Amino acid coded supramolecular self-assembly system as well as preparation method and application thereof

Abstract

The invention discloses an amino acid coded supramolecular self-assembly system as well as a preparation method and application thereof. The amino acid coded supramolecular self-assembly system is formed by self-assembly induced by a reducible oligopeptide derivative through glutathione (GSH); wherein the reducible oligopeptide derivative has a structure as shown in at least any one of the following formulas: # imgabs0. The invention successfully develops an amino acid-coded supramolecular self-assembly system, a targeting anti-tumor prodrug and a nano delivery carrier system, and can be widely applied to the field of biomedicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug delivery and cancer imaging and treatment, and specifically relates to an amino acid-encoded supramolecular self-assembly system and a preparation method and application thereof. Background Art

[0002] Supramolecular self-assembly is a process of spontaneously forming a wide range of highly ordered and complex structures through non-covalent interactions, such as biofilms, polymers, and chromosomes (Science 1991, 254, 5036, 1312-1319). The use of self-assembly strategies to prepare large-scale functional biomaterials with simple small molecule building blocks, this "bottom-up" strategy simplifies the manufacturing process, shortens the synthesis time, and successfully generates a variety of nanostructures, thereby promoting the development of innovative and intelligent probes, drugs, and scaffolds for efficient disease diagnosis, treatment, and post-disease rehabilitation (Chem. Soc. Rev. 2008, 37, 8, 1520-1529). Peptides, especially short peptides, have become attractive candidates for self-assembly building blocks due to their impressive biocompatibility and biodegradability, as well as easy modification and design characteristics (Angew. Chem. Int. Ed. 2022, 61, 5, e202113403).

[0003] Given the impact of morphology, mechanical strength and working mechanism on the performance of self-assembled nanocarriers, constructing a building block platform that can effectively regulate all these key factors is essential for rapid screening of the best molecules for self-assembly and subsequent biological applications (Nat. Nanotechnol. 2016, 11, 7, 585-592). The assembly process of short peptides is mainly determined by non-covalent bonding forces, showing monotonous mechanical properties and limited biological responsiveness (Science 2003, 300, 5619, 625-627). Summary of the invention

[0004] The main purpose of the present invention is to provide an amino acid-encoded supramolecular self-assembly system and a preparation method and application thereof, so as to overcome the deficiencies of the prior art.

[0005] In order to achieve the above-mentioned invention object, the technical solution adopted by the present invention includes:

[0006] The embodiment of the present invention provides an amino acid-encoded supramolecular self-assembly system, wherein the supramolecular self-assembly system is formed by self-assembly induced by GSH reduction of a reducible short peptide derivative; wherein the reducible short peptide derivative has a structure shown in at least one of the following formulae:

[0007]

[0008] The present invention also provides a method for preparing the aforementioned amino acid-encoded supramolecular self-assembly system, which comprises:

[0009] After being reduced by GSH, the reducible short peptide derivatives are assembled to obtain an amino acid-encoded supramolecular self-assembly system.

[0010] The embodiments of the present invention also provide the use of the aforementioned amino acid-encoded supramolecular self-assembly system in bioluminescence imaging, tracing or drug targeted delivery.

[0011] The embodiment of the present invention further provides a targeted anti-tumor prodrug, wherein the targeted anti-tumor prodrug has a structure shown in at least one of the following formulae:

[0012]

[0013] The embodiment of the present invention further provides a nano delivery carrier system, which includes a delivery carrier and a drug loaded on the delivery carrier, wherein the delivery carrier is self-assembled by a reducible short peptide derivative, and the reducible short peptide derivative has a structure shown in at least one of the following formulas:

[0014]

[0015] The embodiment of the present invention further provides a method for preparing the aforementioned nano delivery carrier system, which comprises: co-assembling a reducible short peptide derivative with a drug to form the nano delivery carrier system.

[0016] The embodiment of the present invention further provides a bioluminescent probe, which includes the aforementioned amino acid-encoded supramolecular self-assembly system.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention has successfully developed a tumor-specific delivery, GSH reduction-induced intracellular nanomaterial self-assembly prodrug, wherein CLCBT, CFCBT, CCBT can activate the immune system by inducing tumor cell pyroptosis, CECBT and CKCBT can be used as carriers to improve the performance of loaded drugs, and the biodegradation of nanocarriers produced by CKCBT reduction condensation self-assembly can be used for persistent bioluminescence. These prodrugs have great potential in tumor imaging and treatment. By limiting the thiol or amino group in Cys with other stimulus reaction sites and modifying X with probes or drugs, various multi-morphological and multifunctional nanomedicines can be easily prepared and widely used in the biomedical field. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 Schematic diagram of the use of Cys(SEt)-X-CBT for tumor treatment, drug loading and tumor imaging in a typical embodiment of the present invention;

[0020] Figure 2 This is a diagram showing the hydrogen spectrum analysis result of CECBT in Example 1 of the present invention;

[0021] Figure 3 This is a carbon spectrum analysis result diagram of CECBT in Example 1 of the present invention;

[0022] Figure 4 This is a diagram showing the high-resolution mass spectrometry analysis results of CECBT in Example 1 of the present invention;

[0023] Figure 5 This is a diagram showing the hydrogen spectrum analysis result of CKCBT in Example 1 of the present invention;

[0024] Figure 6 This is a carbon spectrum analysis result diagram of CKCBT in Example 1 of the present invention;

[0025] Figure 7 This is a diagram showing the high-resolution mass spectrometry analysis results of CKCBT in Example 1 of the present invention;

[0026] Figure 8 This is a diagram showing the hydrogen spectrum analysis result of CLCBT in Example 1 of the present invention;

[0027] Fig. 9 This is a graph showing the carbon spectrum analysis results of CLCBT in Example 1 of the present invention;

[0028] Fig.10 This is a diagram showing the high-resolution mass spectrometry analysis results of CLCBT in Example 1 of the present invention;

[0029] Fig.11 This is a diagram showing the hydrogen spectrum analysis result of CFCBT in Example 1 of the present invention;

