Polypeptide-nano-selenium compound and application of composition of polypeptide-nano-selenium compound in tumor resistance

Abstract

The invention discloses a polypeptide-nano-selenium compound and application of a composition of the polypeptide-nano-selenium compound in tumor resistance. The compound is composed of nano-selenium and polypeptide with a specific amino acid sequence Cys-Phe-Leu-Gly-Arg-Glu-Gly-Lys-Trp-Gly-Tyr-Cys, the polypeptide and selenium atoms on the surface of the nano-selenium form coordinate bonds through cysteine residues at two ends, and the problem that the nano-selenium is prone to aggregation and inactivation is effectively solved. The preparation method comprises the following steps: reducing ascorbic acid to prepare nano-selenium, and modifying the polypeptide on the surface of the nano-selenium through a coordination reaction. Experiments show that the change rate of the particle size of the nano-selenium is less than 5% after the compound is stored for 3 months, and the stability is remarkably improved; and the inhibition rate on various tumor cells after 72 hours exceeds 80%. The compound provided by the invention is simple in preparation process and high in safety, can be used as an active component to prepare antitumor drugs in various dosage forms such as tablets, injections and the like, and provides a new effective means and thought for tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology applications and relates to the application of a polypeptide-nanoselenium complex and a composition thereof in anti-tumor applications. Background Art

[0002] Nano-selenium, with its low toxicity and high biological activity, has shown great application potential in many fields such as anti-oxidation, anti-tumor, anti-diabetes, reducing drug toxicity, preventing and improving cardiovascular diseases. However, nano-selenium has high surface energy and is unstable. It is very easy to aggregate during storage and use. Once aggregated, it loses its biological activity, which greatly limits the development of nano-selenium in practical applications. At present, a variety of substances have been tried as stabilizers for nano-selenium, such as polysaccharides and proteins. However, these stabilizers have limited stability-enhancing effects, introduce immunogenicity, and are difficult to accurately control the performance of nano-selenium. They cannot fully meet the needs of nano-selenium in the field of biomedicine, especially in the development of anti-tumor drugs. Therefore, the development of a stabilizer that is efficient, safe, and can significantly improve the stability of nano-selenium has become the key to promoting the application of nano-selenium in fields such as anti-tumor drugs. Summary of the Invention

[0003] In order to overcome the shortcomings of the existing technology, the present invention aims to provide a polypeptide-nanoselenium complex, which effectively solves the problem of easy aggregation and inactivation of nanoselenium through the combination of specific polypeptides and nanoselenium, and significantly improves the stability of nanoselenium; at the same time, based on the complex, its application in anti-tumor drugs is developed to provide a new and effective means for tumor treatment.

[0004] The purpose of the present invention is achieved by adopting the following technical solutions:

[0005] A polypeptide-nanoselenium complex consists of nanoselenium and a polypeptide, the amino acid sequence of the polypeptide is Cys-Phe-Leu-Gly-Arg-Glu-Gly-Lys-Trp-Gly-Tyr-Cys, and the polypeptide is modified on the surface of the nanoselenium to stabilize the nanoselenium structure through the synergistic effect of physical encapsulation and chemical coordination.

[0006] Specifically, the thiol groups of cysteine ​​(Cys) at both ends of the polypeptide chain form coordination bonds (-S-Se-) with the selenium atoms on the surface of the nano-selenium, with a bond energy of about 40 kJ / mol, thereby achieving covalent anchoring of the polypeptide and the nano-selenium; the glycine (Gly) in the chain forms a flexible spatial barrier with its small side chain, and hydrophobic amino acids such as phenylalanine (Phe), leucine (Leu), tryptophan (Trp), and tyrosine (Tyr) form a protective layer on the surface of the nano-selenium through hydrophobic interactions. The benzene rings in the structures of Phe, Trp, and Tyr further increase the spatial steric hindrance, synergistically achieving the dual stability of physical encapsulation and chemical coordination, and inhibiting the aggregation of nano-selenium particles.

[0007] Furthermore, the particle size of the nano-selenium is 10-50 nm. This particle size range helps to improve the biological activity, cellular uptake efficiency and in vivo circulation stability of the nano-selenium.

[0008] Furthermore, the mass ratio of the polypeptide to nano-selenium is (1-5):100. At this ratio, the polypeptide can fully encapsulate and stabilize nano-selenium, while exerting its multiple mechanisms of assisting nano-selenium in anti-tumor effects. When the polypeptide ratio is too low, nano-selenium cannot be effectively encapsulated, resulting in decreased stability of nano-selenium and difficulty in fully exerting the targeting and auxiliary anti-tumor effects of the polypeptide; and when the polypeptide ratio is too high, it will affect the overall particle size and surface properties of the complex, and may even produce some unnecessary side effects, such as increased immunogenicity.

