An amphiphilic molecule, micelle and preparation method and application thereof

Abstract

The present invention discloses an amphiphilic molecule, a micelle, and a preparation method and application thereof. The amphiphilic molecule of the present invention is composed of structural units of mannose, polytyrosine, and fatty amine connected together; wherein, the polytyrosine is selected from a peptide chain polymerized by 2 to 8 tyrosine molecules; the fatty amine is selected from C 16 or C 18 saturated or unsaturated fatty amines. The micelle prepared from the above amphiphilic molecule can be used to prepare a drug for treating melanoma, can be used to inhibit the growth and metastasis of malignant melanoma, but has very high biosafety for normal cells. The melanin endogenously produced in melanoma induced by it can be used for subsequent efficient photothermal therapy, providing a safe and reliable technical means for melanoma treatment and having good clinical application prospects.

Description

Technical Field

[0001] The invention belongs to the field of medicine, and specifically relates to an amphiphilic molecule, micelle and a preparation method and application thereof. Background Art

[0002] Amphiphilic molecules refer to compound molecules that have both hydrophilic and lipophilic properties. Such substances are called "amphiphilic". Amphiphilic molecules have a hydrophilic head and a hydrophobic tail. The hydrophilic head is generally composed of polar groups such as choline and amine salts, while the hydrophobic tail is generally composed of long fatty chains. Amphiphilic molecules can self-assemble into various molecular assemblies in water, such as micelles and vesicles. The structure and properties of the formed assembly are directly related to the structure of the amphiphilic molecules. At present, the self-assembly of amphiphilic molecules has made great progress in nanomaterials and is widely used.

[0003] Micelles are mostly formed by the self-assembly of amphiphilic molecules. When the concentration of amphiphilic molecules reaches a certain value, a large number of ordered molecular aggregates begin to form. In micelles, the hydrophobic groups of amphiphilic molecules aggregate to form the inner core of the micelle, and the hydrophilic polar groups form the outer layer of the micelle. The nano-core-shell structure of micelles makes them suitable for drug loading and delivery. The hydrophobic inner shell can be used to transport hydrophobic drugs such as DNAs, RNAs, proteins, paclitaxel, etc., while the hydrophilic outer shell can improve its stability and biocompatibility when circulating in the body, and can be widely used in the medical field.

[0004] Melanoma is a cancer caused by melanocytes, special pigment cells that are mainly found in the skin and can produce melanin. Melanoma progresses rapidly, with a five-year survival rate of less than 10%, and is difficult to cure. Melanoma can be cured by surgery in the early stages, but once it develops to the metastatic stage, it is difficult to treat, and there is currently a lack of effective drugs and treatment options. Therefore, there is an urgent need to develop new drugs and treatments for the efficient treatment of melanoma. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an amphiphilic molecule that can be prepared into an amphiphilic nano-micelle for the treatment of melanoma.

[0006] The invention also provides a method for preparing the amphiphilic molecule.

[0007] The present invention also provides a micelle having the amphiphilic molecule.

[0008] The invention also provides a method for preparing the micelle.

[0009] The present invention also provides the application of the micelle.

[0010] According to one aspect of the present invention, an amphiphilic molecule is provided, wherein the amphiphilic molecule is composed of mannose, polytyrosine and fatty amine; wherein the polytyrosine is selected from a peptide chain formed by polymerization of 2 to 8 tyrosine molecules; and the fatty amine is selected from C 16 or C 18 Saturated or unsaturated fatty amines.

[0011] According to a specific embodiment of the present invention, at least the following beneficial effects are achieved: the amphiphilic molecule of the present invention comprises hydrophilic mannose and polytyrosine peptide chains, and lipophilic fatty chains; wherein mannose can act on the melanoma site in a targeted manner, and the tyrosine released by the decomposition of the polytyrosine peptide chain, as a synthetic substrate of melanin, can induce the metabolism of mouse melanoma in vitro to produce a large amount of melanin, and induce its differentiation, inhibit its proliferation and migration, have extremely high biosafety, provide a safe and reliable technical means for the treatment of melanoma, and have good clinical application prospects.

