Albumin-paclitaxel twin drug nanoparticles based on dipeptide connexon and application of albumin-paclitaxel twin drug nanoparticles

Abstract

The invention provides albumin-paclitaxel twin drug nanoparticles based on peptide linkers and application of the albumin-paclitaxel twin drug nanoparticles, the linker between paclitaxel and drug molecules is a dipeptide linker, the stability of the linker is higher, and the breakage speed in tumor microenvironment response is higher. The twin drug nanoparticles synthesized by different connexons between paclitaxel and drug molecules are compared and analyzed, and dipeptide connexons capable of effectively improving the anti-tumor effect of the albumin-paclitaxel twin drug nanoparticles are screened, so that efficient co-loading of multiple drugs is realized, accumulation of the drugs is increased, and indiscriminate killing of the drugs on normal cells is reduced; the target of treating and fading tumors is achieved, and the current curative effect bottleneck of malignant tumors is expected to be broken through.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical chemistry and biopharmaceutical technology, and specifically relates to the preparation and application of albumin-paclitaxel twin-drug nanoparticles based on peptide linkers. Background Art

[0002] Malignant tumors, commonly known as cancer, are one of the major public health problems facing the world and pose a huge threat to human health. According to data from the World Health Organization and cancer research institutions in various countries, the incidence and mortality of malignant tumors are high worldwide. Treatment usually 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. Chemotherapy drugs are largely limited in their clinical transformation due to their poor solubility, poor targeting, large toxic side effects and drug resistance.

[0003] As a new type of protein carrier, albumin nanoparticles have multiple 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 to achieve targeted delivery of drugs and further improve the efficacy.

[0004] Since many tumors have metabolic abnormalities and complex microenvironments, if the drug's mechanism of action is not comprehensive enough during monotherapy, it may not be able to effectively inhibit tumor growth and spread. Different drugs have different mechanisms of action, and some drugs may be more suitable for combined use to exert synergistic effects. For example, Abraxane has a good effect when used in combination with anti-tumor drugs such as carboplatin and gemcitabine. Although combination therapy can significantly improve the survival rate of patients and improve the drug resistance of single drug use, there are still many problems that need to be solved, such as low bioavailability of free drugs such as carboplatin and gemcitabine, poor targeting, and obvious toxic side effects.

[0005] Patent document CN117731796A discloses the preparation and application of a type of albumin-paclitaxel twin-drug nanoparticles. The application provides a method of constructing a type of paclitaxel twin-drug using paclitaxel as a target head and coupling it with drug molecules through covalent bonds, which then reacts with albumin to form uniform albumin-paclitaxel twin-drug nanoparticles. The comparison shows that the albumin-paclitaxel twin-drug nanoparticles connected with different drugs all have good anti-tumor effects. However, the albumin-paclitaxel twin-drug nanoparticles provided by the patent have limited ability to inhibit tumor growth, and have certain limitations in terms of drug release and therapeutic effects in the nanoparticles.

[0006] Therefore, providing a method that can effectively enhance the anti-tumor effect of albumin-paclitaxel twin-drug nanoparticles and further enhance the therapeutic effect of the drug is of great significance for breaking through the current therapeutic efficacy bottleneck of malignant tumors. Summary of the invention

[0007] In view of the problems existing in the prior art, the present invention provides albumin-paclitaxel twin-drug nanoparticles based on peptide linkers and applications thereof, compares nanoparticles synthesized with different linkers between paclitaxel and drug molecules, and screens out linkers that can effectively improve the anti-tumor effect of albumin-paclitaxel twin-drug nanoparticles, achieves efficient co-loading of multiple drugs, and successfully targets and delivers them to the tumor area, thereby achieving the purpose of treating and regressing the tumor, and is expected to break through the current efficacy bottleneck of malignant tumors.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] On the one hand, the present invention provides a paclitaxel-derived twin drug, which is composed of paclitaxel and a drug molecule connected by a peptide linker, and the connecting unit of the peptide linker is selected from any two or more of valine, citrulline, alanine, glycine, lysine, phenylalanine, glutamic acid, serine, and aspartic acid.

[0010] The paclitaxel-derived twin drug of the present invention is a chemical bond connecting paclitaxel and an anti-tumor drug through a tumor microenvironment-specific response, wherein the chemical bond is a dipeptide bond. The response groups of the peptide linker include but are not limited to valine-citrulline-p-aminobenzyloxy (Val-Cit-PAB), valine-alanine-p-aminobenzyloxy (Val-Ala-PAB), and glycine-glycine-phenylalanine-glycine (Gly-Gly-Phe-Gly).

[0011] The linker provided by the present invention is a peptide linker that can be cleaved by tissue proteases in tumor tissues. Peptide linkers usually have good stability and solubility, which helps to improve the overall stability of paclitaxel-derived twin drugs. At the same time, the structure and properties of peptide linkers are changed by design and selection to achieve precise regulation of paclitaxel-derived twin drugs and responsive release of anti-tumor drugs, reduce the toxic side effects of drugs on non-tumor cells, and thus improve the efficacy.

[0012] Furthermore, 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 linking unit of the peptide linker is selected from valine and citrulline.

[0013] Preferably, the drug molecule includes any one of Larotrectinib, MRYX1133, Capivasertib, mlN8237, di-ABZI, SRO7, T785, MMAE, and AZD7762, and the chemical structure formula of the above 9 drug molecules is as follows: Figure 1 shown.

