Enzyme response self-assembly polypeptide prodrug loaded with camptothecin as well as preparation and application of enzyme response self-assembly polypeptide prodrug

Abstract

The invention discloses a camptothecin-loaded enzyme response self-assembly polypeptide prodrug as well as a preparation method and application of the camptothecin-loaded enzyme response self-assembly polypeptide prodrug. Belongs to the technical field of polypeptide prodrugs and preparation methods and application, the camptothecin-loaded enzyme response polypeptide prodrug is synthesized through a solid-phase organic synthesis reaction, the camptothecin-loaded enzyme response polypeptide prodrug can specifically recognize and shear with fibroblast activated protein alpha and carboxylesterase, in-situ self-assembly is carried out to form nanofibers, and a small-molecule chemotherapeutic drug camptothecin is released; the response type self-assembly polypeptide prodrug disclosed by the invention is simple to synthesize and high in stability; the polypeptide prodrug can be subjected to high-specificity in-situ self-assembly and camptothecin release under the action of fibroblast activated protein alpha and carboxylesterase overexpressed by tumor cells, so that efficient delivery and long retention of the drug to a tumor area are realized; the invention fills the blank in the aspects of design and preparation method of the polypeptide prodrug capable of responding to two enzyme markers to form a nano self-assembly and camptothecin slow release.

Description

Technical Field

[0001] The invention belongs to the technical field of polypeptide prodrugs, preparation methods and applications, and relates to an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin, and the preparation and application thereof. Background Art

[0002] Pancreatic cancer is a highly lethal malignancy with an overall five-year survival rate of less than 10%. Chemotherapy plays an important role in the clinical treatment of pancreatic cancer. Among the first-line chemotherapy drugs for pancreatic cancer, nuclear-acting drugs (such as gemcitabine, 5-fluorouracil, camptothecin, etc.) exert anti-tumor activity by affecting nucleic acid biosynthesis, damaging DNA or interfering with the transcription process. However, these drugs are often accompanied by serious off-target toxicity and drug resistance. Therefore, developing an effective drug delivery system to accurately deliver such drugs into tumor cells will be beneficial for their efficient function while reducing the off-target toxicity of the drugs. Enzyme-induced self-assembly (EISA) has become an ideal drug delivery method in cancer treatment by combining enzyme catalysis with supramolecular assembly. Enzymes overexpressed in cancer cells are used to trigger the specific self-assembly of peptide-drug conjugates at the tumor site to form nanostructures, thereby achieving selective delivery, accumulation or in situ activation of drugs to the tumor site. However, there is no research on the properties of enzyme-responsive self-assembling peptide prodrugs loaded with camptothecin. Summary of the invention

[0003] In view of the above problems, the present invention aims to provide a polypeptide prodrug loaded with camptothecin for tumor treatment, which has tumor-specific response, can be self-assembled in situ on tumors and can release camptothecin in situ.

[0004] Another object of the present invention is to provide a method for preparing an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin.

[0005] The third object of the present invention is to provide an application of an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin in tumor treatment.

[0006] The technical solution of the present invention is: the enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin described in the present invention has a chemical structure as shown below:

[0007]

[0008] Further, the preparation steps are as follows:

[0009] Step (1): adding camptothecin to a suitable organic solvent to make it well dissolved, then adding succinic anhydride and an acid-binding reagent thereto, and reacting overnight under the protection of an inert gas; purifying by chromatography to obtain camptothecin succinic acid, recorded as P1;

[0010] The inert gas in the synthesis step can be independently selected from: nitrogen, helium, argon, among which nitrogen and argon are preferred;