[0012] In some embodiments of the present invention, the polytyrosine is selected from a peptide chain composed of 3 to 6 tyrosine molecules; the fatty amine is selected from C 16 or C 18 A saturated or unsaturated fatty amine, preferably hexadeceneamine or octadeceneamine (oleylamine).

[0013] In some preferred embodiments of the present invention, the polytyrosine is a tyrosine tetrapeptide formed by polymerization of four tyrosine molecules; the fatty amine is oleylamine. The structural formula of the amphiphilic molecule is as follows:

[0014]

[0015] In the present invention, “C 16 or C 18 The term "saturated or unsaturated fatty amine" refers to a saturated or unsaturated fatty amine with 16 or 18 carbon atoms, mainly including alkane chains and olefin chains.

[0016] According to another aspect of the present invention, a method for preparing the above-mentioned amphiphilic molecule is provided, comprising the following steps: using mannose-polytyrosine and fatty amine as raw materials, carrying out an amide condensation reaction in the presence of a carbodiimide condensation agent and a condensation activator to obtain the amphiphilic molecule; wherein the mannose-polytyrosine is selected from a peptide chain composed of 2 to 8 tyrosine molecules connected by mannose; and the fatty amine is selected from C 16 or C 18 Saturated or unsaturated fatty amines.

[0017] The preparation method according to a specific embodiment of the present invention has at least the following beneficial effects: the amphiphilic molecule of mannose-polytyrosine-fatty amine of the present invention can be efficiently prepared by the method.

[0018] In some embodiments of the present invention, the carbodiimide condensation agent is selected from dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI); and / or the condensation activator is selected from 4-dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt) or N-hydroxysuccinimide (NHS).

[0019] In some preferred embodiments of the present invention, mannose-tyrosine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide are weighed in a molar ratio of (1-1.5):2:2, and dissolved in dimethyl sulfoxide, mixed under inert gas for 0.5-1 hour, and then 5 equivalents of oleylamine are added dropwise to the above solution, reacted at room temperature for 24 hours, and the product is loaded into a dialysis bag (molecular weight cutoff 500-1000), and dialyzed alternately with ultrapure water and ethanol, respectively. Finally, the product is freeze-dried to obtain mannose-tyrosine-oleylamine (M-Tyr-OA) as a white powder.

[0020] In some more preferred embodiments of the present invention, the gas atmosphere is argon and the solvent is of dry grade.

[0021] In some more preferred embodiments of the present invention, the purity of mannose-tyrosine, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is above 99%, and the purity of oleylamine is above 90%.

[0022] According to yet another aspect of the present invention, a micelle is provided, wherein the micelle comprises the above-mentioned amphiphilic molecule.

[0023] According to a specific embodiment of the present invention, at least the following beneficial effects are achieved: an amphiphilic nanomicelle composed of mannose, tyrosine and a lipophilic molecule, wherein the mannose and L-tyrosine tetrapeptide blocks in the amphiphilic molecule are in the outer shell of the micelle, and the oleylamine block is in the inner core of the micelle, which can significantly improve the solubility of tyrosine and efficiently deliver it to melanoma cells and tumors, decomposing and releasing tyrosine; the tyrosine released by its decomposition has no side effects on other tissues and organs of the body, and can participate in cellular glycolysis as a raw material for the metabolism of the central carbon atom, and has extremely high biosafety.

[0024] In some embodiments of the present invention, the micelle has at least one of the following properties: i) the particle size ranges from 20 nm to 200 nm; ii) the micelle particle size dispersion index (PDI) is less than 0.3.

[0025] In some preferred embodiments of the present invention, the micelles have at least one of the following properties: i) the particle size range is about 60 nm; ii) the micelle particle size dispersion index (PDI) is about 0.6; iii) the critical micelle concentration is 0.00828 mg / L.

[0026] According to another aspect of the present invention, a method for preparing the above-mentioned micelles is proposed, comprising the following steps: preparing the amphiphilic molecule into a tetrahydrofuran solution with a concentration of 1 to 20 mg / mL; adding the tetrahydrofuran solution to water at a rate of 1 to 5 mL / h, and stirring to obtain the micelles; wherein the volume ratio of the tetrahydrofuran solution to water is 1:(2 to 8).

