A camptothecin polypeptide nanoformulation with variable morphology, its preparation method and application

Abstract

The present invention discloses a camptothecin polypeptide nanoformulation with variable morphology, its preparation method and application. The camptothecin nanoformulation includes an amphiphilic polypeptide and a camptothecin derivative; the camptothecin derivative is covalently linked to the amphiphilic polypeptide through a disulfide bond. The camptothecin polypeptide nanoformulation has acid responsiveness and morphology transformability. Under normal physiological conditions, the nanoparticle structure is maintained to ensure its permeability at the tumor site; under the slightly acidic conditions of the tumor microenvironment, the morphology of the formulation changes from nanoparticles to nanofibers with a length of not less than 200 nm, which can improve the retention ability of camptothecin, increase its concentration at the tumor site, so as to reduce the administration concentration and achieve efficient killing of cancer cells; the camptothecin polypeptide nanoformulation has specificity, and the overexpressed GSH in cancer cells reduces the disulfide bond to release the original camptothecin drug, which can reduce the toxic side effects of camptothecin on normal cells in the blood circulation and achieve specific killing of cancer cells.

Description

Technical Field

[0001] The present invention belongs to the field of anti-tumor drugs, and particularly relates to a camptothecin polypeptide nano preparation with a variable form, a preparation method and an application thereof. Background Art

[0002] Cancer is currently one of the leading causes of death worldwide, with new cases and deaths increasing annually. Chemotherapy has long been a common treatment for cancer, and the chemotherapy drug camptothecin, a cytotoxic quinoline alkaloid and one of the active ingredients in the traditional Chinese medicine Camptotheca acuminata, has been shown to have a significant anti-tumor effect by inhibiting the activity of topoisomerase I during the S phase of cancer cell DNA replication, thereby blocking DNA synthesis and inducing apoptosis. However, due to its low solubility, camptothecin has difficulty achieving therapeutic concentrations at the lesion site, while high dosing concentrations can lead to significant toxic side effects. Therefore, increasing the concentration of camptothecin at the lesion site to reduce the dosing concentration and mitigate toxic side effects is of great significance.

[0003] With the development of nanotechnology, traditional camptothecin nanoformulations have been improved in terms of increasing drug delivery efficiency. Chinese invention patent CN109172527A discloses a cationic amino acid-modified camptothecin prodrug, its preparation method, nanoparticles, and its application. The camptothecin prodrug contains positively charged amino acids and can interact with negatively charged polymers through electrostatic interaction to form uniformly sized and small nanoparticles. These particles have high drug loading capacity and excellent stability, and have a good killing effect on cancer cells. Another Chinese invention patent, CN107714641A, discloses a method for preparing a camptothecin prodrug-loaded dual-drug supramolecular hydrogel for combined drug use. The camptothecin prodrug is formed into a supramolecular hydrogel through hydrophobic polymerization and self-assembly cross-linking based on the host-guest interaction between α-CD and PEG chains. The hydrogel is then loaded with water-soluble anticancer drugs and can be implanted into the lesion site by injection, showing good biocompatibility.

[0004] However, the size of nanoformulations affects their physical or chemical properties, thus determining their effects in biological systems. Traditional spherical camptothecin nanoformulations are easy to penetrate the tumor site, but their retention is poor, while fibrous camptothecin nanoformulations have a long retention effect, but are difficult to penetrate into the tumor. Due to the influence of tumor heterogeneity, although spherical nanoformulations are easy to penetrate the tumor and distribute quickly, they have poor retention at the lesion site, and although nanofibers with high aspect ratios have good tumor retention effects, they are often difficult to target deeply into the tumor. This has led to certain limitations in the clinical application of traditional camptothecin nanoformulations.

[0005] Therefore, there is an urgent need in this field to provide a camptothecin polypeptide nanoformulation with variable morphological properties, which can not only ensure the high permeability of the nanoformulation, but also enable camptothecin to have long retention, increase its concentration at the tumor site, reduce the dosage concentration, reduce toxic side effects, and achieve the purpose of achieving a highly effective anti-tumor effect through a morphological transformation strategy. Summary of the Invention

[0006] In response to the above problems, the present invention proposes a camptothecin polypeptide nanoformulation with variable morphology, wherein the camptothecin nanoformulation comprises an amphiphilic polypeptide and a camptothecin derivative;

[0007] The camptothecin derivative is covalently linked to the amphiphilic polypeptide via a disulfide bond.

[0008] The amino acid sequence of the amphiphilic polypeptide includes alternatingly connected hydrophilic amino acids, hydrophobic amino acids and an intermediate cysteine;

[0009] Among them, the number of hydrophilic amino acids and hydrophobic amino acids is the same, both less than or equal to six.

[0010] Furthermore, the hydrophilic amino acids include amino acids with positively charged and negatively charged side chain groups, and the number of positively charged hydrophilic amino acids is equal to the number of negatively charged hydrophilic amino acids, and both are less than or equal to three.

[0011] Furthermore, the amino acid with a positively charged side chain group includes any one of arginine, lysine or histidine, and the amino acid with a negatively charged side chain group includes any one of glutamic acid or aspartic acid;

[0012] The hydrophobic amino acid includes any one of tryptophan, phenylalanine, valine, leucine, isoleucine, alanine, proline or methionine.