[0030] Fig.12 This is a carbon spectrum analysis result diagram of CFCBT in Example 1 of the present invention;

[0031] Fig.13 This is a diagram showing the high-resolution mass spectrometry analysis results of CFCBT in Example 1 of the present invention;

[0032] Fig.14 This is a diagram showing the hydrogen spectrum analysis result of CCBT in Example 1 of the present invention;

[0033] Fig.15 This is a carbon spectrum analysis result diagram of CCBT in Example 1 of the present invention;

[0034] Fig.16 This is a diagram showing the high-resolution mass spectrometry analysis results of CCBT in Example 1 of the present invention;

[0035] Fig.17 The transmission electron microscope image, scanning electron microscope image, ultraviolet absorption spectrum and dynamic light scanning measurement diagram of the nanostructure in Example 2 of the present invention;

[0036] Fig.18 This is a schematic diagram of bioluminescence of CECBT, CKCBT, CLCBT, CFCBT and CCBT in Example 3 of the present invention;

[0037] Fig.19 The bioluminescence images of CECBT, CKCBT, CLCBT, CFCBT and CCBT in 4T1 tumor cells in Example 3 of the present invention are shown;

[0038] Fig. 20 The bioluminescence images of CECBT, CKCBT, CLCBT, CFCBT and CCBT in the animal model in Example 3 of the present invention;

[0039] Fig.21 The killing of tumor cells and the regularity diagram of CLCBT, CFCBT and CCBT in Example 4 of the present invention;

[0040] Fig. 22 This is a verification diagram of cell pyroptosis induced by CLCBT, CFCBT and CCBT in Example 5 of the present invention;

[0041] Fig.23 The biological electron microscopy images of CLCBT-CONH2 and CLCBT in Example 6 of the present invention;

[0042] Fig.24 The diagram is a diagram showing the therapeutic effect and biocompatibility evaluation of the CLCBT animal model in Example 7 of the present invention;

[0043] Fig.25 This is a diagram showing the immune function test of the CLCBT animal model in Example 8 of the present invention;

[0044] Fig.26 The electron microscope images, fluorescence change images and intermolecular force calculation images of CECBT and CKCBT loaded with NR and Ce6 in Example 9 of the present invention are shown;

[0045] Fig. 27This is a test diagram of the effect of DOX delivery by CECBT, CKCBT, CLCBT, CFCBT and CCBT on the ability of DOX to kill MDA-MB-231 cells in Example 10 of the present invention;

[0046] Fig.28 This is the cell imaging picture of CKCBT co-assembled with NR or Ce6 in Example 11 of the present invention.

[0047] Fig.29 This is a diagram showing the therapeutic effect of the animal model co-assembled with CKCBT and Ce6 in Example 12 of the present invention. DETAILED DESCRIPTION

[0048] In view of the defects of the prior art, the inventor of this case has proposed the technical solution of the present invention after long-term research and extensive practice. The technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0049] Specifically, as one aspect of the technical solution of the present invention, an amino acid-encoded supramolecular self-assembly system is formed by self-assembly induced by GSH reduction of a reducible short peptide derivative (denoted as: Cys(SEt)-X-CBT); wherein the reducible short peptide derivative has a structure shown in at least one of the following formulas:

[0050]

[0051] Another aspect of the embodiments of the present invention further provides a method for preparing the aforementioned amino acid-encoded supramolecular self-assembly system, which comprises:

[0052] After being reduced by GSH, the reducible short peptide derivatives are assembled to obtain an amino acid-encoded supramolecular self-assembly system.

[0053] In some preferred embodiments, the Cys(SEt)-X-CBT includes: Cys(SEt)-Glu-CBT (CECBT), Cys(SEt)-Lys-CBT (CKCBT), Cys(SEt)-Leu-CBT (CLCBT), Cys(SEt)-Phe-CBT (CFCBT) and Cys(SEt)-CBT (CCBT).

[0054] The structures of the five compounds are shown below:

[0055]

[0056] The preparation processes of the five compounds are as follows:

[0057] Synthesis of CECBT:

[0058]

[0059] Compound A was synthesized using a standard solid phase peptide synthesis (SPPS) protocol and purified using high performance liquid chromatography (HPLC). At 0°C, lyophilized compound A (93 mg, 0.2 mmol) was dissolved in 2 mL of tetrahydrofuran (THF), and isobutyl chloroformate (IBCF, 27 mg, 0.14 mmol) and 4-methylmorpholine (MMP, 74 mg, 0.7 mmol) were added to the THF mixed with A, and the reaction mixture was stirred for 30 minutes. 2-Cyanobenzothiazole (CBT, 53 mg, 0.3 mmol) and additional IBCF (13.5 mg, 0.07 mm1) were then dissolved in THF and added to the mixture of A. The mixture was stirred at 0°C for 8 h, then stirred at room temperature overnight, and compound B was obtained after removing THF by rotary evaporation. B was added to a solution prepared by mixing 9.5 mL of trifluoroacetic acid (TFA), 5% dichloromethane (DCM, 0.5 mL) and triisopropylsilane (TIPS, 40 μL), and stirred for 3 h to remove the Boc group of B. The solution was purified by HPLC to obtain CECBT.

[0060] Synthesis of CKCBT:

[0061]

[0062] Compound C was synthesized using the standard SPPS protocol and purified by HPLC. IBCF (27 mg, 0.14 mmol) was added to a mixture of compound C (101.8 mg, 0.2 mmol) and MMP (74 mg, 0.7 mmol) and 2 mL of THF at 0°C and stirred for 30 minutes. A solution of CBT (62 mg, 0.35 mmol) and additional IBCF (13.5 mg, 0.07 mmol) were added to the mixture, stirred for 8 h, and then stirred overnight at room temperature to obtain D. The Boc group of D was removed with 9.5 mL of TFA, 5% DCM (0.5 mL) and TIPS (40 μL) (reaction 3 h) and purified by HPLC to obtain CKCBT.