[0027] In some preferred embodiments of the present invention, the method comprises the following steps: weighing a certain amount of mannose-tyrosine-oleylamine obtained in step (1), preparing a 5 mL tetrahydrofuran solution (1-5 mg / mL), injecting the solution into 20 mL ultrapure water (2 mL / h) using a peristaltic pump, stirring for 1 hour, then placing the solution into a dialysis bag (molecular weight cutoff 7000), dialyzing with ultrapure water, and concentrating with an ultrafiltration tube to obtain tyrosine nanomicelles (M-Tyr-OANP) of the desired concentration.

[0028] According to another aspect of the present invention, the use of the above micelles in the preparation of melanin-producing drugs is proposed.

[0029] According to another aspect of the present invention, the use of the above micelles in preparing drugs for treating melanoma is proposed.

[0030] In some embodiments of the present invention, the drug for treating melanoma is a drug that kills melanoma through photothermal therapy.

[0031] The present invention has at least the following beneficial effects: the tyrosine released by the decomposition of the micelles of the present invention, as a synthetic substrate of melanin, can induce the metabolism of mouse melanoma in vitro to produce a large amount of melanin, and induce its differentiation, inhibit its proliferation and migration; in addition, after intravenous injection, the micelles can reach the melanoma site under the targeting effect of mannose, and significantly inhibit the growth and metastasis of melanoma in vivo. The micelles of the present invention can achieve the controllable and efficient delivery of tyrosine, and have a significant effect in inhibiting the growth and metastasis of malignant melanoma. At the same time, the tyrosine micelles that are not targeted to the tumor site, the tyrosine released after degradation in vivo has no side effects on the body, and can be used as a raw material for the metabolism of the central carbon atom of the organism, participate in the metabolism of the body, and have excellent biosafety; finally, the large amount of metabolites melanin produced by the micelles inducing melanoma can be used for subsequent treatment (such as photothermal therapy, etc.); therefore, the micelles can be used to inhibit the growth and metastasis of malignant melanoma, and have very high biosafety, providing safe and reliable technical means for the treatment of melanoma, and have good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0033] Figure 1 The NMR spectrum of the mannose-tyrosine tetrapeptide-oleylamine amphiphilic molecule prepared in Example 1 of the present invention, the transmission electron microscopy (TEM) image of the tyrosine micelles formed by self-assembly in water, the hydrated particle size, and the critical micelle concentration image;

[0034] Figure 2 This is a diagram of cell activity after different concentrations of tyrosine micelles and three normal cells were co-cultured for different periods of time in Example 2 of the present invention;

[0035] Figure 3 The expression diagram of the genes related to central carbon source metabolism after Hacat cells were treated under different culture medium conditions for 24 hours in Example 3 of the present invention;

[0036] Figure 4 The figure is a graph showing the experimental results of Example 4 of the present invention, showing that tyrosine micelles are internalized by melanoma cells and can induce the cells to produce a large amount of melanin, wherein a is a confocal fluorescence imaging image, b is a cell imaging image, and c is a graph showing the melanin content in the cells;

[0037] Figure 5 Graphs showing the experimental results of tyrosine micelles inhibiting the proliferation and metastasis of melanoma cells in Example 5 of the present invention; wherein a is a cell activity statistics graph, b is a cell cycle distribution graph, c is a cell wound healing experiment graph, and d is a cell transwell invasion experiment graph;

[0038] Figure 6This is an in vivo fluorescence imaging diagram of the targeting experiment of tyrosine micelles on mouse melanoma in Example 6 of the present invention;

[0039] Figure 7 This is an experimental diagram of tyrosine micelles inhibiting the growth and metastasis of melanoma in mice in Example 7 of the present invention, significantly prolonging the survival of mice, wherein a is a photo of the tumor of the experimental mouse, b is a tumor growth curve, c is a mouse survival rate curve, and d is an immunohistochemistry diagram;

[0040] Figure 8 These are experimental diagrams of tyrosine micelles killing melanoma cells through photothermal therapy according to an embodiment of the present invention, wherein a is a thermal imaging diagram under different laser irradiation times, b is a cell activity statistics diagram, and c is a cell live-dead staining diagram. DETAILED DESCRIPTION

[0041] The following will clearly and completely describe the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by technicians in this field without creative work are all within the scope of protection of the present invention. The test methods used in the embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used are all reagents and materials obtained from commercial channels unless otherwise specified.