[0013] Furthermore, the amino acid sequence of the amphiphilic polypeptide includes the amino acid sequence as described in any one of SEQ ID NOs 1 to 18.

[0014] Furthermore, the amino acid sequence of the amphiphilic polypeptide is shown in SEQ ID NO 14.

[0015] Furthermore, the camptothecin nanoformulation having an amphiphilic polypeptide sequence as shown in SEQ ID NO 14 includes a compound shown in the following structural formula:

[0016]

[0017] Furthermore, the morphology of the variable-morphology camptothecin polypeptide nanoformulation is transformed from nanoparticles to nanofibers under the slightly acidic conditions of the tumor microenvironment;

[0018] Among them, the slightly acidic condition is an aqueous solution with a pH of 6.1 to 6.9;

[0019] The diameter of the nanoparticles is 30 to 100 nm;

[0020] The length of the nanofiber is greater than or equal to 500 nm;

[0021] The width of the nanofiber is 10 to 60 nm;

[0022] The conformation of the nanofiber includes any one of α-helix, β-sheet or β-hairpin or a combination of the two.

[0023] On the other hand, the present invention also provides a method for preparing a morphologically variable camptothecin polypeptide nanoformulation, the preparation method comprising:

[0024] Synthesis of camptothecin derivatives and amphiphilic peptides with alternating hydrophilic and hydrophobic properties;

[0025] coupling a camptothecin derivative with an amphiphilic polypeptide to obtain a camptothecin polypeptide;

[0026] The camptothecin polypeptide is self-assembled under neutral conditions to obtain a camptothecin polypeptide nano preparation with a variable morphology.

[0027] Furthermore, the amphiphilic polypeptide molecules are synthesized using solid phase synthesis technology;

[0028] The camptothecin derivatives are based on camptothecin and react with triphosgene in the presence of an organic base to generate an acyl chloride intermediate, which then reacts with 2-[2-(pyridyl)thio]ethanol to obtain the camptothecin derivatives.

[0029] Furthermore, the camptothecin derivative is coupled with the amphiphilic polypeptide molecule using a thiol-dithiol exchange reaction technology, which specifically includes the following steps:

[0030] Using anhydrous DMSO as solvent, the camptothecin derivative and the amphiphilic polypeptide are mixed under a nitrogen atmosphere and stirred at room temperature for reaction. After the reaction is complete, solid impurities are removed by filtration, and then cold ether is added to precipitate the product, which is then washed and dried to obtain the camptothecin polypeptide.

[0031] Furthermore, the non-covalent interaction force for the self-assembly of the camptothecin polypeptide includes any one or a combination of at least two of hydrogen bonds, hydrophobic interactions, π-π interactions, or van der Waals forces, and the self-assembly of the camptothecin polypeptide is obtained by annealing the camptothecin polypeptide under neutral conditions, naturally cooling it to room temperature, and allowing it to stand;

[0032] in,

[0033] Aqueous solution with a pH of 7.3 to 7.6 under neutral conditions;

[0034] The annealing temperature is 75-85°C and the annealing time is 25-35 minutes;

[0035] The standing time is 10 to 15 hours.

[0036] On the other hand, the camptothecin polypeptide nanoformulation proposed in the present invention can be used in the preparation of anticancer drug; the administration of the camptothecin polypeptide nanoformulation includes any one of tail vein administration, subcutaneous administration or intraperitoneal administration.

[0037] Beneficial effects of the present invention:

[0038] (1) The variable-morphology camptothecin polypeptide nanoformulation proposed in the present invention includes an amphiphilic polypeptide with alternating hydrophilic and hydrophobic properties and camptothecin. Camptothecin is covalently linked to the amphiphilic polypeptide via a disulfide bond to prepare a variable-morphology camptothecin polypeptide nanoformulation. The camptothecin polypeptide nanoformulation has acid responsiveness and morphological transformation properties. Under normal physiological conditions, the nanoparticle structure is maintained to ensure its permeability at the tumor site; under the slightly acidic conditions of the tumor microenvironment, the preparation morphology is transformed from nanoparticles to nanofibers with a length of not less than 200 nm, which can improve the retention capacity of camptothecin and increase its concentration at the tumor site, thereby reducing the dosage concentration and achieving efficient killing of cancer cells.

[0039] (2) The variable-morphology camptothecin polypeptide nanoformulation proposed in the present invention has specificity. GSH overexpressed by cancer cells reduces disulfide bonds to release the original drug camptothecin, which can reduce the toxic side effects of camptothecin on normal cells in the blood circulation and achieve specific killing of cancer cells;

[0040] (3) The preparation method of the variable-morphology camptothecin polypeptide nanoformulation proposed in the present invention is mature and can be mass-produced, which promotes the promotion and use of the product and has broad application prospects.

