Albumin-paclitaxel prodrug nanoparticle based on dipeptide linker and use thereof

Abstract

The application provides albumin-paclitaxel twin drug nanoparticles based on a peptide linker and an application thereof, wherein the linker between the paclitaxel and the drug molecule is a dipeptide linker, the linker has higher stability, and the speed of the linker breaking in a tumor microenvironment response is faster. The application compares and analyzes twin drug nanoparticles synthesized by different linkers between paclitaxel and drug molecules, screens a dipeptide linker capable of effectively improving the antitumor effect of albumin-paclitaxel twin drug nanoparticles, realizes efficient co-loading of multiple drugs, increases drug accumulation, reduces indiscriminate killing of normal cells by drugs, is successfully targeted to a tumor region, achieves the purpose of treating and regressing tumors, and is expected to break through the current efficacy bottleneck of malignant tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of medicinal chemistry and biopharmaceutical technology, and particularly relates to the preparation and application of albumin-paclitaxel twin drug nanoparticles based on peptide linkers. BACKGROUND

[0002] Malignant tumors, commonly known as cancer, are a major public health problem worldwide and pose a significant threat to human health. According to data from the World Health Organization and various national cancer research institutions, the incidence and mortality rates of malignant tumors remain high worldwide. Treatment typically includes surgery, radiotherapy, and / or systemic therapy (chemotherapy, hormone therapy, targeted biological therapy). Among them, chemotherapy, as the main means of treating malignant tumors, still has many problems. The poor solubility, poor targeting, high toxicity and drug resistance of chemotherapy drugs greatly limit their clinical transformation.

[0003] Albumin nanoparticles, as a new type of protein carrier, have many properties such as water solubility, biocompatibility, biodegradability, non-toxicity, and non-immunogenicity. It can improve the stability and bioavailability of drugs, prolong the circulation time of drugs in the body, and improve the drugability, toxicity, and solubility of drugs. In addition, albumin nanoparticles can also bind to albumin binding receptors on the surface of tumor cells, achieving targeted drug delivery and further improving efficacy.

[0004] Due to the presence of metabolic abnormalities and complex microenvironments in tumors, if the mechanism of action of a single drug is not comprehensive enough, it may not be able to effectively inhibit the growth and spread of tumors. Different drugs have different mechanisms of action, and some drugs may be more suitable for combination use to exert synergistic effects, such as Abraxane combined with carboplatin, gemcitabine, and other antitumor drugs, which has good use effect. Although combination therapy can significantly improve patient survival and improve drug resistance of single drug use, there are still many problems to be solved, such as low bioavailability, poor targeting, and obvious toxicity of free drugs such as carboplatin and gemcitabine.

[0005] Patent document CN117731796A discloses the preparation and application of a class of albumin-paclitaxel twin drug nanoparticles. The application provides a class of paclitaxel twin drugs constructed by covalently linking paclitaxel as a target head to a drug molecule, and then reacting with albumin to form uniform albumin-paclitaxel twin drug nanoparticles. The albumin-paclitaxel twin drug nanoparticles connected to different drugs all have good antitumor effects, but the albumin-paclitaxel twin drug nanoparticles provided by the patent have limited ability to inhibit tumor growth, and there are still certain limitations in drug release and therapeutic effect of the nanoparticles.

[0006] Therefore, the method capable of effectively improving the antitumor effect of albumin-paclitaxel twin drug nanoparticles and further improving the drug treatment effect has important significance for breaking the current bottleneck of malignant tumor treatment effect. SUMMARY

[0007] In view of the problems in the prior art, the albumin-paclitaxel twin drug nanoparticles based on a peptide linker and the application thereof are provided, the nanoparticles synthesized by different linkers between paclitaxel and drug molecules are compared, the linker capable of effectively improving the antitumor effect of albumin-paclitaxel twin drug nanoparticles is screened, the efficient co-loading of multiple drugs is realized, the successful targeted delivery to the tumor area is achieved, the purpose of treating and regressing the tumor is achieved, and the current bottleneck of malignant tumor treatment effect is expected to be broken.

[0008] In order to achieve the above-mentioned purpose, the following technical solutions are adopted in the present application:

[0009] In one aspect, the present application provides a paclitaxel derivative twin drug, which is composed of paclitaxel and a drug molecule connected by a peptide linker, and the connection unit of the peptide linker is selected from any two or more combinations of valine, citrulline, alanine, glycine, lysine, phenylalanine, glutamic acid, serine and aspartic acid.

[0010] The paclitaxel derivative twin drug provided in the present application is connected by a chemical bond which is specific to the tumor microenvironment, and the chemical bond is a dipeptide bond. The response group of the peptide linker includes but is not limited to valine-citrulline-p-aminobenzyloxy (Val-Cit-PAB), valine-alanine-p-aminobenzyloxy (Val-Ala-PAB), glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0011] The linker provided in the present application is selected from a peptide linker which can be cut by cathepsin in tumor tissue. The peptide linker generally has good stability and solubility, which helps to improve the overall stability of the paclitaxel derivative twin drug. At the same time, the structure and properties of the peptide linker are changed by design and selection, so as to realize the precise regulation of the paclitaxel derivative twin drug and the responsive release of the antitumor drug, reduce the toxic and side effects of the drug on non-tumor cells, and further improve the curative effect.

[0012] Further, the drug molecule includes any one of a cytotoxic drug, a small molecule targeted drug, an immune checkpoint inhibitor, a hormone drug and an immune agonist, and the connection unit of the peptide linker is selected from valine and citrulline.

[0013] Preferably, the drug molecule comprises any one of Larotrectinib, MRYX1133, Capivasertib, mlN8237, di-ABZI, SRO7, T785, MMAE, AZD7762, and the chemical structure of the nine drug molecules is shown in the general formula (1). Figure 1

[0014] Preferably, the paclitaxel derivative twin drug is synthesized by connecting paclitaxel and an antitumor drug through a peptide linker synthesized by a connecting unit of valine and citrulline, and the chemical structure of the peptide linker is shown in the general formula (1).

[0015]

[0016] In another aspect, the present application provides an albumin-paclitaxel derivative twin drug nanoparticle, comprising the paclitaxel derivative twin drug as described above.

[0017] In some embodiments, the drug molecule of the paclitaxel derivative twin drug comprises any one or more of Larotrectinib, MRYX1133, Capivasertib, mlN8237, di-ABZI, SRO7, T785, MMAE, AZD7762. Preferably, the drug molecule comprises any one of T785, MMAE, AZD7762.

[0018] In some embodiments, the albumin-paclitaxel derivative twin drug nanoparticle comprises one or two or three paclitaxel derivative twin drugs.

[0019] Further, the albumin-paclitaxel derivative twin drug nanoparticle further comprises albumin, and the albumin encapsulates the paclitaxel derivative twin drug.

[0020] Further, the albumin comprises any one or more of bovine serum albumin, human serum albumin, ovalbumin, and recombinant human serum albumin. Preferably, the albumin comprises human serum albumin.

[0021] In another aspect, the present application provides a preparation method of a paclitaxel derivative twin drug, wherein paclitaxel, a peptide linker, and a drug molecule are prepared by chemical reaction, and the connecting unit of the peptide linker is selected from valine and citrulline.

[0022] In some embodiments, the synthesis steps of the paclitaxel derivative twin drug with valine and citrulline as the connecting unit of the peptide linker are shown in the general formula (1). Figure 2 ​As shown. Paclitaxel (PTX) is made into intermediate PTX-Q, Boc-Val-cit molecule is reacted with solvent to make intermediate N3-Val-Cit-PAB, then N3-Val-Cit-PAB is reacted with drug molecule in solvent to make N3-Val-Cit-PAB-T785, then N3-Val-Cit-PAB-T785 is reacted with PTX-Q in solvent to make paclitaxel-based twin drug compound.

[0023] In some embodiments, the solvent used in the synthesis method step includes any one or more of dichloromethane, N,N-dimethylformamide, methanol, ethyl acetate, petroleum ether, chloroform, tetrahydrofuran, acetonitrile, 1,4-dioxane, dimethyl sulfoxide, benzene, toluene, diethyl ether, o-dichlorobenzene; the basic reagent is divided into inorganic base and organic base, the inorganic base includes metal hydroxide, alkali metal, the organic base includes one or more of triethylamine, tributylamine, trioctylamine, N,N-diisopropyl ethylamine, pyridine, 4-dimethylamino pyridine, piperidine, N-methyl morpholine, sodium tert-butoxide, potassium tert-butoxide, diisopropyl amine, tetrahydro pyrrole and triethylene diamine; the condensing agent includes one or more of 1-(3-dimethylamino propyl)-3-ethyl carbodiimide (EDCI), dicyclohexyl carbodiimide (DCC), diisopropyl carbodiimide (DIC), 2-(7-azido-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium hexafluorophosphate (HATU), 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyl uronium tetrafluoroborate (HBTU) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyl uronium tetrafluoroborate (TBTU).