[0063] Synthesis of CLCBT:

[0064]

[0065] Compound E was synthesized using the SPPS scheme, purified by HPLC and freeze-dried. At 0°C, THF (2mL) dissolved compound E (78.8mg, 0.2mmol), then IBCF (27mg, 0.14mmol) and MMP (74mg, 0.7mmpl) were added to the THF mixed with E, and the reaction mixture was stirred for 30 minutes. CBT (53mg, 0.3mmpl) and additional IBCF (13.5mg, 0.07mmol) were then dissolved in THF and added to the mixture of E. The mixture was stirred at 0°C for 8h, then stirred at room temperature overnight, and THF was removed by rotary evaporation to obtain compound F. F was added to a solution prepared by mixing 9.5mL TFA with 5% DCM (0.5mL) and TIPS (40μL), stirred for 3h to remove the Boc group of F, and purified by HPLC to obtain CLCBT.

[0066] Synthesis of CFCBT:

[0067]

[0068] Compound G was synthesized using the SPPS scheme, purified by HPLC and freeze-dried. At 0°C, 2 mL of THF dissolved compound G (85.6 mg, 0.2 mmol), then IBCF (27 mg, 0.14 mmol) and MMP (74 mg, 0.7 mmol) were added to the THF mixed with G, and the reaction mixture was stirred for 30 minutes. CBT (53 mg, 0.3 mmol) and another IBCF (13.5 mg, 0.07 mmol) were then dissolved in THF and added to the mixture of G. The mixture was stirred at 0°C for 8 h, then stirred at room temperature overnight, and THF was removed by rotary evaporation to obtain compound H. H was added to a solution of 9.5 mL of TFA mixed with 5% DCM (0.5 mL) and TIPS (40 μL), stirred for 3 h to remove the Boc group of H, and purified by HPLC to obtain CFCBT.

[0069] Synthesis of CCBT:

[0070]

[0071] At 0°C, 2 mL of THF dissolved compound I (56.2 mg, 0.2 mmol), then IBCF (27 mg, 0.14 mmol) and MMP (74 mg, 0.7 mmol) were added to the THF mixed with I, and the reaction mixture was stirred for 30 minutes. Then CBT (53 mg, 0.3 mmol) and another IBCF (13.5 mg, 0.07 mmol) were dissolved in THF and added to the mixture of I. The mixture was stirred at 0°C for 8 h, then stirred at room temperature overnight, and THF was removed by rotary evaporation to obtain compound J. J was added to a solution of 9.5 mL TFA mixed with 5% DCM (0.5 mL) and TIPS (40 μL), stirred for 3 h to remove the Boc group of J, and purified by HPLC to obtain CCBT.

[0072] Another aspect of the embodiments of the present invention further provides the use of the aforementioned amino acid-encoded supramolecular self-assembly system in bioluminescence imaging, tracing or targeted drug delivery.

[0073] Another aspect of the embodiments of the present invention further provides a targeted anti-tumor prodrug, wherein the targeted anti-tumor prodrug has a structure shown in at least one of the following formulae:

[0074]

[0075] In some preferred embodiments, the targeted anti-tumor prodrug can self-assemble into nanoparticles or nanofibers under the action of GSH.

[0076] Another aspect of the embodiments of the invention further provides a nano delivery carrier system, which includes a delivery carrier and a drug loaded on the delivery carrier, wherein the delivery carrier is formed by self-assembly of a reducible short peptide derivative, and the reducible short peptide derivative has a structure shown in at least one of the following formulas:

[0077]

[0078] In some preferred embodiments, the drug can be any drug and probe that can be co-assembled, such as an antibody.

[0079] In some preferred embodiments, the drug includes any one or more combinations of fluorescent dyes, photosensitizers, contrast agents, imaging agents, and drugs, but is not limited thereto.

[0080] For example, cyanine dyes such as indocyanine green and Cy5.5; photosensitizers such as dihydrochlorin e6 and tetraphenylporphyrin; MRI contrast agents such as gadopentetate dimeglumine, gadoteric acid dimeglumine, gadobutrol, gadomaltitol and gadoversenic acid; PET imaging agents such as fluorodeoxyglucose; 99mSPECT imaging agents such as Tc-methoxyisobutyl isocyanate; chemotherapy drugs such as doxorubicin (DOX), cisplatin, paclitaxel, camptothecin and etoposide; immunotherapy drugs such as pembrolizumab and nivolumab; radiotherapy sensitizers such as sodium glycidazole; anti-rheumatic drugs such as methotrexate and sulfasalazine; anti-inflammatory drugs such as dexamethasone and indomethacin; antibacterial drugs such as penicillin and cephalosporin.

[0081] Another aspect of an embodiment of the present invention further provides a method for preparing the aforementioned nano delivery carrier system, which comprises: co-assembling a reducible short peptide derivative with a drug to form the nano delivery carrier system.

[0082] Another aspect of the embodiments of the present invention further provides a bioluminescent probe, which includes the aforementioned amino acid-encoded supramolecular self-assembly system.

[0083] In the present invention, a reduction-triggered disulfide-protected Cys(R)-X-CBT, i.e., Cys(SEt)-X-CBT, is proposed to demonstrate the controllable formation of multi-morphological nanostructures and the intracellular self-assembly of GSH-induced multifunctional nanocarriers for biomedical applications. Four representative amino acids, Glu, Lys, Leu, and Phe, are selected as X to be inserted between Cys(SEt) and CBT. Glu and Lys represent hydrophilic amino acids, and at pH 7.4, Glu has better water solubility than Lys. Leu and Phe represent hydrophobic amino acids, and the aromatic peptide side chain of Phe confers additional intermolecular π-π interactions. The reduction of the Cys protecting group SEt on Cys(SEt)-X-CBT leads to the formation of Cys-X-CBT. Subsequently, when X is Glu or Lys, CBT-Cys condensation reaction occurs between Cys-X-CBT molecules, leading to the self-assembly of nanovesicles, pearl necklace-like nanostructures, large nanoparticles (compared to the latter), nanowires, and small nanoparticles.

