阅读说明：本技术 一种包含tbFGF的新材料及其用途 (New material containing tbFGF and application thereof ) 是由 陈俐娟 温姣琳 于 2019-06-06 设计创作，主要内容包括：本发明公开了材料PEG-VE以及tbFGF-PEG-VE。其中,及tbFGF-PEG-VE的结构式如下式(II)所示,其中,<Image he="51" wi="67" file="DDA0002087494090000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>表示多肽；n为1000～4000。本发明还提供了前述材料在制备脂质体中的用途,以及制备的脂质体。本发明采用PEG-VE以及tbFGF-PEG-VE修饰脂质体,使得脂质体的性能得到了明显的提升,临床应用前景良好。<Image he="156" wi="700" file="DDA0002087494090000012.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention discloses materials PEG-VE and tbFGF-PEG-VE. Wherein the structural formula of tbFGF-PEG-VE is shown as the following formula (II), represents a polypeptide; n is 1000 to 4000. The invention also provides the application of the material in preparing liposome and the prepared liposome. The invention adopts PEG-VE and tbFGF-PEG-VE to modify liposome, so that the lipidThe performance of the plastid is obviously improved, and the clinical application prospect is good.)

1. A material PEG-VE characterized by: the structural formula is shown as the following formula (I):

wherein R is¹H or C1-C10 alkyl; n is 1000 to 4000.

2. The material PEG-VE according to claim 1, characterized in that: in the formula (I): r¹Is H or methyl; and/or n is 1000-2000.

3. A material tbFGF-PEG-VE characterized by: the structural formula is shown as the following formula (II):

wherein the content of the first and second substances,

4. The material tbFGF-PEG-VE of claim 3, characterized in that: the polypeptide is tbFGF, and the amino acid sequence of the tbFGF is KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTY.

5. The material tbFGF-PEG-VE of claim 3, characterized in that: n in the formula (II) is 1000 to 2000.

6. A small particle size liposome characterized by: the liposome is obtained by modifying a liposome with PEG-VE, wherein the liposome is a closed vesicle with a bilayer structure prepared from phospholipid substances and cholesterol substances, and the VE end of the PEG-VE is embedded into the bilayer structure of the liposome; the structural formula of the PEG-VE is shown as the following formula (I):

wherein R is¹H or C1-C10 alkyl; n is 1000 to 4000.

7. The small particle size liposome of claim 6, wherein: in the formula (I): r¹Is H or methyl; and/or n is 1000-2000.

8. The small particle size liposome of claim 6, wherein: the phospholipid is soybean phosphatidylcholine, lecithin, hydrogenated soybean phospholipid or distearoyl phosphatidylcholine; the cholesterol substance is cholesterol.

9. The small particle size liposome of claim 6, wherein: the molar ratio of PEG-VE, phospholipids and cholesterols is 1: (3-15): (3-15), preferably 1: 8: 8.

10. a liposome with excellent slow release performance is characterized in that: the liposome is obtained by tbFGF-PEG-VE modifying the small-particle-size liposome as claimed in any one of claims 6 to 9, wherein the VE end of tbFGF-PEG-VE is embedded into a bilayer structure of the liposome; the structural formula of the tbFGF-PEG-VE is shown as the following formula (II):

wherein the content of the first and second substances,

11. The liposome of claim 10, wherein: n in the formula (II) is 1000 to 2000.

12. The liposome of claim 10, wherein: the mass ratio of the tbFGF-PEG-VE to the small-particle-size liposome is 1-10: 5-50, preferably 2.4: 10.

13. use of the material of any of claims 1 to 5 for the preparation of liposomes of small particle size, high stability and/or sustained release properties.

Technical Field

The present invention relates to a new material comprising tbFGF and its use.