[0042] In the embodiments of the present invention, the amphiphilic molecules are specifically amphiphilic molecules containing tyrosine. In the following embodiments, the micelles prepared by the amphiphilic molecules are referred to as tyrosine micelles.

[0043] Example 1: Preparation and characterization of tyrosine micelles

[0044] 1.1 Preparation of tyrosine micelles, including the following steps:

[0045] (1) Mannosyl-tyrosine tetrapeptide (M-Tyr), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were weighed in a molar ratio of (1-1.5):2:2, and dissolved in dimethyl sulfoxide, mixed under inert gas for 0.5-1 hour, and then 5 times the equivalent of oleylamine (OA) was added dropwise to the above solution, reacted at room temperature for 24 hours, and the product was loaded into a dialysis bag (molecular weight cutoff 500-1000), and dialyzed alternately with ultrapure water and ethanol, respectively. Finally, the product was lyophilized to obtain mannose-tyrosine tetrapeptide-oleylamine (Man-Tyr-OA) white powder.

[0046] The preparation process of mannose-tyrosine tetrapeptide-oleylamine molecule is as follows Figure 1As shown in a, it is obtained by amide condensation of M-Tyr and OA. The obtained mannose-tyrosine tetrapeptide-oleylamine was subjected to mass spectrometry and nuclear magnetic resonance data analysis, and its mass spectrum is shown as follows Figure 1 As shown in b, the H NMR spectrum is as follows Figure 1 As shown in c, its mass spectrometry and NMR data prove that the structure of the synthesized mannose-tyrosine tetrapeptide-oleylamine is correct.

[0047] (2) Weigh a certain amount of the mannose-tyrosine tetrapeptide-oleylamine obtained in step (1) and prepare 5 mL of a tetrahydrofuran solution with a concentration of 2 mg / mL (in other embodiments, a concentration range of 1 to 5 mg / mL can also be used), inject it into 20 mL of ultrapure water using a peristaltic pump (2 mL / h), stir for 1 hour, then put the solution into a dialysis bag (molecular weight cutoff 7000), dialyze with ultrapure water, and concentrate with an ultrafiltration tube to obtain tyrosine micelles of the desired concentration.

[0048] The preparation process of tyrosine micelles is as follows Figure 1 As shown in middle d, mannose-tyrosine tetrapeptide-oleylamine molecules self-assemble to form micelles through the above steps.

[0049] The chemical formula of the prepared mannose-tyrosine tetrapeptide-oleylamine (Man-Tyr-OA) is: 60 H 83 N5O 13 , the structural formula is:

[0050]

[0051] In this embodiment, C 18 Unsaturated fatty chains can be used as lipophilic fragments, and C 16 unsaturated fatty chain replacement; at the same time, the tyrosine tetrapeptide can also select fragments of other tyrosine polymerization degrees, such as polymer fragments of 2 to 8 tyrosines.

[0052] 1.2 Characterization of tyrosine micelles

[0053] The experimental results are as follows Figure 1 As shown: Mannosyl-tyrosine tetrapeptide-oleylamine was synthesized and successfully prepared into tyrosine micelles. Transmission Electron Microscope (TEM) images show ( Figure 1 e), the particle size range is between 20nm-200nm, the average particle size is about 60nm, and the dynamic light scattering (DLS) diagram of the hydrated particle size ( Figure 1f) shows that the average hydrated particle size is 123nm, and the tyrosine nano-micelle PDI is 0.160, which has good dispersibility. Through testing, the critical micelle concentration of tyrosine micelles is 0.00828mg / L ( Figure 1 g).

[0054] The present invention can also synthesize amphiphilic molecules and micelles of tyrosine polypeptides with different polymerization degrees (such as polypeptides polymerized from 2 to 8 tyrosine molecules) by the above method. In this embodiment, tetrameric tyrosine fragments are used because they can improve the solubility of tyrosine and the synthesis cost is appropriate.

