Platinum drug delivery system based on elastin-like polypeptide and preparation method thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides a platinum drug delivery system based on elastin-like polypeptide and a preparation method thereof.The platinum drug delivery system comprises a carrier and a platinum (IV) prodrug, the tail end of the carrier is coupled with the platinum (IV) prodrug, the platinum (IV) prodrug is a platinum (IV) complex with longitudinal ligands containing carboxyl, and the longitudinal ligands contain carboxyl. The carrier is amphiphilic elastin-like polypeptide, the amino acid sequence of the amphiphilic elastin-like polypeptide is G (VPGX1G) n (VPGX2G) nYKm, and the critical micelle temperature of the amphiphilic elastin-like polypeptide is 25-40 DEG C; according to the elastin-like polypeptide-based platinum drug delivery system and the preparation method thereof, the platinum drug delivery system is subjected to phase transformation in vivo and is self-assembled into micelle particles for transportation, so that the blood circulation time is improved, the tumor accumulation is increased, the targeting property of platinum drugs is improved, and the systemic toxicity is reduced. The patent is subsidized by Key Technology and Industrial Demonstration for Green Biological Manufacturing of Medicinal Polypeptides, and the project number is 2021YFC2103900.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a platinum drug delivery system based on elastin-like polypeptide and a preparation method thereof. Background Art

[0002] Platinum drugs are the most widely used first-line chemotherapy drugs in clinical practice. Currently, the commonly used platinum drugs are cisplatin, carboplatin, nedaplatin, lobaplatin and oxaliplatin. Platinum drugs are an important class of anti-tumor drugs, characterized by containing platinum metal centers. These drugs act on the DNA of cancer cells, forming cross-linked structures with purine bases in DNA, hindering DNA replication and transcription, thereby inhibiting the proliferation and survival of cancer cells. As a cell cycle non-specific drug, platinum drugs are activated by hydration reaction after being taken up by tumor cells, and then form platinum-DNA adducts and cross-linked chains with DNA, thereby directly damaging DNA function and producing cytotoxicity. In addition, platinum drugs may cause cytoplasmic acidification, endoplasmic reticulum stress, RNA transcription disruption, oncogenic protein inhibition, and reduced metabolic plasticity of tumor cells.

[0003] Platinum drugs have the advantages of broad spectrum, high efficiency, synergy with other drugs, increased radiotherapy sensitivity, low cost and good market access. At the same time, platinum drugs also have serious side effects, prone to nephrotoxicity, ototoxicity, gastrointestinal toxicity, neurotoxicity and bone marrow suppression, easy to develop drug resistance, and poor targeting. The toxicity of platinum drugs sometimes increases with the accumulation of treatment, especially after multiple courses of treatment, patients may experience more serious adverse reactions. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a platinum drug delivery system based on elastin-like polypeptide and a preparation method thereof to solve the problems raised in the background technology.

[0005] According to the first aspect of the present disclosure, a platinum drug delivery system based on elastin-like polypeptide is proposed, comprising a carrier and a platinum prodrug, wherein the carrier terminal is coupled with a platinum (IV) prodrug, the platinum (IV) prodrug is a platinum (IV) complex having a longitudinal ligand and a carboxyl group, and the carrier is an amphiphilic elastin-like polypeptide.

[0006] Preferably, the particle size of the platinum drug delivery system is 50-200 nm, and the critical micelle temperature is 25-40°C.

[0007] According to the second aspect of the present disclosure, a method for preparing a platinum drug delivery system based on elastin-like polypeptide is proposed, comprising the following steps:

[0008] S1, mixing a platinum (IV) prodrug with an activator, and performing an oscillation reaction to obtain a carboxyl-activated platinum (IV) prodrug;

[0009] S2, preparing an amphiphilic elastin-like polypeptide;

[0010] S3, mixing the carboxyl-activated platinum (IV) prodrug prepared by S1 and the amphiphilic elastin-like polypeptide prepared by S2, and performing dialysis after oscillating reaction to obtain a platinum drug delivery system based on the elastin-like polypeptide.

[0011] Preferably, the activator is a 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and an N-hydroxysuccinimide solution.

