阅读说明：本技术 一种具有高稳定性的水溶性方酸菁型近红外有机大分子光热剂的合成方法 (Synthetic method of water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent with high stability ) 是由 王红明 王怡刚 夏国民 于 2021-08-13 设计创作，主要内容包括：本发明公开了一种具有高稳定性的水溶性方酸菁型近红外有机大分子光热剂的合成方法,属于生物医学技术领域。首先设计合成了一种传统的1,3-型方酸菁光热剂(SQ1),SQ1具有高的摩尔消光系数,因其水溶性较差以及化学不稳定性,本发明在SQ1的中心四元环的2’位通过共价键连接氨基修饰的PEG-(5000)长链,得到一个1,2,3-型方酸菁大分子光热剂(PSQ)。该光热剂在单分子的状态拥有高的光稳定性、化学稳定性,以及很好的水溶性；同时PSQ大分子是一个两亲性分子,它可以在水中自组装形成粒径、形貌均一的纳米粒子(NPs),纳米粒子在体外的光热性能研究中展示出了优异的光热性质。(The invention discloses a synthesis method of a water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent with high stability, belonging to the technical field of biomedicine. Firstly, a traditional 1, 3-type squaraine photo-thermal agent (SQ1) is designed and synthesized, SQ1 has high molar extinction coefficient, and due to poor water solubility and chemical instability, the invention connects amino modified PEG at the 2' position of a central four-membered ring of SQ1 through a covalent bond 5000 And (3) growing a long chain to obtain the 1,2, 3-type squaraine macromolecular photo-thermal agent (PSQ). The photo-thermal agent has high light stability, chemical stability and good water solubility in a monomolecular state; meanwhile, the PSQ macromolecule is an amphiphilic molecule, and can be self-assembled in water to form Nano Particles (NPs) with uniform particle size and morphology, and the nano particles show excellent photo-thermal properties in-vitro photo-thermal performance research.)

1. A high-stability water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent is characterized in that the structural formula of the photo-thermal agent is as follows:

2. a synthetic method of a water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent with high stability is characterized by comprising the following steps:

1) 1, 8-naphthalimide is taken as a starting raw material to synthesize the 1, 3-type squaraine photo-thermal agent;

2) synthesizing the obtained 1, 3-type squarylium cyanine photo-thermal agent into a high-stability water-soluble 1,2, 3-type squarylium cyanine macromolecular photo-thermal agent;

the reaction is as follows:

3) the 1,2, 3-type squaraine macromolecular photo-thermal agent can be self-assembled in aqueous solution to form nanoparticles.

3. The method for synthesizing the water-soluble squaraine near-infrared organic macromolecular photo-thermal agent with high stability as claimed in claim 2, wherein the method for synthesizing the 1, 3-type squaraine photo-thermal agent in step 1) comprises:

s1, dissolving 1, 8-naphthalimide in N, N-dimethylformamide, slowly adding sodium hydride under the condition of ice-water bath, slowly dropping ethyl iodide after stirring, continuously stirring for 2 hours after the reaction system is slowly heated to room temperature, adding water to quench the reaction, extracting by ethyl acetate, and separating and purifying by using column chromatography to obtain a compound 2, wherein the structure of the compound is as follows:

s2, dissolving the compound 2 in ultra-dry tetrahydrofuran, stirring in an ice water bath, dropwise adding a methyl magnesium chloride/tetrahydrofuran solution, heating the reaction system to 60 ℃ for reaction for 2 hours, adding water for quenching reaction, adding a potassium iodide solution to precipitate an orange-red solid, and performing suction filtration to obtain a compound 3, wherein the structure of the compound is as follows:

s3, dissolving the compound 3 and the squaric acid in n-butanol/toluene in a volume ratio of 1: 1, heating the mixture by using a water separator until reflux reaction is carried out for 3 hours, carrying out vacuum rotary evaporation on the reaction solvent, and then separating and purifying by using column chromatography to obtain a brown product, namely the 1, 3-type squaraine photo-thermal agent, wherein the structure is as follows:

4. the method for synthesizing the highly stable water-soluble squaraine near-infrared organic macromolecular photo-thermal agent according to claim 3, wherein the ratio of the amounts of the 1, 8-naphthalimide, sodium hydride and ethyl iodide in S1 is 5: 25: 6.

5. the method for synthesizing the highly stable water-soluble squaraine near-infrared organic macromolecular photo-thermal agent according to claim 3, wherein the ratio of the amounts of the compound 2, the methyl magnesium chloride/tetrahydrofuran and the potassium iodide in S2 is 1: 3: 0.001.

6. the method for synthesizing the highly stable water-soluble squaraine near-infrared organic macromolecular photo-thermal agent according to claim 3, wherein the ratio of the amount of the compound 3 to the amount of the squaraine substance in S3 is 2: 1.

