Nano-drug for promoting cupric death of tumor cells and preparation method of nano-drug

Abstract

The invention discloses a nano-drug for promoting cupric death of tumor cells and a preparation method of the nano-drug. The nano-drug comprises hyaluronic acid; a dopamine; copper ions; a diethyldithiocarbamic acid-copper conjugate; and a method for preparing the same. A peptide fragment with a targeting function; modifying a thiol ligand; the preparation method comprises the following steps: preparing a diethyl dithiocarbamic acid-copper conjugate, forming a nano-structure inner core, wrapping the conjugate, forming a copper ion protection layer, and purifying and carrying out targeted modification on a nano-drug. The preparation method comprises the following steps of: preparing the diethyl dithiocarbamic acid-copper conjugate, forming a nano-structure inner core, wrapping the conjugate, forming a copper ion protection layer, and carrying out targeted modification on the nano-drug. The copper ions are further fixed through the coordination effect of dopamine and the copper ions, wrapping of the diethyl dithiocarbamic acid-copper conjugate and the thiol-copper coordination effect of the modified thiol ligands, so that a double-coordination mechanism is formed, the loading capacity of the copper ions in the nano-drug is remarkably improved, and the stability of the nano-drug is improved. The copper death of tumor cells can be more effectively triggered, the copper ion loading capacity can be improved, and a material basis is provided for the efficient copper death effect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomedicine, in particular to a kind of nanomedicine for promoting tumor cell copper death and preparation method thereof. BACKGROUND

[0002] At present, although the marketed drug disulfiram has been proved to have the potential to induce tumor cell copper death, it still faces the dilemma of insufficient copper loading in practical application, and it is difficult to effectively induce tumor cell copper death, which limits its effect in anti-tumor treatment. In order to improve this situation, the prior art develops nanomedicine to improve the copper loading, but there are still obvious deficiencies in realizing efficient targeted delivery and stable loading of copper ions.

[0003] The existing nanomedicine improves the copper ion loading to a certain extent through the coordination of dopamine and copper ions and the wrapping of diethyl dithio carbamic acid-copper combination, and realizes the targeted delivery of CD44 receptor high expression tumor cells by using hyaluronic acid. However, this nanomedicine still has the following limitations:

[0004] Single and limited targeting: only relying on the interaction between hyaluronic acid and CD44 receptor to realize targeted delivery, the targeting mechanism is relatively single, and it is difficult to cover all types of tumor cells, resulting in limited recognition and enrichment efficiency of nanomedicine for tumor cells in complex tumor microenvironment, and unable to fully realize precise treatment.

[0005] Limited space for improving copper ion loading: although it has been improved compared with traditional drugs, the copper ion loading still cannot meet the needs of more efficient induction of tumor cell copper death, and when facing some tumor types with higher requirements for copper ion concentration, the treatment effect may not be ideal.

[0006] Therefore, we propose a kind of nanomedicine for promoting tumor cell copper death and preparation method thereof. SUMMARY

[0007] The present application aims to provide a kind of nanomedicine for promoting tumor cell copper death and preparation method thereof, which solves the problems proposed in the background art.

[0008] To achieve the above-mentioned purpose, the present application provides the following technical scheme: a kind of nanomedicine for promoting tumor cell copper death, comprising:

[0009] Hyaluronic acid;

[0010] Dopamine;

[0011] Copper ions;

[0012] Diethyl dithio carbamic acid-copper combination;

[0013] Peptide segment with targeting function;

[0014] modified thiol ligand;

[0015] other pharmaceutically acceptable excipients;

[0016] The diethyl dithiocarbamic acid-copper combination is formed by combining diethyl dithiocarbamic acid as a copper carrier with copper ions, dopamine is used as a copper ion carrier to coordinate with copper ions to form a nano-structured core, and the diethyl dithiocarbamic acid-copper combination is wrapped, the modified thiol ligand is further used to fix copper ions through thiol-copper coordination to form a copper ion protective layer, and the nano-structured outer layer adsorbs hyaluronic acid and a peptide segment with a targeting function.

[0017] As a preferred embodiment of the present application, the other pharmaceutically acceptable excipients are osmotic pressure regulators.

[0018] As a preferred embodiment of the present application, the mass ratio of the peptide segment with a targeting function to the modified thiol ligand is 1:0.5-1:2.