[0024] In another aspect, the present application provides a preparation method of albumin-paclitaxel twin drug nanoparticles, which comprises the following steps:

[0025] Further, the preparation method comprises the following steps:

[0026] (1) mixing human serum albumin with water to obtain an aqueous phase;

[0027] (2) mixing paclitaxel derivative twin drug with organic solvent to obtain an organic phase;

[0028] (3) mixing the aqueous phase with the organic phase, and then obtaining albumin-paclitaxel twin drug nanoparticles through ultrasonic treatment, rotary evaporation and ultrafiltration.

[0029] In some embodiments, the concentration of albumin in the aqueous phase in step (1) is 14.5 mg / ml, the molar ratio of paclitaxel derivative to albumin in step (2) is 4.3:1, the organic phase in step (3) comprises any two or more of dichloromethane, chloroform, ethanol, DMSO, DMF, acetonitrile, and the ultrasonic instrument used in step (4) includes but is not limited to ultrasonic cell disruptor, ultrasonic cleaner.

[0030] In some embodiments, step (3) can use one or two or three different organic phase solutions of paclitaxel derivative to add to the albumin solution, and the subsequent steps can be used to produce albumin-paclitaxel derivative nanoparticles including single, double or triple drug.

[0031] The albumin-paclitaxel derivative nanoparticles obtained by the preparation method provided by the present application have an encapsulation efficiency of 80% to 85% for paclitaxel derivative and a drug loading of 6% to 8%.

[0032] Preferably, the albumin-paclitaxel derivative nanoparticles have an encapsulation efficiency of 84.8% for paclitaxel derivative PTX-VC-T785 and a drug loading of 7.96%.

[0033] In another aspect, the present application provides a use of paclitaxel derivative in the preparation of a medicament for treating cancer, wherein the paclitaxel derivative is formed by linking paclitaxel to a drug molecule via a peptide linker, and the linking unit of the peptide linker is selected from valine and citrulline.

[0034] Further, the cancer includes but is not limited to non-small cell lung cancer, cervical cancer, breast cancer, lung cancer, gastric cancer, melanoma, bladder cancer, intestinal cancer, prostate cancer, pancreatic cancer, esophageal cancer, brain cancer, neural cancer, ovarian cancer, and kidney cancer.

[0035] In another aspect, the present application provides a use of albumin-paclitaxel derivative nanoparticles in the preparation of a medicament for treating cancer, wherein the albumin-paclitaxel derivative nanoparticles comprise paclitaxel derivative and albumin, and the paclitaxel derivative is formed by linking paclitaxel to a drug molecule via a peptide linker, and the linking unit of the peptide linker is selected from valine and citrulline.

[0036] Further, the albumin-paclitaxel derivative nanoparticles are configured for oral administration, respiratory administration, intravenous injection, intra-articular injection, intramuscular injection, or direct injection into tumor / cancer.

[0037] Further, the cancer includes but is not limited to non-small cell lung cancer, cervical cancer, breast cancer, lung cancer, gastric cancer, melanoma, bladder cancer, intestinal cancer, prostate cancer, pancreatic cancer, esophageal cancer, brain cancer, neural cancer, ovarian cancer, and kidney cancer.

[0038] In some embodiments, the present application verifies the difference in anti-tumor effect of the paclitaxel derivative twin drug nanoparticles synthesized by different connecting units through comparative experiments, and the results show that the twin drug nanoparticles synthesized by the peptide connecting units composed of different connecting units can effectively inhibit the growth of pancreatic cancer tumor, and the anti-tumor effect of the twin drug nanoparticles synthesized by the peptide connecting unit composed of valine and citrulline is the best.

[0039] The present application has the following beneficial effects:

[0040] 1. The present application designs a kind of cathepsin-cleavable paclitaxel derivative twin drug in tumor tissue based on paclitaxel, which can realize the responsive release of anti-tumor drugs, reduce the toxic and side effects of drugs on non-tumor cells, and thus improve the curative effect.

[0041] 2. The present application provides a series of paclitaxel twin drug nanoparticles with albumin as a wrapping carrier, which realizes efficient co-loading of various drugs and specific release of the loaded drugs inside tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 It is a general structure diagram of the drug molecule;

[0043] Figure 2 It is a synthesis route diagram of the paclitaxel derivative twin drug PTX-VC-T785 of the present application;

[0044] Figure 3 It is the hydrogen spectrum diagram of the compound PTX-Q in the present application;

[0045] Figure 4 It is the hydrogen spectrum diagram of the compound Boc-Val-cit-PAB in the present application;

[0046] Figure 5 It is the hydrogen spectrum diagram of the compound N3-Val-cit-PAB in the present application;

[0047] Figure 6 It is the hydrogen spectrum diagram of the compound N3-Val-cit-PAB-T785 in the present application;

[0048] Figure 7 It is the hydrogen spectrum diagram of the compound PTX-Val-cit-PAB-T785 in the present application;

[0049] Figure 8 It is the hydrogen spectrum diagram of the compound N3-Val-cit-PAB-MMAE in the present application;

[0050] Figure 9 It is the hydrogen spectrum diagram of the compound PTX-Val-cit-PAB-MMAE in the present application;

[0051] Figure 10 The hydrogen spectrum of compound N3-Val-cit-PAB-AZD in the present application;

[0052] Figure 11 The hydrogen spectrum of compound PTX-Val-cit-PAB-AZD in the present application;

[0053] Figure 12 The synthetic route map of paclitaxel derivative twin drug PTX-VA-T785;

[0054] Figure 13 The synthetic route map of paclitaxel derivative twin drug PTX-GGPG-T785;

[0055] Figure 14 The particle size and surface potential map of albumin-paclitaxel twin drug nanoparticles in the present application;

[0056] Figure 15 The transmission electron microscopy map of albumin-paclitaxel twin drug nanoparticles Nab-PTX (A), Nab-PTX-VC-T785 (B), Nab-PTX-VC-AZD (C), Nab-PTX-VC-MMAE (D) in the present application;

[0057] Figure 16 The cytotoxicity map of paclitaxel combined with antitumor drugs in the present application;

[0058] Figure 17 The tumor volume change (A) and body weight change map (B) of C57 mice with pancreatic cancer after intravenous administration. DETAILED DESCRIPTION

[0059] In order to make the above-mentioned objects, technical solutions and advantages of the present application clearer, further detailed description will be given to the present application combined with examples and drawings, the illustrative embodiments of the present application and the description thereof are only used to explain the present application, and do not limit the present application.

[0060] Unless otherwise specified, the raw materials used in the examples are commercially purchased.

[0061] Example 1, synthesis of intermediate PTX-Q

[0062] PTX (3.0 g, 3.51 mmol, 1.0 eq) was dissolved in super dry DMF (10 ml), 5- hexynoic acid (433 mg, 3.86 mmol, 1.1 eq), EDCI (2.86 g, 14 mmol, 4.0 eq), DMAP (429 mg, 3.51 mmol, 1.0 eq) were weighed and stirred at room temperature for 10 h. TLC was used to track the reaction, after completion of the reaction, column chromatography was used to isolate PTX-Q (2.6 g, 78%),1H NMR spectrum is shown in Figure 3 . 1 HNMR (400 MHz, Chloroform-d) δ 8.13 (dt, J = 7.0, 1.4 Hz, 2H), 7.77 - 7.72 (m, 2H), 7.64 - 7.58 (m, 1H), 7.55 - 7.48 (m, 3H), 7.45 - 7.32 (m, 7H), 6.92 (d, J = 9.2 Hz, 1H), 6.31 - 6.21 (m, 2H), 5.96 (dd, J = 9.2, 3.1 Hz, 1H), 5.68 (d, J = 7.1 Hz, 1H), 5.50 (d, J = 3.1 Hz, 1H), 4.97 (dd, J = 9.7, 2.3 Hz, 1H), 4.44 (dd, J = 11.0, 6.6 Hz, 1H), 4.31 (d, J = 8.5 Hz, 1H), 4.20 (dd, J = 8.4, 1.1 Hz, 1H), 3.81 (d, J = 7.0 Hz, 1H), 2.63 - 2.51 (m, 3H), 2.46 (s, 3H), 2.38 (dd, J = 15.4, 9.3 Hz, 1H), 2.22 (d, J = 1.6 Hz, 3H), 1.98 (t, J = 2.6 Hz, 1H), 1.94 (d, J = 1.4 Hz, 3H), 1.83 (q, J = 7.0 Hz, 3H), 1.65 (s, 3H), 1.43 - 1.33 (m, 1H), 1.29 - 1.20 (m, 6H), 1.13 (s, 3H), 0.07 (s, 1H).