[0014] Preferably, the paclitaxel-derived twin drug is synthesized by connecting paclitaxel and an anti-tumor drug with a peptide linker synthesized from valine and citrulline linking units, and its general chemical structure is shown in formula (1):

[0015]

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

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

[0018] In some embodiments, the albumin-paclitaxel twin-drug nanoparticles include 1, 2, or 3 paclitaxel-derived twin-drugs.

[0019] Furthermore, the albumin-paclitaxel twin-drug nanoparticles also include albumin, and the albumin encapsulates the paclitaxel-derived twin-drug.

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

[0021] On the other hand, the present invention provides a method for preparing a paclitaxel-derived twin drug, wherein paclitaxel, a peptide linker and a drug molecule are prepared by chemical reaction, wherein the linking unit of the peptide linker is selected from valine and citrulline.

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

[0023] In some embodiments, the steps of the synthesis method use a solvent, an alkaline reagent and a condensing agent, and the solvent 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, ether, and o-dichlorobenzene; the alkaline reagent is divided into an inorganic base and an organic base, and the inorganic base includes a metal hydroxide and an alkali metal, and the organic base includes triethylamine, tributylamine, trioctylamine, N,N-diisopropylethylamine, pyridine, 4-dimethylaminopyridine, piperidine, N-methylmorpholine, sodium tert-butoxide, potassium tert-butoxide, diisopropylamine , tetrahydropyrrole and triethylenediamine; the condensing agent includes one or more of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (HBTU) and O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU).

[0024] On the other hand, the present invention provides a method for preparing albumin-paclitaxel twin-drug nanoparticles, wherein the paclitaxel-derived twin-drug is mixed with albumin to prepare the albumin-paclitaxel twin-drug nanoparticles.

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

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

[0027] (2) mixing the paclitaxel-derived twin drug with an organic solvent to prepare an organic phase;

[0028] (3) After the aqueous phase and the organic phase are mixed, the albumin-paclitaxel twin-drug nanoparticles are prepared by ultrasound, rotary evaporation and ultrafiltration.

[0029] In some embodiments, the albumin concentration in the aqueous phase in step (1) is 14.5 mg / ml, and the molar concentration ratio of paclitaxel derivative twin drug: 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, and acetonitrile; and the ultrasonic instrument used in step (4) includes but is not limited to an ultrasonic crusher and an ultrasonic cleaner.

[0030] In some embodiments, in step (3), one, two or three different paclitaxel-derived twin-drug organic phase solutions may be added to the albumin solution, and albumin-paclitaxel twin-drug nanoparticles including single-drug, dual-drug or triple-drug may be prepared through subsequent steps.

[0031] The albumin-paclitaxel twin-drug nanoparticles obtained according to the preparation method provided by the present invention have an encapsulation rate of 80% to 85% for the paclitaxel-derived twin-drug and a drug loading of 6% to 8%.

[0032] Preferably, the encapsulation efficiency of the albumin-paclitaxel twin-drug nanoparticles for the paclitaxel-derived twin-drug PTX-VC-T785 is 84.8%, and the drug loading is 7.96%.

[0033] In another aspect, the present invention provides a use of a paclitaxel-derived twin drug in the preparation of a drug for treating cancer, wherein the paclitaxel-derived twin drug is composed of paclitaxel connected to a drug molecule via a peptide linker, and the connecting unit of the peptide linker is selected from valine and citrulline.

[0034] Furthermore, 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, neurological cancer, ovarian cancer, and kidney cancer.

[0035] On the other hand, the present invention provides a use of albumin-paclitaxel twin-drug nanoparticles in the preparation of drugs for treating cancer, wherein the albumin-paclitaxel twin-drug nanoparticles include a paclitaxel-derived twin-drug and albumin, wherein the paclitaxel-derived twin-drug is composed of paclitaxel connected to a drug molecule via a peptide linker, and the connecting unit of the peptide linker is selected from valine and citrulline.

[0036] Furthermore, the albumin-paclitaxel twin-drug nanoparticles are configured for oral administration, respiratory system administration, intravenous injection, intra-articular injection, intramuscular injection, or direct injection into a tumor or adjacent to the cancer.

[0037] Furthermore, 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, neurological cancer, ovarian cancer, and kidney cancer.

[0038] In some embodiments, the present invention verifies the difference in anti-tumor effect of paclitaxel-derived twin-drug nanoparticles synthesized by peptide linkers synthesized by different linking units through comparative experiments. The results show that twin-drug nanoparticles synthesized by peptide linkers composed of different linking units can effectively inhibit the growth of pancreatic cancer tumors, among which the twin-drug nanoparticles synthesized by peptide linkers synthesized by valine and citrulline have the best anti-tumor effect.

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

[0040] 1. Based on paclitaxel, the present invention designs a class of paclitaxel-derived twin drugs that can be cleaved by tissue proteases in tumor tissues, which can achieve responsive release of anti-tumor drugs, reduce the toxic side effects of drugs on non-tumor cells, and thus improve the efficacy.