[0055] Example 2: Tyrosine nanomicelles have excellent biosafety for normal cells

[0056] 2.1 Detection of proliferation activity of three normal cell lines with tyrosine nanomicelles at 1 mg / mL.

[0057] 1) Cell culture: Human epidermal cells Hacat and mouse mononuclear macrophages RAW264.7 were cultured and passaged in RPMI1640 high-glucose medium containing 10% fetal bovine serum and 1% double antibody, and human embryonic kidney cells Hek293T were cultured and passaged in DMEM high-glucose medium containing 10% fetal bovine serum and 1% double antibody under saturated humidity, 5% CO2, and 37°C.

[0058] 2) Cell proliferation activity assay: Three normal cells in the logarithmic growth phase were taken and made into a cell density of 1×10 5 / mL single cell suspension was inoculated in a 96-well plate, 100μL per well, and cultured at saturated humidity, 5% CO2, and 37°C. After 24h, complete medium containing 0.1mg / mL, 0.5mg / mL, and 1mg / mL tyrosine micelles was added, and the control group was replaced with new normal complete medium. Six replicate wells were added for 24h, 48h, and 72h of continuous culture, and 100μL of CCK-8 cell detection solution was added to each well. After continued culture in an incubator for 1h, the absorbance (OD value) at 405nm was detected by an enzyme marker, and the relative proliferation activity of the control group was calculated as 100%, according to the formula relative proliferation activity of the tyrosine micelle treatment group (%) = OD value of the treatment group ÷ OD value of the control group × 100%, and the relative proliferation activity was calculated.

[0059] The experimental results are as follows Figure 2 As shown, the cell activity of three types of cells after co-culture with tyrosine micelles of different concentrations for different time periods is shown, indicating the safety of tyrosine micelles for normal cells. The results showed that tyrosine micelles have excellent safety for three normal cells. After co-culture with cells for 72 hours within a concentration of 1 mg / mL, there is no significant effect on cell proliferation activity, and there is a certain pro-proliferation activity.

[0060] Example 3: Study on the effect of tyrosine micelles on the expression of genes related to central carbon atom metabolism

[0061] 1) Take Hacat cells in the logarithmic growth phase and use complete medium to make the cell density to 1×10 5 / mL single cell suspension was inoculated into 24-well plates, 500μL per well, and cultured at saturated humidity, 5% CO2, and 37°C. After 24h, the treatment group was replaced with low-glucose culture medium containing 0.1mg / mL tyrosine micelles, and the control group was replaced with new normal culture medium. Three replicates were added for each well, and trizol (RNA extraction reagent) was added to collect cell tissues and extract mRNA after continuing to culture for 24h;

[0062] 2) RT-qPCR detection of the expression levels of mRNA of central carbon atom metabolism-related genes such as Akt1, met, pfkl, pik3r2, pfkm, hk1, Pik3cd, mapk3, pkm, Acss2, and eno3;

[0063] The experimental results are as follows Figure 3 As shown, compared with cells cultured in normal medium, the genes related to central carbon atom metabolism of Hacat cells under low sugar culture were downregulated, indicating that the low sugar environment inhibited cell-related energy metabolism; and after the addition of tyrosine, a carbon source was provided for Hacat cells, glycolysis and gluconeogenesis were promoted, and the expression of related genes was restored. The experimental results show that the tyrosine micelles synthesized in Example 1 can upregulate the expression of genes related to central carbon atom metabolism of normal cells under a low sugar environment; it not only has no obvious toxicity to normal cells, but also can provide a carbon source for cell metabolism under a low sugar environment, upregulate the expression of genes related to central carbon atom metabolism, and participate in cell metabolism as a normal nutrient.

[0064] Example 4: Tyrosine micelles induce melanoma cells to produce large amounts of melanin

[0065] 4.1 Cell phagocytosis assay

[0066] Mouse melanoma B16F10 cells in the logarithmic growth phase were taken and made into a cell density of 1×10 5 / mL single cell suspension was inoculated in a confocal dish, 1mL per well, and cultured under saturated humidity, 5% CO2, and 37°C. After 24 hours, 0.5mg / mL tyrosine micelle culture medium linked to FITC was added, and the culture was continued at 37°C for 2h, 4h, and 6h, and the endocytosis of tyrosine micelles by cells was observed under a confocal microscope.