[0011] Step (2): 2-chlorotrityl chloride resin is swollen in an organic solvent, an L-phenylalanine derivative and an acid binding reagent are added to react, and the protecting group of phenylalanine is removed by using an alkaline lysis solution; an activated L-phenylalanine derivative and an acid binding reagent are added to react, and the protecting group of phenylalanine is removed by using an alkaline lysis solution; an activated L-lysine derivative and an acid binding reagent are added to react, and the protecting group of lysine is removed by using an alkaline lysis solution; an activated L-alanine derivative and an acid binding reagent are added to react, and the protecting group of alanine is removed by using an alkaline lysis solution; an activated L-proline derivative and an acid binding reagent are added to react, and the protecting group of proline acid is removed by using an alkaline lysis solution; an activated glycine derivative and an acid binding reagent are added to react, and the protecting group of glycine is removed by using an alkaline lysis solution; an activated L-threonine derivative and an acid binding reagent are added to react, and the protecting group of threonine is removed by using an alkaline lysis solution; and The activated L-lysine derivative and the acid-binding reagent are reacted, and the protecting group of lysine is removed by using an alkaline lysis solution; the activated L-threonine derivative and the acid-binding reagent are added to react, and the protecting group of threonine is removed by using an alkaline lysis solution; the activated L-aspartic acid derivative and the acid-binding reagent are added to react; the protecting group of the lysine side chain is then removed by using an alkaline lysis solution; the activated P1 and the acid-binding reagent are added to react overnight; then, the polypeptide is cut off from the resin using an acid-containing lysis solution, the product is precipitated using a poor solvent, the supernatant is discarded by freezing centrifugation, and the precipitate is purified by chromatography to obtain a polypeptide loaded with the chemotherapy drug camptothecin: tert-butyloxycarbonyl-aspartic acid (tert-butyloxy)-threonine (tert-butyloxy)-lysine (tert-butyloxycarbonyl)-threonine (tert-butyloxy)-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine, recorded as P2;

[0012] The poor solvents in the synthesis step include: petroleum ether, ethanol, isopropanol, diethyl ether, cyclohexane; among which: diethyl ether and cyclohexane are preferred;

[0013] Step (3): dissolving the activated polypeptide P2, 2-naphthylmethylamine and an acid-binding reagent in an organic solvent for reaction; purifying by chromatography to obtain the polypeptide: tert-butyloxycarbonyl-aspartic acid (tert-butyloxy)-threonine (tert-butyloxy)-lysine (tert-butyloxycarbonyl)-threonine (tert-butyloxy)-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine-naphthaleneacetic acid, denoted as P3;

[0014] Step (4): using an acid lysis solution to remove the protecting group on P3; purifying by chromatography to obtain an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin;

[0015] Furthermore, the organic solvent in the synthesis step can be independently selected from: methanol, isopropanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran; wherein dichloromethane, N,N-dimethylformamide or dimethyl sulfoxide is preferred;

[0016] The acid-binding reagent in the synthesis step can be independently selected from: triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, piperidine, pyridine;

[0017] Preferred among them: triethylamine, 4-dimethylaminopyridine;

[0018] The protecting group in the step can be independently selected from: tert-butoxy, tert-butoxycarbonyl, fluorenylmethoxycarbonyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl;

[0019] The alkaline lysis solution in the synthesis step can be independently selected from: piperidine / methanol (5-30%, v / v), piperidine / N,N-dimethylformamide (5-30%, v / v), piperidine / dichloromethane (5-30%, v / v), pyridine / dichloromethane (20-60%, v / v), pyridine / N,N-dimethylformamide (20-60%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-10%, v / v), hydrazine hydrate / methanol (1-10%, v / v);

[0020] Preferred among them are: piperidine / N,N-dimethylformamide (5-30%, v / v), piperidine / dichloromethane (5-30%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-10%, v / v);

[0021] The amino acid activation in the synthesis step refers to adding the following reagents, the reagents are selected from: O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazole-tetramethyluronium hexafluorophosphate, O-(5-chlorobenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate,

[0022] Among them, preferred are: 1-hydroxybenzotriazole, O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, O-benzotriazole-tetramethyluronium hexafluorophosphate;