Background

Many antineoplastic drugs exhibit many disadvantages in clinical applications, including poor therapeutic effects, dose-limiting toxicity, poor patient tolerance, etc., wherein the development of new targeted drug delivery formulations is promoted due to the large toxic effect on normal tissues caused by poor drug distribution. Recently, the targeted drug delivery system based on the nanotechnology is developed rapidly, for example, micelles, liposomes and solid lipid nanoparticles are widely applied to cancer chemotherapy, and have the effects of improving the solubility of the drug, prolonging the half-life period in vivo and increasing the distribution of the drug in tumor tissues and cells, so that the optimal anti-tumor effect and the minimum toxic and side effects are achieved.

Liposomes (lipopomes) are closed vesicles (hollow) with a bilayer structure prepared from phospholipids and cholesterol, and are artificial membranes with the same structure as skin cell membranes. The hydrophilic head of phospholipid molecules in water is inserted into the water, the hydrophobic tail of the liposome extends to the air, and the spherical liposome with double-layer lipid molecules is formed after stirring, and the diameter is different from 25-1000 nm. The liposome can be used for transgenosis or preparing medicines, and the medicines are delivered into cells by utilizing the characteristic that the liposome can be fused with cell membranes.

Compared with free drugs, the liposome-coated drug can play a certain slow release role, but the slow release effect is still unsatisfactory, and the liposome has larger particle size, is not easy to be absorbed, has poor stability and needs further improvement.

Disclosure of Invention

In order to solve the problems, the invention provides a new material and a liposome modified by the new material, which can optimize the performance of the liposome as a carrier.

Firstly, the invention provides a material PEG-VE, the structural formula is shown as the following formula (I):

wherein R is¹H or C1-C10 alkyl; n is 1000 to 4000.

Further, in the formula (I): r¹Is H or methyl; and/or n is 1000-2000.

The invention also provides a material tbFGF-PEG-VE, the structural formula is shown as the following formula (II):

wherein the content of the first and second substances,

represents a polypeptide; n is 1000 to 4000.

Further, the polypeptide is tbFGF, and the amino acid sequence of the tbFGF is

KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTY(SEQ ID NO.1)。

Further, n in the formula (II) is 1000 to 2000.

The invention also provides a small-particle-size liposome which is obtained by modifying the liposome with PEG-VE, wherein the liposome is a closed vesicle with a bilayer structure prepared from phospholipid substances and cholesterol substances, and the VE end of the PEG-VE is embedded into the bilayer structure of the liposome; the structural formula of the PEG-VE is shown as the following formula (I):

wherein R is¹H or C1-C10 alkyl; n is 1000 to 4000.

Further, in the formula (I): r¹Is H or methyl; and/or n is 1000-2000.

Further, the phospholipid is soybean phosphatidylcholine, lecithin, hydrogenated soybean phospholipid or distearoyl phosphatidylcholine; the cholesterol substance is cholesterol.

Further, the molar ratio of PEG-VE, phospholipids and cholesterols is 1: (3-15): (3-15), preferably 1: 8: 8.

the invention also provides a liposome with excellent slow release performance, which is obtained by modifying the small-particle-size liposome by tbFGF-PEG-VE, wherein the VE end of the tbFGF-PEG-VE is embedded into a bilayer structure of the liposome; the structural formula of the tbFGF-PEG-VE is shown as the following formula (II):

wherein the content of the first and second substances,

represents tbFGF and has the amino acid sequence of

KRLYCKNGGFFLRIHPDGRVDGVREKSDPHIKLQLQAEERGVVSIKGVCANRYLAMKEDGRLLASKCVTDECFFFERLESNNYNTY, respectively; n is 1000 to 4000.

Further, n in the formula (II) is 1000 to 2000.

Further, the mass ratio of the tbFGF-PEG-VE to the small-particle-size liposome is 1-10: 5-50, preferably 2.4: 10.

the invention also provides the application of the material in preparing liposome with small particle size, high stability and/or sustained release performance.

The liposome can be prepared into a drug-containing liposome, and the drug-containing liposome consists of a drug and the modified liposome.

The drug in the drug-containing liposome of the present invention may be any drug.

Preferably, the drug is disposed in a vesicle of a liposome, preferably between bilayer structures.