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

[0042] Figure 1 It is the general formula diagram of drug molecular structure;

[0043] Figure 2 The synthetic route of the paclitaxel-derived twin drug PTX-VC-T785 of the present invention;

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

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

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

[0047] Figure 6 is the hydrogen spectrum of the compound N3-Val-cit-PAB-T785 of the present invention;

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

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

[0050] Fig. 9 is the hydrogen spectrum of the compound PTX-Val-cit-PAB-MMAE in the present invention;

[0051] Fig.10 is the hydrogen spectrum of the compound N3-Val-cit-PAB-AZD of the present invention;

[0052] Fig.11 It is the hydrogen spectrum of the compound PTX-Val-cit-PAB-AZD in the present invention;

[0053] Fig.12 The synthetic route of paclitaxel-derived twin drug PTX-VA-T785;

[0054] Fig.13 The synthetic route map for paclitaxel-derived twin drug PTX-GGPG-T785;

[0055] Fig.14 The particle size and surface potential diagram of the albumin-paclitaxel twin-drug nanoparticles of the present invention;

[0056] Fig.15 Transmission electron microscopy images of the albumin-paclitaxel twin-drug nanoparticles Nab-PTX (A), Nab-PTX-VC-T785 (B), Nab-PTX-VC-AZD (C), and Nab-PTX-VC-MMAE (D) of the present invention;

[0057] Fig.16 This is a cytotoxicity diagram of the combination of paclitaxel and anti-tumor drugs of the present invention;

[0058] Fig.17 Figure 2 shows the changes in tumor volume (A) and body weight (B) of pancreatic cancer-bearing C57 mice after intravenous administration. DETAILED DESCRIPTION

[0059] In order to make the above-mentioned objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with embodiments and drawings. The exemplary implementation modes of the present invention and their description are only used to explain the present invention and are not intended to limit the present invention.

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

[0061] Example 1: Synthesis of intermediate PTX-Q

[0062] PTX (3.0 g, 3.51 mmol, 1.0 eq) was weighed and dissolved in ultra-dry DMF (10 ml), and 5-hexynoic acid (433 mg, 3.86 mmol, 1.1 eq), EDCI (2.86 g, 14 mmol, 4.0 eq), and DMAP (429 mg, 3.51 mmol, 1.0 eq) were weighed and stirred at room temperature for 10 h. The reaction was followed by TLC. After the reaction was completed, PTX-Q (2.6 g, 78%) was separated by column chromatography. The hydrogen spectrum was shown in FIG. Figure 3 shown. 1 HNMR(400MHz,Chloroform-d)δ8.13(dt,J=7.0,1.4Hz,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=9.2Hz ,1H),6.31–6.21(m,2H),5.96(dd,J=9.2,3.1Hz,1H),5.68(d,J=7.1Hz,1H), 5.50(d,J=3.1Hz,1H),4.97(dd,J=9.7,2.3Hz,1H),4.44(dd,J=11.0,6.6Hz,1 H),4.31(d,J=8.5Hz,1H),4.20(dd,J=8.4,1.1Hz,1H),3.81(d,J=7.0Hz,1H) ,2.63–2.51(m,3H),2.46(s,3H),2.38(dd,J=15.4,9.3Hz,1H),2.22(d,J=1.6 Hz,3H),1.98(t,J=2.6Hz,1H),1.94(d,J=1.4Hz,3H),1.83(q,J=7.0Hz,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] Weigh Boc-Val-cit (5.06 g, 13.51 mmol, 1 eq) and dissolve it in a mixed solution of MeOH (60 ml) and CH2Cl2 (30 ml), add p-aminobenzyl alcohol (2.0 g, 16.22 mmol, 1.2 eq) and EEDQ (6.68 g, 27.03 mmol, 2.0 eq), and stir overnight at room temperature in the dark. TLC is used to track the reaction. After the reaction is completed, Boc-Val-cit-PAB (3.3 g, 51%) is separated by column chromatography. 1H NMR(400MHz,DMSO-d6)δ7.98(d,J=7.7Hz,1H),7.56–7.50(m,2H),7.26–7.21(m,2H),6.7 8(d,J=8.9Hz,1H),5.97(t,J=5.9Hz,1H),5.42(s,2H),5.11(t,J=5.7Hz,1H),4.43(d,J= 5.8Hz,2H),4.11(q,J=5.3Hz,1H),3.17(d,J=5.3Hz,3H),2.98(dp,J=31.9,6.6Hz,2H),1.96(q,J=6.8Hz,1H),1.72–1.55(m,2H),1.39(s,9H),0.84(dd,J=16.1,6.7Hz,6H). The hydrogen spectrum is shown in Figure 4 shown.

[0065] Example 3: Synthesis of intermediate N3-Val-Cit-PAB

[0066] Weigh Boc-Val-Cit-PAB (3.3 g, 6.88 mmol, 1.0 eq) and dissolve it in ultra-dry DCM (10 ml), then add trifluoroacetic acid (20 ml) and stir at room temperature for 2.5 h. TLC tracks the reaction. After the reaction is completed, spin dry to obtain Val-Cit-PAB. Val-Cit-PAB (2.3 g, 6.00 mmol, 1.0 eq) is dissolved in a mixed solution of ultra-dry DCM (5 ml) and MeOH (10 ml), then add compounds azidopentanoic acid (1.03 g, 7.2 mmol, 1.2 eq) and EEDQ (2.96 g, 12.0 mmol, 2.0 eq), stir at room temperature in the dark for 10 hours, track the reaction by TLC, and after the reaction is completed, column chromatography is used to separate and obtain N3-Val-Cit-PAB (1.93 mg, 64%). 1HNMR(400MHz,DMSO-d6)δ9.94(d,J=16.0Hz,1H),8.10(d,J=7.5Hz,1H),8.08–8.00(m,1H),7.88(d,J=8.6Hz,1H ),7.60(d,J=4.2Hz,1H),7.58–7.52(m,2H),7.24(d,J=8.3Hz,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). The hydrogen spectrum is shown in Figure 5 shown.