[0084] Bioluminescence is a powerful technique for noninvasive monitoring of biological processes and cell transplantation. Nevertheless, D-luciferin, which is widely used as a bioluminescent probe, is limited in long-term in vivo tracking due to its short half-life. Here, we present a novel approach to accumulate and preserve luciferin inside tumor cells via a supramolecular self-assembly strategy using amino acid-encoded building blocks. We successfully developed a novel amino acid-encoded supramolecular self-assembly system, Cys(SEt)-X-CBT, for the generation of various nanomaterials with different morphologies and bioluminescent properties. GSH reduction-induced self-assembly of Cys(SEt)-X-CBT enabled luciferin to accumulate and be preserved inside tumor cells, thereby providing tumor-targeted bioluminescence enhancement. The Cys(SEt)-X-CBT building block platform utilized a covalent-noncovalent hybrid self-assembly mechanism to generate a variety of luciferin-containing nanostructures in tumor cells after GSH reduction, which exhibited effective tumor-targeted delivery as well as sequence-dependent well-designed morphologies and prolonged bioluminescent properties. Among the selected amino acids (X = Glu, Lys, Leu, Phe), CKCBT exhibited the highest bioluminescent signal, the longest sustained luminescence (72 hours) and excellent biocompatibility in living cells and in orthotopic breast tumor model mice. The amino acid-encoded supramolecular self-assembly of the present invention provides a promising approach for designing programmable bioluminescent probes with customized bioluminescent properties for long-term biological tracking and disease imaging.

[0085] Tumor immunotherapy is revolutionizing the clinical treatment strategies and prognosis of various tumors and is an effective means of tumor treatment. However, the low objective response rate limits its wide application. Therefore, it is necessary to develop new methods to induce a stronger anti-tumor immune response. The present invention proposes a new strategy, using CLCBT, CFCBT and CCBT to form a nanomaterial that integrates the advantages of polycarbonyl cationic charge and hydrophobic self-assembling peptide lipophilicity, targets tumor cell mitochondria, and causes cell pyroptosis through mitochondrial function damage, thereby achieving the effect of inhibiting tumor growth and stimulating anti-tumor immunity. Under the action of abundant GSH in tumor cells, CLCBT, CFCBT and CCBT can self-assemble into nanostructures in situ and target mitochondria to cause mitochondrial structural destruction and functional damage, and then activate Casepase 9 and Casepase 3 in turn, and then cut GSDME, triggering cell pyroptosis, leading to the release of pattern damage-associated molecular patterns (DAMPs), promoting dendritic cell (DC) activation, activating effector T cell activation, and secreting effector cytokines, acting on tumor cells, and finally achieving a strong anti-tumor immune function and successfully inhibiting tumor proliferation. This invention provides a new idea for inducing powerful anti-tumor immunotherapy, and because short peptide derivatives are easy to generate and synthesize and have good biosafety, they provide a basis for later clinical transformation.

[0086] In situ co-assembly of self-assembling peptides and their derivatives with loaded drugs provides a promising strategy for improving drug efficacy and introducing multifunctional components. Nevertheless, the development of a simple, widely applicable and practical nanocarrier system to effectively improve drug performance and promote practical applications still faces great challenges. In the present invention, two reducible short peptide derivatives based on CBT-Cys click reaction were prepared for the delivery of fluorescent dye Nile red (NR) and photosensitizer (Ce6). The nanostructures formed by co-assembly exhibited multiple favorable features for tumor imaging and treatment, including adjustable morphology, enhanced cellular uptake and biodistribution. Ultimately, these nanostructures showed excellent performance at the cellular and animal levels, achieving enhanced tumor fluorescence imaging and significant anti-tumor effects by single laser irradiation and single administration. The present invention proposes a direct and powerful strategy to develop supramolecular delivery systems by in situ co-assembly of CBT-Cys peptide derivatives. We designed two biocompatible and reducible hydrophilic peptide derivatives, CECBT and CKCBT, for the delivery of lipid metabolism-related dye NR and photosensitizer Ce6, respectively. By directly incorporating drugs as nanostructure building blocks, drug loading efficiency was improved. The in situ co-assembly approach enhanced drug targeting, prolonged circulation time, and regulated intermolecular and intramolecular interactions. As a result, it enhanced the fluorescence signal of NR and improved the anticancer effect of Ce6. By modifying the amino or thiol groups on Cys and attaching various drugs and probes to the side chains of the intermediate amino acids, a convenient and biocompatible platform based on CBT-Cys peptide derivatives was provided. Through co-assembly with drug molecules, this platform can form well-defined nanostructures, providing a wide range of application value for nano-delivery of various drugs and enhancing the imaging and therapeutic effects of various diseases. These results indicate that CKCBT can effectively deliver NR and Ce6 to tumor sites, resulting in significantly higher fluorescence signals and minimal accumulation in non-target organs.

[0087] The present invention develops an amino acid-encoded supramolecular self-assembly system to combine covalent and non-covalent interactions to construct nanostructures of different morphologies with bioinspired response mechanisms. These materials have positive effects in tumor imaging, tumor treatment and drug delivery ( Figure 1 ).

[0088] The biodegradation of the nanocarriers produced by Cys(SEt)-X-CBT can be easily tracked by bioluminescence imaging for tumor imaging. Among them, CKCBT exhibited excellent long-lasting bioluminescence signal (up to 72 h) and good biocompatibility.

[0089] The clinical results of chemotherapy and immunotherapy are usually limited by low objective response rates and systemic adverse reactions. Compared with conventional chemotherapy combined with immunotherapy, the tumor-specific pyroptosis inducer-stimulated immunotherapy prodrug (CLCBT, CFCBT, CCBT) developed in the present invention has the following advantages: (1) The prodrug stimulates immunity by inducing tumor cell pyroptosis (a violent form of death), which can turn immune "cold" tumors into "hot" tumors, while enhancing the effect of immunotherapy; (2) The mitochondrial targeting ability of the prodrug and the design that requires GSH activation enable the prodrug to have tumor targeting ability and can reduce other systemic side effects. (3) After GSH reduction, the prodrug is condensed into a polymer, and then self-assembles into large nanomaterials in situ in tumor cells through intermolecular interactions such as π-π stacking, thereby prolonging its accumulation at the tumor site.