[0009] Furthermore, the polypeptide-nanoselenium complex serves as an anti-tumor active ingredient and a pharmaceutically acceptable excipient.

[0010] Furthermore, the pharmaceutically acceptable excipients include but are not limited to fillers, binders, disintegrants, lubricants, solvents, emulsifiers, and suspending agents.

[0011] Furthermore, the dosage form of the anti-tumor drug is tablets, capsules, injections, granules, and oral liquids.

[0012] Another object of the present invention is to provide a method for preparing a polypeptide-nanoselenium complex.

[0013] The specific steps include:

[0014] S1. Preparation of Nanoselenium: Sodium selenite solution and ascorbic acid solution are mixed in a volume ratio of 1:(2-4) and stirred at room temperature for 2-4 hours to produce a nanoselenium solution. This reaction utilizes the reducing property of ascorbic acid to reduce selenium(IV) in the sodium selenite to elemental selenium, which then spontaneously aggregates to form nanoparticles. Controlling the volume ratio of sodium selenite solution to ascorbic acid solution, as well as the reaction time and temperature, effectively regulates the particle size and morphology of the nanoselenium to meet the requirements for subsequent complexation with a polypeptide.

[0015] S2. Preparation of polypeptide-nanoselenium complex: Add the polypeptide to the above-mentioned nanoselenium solution in an amount such that the mass ratio of polypeptide to nanoselenium reaches (1-5):100, stir and react at room temperature for 4-6 hours, and after the reaction is completed, centrifuge, wash and dry to obtain the polypeptide-nanoselenium complex. In this step, the polypeptide and nanoselenium are fully contacted under stirring conditions, and the polypeptide undergoes physical adsorption and chemical coordination with the surface of nanoselenium through its specific amino acid residues, thereby modifying the nanoselenium surface to form a stable complex. Centrifugation can remove unreacted polypeptide and other impurities, washing further purifies the complex, and finally drying to obtain a solid polypeptide-nanoselenium complex that is easy to store and subsequently use.

[0016] The polypeptide chain was prepared using Fmoc solid-phase synthesis, a standard method for preparing short peptides of 10-50 amino acids, characterized by high efficiency, high purity, and scalability. The specific amino acid sequence of the polypeptide chain has been precisely defined by the standard sequence listing, as detailed in the table below.

[0017] Amino acid sequence listing:

[0018]

[0019] The SEQ ID NO: 1, through its unique amino acid arrangement, constructs a three-level functional system of 'coordination anchoring-steric stabilization-targeted activation'. This sequence, in conjunction with the Fmoc solid-phase synthesis method, provides a clear material basis and mechanism of action for the anti-tumor activity described in the subsequent examples.

[0020] Another object of the present invention is to provide a polypeptide-nanoselenium complex and a composition thereof for use in the preparation of anti-tumor drugs.

[0021] The anti-tumor mechanism of the polypeptide-nanoselenium complex has a triple anti-tumor mechanism of "electrostatic targeting-hydrophobic internalization-signal regulation". Among them, the positive charges of arginine (Arg) and lysine (Lys) target the negatively charged components (such as glycoproteins) on the surface of tumor cells through electrostatic attraction; the hydrophobicity and benzene ring structure of Phe, Leu, Trp, and Tyr enhance membrane fusion ability and promote the complex to enter tumor cells through endocytosis; Trp and Tyr activate the JNK signaling pathway by simulating the binding sites of growth factor receptors, induce tumor cell apoptosis, and ultimately enhance the tumor-targeted killing efficiency of nanoselenium through the synergistic effect of multiple amino acids.

[0022] The beneficial effects of the present invention are:

[0023] (1) The present invention selects polypeptides with specific amino acid sequences as stabilizers for nano-selenium. The cysteine ​​residues at both ends of the polypeptide can form stable coordination bonds with selenium atoms on the surface of the nano-selenium, firmly modifying the polypeptide on the surface of the nano-selenium, forming steric hindrance and electrostatic repulsion, effectively preventing aggregation between nano-selenium particles, and significantly improving the stability of the nano-selenium. Experimental testing shows that under the same storage conditions, after 3 months of storage, the particle size change rate of the nano-selenium of the polypeptide-nanoselenium complex of the present invention is less than 5%, while the particle size change rate of the unmodified nano-selenium is over 70%, indicating that the present invention greatly improves the stability problem of nano-selenium.

[0024] (2) The polypeptide-nanoselenium complex of the present invention has excellent anti-tumor activity. The complex can enter tumor cells through the endocytosis of tumor cells, and the nanoselenium is released in the tumor cells to exert its anti-tumor activity. At the same time, the polypeptide can enhance the targeting of the complex to tumor cells, further improving the anti-tumor effect. Experiments show that the inhibition rate of the polypeptide-nanoselenium complex of the present invention on various tumor cells such as lung cancer cells A549, liver cancer cells HepG2, and colon cancer cells HCT1 16 is more than 80% after 72 hours, which is significantly better than simple nanoselenium and polypeptide, showing a good synergistic anti-tumor effect.