[0012] Preferably, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, platinum (IV) prodrug and amphiphilic elastin-like polypeptide is (6-12.5):(6-12.5):(6-10):1.

[0013] Preferably, the temperature of the oscillating reaction in S1 and S3 is 20-40° C., the time of the oscillating reaction in S1 is 10-120 min, and the time of the oscillating reaction in S3 is 2-24 h.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] 1. The platinum drug delivery system based on elastin-like polypeptide of the present invention is a soluble liquid system in vitro, undergoes phase transition in vivo, and self-assembles into micellar particles for transportation, which can improve blood circulation time and increase tumor accumulation.

[0016] 2. The preparation process of the present invention is simple, and the prepared platinum drug delivery system based on elastin-like polypeptide has good biocompatibility and is degradable, and has a wide range of applications.

[0017] 3. In the reducing tumor environment of the present invention, the platinum (IV) prodrug in the platinum drug delivery system based on elastin-like polypeptide will be reduced to cytotoxic platinum (II), which helps to improve the targeting of platinum drugs while reducing systemic toxicity; and the delivery system can be applied to fields such as tumor chemotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of ESI-MS of the platinum (IV) prodrug containing axial carboxyl ligand provided in the examples of the present invention (ESI-MS (negative mode): Calc.=432.96, Found=432.96). DETAILED DESCRIPTION

[0019] The following embodiments of the present invention are described in further detail in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0020] Embodiment 1: Figure 1 As shown, a platinum drug delivery system based on elastin-like polypeptide includes a carrier and a platinum (IV) prodrug, wherein the carrier terminal is coupled with the platinum (IV) prodrug; the platinum (IV) prodrug is c,c,t-[Pt(NH3)2C l 2(OH)(O2CCH2CH2CO2H)], and the carrier is an amphiphilic elastin-like polypeptide.

[0021] The molar ratio of the platinum (IV) prodrug to the amphiphilic elastin-like polypeptide is 10:1; efficient loading of the platinum (IV) prodrug is achieved by controlling the molar ratio of the platinum (IV) prodrug to the amphiphilic elastin-like polypeptide.

[0022] Platinum (IV) prodrugs are platinum (IV) complexes with carboxyl groups as longitudinal ligands, which can be reduced to cytotoxic platinum (II) forms under reducing conditions in vivo.

[0023] The particle size of the platinum drug delivery system is 100-200 nm, and its critical micelle temperature is 37°C.

[0024] A method for preparing a platinum drug delivery system based on elastin-like polypeptides comprises the following steps:

[0025] S1. Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in 1×MES buffer and adjust the pH to 5-6 to obtain an EDC solution; dissolve N-hydroxysuccinimide (NHS) in 1×MES buffer and adjust the pH to 5-6 to obtain an NHS solution; dissolve a platinum (IV) prodrug in 1×MES buffer and adjust the pH to 5-6 to obtain a Pt(IV)-COOH solution;

[0026] The EDC solution was mixed with an equal volume of NHS solution to obtain a mixed solution 1, a Pt(IV)-COOH solution was added to the mixed solution 1 to obtain a mixed solution 2, and the mixed solution 2 was oscillated for reaction.

[0027] S2. A gene sequence encoding an amphiphilic elastin-like polypeptide is synthesized through genetic engineering technology. The specific sequence has been disclosed in another application. The ELP has a specific amino acid sequence and critical micelle temperature, can undergo phase transition in vivo, and self-assemble into micelle particles for transportation.

[0028] S3. Dissolve the amphiphilic ELP in 1×MES buffer to obtain an ELP solution; add the ELP solution to the mixed solution 2 and adjust the pH to 7.2-7.5, oscillate the reaction, and dialyze the product in 1×PBS at 4°C overnight to obtain a platinum drug delivery system based on elastin-like polypeptides.

[0029] In one embodiment, in S1, the molar ratio of EDC to NHS is 1:1, the temperature of the oscillation reaction is 25° C., the time of the oscillation reaction is 15 min, and the oscillation speed is 400 rpm.