[0063] Example 2, synthesis of intermediate Boc-Val-Cit-PAB

[0064] Boc-Val-cit (5.06 g, 13.51 mmol, 1 eq) was dissolved in a mixture of MeOH (60 ml) and CH2Cl2(30 ml), p-aminobenzyl alcohol (2.0 g, 16.22 mmol, 1.2 eq) and EEDQ (6.68 g, 27.03 mmol, 2.0 eq) were added and stirred at room temperature overnight in the dark. TLC was used to track the reaction, after completion of the reaction, column chromatography was used to isolate Boc-Val-cit-PAB (3.3 g, 51%), 1H NMR (400 MHz, DMSO-d6) δ 7.98 (d, J = 7.7 Hz, 1H), 7.56 - 7.50 (m, 2H), 7.26 - 7.21 (m, 2H), 6.78 (d, J = 8.9 Hz, 1H), 5.97 (t, J = 5.9 Hz, 1H), 5.42 (s, 2H), 5.11 (t, J = 5.7 Hz, 1H), 4.43 (d, J = 5.8 Hz, 2H), 4.11 (q, J = 5.3 Hz, 1H), 3.17 (d, J = 5.3 Hz, 3H), 2.98 (dp, J = 31.9, 6.6 Hz, 2H), 1.96 (q, J = 6.8 Hz, 1H), 1.72 - 1.55 (m, 2H), 1.39 (s, 9H), 0.84 (dd, J = 16.1, 6.7 Hz, 6H).1H NMR spectrum is shown in Figure 4

[0065] Example 3, synthesis of intermediate N3-Val-Cit-PAB

[0066] Boc-Val-Cit-PAB (3.3 g, 6.88 mmol, 1.0 eq) was weighed into dry DCM (10 ml) and trifluoroacetic acid (20 ml) was added. The reaction was stirred at room temperature for 2.5 h. The reaction was followed by TLC and upon completion, it was evaporated to dryness. Val-Cit-PAB (2.3 g, 6.00 mmol, 1.0 eq) was dissolved in a mixture of dry DCM (5 ml) and MeOH (10 ml) and compound azidopentanoic acid (1.03 g, 7.2 mmol, 1.2 eq) and EEDQ (2.96 g, 12.0 mmol, 2.0 eq) were added. The reaction was stirred at room temperature for 10 h in the dark. The reaction was followed by TLC and upon completion, it was purified by column chromatography to give N3-Val-Cit-PAB (1.93 mg, 64%). 1 ​HNMR (400 MHz, DMSO-d6) δ 9.94 (d, J = 16.0 Hz, 1H), 8.10 (d, J = 7.5 Hz, 1H), 8.08 - 8.00 (m, 1H), 7.88 (d, J = 8.6 Hz, 1H), 7.60 (d, J = 4.2 Hz, 1H), 7.58 - 7.52 (m, 2H), 7.24 (d, J = 8.3 Hz, 2H), 5.98 (s, 1H), 5.42 (s, 1H), 4.45 - 4.36 (m, 3H), 4.21 (dd, J = 8.7, 6.8 Hz, 1H), 3.17 (s, 1H), 2.98 (dq, J = 26.0, 6.5 Hz, 2H), 2.27 - 2.15 (m, 2H), 1.97 (dt, J = 13.4, 6.7 Hz, 1H), 1.68 (d, J = 8.7 Hz, 1H), 1.61 - 1.46 (m, 5H), 1.42 - 1.32 (m, 2H), 1.28 - 1.21 (m, 1H), 0.90 - 0.81 (m, 6H). Hydrogen spectrum as shown in Figure 5

[0067] Example 4, synthesis of N3-Val-Cit-PAB-T785

[0068] N3-Val-Cit-PAB (100 mg, 0.19 mmol, 1.0 eq) was weighed into dry DMF (4 ml) and triethylamine (0.5 ml) was added. Npcl (119 mg, 0.59 mmol, 3.0 eq) was dissolved in dry DCM (1 ml) and the DCM solution was added dropwise to the DMF solution in an ice bath. The reaction was stirred at room temperature for 4 h. T785 (59 mg, 0.19 mmol, 1.0 eq) was weighed into a centrifuge tube, dissolved in DMF (1 ml) and DIPEA (0.5 ml) was added. The solution was added to the reaction mixture and stirred at room temperature overnight. The reaction was followed by TLC and after completion of the reaction, column chromatography was used to isolate N3-Val-Cit-PAB-T785 (90 mg, 56%). 1 ​H NMR (400 MHz, DMSO-d6): δ = 10.12 (s, 1H), 8.17 (d, J = 7.5 Hz, 1H), 8.06 (d, J = 8.3 Hz, 1H), 7.95 (d, J = 8.6 Hz, 1H), 7.70 (dd, J = 8.4, 1.3 Hz, 1H), 7.61 (d, J = 8.4 Hz, 1H), 7.57 - 7.48 (m, 2H), 7.36 (t, J = 7.7 Hz, 1H), 7.27 (t, J = 5.7 Hz, 1H), 7.24 (d, J = 8.4 Hz, 2H), 6.17 (s, 1H), 5.50 (s, 2H), 4.91 (s, 2H), 4.52 (t, J = 7.7 Hz, 2H), 4.43 - 4.38 (m, 1H), 4.21 (t, J = 8.7 Hz, 1H), 3.99 (q, J = 7.1 Hz, 1H), 3.30 (t, J = 6.4 Hz, 1H), 3.16 (s, 1H), 3.12 - 2.96 (m, 4H), 2.92 (t, J = 7.8 Hz, 1H), 2.35 - 2.12 (m, 2H), 2.05 - 1.91 (m, 1H), 1.82 - 1.71 (m, 4H), 1.65 - 1.29 (m, 10H), 1.27 - 1.10 (m, 4H), 0.94 (t, J = 7.4 Hz, 3H), 0.85 (dd, J = 11.4, 6.7 Hz, 6H);13C NMR (101 MHz, DMSO-d6): δ = 172.6, 171.8, 171.1, 159.5, 156.7, 155.0, 150.8, 139.1, 133.8, 132.3, 129.0, 128.1, 125.9, 123.4, 123.2, 121.1, 119.4, 114.2, 65.4, 60.4, 58.2, 53.5, 50.8, 49.1, 45.8, 45.2, 34.9, 31.8, 30.8, 30.0, 29.8, 29.7, 29.5, 29.5, 29.5, 29.3, 29.2, 29.2, 29.0, 28.6, 28.2, 27.5, 27.2, 27.0, 26.8, 26.6, 23.0, 22.6, 22.4, 19.7, 19.7, 18.7, 18.6, 15.1, 14.4, 14.3, 8.9; LR-ESI-MS: m / z calcd. for [4+H]+: 842.48, found m / z = 842.60; m / z calcd. for [4+2H]2+: 421.74, found m / z = 421.90. The1H NMR spectrum is shown in Figure 6 .

[0069] Example 5, synthesis of PTX-Val-Cit-PAB-T785

[0070] N3-Val-Cit-PAB-T785 (50 mg, 0.059 mmol, 1.0 eq) and PTX-Q (112 mg, 0.118 mg, 2.0 eq) were weighed into DMF (2 ml), TBTA (157 mg, 0.29 mmol, 5.0 eq) was weighed into DMF (1 ml), CuSO4(74 mg, 0.29 mmol, 5.0 eq) was weighed into H2O (1 ml), and sodium ascorbate (117 mg, 0.59 mmol, 10.0 eq) was weighed into H2O (1 ml), and added to the reaction solution, respectively, and stirred at 40 °C overnight. The reaction was tracked by TLC, and after the reaction was completed, column chromatography was used to separate PTX-Val-Cit-PAB-T785 (43 mg, 40.9%). 1H NMR (400 MHz, DMSO-d6) δ 10.04 (s, 1H), 9.28 (d, J = 8.4 Hz, 1H), 8.14 (d, J = 7.5 Hz, 1H), 8.02 (d, J = 8.1 Hz, 1H), 7.99 - 7.94 (m, 2H), 7.89 (d, J = 8.6 Hz, 1H), 7.86 - 7.82 (m, 2H), 7.72 (d, J = 7.9 Hz, 2H), 7.68 - 7.63 (m, 3H), 7.58 (d, J = 8.3 Hz, 2H), 7.55 - 7.51 (m, 1H), 7.49 - 7.41 (m, 7H), 7.30 - 7.21 (m, 4H), 7.17 (p, J = 4.3 Hz, 1H), 6.28 (s, 1H), 6.06 (t, J = 5.9 Hz, 1H), 5.80 (t, J = 9.0 Hz, 1H), 5.53 (t, J = 8.8 Hz, 1H), 5.46 - 5.33 (m, 4H), 4.91 (q, J = 8.0, 5.8 Hz, 4H), 4.64 (s, 1H), 4.50 (t, J = 7.6 Hz, 2H), 4.39 (t, J = 7.4 Hz, 1H), 4.23 (dt, J = 24.2, 7.9 Hz, 3H), 4.13 - 4.07 (m, 1H), 4.03 - 3.96 (m, 2H), 3.57 (d, J = 7.1 Hz, 1H), 3.01 (dd, J = 13.2, 6.6 Hz, 3H), 2.96 - 2.88 (m, 3H), 2.63 - 2.57 (m, 2H), 2.24 (s, 3H), 2.22 - 2.14 (m, 2H), 2.09 (s, 3H), 2.03 - 1.93 (m, 2H), 1.90 (s, 2H), 1.87 - 1.82 (m, 2H), 1.80 - 1.76 (m, 7H), 1.72 (d, J = 7.6 Hz, 2H), 1.59 (td, J = 14.6, 5.4 Hz, 5H), 1.49 (s, 5H), 1.44 (dd, J = 10.4, 4.7 Hz, 4H), 1.34 (d, J = 6.1 Hz, 3H), 1.29 (s, 2H), 1.23 (dd, J = 9.7, 3.4 Hz, 9H), 1.00 (d, J = 11.4 Hz, 6H), 0.94 (t, J = 7.4 Hz, 3H), 0.85 - 0.80 (m, 6H). Hydrogen spectrum as shown in Figure 7

