Preparation method of polyamino acid coordination cisplatin prodrug nanoparticles and application thereof

Abstract

The present invention discloses a preparation method and application of polyamino acid coordinated cisplatin prodrug nanoparticles. First, cisplatin (CDDP), a commonly used chemotherapy drug in clinical practice, is oxidized to Pt(IV) prodrug, and AgNO3 is added to obtain hydrated Pt(IV, Cl ‑ ). γ-PGA-g-H(PH) was obtained by grafting a small molecule histidine methyl ester (H) on the side chain of γ-polyglutamic acid (γ-PGA). The two were coordinated by ester bonds to obtain nanoparticles (PHPt) with a particle size of about 220 nm. This method is simple to operate, time-saving, and the synthesis conditions are mild and controllable. The synthesized nanoparticles have good biocompatibility and greatly reduce the toxic side effects of cisplatin; under the action of ultrasound, they show sonosensitizer characteristics and produce singlet oxygen ( 1 O2) and hydroxyl radicals (·OH); the material consumes overexpressed glutathione (GSH) in the tumor microenvironment to reduce cisplatin resistance, while releasing the chemotherapy drug cisplatin, achieving the effect of sonodynamic-chemotherapy synergistic treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical nanomaterials and its preparation, more specifically, relates to a preparation method and application of a polyamino ligand cisplatin prodrug nanoparticle sonosensitizer. BACKGROUND

[0002] In the past few decades, sonodynamic therapy (SDT) has been widely used due to its high tissue penetration depth, no ionizing radiation and low cost (Biomaterials 2021, 269, 120639). SDT is to use ultrasound (US) to excite sonosensitizers to produce ROS to induce cancer cell apoptosis and / or necrosis. Traditional organic sonosensitizers have poor hydrophilicity and limited bioavailability. Inorganic sonosensitizers also have the disadvantages of high toxicity and easy recombination of electron-hole pairs, so it is urgent to develop sonosensitizers with good biocompatibility and high ROS yield. It has been reported that the change of valence state makes the separation efficiency of electron-hole pairs higher, which promotes the production of ROS in sonodynamic therapy (Angew. Chem. Int. Ed. 2022, 61, e202212021). Therefore, we consider oxidizing cisplatin to Pt(IV) prodrug, hoping that it can produce energy through valence change in the reducing microenvironment of tumor, and then transfer to O2 and H2O, and further produce ROS. At the same time, the high expression of glutathione (GSH) in tumor cells can eliminate ROS, which significantly damages the efficacy of SDT. Therefore, reducing GSH at the tumor site while increasing ROS to amplify oxidative stress is a promising strategy (Adv. Mater. 2022, 34, 2206286).

[0003] Platinum complexes are one of the most widely used chemotherapeutic drugs for the treatment of malignant tumors in clinical practice. According to statistics, about 50% of clinical chemotherapy regimens contain platinum drugs. Its clinical application is mainly limited by toxicity and drug resistance. On the one hand, cisplatin lacks selectivity for normal tissues and cancer cells, leading to a series of side effects such as nephrotoxicity and ototoxicity. On the other hand, sulfur-containing biomolecules (such as GSH or metallothionein) are overexpressed in tumor cells. After cisplatin enters the cells, it rapidly combines with thiols to form stable adducts through Pt-S bonds. The adducts are eventually pumped out of the cells by ATP7A and ATP7B, resulting in drug resistance and inability to exert chemotherapy effects. Synthesizing inert Pt(IV) prodrug nanoparticles from cisplatin is considered to be a promising strategy to reduce drug resistance (ACS Nano 2020, 14, 12, 16984-16996).