[0041] Other features and advantages of the present invention will be described in the following description, and in part will become apparent from the description, or will be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained by the structures pointed out in the description, claims and drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0043] Figure 1This is a diagram showing the working principle of the camptothecin polypeptide nanoformulation with variable form according to the present invention;

[0044] Figure 2 The ESI-MS characterization image of the amphiphilic polypeptide shown in SEQ ID NO 1 prepared in Example 1 of the present invention is shown;

[0045] Figure 3 The ESI-MS characterization image of the amphiphilic polypeptide shown in SEQ ID NO 6 prepared in Example 1 of the present invention is shown;

[0046] Figure 4 The ESI-MS characterization image of the amphiphilic polypeptide shown in SEQ ID NO 15 prepared in Example 1 of the present invention is shown;

[0047] Figure 5 Shown is a picture of the MNR characterization of the camptothecin derivative prepared in Example 2 of the present invention;

[0048] Figure 6 The ESI-MS characterization image of the camptothecin polypeptide prepared in Example 3 of the present invention is shown;

[0049] Figure 7 The figure shows the Fourier transform infrared spectroscopy characterization images of the camptothecin polypeptide nanoformulation with variable morphology described in Example 4 of the present invention at pH 6.5 and 7.4;

[0050] Figure 8 The circular dichroism spectra of the camptothecin polypeptide nanoformulation with variable morphology described in Example 4 of the present invention at pH 6.5 and 7.4 are shown;

[0051] Figure 9A The figure shows a transmission electron microscopy image of the camptothecin polypeptide nanoformulation with variable morphology described in Example 4 of the present invention at pH 7.4 (scale bar = 200 nm);

[0052] Figure 9B The figure shows a transmission electron microscopy image of the camptothecin polypeptide nanoformulation with variable morphology described in Example 4 of the present invention at pH 6.5 (scale bar = 200 nm);

[0053] Figure 10A The figure shows a transmission electron microscopy image of the amphiphilic polypeptide of SEQ ID NO 6 in Example 1 of the present invention at pH 7.4 (scale bar = 200 nm);

[0054] Figure 10BThe figure shows a transmission electron microscopy image of the amphiphilic polypeptide of SEQ ID NO 6 in Example 1 of the present invention at pH 7.4 (scale bar = 200 nm);

[0055] Figure 11A The figure shows a transmission electron microscopy image of the amphiphilic polypeptide of SEQ ID NO 15 in Example 1 of the present invention at pH 7.4 (scale bar = 200 nm);

[0056] Figure 11B The figure shows a transmission electron microscopy image of the amphiphilic polypeptide of SEQ ID NO 15 in Example 1 of the present invention at pH 7.4 (scale bar = 200 nm);

[0057] Figure 12 The drug release curves of the camptothecin polypeptide nanoformulation with variable morphology at different GSH concentrations described in Example 5 of the present invention are shown;

[0058] Figure 13 The results of the cell activity detection experiment after the variable-morphology camptothecin polypeptide nanoformulation described in Example 6 of the present invention was co-incubated with A549 cells are shown in the figure;

[0059] Figure 14 The results of the live-dead staining experiment after the camptothecin polypeptide nanoformulation with variable morphology described in Example 6 of the present invention was co-incubated with A549 cells are shown;

[0060] Figure 15 The figure shows the results of the in vivo inhibition of tumor growth of the variable-morphology camptothecin polypeptide nanoformulation described in Example 7 of the present invention. DETAILED DESCRIPTION

[0061] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0062] This invention utilizes a morphological transformation strategy to transform camptothecin nanoparticles into irregularly curled nanoparticles before reaching the tumor site, ensuring high permeability. Upon reaching the tumor site, the nanoparticles transform into nanofibers in a slightly acidic environment, enhancing drug retention at the tumor site. This morphological transformation during drug delivery allows the nanoparticles to achieve both high permeability and long retention, thereby reducing dosing concentration, minimizing toxic side effects, and enhancing anti-tumor efficacy. This approach is applicable to tumor drug treatment.

[0063] The working principle of the variable-morphology camptothecin peptide nanoformulation is as follows Figure 1 As shown, under normal physiological conditions, the electrostatic repulsion of the hydrophilic amino acid side chains causes the amphiphilic peptide to adopt a random coiled conformation, self-assembling into nanoparticles. This ensures high permeability of the morphologically variable camptothecin peptide nanoparticle formulation at the tumor site. Once delivered to the tumor site, the hydrophilic amino acid side chains protonate in the slightly acidic tumor microenvironment, enhancing the electrostatic interactions between the amphiphilic peptide molecules. The conformation shifts from a random coil to a β-sheet, and further from a small particle structure to a high-aspect-ratio fiber. This improves drug retention and concentration at the tumor site, thereby reducing drug concentration and minimizing toxic side effects.

[0064] The following examples further illustrate the morphologically variable camptothecin polypeptide nanoformulations proposed in the present invention. Where specific techniques or conditions are not specified in the examples, the methods were followed according to those described in literature in the art or according to the product instructions. Reagents or instruments used, where the manufacturer is not specified, are conventional products commercially available through reputable channels.

[0065] The materials used in the embodiments of the present invention were obtained from the following channels, but are not limited to the following channels. All materials can be obtained from regular channels:

[0066] Rink amide 4-methylaniline (MBHA) resin, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Cys(Trt)-OH, and Fmoc-Lys(Boc)-OH were purchased from Bidex Pharmaceutical Technology Co., Ltd.;

[0067] Trifluoroacetic acid, 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, methylphenyl sulfide, and camptothecin were purchased from San Chemical Technology Co., Ltd.;

[0068] N,N-diisopropylethylamine, reduced glutathione (GSH), 2,2'-dithiodipyridine, 2-mercaptoethanol, anisole, triphosgene, and 4-dimethylaminopyridine were purchased from J&K Technology Co., Ltd.;

[0069] Calcein-AM and ethylenediamine homodimer-1 were purchased from Merck;

[0070] A549 cells were purchased from Wuhan Punosai Life Science Technology Co., Ltd.

