Nano drug delivery system and application thereof in anti-tumor aspect

Abstract

The invention relates to a nano drug delivery system and application thereof in the anti-tumor aspect. Specifically, the invention provides the hyaluronic acid-paclitaxel conjugate, and the hyaluronic acid-paclitaxel conjugate can be used for preparing the liposome with an excellent anti-tumor effect.

Description

Technical Field

[0001] The present invention relates to the field of drugs. Specifically, the present invention relates to a nano-drug delivery system and its use in anti-tumor treatment. Background Art

[0002] Tumors seriously endanger human health and survival. The latest statistics show that the number of tumor deaths accounts for more than cerebrovascular diseases and ranks first. Chemotherapy is a basic tumor treatment method, which has advantages such as simple and convenient drug administration. For example, paclitaxel (PTX) is a commonly used anti-tumor drug in clinical practice. It inhibits the proliferation of cancer cells by inducing cell cycle arrest and mitotic catastrophe, and has been widely used in the treatment of breast cancer, ovarian cancer, some head and neck cancers and lung cancers in clinical practice. As a diterpenoid alkaloid compound with anti-cancer activity, paclitaxel has received great attention from botanists, chemists, pharmacologists and molecular biologists, making it a research focus. However, existing anti-cancer drugs such as paclitaxel still have the disadvantage of poor treatment effect, thus limiting their anti-cancer applications. Therefore, how to further develop a drug and method for effective treatment of tumors has always been a hot research topic today.

[0003] Therefore, there is a need in the art to develop a drug for effective treatment of tumors. Summary of the Invention

[0004] The object of the present invention is to provide a liposome with excellent treatment effect on tumors.

[0005] In the first aspect of the present invention, a hyaluronic acid-paclitaxel conjugate is provided, and the hyaluronic acid-paclitaxel conjugate has the following structure:

[0006]

[0007] Preferably, n is 3 - 30, more preferably 5 - 20, still more preferably 5 - 15, still more preferably 8 - 12, still more preferably 9 - 11, and most preferably 10.

[0008] Preferably, m is 1 - 15, more preferably 1 - 10, still more preferably 2 - 8, still more preferably 2 - 5, still more preferably 2 - 4, and most preferably 3.

[0009] Preferably, k is 3 - 30, more preferably 5 - 20, still more preferably 8 - 16, still more preferably 10 - 14, still more preferably 11 - 13, and most preferably 12.

[0010] Preferably, n, m and k are positive integers.

[0011] Preferably, the hyaluronic acid-paclitaxel conjugate has the following structure:

[0012]

[0013] The second aspect of the present invention provides a liposome, which comprises the hyaluronic acid-paclitaxel conjugate as described in the first aspect of the present invention.

[0014] Preferably, the liposome further comprises MMP2-C18 and pHAase.

[0015] Preferably, the structure of the MMP2-C18 is as follows:

[0016]

[0017] Preferably, the pHAase is prepared by the following method, which comprises:

[0018] Reacting hyaluronidase with dimethyl maleic anhydride to obtain pHAase.

[0019] Preferably, the mass ratio of the hyaluronidase to the dimethyl maleic anhydride is 1:5-15, preferably 1:8-12, more preferably 1:10.

[0020] Preferably, the temperature of the reaction is 2-6°C, preferably 4°C.

[0021] Preferably, the reaction time is 2-6 h, preferably 4 h.

[0022] Preferably, the reaction is carried out in HEPES buffer.

[0023] Preferably, the reaction solution after the reaction is dialyzed against pH 7.4 PBS buffer to obtain pHAase.

[0024] Preferably, the pHAase is prepared by the following method, which comprises:

[0025] Dissolve 5 mg of hyaluronidase and 50 mg of dimethyl maleic anhydride in HEPES buffer, keep at 4°C for 4 h, then dialyze against pH 7.4 PBS buffer for 24 h, and lyophilize to obtain pHAase.

[0026] Preferably, the liposome further comprises lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and poloxamer 188.

[0027] Preferably, the lecithin includes egg yolk lecithin.

[0028] Preferably, the liposome comprises the hyaluronic acid-paclitaxel conjugate, MMP2-C18, pHAase, lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 and poloxamer 188 as described in the first aspect of the present invention.

[0029] Optionally, the particle size of the liposome is 60 - 200 nm, preferably 80 - 150 nm, more preferably 100 - 150 nm, even more preferably 105 - 120 nm, and even more preferably 110 - 116 nm.

[0030] Optionally, the potential of the liposome is -4 mV to -20 mV, preferably -6 mV to -20 mV, preferably -8 mV to -15 mV, preferably -10 mV to -14 mV, and preferably -10 mV to -13 mV.

[0031] The third aspect of the present invention provides a method for preparing the liposome as described in the second aspect of the present invention. The method includes the steps:

[0032] (1) Dissolve the hyaluronic acid-paclitaxel conjugate and pHAase as described in the first aspect of the present invention in water to obtain an inner aqueous phase;

[0033] (2) Dissolve lecithin and cholesterol in chloroform to obtain an oil phase;

[0034] (3) Add the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and perform probe sonication to obtain a W / O emulsion;

[0035] (4) Mix egg yolk lecithin methanol solution, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, poloxamer 188 aqueous solution, MMP2-C18 aqueous solution, methanol and water to obtain an outer aqueous phase;

[0036] (5) Add the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), perform probe sonication to obtain a W / O / W emulsion, and stir to evaporate the organic solvent to obtain the liposome.

[0037] Preferably, the method includes the steps:

[0038] (1) Dissolve 2 - 4 mg of the hyaluronic acid-paclitaxel conjugate as described in the first aspect of the present invention and 0.5 - 1.2 mg of pHAase in 40 - 60 μL of water to obtain an inner aqueous phase;

[0039] (2) Mix 50 - 70 μL of 10 - 20 mg / mL lecithin chloroform solution, 90 - 110 μL of 5 - 15 mg / mL cholesterol chloroform solution and 80 - 100 μL of chloroform to obtain an oil phase;

[0040] (3) Add the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and perform probe sonication to obtain a W / O emulsion;

[0041] (4) Mix 120 - 140 μL of 10 - 20 mg / ml lecithin methanol solution, 10 - 20 μL of 5 - 15 mg / mL distearoyl phosphatidylethanolamine - polyethylene glycol 2000 methanol solution, 2.0 - 3.0 μL of 3.0 - 4.0 mg / mL poloxamer 188 aqueous solution, 40 - 60 μL of 1.5 - 2.5 mg / mL MMP2 - C18 aqueous solution, 95 - 120 μL of methanol and 1.0 - 1.4 mL of water to obtain the outer aqueous phase;

[0042] (5) Add the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), and use a probe to ultrasonically emulsify to obtain a W / O / W emulsion. Stir to evaporate the organic solvent to obtain liposomes.

[0043] Preferably, the method includes the steps:

[0044] (1) Dissolve 2.5 - 3.5 mg of the hyaluronic acid - paclitaxel conjugate as described in the first aspect of the present invention and 0.6 - 1.0 mg of pHAase in 45 - 55 μL of water to obtain the inner aqueous phase;

[0045] (2) Mix 60 - 65 μL of 13 - 17 mg / mL lecithin chloroform solution, 93 - 102 μL of 8 - 12 mg / mL cholesterol chloroform solution and 85 - 95 μL of chloroform to obtain the oil phase;

[0046] (3) Add the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and use a probe to ultrasonically emulsify to obtain a W / O emulsion;

[0047] (4) Mix 123 - 132 μL of 13 - 17 mg / ml lecithin methanol solution, 12 - 16 μL of 8 - 12 mg / mL distearoyl phosphatidylethanolamine - polyethylene glycol 2000 methanol solution, 2.3 - 2.7 μL of 3.3 - 3.7 mg / mL poloxamer 188 aqueous solution, 50 - 55 μL of 1.8 - 2.2 mg / mL MMP2 - C18 aqueous solution, 100 - 115 μL of methanol and 1.1 - 1.3 mL of water to obtain the outer aqueous phase;

[0048] (5) Add the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), and use a probe to ultrasonically emulsify to obtain a W / O / W emulsion. Stir to evaporate the organic solvent to obtain liposomes.