[0071] Example 6, synthesis of N3-Val-Cit-PAB-MMAE

[0072] ​Vac-Cit-PAB-MMAE (150 mg, 0.13 mmol, 1.0 eq) was dissolved in super dry DMF (2 mL), then compound azido valeric acid (22 mg, 0.15 mmol, 2.0 eq), EDCI (101 mg, 0.53 mmol, 5.0 eq) and DMAP (16 mg, 0.13 mmol, 1.0 eq) were added, and stirred at 40 °C for 10 h. The reaction was tracked by TLC, and after the reaction was completed, column chromatography was used to isolate N3-Vac-Cit-PAB-MMAE (121 mg, 72.7 %). 1H NMR (400 MHz, DMSO-d6): δ = 10.01 (br, 1H), 8.33 (br, 1H), 8.13 (d, J = 7.6 Hz, 1H), 7.92-7.87 (m, 1H), 7.66-7.58 (m, 3H), 7.35-7.24 (m, 6H), 7.20–7.16 (m, 1H), 5.99 (t, J = 5.9 Hz, 1H), 5.43 (d, J = 4.9 Hz, 2H), 5.22-4.88 (m, 2H), 4.69 (br, 1H), 4.54-4.34 (m, 3H), 4.33-4.16 (m, 2H), 4.12-3.88 (m, 2H), 3.78 (dd, J = 9.4, 2.3 Hz, 1H), 3.58 (br, 2H), 3.53-3.41 (m, 1H), 3.31 (d, J = 6.16 Hz, 2H), 3.25-3.16 (m, 8H), 3.12 (s, 2H), 3.06-2.93 (m, 4H), 2.89-2.84 (m, 3H), 2.42 (d, J = 16.0 Hz, 1H), 2.37-2.18 (m, 4H), 2.16-2.06 (m, 2H), 2.03-1.91 (m, 2H), 1.87-1.66 (m, 4H), 1.54 (m, 6H), 1.44-1.20 (m, 5H), 1.17-0.94 (m, 6H), 0.91-0.63 (m, 21H);13C NMR (101 MHz, DMSO-d6): δ = 172.8, 172.7, 172.5, 171.7, 171.1, 170.3, 170.3, 169.2, 159.3, 144.1, 139.1, 132.2, 128.6, 128.3, 128.2, 127.2, 127.1, 126.9, 126.9, 119.4, 119.2, 85.9, 82.1, 79.1, 75.2, 66.5, 61.4, 60.7, 59.1, 58.6, 58.0, 57.6, 57.6, 55.4, 54.6, 53.6, 50.9, 50.8, 50.2, 49.6, 47.7, 46.7, 44.2, 43.6, 35.5, 34.9, 32.3, 32.0, 30.9, 30.4, 29.8, 28.2, 27.3, 25.8, 24.8, 23.6, 23.0, 19.7, 19.4, 19.2, 19.0, 18.8, 18.7, 16.3, 16.1, 15.9, 15.7, 15.5, 10.9, 10.8; LR-ESI-MS: m / z calcd. for [2+H]+: 1248.77, found m / z = 1248.80; m / z calcd. for [2+2H]2+: 624.89, found m / z = 624.80. Hydrogen spectrum as shown. Figure 8

[0073] Example 7, Synthesis of PTX-Val-Cit-PAB-MMAE

[0074] N3-Val-Cit-PAB-MMAE (120 mg, 0.096 mmol, 1.0 eq) and PTX-Q (182 mg, 0.19 mmol, 2.0 eq) were weighed into DMF (2 ml), TBTA (254 mg, 0.48 mmol, 5.0 eq) was weighed into DMF (2 ml), CuSO4(119 mg, 0.29 mmol, 5.0 eq) was weighed into H2O (1 ml), and sodium ascorbate (190 mg, 0.96 mmol, 10.0 eq) was weighed into H2O (1 ml), and added to the reaction mixture, respectively. The reaction was stirred at 40 °C overnight. TLC was used to track the reaction, and after the reaction was completed, column chromatography was used to isolate PTX-N3-Val-Cit-PAB-MMAE (97 mg, 46%). 1 ​H NMR (400 MHz, DMSO-d6) δ 10.03 (s, 1H), 9.26 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 7.3 Hz, 1H), 8.00 - 7.96 (m, 2H), 7.93 - 7.88 (m, 1H), 7.87 - 7.82 (m, 2H), 7.72 (d, J = 7.0 Hz, 2H), 7.68 - 7.63 (m, 2H), 7.61 - 7.52 (m, 3H), 7.49 - 7.41 (m, 6H), 7.33 - 7.27 (m, 5H), 7.25 (dd, J = 7.8, 2.2 Hz, 2H), 7.18 (td, J = 6.7, 6.0, 3.0 Hz, 3H), 6.29 (s, 1H), 6.01 (s, 1H), 5.80 (dd, J = 7.6, 2.8 Hz, 1H), 5.54 (d, J = 8.9 Hz, 1H), 5.44 - 5.41 (m, 2H), 5.39 - 5.34 (m, 2H), 5.04 (s, 1H), 4.95 - 4.91 (m, 2H), 4.64 (s, 1H), 4.50 - 4.46 (m, 1H), 4.49 - 4.34 (m, 3H), 4.27 (dd, J = 8.6, 6.2 Hz, 2H), 4.21 (dd, J = 8.8, 6.9 Hz, 2H), 4.14 - 4.07 (m, 2H), 4.05 - 3.93 (m, 4H), 3.58 (d, J = 7.1 Hz, 2H), 3.23 (d, J = 6.3 Hz, 3H), 3.18 (d, J = 10.5 Hz, 3H), 3.12 (s, 2H), 2.97 (s, 2H), 2.89 - 2.86 (m, 2H), 2.84 (d, J = 4.5 Hz, 1H), 2.61 (t, J = 7.6 Hz, 2H), 2.24 (s, 3H), 2.12 (s, 1H), 2.10 (s, 4H), 2.00 - 1.95 (m, 2H), 1.78 - 1.76 (m, 3H), 1.49 (s, 4H), 1.31 (s, 2H), 1.05 - 1.01 (m, 5H), 0.77 - 0.74 (m, 5H). Hydrogen spectrum as shown in Figure 9

[0075] Example 8, synthesis of N3-Val-Cit-PAB-AZD

[0076] ​N3-Val-Cit-PAB (100 mg, 0.19 mmol, 1.0 eq) was weighed into dry DMF (4 ml) and triethylamine (0.5 ml) was added. Npcl (119 mg, 0.59 mmol, 3.0 eq) was dissolved in dry DCM (1 ml) and the DCM solution was added dropwise to the DMF solution in an ice bath. The reaction was stirred at room temperature for 4 h. AZD (68 mg, 0.19 mmol, 1.0 eq) was weighed into a centrifuge tube, dissolved in DMF (1 ml) and DIPEA (0.5 ml) was added. This was added to the reaction mixture after the powder had dissolved and the reaction was stirred at room temperature overnight. The reaction was followed by TLC and after completion the product was isolated by column chromatography to give N3-Val-Cit-PAB-AZD (90 mg, 53%). 1 H NMR (400 MHz, DMSO-d6) δ 10.05 (s, 1H), 8.30 (s, 1H), 8.19 - 7.77 (m, 3H), 7.54 - 7.41 (m, 3H), 7.32 - 7.21 (m, 3H), 6.72 - 6.66 (m, 1H), 6.06 (t, J = 6.0 Hz, 1H), 5.45 (s, 1H), 5.09 - 4.93 (m, 2H), 4.45 - 4.35 (m, 1H), 4.24 - 4.18 (m, 1H), 3.94 (ddd, J = 35.0, 10.5, 5.2 Hz, 2H), 3.79 (d, J = 9.4 Hz, 1H), 3.44 - 3.40 (m, 1H), 3.12 - 2.88 (m, 3H), 2.80 - 2.68 (m, 2H), 2.21 (dq, J = 18.0, 7.4 Hz, 2H), 2.01 - 1.65 (m, 5H), 1.61 - 1.45 (m, 6H), 1.35 (d, J = 11.9 Hz, 6H), 0.89 - 0.80 (m, 7H).1H NMR spectrum is shown in Figure Figure 10