[0090] There are still great challenges in developing a simple, widely applicable and practical nanocarrier system to effectively improve drug performance and promote practical applications. The two reducible short peptide derivatives CECBT and CKCBT based on CBT-Cys click reaction in the present invention are used to deliver fluorescent dye Nile red (NR) and photosensitizer (Ce6). The nanostructures formed by co-assembly show a variety of favorable features for tumor imaging and treatment, including adjustable morphology, enhanced cellular uptake and biodistribution. Ultimately, these nanostructures show excellent performance at the cellular and animal levels, achieving enhanced tumor fluorescence imaging and significant anti-tumor effects through single laser irradiation and single administration.

[0091] In summary, the present invention has developed a tumor-specific delivery, GSH reduction-induced intracellular nanomaterial self-assembly prodrug. Among them, CLCBT, CFCBT, CCBT can activate immunotherapy by inducing tumor cell pyroptosis, CECBT and CKCBT can be used as carriers to improve the performance of loaded drugs, and the biodegradation release of the nanocarrier produced by the reduction condensation self-assembly of CKCBT can be used for persistent bioluminescence. These prodrugs have great potential in tumor imaging and treatment. By limiting the thiol or amino group in Cys with other stimulus reaction sites and modifying X with probes or drugs, various multi-morphological and multifunctional nanomedicines can be easily prepared and widely used in the biomedical field.

[0092] The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and drawings. This embodiment is implemented on the premise of the technical solution of the invention, and a detailed implementation method and specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0093] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.

[0094] Example 1

[0095] Synthesis of CECBT:

[0096]

[0097] Compound A was synthesized using the standard SPPS protocol and purified by HPLC. At 0°C, lyophilized compound A (93 mg, 0.2 mmol) was dissolved in 2 mL of THF, and IBCF (27 mg, 0.14 mmol) and MMP (74 mg, 0.7 mmol) were added to the THF mixed with A, and the reaction mixture was stirred for 30 minutes. CBT (53 mg, 0.3 mmol) and additional IBCF (13.5 mg, 0.07 mmol) were then dissolved in THF and added to the mixture of A. The mixture was stirred at 0°C for 8 h, then stirred at room temperature overnight, and compound B was obtained after THF was removed by rotary evaporation. B was added to a solution prepared by mixing 9.5 mL of TFA with 5% DCM (0.5 mL) and TIPS (40 μL), stirred for 3 h to remove the Boc group of B, and purified by HPLC to obtain CECBT; Figure 2 , Figure 3 , Figure 4 They are the hydrogen nuclear magnetic resonance spectrum, carbon spectrum and high-resolution mass spectrum of CECBT;

[0098] Synthesis of CKCBT:

[0099]

[0100] Compound C was synthesized using the standard SPPS protocol and purified by HPLC. At 0°C, IBCF (27 mg, 0.14 mmol) was added to a mixture of compound C (101.8 mg, 0.2 mmol) and MMP (74 mg, 0.7 mmol) and 2 mL of THF, and the reaction was stirred for 30 minutes. A solution of CBT (62 mg, 0.35 mmol) and additional IBCF (13.5 mg, 0.07 mmol) were added to the mixture, and stirring was continued for 8 h, and then stirred at room temperature overnight to obtain D. The Boc group of D was removed with 9.5 mL of TFA, 5% DCM (0.5 mL) and TIPS (40 μL) (reaction 3 h) and then purified by HPLC to obtain CKCBT; Figure 5 , Figure 6 , Figure 7 They are the hydrogen nuclear magnetic resonance spectrum, carbon spectrum and high-resolution mass spectrum of CKCBT;

[0101] Synthesis of CLCBT:

[0102]

[0103] Compound E was synthesized using the SPPS scheme, purified by HPLC and freeze-dried. At 0°C, THF (2mL) dissolved compound E (78.8mg, 0.2mmol), and then IBCF (27mg, 0.14mmol) and MMP (74mg, 0.7mmol) were added to the THF mixed with E, and the reaction mixture was stirred for 30 minutes. Then 2-cyanobenzothiazole (CBT, 53mg, 0.3mmol) and additional IBCF (13.5mg, 0.07mmol) were dissolved in THF and added to the mixture of E. The mixture was stirred at 0°C for 8h, then stirred at room temperature overnight, and compound F was obtained after THF was removed by rotary evaporation. F was added to a solution prepared by mixing 9.5mL TFA with 5% DCM (0.5mL) and TIPS (40μL), stirred for 3h to remove the Boc group of F, and purified by HPLC to obtain CLCBT; Figure 8 , Fig. 9 , Fig.10 They are the hydrogen nuclear magnetic resonance spectrum, carbon spectrum and high-resolution mass spectrum of CLCBT;

[0104] Synthesis of CFCBT:

[0105]

[0106] Compound G was synthesized using the SPPS scheme, purified by HPLC and freeze-dried. At 0°C, 2 mL of THF was used to dissolve compound G (85.6 mg, 0.2 mmol), and then IBCF (27 mg, 0.14 mmol) and MMP (74 mg, 0.7 mmol) were added to the THF mixed with G, and the reaction mixture was stirred for 30 minutes. CBT (53 mg, 0.3 mmol) and another IBCF (13.5 mg, 0.07 mmol) were then dissolved in THF and added to the mixture of G. The mixture was then stirred at 0°C for 8 h, then stirred at room temperature overnight, and compound H was obtained after THF was removed by rotary evaporation. H was added to a solution prepared by mixing 9.5 mL of TFA with 5% DCM (0.5 mL) and TIPS (40 μL), stirred for 3 h to remove the Boc group of H, and purified by HPLC to obtain CFCBT; Fig.11 , Fig.12 , Fig.13 They are the hydrogen nuclear magnetic resonance spectrum, carbon spectrum and high-resolution mass spectrum of CFCBT;

[0107] Synthesis of CCBT:

[0108]

[0109] At 0°C, 2 mL of THF dissolved compound I (56.2 mg, 0.2 mmol), then IBCF (27 mg, 0.14 mmol) and MMP (74 mg, 0.7 mmol) were added to the THF mixed with I, and the reaction mixture was stirred for 30 minutes. Then CBT (53 mg, 0.3 mmol) and another IBCF (13.5 mg, 0.07 mmol) were dissolved in THF and added to the mixture of I. The mixture was stirred at 0°C for 8 hours, then stirred at room temperature overnight, and THF was removed by rotary evaporation to obtain compound J. J was added to a solution prepared by mixing 9.5 mL of TFA with 5% DCM (0.5 mL) and TIPS (40 μL), stirred for 3 hours to remove the Boc group of J, and purified by HPLC to obtain CCBT; Fig.14 , Fig.15 , Fig.16 They are the nuclear magnetic resonance hydrogen spectrum, carbon spectrum and high-resolution mass spectrum of CCBT respectively.