[0019] As a preferred embodiment of the present application, the peptide segment with a targeting function is an RGD peptide segment.

[0020] As a preferred embodiment of the present application, the modified thiol ligand is a mercaptopropionic acid modified polyethylene glycol.

[0021] The present application also relates to a preparation method of a nano-drug for promoting copper death of tumor cells, comprising the following method steps:

[0022] Step one: preparation of a diethyl dithiocarbamic acid-copper combination: sodium diethyl dithiocarbamate and copper chloride are dissolved in deionized water according to a certain molar ratio, sodium diethyl dithiocarbamate:copper chloride=2:1, stirring at room temperature for 30 minutes, centrifugal separation to collect the precipitate, and obtaining the diethyl dithiocarbamic acid-copper combination;

[0023] Step two: formation of a nano-structured core: a certain amount of dopamine hydrochloride and copper chloride are weighed, the mass ratio of dopamine hydrochloride to copper chloride is dopamine hydrochloride:copper chloride=1.5:1, deionized water is added for dissolution, and coordination stirring is performed for 1 hour, a Tris solution with pH=8.5 is added, the volume ratio of deionized water to the Tris solution is 1-6:2, and the mass / volume ratio mg / mL of dopamine hydrochloride to the Tirs buffer is 1:1;

[0024] Step three: the wrapping of the combination and the formation of the copper ion protection layer: the diethyl dithiocarbamic acid-copper combination is dissolved in dimethyl sulfoxide (DMSO), the mass-volume ratio mg / μL of diethyl dithiocarbamic acid-copper and DMSO is 1:100, the above-mentioned reaction solution of dopamine hydrochloride and copper chloride is quickly added, the mass ratio of diethyl dithiocarbamic acid-copper combination and dopamine hydrochloride is diethyl dithiocarbamic acid-copper combination:dopamine hydrochloride = 1:10, and the modified thiol ligand is added at the same time, the reaction solution is stirred for 10 hours to form a nanostructure with a copper ion protection layer;

[0025] Step four: purification and targeted modification of the nano drug: the dialysis bag is dialyzed in water overnight, then a certain amount of hyaluronic acid and a peptide segment with a targeting function are added and stirred for 2 hours, and the nano drug is obtained by high-speed centrifugal purification.

[0026] As a preferred embodiment of the present application, the stirring temperature is 25-37℃, and the stirring speed is 200-500rpm.

[0027] As a preferred embodiment of the present application, the molecular weight cut-off of the dialysis bag is 8000-14000, and the dialysis time is 12-24 hours.

[0028] As a preferred embodiment of the present application, the speed of high-speed centrifugation is 10000-15000rpm, and the centrifugation time is 15-30 minutes.

[0029] Compared with the prior art, the present application has the following advantages:

[0030] The present application forms a double coordination mechanism through the coordination of dopamine and copper ions and the wrapping of diethyl dithiocarbamic acid-copper combination, and further fixes the copper ions through the thiol-copper coordination of the modified thiol ligand, significantly improves the copper ion loading in the nano drug, can more effectively induce tumor cell copper death, is conducive to improving the copper ion loading, and provides a material basis for efficient copper death effect.

[0031] On the basis of adsorbing hyaluronic acid on the outer layer of the nanostructure, the peptide segment with a targeting function is further introduced, which can specifically recognize the receptors on the surface of tumor cells, realize the synergistic effect of active targeting and passive targeting, improve the targeting of the nano drug to tumor cells, reduce the damage to normal cells, is conducive to improving the enrichment efficiency of the nano drug in tumor tissues compared with a single targeting carrier, and is conducive to enhancing the selectivity and effectiveness of treatment.

[0032] The copper ion protection layer formed by the modified thiol ligand effectively prevents the leakage of copper ions in the in-vivo circulation, ensures the stability of the copper ions before the drug reaches the tumor site, and is beneficial to improve the treatment effect and safety of the drug. BRIEF DESCRIPTION OF DRAWINGS

[0033] Other features, objects, and advantages of the application will become more apparent from the following detailed description of non-limiting embodiments, when read in conjunction with the accompanying drawings:

[0034] Figure 1 A flow chart of a preparation method of a nano drug for promoting copper death of tumor cells. DETAILED DESCRIPTION

[0035] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application is further described below in conjunction with specific embodiments.