[0049] Preferably, the method includes the steps:

[0050] (1) Dissolve 3.0 mg of the hyaluronic acid - paclitaxel conjugate as described in the first aspect of the present invention and 0.8 mg of pHAase in 50 μL of water to obtain the inner aqueous phase;

[0051] (2) Mix 63.75 μL of 15 mg / mL lecithin chloroform solution, 97.2 μL of 10 mg / mL cholesterol chloroform solution and 89.05 μL of chloroform to obtain an oil phase;

[0052] (3) Add the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and perform probe sonication to obtain a W / O emulsion;

[0053] (4) Mix 127.5 μL of 15 mg / ml lecithin methanol solution, 14.2 μL of 10 mg / mL distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, 2.5 μL of 3.5 mg / mL poloxamer 188 aqueous solution, 52.5 μL of 2 mg / mL MMP2-C18 aqueous solution, 106.6 μL of methanol and 1.1975 mL of water to obtain an outer aqueous phase;

[0054] (5) Add the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), perform probe sonication to obtain a W / O / W emulsion, and stir to evaporate the organic solvent to obtain liposomes.

[0055] Preferably, in step (3), the intensity of the sonication is 200 - 400 W, more preferably 250 - 350 W, still more preferably 280 - 320 W, and most preferably 300 W.

[0056] Preferably, in step (3), the emulsification time is 2 - 8 min, more preferably 4 - 6 min, and most preferably 5 min.

[0057] Preferably, in step (5), the intensity of the sonication is 200 - 400 W, more preferably 250 - 350 W, still more preferably 280 - 320 W, and most preferably 300 W.

[0058] Preferably, in step (5), the emulsification time is 2 - 8 min, more preferably 4 - 6 min, and most preferably 5 min.

[0059] Preferably, in step (5), the stirring time is 10 - 15 h, more preferably 11 - 13 h, and most preferably 12 h.

[0060] The fourth aspect of the present invention provides a composition, which comprises the hyaluronic acid-paclitaxel conjugate as described in the first aspect of the present invention or the liposome as described in the second aspect of the present invention.

[0061] Preferably, the composition further comprises a PD-1 inhibitor.

[0062] Preferably, the PD-1 inhibitor comprises a polypeptide or an antibody.

[0063] Preferably, the PD-1 inhibitor includes a small molecule compound, a protein or a gene.

[0064] Preferably, the PD-1 inhibitor includes a protein antibody.

[0065] Preferably, the PD-1 inhibitor includes InVivo MAb anti-mouse PD-1.

[0066] Preferably, the InVivo MAb anti-mouse PD-1 is Cat.No BE0273, from biocell, USA.

[0067] Preferably, the weight ratio of paclitaxel equivalent in the liposome to the PD-1 inhibitor is 1:(0.001 - 1000), preferably 1:(0.01 - 100), more preferably 1:(0.05 - 50), more preferably 1:(0.08 - 30), more preferably 1:(0.1 - 20), more preferably 1:(0.1 - 10), more preferably 1:(0.3 - 8), more preferably 1:(0.5 - 5), more preferably 1:(0.5 - 3), more preferably 1:(0.5 - 2), more preferably 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), and most preferably 1:1.

[0068] Preferably, the composition is a composition for preventing and / or treating tumors.

[0069] Preferably, the tumors include lung cancer and / or liver cancer.

[0070] Preferably, the composition is a pharmaceutical composition.

[0071] Preferably, the composition further includes a pharmaceutically acceptable carrier.

[0072] Preferably, the composition is a solid preparation, a liquid preparation or a semi-solid preparation.

[0073] Preferably, the composition is an injection preparation or an oral preparation.

[0074] Preferably, the injection preparation is an intravascular injection preparation.

[0075] Preferably, the injection preparation is an intravenous injection preparation, an intratumoral injection preparation, an intratumoral vascular injection preparation or a tumor microenvironment injection preparation.

[0076] Preferably, the content of the liposome is 0.001 - 99.9 wt%, preferably 0.1 - 99 wt%, more preferably 1 - 99 wt%, more preferably 1 - 90 wt%, more preferably 10 - 90 wt%, more preferably 20 - 80 wt%, more preferably 30 - 70 wt%, more preferably 20 - 40 wt%, based on the weight of the composition.

[0077] Preferably, the content of the PD-1 inhibitor is 0.001 - 99.9 wt%, preferably 0.1 - 99 wt%, more preferably 1 - 99 wt%, more preferably 1 - 90 wt%, more preferably 10 - 90 wt%, more preferably 20 - 80 wt%, more preferably 30 - 70 wt%, more preferably 20 - 40 wt%, based on the weight of the composition.

[0078] The fifth aspect of the present invention provides a pharmaceutical composition, which comprises:

[0079] (i) a liposome as described in the second aspect of the present invention; and

[0080] (ii) a PD-1 inhibitor.

[0081] Preferably, the weight ratio of the paclitaxel equivalent in the liposome to the PD-1 inhibitor is 1:(0.001 - 1000), preferably 1:(0.01 - 100), more preferably 1:(0.05 - 50), more preferably 1:(0.08 - 30), more preferably 1:(0.1 - 20), more preferably 1:(0.1 - 10), more preferably 1:(0.3 - 8), more preferably 1:(0.5 - 5), more preferably 1:(0.5 - 3), more preferably 1:(0.5 - 2), more preferably 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), most preferably 1:1.

[0082] Preferably, the PD-1 inhibitor includes a polypeptide or an antibody.

[0083] Preferably, the PD-1 inhibitor includes a small molecule compound, a protein or a gene.

[0084] Preferably, the PD-1 inhibitor includes a protein antibody.

[0085] Preferably, the PD-1 inhibitor includes InVivo MAb anti-mouse PD-1.

[0086] Preferably, the InVivo MAb anti-mouse PD-1 is Cat.No BE0273, from bioxcell, USA.

[0087] Preferably, the pharmaceutical composition is a composition for preventing and / or treating tumors.

[0088] Preferably, the tumor includes lung cancer and / or liver cancer.

[0089] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0090] Preferably, the composition is a solid preparation, a liquid preparation or a semi-solid preparation.

[0091] Preferably, the composition is an injection preparation or an oral preparation.

[0092] Preferably, the injection preparation is an intravascular injection preparation.

[0093] Preferably, the injection preparation is an intravenous injection preparation, an intratumoral injection preparation, an intratumoral blood vessel injection preparation or a tumor microenvironment injection preparation.

[0094] Preferably, the content of the liposome is 0.001 - 99.9 wt%, preferably 0.1 - 99 wt%, more preferably 1 - 99 wt%, more preferably 1 - 90 wt%, more preferably 10 - 90 wt%, more preferably 20 - 80 wt%, more preferably 30 - 70 wt%, more preferably 20 - 40 wt%, based on the weight of the composition.

[0095] Preferably, the content of the PD-1 inhibitor is 0.001 - 99.9 wt%, preferably 0.1 - 99 wt%, more preferably 1 - 99 wt%, more preferably 1 - 90 wt%, more preferably 10 - 90 wt%, more preferably 20 - 80 wt%, more preferably 30 - 70 wt%, more preferably 20 - 40 wt%, based on the weight of the composition.

[0096] The sixth aspect of the present invention provides an active ingredient combination, and the active ingredient combination comprises the following components:

[0097] (a) A first active ingredient, and the first active ingredient comprises the liposome as described in the second aspect of the present invention; and

[0098] (b) A second active ingredient, and the second active ingredient comprises a PD-1 inhibitor.

[0099] Preferably, the weight ratio of paclitaxel equivalent in the liposome to the PD-1 inhibitor is 1:(0.001 - 1000), preferably 1:(0.01 - 100), more preferably 1:(0.05 - 50), more preferably 1:(0.08 - 30), more preferably 1:(0.1 - 20), more preferably 1:(0.1 - 10), more preferably 1:(0.3 - 8), more preferably 1:(0.5 - 5), more preferably 1:(0.5 - 3), more preferably 1:(0.5 - 2), more preferably 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), and most preferably 1:1.

[0100] Preferably, the PD-1 inhibitor includes a polypeptide or an antibody.

[0101] Preferably, the PD-1 inhibitor includes a small molecule compound, a protein, or a gene.

[0102] Preferably, the PD-1 inhibitor includes a protein antibody.

[0103] Preferably, the PD-1 inhibitor includes InVivo MAb anti-mouse PD-1.

[0104] Preferably, the InVivo MAb anti-mouse PD-1 is Cat.No BE0273, from bioxcell, USA.

[0105] Preferably, the combination of active ingredients is a combination of active ingredients for preventing and / or treating tumors.

[0106] Preferably, the tumor includes lung cancer and / or liver cancer.