[0004] In the present application, we synthesized a polyamino acid ligand cisplatin prodrug nanoparticle PHPt for sonodynamic-chemotherapy combination therapy. First, we oxidized the clinically commonly used chemotherapy drug cisplatin (Cisplatin, CDDP) to Pt(IV) prodrug, added AgNO3 to obtain hydrated Pt(IV, Cl- ). The gamma-PGA-g-H (PH) is obtained by grafting small molecule histidine methyl ester (H) on the side chain of gamma-polyglutamic acid (gamma-PGA). The two are coupled by ester bond coordination to obtain nanoparticles (PHPt) with a particle size of about 220 nm. The method is simple to operate, short in time consumption, and mild and controllable in synthesis conditions. The PHPt has good biocompatibility, reduces the toxic side effects of cisplatin; under ultrasonic conditions, the PHPt shows the characteristics of a sonosensitizer, generating singlet oxygen ( 1 O2) and hydroxyl radicals (·OH); the material can react with GSH to release the chemotherapeutic drug cisplatin, which can amplify oxidative stress on the one hand and reduce cisplatin resistance on the other hand, so as to achieve the effect of sonodynamic-chemotherapy synergistic treatment. SUMMARY

[0005] The purpose of the application is to provide a polyamino acid coordinated cisplatin prodrug nanoparticle with the characteristics of a sonosensitizer for sonodynamic-chemotherapy synergistic treatment of tumors. That is, the gamma-PGA grafted polymer is used as a carrier, and is coordinated with hydrated Pt (IV, Cl - ) to realize the synergistic treatment of sonodynamic-chemotherapy.

[0006] A preparation method of a polyamino acid coordinated cisplatin prodrug nanoparticle, characterized by the preparation method of PH, which comprises the following steps: 0.8 mmol of gamma-polyglutamic acid (gamma-PGA) is dissolved in a 50 mM, 16 mL NaHCO3 solution; 0.8 mmol of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and 0.8 mmol of N-hydroxysuccinimide (NHS) are sequentially added to the above solution under ice bath conditions, and reacted for 10 min; 0.8 mmol of histidine methyl ester (H) is added to the above solution, and reacted for 1 h under ice bath conditions; after 1 h, the ice bath is removed, and the reaction is carried out at room temperature for 12 h; dialysis is continued for 2 days, and the water is changed every 3 h; the sample is transferred from the dialysis bag to a 10 mL centrifuge tube, the sample is stored in liquid nitrogen, and dried in a vacuum freeze dryer for 36 h to obtain gamma-PGA-g-H (PH) powder.

[0007] hydrated Pt (IV, Cl -) comprises the following steps: suspending 120 mg of CDDP in 22.5 mL of water, then preparing an equal volume of 2.67 mg / mL NCS solution, adding the solution, reacting at room temperature for 24 h, washing with ethanol and ether, and vacuum drying to obtain Pt(IV); dissolving 9.4 mg of Pt(IV) in 8 mL of water, adding 13.6 mg of AgNO3, reacting at room temperature for 24 h, centrifuging at 12000 rpm for 10 min, and filtering with a 0.22 μm filter membrane to obtain hydrated Pt(IV,Cl - ) solution.

[0008] Then, 3.5 mg of PH was dissolved in 1 mL of water, and 1.178 mL of hydrated Pt(IV) solution was added. The mixture was reacted at room temperature for 5 h, and centrifuged at 14,000 rpm for 20 min to remove the supernatant to obtain PHPt.

[0009] The obtained nanoparticles have the following characteristics:

[0010] (1) Has uniform size, with a particle size of approximately 220 nm;

[0011] (2) It has good sonosensitizer properties and produces a large amount of active oxygen under ultrasound;

[0012] (3) Has GSH depletion effect, inhibiting cisplatin resistance

[0013] (4) Good biocompatibility, reducing the toxic side effects of cisplatin;

[0014] (5) Have good in vitro cell therapy effects; BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : The nuclear magnetic hydrogen spectrum analysis spectrum of pH of the present invention.

[0016] Figure 2 : H-NMR spectrum analysis of Pt(IV) of the present invention.

[0017] Figure 3 :The nano-microspheres of the present invention produce ultrasonic catalysis 1 O2 diagram.

[0018] Figure 4 : The ultrasonic catalytic generation of ·OH by the nano-microspheres of the present invention.

[0019] Figure 5 : Particle size diagram of nanoparticles of the present invention.

[0020] Figure 6 : Transmission electron microscope image of the nanospheres of the present invention.