[0067] 4.2 Detection of melanin content in melanoma cells

[0068] Mouse melanoma B16F10 cells in the logarithmic growth phase were taken and made into a cell density of 1×10 5 / mL single cell suspension was inoculated into 6-well plates, 1mL per well, and cultured at saturated humidity, 5% CO2, and 37°C. After 24 hours, medium containing (0, 0.5, and 1 mg / mL) tyrosine micelles was added, and the cells (about 1×10 6 ) and dissolved in 100 μL of 1 M NaOH-10% DMSO solution, placed at 80°C for 2 hours, and the content of melanin in melanocytes was determined by measuring the absorbance at 405 nm and comparing the results with the standard curve generated by synthetic melanin (Sigma, M8631).

[0069] The experimental results are as follows Figure 4 As shown, from Figure 4 a As can be seen, due to the amphiphilicity of tyrosine micelles, they can be internalized by melanoma cells after being co-cultured with them for 2 hours, and the amount of endocytosis increases over time. Tyrosine released by the decomposition of tyrosine micelles after entering the cells can be used as a substrate for melanin synthesis, inducing melanocytes to produce a large amount of melanin. The light microscopic photo of tyrosine micelles (1 mg / mL) co-cultured with melanoma cells for 72 hours is shown in the figure. Figure 4 As shown in b, compared with the control group cells, there was a significant increase ( Figure 4 c) The results showed that tyrosine micelles can be efficiently internalized by melanoma cells in about 2 hours and can induce melanoma cells to produce a large amount of melanin.

[0070] Example 5: Ability of tyrosine micelles to inhibit proliferation and metastasis of mouse melanoma cells in vitro

[0071] 5.1 Tyrosine micelles inhibit melanoma cell proliferation

[0072] 1) Cell proliferation activity detection: B16F10 cells of the same concentration as in 1 were inoculated into 96-well plates, 100 μL per well. After 24 hours, complete medium containing 0.1 mg / mL, 0.5 mg / mL, and 1 mg / mL tyrosine micelles was added, and the control group was replaced with new normal complete medium. Six replicate wells were cultured for 24 hours, 48 ​​hours, and 72 hours, respectively. 100 μL CCK-8 cell detection solution was added to each well. After culturing in an incubator for 1 hour, the absorbance (OD value) at 405 nm was detected by an ELISA instrument. The relative proliferation activity of the control group was 100%, and the relative proliferation activity was calculated according to the formula: relative proliferation activity of tyrosine micelle treatment group (%) = OD value of treatment group ÷ OD value of control group × 100%.

[0073] 2) Cell cycle detection:

[0074] B16F10 cells (about 5×10 5 Cells / well) were seeded in 6-well plates. After 24 hours, the medium was replaced with fresh medium containing 0, 0.5 or 1 mg / mL tyrosine micelles. After further incubation at 37 degrees for 48 hours, the cells were collected by trypsin digestion and then fixed with cold ethanol (70%) for 2 hours. The cells were stained with PI and analyzed using an analytical flow cytometer to detect fluorescence at 530 nm to analyze cell cycle distribution. Each experiment was repeated three times.

[0075] 5.2 Analysis of cell migration and invasion using wound healing and transwell assays

[0076] 1) Wound healing experiment:

[0077] B16F10 cells (about 5×10 5 cells / well) were seeded in 6 cm 2 After 24 hours, replace the culture medium with fresh culture medium containing 0, 0.5 or 1 mg / mL tyrosine micelles in a small dish. Continue incubation at 37 degrees for 48 hours. Aspirate the culture medium and use a 200μL sterilized pipette tip to draw a vertical line with a ruler, keeping the force consistent. Wash the scratched cells 2-3 times with PBS, replace the serum-free culture medium, and place in a 37-degree incubator. Take pictures and observe after 12, 24, and 48 hours. Use ImageJ software to quantify and statistically analyze the wound healing rate. Select 3 fields of view for each sample and take pictures, with 3 replicates per group.