[0107] Preferably, in the combination of active ingredients, at least one active ingredient is independent.

[0108] Preferably, in the combination of active ingredients, the first active ingredient and the second active ingredient are independent of each other.

[0109] The seventh aspect of the present invention provides a medicine box, which includes:

[0110] (A) A first preparation containing a first active ingredient, and the first active ingredient includes the liposome as described in the second aspect of the present invention; and

[0111] (B) A second preparation containing a second active ingredient, and the second active ingredient includes a PD-1 inhibitor.

[0112] Preferably, the weight ratio of the paclitaxel equivalent in the liposome to the PD-1 inhibitor is 1:(0.001 - 1000), preferably 1:(0.01 - 100), more preferably 1:(0.05 - 50), more preferably 1:(0.08 - 30), more preferably 1:(0.1 - 20), more preferably 1:(0.1 - 10), more preferably 1:(0.3 - 8), more preferably 1:(0.5 - 5), more preferably 1:(0.5 - 3), more preferably 1:(0.5 - 2), more preferably 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), and most preferably 1:1.

[0113] Preferably, the PD-1 inhibitor includes a polypeptide or an antibody.

[0114] Preferably, the PD-1 inhibitor includes a small molecule compound, a protein, or a gene.

[0115] Preferably, the PD-1 inhibitor includes a protein antibody.

[0116] Preferably, the PD-1 inhibitor includes InVivo MAb anti-mouse PD-1.

[0117] Preferably, the InVivo MAb anti-mouse PD-1 is Cat.No BE0273, from bioxcell, USA.

[0118] Preferably, the kit is a kit for preventing and / or treating tumors.

[0119] Preferably, the tumor includes lung cancer and / or liver cancer.

[0120] Preferably, the first preparation and the second preparation are independent preparations.

[0121] Preferably, the first preparation and the second preparation are combined preparations.

[0122] Preferably, the kit, food box, health product box, or cosmetic box further includes an instruction manual.

[0123] Preferably, the instruction manual indicates that the first preparation and the second preparation are used in combination for preventing and / or treating tumors.

[0124] The eighth aspect of the present invention provides the use of a hyaluronic acid-paclitaxel conjugate as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, a composition as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention, an active ingredient combination as described in the sixth aspect of the present invention, or a kit as described in the seventh aspect of the present invention, for preparing a drug for preventing and / or treating tumors.

[0125] Preferably, the tumor includes tumors of humans or non-human mammals.

[0126] Preferably, the non-human mammals include mice, dogs, cats or pigs.

[0127] Preferably, the mice include mice and rats.

[0128] Preferably, the mice include C57BL / 6 mice.

[0129] Preferably, the tumor includes lung cancer or liver cancer.

[0130] Preferably, the tumor cells of the tumor include LLC cells.

[0131] Preferably, the dosage form of the drug is a solid preparation, a liquid preparation or a semi-solid preparation.

[0132] Preferably, the dosage form of the drug is an injection preparation or an oral preparation.

[0133] Preferably, the injection preparation is an intravascular injection preparation.

[0134] Preferably, the injection preparation is an intravenous injection preparation, an intratumoral injection preparation, an intratumoral vascular injection preparation or an intratumoral microenvironment injection preparation.

[0135] The ninth aspect of the present invention provides a method for inhibiting tumor cells, and the method includes the steps of:

[0136] Contacting tumor cells with a hyaluronic acid-paclitaxel conjugate as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, a composition as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention, or an active ingredient as described in the sixth aspect of the present invention, so as to inhibit tumor cells.

[0137] Preferably, the tumor includes tumors of humans or non-human mammals.

[0138] Preferably, the non-human mammals include mice, dogs, cats or pigs.

[0139] Preferably, the mice include mice and rats.

[0140] Preferably, the mice include C57BL / 6 mice.

[0141] Preferably, the tumor includes lung cancer or liver cancer.

[0142] Preferably, the tumor cells of the tumor include LLC cells.

[0143] Preferably, the methods described herein include non-therapeutic and non-diagnostic methods.

[0144] Preferably, the methods described herein include in vitro methods.

[0145] Preferably, the contacting described herein includes in vitro contacting.

[0146] Preferably, the contacting described herein includes contacting in an in vitro culture medium.

[0147] A tenth aspect of the present invention provides a method for preventing and / or treating tumors, the method including administering to a subject in need thereof a hyaluronic acid-paclitaxel conjugate as described in the first aspect of the present invention, a liposome as described in the second aspect of the present invention, a composition as described in the fourth aspect of the present invention, a pharmaceutical composition as described in the fifth aspect of the present invention, a combination of active ingredients as described in the sixth aspect of the present invention, or a kit as described in the seventh aspect of the present invention, thereby preventing and / or treating tumors.

[0148] Preferably, the subject is a human or non-human mammal.

[0149] Preferably, the tumors include human or non-human mammal tumors.

[0150] Preferably, the non-human mammals include mice, dogs, cats, or pigs.

[0151] Preferably, the mice include mice and rats.

[0152] Preferably, the mice include C57BL / 6 mice.

[0153] Preferably, the tumors include lung cancer or liver cancer

[0154] Preferably, the tumor cells of the tumors include LLC cells.

[0155] Preferably, the administration is by injection or oral administration.

[0156] Preferably, the injection administration is by intravascular injection.

[0157] Preferably, the injection administration is by intravenous injection, intratumoral injection, intratumor vascular injection, or intratumor microenvironment injection.

[0158] Within the scope of the present invention, the above-described technical features of the present invention and the technical features specifically described hereinafter can be combined with each other to form new or preferred technical solutions. BRIEF DESCRIPTION OF THE DRAWINGS

[0159] Figure 1FT-IR spectra of hyaluronic acid (HA), HA-ADH-SS-PTX (HSP), HA-ADH-CC-PTX (HCP) and HA-ADH.

[0160] Figure 2 1H NMR spectrum of HA-ADH 1

[0161] Figure 3 1H NMR spectrum of PTX-SS-COOH 1

[0162] Figure 4 1H NMR spectrum of HA-ADH-SS-PTX (HSP) conjugate 1

[0163] Figure 5 1H NMR spectrum of PTX-CC-COOH 1

[0164] Figure 6 1H NMR spectrum of HA-ADH-CC-PTX (HCP) 1

[0165] Figure 7 1H NMR spectrum of N-octadecyl maleimide (C18-MAL) 1

[0166] Figure 8 1H NMR spectrum of MMP2-C18 1

[0167] Figure 9 Relative viscosity of each group. * P < 0.05, ** P < 0.01 vs HA. ## P < 0.01, #### P < 0.0001.

[0168] Figure 10 Flow cytometry detection of CRT expression in tumor tissues of different treatment groups.

[0169] Figure 11 Percentage of CRT + cells in tumor tissues of different treatment groups, compared with the control group, ***P < 0.001, ****P < 0.0001; # P < 0.05. ​​​​​​​

[0170] Figure 12 CD80 in tumor tissues of different treatment groups + CD86 + Flow cytometry detection of mature DCs (Dendritic cells).

[0171] Figure 13 CD80 in tumor tissues of different treatment groups + CD86 + Percentage of mature DCs (Dendritic cells), compared with the control group (Saline group), ***P<0.001,****P<0.0001; # P<0.05.

[0172] Figure 14 CD-8 in tumor tissues of different treatment groups + Flow cytometry detection of T cells

[0173] Figure 15 CD-8 in tumor tissues of different treatment groups + Percentage of T cells, compared with the control group, **P<0.01,***P<0.001,****P<0.0001; # P<0.05.

[0174] Figure 16 CD25 in tumor tissues of different treatment groups + FOXp3 + Flow cytometry detection of Treg cells

[0175] Figure 17 CD25 in tumor tissues of different treatment groups + FOXp3 + Percentage of Treg cells, compared with the control group, **P<0.01,***P<0.001; ## P<0.01.

[0176] Figure 18 Changes in tumor volume of mice in different treatment groups over time *** P<0.001, **** P<0.0001.

[0177] Figure 19 CD25 in tumor tissues of different treatment groups + FOXp3 + Amount of Treg cells, compared with the Saline group, **P<0.01,***P<0.001; ## P<0.01. Detailed implementation mode

[0178] The present invention has developed a hyaluronic acid-paclitaxel conjugate, and the hyaluronic acid-paclitaxel conjugate can be used to prepare liposomes with excellent anti-tumor effects. In addition, the combination of the liposomes and PD-1 inhibitors of the present invention has excellent synergistic effects in anti-tumor.