[0036] The present application provides a nano drug for promoting copper death of tumor cells, characterized in that it comprises:

[0037] Hyaluronic acid;

[0038] Dopamine;

[0039] Copper ions;

[0040] Diethyl dithiocarbamic acid-copper complex;

[0041] Peptide segment with targeting function;

[0042] Modified thiol ligand;

[0043] Other pharmaceutically acceptable excipients;

[0044] The diethyl dithiocarbamic acid-copper complex is formed by combining diethyl dithiocarbamic acid as a copper carrier with copper ions, dopamine as a copper ion carrier is coordinated with copper ions to form a nano structure core, and the diethyl dithiocarbamic acid-copper complex is wrapped, the modified thiol ligand further fixes the copper ions through thiol-copper coordination to form a copper ion protection layer, and the outer layer of the nano structure absorbs hyaluronic acid and a peptide segment with targeting function.

[0045] The other pharmaceutically acceptable excipients are osmotic pressure regulators, the mass ratio of the peptide segment with targeting function to the modified thiol ligand is 1:0.5-1:2, the peptide segment with targeting function is an RGD peptide segment, and the modified thiol ligand is a polyethylene glycol modified with mercaptopropionic acid.

[0046] Example 1

[0047] Preparation of diethyl dithiocarbamic acid-copper complex: 34.2 mg of sodium diethyl dithiocarbamate and 13.5 mg of copper chloride were respectively dissolved in deionized water, mixed and stirred at room temperature for 30 minutes, and the precipitate was collected by centrifugal separation to obtain the diethyl dithiocarbamic acid-copper complex;

[0048] Formation of nanostructured core: 20 mg of dopamine hydrochloride and 13.3 mg of copper chloride were weighed and dissolved in 30 ml of deionized water, and the reaction was stirred for 1 hour. A Tris solution with pH = 8.5 (volume ratio of deionized water to Tris solution = 3:2) was added, and the mass / volume ratio of dopamine hydrochloride to Tris buffer was 1:1 mg / mL;

[0049] Encapsulation of the complex and formation of copper ion protective layer: 2 mg of diethyl dithiocarbamic acid-copper complex was dissolved in 200 microliters of DMSO (mass / volume ratio of diethyl dithiocarbamic acid-copper to DMSO = 1:100 mg / μL), and the above-mentioned reaction solution of dopamine hydrochloride and copper chloride was quickly added. The mass ratio of diethyl dithiocarbamic acid-copper complex to dopamine hydrochloride was 1:10, and 10 mg of mercaptopropionic acid-modified polyethylene glycol (the mass ratio of the RGD peptide segment with a targeting function to the modified thiol ligand was 1:1) was added at the same time. The reaction solution was stirred at 300 rpm at 30°C for 10 hours to form a nanostructure with a copper ion protective layer;

[0050] Purification and targeting modification of nanomedicine: The nanomedicine was purified by dialysis in water for 18 hours using a dialysis bag with a molecular weight cutoff of 10,000, followed by the addition of 20 mg of hyaluronic acid and 10 mg of RGD peptide segment and stirring for 2 hours. The nanomedicine was purified by high-speed centrifugation at 12,000 rpm for 20 minutes.

[0051] Example 2

[0052] Preparation of diethyl dithiocarbamic acid-copper complex: 34.2 mg of sodium diethyl dithiocarbamate and 13.5 mg of copper chloride were respectively dissolved in deionized water, mixed and stirred at room temperature for 30 minutes, and the precipitate was collected by centrifugal separation to obtain the diethyl dithiocarbamic acid-copper complex;

[0053] Formation of nanostructured core: 20 mg of dopamine hydrochloride and 13.3 mg of copper chloride were weighed and dissolved in 30 ml of deionized water, and the reaction was stirred for 1 hour. A Tris solution with pH = 8.5 (volume ratio of deionized water to Tris solution = 2:1) was added, and the mass / volume ratio of dopamine hydrochloride to Tris buffer was 1:1 mg / mL;