[0067] Example 4: Synthesis of N3-Val-Cit-PAB-T785

[0068] Weigh N3-Val-Cit-PAB (100 mg, 0.19 mmol, 1.0 eq) and dissolve it in ultra-dry DMF (4 ml), then add triethylamine (0.5 ml), dissolve Npcl (119 mg, 0.59 mmol, 3.0 eq) in ultra-dry DCM (1 ml), add the DCM solution dropwise to DMF in an ice bath, and react at room temperature for 4 hours, then weigh T785 (59 mg, 0.19 mmol, 1.0 eq) in a centrifuge tube, dissolve it in DMF (1 ml), and add DIPEA (0.5 ml), repeat the powder dissolution and add it to the reaction solution, and stir at room temperature overnight. TLC tracks the reaction, and after the reaction is completed, column chromatography is used to separate N3-Val-Cit-PAB-T785 (90 mg, 56%). 11H 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. The 1H NMR spectrum is as shown in Figure 6 shown below.

[0069] Example 5: Synthesis of PTX-Val-Cit-PAB-T785

[0070] Weigh N3-Val-Cit-PAB-T785 (50 mg, 0.059 mmol, 1.0 eq) and PTX-Q (112 mg, 0.118 mg, 2.0 eq) and dissolve them in DMF (2 ml), then weigh TBTA (157 mg, 0.29 mmol, 5.0 eq) and dissolve them in DMF (1 ml), CuSO4 (74 mg, 0.29 mmol, 5.0 eq) and dissolve them in H2O (1 ml), and sodium ascorbate (117 mg, 0.59 mmol, 10.0 eq) and dissolve them in H2O (1 ml), and add them to the reaction solution and stir overnight at 40°C. TLC is used to track the reaction. After the reaction is completed, PTX-Val-Cit-PAB-T785 (43 mg, 40.9%) is separated by column chromatography. 1H NMR (400MHz, DMSO-d6) δ10.04(s,1H),9.28(d,J=8.4Hz,1H),8.14(d,J=7.5Hz,1H),8.02(d,J=8.1Hz,1H),7.99–7.94(m,2H), 7.89(d,J=8.6Hz,1H),7.86–7.82(m,2H),7.72(d,J=7.9Hz,2H),7.68–7.63(m,3H),7.58(d,J=8.3Hz,2H),7.55–7.51(m,1H), 7.49–7.41(m,7H),7.30–7.21(m,4H),7.17(p,J=4.3Hz,1H),6.28(s,1H),6.06(t,J=5.9Hz,1H),5.80(t,J=9.0Hz,1H),5.53( t,J=8.8Hz,1H),5.46–5.33(m,4H),4.91(q,J=8.0,5.8Hz,4H),4.64(s,1H),4.50(t,J=7.6Hz,2H),4.39(t,J=7.4Hz,1H),4.2 3(dt,J=24.2,7.9Hz,3H),4.13–4.07(m,1H),4.03–3.96(m,2H),3.57(d,J=7.1Hz,1H),3.01(dd,J=13.2,6.6Hz,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=7.6Hz,2H),1.59(td,J=14.6,5.4Hz,5H),1.49(s,5H),1.44(dd,J=10.4,4.7Hz,4H),1.34(d,J=6.1Hz,3H),1.29(s,2H),1.23(dd,J=9.7,3.4Hz,9H),1.00(d,J=11.4Hz,6H),0.94(t,J=7.4Hz,3H),0.85–0.80(m,6H). The hydrogen spectrum is shown in Figure 7 shown.

[0071] Example 6: Synthesis of N3-Val-Cit-PAB-MMAE

[0072] Vac-Cit-PAB-MMAE (150 mg, 0.13 mmol, 1.0 eq) was weighed and dissolved in ultra-dry DMF (2 mL), and then azidopentanoic 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 hours. The reaction was followed by TLC, and after the reaction was completed, N3-Vac-Cit-PAB-MMAE (121 mg, 72.7%) was obtained by column chromatography. 1H NMR(400MHz,DMSO-d6):δ=10.01(br,1H),8.33(br,1H),8.13(d,J=7.6Hz,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=5.9Hz,1H),5.43(d,J=4.9Hz,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=9.4,2.3Hz,1H),3.58(br,2H),3.53-3.41(m,1H),3.31(d,J=6.16Hz,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=16.0Hz,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(101MHz,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 / zcalcd.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 shown.