[0110] Example 2

[0111] The transmission electron microscope images, scanning electron microscope images, ultraviolet absorption spectra and dynamic light scanning measurement images of the nanostructures formed after 100 μM CECBT (a), CKCBT (b), CLCBT (c), CFCBT (d) and CCBT (e) prepared in Example 1 were incubated with 400 μM in a buffer solution containing a reducing agent at 37°C for 1 hour. Fig.17 Shown; scale bar represents 200 nm.

[0112] Example 3

[0113] 4T1-fLuc cells (1×10 5 / well) and HCT116-fLuc cells (1×10 5 / well) were inoculated into 96-well plates and allowed to adhere overnight. Subsequently, the cell culture medium was removed and the cells were treated with Cys(SEt)-X-CBT at a concentration of 150μM (100μL culture medium / well). Among them, the inhibitor group incubated the cells with protease inhibitor cocktail (protease inhibitor cocktail, PIC, v / v, 1:100, i.e. 1μL 100X PIC diluted with 99μL saline) for half an hour before adding the probe. Animal imaging used an orthotopic breast cancer tumor model. 3×10 6 A cell suspension of 4T1-fLuc cells was injected under the fourth mammary pad on the right side of 6-week-old Balb / c mice to construct an orthotopic breast cancer model in mice. 3Cys(SEt)-X-CBT and NH2-Luc (50 μmol / kg, 2% DMSO and 2% Tween-80) were injected intraperitoneally into randomized tumor-bearing Balb / c mice. BLI images of cells were taken at different time points using an in vivo optical imaging system (ChemScope 6100, Clinx, Shanghai, China) equipped with a cooled charge-coupled device (CCD) camera. BL images of animals were obtained by taking pictures on another in vivo optical imaging system (SIIPreclinical, SINSAGE, USA). Animals were anesthetized using isoflurane gas (1.5%, 1 L / min). A circular region of interest was delineated in the tumor area on the luminescent image, and finally the BL intensity within the region of interest was quantitatively measured using Clinx software (cells) and Image J software (animals).

[0114] After the CECBT, CKCBT, CLCBT, CFCBT and CCBT prepared in Example 1 enter tumor cells, they are reduced by GSH, and then undergo a click condensation reaction to form oligomers and self-assemble to form nanostructures of different morphologies. The nanostructures are hydrolyzed by intracellular proteases to form fluorescein, thereby achieving bioluminescent imaging capabilities, wherein: Fig.18 Schematic diagram of bioluminescence of CECBT, CKCBT, CLCBT, CFCBT and CCBT; Fig.19 Bioluminescence images of CECBT, CKCBT, CLCBT, CFCBT and CCBT in 4T1 tumor cells; Fig. 20 Bioluminescence images of CECBT, CKCBT, CLCBT, CFCBT, and CCBT in animal models.

[0115] Example 4

[0116] Human breast cancer MDA-MB-231 cells and mouse breast cancer EMT-6 cells, and normal human mammary epithelial cells (MCF-10A cells and HBL-100 cells) were seeded in 96-well plates at a density of 5000 per well. After incubation for a predetermined time, different concentrations of Cys(SEt)-X-CBT (0 to 160 μM) were added to the corresponding wells. After incubation for a predetermined time, 10 μL of CCK-8 reagent was added to each well and incubated for 30 minutes. The absorbance value at 450 nm was detected using an enzyme-linked immunosorbent assay reader.

[0117] The effects of CLCBT, CFCBT, CCBT and the non-assembly control compound CLCBT-CONH2 on the cell viability of different breast cancer cell lines (MDA-MB-231 and EMT-6) were detected. The IC50 values ​​of CLCBT, CFCBT and CCBT in MDA-MB-231 cells were 18.81μM, 17.1μM and 68.89μM, respectively. In EMT-6 cells, they were 21.31μM, 15.42μM and 29.47μM, respectively. The IC50 values ​​of CLCBT-CONH2 on MDA-MB-231 and EMT-6 tumor cells were 294.3μM and 162.8μM, respectively. The effects of CLCBT, CFCBT and CCBT on the cell viability of normal human breast epithelial cells (MCF-10A cells and HBL-100 cells) were detected. The IC50 values ​​of CLCBT, CFCBT and CCBT molecules for MCF-10A and HBL-100 cells were 80.3μM, 101.2μM, 256.9μM and 60.8μM, 75.2μM, 117.6μM, respectively (e.g. Fig.21 ).

[0118] Example 5

[0119] EMT-6 cells and MDA-MB-231 cells incubated with PBS, 35μM CLCBT, 35μM CFCBT and 60μM CCBT for 12 hours were collected by centrifugation, and the cells were lysed with RIPA solution containing phenylmethylsulfonyl fluoride (1mM). The concentration of intracellular proteins was quantitatively determined by the bicinchoninic acid (BCA) method. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane. Next, the PVDF membrane was blocked with skim milk (wt, 5%). The PVDF membrane was incubated with the corresponding primary antibody for 12 hours (4°C). Subsequently, the PVDF membrane was rewarmed and the membrane was washed 3 times with PBS buffer containing Tween-20. The membrane was then incubated with the secondary antibody at room temperature for 1 hour, and finally with The CLx infrared imaging system was used to detect protein expression levels.

