阅读说明：本技术 一种水溶性聚卟啉类无载体纳米药物的制备方法 (Preparation method of water-soluble polyporphyrin carrier-free nano-drug ) 是由 郑楠 宋汪泽 杜梦 郑玉斌 于 2021-09-06 设计创作，主要内容包括：本发明属于高分子材料,生物医药材料及光动力、声动力治疗技术领域,提供了一种水溶性聚卟啉类无载体纳米药物的制备方法。本发明的制备方法得到的水溶性聚卟啉类无载体纳米药物的数均分子量范围为5000g/mol-30000g/mol,在水中溶解度可达50mg/mL,单线态氧产率为四羧基苯基卟啉的1.5-3倍。该聚合物可直接溶解于水中,在浓度为0.01mg/mL-10mg/mL的范围内,可以无载体的形式自发形成稳定的纳米粒,粒径范围为100nm-200nm。与普通的聚卟啉相比,该种聚卟啉具有水溶性,提高了生物相容性。该种聚卟啉可以在水中自组装成纳米颗粒,无需其他载体,具有潜在的体内递送效果。(The invention belongs to the technical field of high polymer materials, biomedical materials and photodynamic and sonodynamic treatment, and provides a preparation method of a water-soluble polyporphyrin carrier-free nano-drug. The number average molecular weight range of the water-soluble polyporphyrin carrier-free nano-drug prepared by the preparation method is 5000g/mol-30000g/mol, the solubility in water can reach 50mg/mL, and the singlet oxygen yield is 1.5-3 times of that of tetracarboxyphenyl porphyrin. The polymer can be directly dissolved in water, and can spontaneously form stable nanoparticles with a particle size range of 100nm-200nm in a carrier-free form within a concentration range of 0.01mg/mL-10 mg/mL. Compared with the common polyporphyrin, the polyporphyrin has water solubility and improved biocompatibility. The polyporphyrin can be self-assembled into nanoparticles in water, does not need other carriers, and has a potential in vivo delivery effect.)

1. A preparation method of a water-soluble carrier-free polyporphyrin nano-drug is characterized by comprising the following steps:

weighing 1 molar equivalent of tetracarboxyphenylporphyrin, 4-10 molar equivalents of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 4-10 molar equivalents of p-dimethylaminopyridine, dissolving in chloroform, tetrahydrofuran or N, N-dimethylformamide, controlling the monomer concentration range of the tetracarboxyphenylporphyrin to be 0.1M-5M, heating to 40-90 ℃, and stirring for 10-30 min; then, adding 1.8-2.2 molar equivalents of diol monomer containing a plurality of ether bonds into the solution, heating the reaction system to 90-140 ℃, and continuing to react for 12-144 h; after the reaction is finished, settling the reaction solution in normal hexane or diethyl ether (the volume of the normal hexane or the diethyl ether is 10-50 times of the volume of the mixed solution), centrifugally collecting precipitate, and vacuum-drying overnight to remove residual solvent; dissolving the precipitate in DMSO and H at a volume ratio of 1/10-0/10₂Dialyzing in deionized water for 12-48h by using a dialysis bag with the molecular weight cutoff of 300-3000Da in a solvent of O, and freeze-drying in a freeze dryer to obtain a purple solid P-nO, wherein n is 1-20; the number average molecular weight of the polyporphyrin ranges from 5000g/mol to 30000g/mol, the solubility in water can reach 50mg/mL, and the singlet oxygen yield is 1.5 to 3 times of that of tetracarboxyphenyl porphyrin; the polymer can be directly dissolved in water, and can spontaneously form stable nanoparticles with a particle size range of 100nm-200nm in a carrier-free manner within a concentration range of 0.01mg/mL-10 mg/mL.

Technical Field

The invention belongs to the technical field of high polymer materials, biomedical materials and photodynamic and sonodynamic treatment, and relates to a preparation method of water-soluble polyporphyrin and application of the water-soluble polyporphyrin in photoacoustic photodynamic treatment of tumors.