[0071] Example 1 Synthesis of amphiphilic polypeptide sequences

[0072] In this example, amphiphilic polypeptide sequences are selected.

[0073] The amphiphilic polypeptide sequence needs to contain hydrophilic amino acids, hydrophobic amino acids and cysteine. The hydrophobic amino acids include any one of tryptophan (W), phenylalanine (F), valine (V), leucine (L), isoleucine (I), alanine (A), proline (P) or methionine (M). Too strong or too weak hydrophobicity is not conducive to the morphological regulation properties of polypeptide self-assembly. Since valine is more hydrophobic than tryptophan, alanine, proline and methionine, but weaker than phenylalanine, leucine and isoleucine, valine is preferred. The amino acids include amino acids with positively charged and negatively charged side chains, the amino acids with positively charged side chains include any one of arginine (R), lysine (K) or histidine (H), and the amino acids with negatively charged side chains include any one of glutamic acid (E) or aspartic acid (D); in the process of selecting the amphiphilic polypeptide sequence, the number of hydrophilic amino acids and hydrophobic amino acids is equal, and no more than six each; the number of amino acids with positively charged and negatively charged side chains is equal, and no more than three each; according to the above requirements, three groups of amphiphilic polypeptide sequences of SEQ ID NOs 1 to 18 are designed, and multiple sequences with net charges of -1.8, -0.9, 0.0 and 1.0 at pH = 7 are designed, respectively. The specific sequences are shown in the following table:

[0074]

[0075] The amphiphilic polypeptides of SEQ ID NOs 1 to 11 all have a net charge of 1.0 at pH 7, an average hydrophilicity of 0.4-0.6, and a hydrophilic residue ratio of 40-46%. The amphiphilic polypeptide sequences of SEQ ID NOs 12 to 14 all have a net charge of -1.8 at pH 7, an average hydrophilicity of -0.1, and a hydrophilic residue ratio of 23%. The amphiphilic polypeptide sequences of SEQ ID NOs 15 to 16 all have a net charge of -0.9 at pH 7, an average hydrophilicity of -0.2, and a hydrophilic residue ratio of 22%. The amphiphilic polypeptide sequences of SEQ ID NOs 17 to 18 all have a net charge of 0.0 at pH 7, an average hydrophilicity of -0.2, and a hydrophilic residue ratio of 22%. The above combinations are merely illustrative and are not exhaustive of all eligible amphiphilic polypeptide sequences.

[0076] The amphiphilic polypeptide was synthesized using solid phase synthesis technology. This example uses the amphiphilic polypeptide sequence of SEQ ID NO 14 as an example to illustrate the synthesis method.

[0077] 1.1 Deprotection of protecting groups

[0078] MBHA resin with a modification density of 0.436 mmol / g was used to remove the Fmoc protecting group from the N-terminus of the MBHA resin using a mixture of piperidine and N,N-dimethylformamide in a 1:3 volume ratio. A small amount of MBHA resin was then subjected to a ninhydrin color development reaction. If the solution is colorless, the α-amino protecting group has not been removed and the deprotection step needs to be repeated. If the solution turns blue-purple, the α-amino protecting group has been removed and amino acid condensation reactions can proceed.

[0079] 1.2 Condensation reaction

[0080] Weigh 4 times the molar weight of valine and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate, weigh 4 times the molar weight of N,N-diisopropylethylamine, add N,N-dimethylformamide, and stir to dissolve. Transfer the solution to a peptide synthesis tube and react for 1 hour. After the reaction, remove the solvent and wash the resin three times with N,N-dimethylformamide and dichloromethane. A small amount of MBHA resin is then used for ninhydrin color development to verify the success of the amide condensation reaction. If successful, repeat the deprotection and condensation reaction for each amino acid, and then sequentially couple all remaining amino acids via amide condensation. If the reaction is unsuccessful, add another 4 times the molar weight of amino acid and continue the reaction until the reaction is successful, then continue with the next amino acid, and continue until all remaining amino acids have been coupled via amide condensation to obtain the MBHA resin peptide.

[0081] 1.3 Cutting

[0082] A cleavage agent was added to a synthesis tube. The cleavage agent was composed of a mixture of trifluoroacetic acid: methyl phenyl sulfide: anisole: 1,2-ethanedithiol in a volume ratio of 90:5:3:2. The amphiphilic polypeptide was cleaved from the MBHA resin. The reaction was allowed to proceed for 2.5 hours. After the reaction, the cleavage solution was filtered and concentrated using a rotary evaporator to a viscous state. Cold ether was then added to precipitate a crude product, which was then washed and dried to obtain the amphiphilic polypeptide represented by SEQ ID NO 14.

[0083] The obtained amphiphilic polypeptide represented by SEQ ID NO 14 was characterized by ESI-MS (electrospray mass spectrometry), and the obtained mass spectrum was as shown below: Figure 2 As shown by Figure 2 It can be seen that the mass spectrum of the obtained amphiphilic polypeptide is:

[0084] C 45H 82 N 12 O13S[M+H] + :1032.34.

[0085] According to the above method, the amphiphilic polypeptide sequences of SEQ ID NOs. 1 to 16 were synthesized respectively, and the obtained amphiphilic polypeptides were characterized by ESI-MS (electrospray mass spectrometry). For example, the mass spectrometry results of the amphiphilic polypeptides represented by SEQ ID NOs. 6 and 15 are shown in Figures 3 and 4, respectively. Figure 4 As shown, the mass spectrometry results showed that the above sequence was correctly synthesized.