[0179] Term

[0180] As used herein, the terms "comprising", "including" and "containing" can be used interchangeably, including not only open definitions, but also semi-closed and closed definitions. In other words, the said terms include "consisting of" and "consisting essentially of".

[0181] As used herein, the term "HEPES" refers to 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid.

[0182] As used herein, the term "PBS" refers to phosphate buffer saline.

[0183] As used herein, the English name of paclitaxel is paclitaxel, abbreviated as PTX, and the CAS registration number is 33069-62-4.

[0184] As used herein, the English of hyaluronic acid is hyaluronic acid, abbreviated as HA.

[0185] As used herein, the English of distearoylphosphatidylethanolamine-polyethylene glycol 2000 is Distearoylphosphatidyl ethanolamine-polyethylene glycol 2000, abbreviated as DSPE-PEG 2000.

[0186] As used herein, PD-1 refers to programmed cell death protein 1.

[0187] As used herein, the CAS registration number of dimethyl maleic anhydride is 766-39-2.

[0188] As used herein, the abbreviation of hyaluronidase is HAase, and the CAS registration number is 37326-33-3.

[0189] As used herein, LLC cells refer to lung cancer cells.

[0190] As used herein, the 15 mg / mL egg yolk lecithin chloroform solution means that the concentration of egg yolk lecithin in the egg yolk lecithin chloroform solution is 15 mg / mL; the 10 mg / mL cholesterol chloroform solution means that the concentration of cholesterol in the cholesterol chloroform solution is 10 mg / mL; the 15 mg / ml egg yolk lecithin methanol solution means that the concentration of egg yolk lecithin in the egg yolk lecithin methanol solution is 15 mg / ml; the 10 mg / mL distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution means that the concentration of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 in the distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution is 10 mg / mL; the 3.5 mg / mL poloxamer 188 aqueous solution means that the concentration of poloxamer 188 in the poloxamer 188 aqueous solution is 3.5 mg / mL; the 2 mg / mL MMP2-C18 aqueous solution means that the concentration of MMP2-C18 in the MMP2-C18 aqueous solution is 2 mg / mL; and so on.

[0191] In the present invention, the term "prevent" refers to a method of preventing the onset of a disease and / or its attendant symptoms or protecting a subject from acquiring a disease.

[0192] "Treatment" as used in the present invention includes delaying and terminating the progression of a disease, or eliminating the disease, and does not require 100% inhibition, elimination, and reversal. In some embodiments, the hyaluronic acid-paclitaxel conjugate of the present invention reduces, inhibits, and / or reverses a tumor by, for example, at least about 30%, at least about 50%, or at least about 80%, or 100% compared to the level observed in the absence of the liposomes of the present invention.

[0193] Hyaluronic acid-paclitaxel conjugate

[0194] The present invention provides a hyaluronic acid-paclitaxel conjugate having excellent therapeutic effects on tumors. Representatively, the hyaluronic acid-paclitaxel conjugate has the following structure:

[0195]

[0196] Preferably, n is 3 - 30, more preferably 5 - 20, still more preferably 5 - 15, still more preferably 8 - 12, still more preferably 9 - 11, and most preferably 10.

[0197] Preferably, m is 1 - 15, more preferably 1 - 10, still more preferably 2 - 8, still more preferably 2 - 5, still more preferably 2 - 4, and most preferably 3.

[0198] Preferably, k is 3 - 30, more preferably 5 - 20, still more preferably 8 - 16, still more preferably 10 - 14, still more preferably 11 - 13, and most preferably 12.

[0199] Specifically, the hyaluronic acid-paclitaxel conjugate is as described in the first aspect of the present invention above.

[0200] Liposomes and their preparation methods

[0201] The present invention provides a liposome, and the liposome includes the hyaluronic acid-paclitaxel conjugate described in the present invention.

[0202] In a preferred embodiment of the present invention, the liposome includes a hyaluronic acid-paclitaxel conjugate, MMP2-C18, pHAase, lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and poloxamer 188.

[0203] Specifically, the liposome described in the present invention is as described in the second aspect of the present invention above.

[0204] The present invention also provides a method for preparing the liposome described in the present invention. In a preferred embodiment of the present invention, the method for preparing the liposome includes the steps of:

[0205] (1) Dissolve the hyaluronic acid-paclitaxel conjugate and pHAase described in the first aspect of the present invention in water to obtain an inner aqueous phase;

[0206] (2) Dissolve lecithin and cholesterol in chloroform to obtain an oil phase;

[0207] (3) Add the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and perform probe sonication to obtain a W / O emulsion;

[0208] (4) Mix an egg yolk lecithin methanol solution, a distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, a poloxamer 188 aqueous solution, an MMP2-C18 aqueous solution, methanol, and water to obtain an outer aqueous phase;

[0209] (5) Add the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), perform probe sonication to obtain a W / O / W emulsion, and stir to evaporate the organic solvent to obtain the liposome.

[0210] Specifically, the method for preparing the liposome described in the present invention is as described in the third aspect of the present invention above.

[0211] Use

[0212] The present invention provides a use of the hyaluronic acid-paclitaxel conjugate or liposome described in the present invention for preparing a drug for preventing and / or treating tumors.

[0213] In a preferred embodiment of the present invention, the tumor includes tumors of humans or non-human mammals.

[0214] Preferably, the non-human mammals include mice, dogs, cats or pigs.

[0215] Preferably, the mice include mice and rats.

[0216] Preferably, the mice include C57BL / 6 mice.

[0217] In a preferred embodiment of the present invention, the tumor includes (but is not limited to) lung cancer or liver cancer

[0218] Preferably, the tumor cells of the tumor include LLC cells.

[0219] Composition

[0220] The composition according to the present invention is preferably a pharmaceutical composition. The composition according to the present invention may also include a pharmaceutically acceptable carrier.

[0221] As used herein, "pharmaceutically acceptable carrier" refers to one or more compatible solid, semi-solid, liquid or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" means that the components in the pharmaceutical composition and the active ingredient of the drug and their mutual admixture do not significantly reduce the drug efficacy.

[0222] It should be understood that in the present invention, the pharmaceutically acceptable carrier is not particularly limited, and the commonly used materials in the art can be selected, or prepared by conventional methods, or obtained from the market. Some examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium dodecyl sulfate), buffers, chelating agents, thickening agents, pH regulators, transdermal promoters, coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, bacteriostatic agents, pyrogen-free water, etc.

[0223] In the present invention, the dosage forms of the composition include but are not limited to oral preparations or injection preparations. Representatively, the dosage forms of the composition include but are not limited to tablets, injections, infusions, solutions, microspheres.

[0224] Representatively, the injection preparations include but are not limited to intravenous injection preparations, intratumoral injection preparations, intratumoral vascular injection preparations or tumor microenvironment injection preparations.

[0225] The composition of the present invention may include liposomes and PD-1 inhibitors.

[0226] In a preferred embodiment of the present invention, the weight ratio of paclitaxel equivalent in the liposome to the PD-1 inhibitor is 1:(0.001 - 1000), preferably 1:(0.01 - 100), more preferably 1:(0.05 - 50), more preferably 1:(0.08 - 30), more preferably 1:(0.1 - 20), more preferably 1:(0.1 - 10), more preferably 1:(0.3 - 8), more preferably 1:(0.5 - 5), more preferably 1:(0.5 - 3), more preferably 1:(0.5 - 2), more preferably 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), and most preferably 1:1.

[0227] The pharmaceutical preparation should be matched with the administration method. The preferred administration methods are oral administration and injection (such as intravenous injection). When in use, a therapeutically effective amount of the drug is administered to the desired subject (such as a human or non-human mammal). As used herein, the term "therapeutically effective amount" refers to an amount that produces a function or activity in humans and / or animals and is acceptable to humans and / or animals. Those of ordinary skill in the art should understand that the "therapeutically effective amount" may vary depending on the form of the pharmaceutical composition, the administration route, the excipients of the drug used, the severity of the disease, and the combination with other drugs, etc., which are all within the scope of the skills of a skilled physician / researcher.

[0228] The main excellent technical effects of the present invention include:

[0229] 1. The present invention develops a hyaluronic acid-paclitaxel conjugate, and the hyaluronic acid-paclitaxel conjugate can be used to prepare liposomes with excellent anti-tumor effects.