Background

Cancer is one of the leading causes of morbidity and mortality in the world, and the traditional tumor treatment means comprises surgical excision, radiotherapy and chemotherapy, can achieve the effect of treating tumors to a certain extent, but has the defects of easy relapse, large toxic and side effects, poor selectivity, invasiveness and the like. Compared with the traditional method for treating the tumor, the photodynamic therapy and the sonodynamic therapy are novel and non-invasive methods for treating the tumor, have small toxic and side effects, good repeatability and selectivity and non-invasive or minimally invasive properties, and are considered as potential new means for treating the tumor. Currently, photodynamic or sonodynamic therapy has become the clinical treatment modality for a variety of diseases, including cancer.

Photodynamic therapy requires the combined action of light, oxygen and a photosensitizer, while sonodynamic requires the therapeutic effect to be exerted by means of a sonosensitizer, and the photosensitizer or sonosensitizer is the core of photodynamic or sonodynamic therapy. The currently developed optical and acoustic sensitizers mainly include porphyrins, phthalocyanines, condensed rings, boron dipyrromethenes and the like, and porphyrin derivatives are most widely applied to photodynamic or sonodynamic. Tetraphenylporphyrin (TPP) is a typical second generation photosensitizer, efficiently produces singlet oxygen, and has relatively low dark toxicity. However, since TPP is a planar rigid conjugated structure, the photosensitizer tends to aggregate with increasing concentration due to the strongly hydrophobic pi-pi stacking effect in aqueous media, and aggregation-induced quenching (ACQ) effect is easily induced by aggregation, which may greatly reduce singlet oxygen quantum yield and weaken the efficacy of photodynamic light. In addition, TPP is a small molecule that is poorly water soluble, is not conducive to drug delivery, and has a short circulation time in vivo, which limits its in vivo applications.

In order to overcome the problems, a water-soluble carrier-free porphyrin polymer nano-drug is synthesized by introducing a flexible chain design, can be self-assembled into nano-particles in water, and is tested for singlet oxygen yield, nano-particle stability and in-vitro light dark toxicity. The invention solves the problems that the photosensitizer with a macrocyclic planar conjugated structure is easy to generate aggregation-induced quenching and has poor water solubility. The polyporphyrin structure designed by the invention can also be used as a sonosensitizer for the sonodynamic treatment of tumors.

Disclosure of Invention

The invention aims to mainly provide a preparation method of a water-soluble polyporphyrin carrier-free nano-drug.

The technical scheme of the invention is as follows:

a preparation method of a water-soluble carrier-free polyporphyrin nano-drug comprises the following steps:

weighing 1 molar equivalent of tetracarboxyphenylporphyrin, 4-10 molar equivalents of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDCI) and 4-10 molar equivalents of p-Dimethylaminopyridine (DMAP) dissolved in chloroform (CHCl)₃) Controlling the monomer concentration range of the tetra-carboxyl phenyl porphyrin to be 0.1-5M in Tetrahydrofuran (THF) or N, N-Dimethylformamide (DMF), heating to 40-90 ℃, and stirring for 10-30 min; then, adding 1.8-2.2 molar equivalents of diol monomer containing a plurality of ether bonds into the solution, heating the reaction system to 90-140 ℃, and continuing to react for 12-144 h; after the reaction is finished, the reaction solution is settled in normal hexane or diethyl ether (the volume of the normal hexane or the diethyl ether is 10-50 times of the volume of the mixed solution), and the precipitate is collected by centrifugation and is dried overnight under vacuum to remove the residual solvent. Dissolving the precipitate in DMSO and H at a volume ratio of 1/10-0/10₂And D, dissolving the mixture in a solvent of O, dialyzing the mixture in deionized water for 12 to 48 hours by using a dialysis bag with the molecular weight cut-off (MWCO) of 300-3000Da, and freeze-drying the mixture in a freeze dryer to obtain the final product, namely the purple solid P-nO, wherein n is 1 to 20. The number average molecular weight of the polyporphyrin is 5000g/mol-30000g/mol, the solubility in water can reach 50mg/mL, and the singlet oxygen yield is 1.5-3 times of that of tetracarboxyphenyl porphyrin. The polymer can be directly dissolved in water, can spontaneously form stable nanoparticles in a carrier-free form within the concentration range of 0.01-10 mg/mL, and has a particle size range100nm-200nm。

The reaction route is as follows:

the invention has the beneficial effects that:

(1) the invention provides a preparation method of water-soluble polyporphyrin, which has high universality and provides technical reference for synthesizing high molecular weight porphyrin polymer.