[0054] The formation of the copper ion protective layer: 2 mg of diethyl dithio carbamic acid-copper complex was dissolved in 200 microliters of DMSO (the mass / volume ratio of diethyl dithio carbamic acid-copper and DMSO was 1:100 mg / μL), and then quickly added to the reaction solution of the above-mentioned dopamine hydrochloride and copper chloride. The mass ratio of diethyl dithio carbamic acid-copper complex to dopamine hydrochloride was 1:10 (diethyl dithio carbamic acid-copper complex:dopamine hydrochloride = 1:10), and 5 mg of mercaptopropionic acid-modified polyethylene glycol (the mass ratio of the RGD peptide segment with a targeting function to the modified thiol ligand was 1:0.5) was added at the same time. The reaction solution was stirred at 400 rpm at 25°C for 10 hours to form a nanostructure with a copper ion protective layer.

[0055] Purification and targeted modification of the nano-drug: the dialysis bag with a molecular weight cut-off of 12000 was used to dialyze in water for 20 hours, and then 20 mg of hyaluronic acid and 5 mg of RGD peptide segment were added and stirred for 2 hours. The nano-drug was purified by high-speed centrifugation at 13000 rpm for 15 minutes.

[0056] Comparative example

[0057] Preparation of diethyl dithio carbamic acid-copper complex: 34.2 mg of sodium diethyl dithio carbamate and 13.5 mg of copper chloride were dissolved in deionized water, respectively, and then mixed and stirred at room temperature for 30 minutes. The precipitate was collected by centrifugal separation to obtain diethyl dithio carbamic acid-copper complex.

[0058] Formation of the core of the nanostructure: 20 mg of dopamine hydrochloride and 13.3 mg of copper chloride were weighed and dissolved in 30 ml of deionized water, and then coordinated and stirred for 1 hour. A Tris solution with a pH of 8.5 (the volume ratio of deionized water to Tris solution was 3:2) was added, and the mass / volume ratio of dopamine hydrochloride to Tirs buffer was 1:1 mg / mL.

[0059] Encapsulation of the complex: 2 mg of diethyl dithio carbamic acid-copper complex was dissolved in 200 microliters of DMSO (the mass / volume ratio of diethyl dithio carbamic acid-copper and DMSO was 1:100 mg / μL), and then quickly added to the reaction solution of the above-mentioned dopamine hydrochloride and copper chloride. The mass ratio of diethyl dithio carbamic acid-copper complex to dopamine hydrochloride was 1:10 (diethyl dithio carbamic acid-copper complex:dopamine hydrochloride = 1:10), and the reaction solution was stirred at 300 rpm at 30°C for 10 hours to form a nanostructure.

[0060] Purification and targeted modification of the nano-drug: the dialysis bag with a molecular weight cut-off of 10000 was used to dialyze in water for 18 hours, and then 20 mg of hyaluronic acid was added and stirred for 2 hours. The nano-drug was purified by high-speed centrifugation at 12000 rpm for 20 minutes.

[0061] The drugs obtained from the above-mentioned examples 1-2 and comparative examples were tested, and the test data are as follows:

[0062]

[0063]

[0064] In summary, the present application forms a double coordination mechanism by the coordination of dopamine and copper ions and the wrapping of diethyl dithiocarbamic acid-copper combination, and further fixes the copper ions through the thiol-copper coordination of the modified thiol ligand, significantly improves the copper ion loading in the nanodrug, can more effectively induce tumor cell copper death, is conducive to improving the copper ion loading, and provides a material basis for efficient copper death effect; on the basis of the adsorption of hyaluronic acid on the outer layer of the nano structure, the peptide segment with targeting function is further introduced, which can specifically recognize the receptors on the surface of tumor cells, realize the synergistic effect of active targeting and passive targeting, improve the targeting of the nanodrug to tumor cells, reduce the damage to normal cells, and is conducive to improving the enrichment efficiency of the nanodrug in tumor tissues compared with single-targeting carriers, and is conducive to enhancing the selectivity and effectiveness of treatment; the copper ion protection layer formed by the modified thiol ligand effectively prevents the leakage of copper ions in the body circulation, ensures the stability of the copper ions before the drug reaches the tumor site, and is conducive to improving the therapeutic effect and safety of the drug.

[0065] The above shows and describes the basic principles and main features of the present application and the advantages of the present application, and it is obvious to those skilled in the art that the present application is not limited to the details of the above-mentioned exemplary embodiments, and the present application can be realized in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application.