[0230] 2. The liposomes of the present invention have excellent anti-tumor effects and can significantly enhance the immune response ability against tumors.

[0231] 3. The combination of the liposomes of the present invention and the PD-1 inhibitor has excellent synergistic effects in inhibiting tumors and enhancing the immune response ability against tumors. Therefore, the combination of the liposomes of the present invention and the PD-1 inhibitor can significantly enhance the therapeutic effect on tumors.

[0232] The following further elaborates the present invention in conjunction with specific embodiments. It should be understood that the following specific embodiments are based on the present technical solution and give detailed implementation manners and specific operation processes, but the protection scope of the present invention is not limited to this embodiment.

[0233] Example 1

[0234] 1. Reagents

[0235] The English name of paclitaxel is paclitaxel, abbreviated as PTX, and the CAS registration number is 33069-62-4.

[0236] The English name of hyaluronic acid is hyaluronic acid, abbreviated as HA.

[0237] The English name of distearoyl phosphatidylethanolamine-polyethylene glycol 2000 is Distearoyl phosphatidylethanolamine-polyethylene glycol 2000, abbreviated as DSPE-PEG 2000.

[0238] PD-1 refers to programmed cell death protein 1.

[0239] The CAS registry number of dimethyl maleic anhydride is 766-39-2.

[0240] The abbreviation of hyaluronidase is HAase, and its CAS registry number is 37326-33-3.

[0241] 2. Synthesis of materials

[0242] 2.1 Synthesis of HA-ADH-SS-PTX (HSP)

[0243] 2.1.1 Synthesis of HA-ADH

[0244]

[0245] Weigh 200 mg of hyaluronic acid (HA, the Fourier transform infrared (FT-IR) spectrum is as Figure 1 shown) and dissolve it in purified water. Add 345.5 mg of adipic dihydrazide (ADH), adjust the pH to 5.0, add N,N'-dicyclohexylcarbodiimide (DCC) as a catalyst, and react at room temperature for 4 h. Then adjust the pH to 9.0 with NaOH solution to terminate the reaction. Dialyze the reaction solution with deionized water for 48 h and freeze-dry to obtain a white solid HA-ADH. The Fourier transform infrared (FT-IR) spectrum of the synthesized HA-ADH is as Figure 1 shown, and the nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) of the synthesized HA-ADH is as Figure 2 shown.

[0246] 2.1.2 Synthesis of PTX-SS-COOH

[0247]

[0248] Weigh 2,2'-dithiobisacetic acid (DSDD) and dissolve it in dichloromethane. Then weigh a catalytic amount of catalysts N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP) (molar ratio 2:1) and dissolve them in dichloromethane. Drop the catalyst solution into the DSDD solution. After activation for 2 hours, add the dichloromethane solution of paclitaxel (PTX) (molar ratio of DSDD:PTX is 2:1), stir at room temperature overnight, and monitor the reaction result by TLC. Extract the reaction solution with saturated brine, take the organic solution layer and vacuum-dry it, and purify it by silica gel column chromatography to obtain PTX-SS-COOH. The nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) spectrum of the synthesized PTX-SS-COOH is as Figure 3 shown.

[0249] 2.1.3 Synthesis of HA-ADH-SS-PTX

[0250]

[0251] Weigh 30 mg of PTX-SS-COOH and dissolve it in dimethyl sulfoxide. After adding 2-fold molar amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) and activating for 1 h, drop it into the dimethyl sulfoxide solution of HA-ADH (50 mg). Under nitrogen protection, stir and react at 37 °C. Precipitate the reaction solution with ether, centrifuge to collect the precipitate, redissolve it in water, transfer it to a dialysis bag and dialyze for 48 h, and then freeze-dry to obtain the HA-ADH-SS-PTX (HSP) conjugate. The Fourier transform infrared (FT-IR) spectrum of the synthesized HA-ADH-SS-PTX (HSP) conjugate is as Figure 1 shown, and the nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) spectrum of the synthesized HA-ADH-SS-PTX (HSP) conjugate is as Figure 4 shown.

[0252] 2.2 Synthesis of HA-ADH-CC-PTX (HCP)

[0253] 2.2.1 Synthesis of HA-ADH

[0254]

[0255] Weigh hyaluronic acid (HA, the Fourier transform infrared (FT-IR) spectrum of which is as Figure 1As shown in the figure, 200 mg was dissolved in purified water, 345.5 mg of adipic dihydrazide (ADH) was added, the pH was adjusted to 5.0, N,N'-dicyclohexylcarbodiimide (DCC) was added for catalysis, and the reaction was carried out at room temperature for 4 h. The reaction was terminated by adjusting the pH to 9.0 with NaOH solution. The reaction solution was dialyzed with deionized water for 48 h and freeze-dried to obtain a white solid HA-ADH. The Fourier transform infrared (FT-IR) spectrum of the synthesized HA-ADH is as shown in Figure 1 shown, and the nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) of the synthesized HA-ADH is as shown in Figure 2 shown.

[0256] 2.2.2 Synthesis of PTX-CC-COOH

[0257]

[0258] Weigh 25 mg of paclitaxel (PTX) into a round-bottom flask, dissolve it in pyridine, then add 30 mg of succinic anhydride (SA) and react at room temperature. The reaction result was monitored by TLC. The reaction solution was rotary evaporated and washed with water. The separated precipitate was dissolved in acetone, precipitated with water and filtered. The filter cake was dried under vacuum overnight and purified by silica gel column chromatography to obtain PTX-CC-COOH. The nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) of the synthesized PTX-CC-COOH is as shown in Figure 5 shown.

[0259] 2.2.3 Synthesis of HA-ADH-CC-PTX

[0260]

[0261] Weigh 30 mg of PTX-CC-COOH and dissolve it in dimethyl sulfoxide. After activating it with 2-fold molar amount of 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) for 1 h, it was dropped into the dimethyl sulfoxide solution containing 50 mg of HA-ADH. Under nitrogen protection, the reaction was stirred at 37 °C. The reaction solution was precipitated with ether, the precipitate was collected by centrifugation, redissolved in water, transferred to a dialysis bag and dialyzed for 48 h, and then freeze-dried to obtain the HA-ADH-CC-PTX (HCP) conjugate. The Fourier transform infrared (FT-IR) spectrum of the synthesized HA-ADH-CC-PTX (HCP) conjugate is as shown in Figure 1 shown, and the nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) of the synthesized HA-ADH-CC-PTX (HCP) is as shown in Figure 6 shown.

[0262] 2.3. Synthesis of MMP2-C18

[0263] 2.3.1 C18-MAL Synthesis

[0264]

[0265] Weigh 23.53 mg of maleic anhydride (MAL) and 0.83 mg of p-toluenesulfonic acid as catalyst into a flask. Subsequently, add toluene as a solvent and heat for reaction. Then slowly add 53.9 mg of octadecylamine (C18) and raise the temperature for reaction for 16 h. After the reaction is completed, add petroleum ether, and then extract the reaction solution with an alkaline solution. Take the petroleum ether layer and distill it under reduced pressure to obtain white powdery N-octadecylmaleimide (C18-MAL). The nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) of the synthesized N-octadecylmaleimide (C18-MAL) is as Figure 7 shown.

[0266] 2.3.2 MMP2-C18 Synthesis

[0267]

[0268] Weigh an appropriate amount of tris(2-carboxyethyl)phosphine (TCEP), and prepare a 10 mM TCEP deionized aqueous solution. Dissolve the MMP-2 responsive polypeptide in the TCEP solution, and dissolve C18-MAL in tetrahydrofuran. Subsequently, add the TCEP solution dissolving the MMP-2 responsive polypeptide to the tetrahydrofuran solution dissolving C18-MAL, and adjust the pH to alkaline with sodium hydroxide and react at room temperature for 24 h. Remove the organic solvent by rotary evaporation, and finally transfer it to a dialysis bag and dialyze for 24 h to remove TCEP, and then freeze-dry to obtain the MMP-2 responsive amphiphile MMP2-C18. The nuclear magnetic resonance hydrogen spectrum ( 1 1H NMR) of the synthesized MMP2-C18 is as Figure 8 shown.

[0269] 2.4. pHAase Synthesis

[0270] Dissolve 5 mg of hyaluronidase (HAase, CAS: 37326-33-3) and 50 mg of dimethyl maleic anhydride (DMA) in HEPES buffer, keep it at 4 °C for 4 h, then dialyze with pH 7.4 PBS buffer for 24 h and freeze-dry to obtain pHAase.