[0025] (3) The preparation method of the polypeptide-nanoselenium complex of the present invention is simple, the conditions are mild, and it is easy to produce on a large scale; and the polypeptide used is artificially synthesized, with a clear source, controllable quality, and high safety, avoiding the problems such as immunogenicity that may be caused by traditional stabilizers, and providing reliable guarantee for its application in anti-tumor drugs. DETAILED DESCRIPTION

[0026] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail with reference to the following specific embodiments.

[0027] Example 1

[0028] Preparation of a polypeptide-nanoselenium complex:

[0029] (1) Preparation of nano-selenium: 10 mL of a 0.1 mol / L sodium selenite solution was mixed with 20 mL of a 0.2 mol / L ascorbic acid solution, and the mixture was stirred at room temperature at a speed of 200 r / min for 2 h to obtain a nano-selenium solution.

[0030] (2) Preparation of polypeptide-nanoselenium complex: 10 mg of a polypeptide having the amino acid sequence Cys-Phe-Leu-Gly-Arg-Glu-Gly-Lys-Trp-Gly-Tyr-Cys was added to the above-mentioned nanoselenium solution so that the mass ratio of polypeptide to nanoselenium was 1:100. The mixture was stirred at room temperature at a speed of 200 r / min for 4 h. After the reaction, the reaction solution was centrifuged at a speed of 8000 r / min for 10 min, the supernatant was discarded, and the precipitate was washed three times with deionized water and then dried in vacuo at 60°C to obtain a polypeptide-nanoselenium complex.

[0031] Example 2

[0032] Preparation of a polypeptide-nanoselenium complex:

[0033] (1) Preparation of nano-selenium: 10 mL of a 0.1 mol / L sodium selenite solution was mixed with 30 mL of a 0.3 mol / L ascorbic acid solution, and the mixture was stirred at room temperature at a speed of 250 r / min for 3 h to obtain a nano-selenium solution.

[0034] (2) Preparation of polypeptide-nanoselenium complex: 25 mg of a polypeptide having the amino acid sequence Cys-Phe-Leu-Gly-Arg-Glu-Gly-Lys-Trp-Gly-Tyr-Cys was added to the above-mentioned nanoselenium solution so that the mass ratio of polypeptide to nanoselenium was 2.5:100. The mixture was stirred at 250 r / min at room temperature for 5 h. After the reaction, the reaction solution was centrifuged at 9000 r / min for 12 min, the supernatant was discarded, and the precipitate was washed three times with deionized water and then dried in vacuo at 65°C to obtain a polypeptide-nanoselenium complex.

[0035] Example 3

[0036] Preparation of a polypeptide-nanoselenium complex:

[0037] (1) Preparation of nano-selenium: 10 mL of a 0.1 mol / L sodium selenite solution was mixed with 40 mL of a 0.4 mol / L ascorbic acid solution, and the mixture was stirred at room temperature at a speed of 300 r / min for 4 h to obtain a nano-selenium solution.

[0038] (2) Preparation of polypeptide-nanoselenium complex: 50 mg of a polypeptide having the amino acid sequence Cys-Phe-Leu-Gly-Arg-Glu-Gly-Lys-Trp-Gly-Tyr-Cys was added to the above-mentioned nanoselenium solution so that the mass ratio of polypeptide to nanoselenium was 5:100. The mixture was stirred at 300 r / min for 6 h at room temperature. After the reaction, the reaction solution was centrifuged at 10,000 r / min for 15 min, the supernatant was discarded, and the precipitate was washed three times with deionized water and then dried in vacuo at 70°C to obtain a polypeptide-nanoselenium complex.

[0039] Example 4

[0040] Preparation of an anti-tumor drug tablet:

[0041] 100 g of the polypeptide-nanoselenium complex prepared in Example 2, 150 g of lactose, 100 g of starch, and 20 g of sodium carboxymethyl starch were mixed evenly, and an appropriate amount of 5% hydroxypropyl methylcellulose ethanol solution was added to prepare a soft material. The mixture was passed through a 20-mesh sieve and granulated. The mixture was dried at 60°C, and 5 g of magnesium stearate was added after the granulation. The mixture was mixed evenly and pressed into tablets to obtain anti-tumor drug tablets.

[0042] Example 5

[0043] Preparation of an anti-tumor drug injection:

[0044] 50 g of the polypeptide-nanoselenium complex prepared in Example 2 was dissolved in an appropriate amount of water for injection, 9 g of sodium chloride was added to adjust the osmotic pressure, the pH was adjusted to 7.0-7.5 with sodium hydroxide solution, and then 0.5 g of sodium bisulfite was added as an antioxidant. After stirring evenly, the mixture was filtered, sealed in an ampoule, and sterilized at 121°C for 30 min to obtain an anti-tumor drug injection.