[0021] Figure 7 : Cell therapy diagram of nanospheres of the present invention. DETAILED DESCRIPTION

[0022] The present application is further illustrated by the following examples, but the present application is not limited to the following examples. The concentration, volume, etc. of the solution used can be adjusted as needed.

[0023] Example 1

[0024] (1) 0.8 mmol of γ-PGA was dissolved in 50 mM, 16 mL NaHCO3solution, and placed in a 20 mL reaction bottle, and stirred at room temperature until completely dissolved.

[0025] (2) The reaction bottle was placed in a 100 mL beaker, and an appropriate amount of ice was added to the ice bath. 0.8 mmol of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) was first added to the reaction bottle, followed by 0.8 mmol of N-hydroxysuccinimide (NHS), and the reaction was carried out under ice bath conditions for 10 min.

[0026] (3) 0.8 mmol of histidine methyl ester (H) was added to the reaction bottle, and the reaction was carried out under ice bath conditions for 1 h; after 1 h, the ice bath was removed, and the reaction was carried out at room temperature for 12 h.

[0027] (4) The sample in the reaction bottle was completely transferred to a dialysis bag, and the two ends were clamped with clamps and placed in a beaker for dialysis, and the water was changed every 3 h, and the dialysis was continued for 2 days.

[0028] (5) The sample was transferred from the dialysis bag to a 10 mL centrifuge tube, and the sample was frozen with liquid nitrogen, and placed in a vacuum freeze dryer for 36 h to obtain γ-PGA-g-H (PH).

[0029] The obtained PH was dissolved in 550 μL of deuterium water, and the grafting rate of each small molecule amino acid was calculated by nuclear magnetic resonance hydrogen spectrum (H Nuclear Magnetic Resonance, H NMR). 1 H Nuclear Magnetic Resonance, 1 H NMR) of the PH grafting polymer H was 31%.

[0030] (6) 120 mg of CDDP was suspended in 22.5 mL of water, and then an equal volume of 2.67 mg / mL NCS solution was prepared and added to the above solution, and the reaction was carried out at room temperature for 24 h, and then washed with ethanol and diethyl ether, and vacuum dried to obtain Pt(IV).

[0031] The obtained Pt(IV) was dissolved in 550 μL of deuterium dimethyl sulfoxide (Methyl sulfoxide-d6, DMSO-d6), and the grafting rate of each small molecule amino acid was calculated by nuclear magnetic resonance hydrogen spectrum (H Nuclear Magnetic Resonance, 1 H NMR).1 H NMR) showed that Pt(IV) was successfully synthesized.

[0032] (7) 9.4 mg of Pt(IV) was dissolved in 8 mL of water, 13.6 mg of AgNO3 was added, and the mixture was reacted at room temperature for 24 h. The mixture was centrifuged at 12000 rpm for 10 min and filtered through a 0.22 μm filter membrane to obtain hydrated Pt(IV,Cl). - ) solution.

[0033] (8) 3.5 mg of PH was dissolved in 1 mL of water, and 1.178 mL of hydrated Pt(IV) solution was added. The mixture was reacted at room temperature for 5 h, and the supernatant was removed by centrifugation to obtain PHPt-1. The hydrated particle size was measured by Mastersize 3000 dynamic light scattering (DLS) (Malvern Instruments, UK) to be 219.8 nm, and the PDI was 0.260.

[0034] (9) 3.5 mg of PH was dissolved in 1 mL of water, and 1.473 mL of hydrated Pt(IV) solution was added. The mixture was reacted at room temperature for 5 h, and the supernatant was removed by centrifugation to obtain PHPt-2. The hydrated particle size was measured by Mastersize 3000 dynamic light scattering (DLS) (Malvern Instruments, UK) to be 193.5 nm, and the PDI was 0.294.

[0035] (10) Electron Paramagnetic Resonance Spectrometer (EPR) detects singlet oxygen ( 1 O2): 10 μL TEMP was added to 100 μL H2O, PHPt-1 (200 μg / mL), and PHPt-2 (200 μg / mL), respectively, and then sonicated (1.0 MHz, 1.5 W cm -2 , 2min). 1 The O2 signal is revealed by ESR spectroscopy. Figure 3 The data showed that compared with PHPt-2, PHPt-1 can produce stronger 1 O2 signal with remarkable acoustic dynamic performance.