[0271] The modification rate of primary amine groups in hyaluronidase by dimethyl maleic anhydride measured by the fluorescamine method is 78.44 ± 0.025%

[0272] pH-responsive activity of pHAase: HA degradation experiment: Equal volumes of the following solutions were added to a 2 mg / mL + HA (10 kDa) solution: 2 mg / mL HAase (pH 7.4), 2 mg / mL HAase (pH 6.0), pHAase (pH 7.4), pHAase (pH 6.0), HSP + pHAase (pH 6.0). After incubation for 30 min, the viscosity change of the HA solution was measured.

[0273] Weighed 10 mg of HA, HAase, or pHAase were separately dissolved in 5 mL of PBS buffer at pH 7.4 or 6.0. Heating was used to completely dissolve HA. The HA solution was mixed with the HAase or pHAase solution and incubated at 37 °C for 30 min, after which the viscosity was measured. Using the viscosity of HA as a control, the relative viscosity changes of each group were obtained.

[0274] It can be seen from Figure 9 that under the condition of pH 7.4, the enzyme activity of pHAase decreases, its ability to degrade HA is weak, and HA still has a relatively large viscosity. However, when the pH is reduced to 6.0, the activity of pHAase is activated, its ability to degrade HA is enhanced, and the viscosity of HA shows a significant decrease.

[0275] 3. Preparation of liposomes

[0276] 3.1 Preparation of HSP / pHAase@MLip liposomes

[0277] (1) Dissolved 3 mg of HA-ADH-SS-PTX (HSP) and 0.8 mg of pHAase in 50 μL of water to obtain the inner aqueous phase.

[0278] (2) Mixed 63.75 μL of 15 mg / mL egg yolk lecithin chloroform solution, 97.2 μL of 10 mg / mL cholesterol chloroform solution, and 89.05 μL of chloroform to obtain the oil phase.

[0279] (3) Added the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and emulsified it with a probe sonicator (sonication intensity: 300 W) for 5 min to obtain a W / O emulsion.

[0280] (4) Mixed 127.5 μL of 15 mg / ml egg yolk lecithin methanol solution, 14.2 μL of 10 mg / mL distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, 2.5 μL of 3.5 mg / mL poloxamer 188 aqueous solution, 52.5 μL of 2 mg / mL MMP2-C18 aqueous solution, 106.6 μL of methanol, and 1.1975 mL of water to obtain the outer aqueous phase.

[0281] (5) Add the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), and emulsify it with a probe ultrasonic wave (ultrasonic intensity: 300 W) for 5 min to obtain a W / O / W emulsion. Stir for 12 h to volatilize the organic solvent to obtain the HSP / pHAase@MLip liposome dispersion.

[0282] 3.2. Preparation of HCP / pHAase@MLip Liposomes

[0283] The preparation of HCP / pHAase@MLip liposomes is the same as that of "3.1 Preparation of HSP / pHAase@MLip liposomes", and the differences are as follows:

[0284] In step (1), dissolve 3 mg of HA-ADH-CC-PTX (HCP) and 0.8 mg of pHAase in 50 μL of water to obtain the inner aqueous phase.

[0285] 3.3. Preparation of HSP@MLip Liposomes

[0286] The preparation of HSP@MLip liposomes is the same as that of "3.1 Preparation of HSP / pHAase@MLip liposomes", and the differences are as follows:

[0287] In step (1), dissolve 3 mg of HA-ADH-SS-PTX (HSP) in 50 μL of water to obtain the inner aqueous phase.

[0288] 3.4. Preparation of pHAase@MLip Liposomes

[0289] The preparation of pHAase@MLip liposomes is the same as that of "3.1 Preparation of HSP / pHAase@MLip liposomes", and the differences are as follows:

[0290] In step (1), dissolve 0.8 mg of pHAase in 50 μL of water to obtain the inner aqueous phase.

[0291] 3.5. Preparation of HSP / pHAase@Lip Liposomes

[0292] The preparation of HSP / pHAase@Lip liposomes is the same as that of "3.1 Preparation of HSP / pHAase@MLip liposomes", and the differences are as follows:

[0293] (4) Mix 127.5 μL of 15 mg / ml egg yolk lecithin methanol solution, 14.2 μL of 10 mg / mL distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, 2.5 μL of 3.5 mg / mL poloxamer 188 aqueous solution, 52.5 μL of 2 mg / mL C18 aqueous solution, 106.6 μL of methanol and 1.1975 mL of water to obtain the outer aqueous phase.

[0294] The zeta potential and particle size of different liposomes are shown in Table 1 below:

[0295] Table 1 Zeta potential and particle size of different liposomes

[0296]

[0297] 4. Immune response

[0298] 5×10 5 LLC cells (lung cancer cells) were inoculated subcutaneously into the right side of C57BL / 6 mice. When the tumors grew to 50 mm 3 (recorded as the 1st day), the established LLC tumor-bearing mice were randomly divided into a Saline group, a Taxol group, an HSP@MLip group, an HCP / pHAase@MLip group, an HSP / pHAase@Lip group, and an HSP / pHAase@MLip group, with 6 mice in each group. Each group was intravenously injected with the same volume of the following drugs via the tail vein on the 1st, 4th, 7th, and 10th days:

[0299] Saline group: normal saline;

[0300] Taxol group: Taxol (Paclitaxel Injection), where the administration dose of paclitaxel (PTX) was 5.0 mg / kg;

[0301] HSP@MLip group: physiological saline dispersion of HSP@MLip liposomes, where the equivalent administration dose of paclitaxel (PTX) was 5.0 mg / kg;

[0302] HCP / pHAase@MLip group: physiological saline dispersion of HCP / pHAase@MLip liposomes, where the equivalent administration dose of paclitaxel (PTX) was 5.0 mg / kg;

[0303] HSP / pHAase@Lip group: physiological saline dispersion of HSP / pHAase@Lip liposomes, where the equivalent administration dose of paclitaxel (PTX) was 5.0 mg / kg;

[0304] HSP / pHAase@MLip group: physiological saline dispersion of HSP / pHAase@MLip liposomes, where the equivalent administration dose of paclitaxel (PTX) was 5.0 mg / kg;

[0305] On the 12th day, mouse tumor tissues were collected to prepare single-cell suspensions of tumor tissues, and the changes in immune cell populations were analyzed by FCM (flow cytometry) to evaluate the immunological effects of drug treatments in each group. The evaluation methods and results are as follows:

[0306] Preparation of single-cell suspension from tumor tissues: First, wash with pre-cooled PBS, and then cut the tumor tissues of each group into tumor tissue blocks with a size of 2-4 mm. After thoroughly grinding the tumor tissue blocks, add 3 mL of DMEM medium containing collagenase IV (1 mg / mL) and DNase I (0.2 mg / mL) to each group as an enzyme digestion solution, and further digest in a constant temperature shaking incubator at 37 °C for 1 h. Subsequently, gently pipette the tissue suspension and filter it through a 70-μm cell sieve to remove aggregated cell clusters and undigested tissues, thus successfully preparing a single-cell suspension. Centrifuge and wash the obtained single-cell suspension twice with pre-cooled PBS (centrifuge at 300 g for 7 min at 4 °C), discard the supernatant, and obtain the cell pellet at the bottom layer. Resuspend the cell pellet with 0.5 mL of PBS, count the cells, and observe the cell status. Finally, adjust the cell concentration to 1×10 7 / mL to set up blank tubes, single-staining tubes, and sample tubes, with 1×10 6 cells in each tube.

[0307] 1) Evaluation of ICD induction: Detection of CRT (Calreticulin)

[0308] Evaluate the ICD (immunogenic cell death) induction ability of different drugs by detecting the change in the level of Calreticulin (CRT) on the surface of tumor cells. Collect cell samples from each group, resuspend them with 50 μL of PBS, and then add the PE-labeled CRT antibody (anti-CRT-PE) diluted 1:50, and incubate on ice for 30 min. After incubation, centrifuge to collect the cells, resuspend the cells with 50 μL of PBS, and detect them by FCM to analyze the proportion of CRT + cells. The results are as shown in Figure 10 and Figure 11 .

[0309] It can be seen from Figure 10 and Figure 11 that after treatment with each drug, the CRT+ cell population in the tumor tissues increased significantly, but the increase in the CRT + cell population in the tumor tissues treated with HSP / pHAase@MLip liposomes was the most significant.