[0045] Experimental Example 1

[0046] Stability test of peptide-nanoselenium complex:

[0047] The polypeptide-nanoselenium complexes prepared in Examples 1-3 and unmodified nanoselenium were prepared into aqueous solutions with a concentration of 1 mg / mL, respectively, and stored at room temperature for 3 months. Dynamic light scattering (DLS) was used to measure the change in the particle size of the nanoselenium before and after storage. The results are shown in the following table:

[0048] sample Initial particle size (nm) Initial particle size (nm) Initial particle size (nm) Example 1 20 21 5％ Example 2 22 23 4.5％ Example 3 23 26 4％ Unmodified nanoselenium 20 35 75％

[0049] The experimental results show that the polypeptide-nanoselenium complex prepared by the present invention has good stability and can effectively inhibit the aggregation of nanoselenium.

[0050] Experimental Example 2

[0051] Antitumor activity test of peptide-nanoselenium complex

[0052] (1) Cell culture: Lung cancer cells A549, liver cancer cells HepG2, and colon cancer cells HCT116 were cultured in RPMI1640 medium containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin) in an incubator at 37°C and 5% CO2.

[0053] (2) Grouping and treatment: Tumor cells in the logarithmic growth phase were inoculated into 96-well plates, with 1×10 4 Cells were cultured for 24 hours and then divided into the following groups: a control group (added with an equal volume of culture medium), a nanoselenium group (added with a nanoselenium solution at a concentration of 10 μg / mL), a polypeptide group (added with a polypeptide solution at a concentration of 10 μg / mL), and a polypeptide-nanoselenium complex group (added with a polypeptide-nanoselenium complex solution prepared in Example 2 at a concentration of 10 μg / mL). Six replicate wells were set up in each group. After further culture for 72 hours, the cell survival rate was detected by the MTT assay, and the cell inhibition rate was calculated. The results are shown in the following table:

[0054]

[0055]

[0056] The experimental results show that the polypeptide-nanoselenium complex of the present invention has significant anti-tumor activity, and its effect is significantly better than that of simple nanoselenium and polypeptide.

[0057] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations that come within the meaning and range of equivalents of the claims be embraced therein.

[0058] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This description is for clarity only. Those skilled in the art should consider this specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art. Technical details not described in detail in this invention can be implemented by any existing technology in the art. In particular, all technical features not described in detail in this invention can be implemented by any existing technology.

Claims

1. A polypeptide-nanoselenium complex, characterized in that: The complex consists of nano-selenium and a polypeptide, the amino acid sequence of the polypeptide is Cys-Phe-Leu-Gly-Arg-Glu-Gly-Lys-Trp-Gly-Tyr-Cys, and the polypeptide is modified on the surface of the nano-selenium.

2. The polypeptide-nanoselenium complex according to claim 1, characterized in that The cysteine ​​residues at both ends of the polypeptide form coordination bonds (-S-Se-) with selenium atoms on the surface of the nano-selenium.

3. The polypeptide-nanoselenium complex according to claim 1, characterized in that The particle size of the nano-selenium is 10-50 nm.

4. The polypeptide-nanoselenium complex according to claim 1, characterized in that The mass ratio of the polypeptide to nano-selenium is (1-5):

100.

5. The polypeptide-nanoselenium complex according to claim 1, characterized in that The polypeptide-nanoselenium complex serves as an anti-tumor active ingredient and a pharmaceutically acceptable auxiliary material.

6. The polypeptide-nanoselenium complex according to claim 5, characterized in that The pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, lubricants, solvents, emulsifiers, and suspending agents.

7. The polypeptide-nanoselenium complex according to claim 5, characterized in that The dosage forms of the anti-tumor drug are tablets, capsules, injections, granules and oral liquids.

8. A method for preparing the polypeptide-nanoselenium complex according to any one of claims 1 to 7, characterized in that: The following steps are involved: S1. Preparation of nano-selenium: Mix sodium selenite solution and ascorbic acid solution in a volume ratio of 1:(2-4), and stir at room temperature for 2-4 hours to obtain a nano-selenium solution; S2. Preparation of polypeptide-nanoselenium complex: Add the polypeptide to the above-mentioned nanoselenium solution in an amount such that the mass ratio of polypeptide to nanoselenium reaches (1-5):100, stir and react at room temperature for 4-6 hours, and after the reaction is completed, obtain the polypeptide-nanoselenium complex by centrifugal separation, washing, and drying.

9. Use of the polypeptide-nanoselenium complex and composition thereof according to any one of claims 1 to 8 in the preparation of anti-tumor drugs.

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