[0077] Example 9, synthesis of PTX-Val-Cit-PAB-AZD

[0078] ​N3-Val-Cit-PAB-AZD (50 mg, 0.056 mmol, 1.0 eq) and PTX-Q (106 mg, 0.112 mmol, 2.0 eq) were dissolved in DMF (1 ml), TBTA (148 mg, 0.27 mmol, 5.0 eq) was dissolved in DMF (1 ml), CuSO4(69 mg, 0.27 mmol, 5.0 eq) was dissolved in H2O (1 ml), sodium ascorbate (110 mg, 0.55 mmol, 10.0 eq) was dissolved in H2O (1 ml), and then added to the reaction solution, respectively, and stirred at 40 °C overnight. The reaction was tracked by TLC, and after the reaction was completed, column chromatography was used to separate PTX-Val-Cit-PAB-AZD (52 mg, 50.9%). 1H NMR (400 MHz, DMSO-d6) δ 10.09 (s, 1H), 9.31 (d, J = 8.4 Hz, 1H), 8.30 (s, 1H), 8.16 (d, J = 7.5 Hz, 1H), 8.04 (d, J = 7.6 Hz, 1H), 8.00 - 7.97 (m, 2H), 7.91 (d, J = 8.7 Hz, 1H), 7.88 - 7.84 (m, 2H), 7.73 (d, J = 7.8 Hz, 2H), 7.69 - 7.60 (m, 4H), 7.57 - 7.51 (m, 2H), 7.49 (q, J = 1.5 Hz, 2H), 7.46 (d, J = 4.3 Hz, 6H), 7.30 (d, J = 8.2 Hz, 2H), 7.25 - 7.21 (m, 1H), 7.20 - 7.16 (m, 1H), 6.71 - 6.66 (m, 1H), 6.30 (s, 1H), 6.08 (d, J = 5.8 Hz, 1H), 5.82 (t, J = 9.0 Hz, 1H), 5.54 (t, J = 8.8 Hz, 1H), 5.47 - 5.38 (m, 4H), 5.01 (d, J = 3.7 Hz, 2H), 4.94 (dd, J = 9.3, 4.5 Hz, 2H), 4.65 (s, 1H), 4.39 (dt, J = 7.9, 4.0 Hz, 1H), 4.27 (t, J = 7.0 Hz, 2H), 4.23 - 4.19 (m, 1H), 4.13 (dd, J = 10.7, 6.8 Hz, 2H), 4.04 - 3.98 (m, 2H), 3.93 - 3.86 (m, 1H), 3.77 (d, J = 7.4 Hz, 1H), 3.59 (d, J = 7.1 Hz, 1H), 3.19 - 3.14 (m, 2H), 2.97 (ddt, J = 26.1, 13.0, 6.5 Hz, 3H), 2.77 (tt, J = 12.1, 5.0 Hz, 2H), 2.65 - 2.60 (m, 2H), 2.46 (dd, J = 7.4, 2.5 Hz, 2H), 2.25 (s, 4H), 2.11 (d, J = 5.9 Hz, 5H), 2.00 - 1.94 (m, 2H), 1.86 (t, J = 7.4 Hz, 3H), 1.81 - 1.75 (m, 6H), 1.61 (td, J = 11.9, 3.5 Hz, 4H), 1.50 (s, 4H), 1.47 - 1.43 (m, 4H), 1.40 - 1.38 (m, 4H), 1.36 (q, J = 2.1 Hz, 2H), 1.30 (s, 2H), 1.26 (s, 2H), 1.23 (d, J = 2.6 Hz, 4H), 1.01 (d, J = 11.2 Hz, 6H), 0.86 - 0.81 (m, 6H). The1H NMR spectrum is shown in Figure Figure 11 .

[0079] Example 10, synthesis of PTX-Val-Ala-PAB-T785

[0080] Val-Ala-PAB (3.0 g, 10.2 mmol, 1.0 eq) was dissolved in a mixture of super dry DCM (10 mL) and MeOH (20 ml), then compound azido valeric acid (12.2 mmol, 1.75 g, 1.2 eq) and EEDQ (5.0 g, 20.4 mmol, 2.0 eq) were added, stirred at room temperature for 10 hours in the dark, TLC tracking reaction, after the reaction was completed, column chromatography separation to obtain N3-Val-Ala-PAB (3.4 g, 79.8%).

[0081] N3-Val-Ala-PAB (200 mg, 0.47 mmol, 1.0 eq) was weighed into super dry DMF (4 mL), then triethylamine (0.5 ml) was added, Npcl (283.4 mg, 1.41 mmol, 3.0 eq) was dissolved in super dry DCM (2 ml), the DCM solution was added dropwise to the DMF in an ice bath, and the reaction was carried out at room temperature for 4 h, T785 (146.1 mg, 0.47 mmol, 1.0 eq) was weighed into a centrifuge tube, dissolved in DMF (1 ml), and DIPEA (0.5 ml) was added, and after the powder was dissolved, it was added to the reaction solution, and stirred at room temperature overnight. TLC tracking reaction, after the reaction was completed, column chromatography separation to obtain N3-Val-Ala-PAB-T785 (186 mg, 52.5%).

[0082] N3-Val-Ala-PAB-T785 (186 mg, 0.24 mmol, 1.0 eq) and PTX-Q (233 mg, 0.48 mg, 2.0 eq) were weighed into DMF (2 mL), TBTA (636 mg, 1.2 mmol, 5.0 eq) was weighed into DMF (1 ml), CuSO4 (298 mg, 1.2 mmol, 5.0 eq) was dissolved in H2O (2 ml), and sodium ascorbate (475 mg, 2.4 mmol, 10.0 eq) was dissolved in H2O (1 ml), and added to the reaction solution, respectively, stirred at 40°C overnight. TLC tracking reaction, after the reaction was completed, column chromatography separation to obtain PTX-Val-Ala-PAB-T785 (207 mg, 50.7%). 1H NMR (500 MHz, Chloroform-d) δ 8.18 (s, 1H), 8.17 - 8.13 (m, 2H), 8.10 (s, 1H), 7.76 - 7.71 (m, 2H), 7.67 (s, 1H), 7.64 - 7.57 (m, 2H), 7.55 - 7.46 (m, 3H), 7.45 - 7.37 (m, 5H), 7.35 - 7.26 (m, 6H), 7.22 - 7.08 (m, 4H), 7.03 (s, 1H), 6.85 (s, 2H), 6.54 (p, J = 1.0 Hz, 1H), 6.23 - 6.09 (m, 2H), 5.82 - 5.73 (m, 1H), 5.70 (s, 1H), 5.22 (s, 1H), 5.15 - 5.02 (m, 2H), 4.91 (s, 1H), 4.62 (d, J = 6.0 Hz, 1H), 4.47 (s, 1H), 4.29 (s, 1H), 4.25 - 4.16 (m, 4H), 4.15 - 4.10 (m, 2H), 3.97 (d, J = 4.9 Hz, 1H), 3.87 (s, 1H), 3.09 (d, J = 7.7 Hz, 3H), 2.98 - 2.81 (m, 4H), 2.50 (s, 3H), 2.41 - 2.14 (m, 11H), 1.94 (s, 2H), 1.89 (d, J = 0.9 Hz, 3H), 1.87 - 1.73 (m, 5H), 1.71 - 1.61 (m, 5H), 1.60 - 1.36 (m, 9H), 1.18 (d, J = 24.9 Hz, 6H), 0.90 (s, 3H), 0.77 (d, J = 24.9 Hz, 6H). The procedure of synthesis is shown in Figure 12

[0083] Example 11, Synthesis of PTX-Gly-Gly-Phe-Gly-T785

[0084] Boc-Gly-Gly-Phe-Gly (4.0 g, 9.17 mmol, 1 eq) was dissolved in a mixture of MeOH (60 ml) and CH2Cl2(30 mL), p-aminobenzyl alcohol (1.35 g, 11.0 mmol, 1.2 eq) and EEDQ (4.52 g, 18.3 mmol, 2.0 eq) were added, and stirred at room temperature overnight in the dark. TLC tracking of the reaction, after the reaction was completed, column chromatography was used to separate to obtain Boc-Gly-Gly-Phe-Gly-PAB (2.9 g, 59.1%).

[0085] ​Boc-Gly-Gly-Phe-Gly-PAB (2.9 g, 5.36 mmol, 1.0 eq) was dissolved in super dry DCM (10 mL), trifluoroacetic acid (20 ml) was added, and the mixture was stirred at room temperature for 2.5 h. The reaction was tracked by TLC, and after the reaction was completed, it was concentrated to obtain Gly-Gly-Phe-Gly-PAB. Gly-Gly-Phe-Gly-PAB was dissolved in a mixture of super dry DCM (5 mL) and MeOH (10 ml), and compound azido valeric acid (6.4 mmol, 0.91 g, 1.2 eq) and EEDQ (2.63 g, 10.7 mmol, 2.0 eq) were added, and the mixture was stirred at room temperature for 10 h in the dark. The reaction was tracked by TLC, and after the reaction was completed, column chromatography was used to separate N3-Gly-Gly-Phe-Gly-PAB (2.1 g, 70%).