[0310] 2) Antitumor immune response mediated by ICD

[0311] For the prepared single-cell suspension of tumor tissues, first add 1 μL of the live / dead dye FVS780 to the cells in each sample tube. Using 1 mL of PBS as the staining medium, incubate in the dark at room temperature for 15 min, then centrifuge at 300 g for 6 min, discard the supernatant, and collect the cell pellet at the bottom. Subsequently, add 1 mL of PBS containing 0.5% BSA to wash the obtained cell pellet to inactivate the free dye. Subsequently, add FITC Rat Anti-Mouse CD45, PE-Cy TM 7 Hamster Anti-Mouse CD11c, BV605 Hamster Anti-Mouse CD80, and BV421 Rat Anti-Mouse CD86 for cell surface staining. The dosage of each antibody is 1 μg / sample, and only the corresponding antibody is added for staining in the single-staining tube. Incubate on ice in the dark for 30 min, centrifuge, discard the supernatant, collect the cell pellet at the bottom, add an appropriate amount of PBS to resuspend the cell pellet, and perform FCM detection: Analyze the proportion of CD80 + CD86 + mature DCs (Dendritic cells) under the CD45+CD11+ logic gate. The results are as Figure 12 、 Figure 13 shown.

[0312] It can be seen from Figure 12 and Figure 13 that after treatment with HSP / pHAase@MLip liposomes, the proportion of CD80+CD86+ mature DCs in tumor tissues is the highest.

[0313] 3) CD8 + T cell detection

[0314] For the prepared single-cell suspension of tumor tissues, first add 1 μL of the live / dead dye FVS780 to the cells in each sample tube. Using 1 mL of PBS as the staining medium, incubate in the dark at room temperature for 15 min, then centrifuge at 300 g for 6 min, discard the supernatant, and collect the cell pellet at the bottom. Subsequently, add 1 mL of PBS containing 0.5% BSA to wash the obtained cell pellet to inactivate the free dye. Subsequently, add FITC Rat Anti-Mouse CD45, BV510 Hamster Anti-Mouse CD3e, BV421 RatAnti-Mouse CD25, APC Rat Anti-Mouse CD4, PE-Cy TMFor cell surface staining with 7 Rat Anti-Mouse CD8a, the dosage of each antibody was 1 μg / sample, and only the corresponding antibody was added to the single-staining tube. Incubate in the dark on ice for 30 min, centrifuge to discard the supernatant, and collect the cell pellet at the bottom layer. Then add 1 mL of freshly prepared 1×Fix / Perm Buffer working solution, incubate in the dark on ice for 45 min for fixation and permeabilization. After centrifugation again, add 1 mL of 1×Perm / Wash Buffer to wash the obtained cell pellet, centrifuge to discard the supernatant, and collect the cell pellet at the bottom layer. Subsequently, resuspend the obtained cell pellet with 100 μL of 1×Perm / Wash Buffer, add 1 μg of PE anti-FOXp3, incubate in the dark on ice for 45 min, centrifuge and discard the supernatant, add an appropriate amount of PBS to resuspend the cell pellet, and perform FCM detection: at CD45 + CD3 + Analyze CD8 under the logic gate + of T cells, and the results are as Figure 14 and Figure 15 shown.

[0315] From Figure 14 and Figure 15 it can be seen that after treatment with HSP / pHAase@MLip liposomes, the number of CD8 + T cells in the tumor tissue increased most significantly, far higher than other groups; it can up-regulate the intratumoral accumulation of cytotoxic T cells and mediate anti-tumor immune responses.

[0316] 4) Treg cells (Regulatory cells)

[0317] For the single-cell suspension of tumor tissue prepared, first add 1 μL of live / dead dye FVS780 to stain the cells in each sample tube. Using 1 mL of PBS as the staining medium, incubate in the dark at room temperature for 15 min, then centrifuge at 300 g for 6 min, discard the supernatant, and collect the cell pellet at the bottom layer. Subsequently, add 1 mL of PBS containing 0.5% BSA to wash the obtained cell pellet to inactivate the free dye. Subsequently, add FITC Rat Anti-Mouse CD45, BV510 Hamster Anti-Mouse CD3e, BV421 RatAnti-Mouse CD25, APC Rat Anti-Mouse CD4, PE-Cy TMFor cell surface staining with 7 Rat Anti-Mouse CD8a, the dosage of each antibody was 1 μg / sample, and only the corresponding antibody was added to the single-staining tube. Incubate in the dark on ice for 30 min, centrifuge to discard the supernatant, and collect the cell pellet at the bottom. Then add 1 mL of freshly prepared 1×Fix / Perm Buffer working solution, incubate in the dark on ice for 45 min for fixation and permeabilization. After centrifugation again, add 1 mL of 1×Perm / Wash Buffer to wash the obtained cell pellet, centrifuge to discard the supernatant, and collect the cell pellet at the bottom. Subsequently, resuspend the obtained cell pellet with 100 μL of 1×Perm / Wash Buffer, add 1 μg of PE anti-FOXp3, incubate in the dark on ice for 45 min, centrifuge and discard the supernatant, add an appropriate amount of PBS to resuspend the cell pellet, and analyze CD25 + CD3 + CD4 + under the logic gate + FOXp3 + for Treg cells, and the results are as Figure 16 and Figure 17 shown

[0318] From Figure 16 and Figure 17 it can be seen that after treatment with HSP / pHAase@MLip liposomes, the number of Treg cells in the tumor tissue was the lowest

[0319] From the above immune response results, it can be seen that treatment with HSP / pHAase@MLip liposomes can significantly enhance the immune response ability against tumors

[0320] 5. Investigation of the tumor suppression effect and immune response effect of different drugs and their combinations

[0321] Agent: InVivo MAb anti-mouse PD-1: Cat.No BE0273, bioxcell, USA

[0322] Experimental method

[0323] Inoculate LLC cells (lung cancer cells) subcutaneously into the right side of the back of C57BL / 6 mice. When the tumor grows to 50 mm 3 (recorded as day 0), randomly divide the established LLC tumor-bearing mice into a Saline group, a HSP / pHAase@MLip group, an anti-PD-1 group, and a HSP / pHAase@MLip + anti-PD-1 group, with 5 mice in each group. Each group was intravenously injected with the same volume of the following drugs at the tail vein on days 0, 3, 6, 9, and 12:

[0324] Saline group: normal saline​

[0325] HSP / pHAase@MLip group: A physiological saline dispersion of HSP / pHAase@MLip liposomes, where the equivalent dose of paclitaxel (PTX) is 5.0 mg / kg;

[0326] anti-PD-1 group: A physiological saline dispersion of InVivo MAb anti-mouse PD-1, and the dosage of InVivo MAb anti-mouse PD-1 is 5.0 mg / kg;

[0327] HSP / pHAase@MLip + anti-PD-1 group: A physiological saline dispersion of HSP / pHAase@MLip liposomes and InVivo MAb anti-mouse PD-1, where the equivalent dose of paclitaxel (PTX) is 5.0 mg / kg, and the dosage of InVivo MAb anti-mouse PD-1 inhibitor is 5.0 mg / kg.

[0328] On days 0, 3, 6, 9, 12, and 15, the long diameter (a) and short diameter (b) of the tumor were measured with vernier calipers. According to the formula: tumor volume (V, mm 3 ) = 0.5 × a × b 2 , the tumor volume was calculated, and the change of tumor volume over time was plotted. On day 15, mouse tumor tissues were taken, weighed, and then single-cell suspensions of tumor tissues were prepared. The changes in the Treg cell (regulatory T cells) population in the tumor tissues were analyzed by FCM (flow cytometry) to evaluate the immunological effects of drug treatments in each group.

[0329] The changes in the tumor volume of mice in different treatment groups over time are as Figure 18 shown, and the tumor weights of mice in different treatment groups on day 15 are shown in Table 2 below.

[0330] Table 2 Tumor weights of mice in different treatment groups on day 15

[0331]

[0332] Note: Compared with the Saline group, "*" indicates p < 0.05, "**" indicates p < 0.01, and "****" indicates p < 0.0001.

[0333] From Table 2 and Figure 18As can be seen, the HSP / pHAase@MLip liposomes prepared in Example 1 have excellent in vivo anti-tumor effects. In particular, from the tumor volumes and tumor weights of the HSP / pHAase@MLip group, anti-PD-1 group, and HSP / pHAase@MLip + anti-PD-1 group, it can be seen that the combination of HSP / pHAase@MLip liposomes and anti-PD-1 inhibitor has an excellent synergistic inhibitory effect on tumor growth. Therefore, the combination of HSP / pHAase@MLip liposomes and anti-PD-1 inhibitor can significantly enhance the therapeutic effect on tumors.