[0023] The acid lysis solution in the synthesis step can be independently selected from: trifluoroacetic acid / methanol (1-99%, v / v), trifluoroacetic acid / N,N-dimethylformamide (1-99%, v / v), trifluoroacetic acid / dichloromethane (1-99%, v / v), wherein trifluoroacetic acid / N,N-dimethylformamide (1-99%, v / v), trifluoroacetic acid / dichloromethane (1-99%, v / v) are preferred;

[0024] Furthermore, the characteristic structures of the compounds P1, P2 and P3 synthesized above are:

[0025]

[0026] Furthermore, the use of a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug prepared by the preparation method in anti-tumor treatment;

[0027] The tumors include digestive system cancer, leukemia, lung cancer, bladder cancer, etc.

[0028] The beneficial effects of the present invention are as follows: the present invention designs and synthesizes a fibroblast activation protein α and a carboxylesterase-responsive polypeptide-camptothecin conjugate based on the strategy of tandem enzyme-controlled self-assembly and drug release to achieve precise delivery and sustained release of the chemotherapy drug camptothecin, and enhances the accumulation of the drug in the tumor area and the entry of camptothecin into the nucleus of cancer cells through the in situ assembly of nanofibers in cells, which is expected to enhance the chemotherapy effect of chemotherapy drugs; the present invention intends to develop a new type of polypeptide-camptothecin conjugate to improve the water solubility and stability of camptothecin, reduce its toxic side effects on normal tissues, and enhance the specific accumulation of the drug in tumor cells and specific intelligent sustained release. The polypeptide-camptothecin conjugate is simple to synthesize, and because the polypeptide contains a segment that can be specifically recognized and sheared by fibroblast activation protein α, the hydrophilic portion of the polypeptide can be slowly cleaved off by the fibroblast activation protein α, and converted into a relatively hydrophobic polypeptide-camptothecin conjugate and assembled into nanofibers in cells, thereby prolonging the residence time of the drug at the tumor site; in the present invention, the polypeptide and camptothecin are connected by an ester bond, which can be hydrolyzed by the action of carboxylesterase in the cell, so that camptothecin is slowly released from the polypeptide side chain, and the polypeptide fragment after enzymatic cleavage can promote camptothecin to enter the cell nucleus to exert a chemotherapy effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is the synthesis route diagram of the present invention;

[0030] Figure 2 is the mass spectrum of the pure compound P1 synthesized in Example 1 of the present invention;

[0031] Figure 3 is the mass spectrum of the pure compound P2 synthesized in Example 2 of the present invention;

[0032] Figure 4 is the mass spectrum of the pure compound P3 synthesized in Example 2 of the present invention;

[0033] Figure 5 is the mass spectrum of the pure compound P4 synthesized in Example 2 of the present invention;

[0034] Figure 6is the hydrogen nuclear magnetic resonance spectrum of the pure compound P4 synthesized in Example 2 of the present invention;

[0035] Figure 7 is the carbon NMR spectrum of the pure compound P4 synthesized in Example 2 of the present invention;

[0036] Figure 8 This is a schematic diagram of the transmission electron microscopy characterization results of the pure compound P4 synthesized in Example 3 of the present invention after the fibroblast activation protein α and carboxylesterase recognition and cleavage are completed in an in vitro environment;

[0037] Fig. 9 It is a schematic diagram of the results of HPLC analysis of the pure compound P4 synthesized in Example 3 of the present invention before and after the recognition and cleavage of fibroblast activation protein α and carboxylesterase in an in vitro environment. DETAILED DESCRIPTION

[0038] The specific technical scheme of the present invention is further described in detail below in conjunction with specific examples; specifically, as shown in the figure.