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

[0074] Weigh N3-Val-Cit-PAB-MMAE (120 mg, 0.096 mmol, 1.0 eq) and PTX-Q (182 mg, 0.19 mmol, 2.0 eq) and dissolve them in DMF (2 ml), then weigh TBTA (254 mg, 0.48 mmol, 5.0 eq) and dissolve them in DMF (2 ml), CuSO4 (119 mg, 0.29 mmol, 5.0 eq) and dissolve them in H2O (1 ml), and sodium ascorbate (190 mg, 0.96 mmol, 10.0 eq) in H2O (1 ml), and add them to the reaction solution and stir overnight at 40°C. TLC is used to track the reaction. After the reaction is completed, PTX-N3-Val-Cit-PAB-MMAE (97 mg, 46%) is obtained by column chromatography. 1H NMR (400MHz, DMSO-d6) δ10.03(s,1H),9.26(d,J=8.5Hz,1H),8.14(d,J=7.3Hz,1H),8.00–7.96(m,2H),7.93–7.8 8(m,1H),7.87–7.82(m,2H),7.72(d,J=7.0Hz,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=7.8,2.2Hz,2H),7.18(td,J=6.7,6.0,3.0Hz,3H),6.29(s,1H),6.01(s,1H),5.80(dd, J=7.6,2.8Hz,1H),5.54(d,J=8.9Hz,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=8.6,6.2Hz,2H),4.21(dd,J=8.8,6.9Hz,2H),4.14 –4.07(m,2H),4.05–3.93(m,4H),3.58(d,J=7.1Hz,2H),3.23(d,J=6.3Hz,3H),3.18(d,J=10.5Hz,3H),3.12(s,2H ),2.97(s,2H),2.89–2.86(m,2H),2.84(d,J=4.5Hz,1H),2.61(t,J=7.6Hz,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). The hydrogen spectrum is shown in Fig. 9 shown.

[0075] Example 8: Synthesis of N3-Val-Cit-PAB-AZD

[0076] Weigh N3-Val-Cit-PAB (100 mg, 0.19 mmol, 1.0 eq) and dissolve it in ultra-dry DMF (4 ml), then add triethylamine (0.5 ml), dissolve Npcl (119 mg, 0.59 mmol, 3.0 eq) in ultra-dry DCM (1 ml), add the DCM solution dropwise to DMF in an ice bath, and react at room temperature for 4 hours, then weigh AZD (68 mg, 0.19 mmol, 1.0 eq) in a centrifuge tube, dissolve it in DMF (1 ml), and add DIPEA (0.5 ml), repeat the powder dissolution and add it to the reaction solution, and stir at room temperature overnight. TLC tracks the reaction, and after the reaction is completed, column chromatography is used to separate N3-Val-Cit-PAB-AZD (90 mg, 53%). 1 H NMR(400MHz,DMSO-d6)δ10.05(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=6.0Hz,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). The hydrogen spectrum is shown in Fig.10 shown.

[0077] Example 9: Synthesis of PTX-Val-Cit-PAB-AZD

[0078] Weigh N3-Val-Cit-PAB-AZD (50 mg, 0.056 mmol, 1.0 eq) and PTX-Q (106 mg, 0.112 mmol, 2.0 eq) and dissolve them in DMF (1 ml), then weigh TBTA (148 mg, 0.27 mmol, 5.0 eq) and dissolve them in DMF (1 ml), CuSO4 (69 mg, 0.27 mmol, 5.0 eq) and dissolve them in H2O (1 ml), and sodium ascorbate (110 mg, 0.55 mmol, 10.0 eq) and dissolve them in H2O (1 ml), and add them to the reaction solution and stir overnight at 40°C. TLC was used to track the reaction. After the reaction was completed, column chromatography was used to separate PTX-Val-Cit-PAB-AZD (52 mg, 50.9%). 11H 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). The 1H NMR spectrum is as shown in Fig.11 shown below.

[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 mixed solution of ultra-dry DCM (10 mL) and MeOH (20 ml), and then compounds azidovaleric acid (12.2 mmol, 1.75 g, 1.2 eq) and EEDQ (5.0 g, 20.4 mmol, 2.0 eq) were added. The mixture was stirred in the dark at room temperature for 10 hours and the reaction was monitored by TLC. After the reaction was completed, N3-Val-Ala-PAB (3.4 g, 79.8%) was obtained by column chromatography.

[0081] Weigh N3-Val-Ala-PAB (200 mg, 0.47 mmol, 1.0 eq) and dissolve it in ultra-dry DMF (4 mL), then add triethylamine (0.5 ml), dissolve Npcl (283.4 mg, 1.41 mmol, 3.0 eq) in ultra-dry DCM (2 ml), add the DCM solution dropwise to DMF in an ice bath, and react at room temperature for 4 h, weigh T785 (146.1 mg, 0.47 mmol, 1.0 eq) in a centrifuge tube, dissolve it in DMF (1 ml), and add DIPEA (0.5 ml), repeat the powder dissolution and add it to the reaction solution, and stir at room temperature overnight. TLC tracks the reaction, and after the reaction is completed, column chromatography is used to separate N3-Val-Ala-PAB-T785 (186 mg, 52.5%).

[0082] Weigh N3-Val-Ala-PAB-T785 (186 mg, 0.24 mmol, 1.0 eq) and PTX-Q (233 mg, 0.48 mg, 2.0 eq) and dissolve them in DMF (2 mL), then weigh TBTA (636 mg, 1.2 mmol, 5.0 eq) and dissolve them in DMF (1 ml), CuSO4 (298 mg, 1.2 mmol, 5.0 eq) and dissolve them in H2O (2 ml), and sodium ascorbate (475 mg, 2.4 mmol, 10.0 eq) and dissolve them in H2O (1 ml), and add them to the reaction solution and stir overnight at 40 ° C. TLC was used to track the reaction. After the reaction was completed, column chromatography was used to separate PTX-Val-Ala-PAB-T785 (207 mg, 50.7%). 1H NMR(500MHz,Chloroform-d)δ8.18(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=1.0Hz,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=6.0Hz,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=4.9Hz,1H),3.87(s,1H),3.09(d,J=7.7Hz,3H),2.98–2.81(m,4H),2.50(s,3H),2.4 1–2.14 (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 synthesis process is as follows Fig.12 shown.