[0334] On the 15th day, the measurement methods and results of the Treg cell population in the tumor tissues isolated from different groups are as follows:

[0335] Preparation of single-cell suspension of tumor tissue: First, wash with pre-cooled PBS, and then cut the tumor tissues of each group into tumor tissue blocks with a size of 2 - 4 mm. After thoroughly grinding the tumor tissue blocks, add 3 mL of DMEM medium containing collagenase IV (1 mg / mL) and DNase I (0.2 mg / mL) to each group as an enzyme digestion solution, and further digest in a 37°C constant temperature shaking incubator for 1 h. Subsequently, gently pipette the tissue suspension, and pass it through a 70 μm cell sieve to remove aggregated cell clusters and undigested tissues, that is, a single-cell suspension is successfully prepared. Centrifuge and wash the obtained single-cell suspension twice with pre-cooled PBS (centrifuge at 4°C, 300 g for 7 min), discard the supernatant, and obtain the cell pellet at the bottom layer. Resuspend the cell pellet with 0.5 mL PBS, count and observe the cell status. Finally, adjust the cell concentration to 1×10 7 / mL to set up blank tubes, single-staining tubes, and sample tubes, with 1×10 6 cells in each tube.

[0336] For the prepared single-cell suspension of tumor tissue, first add 1 μL of the live / dead dye FVS780 to the cells in each sample tube. Using 1 mL of PBS as the staining medium, incubate in the dark at room temperature for 15 min, then centrifuge at 300 g for 6 min, discard the supernatant, and collect the cell pellet at the bottom. Subsequently, add 1 mL of PBS containing 0.5% BSA to wash the obtained cell pellet to inactivate the free dye. Then, add anti-CD45, CD3, CD25, CD4, and CD8 to the 100 μL PBS system for cell surface staining. The dosage of each antibody is 1 μg / sample, and only the corresponding antibody is added to the single-staining tube for staining. Incubate on ice in the dark for 30 min, centrifuge, discard the supernatant, and collect the cell pellet at the bottom. Add 1 mL of freshly prepared 1×Fix / Perm Buffer working solution, incubate on ice in the dark for 45 min for fixation and permeabilization. After centrifugation again, add 1 mL of 1×Perm / Wash Buffer to wash the obtained cell pellet, centrifuge, discard the supernatant, and collect the cell pellet at the bottom. Subsequently, resuspend the obtained cell pellet with 100 μL of 1×Perm / Wash Buffer, add 1 μg of anti-FOXp3, incubate on ice in the dark for 45 min, centrifuge, discard the supernatant, add an appropriate amount of PBS to resuspend the cell pellet, and analyze the Treg cells of CD45 + CD3 + CD4 + Analyze CD25 under the logic gate + FOXp3 + , and the results are as Figure 19 shown.

[0337] From Figure 19 , it can be seen that compared with the reduction of the number of Treg cells by the single HSP / pHAase@MLip liposome or the single anti-PD-1 inhibitor, the combination of the HSP / pHAase@MLip liposome and the anti-PD-1 inhibitor has a synergistic effect in reducing the number of Treg cells. Therefore, the combination of the HSP / pHAase@MLip liposome and the anti-PD-1 inhibitor can synergistically enhance the immune response ability against tumors.

[0338] The above is the implementation scheme designed by the present invention for a case. It should be noted that for those of ordinary skill in the art, several improvements can be made without departing from the principle of the present invention, and these improvements should also be regarded as the protection scope of the present invention.

Claims

1. A hyaluronic acid-paclitaxel conjugate, characterized in that, The hyaluronic acid-paclitaxel conjugate has the following structure: n is from 3 to 30, preferably from 5 to 20, more preferably from 5 to 15, still more preferably from 8 to 12, even more preferably from 9 to 11, and most preferably 10; m is from 1 to 15, preferably from 1 to 10, more preferably from 2 to 8, still more preferably from 2 to 5, even more preferably from 2 to 4, and most preferably 3; k is from 3 to 30, preferably from 5 to 20, more preferably from 8 to 16, still more preferably from 10 to 14, even more preferably from 11 to 13, and most preferably 12.

2. A liposome, characterized in that, The liposome comprises the hyaluronic acid-paclitaxel conjugate as described in claim 1.

3. The liposome according to claim 2, characterized in that, The liposome comprises the hyaluronic acid-paclitaxel conjugate as described in claim 1, MMP2-C18, pHAase, lecithin, cholesterol, distearoyl phosphatidylethanolamine-polyethylene glycol 2000, and poloxamer 188.

4. A method for preparing the liposome according to claim 2, characterized in that, The method comprises the steps of: (1) Dissolving the hyaluronic acid-paclitaxel conjugate as described in claim 1 and pHAase in water to obtain an inner aqueous phase; (2) Dissolving lecithin and cholesterol in chloroform to obtain an oil phase; (3) Adding the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and performing probe sonication to obtain a W / O emulsion; (4) Mixing egg yolk lecithin methanol solution, distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, poloxamer 188 aqueous solution, MMP2-C18 aqueous solution, methanol, and water to obtain an outer aqueous phase; (5) Adding the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), and performing probe sonication to obtain a W / O / W emulsion, and stirring to evaporate the organic solvent to obtain the liposome.

5. The method according to claim 4, characterized in that, The method comprises the steps of: (1) Dissolving 2-4 mg of the hyaluronic acid-paclitaxel conjugate as described in claim 1 and 0.5-1.2 mg of pHAase in 40-60 μL of water to obtain an inner aqueous phase; (2) Mixing 50-70 μL of 10-20 mg / mL lecithin chloroform solution, 90-110 μL of 5-15 mg / mL cholesterol chloroform solution, and 80-100 μL of chloroform to obtain an oil phase; (3) Adding the inner aqueous phase prepared in step (1) to the oil phase prepared in step (2), and performing probe sonication to obtain a W / O emulsion; (4) Mixing 120-140 μL of 10-20 mg / ml lecithin methanol solution, 10-20 μL of 5-15 mg / mL distearoyl phosphatidylethanolamine-polyethylene glycol 2000 methanol solution, 2.0-3.0 μL of 3.0-4.0 mg / mL poloxamer 188 aqueous solution, 40-60 μL of 1.5-2.5 mg / mL MMP2-C18 aqueous solution, 95-120 μL of methanol, and 1.0-1.4 mL of water to obtain an outer aqueous phase; (5) Adding the W / O emulsion prepared in step (3) to the outer aqueous phase prepared in step (4), and performing probe sonication to obtain a W / O / W emulsion, and stirring to evaporate the organic solvent to obtain the liposome.

6. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the liposome as described in claim 2; and (ii) a PD-1 inhibitor.

7. A medicine box, characterized in that, The kit comprises: (A) A first preparation containing a first active ingredient, the first active ingredient including the liposome as described in claim 2; and (B) A second preparation containing a second active ingredient, the second active ingredient including a PD-1 inhibitor.

8. The pharmaceutical composition according to claim 6 or the kit according to claim 7, characterized in that, The weight ratio of the paclitaxel equivalent in the liposome to the PD-1 inhibitor is 1:(0.001 - 1000), preferably 1:(0.01 - 100), more preferably 1:(0.05 - 50), more preferably 1:(0.08 - 30), more preferably 1:(0.1 - 20), more preferably 1:(0.1 - 10), more preferably 1:(0.3 - 8), more preferably 1:(0.5 - 5), more preferably 1:(0.5 - 3), more preferably 1:(0.5 - 2), more preferably 1:(0.5 - 1.5), more preferably 1:(0.8 - 1.2), most preferably 1:

1.

9. Use of a hyaluronic acid-paclitaxel conjugate as described in claim 1, a liposome as described in claim 2, a pharmaceutical composition as described in claim 6, or a kit as described in claim 7, characterized in that, For preparing a medicament for preventing and / or treating tumors.

10. A method for inhibiting tumor cells, characterized in that, The method includes the steps of: Contacting tumor cells with the hyaluronic acid-paclitaxel conjugate as described in claim 1, the liposome as described in claim 2, or the pharmaceutical composition as described in claim 6 of the present claim, so as to inhibit the tumor cells.

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