[0120] We examined whether CLCBT, CFCBT, and CCBT induced changes in the expression level of GSDME-N protein. Compared with the control compound (CLCBT-CONH2), GSDME was cleaved (e.g., Fig. 22 ).

[0121] Example 6

[0122] 35μM CLCBT and 35μM CLCBT-CONH2 were incubated with EMT-6 cells for 3 hours respectively. The treated cells were collected and washed three times with PBS by low-speed centrifugation. The collected EMT-6 cells were then resuspended in electron microscope fixative and fixed at room temperature for 1 hour. After that, the cells were fixed with 2.5% glutaraldehyde at 4°C for 12 hours. The cells were then fixed in 1% osmium tetroxide at room temperature for 1 hour. Subsequently, the cells were graded and dehydrated (ethanol) and embedded in epoxy resin materials. The embedded samples were then cut into 80nm slices with the help of a microtome, and then the samples were fished out with a copper mesh. The copper mesh was stained with uranyl acetate and lead citrate, and finally imaged under TEM.

[0123] Among them, CLCBT accumulated in mitochondria and destroyed mitochondrial structure, mitochondrial membrane fragmentation, mitochondrial cristae rupture or disappearance, while these phenomena were not observed in the biological electron microscope of the control compound CLCBT-CONH2 ( Fig.23 ).

[0124] Example 7

[0125] Will contain 3×10 6 The cell suspension of EMT-6 cells was injected under the fourth mammary pad of the right side of 6-week-old Balb / c mice (each mouse weighed about 18-20 grams) to construct an orthotopic breast cancer model in mice. On the 9th day after modeling (the tumor volume grew to about 80mm 3 ), PBS, CLCBT and CLCBT-CONH2 (injected once every 2 days, each injection of 22.5 mg / kg) were injected into the mice through the tail vein for 10 consecutive injections. The tumor volume and body weight of the mice were monitored every 2 days for 2 weeks. The tumor growth in the CLCBT group was detected by bioluminescence. On the 14th day, the mice were euthanized. The tumors of all mice were dissected and separated, and the dissected tumors were weighed and photographed. Among them, CLCBT showed good biocompatibility and excellent anti-tumor therapeutic effects. After CLCBT acted on the mouse orthotopic tumor model, it significantly inhibited tumor proliferation without causing significant changes in the mouse body weight, liver and kidney function and blood routine ( Fig.24 )

[0126] Example 8

[0127] On the 17th day after modeling, the tumors of the modeling Balb / c mice were dissected and cut into pieces, and then the tumors were fully digested and processed to a single cell suspension with a digestion working solution containing 2mg / mL collagenase D, 100μg / mL DNase I and 1% fetal bovine serum. The above suspension was filtered through a filter (diameter 70μm) and then treated with buffer for 5 minutes. After that, the single cell suspension was prepared by washing twice with PBS (containing 2% fetal bovine serum). The single cell suspension was incubated with anti-mouse CD16 / CD32 antibodies at 4°C for 30 minutes, and then incubated with anti-CD45, CD8-a and CD3 antibodies for 30 minutes. Dead cells were then excluded with fixed active stain 660 with added antibodies. Finally, the cells were washed twice and resuspended in PBS containing 2% fetal bovine serum for flow cytometry detection. Cytokine detection was performed using an enzyme-linked immunosorbent assay kit.

[0128] Among them, CLCBT changes the tumor immune environment, effectively promotes lymphocyte infiltration, secretes cytokines, and activates anti-tumor immunity ( Fig.25 )

[0129] Example 9

[0130] Among them, the morphology and fluorescence spectrum of the nanostructures formed by the co-assembly of CECBT or CKCBT with NR or Ce6 changed, and the existence of non-covalent forces in the co-assembly process was confirmed by first-principles calculations. After 100 μM CECBT or CKCBT was incubated with 10 μM NR or Ce6 in PBS containing a reducing agent for 1 hour at 37°C, the results of TEM and SEM image analysis showed that the co-incubation of CECBT with NR or Ce6 led to the formation of nanoparticles, while the co-incubation of CKCBT with NR or Ce6 led to the formation of nanofiber assemblies. CECBT, CKCBT, NR, Ce6, CECBT+NR, CKCBT+NR, CECBT+Ce6 and CKCBT+Ce6 (the equivalent concentration of CECBT or CKCBT was 100 μM, and the equivalent concentration of NR or Ce6 was 10 μM) were dissolved in PBS (2% DMSO) containing 400 μM TCEP. The mixture was then incubated at 37 °C for 1 hour to allow for proper interaction. Subsequently, the fluorescence spectra of the incubated samples were measured. For NR, the measurement range was from 625 nm to 800 nm, and the excitation wavelength was 550 nm. For Ce6, the measurement range was from 600 nm to 750 nm, and the excitation wavelength used was 410 nm. Compared with free NR, both CECBT and CKCBT showed significantly enhanced fluorescence after co-incubation with NR. Compared with free Ce6, CKCBT exhibited obvious aggregation fluorescence quenching effect after co-incubation with Ce6. First-principles calculations and MD simulation results indicate that the energy of the intermolecular non-bonded interaction between CKCBT and NR or Ce6 is stronger than that between CECBT and NR or Ce6, thereby promoting the co-assembly process ( Fig.26 ).

[0131] Example 10

[0132] In addition, the cell counting kit-8 (CCK-8) assay was used to evaluate the effects of DOX delivered by CECBT, CKCBT, CLCBT, CFCBT, and CCBT on the cytotoxicity of MDA-MB-231 cells. Cells growing in logarithmic phase were plated at 5 × 10 3 The cells were seeded at a density of 100 μL into a 96-well cell culture plate and incubated overnight. Then, 100 μL of DMEM medium containing 2 μM DOX and 20 μM CECBT, CKCBT, CLCBT, CFCBT, and CCBT were added to the corresponding wells and incubated with MDA-MB-231 cells for 12 hours. Subsequently, 10 μL of CCK-8 was added to each well and incubated with the cells for 1 hour. The absorbance at 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader (BioTek Synergy H1). Fig. 27 ).