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

[0084] Weigh Boc-Gly-Gly-Phe-Gly (4.0 g, 9.17 mmol, 1 eq) and dissolve it in a mixed solution of MeOH (60 ml) and CH2Cl2 (30 mL), add p-aminobenzyl alcohol (1.35 g, 11.0 mmol, 1.2 eq) and EEDQ (4.52 g, 18.3 mmol, 2.0 eq), and stir overnight at room temperature in the dark. TLC is used to track the reaction. After the reaction is completed, column chromatography is used to separate Boc-Gly-Gly-Phe-Gly-PAB (2.9 g, 59.1%).

[0085] Weigh Boc-Gly-Gly-Phe-Gly-PAB (2.9 g, 5.36 mmol, 1.0 eq) and dissolve it in ultra-dry DCM (10 mL), then add trifluoroacetic acid (20 ml), and stir at room temperature for 2.5 h. TLC is used to track the reaction. After the reaction is completed, spin dry to obtain Gly-Gly-Phe-Gly-PAB. Gly-Gly-Phe-Gly-PAB is dissolved in a mixed solution of ultra-dry DCM (5 mL) and MeOH (10 ml), and then compounds azidopentanoic acid (6.4 mmol, 0.91 g, 1.2 eq) and EEDQ (2.63 g, 10.7 mmol, 2.0 eq) are added. Stir for 10 hours at room temperature in the dark, and TLC is used to track the reaction. After the reaction is completed, column chromatography is used to separate and obtain N3-Gly-Gly-Phe-Gly-PAB (2.1 g, 70%).

[0086] Weigh N3-Gly-Gly-Phe-Gly-PAB (150 mg, 0.26 mmol, 1.0 eq) and dissolve it in ultra-dry DMF (4 mL), then add triethylamine (0.5 ml), dissolve Npcl (156 mg, 0.78 mmol, 3.0 eq) in ultra-dry DCM (1 ml), add the DCM solution dropwise to DMF in an ice bath, and react at room temperature for 4 h, weigh T785 (80 mg, 0.26 mmol, 1.0 eq) in a centrifuge tube, dissolve it in DMF (1 ml), and add DIPEA (0.5 ml), repeat the powder dissolution and add it to the reaction solution, and stir at room temperature overnight. TLC tracks the reaction, and after the reaction is completed, column chromatography is used to separate N3-Gly-Gly-Phe-Gly-PAB-T785 (112 mg, 47%).

[0087] Weigh 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) and dissolve them in DMF (2 mL), then weigh TBTA (143 mg, 0.27 mmol, 5.0 eq) and dissolve them in DMF (1 ml), CuSO4 (67 mg, 0.27 mmol, 5.0 eq) and dissolve them in H2O (1 ml), and sodium ascorbate (106.9 mg, 0.54 mmol, 10.0 eq) and dissolve them in H2O (1 ml), and add them to the reaction solution and stir at 40°C overnight. TLC was used to track the reaction. After the reaction was completed, column chromatography was used to separate PTX-Gly-Gly-Phe-Gly-T785 (47 mg, 46.6%). 1H NMR(500MHz,Chloroform-d)δ8.18(s,1H),8.16(d,J=2.0Hz,1H),8.14(d,J=2.0Hz,1H),8.03(d,J=3.1Hz ,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,3 H),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=1.0Hz,1H),6.21(d,J=1.0Hz,1H),5.88–5.57(m,2H),5.22(s,1H),5.04– 4.83(m,3H),4.62(d,J=6.0Hz,1H),4.30–3.95(m,12H),3.89–3.75(m,3H),3.37(dt,J=12.3,0.7Hz,1H), 3.30(d,J=12.3Hz,1H),3.18(d,J=12.4Hz,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 synthesis process is as follows. Fig.13 shown.

[0088] Example 12. Preparation of albumin paclitaxel nanoparticles (Nab-PTX)

[0089] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 0.8 mg paclitaxel 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of pausing for 15 seconds every 30 seconds of ultrasound. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered for 15 minutes using a 100KD ultrafiltration tube at 4000 rpm to obtain the nanoparticle complex Nab-PTX.

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

[0091] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 1.65 mg PTX-Val-Cit-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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of pausing for 15 seconds every 30 seconds of ultrasonication. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain a single drug nanoparticle complex Nab-PTX-VC-T785.

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

[0093] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 2.06 mg PTX-Val-Cit-PAB-MMAE 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of pausing for 15 seconds every 30 seconds of ultrasonication. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain a single drug nanoparticle complex Nab-PTX-VC-MMAE.

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

[0095] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 1.72 mg PTX-Val-Cit-PAB-AZD 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of pausing for 15 seconds every 30 seconds of ultrasound. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain a single drug nanoparticle complex Nab-PTX-VC-AZD.

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

[0097] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of pausing for 15 seconds every 30 seconds of ultrasound. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain a single drug nanoparticle complex Nab-PTX-VA-T785.

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

[0099] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 1.35 mg PTX-Gly-Gly-Phe-Gly-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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of pausing for 15 seconds every 30 seconds of ultrasound. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain a single drug nanoparticle complex Nab-PTX-GGPG-T785.

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

[0101] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 0.835 mg PTX-Val-Cit-PAB-T785 and 0.86 mg PTX-Val-Cit-PAB-ZAD were 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of 30 seconds of ultrasound and 15 seconds of pause. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40 ° C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain the dual-drug nanoparticle complex Nab-PTX-VC-T785@ZAD.