[0078] 2) Transwell experiment

[0079] Dilute matrigel gel at 1:10 with serum-free medium and coat it on the bottom membrane of the transwell chamber. Place it at 37 degrees for 1 hour to allow matrigel to polymerize into gel and rehydrate it before use. 5 cells / well) were seeded in 6 cm 2 After 24 hours, the medium was replaced with fresh medium containing 0, 0.5 or 1 mg / mL tyrosine micelles. The cells were incubated at 37°C for 48 hours. The cells were harvested by trypsinization and made into 1×10 cells / mL culture medium with serum-free medium. 6 10 cells / mL suspension, add 200 μL of cell suspension to the upper chamber of each transwell well, and add 1 mL of complete medium to the lower chamber. Continue to culture in a 37°C incubator for 72 hours, and count the number of cells on the transwell chamber membrane by crystal violet staining.

[0080] The experimental results are as follows Figure 5As shown in the figure, tyrosine micelles can significantly inhibit the proliferation activity of melanoma cells in a concentration- and time-dependent manner. Through cycle detection, it can be seen that tyrosine micelles can inhibit the cell cycle of co-cultured melanoma cells in the G1 phase, thereby inhibiting their proliferation ( Figure 5 b); The results of cell wound healing and transwell experiments showed that the migration and invasion abilities of melanoma cells were significantly reduced after tyrosine treatment ( Figure 5 c and d). The experimental results show that tyrosine micelles can significantly inhibit the proliferation and metastasis of mouse melanoma cells B16F10.

[0081] Example 6: Study on the targeting of tyrosine micelles to melanoma in mice

[0082] Three C57 mice of about 6 weeks old were selected and depilated. Each mouse was inoculated with 1×10 6 Two weeks later, each mouse was intravenously injected with 100 μL of 5 mg / mL tyrosine micelles linked to Cy5.5, and the enrichment of tyrosine micelles in the tumor site was observed by mouse in vivo imaging at 3, 6, and 12 hours.

[0083] The experimental results are as follows Figure 6 As shown: tyrosine micelles are enriched in the tumor site at around 3 hours and increase with time. Strong fluorescence intensity can be observed in the tumor site at 12 hours, indicating that tyrosine micelles connected with mannose have a good targeting effect on melanoma.

[0084] Example 7: Tyrosine micelles inhibit the growth and metastasis of melanoma in mice

[0085] Ten C57 mice of about 6 weeks old were randomly divided into two groups, namely the control group and the tyrosine treatment group. After depilation, each mouse was inoculated with about 1×10 6 melanoma cells, and when the tumor grows to 80 mm 3 Around 100 μL of PBS was intravenously injected into the control group every other day, and 100 μL of 5 mg / mL tyrosine nano-micelles was intravenously injected into the tyrosine group every other day. The tumor size was recorded every 3 days, and the survival curve of the mice was recorded. On the other 15th day, one tumor was taken from each group of mice, fixed and embedded for immunohistochemistry, and the expression of metastasis-related proteins MMP2 and MMP9 was observed.

[0086] The experimental results are as follows Figure 7 As shown, compared with the control group, the tumor growth of mice in the tyrosine treatment group was significantly inhibited and the survival period was significantly prolonged ( Figure 7 a, b and c). The results of immunohistochemistry showed that the expression of MMP2 and MMP9 in the treated mice was significantly reduced ( Figure 7d). The experimental results showed that tyrosine micelles have the function of inhibiting the growth and metastasis of melanoma in mice.

[0087] Example 8: Tyrosine micelles kill melanoma cells via photothermal therapy

[0088] Mouse melanoma B16F10 cells in the logarithmic growth phase were taken and made into a cell density of 1×10 5 / mL single cell suspension was inoculated in a 96-well plate, 100μL per well. After 24h, complete medium containing 0.5mg / mL and 1mg / mL tyrosine micelles was added, and the control group was replaced with new normal complete medium. Six replicates were plated and cultured for 48h. Each well of the tyrosine treatment group was irradiated with 808nm laser for 5min (0.28w / cm 2 ), and the temperature in the wells was detected by a thermal imager. The cells were then placed in a 37°C incubator for 6 h, and 100 μL CCK-8 cell detection solution was added to each well to detect cell activity.