Preferably, the drug is an anti-tumor drug, preferably paclitaxel, docetaxel (i.e., docetaxel).

Preferably, the drug-containing liposome is prepared according to the following method: dissolving PEG-VE, medicine, phospholipid and cholesterol in organic solvent to obtain organic solution, adding water, mixing, rotary steaming, and filtering to obtain PEG-VE modified liposome containing medicine.

Preferably, the method further comprises the steps of: and (3) taking the drug-containing liposome modified by the PEG-VE, adding the tbFGF-PEG-VE, dissolving, uniformly mixing and incubating to obtain the drug-containing liposome modified by the tbFGF-PEG-VE.

The invention also provides a drug-containing liposome preparation, which is characterized in that: the preparation is prepared from the drug-containing liposome and pharmaceutically acceptable auxiliary materials or auxiliary components.

Preferably, the excipient is a lyoprotectant, preferably sucrose, mannitol, glucose, maltose.

The invention also provides a method for preparing the drug-containing liposome, which is characterized by comprising the following steps: the method comprises the following steps: preparing PEG-VE modified drug-containing liposome from PEG-VE, drugs, phospholipids and cholesterol by liposome preparation method.

The liposome of the present invention can be prepared by any method known in the art for preparing liposomes. The preparation method of the liposome is various and is selected according to the property or requirement of the medicine. (1) Film dispersion method: dissolving phospholipid membrane material in chloroform or other organic solvent, adding liposoluble medicine, removing solvent under reduced pressure to form lipid film, adding buffer solution or other solution containing no or water soluble medicine, and shaking to form multilayer liposome. (2) An injection method: dissolving lipid material such as phospholipid and liposoluble medicine in organic solvent (oil phase), injecting the oil phase into water phase (with or without water-soluble medicine) at uniform speed, and removing organic solvent to obtain liposome. The method can be classified into ethanol or tert-butanol injection method, ether injection method, etc. according to the solvent. (3) A reverse evaporation method: dissolving lipid material such as phospholipid in organic solvent such as chloroform, mixing with buffer solution containing medicine at a certain ratio, emulsifying, and vacuum evaporating to remove organic solvent to obtain liposome. The method is suitable for preparing liposome of water soluble medicine and macromolecular active substance, such as insulin, etc., and can improve entrapment rate. The liposome formed by the dispersion of the methods is a multi-chamber liposome, and the single-chamber liposome with small and uniform particle size can be obtained by an ultrasonic method, a homogenization method and an extrusion method.

Preferably, the liposome is prepared by: dissolving PEG-VE, medicine, phospholipid and cholesterol in organic solvent to obtain organic solution, adding water or sugar-containing water solution, mixing, rotary steaming, and filtering to obtain PEG-VE modified liposome containing medicine.

Preferably, the organic solvent is an alcohol solvent, preferably ethanol; and/or, the organic solvent dissolves PEG-VE, drugs, phospholipids and cholesterols in the following modes: and/or adding PEG-VE, medicine, phospholipid matter and cholesterol matter into organic solvent, heating and dissolving; and/or the uniformly mixing mode is stirring; the filtration mode is a 0.22 μm filter membrane.

Preferably, the method further comprises the following steps: and (3) taking the drug-containing liposome modified by the PEG-VE, adding tbFGF-PEG-VE, dissolving, uniformly mixing and incubating to obtain the drug-containing liposome modified by the tbFGF-PEG-VE and the PEG-VE.

Preferably, the dissolving is with water or an aqueous solution containing sugar; and/or the incubation is a water bath incubation at 37 ℃ for 30 min.

The invention adopts PEG-VE and tbFGF-PEG-VE to modify the liposome, so that the performance of the liposome is obviously improved, on one hand, the particle size of the liposome is reduced, the liposome is easy to be absorbed by organisms, on the second hand, the stability of the liposome is improved, and on the other hand, the slow release effect of the liposome is enhanced, compared with the unmodified liposome, the drug release speed of the liposome modified by the invention is obviously reduced, and the absorption and utilization of the drug are facilitated; the docetaxel is adopted as a medicament for verification, the antitumor effect of the liposome modified by the invention is obviously superior to that of an unmodified liposome, the side effect is reduced, and meanwhile, the liposome is used as a medicament carrier, so that the multidrug resistance of tumor cells can be reduced. Has good clinical application prospect.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1mPEG₂₀₀₀-synthesis reaction scheme of VE.