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

[0103] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 0.835 mg PTX-Val-Cit-PAB-T785 and 1.03 mg PTX-Val-Cit-PAB-MMAE were 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of 30 seconds of ultrasound and 15 seconds of pause. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40 ° C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain the dual-drug nanoparticle complex Nab-PTX-VC-T785@MMAE.

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

[0105] First, a 14.5 mg / ml human serum albumin aqueous solution was prepared; 0.86 mg PTX-Val-Cit-PAB-T785 and 1.03 mg PTX-Val-Cit-PAB-MMAE were 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set the amplitude to 40%, and treated it continuously for 6 minutes according to the rule of 30 seconds of ultrasound and 15 seconds of pause. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40 ° C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered at 4000 rpm for 15 minutes using a 100KD ultrafiltration tube to obtain the dual-drug nanoparticle complex Nab-PTX-VC-MMAE@AZD.

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

[0107] First, a 14.5 mg / ml aqueous solution of human serum albumin was prepared; 0.46 mg PTX-Val-Cit-PAB-T785, 0.69 mg PTX-Val-Cit-PAB-MMAE and 0.57 mg PTX-Val-Cit-PAB-AZD were 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 phase solution. In order to obtain the nanosuspension, we used a 650W probe ultrasonic crusher equipped with a 2 mm amplitude rod and set to 40% amplitude, and treated it continuously for 6 minutes according to the rule of 30 seconds of ultrasound and 15 seconds of pause. Then we placed the obtained nanosuspension on a rotary evaporator and rotary evaporated it for 30 minutes under vacuum control conditions of 40°C and 533 mbar; finally, the rotary evaporated solution was ultrafiltered using a 100KD ultrafiltration tube at 4000rpm for 15 minutes to obtain the three-drug nanoparticle complex Nab-PTX-VC-MMAE@T785@AZD.

[0108] Experimental Example 1: Hydration Particle Size and Surface Potential of Albumin-Paclitaxel-Derived Twin-Drug Nanoparticles

[0109] The nanoparticles obtained in Examples 12-21 were diluted 10 times with deionized water, respectively, and the particle size, distribution and PDI of albumin-paclitaxel and albumin-paclitaxel twin-drug nanoparticles were measured using a Malvern Zetasizer Nano ZS nanoparticle size potentiometer, and their surface potential was measured. The specific results are shown in Table 1, where 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. Fig.14 .

[0110] Table 1 Hydration particle size and surface potential of albumin-paclitaxel-derived twin-drug nanoparticles

[0111]

[0112]

[0113] The results showed that except for Nab-PTX with a hydrated particle size of 164.0 nm, the particle sizes of the other albumin-bound paclitaxel-derived twin drugs were between 90 and 160 nm; the PDIs were all less than 0.24, indicating that the nanoparticles had uniform particle size and uniform molecular weight distribution.

[0114] Experimental Example 2: Transmission electron microscopy results of albumin-paclitaxel-derived twin-drug nanoparticles

[0115] Place the TEM grid on the TEM filter paper, and use a pipette to draw an appropriate amount of albumin nanoparticle sample and drop it on the grid. In order to make the sample deposit on the grid better, the grid can be hydrophilized or the grid surface can be treated with an oxygen plasma cleaner. Choose a suitable heavy metal salt solution as a stain, such as phosphotungstic acid (PTA) or uranyl acetate. Here, uranyl acetate (2%) is used as a stain. Finally, use a biological transmission electron microscope to observe and photograph the morphology of the nanoparticles.

[0116] like Fig.15 As shown, transmission electron microscopy results show that the synthesized nanoparticles of different paclitaxel-derived twin drugs are spherical, uniform in size and stable in structure.

[0117] Experimental Example 3: Drug loading rate of albumin-paclitaxel-derived twin-drug nanoparticles

[0118] This example further analyzes the encapsulation efficiency and drug loading of paclitaxel-derived twin drugs PTX-VC-T785, PTX-VA-T785, and PTX-GGPG-T785 in the albumin paclitaxel-derived twin drug nanoparticles Nab-PTX-VC-T785, Nab-PTX-VA-T785, and Nab-PTX-GGPG-T785. 1.2 mg, 1.5 mg, and 2 mg of PTX-VC-T785, 1.2 mg, 1.5 mg, and 2 mg of PTX-VA-T785, and 1.2 mg, 1.5 mg, and 2 mg of PTX-GGPG-T785 were respectively weighed and dissolved in 1 ml of DMSO, 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, and 1.35 mg of PTX-GGPG-T785 were weighed and prepared according to the methods of Examples 13, 16, and 17, respectively, 12.5 uL of sample was added to DMSO, ultrasonicated for 10 minutes, and centrifuged at 8000 rpm for 5 minutes, and the supernatant was taken to measure the drug mass in the nanoparticles.

[0119] Nab-PTX-VC-T785, Nab-PTX-VA-T785 and Nab-PTX-GGPG-T785 are three kinds of nanoparticles. 50μL and 100μL of each nanoparticle were taken respectively, and DMSO was added to make the volume to 500uL. After ultrasonication for 10 minutes, centrifugation was performed at 8000rpm for 5 minutes, and the supernatant was taken and injected into high-performance liquid chromatography for detection. The injection volume was 10μL. Chromatographic conditions: The chromatographic column was a WatersXBridge-C18 column (4.6×250mm, 5μm), acetonitrile: water (65 / 35, v / v) was used as the mobile phase, the detection wavelength was 254nm, and the column temperature was 40°C. The encapsulation efficiency of the drug was calculated as follows: the drug loading and encapsulation rate:

[0120] Encapsulation efficiency (%) = drug mass in nanoparticles / actual dosage × 100%

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

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

[0123] Experimental Example 4: Cytotoxicity Assay

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

[0125] like Fig.16 As shown, the combination of paclitaxel and two drugs significantly improves the inhibition rate of cancer cells compared with paclitaxel alone or free single drug.