[0036] Similarly, hydroxyl radicals (·OH) were detected by EPR: 10 μL DMPO was added to 100 μL EtOH, PHPt-1 (200 μg / mL), and PHPt-2 (200 μg / mL), respectively, and then sonicated (1.0 MHz, 1.5 W cm -2 , 2 min), the signal of ·OH was shown by EPR spectrum. Figure 4Data show that PHPt-1 can produce stronger ·OH signal under the action of ultrasound compared to PHPt-2, and has significant sonodynamic performance.

[0037] In summary, PHPt-1 was selected for subsequent experimental study

[0038] Figure 5 Data show that the obtained PHPt-1 nanospheres have a particle size of about 220 nm.

[0039] Figure 6 Transmission electron microscopy shows that the obtained PHPt-1 nanoparticles are spherical.

[0040] (11) MTT method for detecting cell activity: in the cell therapy experiment, 4T1 cells were seeded in a 96-well plate at 10000 cells per well and cultured for 24 h, then co-cultured with different concentrations of PHPt, cisplatin (CDDP) for 12 h, then treated with a 1.0 MHz, 50% duty cycle, 1.0 W cm -2 ultrasound instrument for 2 min, then cultured for 12 h, and the cell viability was checked by MTT assay. In the cell therapy experiment, the cells were treated with chemotherapy alone, ultrasound therapy alone, and chemotherapy combined with ultrasound therapy, Figure 7 The data results show that ultrasound therapy combined with chemotherapy has a significant therapeutic effect. The reason is that after the material enters the cells, a large amount of active oxygen is generated under the action of ultrasound, which destroys the intracellular redox balance and consumes GSH to release cisplatin, inhibits cisplatin resistance, and improves the chemotherapy effect of cisplatin, achieving the effect of sonodynamic-chemotherapy synergistic therapy.

Claims

1. A process for the preparation of a polyamino acid-coordinated cisplatin prodrug, characterized by, Cisplatin (CDDP) was reacted with N-chlorosuccinimide (NCS) to obtain Pt(IV), and then AgNO3 was added to obtain a Pt(IV,Cl - ) solution; a polyglutamic acid grafted polymer (γ-PGA-g-H, PH) was reacted with the Pt(IV,Cl - ) solution in coordination, and the supernatant was removed by centrifugation to obtain polyamino acid coordinated Pt(IV) nanoparticles PHPt. The preparation method of the PH comprises the following steps: 1) dissolving γ-polyglutamic acid (γ-PGA) in a NaHCO3 solution; 2) adding N-(3-dimethylaminopropyl)-N-ethyl carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) into the above solution in sequence under ice bath condition, and reacting for 10 min; 3) adding histidinemethyl ester (H) into the above solution to synthesize a PH polyglutamic acid grafted polymer; the concentration of the NaHCO3 is 50 mM, and the molar ratio of γ-PGA: NaHCO3: EDC: NHS: histidinemethyl ester is 1:1:1:1:1; The preparation method of the hydrated Pt(IV, Cl - ) includes the following steps: 1) suspending CDDP in water, then preparing an equal volume of NCS solution, adding the above solution, reacting at room temperature for 24 hours, washing with ethanol and diethyl ether, and drying in vacuum to obtain Pt(IV); the molar ratio of CDDP:NCS is 1:1.125; 2) dissolving Pt(IV) in water, adding AgNO3, reacting at room temperature for 24 hours, centrifuging, and filtering through a 0.22 μm filter to obtain a hydrated Pt(IV, Cl - ) solution; the molar ratio of Pt(IV):AgNO3 is 1:

3. 3.5 mg of the PH is dissolved in 1 mL of water, 1.473 mL of a hydrated Pt(IV) solution is added, and reaction is carried out at room temperature for 5 h; the supernatant is removed by centrifugation to obtain PHPt.

2. The method of claim 1, wherein: The centrifugal speed is 12000-14000 rpm, and the centrifugal time is 10-20 min.

3. A polyamino acid coordination cisplatin prodrug prepared by the preparation method according to claim 1.

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