[0039] Example 1: Synthesis of pure compound P1 camptothecin succinate, wherein the structure of pure compound P1 is:

[0040]

[0041] The synthetic route of the first compound P1 in this example is as follows:

[0042] Add camptothecin to a suitable organic solvent to make it well dissolved, then add excess succinic anhydride and acid binding reagent thereto, and react overnight under the protection of inert gas; purify by chromatography to obtain camptothecin succinic acid, recorded as P1;

[0043] The organic solvent in the synthesis step can be independently selected from: methanol, isopropanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, among which dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide are preferred; the acid binding reagent in the synthesis step can be independently selected from: triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, piperidine, pyridine, among which triethylamine and 4-dimethylaminopyridine are preferred; the inert gas in the synthesis step can be independently selected from: nitrogen, helium, argon, among which nitrogen and argon are preferred;

[0044] The pure compound P1 was analyzed by electrospray ionization mass spectrometry using liquid chromatography-mass spectrometry to obtain Figure 2 The mass spectrum shown is:

[0045] Figure 2 is the mass spectrum of the first compound P1 synthesized in this example; Figure 2It can be seen that the mass spectrum result of the first compound P1 is obsvd.ESI-MS[(M+H) + ]:m / z 449.1.

[0046] Example 2: A method for synthesizing pure compounds P2: tert-butyloxycarbonyl-aspartic acid (tert-butyloxy)-threonine (tert-butyloxy)-lysine (tert-butyloxycarbonyl)-threonine (tert-butyloxy)-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine, P3: tert-butyloxycarbonyl-aspartic acid (tert-butyloxy)-threonine (tert-butyloxycarbonyl)-lysine (tert-butyloxycarbonyl)-threonine (tert-butyloxy)-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine-naphthaleneacetic acid, and P4: aspartic acid-threonine-lysine-threonine-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine-naphthaleneacetic acid, wherein the structures of pure compounds P2, P3 and P4 are respectively:

[0047]

[0048] The synthetic routes of compound P2, compound P3 and compound P4 in this example are as follows:

[0049] The 2-chlorotrityl chloride resin is swollen in an organic solvent, an L-phenylalanine derivative and an acid binding reagent are added to react, and the protecting group of phenylalanine is removed by using an alkaline lysis solution; an activated L-phenylalanine derivative and an acid binding reagent are added to react, and the protecting group of phenylalanine is removed by using an alkaline lysis solution; an activated L-lysine derivative and an acid binding reagent are added to react, and the protecting group of lysine is removed by using an alkaline lysis solution; an activated L-alanine derivative and an acid binding reagent are added to react, and the protecting group of alanine is removed by using an alkaline lysis solution; an activated L-proline derivative and an acid binding reagent are added to react, and the protecting group of proline acid is removed by using an alkaline lysis solution; an activated glycine derivative and an acid binding reagent are added to react, and the protecting group of glycine is removed by using an alkaline lysis solution; an activated L-threonine derivative and an acid binding reagent are added to react, and the protecting group of threonine is removed by using an alkaline lysis solution; an activated L-lysine derivative and acid binding reagent react, use alkaline lysis solution to remove the protecting group of lysine; add activated L-threonine derivative and acid binding reagent to react, use alkaline lysis solution to remove the protecting group of threonine; add activated L-aspartic acid derivative and acid binding reagent to react; then use alkaline lysis solution to remove the protecting group of lysine side chain; add activated P1 and acid binding reagent to react overnight; then, use acid lysis solution to cut the polypeptide from the resin, use poor solvent to precipitate the product, freeze centrifuge and discard the supernatant, and purify the precipitate by chromatography to obtain a polypeptide loaded with the chemotherapy drug camptothecin: tert-butyloxycarbonyl-aspartic acid (tert-butyloxy)-threonine (tert-butyloxy)-lysine (tert-butyloxycarbonyl)-threonine (tert-butyloxy)-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine, denoted as P2;

[0050] The poor solvents in the synthesis step include: petroleum ether, ethanol, isopropanol, diethyl ether, cyclohexane; among which: diethyl ether and cyclohexane are preferred;