[0086] N3-Gly-Gly-Phe-Gly-PAB (150 mg, 0.26 mmol, 1.0 eq) was dissolved in super dry DMF (4 mL), and triethylamine (0.5 ml) was added. Npcl (156 mg, 0.78 mmol, 3.0 eq) was dissolved in super dry DCM (1 ml), and the DCM solution was added dropwise to the DMF solution in an ice bath. The reaction was stirred at room temperature for 4 h. T785 (80 mg, 0.26 mmol, 1.0 eq) was weighed in a centrifuge tube, dissolved in DMF (1 ml), and DIPEA (0.5 ml) was added. After the powder was dissolved, it was added to the reaction solution, and the mixture was stirred at room temperature overnight. The reaction was tracked by TLC, and after the reaction was completed, column chromatography was used to separate N3-Gly-Gly-Phe-Gly-PAB-T785 (112 mg, 47%).

[0087] N3-Gly-Gly-Phe-Gly-PAB-T785 (50 mg, 0.054 mmol, 1.0 eq) and PTX-Q (112 mg, 0.108 mg, 2.0 eq) were dissolved in DMF (2 mL). TBTA (143 mg, 0.27 mmol, 5.0 eq) was dissolved in DMF (1 ml), CuSO4 (67 mg, 0.27 mmol, 5.0 eq) was dissolved in H2O (1 ml), and sodium ascorbate (106.9 mg, 0.54 mmol, 10.0 eq) was dissolved in H2O (1 ml). The above solutions were added to the reaction solution, and the mixture was stirred at 40°C overnight. The reaction was tracked by TLC, and after the reaction was completed, column chromatography was used to separate PTX-Gly-Gly-Phe-Gly-T785 (47 mg, 46.6%). 1H NMR (500 MHz, Chloroform-d) δ 8.18 (s, 1H), 8.16 (d, J = 2.0 Hz, 1H), 8.14 (d, J = 2.0 Hz, 1H), 8.03 (d, J = 3.1 Hz, 2H), 8.00 (s, 1H), 7.97 - 7.96 (m, 2H), 7.77 - 7.70 (m, 2H), 7.67 (s, 1H), 7.72 - 7.56 (m, 3H), 7.55 - 7.47 (m, 3H), 7.44 (s, 1H), 7.42 - 7.38 (m, 4H), 7.35 - 7.29 (m, 5H), 7.29 (s, 1H), 7.25 - 7.19 (m, 7H), 7.17 (s, 1H), 7.11 (s, 1H), 7.03 (s, 1H), 6.54 (p, J = 1.0 Hz, 1H), 6.21 (d, J = 1.0 Hz, 1H), 5.88 - 5.57 (m, 2H), 5.22 (s, 1H), 5.04 - 4.83 (m, 3H), 4.62 (d, J = 6.0 Hz, 1H), 4.30 - 3.95 (m, 12H), 3.89 - 3.75 (m, 3H), 3.37 (dt, J = 12.3, 0.7 Hz, 1H), 3.30 (d, J = 12.3 Hz, 1H), 3.18 (d, J = 12.4 Hz, 1H), 3.08 (s, 1H), 2.98 - 2.82 (m, 5H), 2.50 (s, 3H), 2.40 - 2.31 (m, 4H), 2.29 (d, J = 13.0 Hz, 1H), 2.24 (s, 1H), 2.19 (d, J = 8.8 Hz, 5H), 1.94 (s, 2H), 1.89 (d, J = 1.0 Hz, 3H), 1.84 - 1.75 (m, 4H), 1.72 - 1.60 (m, 7H), 1.57 (s, 2H), 1.39 (s, 2H), 1.18 (d, J = 24.9 Hz, 6H), 0.90 (s, 3H). The procedure of synthesis is as Figure 13 indicated.

[0088] Example 12, Preparation of Albumin Paclitaxel Nanoparticle (Nab-PTX)

[0089] First, an aqueous solution of human serum albumin was prepared at a concentration of 14.5 mg / ml; 1.65 mg of PTX-Val-Cit-PAB-T785 was dissolved in a mixed solvent consisting of 52.2 μL of chloroform and 5.8 μL of ethanol to form an organic phase. Then, 58 μL of the organic phase was added to 1 ml of the aqueous solution. To obtain the nanosuspension, we used a probe sonicator at 650 W, equipped with a 2 mm amplitude rod and set at 40% amplitude, and processed it continuously for 6 min, with 15 s of pause after each 30 s of sonication. Subsequently, the resulting nanosuspension was placed in a rotary evaporator and evaporated at 40°C and 533 mbar for 30 min; finally, the evaporated solution was ultrafiltered at 4000 rpm for 15 min using a 100 KD ultrafilter tube to obtain the nanoparticle complex Nab-PTX-VC-T785.

[0090] Example 13, Preparation of Albumin-PTX-Val-Cit-PAB-T785 Nanoparticles

[0091] First, an aqueous solution of human serum albumin was prepared at a concentration of 14.5 mg / ml; 1.65 mg of PTX-Val-Cit-PAB-T785 was dissolved in a mixed solvent consisting of 52.2 μL of chloroform and 5.8 μL of ethanol to form an organic phase. Then, 58 μL of the organic phase was added to 1 ml of the aqueous solution. To obtain the nanosuspension, we used a probe sonicator at 650 W, equipped with a 2 mm amplitude rod and set at 40% amplitude, and processed it continuously for 6 min, with 15 s of pause after each 30 s of sonication. Subsequently, the resulting nanosuspension was placed in a rotary evaporator and evaporated at 40°C and 533 mbar for 30 min; finally, the evaporated solution was ultrafiltered at 4000 rpm for 15 min using a 100 KD ultrafilter tube to obtain the nanoparticle complex Nab-PTX-VC-T785.

[0092] Example 14, Preparation of Albumin-PTX-Val-Cit-PAB-MMAE Nanoparticles

[0093] Firstly, an aqueous solution of human serum albumin was prepared with a concentration of 14.5 mg / ml; 2.06 mg of PTX-Val-Cit-PAB-MMAE was dissolved in a mixed solvent consisting of 52.2 μL of chloroform and 5.8 μL of ethanol to form an organic phase. Then 58 μL of the organic phase was added to 1 ml of the aqueous solution. To obtain the nanosuspension, we used a probe sonicator with a power of 650 W, equipped with an amplitude rod of 2 mm and set at an amplitude of 40%, and processed continuously for 6 minutes, with a pause of 15 seconds for each 30 seconds of sonication. Subsequently, the nanosuspension obtained was placed in a rotary evaporator, under the control of a vacuum of 533 mbar at 40°C for 30 minutes; finally, the solution after rotary evaporation was ultrafiltered with a 100 KD ultrafilter at 4000 rpm for 15 minutes to obtain the single drug nanoparticle complex Nab-PTX-VC-MMAE.

[0094] Example 15, Preparation of Albumin-PTX-Val-Cit-PAB-AZD Nanoparticles

[0095] Firstly, an aqueous solution of human serum albumin was prepared with a concentration of 14.5 mg / ml; 2.06 mg of PTX-Val-Cit-PAB-MMAE was dissolved in a mixed solvent consisting of 52.2 μL of chloroform and 5.8 μL of ethanol to form an organic phase. Then 58 μL of the organic phase was added to 1 ml of the aqueous solution. To obtain the nanosuspension, we used a probe sonicator with a power of 650 W, equipped with an amplitude rod of 2 mm and set at an amplitude of 40%, and processed continuously for 6 minutes, with a pause of 15 seconds for each 30 seconds of sonication. Subsequently, the nanosuspension obtained was placed in a rotary evaporator, under the control of a vacuum of 533 mbar at 40°C for 30 minutes; finally, the solution after rotary evaporation was ultrafiltered with a 100 KD ultrafilter at 4000 rpm for 15 minutes to obtain the single drug nanoparticle complex Nab-PTX-VC-MMAE.

[0096] Example 16, Preparation of Albumin-PTX-Val-Ala-PAB-T785 Nanoparticles

[0097] Firstly, an aqueous solution of human serum albumin was prepared with a concentration of 14.5 mg / ml; 1.58 mg PTX-Val-Ala-PAB-T785 was dissolved in a mixed solvent consisting of 52.2 μL chloroform and 5.8 μL ethanol to form an organic phase. Then 58 μL of the organic phase was added to 1 ml of the aqueous solution. To obtain the nanosuspension, we used a probe sonicator with a power of 650 W, equipped with an amplitude rod of 2 mm and set at an amplitude of 40%, and processed continuously for 6 minutes, with a pause of 15 seconds after each 30 seconds of sonication. Subsequently, the resulting nanosuspension was placed in a rotary evaporator under the control of a vacuum of 533 mbar at 40°C for 30 minutes; finally, the solution after rotary evaporation was ultrafiltered with a 100 KD ultrafilter at 4000 rpm for 15 minutes to obtain the single drug nanoparticle complex Nab-PTX-VA-T785.