[0086] Example 2 Synthesis of Camptothecin Derivatives

[0087] The steps for synthesizing camptothecin derivatives include:

[0088] 2.1 Synthetic intermediates

[0089] Under nitrogen atmosphere, 2,2'-dithiodipyridine (Formula a) was dissolved in methanol, and then 2-mercaptoethanol was added. The reaction mixture was added dropwise to the solution, and the reaction liquid was stirred at room temperature for 2 hours after the color turned yellow. After the reaction was completed, the mixture was separated by column chromatography to obtain a pure intermediate 2-[2-(pyridyl)thio]ethanol. The structural formula of the 2-[2-(pyridyl)thio]ethanol is shown in Formula b.

[0090] The specific reaction formula is as follows:

[0091]

[0092] 2.2 Synthesis of camptothecin derivatives

[0093] The original drug of camptothecin reacts with triphosgene in an organic solvent in the presence of an organic base (such as 4-dimethylaminopyridine) for 15 minutes to generate an acyl chloride intermediate. The acyl chloride intermediate reacts with the 2-[2-(pyridyl)thio]ethanol for 16 hours. After the reaction, the reaction solution is washed twice with a saturated NaCl solution, concentrated, and then dissolved with hot methanol. The solution is stored at 4°C for 48 hours to obtain an off-white precipitate. The off-white solid is collected by filtration and washed three times with methanol to obtain a camptothecin derivative containing a disulfide bond. The structural formula of the camptothecin derivative is shown in Formula C.

[0094] The specific reaction formula is as follows:

[0095]

[0096] The obtained camptothecin derivatives were characterized by MNR (nuclear magnetic resonance spectroscopy), and the results were as follows: Figure 5 As shown by Figure 5 It can be seen that the NMR spectrum of the obtained camptothecin derivative is1 H NMR (400MHz, DMSO) δ8.69 (s, 1H), 8.39 (dd, J=4.8, 0.8Hz, 1H), 8.14 (t, J=7.8Hz, 2H), 7.85 (dd, J=11.8, 4.7Hz, 1H), 7.79–7.65 (m, 3H), 7.15 (ddd, J=7.3, 4.8, 0.9Hz, 1H), 7.10 (s, 1H), 5.52 (d, J=1.2Hz, 2H), 5.30 (s, 2H), 4.33 (t, J=6.0Hz, 2H), 3.15 (t, J=6.1Hz, 2H), 2.18 (qt, J=14.5, 7.2Hz, 2H), 0.92 (t, J=7.4Hz, 3H).

[0097] Example 3

[0098] The camptothecin derivatives and amphiphilic polypeptides are synthesized into a variable-form camptothecin polypeptide nano-preparation by using the thiol-dithiol exchange reaction technology.

[0099] This example uses the amphiphilic polypeptide shown in SEQ ID NO 14 synthesized in Example 1 and the camptothecin derivative synthesized in Example 2 as examples to illustrate the synthesis process of the camptothecin polypeptide nanoformulation.

[0100] A camptothecin polypeptide nanoformulation with a variable form, comprising an amphiphilic polypeptide and camptothecin;

[0101] The amino acid sequence of the amphiphilic polypeptide is shown in SEQ ID NO 14.

[0102] SEQ ID NO14: VKVECKVEV-NH2.

[0103] The molecular structure of the variable-morphology camptothecin polypeptide nanoformulation is shown in Formula I:

[0104]

[0105] The method comprises the following steps: using anhydrous DMSO as a solvent, mixing a camptothecin derivative and an amphiphilic polypeptide under a nitrogen atmosphere, stirring at room temperature for 24 hours, filtering to remove solid impurities, then adding cold ether to precipitate the product, washing and drying to obtain a camptothecin polypeptide.

[0106] The obtained camptothecin peptide was characterized by ESI-MS. Figure 6 shown.

[0107] Depend on Figure 6 It can be seen that the mass spectrum of the obtained camptothecin polypeptide is:

[0108] C45 H 82 N 12 O 13 S[M+H] + :1483.24.

[0109] The camptothecin polypeptide is self-assembled, wherein the non-covalent bond interaction force of the self-assembly includes any one of hydrogen bond, hydrophobic interaction, π-π interaction or van der Waals force, or a combination of at least two thereof;

[0110] Dissolve the camptothecin polypeptide in a neutral aqueous solution with a pH of 7.3 to 7.6;

[0111] Annealing is performed at 75-85° C. for 25-35 minutes, then naturally cooled to room temperature, and allowed to stand for 10-15 hours to obtain a camptothecin polypeptide nanoformulation with a variable morphology.

[0112] In this embodiment, the pH of the neutral aqueous solution is 7.4, the annealing temperature is 80° C., the annealing time is 30 min, and the standing time is 12 h.

[0113] Example 4

[0114] An experiment was conducted to verify whether the camptothecin polypeptide nanoformulation prepared in Example 3 undergoes a morphological transformation under slightly acidic conditions. The conformation of the camptothecin polypeptide nanoformulation includes any one of α-helix, β-sheet, or β-hairpin, or a combination of two.