[0051] The activated polypeptide P2, 2-naphthylmethylamine and an acid-binding agent are dissolved in an organic solvent for reaction; after purification by chromatography, a polypeptide is obtained: tert-butyloxycarbonyl-aspartic acid (tert-butyloxy)-threonine (tert-butyloxy)-lysine (tert-butyloxycarbonyl)-threonine (tert-butyloxy)-glycine-proline-alanine-lysine (camptothecin succinate)-phenylalanine-phenylalanine-naphthaleneacetic acid, denoted as P3;

[0052] Using an acid lysis solution to remove the protecting group on P3; after purification by chromatography, an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin is obtained;

[0053] Further, the organic solvent in the synthesis step can be independently selected from: methanol, isopropanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide, tetrahydrofuran, preferably: dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide; the acid binding reagent in the synthesis step can be independently selected from: triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, piperidine, pyridine, preferably: triethylamine, 4-dimethylaminopyridine; the protecting group in the step can be independently selected from: tert-butoxy, tert-butoxycarbonyl, fluorenylmethoxycarbonyl, 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl The alkaline lysis solution in the synthesis step can be independently selected from: piperidine / methanol (5-30%, v / v), piperidine / N,N-dimethylformamide (5-30%, v / v), piperidine / dichloromethane (5-30%, v / v), pyridine / dichloromethane (20-60%, v / v), pyridine / N,N-dimethylformamide (20-60%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-10%, v / v), hydrazine hydrate / methanol (1-10%, v / v) wherein preferably: piperidine / N,N-dimethylformamide (5-30%, v / v), piperidine / dichloromethane (5-30%, v / v), pyridine / dichloromethane (20-60%, v / v), pyridine / N,N-dimethylformamide (20-60%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-10%, v / v), hydrazine hydrate / methanol (1-10%, v / v) Chloromethane (5-30%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-10%, v / v); the amino acid activation in the synthesis step refers to the addition of the following reagents, the reagents are selected from: O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazole-tetramethyluronium hexafluorophosphate, O-(5-chlorobenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, etc., among which 1-hydroxybenzotriazole, O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, etc. are preferred. Fluorophosphate, O-benzotriazole-tetramethyluronium hexafluorophosphate; the acid lysis solution in the synthesis step can be independently selected from: trifluoroacetic acid / methanol (1-99%, v / v), trifluoroacetic acid / N,N-dimethylformamide (1-99%, v / v), trifluoroacetic acid / dichloromethane (1-99%, v / v), wherein trifluoroacetic acid / N,N-dimethylformamide (1-99%, v / v), trifluoroacetic acid / dichloromethane (1-99%, v / v) are preferred; the amidation reaction, alkaline lysis reaction, and acid lysis reaction in the synthesis step are reacted under the following conditions: 0.5-10 hours at room temperature;

[0054] The pure compounds P2, P3 and P4 were analyzed by electrospray ionization mass spectrometry using liquid chromatography-mass spectrometry to obtain Figure 3 , Figure 4 and Figure 5 The mass spectrum shown is:

[0055] Figure 3 is the mass spectrum of compound P2 synthesized in this example; Figure 4is the mass spectrum of compound P3 synthesized in this example; Figure 5 is the mass spectrum of compound P4 synthesized in this example; Figure 3 It can be seen that the mass spectrum result of compound P2 is obsvd.ESI-MS[(MH) - ]:m / z 1907.2; Figure 4 It can be seen that the mass spectrum result of compound P3 is obsvd.ESI-MS[(M+2H) 2+ ] / 2: m / z 1025.6, [(M-Boc+2H) 2+ ] / 2: m / z 975.1; by Figure 5 It can be seen that the mass spectrum result of compound P4 is obsvd.ESI-MS[(M+H) + ]:m / z1680.6,[(M+2H) 2+ ] / 2:m / z 841.0.

