation inhibition degree has similar effect with that of dexamethasone which is a clinical common hormone drug. In FIG. 2, it is shown that dermal injection of free melittin was effective in inhibiting the swelling of inflammation caused by allergy in a model of chronic allergic skin disease induced by multiple haptens in a C57/BL6 mouse. FIG. 3 shows HE staining results of ear skin sections, which shows that melittin dermal injection can effectively inhibit swelling, inflammatory cell infiltration, and abnormal proliferation of epidermal tissue caused by allergic skin diseases.

Example 4 comparison of toxicity studies of melittin-carrying nanoparticles and free melittin in vivo

The toxicity study of melittin-carrying nanoparticles of example 2 and free melittin of example 1 applied to living bodies is compared with fig. 4, fig. 5. FIG. 4 is a comparison of the results of HE staining of tissue sections of melittin-loaded nanoparticles injected into skin at 1mg/mL, with free melittin causing more severe inflammatory cell infiltration and loss of tissue and cell structure, whereas the melittin-loaded nanoparticles were infiltrated with only a small amount of inflammatory cells and did not destroy the tissue cells. A comparison of the swelling of the mouse ears after 12h intradermal injection with melittin carrying nanoparticles and free melittin at a concentration of 1mg/mL is shown in FIG. 5: when the nano-particles carrying the melittin are injected into ears at a concentration of 1mg/mL, swelling of about 50 micrometers can be caused; while the 1mg/mL concentration of free melittin administered in the ear resulted in swelling of around 150 μm. Figure 4 is a pictorial representation of comparative mouse skin lesions following intradermal injection of melittin-carrying nanoparticles and free melittin, respectively, in the abdomen of a living subject. Free melittin causes visible skin lesions and persistent swelling, while the nanoparticles carrying melittin do not.

Example 5: studies of melittin-carrying nanoparticles for the in vivo treatment of chronic allergic skin diseases

The melittin-carrying nanoparticles of example 2 were safely used in the study of the in vivo treatment of chronic allergic skin diseases, see fig. 5 and 6. FIG. 7 HE staining results of ear skin sections show that melittin nanoparticles are effective in inhibiting swelling, inflammatory cell infiltration, and abnormal proliferation of epidermal tissue caused by allergic skin diseases. FIG. 12 shows that melittin-carrying nanoparticles are effective in reducing the expression levels of the pro-inflammatory relevant cytokines IFN-. gamma.and IL-4 in serum.

Example 6: studies that melittin-carrying nanoparticles are effective in accumulating in cutaneous draining lymph nodes and inhibiting hapten-specific CD8+ T cell proliferation and inhibiting the release of cellular inflammatory factors

The melittin-carrying nanoparticles of example 2 were effective in accumulating in cutaneous draining lymph nodes and inhibiting hapten-specific CD8+ T cell proliferation and inhibiting the release of cellular inflammatory factors for studies see fig. 8, 9, 10 and 11. As shown in fig. 8, the melittin-carrying nanoparticles were injected intradermally on both sides of the caudal root and the core was loaded with the fluorescent dye DIR-BOA, and the results showed that at 12h, a large amount of fluorescent dye signals were accumulated in the cutaneous draining lymph nodes, and dissected mice found that strong fluorescent signals were found only in the inguinal and axillary draining lymph nodes of the mouse skin, and the liver, demonstrating that the melittin-carrying nanoparticles could enter the lymphatic system to regulate immune response and have less burden on the viscera. FIGS. 9 and 10 show that the melittin-carrying nanoparticles are effective in inhibiting allergen-specific CD8+ T cell proliferation in lymph nodes; FIG. 11 shows that the melittin-carrying nanoparticles were incubated with allergen-stimulated lymphocytes and cell supernatants were examined, which effectively inhibited the expression of the inflammation-associated cytokine IFN-. gamma..

Example 7: clinical application of three types of composition substances (phospholipid, cholesterol ester and polypeptide) carrying melittin nanoparticles

The three classes of constituent substances (phospholipids, cholesterol oleate, polypeptides) of melittin-carrying nanoparticles of example 2 have been used clinically.

Phospholipids have been used in clinical therapy, liposomes being a good example. Doxil is a drug that uses liposome-encapsulated doxorubicin to treat Kaposi's sarcoma and has been approved by the FDA in the United states for the treatment of ovarian cancer and multiple myeloma.

Cholesterol is a substance existing in human bodies and has good biological safety.

There are also many applications of polypeptides in clinical therapy, for example: the cervus and cucumis polypeptide is a good medicine for treating fracture. The bone induction polypeptide biological factor as the main component of the medicine can effectively promote the synthesis of bone-derived growth factors influencing bone formation and absorption in an organism. The cervus and cucumis polypeptide injection can obviously promote bone healing and formation of new bones, regulate bone metabolism, has the effects of resisting inflammation, relieving pain and the like, has definite curative effect on fracture nonunion osteoporosis, has no obvious adverse reaction, and has excellent curative effect on rheumatoid arthritis soft tissue injury.

The present invention is not limited to the above-described embodiments, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and such modifications and improvements are also considered to be within the scope of the present invention. Those not described in detail in this specification are within the skill of the art.

Sequence listing

<110> university of science and technology in Huazhong

EZHOU INSTITUTE OF INDUSTRIAL TECHNOLOGY HUAZHONG University OF SCIENCE AND TECHNOLOGY

<120> application of melittin and application of nano-particles carrying melittin

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Asp Trp Phe Lys Ala Phe Tyr Asp Lys Val Ala Glu Lys Phe Lys Glu

1 5 10 15

Ala Phe

<210> 2

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu

1 5 10 15

Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln

20 25

Claims

1. Use of melittin-carrying nanoparticles, characterized in that: the melittin-carrying nanoparticles consist of melittin-carrying polypeptides and phospholipids, cholesterol esters; the polypeptide carrying melittin is formed by connecting alpha helical polypeptide, a connecting sequence and melittin in series in a covalent bond mode; the nanoparticles carrying melittin are used for preparing a medicament for treating chronic contact allergic dermatitis, and the medicament can inhibit antigen-specific T cell proliferation and cell inflammatory factor release in lymph nodes;

the phospholipid is dimyristoyl phosphatidylcholine (DMPC);

the amino acid sequence of the alpha-helical polypeptide is DWFKAFYDKVAEKFKEAF;

the amino acid sequence of the melittin is GIGAVLKVLTTGLPALISWIKRKRQQ;

the amino acid sequence of the linker sequence is GSG.