[0030] In one embodiment, in S3, the final molar ratio of EDC, NHS, platinum (IV) prodrug and amphiphilic ELP is 10:10:8:0.8, the oscillation reaction temperature is 25°C, the oscillation reaction time is 24h, and the oscillation speed is 400rpm. By controlling the incubation temperature and time, the loading efficiency of the platinum (IV) prodrug is improved.

[0031] Example 2: A platinum drug delivery system based on elastin-like polypeptide, comprising a carrier and a platinum (IV) prodrug, wherein the carrier end is coupled with the platinum (IV) prodrug; the platinum (IV) prodrug is c,c,t-[Pt(NH3)2C l 2(O2CCH2CH2CO2H)2], and the carrier is an amphiphilic elastin-like polypeptide.

[0032] The molar ratio of the platinum (IV) prodrug to the amphiphilic elastin-like polypeptide is 6:1; efficient loading of the platinum (IV) prodrug is achieved by controlling the molar ratio of the platinum (IV) prodrug to the amphiphilic elastin-like polypeptide.

[0033] In one embodiment, the particle size of the platinum drug delivery system is 100-200 nm, and the critical micelle temperature is 30°C.

[0034] A method for preparing a platinum drug delivery system based on elastin-like polypeptides comprises the following steps:

[0035] S1. Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in 1×MES buffer and adjust the pH to 5-6 to obtain an EDC solution; dissolve N-hydroxysuccinimide (NHS) in 1×MES buffer and adjust the pH to 5-6 to obtain an NHS solution; dissolve a platinum (IV) prodrug in 1×MES buffer and adjust the pH to 5-6 to obtain a Pt(IV)-COOH solution;

[0036] The EDC solution was mixed with an equal volume of NHS solution to obtain a mixed solution 1, a Pt(IV)-COOH solution was added to the mixed solution 1 to obtain a mixed solution 2, and the mixed solution 2 was oscillated for reaction;

[0037] S2. A gene sequence encoding an amphiphilic elastin-like polypeptide is synthesized through genetic engineering technology. The specific sequence has been disclosed in another application. The ELP has a specific amino acid sequence and critical micelle temperature, can undergo phase transition in vivo, and self-assemble into micelle particles for transportation.

[0038] S3. Dissolve the amphiphilic ELP in 1×MES buffer to obtain an ELP solution; add the ELP solution to the mixed solution 2 and adjust the pH to 7.2-7.5, oscillate the reaction, and dialyze the product in 1×PBS at 4°C overnight to obtain a platinum drug delivery system based on elastin-like polypeptides.

[0039] In one embodiment, in S1, the molar ratio of EDC to NHS is 1.2:1, the temperature of the oscillation reaction is 25° C., the time of the oscillation reaction is 15 min, and the oscillation speed is 400 rpm.

[0040] In one embodiment, in S3, the final molar ratio of EDC, NHS, platinum (IV) prodrug and amphiphilic ELP is 9:7.5:6:1, the oscillation reaction temperature is 25°C, the oscillation reaction time is 12h, and the oscillation speed is 400rpm. By controlling the incubation temperature and time, the loading efficiency of the platinum (IV) prodrug is improved.

[0041] Embodiment 3: Figure 1 As shown, a platinum drug delivery system based on elastin-like polypeptides includes a carrier and a platinum (IV) prodrug, wherein the carrier terminal is coupled with the platinum (IV) prodrug; the platinum (IV) prodrug is c,c,t-[Pt(NH3)2C l 2(CO2C6H5)(CO2C3H6COOH)]; the carrier is an amphiphilic elastin-like polypeptide;

[0042] The molar ratio of the platinum (IV) prodrug to the amphiphilic elastin-like polypeptide is 8:1; efficient loading of the platinum (IV) prodrug is achieved by controlling the molar ratio of the platinum (IV) prodrug to the amphiphilic elastin-like polypeptide.

[0043] In one embodiment, the particle size of the platinum drug delivery system is 50-100 nm, and the critical micelle temperature thereof is 40°C.