[0115] (1) Fourier transform infrared spectroscopy characterization

[0116] The same concentration of camptothecin polypeptide nanoparticles were dissolved in pH neutral conditions (aqueous solution with a pH of 7.3 to 7.6) and slightly acidic conditions (aqueous solution with a pH of 6.1 to 6.9), respectively. In this embodiment, the neutral condition was an aqueous solution with a pH of 7.4, and the slightly acidic condition was an aqueous solution with a pH of 6.5. The results were characterized by Fourier transform infrared spectroscopy. Figure 7 shown.

[0117] Depend on Figure 7 It can be seen that at pH 7.4, the Fourier transform infrared spectrum of the camptothecin polypeptide nanoformulation showed a peak at 1646 cm －1 There is an absorption band related to the vibration absorption of amide I bond at 1624 cm, indicating that the camptothecin polypeptide nanoformulation is a random coil structure under this condition. At pH 6.5, the Fourier transform infrared spectrum of the camptothecin polypeptide nanoformulation with the same concentration shows an absorption band at 1624 cm －1 and 1691cm －1 There are two absorption bands related to the vibration absorption of amide I bond, indicating that the camptothecin polypeptide nanoformulation formed an antiparallel β-sheet structure under this condition.

[0118] (2) Circular dichroism spectroscopy characterization

[0119] The conformation of the camptothecin polypeptide nanoformulation in aqueous solution at pH 7.4 and 6.5 was further characterized using circular dichroism spectroscopy. Figure 8 shown.

[0120] Depend on Figure 8 It can be seen that at a pH of 7.4, the circular dichroism spectrum of the camptothecin polypeptide nanoformulation showed a negative characteristic peak at 199nm, confirming that the camptothecin polypeptide nanoformulation was in a random coil conformation under this condition. At a pH of 6.5, the circular dichroism spectrum of the camptothecin polypeptide nanoformulation of the same concentration showed a negative characteristic peak at 217nm, confirming that the camptothecin polypeptide nanoformulation was in a β-sheet conformation under this condition, indicating that the conformation of the camptothecin polypeptide nanoformulation changed from random coil to β-sheet under slightly acidic conditions.

[0121] (3) Electron microscopy characterization

[0122] Transmission electron microscopy observation of the nanostructure of the camptothecin polypeptide nanoformulation in aqueous solutions at pH 7.4 and 6.5 showed that the diameter of the nanoparticles was 30-100 nm, preferably 50-70 nm; after conversion into nanofibers, the length of the nanofibers was greater than or equal to 500 nm, and the width of the nanofibers was 10-60 nm, preferably 20-40 nm.

[0123] The nanostructures of the camptothecin polypeptide nanoformulations in this embodiment are as follows: Figure 9A and Figure 9B As shown. Figure 9A It can be seen that at pH 7.4, the camptothecin polypeptide nanoparticles aggregate to form nanoparticles with a diameter of about 60 nm, or 50 to 70 nm. Figure 9B It can be seen that under slightly acidic conditions of pH 6.5, the camptothecin polypeptide nanoformulation self-assembles to form nanofibers with a length of not less than 500 nm and a width of about 25 nm, proving that the nanostructure of the camptothecin polypeptide nanoformulation undergoes a transformation under slightly acidic conditions.

[0124] In addition, this example also performed electron microscopic characterization on the nanostructures of camptothecin polypeptide nanoformulations of SEQ ID NO6 and SEQ ID NO15 under different pH environments. The electron microscopic results of the amphiphilic polypeptide represented by SEQ ID NO6 in an aqueous solution with a pH of 7.4 and an aqueous solution with a slightly acidic pH of 6.5 are shown as follows: Figure 10A and 10B As shown; the amphiphilic polypeptide shown in SEQ ID NO15 is in an aqueous solution with a pH of 7.4 and an aqueous solution with a slightly acidic pH of 6.5, and the results are as follows: Figure 11A and11B shown by Figure 11A and 11B It can be seen that the conformational nanostructure of the synthesized camptothecin polypeptide nanoformulation is transformed from nanoparticles to nanofibers under slightly acidic conditions.

[0125] Example 5

[0126] This example verifies that the camptothecin polypeptide nanoformulation prepared in Example 3 releases the original camptothecin drug under the reduction of disulfide bonds by GSH.

[0127] The camptothecin polypeptide nanoformulation was dissolved in PBS buffer solution, and different concentrations of GSH were added. The drug release curve was obtained by detecting the remaining content of the camptothecin polypeptide nanoformulation. The results are as follows: Figure 12 shown.

[0128] from Figure 12 As can be seen, in the absence of GSH, less than 5% of the original camptothecin drug was released from the camptothecin peptide nanoformulation after 14 hours, indicating that the disulfide bonds are relatively stable in the absence of GSH, preventing the release of the original camptothecin drug. Adding GSH to the camptothecin peptide nanoformulation resulted in a gradual release of the original camptothecin drug, with the release rate accelerating with increasing GSH concentration. In the presence of 2mM GSH, approximately 53% of the original camptothecin drug was released after 14 hours; in the presence of 4mM GSH, approximately 62% was released after 14 hours; in the presence of 6mM GSH, approximately 70% was released after 14 hours; in the presence of 8mM GSH, approximately 83% was released after 14 hours; and in the presence of 10mM GSH, approximately 90% was released after 14 hours.

[0129] Example 6

[0130] This example verifies the killing effect of the camptothecin polypeptide nanoformulation prepared in Example 3 on cancer cells.