[0056] The pure compound synthesized in this example was analyzed using NMR software to obtain Figure 6 and Figure 7 NMR spectra shown;

[0057] Figure 6 is the hydrogen nuclear magnetic resonance spectrum of the pure compound P4 synthesized in this example; Figure 7 is the carbon NMR spectrum of the pure compound P4 synthesized in this example. Figure 6 It can be seen that the H NMR spectrum of compound P4 (d6-dimethyl sulfoxide, 500 MHz): δ8.64 (s, 1H), 8.49–8.38 (m, 1H), 8.15 (dd, J=28.1, 19.8 Hz, 5H), 7.89–7.78 (m, 10H), 7.69–7.62 (m, 2H), 7.55–7.45 (m, 4H), 7.25–7.12 (m, 10H), 5.47 (s, 2H), 5.24 (s, 2H), 4.57–4.21 (m, 12H), 4. 06–3.98 (m, 3H), 3.52–3.39 (m, 2H), 2.91 (s, 14H), 2.76–2.69 (m, 4H), 2.35 (d, J=15.9Hz, 2H), 2.15–2.10 (m, 2H), 1.7 7 (d, J=47.0Hz, 2H), 1.54 (s, 3H), 1.32 (s, 4H), 1.16 (q, J=9.2, 8.5Hz, 4H), 1.08–0.99 (m, 6H), 0.91 (t, J=7.4Hz, 3H). Depend on Figure 7It can be seen that the carbon NMR spectrum of compound P4 (d6-dimethyl sulfoxide, 151 MHz): δ 172.60, 172.06, 172.04, 171.64, 171.16, 171.11, 170.52, 170.42, 169.80, 168.43, 167.76, 167.44, 167.33, 161.47, 157.02, 152.82, 148 .37, 146.37, 145.97, 137.99, 137.91, 137.11, 135.53, 133.32, 133.28, 132.84, 132.51, 130.88, 130.19, 129.63, 129.59, 129.40, 129.00, 128.86, 128.57, 128.42, 128.40, 128. 24, 128.13, 128.09, 127.96, 127.94, 126.98, 126.67, 126.57, 126.26, 126.19, 125.70, 1 19.22, 95.72, 76.31, 67.10(2C), 66.71, 59.78(2C), 58.94(2C), 58.42(2C), 54.69, 54.0 6, 50.64, 49.29, 48.75, 48.44(2C), 46.35, 42.73, 38.18, 37.87, 36.03, 31.69, 30.76, 30 .10, 29.68, 29.42, 29.24, 27.11, 24.61, 23.04, 22.60, 22.04, 20.06, 19.92, 18.18, 8.03.

[0058] Example 3: Detection of enzyme-responsive self-assembly of peptide-camptothecin conjugates and drug camptothecin release under in vitro conditions

[0059] In the detection experiment of this embodiment, the fourth pure compound P4 with a concentration of 50 μmol / L was used. 100 μL of 20 mg / L fibroblast activation protein α and 100 μL of 1000 units / mL carboxylesterase from pig liver were added to a phosphate buffer containing a concentration of 200 mmol / L. The mixture was incubated at 37 degrees Celsius for 24 hours to complete the recognition and cleavage of fibroblast activation protein α and carboxylesterase.

[0060] Figure 8 The transmission electron microscopy characterization results of the compound P4 synthesized in Example 3 after the compound P4 was recognized and cleaved by fibroblast activation protein α and carboxylesterase in vitro; Figure 8 The transmission electron microscopy characterization results show that under the action of fibroblast activation protein α and carboxylesterase, P4 molecules self-assemble to form a nanofiber structure.