(2) Compared with monomer small molecular porphyrin, the singlet oxygen yield of the polyporphyrin can be improved to about 2 to 3 times.

(3) Compared with the common polyporphyrin, the polyporphyrin has water solubility and improved biocompatibility.

(4) Compared with the common polyporphyrin, the polyporphyrin can be self-assembled into nanoparticles in water, does not need other carriers, and has potential in vivo delivery effect.

Drawings

FIG. 1 shows the hydrogen nuclear magnetization of polyporphyrin P-3O.

FIG. 2 is the carbon nuclear magnetism of polyporphyrin P-3O.

FIG. 3 is the hydrogen nuclear magnetization of polyporphyrin P-5O.

FIG. 4 is a dilution stability characterization of polyporphyrin P-5O.

FIG. 5 is a lyophilization stability characterization of polyporphyrin P-5O.

FIG. 6 shows phototoxicity and sonotoxicity of polyporphyrin P-5O.

Detailed Description

The following further describes a specific embodiment of the present invention with reference to the drawings and technical solutions.

Example 1: synthesis of water-soluble polyporphyrin P-3O

Tetracarboxyphenylporphyrin (25mg, 0.032mmol), EDCI (49mg, 0.256mmol) and DMAP (15.6mg, 0.128mmol) were weighed out accurately and dissolved in 0.2mL-2mL of DMMF, heated to 60 deg.C-80 deg.C and stirred for 10 min. Then, a mixed solution of tetraethylene glycol (12.4mg, 0.064mmol) and 0.1mL-0.5mL of DMMF is added, and the temperature is raised to 100-140 ℃ to continue the reaction for 24-48 h. After the reaction is finishedThe reaction solution was precipitated in 15mL of ether, the precipitate was collected by centrifugation, and the residue of ether was removed by vacuum drying overnight. Dissolving the precipitate in a volume ratio of 10: 1H₂And D, dissolving the mixture in a mixed solvent of O and DMSO, dialyzing the mixture in deionized water for 24 hours by using a dialysis bag with the molecular weight cut-off (MWCO) of 500, and freeze-drying the mixture in a freeze dryer to obtain the final product, namely the purple solid P-3O. The nuclear magnetic characterization of the obtained polyporphyrin is shown in FIG. 1. The nuclear magnetic characterization of the obtained polyporphyrin is shown in FIG. 2.

Example 2: synthesis of water-soluble polyporphyrin P-5O

Tetracarboxyphenylporphyrin (25mg, 0.032mmol), EDCI (49mg, 0.256mmol) and DMAP (15.6mg, 0.128mmol) were weighed out accurately and dissolved in 0.2mL-2mL of DMMF, heated to 60 deg.C-80 deg.C and stirred for 10 min. Then, adding a mixed solution of hexaethylene glycol (17.6mg, 0.064mmol) and 0.1mL-0.5mL of DMMF, heating to 100-140 ℃, and continuing the reaction for 24-48 h. After the reaction was complete, the reaction solution was allowed to settle in 15mL of diethyl ether, the precipitate was collected by centrifugation, and the residual diethyl ether was removed by vacuum drying overnight. Dissolving the precipitate in a volume ratio of 10: 1H₂And D, dissolving the mixture in a mixed solvent of O and DMSO, dialyzing the mixture in deionized water for 24 hours by using a dialysis bag with the molecular weight cut-off (MWCO) of 500, and freeze-drying the mixture in a freeze dryer to obtain the final product, namely the purple solid P-5O. The nuclear magnetic characterization of the obtained polyporphyrin is shown in FIG. 3.