[0131] A549 cells were selected to investigate the killing effect of camptothecin peptide nanoformulation on cancer cells. A549 cells were seeded in 96-well culture plates, with 1×10 4 Cells were cultured for 24 hours to allow attachment. Different concentrations of camptothecin stock, camptothecin peptide nanoformulations, or blank culture medium were added to each well and incubated for an additional 48 hours. The culture medium was removed and replaced with culture medium containing CCK-8 detection reagent, and incubated for 2 hours. Finally, the 96-well plate was placed in a microplate reader and the absorbance of each well was measured at 450 nm.

[0132] The relative cell activity was obtained by comparing the absorbance of cells in the camptothecin peptide nanoformulation group and blank cells. Figure 13 shown.

[0133] Depend on Figure 13 It can be seen that the IC of camptothecin original drug to A549 cells 50 (half inhibitory concentration) was 2.79 μM, while the IC 50 The results showed that the killing effect of the camptothecin peptide nanoparticle preparation on A549 cells was comparable to that of the original camptothecin drug, indicating that the camptothecin peptide nanoparticle preparation had a good killing effect on A549 cells.

[0134] In order to further verify the killing effect of camptothecin peptide nanoformulation on cancer cells, A549 cell live-dead staining experiment was performed.

[0135] A549 cells were seeded in 96-well culture plates at 1×10 4 cells, 3 μM camptothecin original drug and camptothecin peptide nanoformulation were added to each well and incubated for 48 h. Then, a mixture of 2 μM calcein-AM and 4 μM ethylenediamine homodimer-1 was added and incubated for 30 min to stain live cells and dead cell colonies, respectively.

[0136] Live and dead cells were observed using a laser confocal microscope. The excitation wavelength of calcein-AM was 450-490 nm, and the excitation wavelength of ethylenediamine homodimer-1 was 510-560 nm. The results are shown in Figure 14.

[0137] Depend on Figure 14 It can be seen that the fluorescence intensity of A549 dead cells treated with camptothecin original drug and camptothecin polypeptide nanoformulation is comparable, which fully proves that the morphologically variable camptothecin polypeptide nanoformulation designed by the present invention has a good killing effect on cancer cells.

[0138] Example 7

[0139] This example verifies the anti-tumor effect of the camptothecin polypeptide nanoformulation prepared in Example 3.

[0140] Female Balb / C nude mice weighing 18-20 g and aged 6-8 weeks were randomly divided into three groups, with 8 mice in each group. 5×10 4 A549 cells, and when the tumor volume is about 100 mm 3 Start the experiment.

[0141] A camptothecin polypeptide nanoformulation without morphological transformation but with the same amino acid sequence was used as a control sample. The morphologically transformable camptothecin polypeptide nanoformulation, the control sample, or PBS was injected into mice via tail vein every 2 days (day 1, 4, 7, 10, and 13) (~9 mg / kg sample, administration volume of 200 μL). During the treatment, the body weight of mice in each group was recorded every 2 days, and the tumor volume was measured with a vernier caliper. The tumor volume was calculated according to the following formula: V = (W 2 × L) / 2, where W is the width of the tumor and L is the length of the tumor. The data were recorded for 24 consecutive days. The tumor growth curve was drawn with tumor volume as the ordinate and time as the abscissa. The results are shown in the figure below. Figure 15 shown.

[0142] Depend on Figure 15 It can be seen that the tumor volume of mice administered with the variable-morphology camptothecin polypeptide nanoformulation is the smallest, indicating that the nanofibers formed by the variable-morphology camptothecin polypeptide nanoformulation under the slightly acidic conditions of the tumor microenvironment improve the retention effect of camptothecin in cancer cells, thereby enhancing the anti-tumor effect, so as to achieve the purpose of reducing the dosage concentration and reducing toxic side effects, proving that the anti-tumor effect of camptothecin polypeptide nanoformulation can be enhanced by morphological transformation properties.

[0143] From the results of the above examples, it can be seen that the variable-morphology camptothecin polypeptide nanoformulation provided by the present invention has acid responsiveness, specificity and morphological transformation. Under normal physiological conditions, the nanoparticle structure is maintained to ensure its permeability at the tumor site. Under the slightly acidic conditions of the tumor microenvironment, the camptothecin polypeptide nanoformulation is transformed from nanoparticles to nanofibers, which can improve the retention capacity of camptothecin and increase its concentration at the tumor site, thereby reducing the dosage concentration and achieving efficient killing of cancer cells. The preparation method is mature and efficient and has broad application prospects.