[0061] Fig. 9 The results of HPLC analysis of the compound P4 synthesized in Example 3 before and after the recognition and cleavage by fibroblast activation protein α and carboxylesterase in vitro; Curve a is the chromatographic analysis result of the fourth compound P4, and its chromatographic peak a1 represents the fourth compound P4; chromatographic peak b1 represents the polypeptide compound (Ala-Lys(SA-CPT)-Phe-Phe-Nap) after the compound P4 is recognized and cleaved by the fibroblast activation protein α enzyme; chromatographic peak c1 represents the self-assembled polypeptide skeleton (Ala-Lys(SA)-Phe-Phe-Nap) of the compound P4 recognized and cleaved by the fibroblast activation protein α enzyme and carboxylesterase; chromatographic peak d1 represents the chemotherapeutic drug camptothecin (CPT); Curve e is the chromatographic analysis result obtained by incubating the fourth compound P4 with fibroblast activation protein α and carboxylesterase for 24 hours, wherein the retention time of chromatographic peak e1 is the same as that of chromatographic peak d1 of curve d, proving that pure compound P4 can be recognized and cleaved by carboxylesterase in vitro and release the chemotherapeutic drug camptothecin (CPT); the retention time of chromatographic peak e2 is the same as that of chromatographic peak c1 of curve c, proving that pure compound P4 is recognized and cleaved by fibroblast activation protein α and carboxylesterase in vitro to produce a self-assembled polypeptide skeleton; the retention time of chromatographic peak e3 is the same as that of chromatographic peak a1 of curve a, and the retention time of chromatographic peak e4 is the same as that of chromatographic peak b1 of curve b, indicating that a small amount of compound P4 remains after enzyme recognition and cleavage for 24 hours.

[0062] It can be seen from the results of the above embodiments and their detection experiments that the enzyme-responsive self-assembling polypeptide prodrug loaded with the chemotherapeutic drug camptothecin of the present invention is obtained by simple organic synthesis, is easy to prepare, and can specifically recognize and shear with fibroblast activation protein α and carboxylesterase, self-assemble in situ to form nanofibers, and release the chemotherapeutic small molecule drug camptothecin; in summary, by utilizing the responsive self-assembling polypeptide loaded with chemotherapeutic small molecule drugs of the present invention, highly specific in situ self-assembly of tumor cells and release of chemotherapeutic drugs controlled by fibroblast activation protein α and carboxylesterase dual enzymes are achieved, thereby achieving efficient delivery and long retention of drugs to the tumor area; the present invention fills the gap in polypeptide prodrugs that can respond to fibroblast activation protein α and carboxylesterase and self-assemble in situ into nanofibers and sustained-release chemotherapeutic small molecule drugs and their preparation methods, and realizes the detection experiment of polypeptide prodrug enzyme response to self-assemble into fibers and release chemotherapeutic small molecule drugs under in vitro conditions.

Claims

1. An enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin, characterized in that: Its chemical structure is shown below:

2. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 1, characterized in that: The preparation steps are as follows: Step (1): dissolving camptothecin in an organic solvent, then adding succinic anhydride and an acid-binding reagent thereto, and reacting overnight under the protection of an inert gas; purifying by chromatography to obtain camptothecin succinic acid, recorded as P1; Step (2): swell 2-chlorotrityl chloride resin in an organic solvent, add phenylalanine derivative and acid binding reagent to react, and use alkaline lysis solution to remove the protecting group of phenylalanine; add activated phenylalanine derivative and acid binding reagent to react, and use alkaline lysis solution to remove the protecting group of phenylalanine; repeat the above amide condensation and alkaline lysis deprotection steps, and add lysine derivative, alanine derivative, proline derivative, glycine derivative, threonine derivative, lysine derivative, threonine derivative, aspartic acid derivative in each repeated step; then use alkaline lysis solution to remove the protecting group of lysine side chain; add activated P1 and acid binding reagent to react overnight; Subsequently, the peptide was cut from the resin using an acid lysis solution, the product was precipitated using a poor solvent, the supernatant was discarded by freezing centrifugation, and the precipitate was purified by chromatography to obtain a peptide loaded with the chemotherapeutic drug camptothecin, which was recorded as P2; Step (3): dissolving the activated polypeptide P2, 2-naphthylmethylamine and an acid-binding agent in an organic solvent for reaction; purifying by chromatography to obtain a polypeptide, which is designated as P3; Step (4): using an acid lysis solution to remove the protecting group on P3; and obtaining an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin after purification by chromatography.

3. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: The organic solvent in the synthesis step is selected from: methanol, isopropanol, dichloromethane, N,N-dimethylformamide, dimethyl sulfoxide or tetrahydrofuran.

4. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: The acid-binding reagent in the synthesis step is selected from: triethylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, piperidine or pyridine.

5. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: The protecting group in the synthesis step is selected from: tert-butoxy, tert-butoxycarbonyl, fluorenylmethoxycarbonyl or 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl.

6. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: The alkaline lysis solution in the synthesis step is selected from: piperidine / methanol (5-30%, v / v), piperidine / N,N-dimethylformamide (5-30%, v / v), piperidine / dichloromethane (5-30%, v / v), pyridine / dichloromethane (20-60%, v / v), pyridine / N,N-dimethylformamide (20-60%, v / v), hydrazine hydrate / N,N-dimethylformamide (1-10%, v / v) or hydrazine hydrate / methanol (1-10%, v / v).

7. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: The amino acid activation in the synthesis step is carried out by adding the following reagent, which is selected from: O-(7-azabenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate, 1-hydroxybenzotriazole, O-benzotriazole-tetramethyluronium hexafluorophosphate or O-(5-chlorobenzotriazole-1-yl)-bis(dimethylamino)carbonium hexafluorophosphate.

8. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: The acid lysis solution in the synthesis step is selected from: trifluoroacetic acid / methanol (1-99%, v / v), trifluoroacetic acid / N,N-dimethylformamide (1-99%, v / v) or trifluoroacetic acid / dichloromethane (1-99%, v / v).

9. The method for preparing a camptothecin-loaded enzyme-responsive self-assembling polypeptide prodrug according to claim 2, characterized in that: In step (1), the inert gas is nitrogen; In step (2), the repeated amide condensation and alkaline cleavage deprotection steps are: adding activated N-fluorenylmethoxycarbonyl-L-phenylalanine and reacting with an acid binding agent, using 20% ​​piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of phenylalanine; adding activated N-fluorenylmethoxycarbonyl-N'-[1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl]-L-lysine and reacting with an acid binding agent, using 20% ​​piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of phenylalanine; piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of lysine; add activated N-fluorenylmethoxycarbonyl-L-alanine and an acid binding agent to react, use 20% piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of alanine; add activated N-tert-butylfluorenylmethoxycarbonyl-L-proline and an acid binding agent to react, use 20% piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of proline acid; add activated N-fluorenylmethoxycarbonyl-glycine and an acid binding agent to react, use 20% piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of proline acid; The N-fluorenylmethoxycarbonyl protecting group of glycine was removed by using 20% ​​piperidine; activated N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine was added to react with an acid-binding agent, and the N-fluorenylmethoxycarbonyl protecting group of threonine was removed by using 20% ​​piperidine; activated N-fluorenylmethoxycarbonyl-N-tert-butyloxycarbonyl-L-lysine was added to react with an acid-binding agent, and the N-fluorenylmethoxycarbonyl protecting group of lysine was removed by using 20% ​​piperidine. ; adding activated N-fluorenylmethoxycarbonyl-O-tert-butyl-L-threonine and reacting with an acid binding agent, using 20% ​​piperidine to remove the N-fluorenylmethoxycarbonyl protecting group of threonine; adding activated N-(tert-butyloxycarbonyl)-L-aspartic acid-4-tert-butyl ester and reacting with an acid binding agent; subsequently, using 2% hydrazine hydrate solution to remove the 1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl protecting group of the lysine side chain; The poor solvent is ether.

10. Use of an enzyme-responsive self-assembling polypeptide prodrug loaded with camptothecin prepared by the preparation method according to any one of claims 1 to 9 in anti-tumor treatment; The tumors include digestive system cancer, leukemia, lung cancer and bladder cancer.

Images