[0133] Embodiment 11

[0134] The cell imaging capabilities of CKCBT, NR, Ce6, CKCBT+NR, and CKCBT+Ce6 were studied. First, 4T1 tumor cells (3×10 5 ) were inoculated into a 6-cm glass-bottom culture dish. After 12 hours, the culture medium was discarded, and the cells were incubated with fresh culture medium containing 100μM CKCBT, 10μM NR, 10μM Ce6, 100μM CKCBT+10μM NR and 100μM CKCBT+10μM Ce6 for 3 hours respectively. After the culture period, the cells were gently rinsed three times with PBS. The cells were then incubated with the nuclear dye 4′,6-diamidino-2-phenylindole for 30 minutes. Afterwards, the cells were gently rinsed again with PBS three times, and fixed with 4% paraformaldehyde at room temperature for 30 minutes. Finally, a fluorescent inverted microscope was used to take cell imaging pictures, and the fluorescent images were quantitatively analyzed using ImageJ software ( Fig.28 ).

[0135] Example 12

[0136] When the mammary tumor volume of the model mice grew to about 120 mm 3 Afterwards, the patients were randomly divided into 8 groups (n=6 in each group) and received the following treatments: (I) intratumoral injection of 100 μL NS; (II) intratumoral injection of 100 μL NS plus laser irradiation (660 nm, 0.6 W cm -2 , 10 min); (III) intratumoral injection of 100 μL NS (2% DMSO and 2% Tween-80) containing 2 mM CKCBT; (IV) intratumoral injection of NS (2% DMSO and 2% Tween-80) containing 2 mM CKCBT + laser irradiation (660 nm, 0.6 W cm -2 , 10 min); (V) intratumoral injection of 100 μL NS (2% DMSO and 2% Tween-80) containing 200 μM Ce6; (VI) intratumoral injection of 100 μL NS (2% DMSO and 2% Tween-80) containing 200 μM Ce6 + laser irradiation (660 nm, 0.6 W cm -2 , 10 min); (VII) intratumoral injection of 100 μL NS containing CKCBT+Ce6 (2 mM CKCBT and 200 μM Ce6, 2% DMSO and 2% Tween-80); (VIII) intratumoral injection of 100 μL NS containing CKCBT+Ce6 (2 mM CKCBT and 200 μM Ce6, 2% DMSO and 2% Tween-80) + laser irradiation (660 nm, 0.6 W cm -2, 10 minutes). The tumor volume and body weight of the mice were continuously monitored for 2 weeks (monitored every 2 days). On the 14th day, the mice were euthanized. The tumors and major organs (heart, liver, spleen, lung and kidney) of all mice were dissected and separated, and the dissected tumors were weighed and photographed.

[0137] Among them, CKCBT as a carrier co-assembled with Ce6 enhanced the anti-tumor effect of Ce6 in animal models. The most significant reduction in average tumor volume was observed in the CKCBT+Ce6 group treated with laser irradiation. TUNEL staining results further showed that co-injection of CKCBT and Ce6 followed by excitation irradiation resulted in the most cell death and the most obvious apoptosis ( Fig.29 ).

[0138] In addition, the inventors of this case also referred to the aforementioned embodiments and conducted experiments with other raw materials, process operations, and process conditions described in this specification, and obtained relatively ideal results.

[0139] It should be understood that the technical solution of the present invention is not limited to the above-mentioned specific implementation cases. Any technical deformation made according to the technical solution of the present invention without departing from the scope of protection of the purpose of the present invention and the claims shall fall within the protection scope of the present invention.

Claims

1. An amino acid-encoded supramolecular self-assembly system, characterized in that: The supramolecular self-assembly system is formed by self-assembly induced by GSH reduction of a reducible short peptide derivative; wherein the reducible short peptide derivative has a structure shown in at least one of the following formulas:

2. The method for preparing the amino acid-encoded supramolecular self-assembly system according to claim 1, characterized in that: include: After being reduced by GSH, the reducible short peptide derivatives are assembled to obtain an amino acid-encoded supramolecular self-assembly system.

3. Use of the amino acid-encoded supramolecular self-assembly system according to claim 1 in bioluminescence imaging, tracing or targeted drug delivery.

4. A targeted anti-tumor prodrug, characterized in that: The targeted anti-tumor prodrug has a structure shown in at least one of the following formulae:

5. The targeted anti-tumor prodrug according to claim 4, characterized in that: The targeted anti-tumor prodrug can self-assemble to form nanoparticles or nanofibers under the action of GSH.

6. A nano delivery carrier system, characterized in that: The invention comprises a delivery vector and a drug loaded on the delivery vector, wherein the delivery vector is formed by self-assembly of a reducible short peptide derivative, and the reducible short peptide derivative has a structure shown in at least one of the following formulas:

7. The nano-delivery carrier system according to claim 6, characterized in that: The drug includes any one or more combinations of fluorescent dyes, photosensitizers, contrast agents, imaging agents, and drugs.

8. The nano-delivery carrier system according to claim 7, characterized in that: The fluorescent dye includes indocyanine green and / or Cy5.5; And / or, the photosensitizer includes dihydrochlorin e6 and / or tetraphenylporphyrin; And / or, the contrast agent includes any one or more combinations of gadopentetate dimeglumine, gadoterate dimeglumine, gadobutrol, gadomaltitol, and gadoversenic acid; And / or, the imaging agent comprises fluorodeoxyglucose and / or 99m Tc-methoxyisobutyl isocyanide; And / or, the drug includes any one or more combinations of chemotherapeutic drugs, immune drugs, radiosensitizers, anti-rheumatic drugs, anti-inflammatory drugs, and antibacterial drugs.

9. The method for preparing the nano-delivery carrier system according to any one of claims 6 to 8, characterized in that: include: The reducible short peptide derivative is co-assembled with the drug to form the nano delivery carrier system.

10. A bioluminescent probe, characterized in that: The invention comprises the amino acid-encoded supramolecular self-assembly system according to claim 1.

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