FIG. 2COOH-PEG₂₀₀₀-synthesis reaction scheme of VE.

FIG. 3tbFGF and VE-PEG₂₀₀₀Schematic diagram of-COOH micelle coupling process.

FIG. 4mPEG₂₀₀₀of-VE¹H NMR spectrum.

FIG. 5VE-PEG₂₀₀₀of-OH¹H NMR spectrum.

FIG. 6VE-PEG₂₀₀₀of-COOH¹H NMR spectrum.

FIG. 7HO-PEG₂₀₀₀-OH(A)、mPEG₂₀₀₀-VE(B)、HO-PEG₂₀₀₀-VE (C) and HOOC-PEG₂₀₀₀-infrared absorption spectrum of VE (D).

FIG. 8tbFGF-PEG₂₀₀₀-infrared absorption spectrum of VE.

FIG. 9HO-PEG₂₀₀₀-OH(A)、mPEG₂₀₀₀-VE(B)、HO-PEG₂₀₀₀-VE (C) and HOOC-PEG₂₀₀₀DSC plot of VE (D).

FIG. 10tbFGF and VE-PEG₂₀₀₀-COOH-coupled SDS-PAGE gel electrophoresis (A) free tbFGF solution (B) tbFGF with VE-PEG₂₀₀₀-COOH mixed solution (C) VE-PEG₂₀₀₀tbFGF solution.

FIG. 11 schematic diagram of DTX-tbFGF-LPs liposome preparation.

FIG. 12 is a DTX-PEG-LPs picture (A) of DTX-PEG-LPs lyophilized powder (B) of DTX-PEG-LPs lyophilized powder.

Figure 13 particle size distribution diagram of docetaxel liposomes (a) DTX-PEG-LPs solution before lyophilization (B) DTX-PEG-LPs solution after lyophilization (C) DTX-tbFGF-LPs solution).

Figure 14 transmission electron micrograph of docetaxel liposomes (a) DTX-PEG-LPs solution reconstituted after lyophilization (B) DTX-tbFGF-LPs solution (scale: 100 nm).

Figure 15 docetaxel standard plots.

Figure 16 blank liposome liquid chromatogram.

Figure 17 different in vitro release profiles of DTX liposomes.

Figure 18 effect of docetaxel on LL/2 cell growth inhibition free DTX and effect of different DTX liposomes on LL/2 cells 48h (a) and 72h (b) on cell growth inhibition rate.

Figure 19 effect of docetaxel on B16 cell growth inhibition free DTX and effect of different DTX liposomes on B16 cells 48h (a) and 72h (B) effect on cell growth inhibition rate.

FIG. 20 fluorescent micrograph of coumarin-6 acting on B16 cells for 2 h.

FIG. 21 flow cytometric analysis of B16 cells after 2h of coumarin 6 treatment.

Figure 22 photographs of the appearance of mouse tumors after treatment with different DTX LPs.

Figure 23 change in body weight of mice during different docetaxel liposome treatments.

Figure 24 tumor volume change during different docetaxel liposome treatments.

FIG. 25H & E staining results of various groups of tumor tissues.

FIG. 26H & E staining results for normal tissues in each group (A) saline group (B) blank liposome group (C) Free DTX (D) DTX LPs (E) DTX-PEG-LPs (F) DTX-tbFGF-LPs.

FIG. 27 CD31 staining results for various groups of tumor tissues.

Fig. 28 MVD counts for each group of tumor tissues.

Figure 29 results of biochemical indicators of mouse serum after treatment with different docetaxel liposomes.

FIG. 30 shows the inhibition effect of the liposome of the present invention on P-gp enzyme.

Detailed Description