[0066] In addition, it should be understood that although the present specification is described in terms of embodiments, not every embodiment contains only one independent technical solution, and the description manner of the specification is only for the sake of clarity, and those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that those skilled in the art can understand.

Claims

1. A nanomedicine that promotes copper death in tumor cells, characterized in that, include: Hyaluronic acid; Dopamine; Copper ions; Diethyldithiocarbamate-copper conjugate; Peptides with targeting functions; Modified thiol ligands; Other pharmaceutically acceptable excipients; Among them, the diethyldithiocarbamate-copper conjugate is formed by diethyldithiocarbamate as a copper carrier and copper ions. Dopamine acts as a copper ion carrier and coordinates with copper ions to form a nanostructure core, which encapsulates the diethyldithiocarbamate-copper conjugate. Modified thiol ligands further fix copper ions through thiol-copper coordination to form a copper ion protective layer. The outer layer of the nanostructure adsorbs hyaluronic acid and peptides with targeting functions.

2. The nanomedicine for promoting copper death of tumor cells according to claim 1, characterized in that: Other pharmaceutically acceptable excipients are osmotic pressure regulators.

3. The nanomedicine for promoting copper death of tumor cells according to claim 1, characterized in that: The mass ratio of the targeted peptide to the modified thiol ligand is 1:0.5-1:

2.

4. The nanomedicine for promoting copper death of tumor cells according to claim 1, characterized in that: The peptide with targeting function is the RGD peptide.

5. The nanomedicine for promoting copper death of tumor cells according to claim 1, characterized in that: The modified thiol ligand is polyethylene glycol modified with mercaptopropionic acid.

6. A method for preparing a nanomedicine that promotes copper death in tumor cells, applicable to the nanomedicine for promoting copper death in tumor cells according to any one of claims 1-5, characterized in that: The methods and steps include the following: Step 1: Preparation of diethyldithiocarbamate-copper conjugate: Sodium diethyldithiocarbamate and copper chloride were dissolved in deionized water at a certain molar ratio of sodium diethyldithiocarbamate:copper chloride = 2:

1. The mixture was stirred at room temperature for 30 minutes, and the precipitate was collected by centrifugation to obtain diethyldithiocarbamate-copper conjugate. Step 2: Formation of the nanostructure core: Weigh a certain amount of dopamine hydrochloride and copper chloride, with a mass ratio of dopamine hydrochloride to copper chloride of 1.5:

1. Dissolve in deionized water, stir and react for 1 hour, then add Tris solution with pH=8.5, with a volume ratio of deionized water to Tris solution of 1-6:

2. The mass / volume ratio of dopamine hydrochloride to Tris buffer (mg / mL) is 1:

1. Step 3: Encapsulation of the conjugate and formation of the copper ion protective layer: The diethyldithiocarbamate-copper conjugate is dissolved in dimethyl sulfoxide (DMSO) at a mass-to-volume ratio of 1:100 mg / μL. This solution is then rapidly added to the above reaction solution of dopamine hydrochloride and copper chloride, with a mass ratio of 1:10 between the diethyldithiocarbamate-copper conjugate and dopamine hydrochloride. Simultaneously, a modified thiol ligand is added, and the reaction solution is stirred for 10 hours to form a nanostructure with a copper ion protective layer. Step 4: Purification and targeted modification of nanomedicine: Dialyze the nanomedicine overnight in water using a dialysis bag, then add a certain amount of hyaluronic acid and a peptide with targeting function and stir for 2 hours. Purify the nanomedicine by high-speed centrifugation.

7. The method for preparing a nanomedicine that promotes copper death in tumor cells according to claim 6, characterized in that: The temperature of the stirring reaction is 25-37℃, and the stirring speed is 200-500 rpm.

8. The method for preparing a nanomedicine that promotes copper death in tumor cells according to claim 6, characterized in that: The dialysis bag has a molecular weight cutoff of 8,000-14,000 and a dialysis time of 12-24 hours.

9. The method for preparing a nanomedicine that promotes copper death in tumor cells according to claim 6, characterized in that: The high-speed centrifuge operates at a speed of 10,000-15,000 rpm for 15-30 minutes.

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