[0098] Example 17, Preparation of Albumin-PTX-Gly-Gly-Phe-Gly-T785 Nanoparticles

[0099] Firstly, an aqueous solution of human serum albumin was prepared with a concentration of 14.5 mg / ml; 1.58 mg PTX-Val-Ala-PAB-T785 was dissolved in a mixed solvent consisting of 52.2 μL chloroform and 5.8 μL ethanol to form an organic phase. Then 58 μL of the organic phase was added to 1 ml of the aqueous solution. To obtain the nanosuspension, we used a probe sonicator with a power of 650 W, equipped with an amplitude rod of 2 mm and set at an amplitude of 40%, and processed continuously for 6 minutes, with a pause of 15 seconds after each 30 seconds of sonication. Subsequently, the resulting nanosuspension was placed in a rotary evaporator under the control of a vacuum of 533 mbar at 40°C for 30 minutes; finally, the solution after rotary evaporation was ultrafiltered with a 100 KD ultrafilter at 4000 rpm for 15 minutes to obtain the single drug nanoparticle complex Nab-PTX-VA-T785.

[0100] Example 18, Preparation of Albumin-PTX-Val-Cit-PAB-T785@AZD Nanoparticles

[0101] Firstly, prepare the human serum albumin aqueous solution with the concentration of 14.5 mg / ml; dissolve 0.835 mg PTX-Val-Cit-PAB-T785 and 0.86 mg PTX-Val-Cit-PAB-ZAD in the mixed solvent consisting of 52.2 μL chloroform and 5.8 μL ethanol to form the organic phase. Then add 58 μL of the organic phase to 1 ml of the aqueous solution. In order to obtain the nanosuspension, we use the probe sonicator with the power of 650 W, equipped with a 2 mm amplitude rod and set the amplitude to 40%, and continuously treat for 6 minutes according to the rule of 30 seconds of ultrasonic treatment and 15 seconds of pause. Subsequently, we place the obtained nanosuspension on the rotary evaporator under the conditions of 40°C and 533 mbar vacuum control for 30 minutes; finally, the solution after rotary evaporation is ultrafiltered by 100 KD ultrafiltration tube at 4000 rpm for 15 minutes to obtain the dual-drug nanoparticle composite Nab-PTX-VC-T785@ZAD.

[0102] Example 19, Preparation of Albumin-PTX-Val-Cit-PAB-T785@MMAE Nanoparticles

[0103] Firstly, prepare the human serum albumin aqueous solution with the concentration of 14.5 mg / ml; dissolve 0.835 mg PTX-Val-Cit-PAB-T785 and 1.03 mg PTX-Val-Cit-PAB-MMAE in the mixed solvent consisting of 52.2 μL chloroform and 5.8 μL ethanol to form the organic phase. Then add 58 μL of the organic phase to 1 ml of the aqueous solution. In order to obtain the nanosuspension, we use the probe sonicator with the power of 650 W, equipped with a 2 mm amplitude rod and set the amplitude to 40%, and continuously treat for 6 minutes according to the rule of 30 seconds of ultrasonic treatment and 15 seconds of pause. Subsequently, we place the obtained nanosuspension on the rotary evaporator under the conditions of 40°C and 533 mbar vacuum control for 30 minutes; finally, the solution after rotary evaporation is ultrafiltered by 100 KD ultrafiltration tube at 4000 rpm for 15 minutes to obtain the dual-drug nanoparticle composite Nab-PTX-VC-T785@MMAE.

[0104] Example 20, Preparation of Albumin-PTX-Val-Cit-PAB-MMAE@AZD Nanoparticles

[0105] Firstly, prepare a human serum albumin aqueous solution with a concentration of 14.5 mg / ml; dissolve 0.86 mg PTX-Val-Cit-PAB-T785 and 1.03 mg PTX-Val-Cit-PAB-MMAE in a mixed solvent consisting of 52.2 μL chloroform and 5.8 μL ethanol to form an organic phase. Then add 58 μL of the organic phase to 1 ml of the aqueous solution. To obtain a nanosuspension, we use a 650 W probe-type ultrasonic disrupter, equipped with a 2 mm amplitude rod and set to 40% amplitude, and continuously process for 6 minutes according to the rule of 30 seconds of ultrasonic treatment and 15 seconds of pause. Subsequently, we place the obtained nanosuspension on a rotary evaporator and rotary evaporate for 30 minutes under the control of a vacuum of 533 mbar at 40°C; finally, we ultrafilter the rotary evaporated solution with a 100 KD ultrafilter tube at 4000 rpm for 15 minutes to obtain the dual-drug nanoparticle composite Nab-PTX-VC-MMAE@AZD.

[0106] Example 21, Preparation of Albumin-PTX-Val-Cit-PAB-AZD@MMAE@T785 Nanoparticles

[0107] Firstly, prepare a human serum albumin aqueous solution with a concentration of 14.5 mg / ml; dissolve 0.86 mg PTX-Val-Cit-PAB-T785 and 1.03 mg PTX-Val-Cit-PAB-MMAE in a mixed solvent consisting of 52.2 μL chloroform and 5.8 μL ethanol to form an organic phase. Then add 58 μL of the organic phase to 1 ml of the aqueous solution. To obtain a nanosuspension, we use a 650 W probe-type ultrasonic disrupter, equipped with a 2 mm amplitude rod and set to 40% amplitude, and continuously process for 6 minutes according to the rule of 30 seconds of ultrasonic treatment and 15 seconds of pause. Subsequently, we place the obtained nanosuspension on a rotary evaporator and rotary evaporate for 30 minutes under the control of a vacuum of 533 mbar at 40°C; finally, we ultrafilter the rotary evaporated solution with a 100 KD ultrafilter tube at 4000 rpm for 15 minutes to obtain the dual-drug nanoparticle composite Nab-PTX-VC-MMAE@AZD.

[0108] Experimental Example 1, Hydrated Particle Size and Surface Potential of Albumin Paclitaxel Derivative Twin-Drug Nanoparticles

[0109] The nanoparticles obtained in the above Examples 12-21 were diluted 10 times with deionized water, and the particle size and distribution and PDI of the albumin paclitaxel and albumin-paclitaxel conjugate nanoparticles were measured using a Malvern Zetasizer Nano ZS nanoparticle size potential instrument, and the surface potential was measured, and the specific results are shown in Table 1, wherein the hydrated particle size and surface potential of the nanoparticles Nab-PTX, Nab-PTX-VC-T785, Nab-PTX-VC-MMAE, Nab-PTX-VC-AZD, Nab-PTX-VA-T785, Nab-PTX-GGPG-T785, Nab-PTX-VC-T785@MMAE, Nab-PTX-VC-T785@ZAD, and Nab-PTX-VC-MMAE@AZD are shown in Table 1. Figure 14 .

[0110] Table 1 Hydrated particle size and surface potential of albumin paclitaxel derivative conjugate nanoparticles

[0111]

[0112]

[0113] The results show that, in addition to the hydrated particle size of Nab-PTX being 164.0 nm, the remaining albumin-bound paclitaxel derivative conjugate nanoparticles are between 90-160 nm; the PDI is less than 0.24, indicating that the nanoparticle particle size is uniform and the molecular weight distribution is uniform.

[0114] Experimental Example 2 Transmission electron microscopy results of albumin paclitaxel derivative conjugate nanoparticles

[0115] The TEM grid was placed on a TEM special filter paper, and an appropriate amount of albumin nanoparticle sample was taken with a pipette and dropped on the grid. In order to make the sample better deposited on the grid, the grid can be hydrophilic treated or the grid surface can be treated with an oxygen plasma cleaner. A suitable heavy metal salt solution is selected as a staining agent, such as phosphotungstic acid (PTA) or uranyl acetate, etc. Here, uranyl acetate (2%) was used as a staining agent. Finally, a biological transmission electron microscope was used to observe and take pictures of the morphology of the nanoparticles.

[0116] As shown in Figure 15 , the transmission electron microscopy results show that the synthesized nanoparticles of different paclitaxel derivative conjugates all exhibit spherical shape, uniform size, and stable structure.