[0044] A method for preparing a platinum drug delivery system based on elastin-like polypeptides comprises the following steps:

[0045] S1. Dissolve 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC) in 1×MES buffer and adjust the pH to 5-6 to obtain an EDC solution; dissolve N-hydroxysuccinimide (NHS) in 1×MES buffer and adjust the pH to 5-6 to obtain an NHS solution; dissolve a platinum (IV) prodrug in 1×MES buffer and adjust the pH to 5-6 to obtain a Pt(IV)-COOH solution;

[0046] The EDC solution was mixed with an equal volume of NHS solution to obtain a mixed solution 1, a Pt(IV)-COOH solution was added to the mixed solution 1 to obtain a mixed solution 2, and the mixed solution 2 was oscillated for reaction;

[0047] S2. A gene sequence encoding an amphiphilic elastin-like polypeptide is synthesized through genetic engineering technology. The specific sequence has been disclosed in another application. The ELP has a specific amino acid sequence and critical micelle temperature, can undergo phase transition in vivo, and self-assemble into micelle particles for transportation.

[0048] S3. Dissolve the amphiphilic ELP in 1×MES buffer to obtain an ELP solution; add the ELP solution to the mixed solution 2 and adjust the pH to 7.2-7.5, oscillate the reaction, and dialyze the product in 1×PBS at 4°C overnight to obtain a platinum drug delivery system based on elastin-like polypeptides.

[0049] In one embodiment, in S1, the molar ratio of EDC to NHS is 1.2:1, the temperature of the oscillation reaction is 25° C., the time of the oscillation reaction is 30 min, and the oscillation speed is 400 rpm.

[0050] In one embodiment, in S3, the final molar ratio of EDC, NHS, platinum (IV) prodrug and amphiphilic ELP is 12:10:8:1, the oscillation reaction temperature is 25°C, the oscillation reaction time is 6h, and the oscillation speed is 400rpm. By controlling the incubation temperature and time, the loading efficiency of the platinum (IV) prodrug is improved.

[0051] Additionally, in order to provide a concise description of exemplary embodiments, all features of an actual embodiment (ie, those features that are not relevant to the best mode presently contemplated for carrying out the invention or those that are not relevant to implementing the invention) may not be described.

[0052] It will be appreciated that in the development of any actual implementation, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will be a routine task of design, fabrication, and production for those of ordinary skill having the benefit of this disclosure without undue experimentation.

[0053] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A platinum drug delivery system based on elastin-like polypeptides, characterized in that: The invention comprises a carrier and a platinum prodrug. The terminal of the carrier is coupled with a platinum (IV) prodrug. The platinum (IV) prodrug is a platinum (IV) complex with a longitudinal ligand containing a carboxyl group. The carrier is an amphiphilic elastin-like polypeptide.

2. A platinum drug delivery system based on elastin-like polypeptide according to claim 1, characterized in that: The particle size of the platinum drug delivery system is 50-200 nm, and the critical micelle temperature is 25-40° C.

3. A method for preparing a platinum drug delivery system based on elastin-like polypeptides, applied to a platinum drug delivery system based on elastin-like polypeptides as claimed in any one of claims 1 to 2, characterized in that: The following steps are involved: S1, mixing a platinum (IV) prodrug with an activator, and performing an oscillation reaction to obtain a carboxyl-activated platinum (IV) prodrug; S2, preparing an amphiphilic elastin-like polypeptide; S3, mixing the carboxyl-activated platinum (IV) prodrug prepared by S1 and the amphiphilic elastin-like polypeptide prepared by S2, and performing dialysis after oscillating reaction to obtain a platinum drug delivery system based on the elastin-like polypeptide.

4. A method for preparing a platinum drug delivery system based on elastin-like polypeptides as claimed in claim 3, characterized in that: The activators are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide solution and N-hydroxysuccinimide solution.

5. A platinum drug delivery system based on elastin-like polypeptide and a preparation method thereof as claimed in claim 4, characterized in that: The molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, platinum (IV) prodrug and amphiphilic elastin-like polypeptide is (6-12.5):(6-12.5):(6-10):

1.

6. A platinum drug delivery system based on elastin-like polypeptide and a preparation method thereof as claimed in claim 3, characterized in that: The temperature of the oscillating reaction in S1 and S3 is 20-40° C., the time of the oscillating reaction in S1 is 10-120 min, and the time of the oscillating reaction in S3 is 2-24 h.

Images