[0144] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention. Sequence Listing <110> Anhui University of Traditional Chinese Medicine <120> A morphologically variable camptothecin polypeptide nanoformulation, preparation method, and application thereof <160> 18 <170> SIPOSequenceListing 1.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <400> 1 Val Arg Cys Glu Val 1 5 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Val Arg Val Glu Cys Arg Val Glu Val 1 5 <210> 3 <211> 13 <212> PRT <213> Artificial Sequence <400> 3 Val Arg Val Glu Val Arg Cys Glu Val Arg Val Glu Val 1 5 10 <210> 4 <211> 5 <212> PRT <213> Artificial Sequence <400> 4 Val Arg Cys Asp Val 1 5 <210> 5 <211> 9 <212> PRT <213> Artificial Sequence <400> 5 Val Arg Val Asp Cys Arg Val Asp Val 1 5 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <400> 6 Val Arg Val Asp Val Arg Cys Asp Val Arg Val Asp Val 1 5 10 <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <400> 7 Val Lys Cys Glu Val 1 5 <210> 8 <211> 13 <212> PRT <213> Artificial Sequence <400> 8 Val Lys Val Glu Val Lys Cys Glu Val Lys Val Glu Val 1 5 10 <210> 9 <211> 5 <212> PRT <213> Artificial Sequence <400> 9 Val Lys Cys Asp Val 1 5 <210> 10 <211> 9 <212> PRT <213> Artificial Sequence <400> 10 Val Lys Val Asp Cys Lys Val Asp Val 1 5 <210> 11 <211> 13 <212> PRT <213> Artificial Sequence <400> 11 Val Lys Val Asp Val Lys Cys Asp Val Lys Val Asp Val 1 5 10 <210> 12 <211> 13 <212> PRT <213> Artificial Sequence <400> 12 Val His Val Asp Val His Cys Asp Val His Val Asp Val 1 5 10 <210> 13 <211> 9 <212> PRT <213> Artificial Sequence <400> 13 Val Lys Val Glu Cys Lys Val Glu Val 1 5 <210> 14 <211> 13 <212> PRT <213> Artificial Sequence <400> 14 Val His Val Glu Val His Cys Glu Val His Val Glu Val 1 5 10 <210> 15 <211> 9 <212> PRT <213> Artificial Sequence <400> 15 Val His Val Glu Cys His Val Glu Val 1 5 <210> 16 <211> 9 <212> PRT <213> Artificial Sequence <400> 16 Val His Val Asp Cys His Val Asp Val 1 5 <210> 17 <211> 5 <212> PRT <213> Artificial Sequence <400> 17 Val His Cys Glu Val 1 5 <210> 18 <211> 5 <212> PRT <213> Artificial Sequence <400> 18 Val His Cys Asp Val 1 5

Claims

1. A camptothecin polypeptide nano - preparation with variable morphology, characterized in that, The camptothecin polypeptide nano - preparation comprises a compound shown in the following structural formula: 。 2. The camptothecin polypeptide nano - preparation according to claim 1, wherein, The morphology of the variable - morphology camptothecin polypeptide nano - preparation changes from nanoparticles to nanofibers under the slightly acidic conditions of the tumor microenvironment. Among them, the slightly acidic condition is an aqueous solution with a pH of 6.1 - 6.

9. The diameter of the nanoparticles is 30 - 100 nm. The length of the nanofibers is greater than or equal to 500 nm. The width of the nanofibers is 10 - 60 nm. The conformation of the nanofibers includes any one or a combination of two of α - helix, β - sheet or β - hairpin.

3. A preparation method of the variable - morphology camptothecin polypeptide nano - preparation according to claim 1 or 2, characterized in that The preparation method includes: Synthesize a camptothecin derivative and an amphiphilic polypeptide; the amino acid sequence of the amphiphilic polypeptide is shown in SEQ ID NO14, and the structural formula of the camptothecin derivative is as follows: Couple the camptothecin derivative with the amphiphilic polypeptide to obtain a camptothecin polypeptide. Self - assemble the camptothecin polypeptide under neutral conditions to obtain a variable - morphology camptothecin polypeptide nano - preparation.

4. According to the preparation method of the camptothecin polypeptide nano - preparation described in claim 3, characterized in that The amphiphilic polypeptide molecule is synthesized by solid - phase synthesis technology. The camptothecin derivative uses camptothecin as a basic reactant, reacts with triphosgene in the presence of an organic base to generate an acyl chloride intermediate, and the acyl chloride intermediate then reacts with 2 - [2 - (pyridyl) disulfanyl] ethanol to obtain the camptothecin derivative.

5. According to the preparation method of the camptothecin polypeptide nano - preparation described in claim 4, characterized in that The camptothecin derivative is coupled with the amphiphilic polypeptide molecule by a thiol - disulfide exchange reaction technology, which specifically includes the following steps: Using anhydrous DMSO as a solvent, mix the camptothecin derivative and the amphiphilic polypeptide under a nitrogen atmosphere, stir at room temperature for reaction, after the reaction is complete, filter to remove solid impurities, then add cold ether to precipitate the product, wash and dry to obtain the camptothecin polypeptide.

6. According to the preparation method of the camptothecin polypeptide nano - preparation described in claim 3, characterized in that The non - covalent interaction forces for the self - assembly of the camptothecin polypeptide include any one or a combination of at least two of hydrogen bonds, hydrophobic interactions, π - π interactions or van der Waals forces. The self - assembly of the camptothecin polypeptide is obtained by annealing the camptothecin polypeptide under neutral conditions, naturally cooling to room temperature and standing still. Among them, The neutral condition is an aqueous solution with a pH of 7.3 - 7.

6. The annealing temperature is 75 - 85 °C, and the annealing time is 25 - 35 min. The standing time is 10 - 15 h.

7. An application of the camptothecin polypeptide nano - preparation according to claim 1 or 2 or the camptothecin polypeptide nano - preparation prepared by the preparation method described in any one of claims 4 - 6 in the preparation of a drug against A549 cells; The administration methods of the camptothecin polypeptide nano - preparation include any one of tail - vein injection, subcutaneous injection or intraperitoneal injection.

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