[0117] Experimental Example 3 Drug loading rate of albumin paclitaxel derivative conjugate nanoparticles

[0118] The present example further analyzes the encapsulation efficiency and drug loading of paclitaxel derivative twin drugs PTX-VC-T785, PTX-VA-T785, PTX-GGPG-T785 in albumin paclitaxel derivative twin drug nanoparticles Nab-PTX-VC-T785, Nab-PTX-VA-T785 and Nab-PTX-GGPG-T785. 1.2 mg, 1.5 mg, 2 mg of PTX-VC-T785, 1.2 mg, 1.5 mg, 2 mg of PTX-VA-T785, 1.2 mg, 1.5 mg, 2 mg of PTX-GGPG-T785 were weighed and dissolved in 1 ml of DMSO, respectively, and detected by high performance liquid chromatography to prepare a standard curve; 1.65 mg of PTX-VC-T785, 1.58 mg of PTX-VA-T785, 1.35 mg of PTX-GGPG-T785 were weighed and albumin nanoparticles were prepared according to the methods of Examples 13, 16 and 17, respectively, 12.5 uL of the sample was added to DMSO, ultrasonic treatment was performed for 10 minutes, centrifugation was performed at 8000 rpm for 5 minutes, and the supernatant was measured for the mass of the drug in the nanoparticles.

[0119] Nab-PTX-VC-T785, Nab-PTX-VA-T785 and Nab-PTX-GGPG-T785 three kinds of nanoparticles, according to each kind of nanoparticle respectively take 50 uL and 100 uL, add DMSO constant volume to 500 uL, ultrasonic treatment for 10 minutes, centrifugation at 8000 rpm for 5 minutes, take supernatant and inject into high performance liquid chromatography for detection, the injection volume is 10 uL. Chromatographic conditions: the chromatographic column is Waters XBridge-C18 column (4.6 x 250 mm, 5 um), acetonitrile: water (65 / 35, v / v) is used as the mobile phase, the detection wavelength is 254 nm, and the column temperature is 40℃. The drug encapsulation efficiency is calculated by drug loading and encapsulation efficiency as follows:

[0120] Encapsulation efficiency (%) = mass of drug in nanoparticles / actual drug loading x 100%

[0121] Drug loading (%) = mass of drug in nanoparticles / total mass of nanoparticles x 100%

[0122] The results show that the total mass of the Nab-PTX-VC-T785 nanoparticles is 17.582, the mass of PTX-VC-T785 in the nanoparticles is 1.4 mg, and the encapsulation rate is calculated to be 84.8% and the drug loading is 7.96%; the total mass of the Nab-PTX-VA-T785 nanoparticles is 18.245, the mass of PTX-VA-T785 in the nanoparticles is 1.3 mg, and the encapsulation rate is calculated to be 82.27% and the drug loading is 7.12%; the total mass of the Nab-PTX-GGPG-T785 nanoparticles is 16.525, the mass of PTX-GGPG-T785 in the nanoparticles is 1.1 mg, and the encapsulation rate is calculated to be 81.5% and the drug loading is 6.65%.

[0123] Experimental Example 4, Cell toxicity assay

[0124] In order to evaluate the toxic effect of paclitaxel (PTX) combined with other anti-tumor drugs on BxPC-3 pancreatic cancer cells, this embodiment uses CCK8 method to conduct experiments. First, pancreatic cancer cells are inoculated into 96-well plates at a density of 1000 cells per well and cultured overnight under suitable conditions to ensure that the cells can adhere and reach a stable growth state. Then different concentrations of paclitaxel, different concentrations of anti-tumor drugs, paclitaxel (1 μM) + different concentrations of anti-tumor drugs are added to the wells respectively; 72 hours later, 10 μL of CCK8 working solution is added to each well; incubate in the cell incubator for 1-2 hours; use the enzyme label instrument to detect the absorbance value (450 nm) of each well and calculate the viability.

[0125] As shown in Figure 16 , the inhibition rate of paclitaxel double-drug combination on cancer cells is significantly improved compared with paclitaxel single drug and free single drug.

[0126] Experimental Example 5, In vivo anti-tumor effect of albumin-paclitaxel twin drug

[0127] In order to further evaluate the anti-tumor effect of PTX and different anti-tumor drug prepared twin drug nanoparticles in vivo, as well as the anti-tumor effect of different peptide linker prepared twin drug nanoparticles in vivo, KPC pancreatic cancer cell suspension is mixed uniformly with Matrigel at a ratio of 1:1, and then injected into the right axillary of C57BL / 6 mice, and when the tumor volume reaches 100 mm 3Around 1000 mice were randomly divided into groups of 6 each and subjected to the treatments shown in Table 2. The treatments in Table 2 were also compared with the antitumor effect of the albumin-paclitaxel twin drug Nab-PTX-SS-T785 in Chinese Patent Application No. CN117731796A, entitled "Preparation and Application of a Class A Albumin-Paclitaxel Twin Drug Nanoparticles" (Prior Document 1). Nab-PTX-SS-T785 twin drug nanoparticles were prepared according to the preparation method in Prior Document 1, and the treatment methods were the same as for the other treatment groups. Tumor volume and mouse weight were recorded every two days. The results are shown in Table 2. Figure 17 As shown.

[0128] Table 2. Medication categories and treatment methods for each group

[0129] Group Treatment Medicines and content (dose: 200 μL / time) PBS injection once every 3 days PBS Nab-PTX injection once every 3 days PTX: 15 mg / kg Nab-PTX-VC-MMAE injection once every 3 days PTX-VC-MMAE: 38.5 mg / kg Nab-PTX-VC-T785 injection once every 3 days PTX-VC-T785: 31.4 mg / kg Nab-PTX-VC-AZD injection once every 3 days PTX-VC-AZD: 32.3 mg / kg Nab-PTX-VA-T785 injection once every 3 days PTX-VA-T785: 29.8 mg / kg Nab-PTX-GGPG-T785 injection once every 3 days PTX-GGPG-T785: 30.5 mg / kg Nab-PTX-SS-T785 injection once every 3 days PTX-SS-T785: 5 mg / kg

[0130] like Figure 17 As shown, paclitaxel, when combined with various antitumor drugs to prepare twinned nanoparticles, exhibits a significant inhibitory effect on tumor growth. However, the body weight of tumor-bearing mice treated with different nanoparticles did not change significantly. Compared with the twinned nanoparticles in Reference Document 1, it can be seen that the tumor volume change of the present invention is smaller, regardless of whether the same peptide linker is connected to different antitumor drugs or different peptide linkers are connected to the same antitumor drug. This indicates that the twinned nanoparticles provided by the present invention have a better effect on inhibiting tumor growth in vivo. Meanwhile, the antitumor effects of nanoparticles prepared with different peptide linkers or nanoparticles linked with different antitumor drugs varied. Among them, the tumor volume did not change significantly from day 0 to day 24 after treatment with the three nanoparticles Nab-PTX-VC-T785, Nab-PTX-VA-T785, and Nab-PTX-GGPG-T785. The twin-drug nanoparticle Nab-PTX-VC-T785 showed the smallest tumor volume increase at day 24, indicating that Nab-PTX-VC-T785 had the best inhibitory effect on the growth of pancreatic cancer tumors.

[0131] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Albumin-paclitaxel conjugate nanoparticles, characterized in that, The application relates to a paclitaxel derivative conjugate and albumin, wherein the albumin encapsulates the paclitaxel derivative conjugate, the paclitaxel derivative conjugate is formed by connecting paclitaxel with a drug molecule through a peptide linker, the connecting unit of the peptide linker is selected from any one of valine-citrulline-p-aminobenzyloxy (Val-Cit-PAB), valine-alanine-p-aminobenzyloxy (Val-Ala-PAB) and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly), and the drug molecule comprises any one of T785, MMAE and AZD7762.

2. The albumin-paclitaxel conjugate nanoparticle of claim 1, wherein, The albumin comprises any one or more of bovine serum albumin, human serum albumin, ovalbumin and recombinant human serum albumin.

3. A method of preparing albumin-paclitaxel conjugate nanoparticles as claimed in any one of claims 1-2, wherein, The paclitaxel derivative conjugate is mixed with the albumin to prepare albumin-paclitaxel conjugate nanoparticles.

4. The production method according to claim 3, wherein The application comprises the following steps: (1) mixing human serum albumin with water to prepare an aqueous phase; (2) mixing a paclitaxel derivative conjugate with an organic solvent to prepare an organic phase; (3) mixing the aqueous phase with the organic phase, and then preparing albumin-paclitaxel conjugate nanoparticles through ultrasonic treatment, rotary evaporation and ultrafiltration.

5. Use of the albumin-paclitaxel conjugate nanoparticle of any one of claims 1-2 in the manufacture of a medicament for the treatment of cancer, wherein the albumin-paclitaxel conjugate nanoparticle is administered in combination with a chemotherapeutic agent. The cancer comprises cervical cancer, breast cancer, lung cancer, gastric cancer, melanoma, bladder cancer, intestinal cancer, prostate cancer, pancreatic cancer, esophageal cancer, neural cancer, ovarian cancer and renal cancer.

6. Use of albumin-paclitaxel conjugate nanoparticles as claimed in claim 5 for the preparation of a medicament for the treatment of cancer. The lung cancer is non-small cell lung cancer, and the neural cancer is brain cancer.

Images