Example 3: synthesis of water-soluble polyporphyrin P-11O

Tetracarboxyphenylporphyrin (25mg, 0.032mmol), EDCI (49mg, 0.256mmol) and DMAP (15.6mg, 0.128mmol) were weighed accurately into 1mL of chloroform and heated to 60 deg.C-80 deg.C and stirred for 10 min. Then, a mixed solution of polyethylene glycol (32mg, 0.064mmol) having a molecular weight of 500Da and 1mL of chloroform was added, and the reaction was continued for 24 to 48 hours while elevating the temperature to 70 ℃. After the reaction is finished, the reaction solution is settled in 30mL of normal hexane, the precipitate is collected by centrifugation, and the residual normal hexane is removed by vacuum drying overnight. Dissolve the precipitate in 1mL H₂And O, dialyzing in deionized water by using a dialysis bag with the molecular weight cut-off (MWCO) of 1000 for 24 hours, and freeze-drying in a freeze dryer to obtain the final product, namely the purple solid P-11O.

Example 4: synthesis of water-soluble polyporphyrin P-18O

Tetracarboxyphenylporphyrin (25mg, 0.032mmol), EDCI (49mg, 0.256mmol) and DMAP (15.6mg, 0.128mmol) were weighed accurately into 1mL tetrahydrofuran and heated to 60 deg.C-80 deg.C with stirring for 10 min. Then, a mixed solution of polyethylene glycol (51.2mg, 0.064mmol) having a molecular weight of 800Da and 1mL of tetrahydrofuran was added, and the reaction was continued for 24 to 48 hours while elevating the temperature to 70 ℃. After the reaction is finished, the reaction solution is settled in 30mL of normal hexane, the precipitate is collected by centrifugation, and the residual normal hexane is removed by vacuum drying overnight. Dissolving the precipitate in 1mLH₂And O, dialyzing in deionized water by using a dialysis bag with the molecular weight cut-off (MWCO) of 1000 for 24 hours, and freeze-drying in a freeze dryer to obtain the final product, namely the purple solid P-11O.

Example 5: stability characterization of Water-soluble Polyporphyrin nanoparticles

Firstly, directly dissolving the polyporphyrin P-5O in deionized water to prepare a mother solution with the concentration of 0.5mg/mL, standing for 1h at 37 ℃, and self-assembling into nanoparticles.

Stability to diluent: the nanoparticles were diluted 5-fold, 10-fold, 20-fold, 50-fold, 100-fold with serum buffer (FBS: 10%) at room temperature, and then the particle size change was measured by DLS, and the resulting particle size was shown in FIG. 4.

Freeze-drying stability: the particle size of the nanoparticles was measured by DLS before and after lyophilization in PBS buffer, and the obtained particle size was shown in fig. 5.

Example 6: photoacoustic toxicity characterization of Water-soluble Polyporphyrins

Phototoxicity: the MTT method is adopted to determine the cytotoxicity of the polyporphyrin nanoparticles on mouse liver cancer cells (Hep 1-6). Hep1-6 cells at 1X 10⁴Cell/well density was seeded in 96-well plates and incubated in cell culture dishes containing 10% FBS. The medium was changed and 50 μ M nanoparticles were added. After 24 hours of incubation, a 650nm laser (10 mW/cm) was used²) The well plate was irradiated for 30 minutes. Cells were incubated for an additional 20 hours before MTT assay to assess cell viability.

Sonotoxicity: the MTT method is adopted to determine the cytotoxicity of the polyporphyrin nanoparticles on mouse liver cancer cells (Hep 1-6). Hep1-6 cells at 1X 10⁴Cell/well Density seeded in 96-well plates incubated in cell culture dishes containing 10% FBSIn (1). The medium was changed and 50 μ M nanoparticles were added. After 24 hours of incubation, the cells were sonicated (1MHz,700 mW/cm) using a sonicator²) The cells were treated for 10 minutes and incubated for an additional 20 hours before MTT assay to assess cell viability.