[0126] Experimental Example 5: In vivo antitumor effect of albumin-paclitaxel twin drugs

[0127] To further evaluate the in vivo antitumor effects of twin-drug nanoparticles prepared with PTX and different antitumor drugs, as well as the in vivo antitumor effects of twin-drug nanoparticles prepared with different peptide linkers, KPC pancreatic cancer cell suspension was mixed with matrix gel at a ratio of 1:1 and injected into the right axilla of C57BL / 6 mice. The tumor volume reached 100 mm 3The tumor-bearing mice were randomly divided into groups, with 6 mice in each group, and the treatments shown in Table 2 were performed respectively. The treatments shown in Table 2 were compared with the anti-tumor effect of albumin-paclitaxel twin drug Nab-PTX-SS-T785 in the Chinese patent with application number "CN117731796A" and invention name "Preparation and application of a type of albumin-paclitaxel twin drug nanoparticles" (comparative document 1). Nab-PTX-SS-T785 twin drug nanoparticles were prepared according to the preparation method of comparative document 1, and the treatment method was the same as that of other treatment groups. The tumor volume and mouse weight were recorded once every two days. The results are as follows Fig.17 shown.

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

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

[0130] like Fig.17 As shown, the twin-drug nanoparticles prepared from paclitaxel and a variety of anti-tumor drugs have a significant inhibitory effect on tumor growth, but there is no significant change in the weight of tumor-bearing mice after treatment with different nanoparticles. Compared with the twin-drug nanoparticles of comparative document 1, it can be seen that whether the same peptide linker is connected to different anti-tumor drugs or different peptide linkers are connected to the same anti-tumor drug, the volume change of the tumor provided by the present invention is smaller than that of comparative document 1, indicating that the twin-drug nanoparticles provided by the present invention have a better effect in inhibiting tumor growth in vivo. At the same time, there are certain differences in the anti-tumor effects of nanoparticles prepared by different peptide linkers or connected with different anti-tumor drugs. After treatment with the three nanoparticles Nab-PTX-VC-T785, Nab-PTX-VA-T785 and Nab-PTX-GGPG-T785, the tumor volume did not change significantly from the 0th day to the 24th day. The twin-drug nanoparticle Nab-PTX-VC-T785 had the smallest tumor volume increase on the 24th day, indicating that Nab-PTX-VC-T785 has the best inhibitory effect on the growth of pancreatic cancer tumors.

[0131] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A paclitaxel-derived twin drug, characterized in that: The paclitaxel-derived twin drug is composed of paclitaxel and a drug molecule connected by a peptide linker, and the connecting unit of the peptide linker is selected from a combination of any two or more of valine, citrulline, alanine, glycine, lysine, phenylalanine, glutamic acid, serine, and aspartic acid.

2. The paclitaxel-derived twin drug according to claim 1, characterized in that: 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 linking unit of the peptide linker is selected from valine and citrulline.

3. An albumin-paclitaxel twin-drug nanoparticle, characterized in that: Including the paclitaxel-derived twin drug as described in any one of claims 1-2.

4. The albumin-paclitaxel twin-drug nanoparticles according to claim 3, characterized in that: Also included is albumin, which encapsulates the paclitaxel-derived twin drug.

5. The albumin-paclitaxel twin-drug nanoparticles according to claim 4, characterized in that: The albumin includes any one or more of bovine serum albumin, human serum albumin, ovalbumin, and recombinant human serum albumin.

6. A method for preparing a paclitaxel-derived twin drug, characterized in that: Paclitaxel, a peptide linker and a drug molecule are prepared by chemical reaction, wherein the linking unit of the peptide linker is selected from valine and citrulline.

7. A method for preparing albumin-paclitaxel twin-drug nanoparticles, characterized in that: The paclitaxel-derived twin-drug nanoparticles are prepared by mixing the paclitaxel-derived twin-drug according to claim 1 or 2 with albumin.

8. The preparation method according to claim 7, characterized in that: The following steps are involved: (1) mixing human serum albumin with water to prepare an aqueous phase; (2) mixing the paclitaxel-derived twin drug with an organic solvent to prepare an organic phase; (3) After the aqueous phase and the organic phase are mixed, the albumin-paclitaxel twin-drug nanoparticles are prepared by ultrasound, rotary evaporation and ultrafiltration.

9. Use of a paclitaxel-derived twin drug in the preparation of a drug for treating cancer, characterized in that: The paclitaxel-derived twin drug is composed of paclitaxel connected to a drug molecule via a peptide linker, and the linking unit of the peptide linker is selected from valine and citrulline.

10. Use of albumin-paclitaxel twin-drug nanoparticles in preparing drugs for treating cancer, characterized in that: The albumin-paclitaxel twin-drug nanoparticles include a paclitaxel-derived twin-drug and albumin. The paclitaxel-derived twin-drug is composed of paclitaxel connected to a drug molecule via a peptide linker, and the linking unit of the peptide linker is selected from valine and citrulline.

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