[0089] The experimental results are as follows Figure 8 As shown, compared with the control group, the temperature of the cells in the tyrosine treatment group rose to about 50 degrees after 808nm laser irradiation for 5 minutes ( Figure 8 a). At this temperature, the activity of melanoma cells decreased significantly, and a large number of cells died. Compared with the cell activity of the control group, the laser treatment group, and the tyrosine treatment group, there was a very significant statistical difference ( Figure 8 b and c). The experimental results show that the melanin produced by tyrosine micelles induced melanoma has the ability of photothermal conversion under near-infrared laser irradiation, and the thermal effect can effectively kill melanoma cells. Therefore, the melanin produced by tyrosine micelles induced melanoma can be used as a photothermal agent to kill melanoma cells through photothermal therapy.

[0090] The micelles of this embodiment are formed by self-assembly of a tyrosine amphiphilic molecule in an aqueous solution, the amphiphilic molecule is composed of hydrophilic mannose, L-tyrosine tetrapeptide and lipophilic oleylamine, the mannose and L-tyrosine tetrapeptide blocks in the amphiphilic molecule are in the outer shell of the micelle, and the oleylamine block is in the inner core of the micelle. The experimental results show that the tyrosine released by the decomposition of the tyrosine micelle, as a synthetic substrate of melanin, can induce the metabolism of mouse melanoma in vitro to produce a large amount of melanin, induce its differentiation, inhibit its proliferation and migration; in addition, after intravenous injection, the tyrosine micelle can reach the melanoma site under the targeting action of mannose, and significantly inhibit the growth and metastasis of melanoma in vivo. The tyrosine micelles of the present invention can achieve controllable and efficient delivery of tyrosine, and have a significant effect in inhibiting the growth and metastasis of malignant melanoma. At the same time, the tyrosine micelles that are not targeted to the tumor site release tyrosine after degradation in the body, which has no side effects on the body and can be used as a raw material for the metabolism of the central carbon atom of the organism, participating in the metabolism of the body, and having excellent biosafety. Finally, the large amount of melanin metabolites produced by melanoma induced by tyrosine micelles can be used for subsequent treatments (such as photothermal therapy, etc.).

[0091] In summary, the tyrosine micelles can be used to inhibit the growth and metastasis of malignant melanoma, and have very high biosafety, providing a safe and reliable technical means for the treatment of melanoma, and have good application prospects.

[0092] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An amphiphilic molecule, characterized in that The structural formula of the amphiphilic molecule is shown below:

2. The method for preparing an amphiphilic molecule according to claim 1, characterized in that: The following steps are involved: Using mannose-polytyrosine and fatty amine as raw materials, in the presence of a carbodiimide condensation agent and a condensation activator, an amide condensation reaction is carried out to obtain the amphiphilic molecule; Wherein, the mannose-polytyrosine is selected from a peptide chain formed by polymerization of four tyrosine molecules connected by mannose; the fatty amine is selected from oleylamine; The carbodiimide condensing agent is selected from dicyclohexylcarbodiimide, diisopropylcarbodiimide or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide.

3. The preparation method according to claim 2, characterized in that: The condensation activator is selected from 4-dimethylaminopyridine, 1-hydroxybenzotriazole or N-hydroxysuccinimide.

4. A micelle, characterized in that The micelle comprises the amphiphilic molecule as claimed in claim 1.

5. The micelle according to claim 4, characterized in that The micelle has at least one of the properties shown in the following i) to iii): i) The particle size ranges from 20 to 200 nm; ii) the dispersion index of the micelle hydrated particle size is less than 0.3; iii) The critical micelle concentration is 0.00828 mg / L.

6. The method for preparing micelles according to claim 4 or 5, characterized in that: The following steps are involved: The amphiphilic molecule is prepared into a tetrahydrofuran solution with a concentration of 1 to 5 mg / mL; Adding the tetrahydrofuran solution into water at a rate of 1 to 5 mL / h and stirring to obtain the micelles; Wherein, the volume ratio of the tetrahydrofuran solution to water is 1:(2-8).

7. Use of the micelle according to claim 4 or 5 in preparing a drug for treating melanoma.

8. The use according to claim 7, characterized in that The melanoma treatment drug is a drug that kills melanoma through photothermal therapy.

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