7. the method for synthesizing the water-soluble squaraine near-infrared organic macromolecular photo-thermal agent with high stability according to claim 2, wherein the method for synthesizing the 1,2, 3-type squaraine macromolecular photo-thermal agent in the step 2) comprises the following steps:

dissolving the 1, 3-type squarylium cyanine photo-thermal agent obtained in the step 1) in dichloromethane, slowly dropwise adding methyl trifluoromethanesulfonate, stirring at room temperature for 6 hours, adding a potassium iodide solution for extraction, performing vacuum rotary evaporation on the dichloromethane solution, and performing column chromatography separation and purification to obtain a brown product; the brown product obtained is reacted with NH₂-PEG₅₀₀₀Dissolving in dichloromethane, stirring at room temperature for 6h, performing vacuum rotary evaporation on the reaction solvent, and performing column chromatography separation and purification to obtain a brown oily liquid, namely the 1,2, 3-type squaraine macromolecular photo-thermal agent, wherein the structure is as follows:

8. the method for synthesizing the highly stable water-soluble squaraine near-infrared organic macromolecular photo-thermal agent according to claim 7, wherein the ratio of the amount of the 1, 3-type squaraine photo-thermal agent to the amount of the methyl trifluoromethanesulfonate is 1: 3; the brown product is reacted with NH₂-PEG₅₀₀₀The ratio of the amounts of substances is 1: 1.

9. the method for synthesizing the high-stability water-soluble squaraine near-infrared organic macromolecular photo-thermal agent according to claim 2, wherein the self-assembly in the step 3) comprises the following specific operations:

dissolving the 1,2, 3-type squaraine macromolecular photo-thermal agent obtained in the step 2) in dimethyl sulfoxide, slowly dripping deionized water under vigorous stirring, then stirring overnight at room temperature, and dialyzing to obtain a nanoparticle solution.

10. The method for synthesizing the highly stable water-soluble squaraine near-infrared organic macromolecular photo-thermal agent according to claim 9, wherein the mass-to-volume ratio of the 1,2, 3-type squaraine macromolecular photo-thermal agent to the dropped deionized water is 20: 1.8, mg: mL; deionized water is used for dialysis, the molecular weight cutoff is 3500, and the deionized water is replaced every 6 hours.

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to a synthesis method of a water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent with high stability.

Background

Photothermal therapy (PTT) is a typical photon-triggered therapy to kill tumor cells by local hyperthermia generated by photothermal agents (PTAs) under visible or Near Infrared (NIR) laser. It exploits the sensitivity of cells to heat to induce apoptosis or increase sensitivity to radiation or chemotherapy. Compared to traditional cancer treatment modalities, such as surgery, radiation therapy and chemotherapy, PTT is very attractive because it can have a high degree of intrinsic specificity and a low invasive burden. PTT is also, in principle, a cancer treatment with relatively little damage to surrounding healthy tissue. This is because thermal effects are only produced when NIR laser light is applied, and at the same time only when PTAs are present. By appropriate design, PTAs can be directed specifically to the cancer site, further increasing selectivity. Given that molecular design plays such a critical role in the success of PTT, it is not surprising that considerable effort has been devoted to the development of new sensitizers. To date, such efforts have mostly focused on near-infrared light-triggered inorganic materials, including transition metal nanoparticles, sulfide nanoparticles, gold nanoparticles, and platinum nanoparticles. While these systems generally exhibit good absorption characteristics, excellent light-to-heat conversion efficiency, and good light stability, they lack biodegradability and potential long-term toxicity, which present obstacles to effective clinical transformation efforts. In recent years, small organic molecules have attracted more and more attention as potential substitutes for nanomaterials in the PTT field. These molecular sensitizers typically function by absorbing photons generated by near infrared radiation. One potential advantage of organic PTT sensitizers is that unlike inorganic materials, photothermal materials based on small organic molecules are designed with safety and substrate-building issues in mind. Their characteristics can be further fine-tuned by specialized synthesis. For example, small organic molecule-based photothermal agents may be further modified to perform therapeutic functions.

Squaraines (SQs), which are generally composed of a central electron-deficient four-membered ring and two electron donors, in the form of resonance-stable homogeneous donor-acceptor-donors (D-a-D), have been widely used in the fields of bioimaging, ion detection, photodynamic therapy, photovoltaics and nonlinear optics. These dyes exhibit low rates of interstitial crossing (ISC) and high molar absorptivity (. epsilon. > 10) in the near infrared region⁵L·mol^-1·cm^-1). Therefore, SQ derivatives are a promising class of PTAs with their unique optical properties and good photostability. However, the inherent chemical instability and poor water solubility of SQ prevents its use in biological and medical applications. Therefore, there is a potential to develop a photothermal agent based on SQ small molecules, which has high stability and good water solubility.

Disclosure of Invention

The invention aims to provide a synthesis method of a water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent with high stability, which has high photo-thermal conversion efficiency and good photo, thermal and chemical stability.

The invention is realized by the following technical scheme:

a synthetic method of a water-soluble squarylium cyanine near-infrared organic macromolecular photo-thermal agent with high stability comprises the following steps:

1) synthesizing a 1, 3-type squaraine photo-thermal agent (SQ 1);

2) synthesizing a high-stability water-soluble 1,2, 3-type squarylium cyanine macromolecular photo-thermal agent (PSQ);

3) the PSQ self-assembles in aqueous solution to form nanoparticles.

Further, the synthesis method of SQ1 in step 1) includes:

s1, dissolving 1, 8-naphthalimide (compound 1) in N, N-dimethylformamide, slowly adding sodium hydride under the condition of ice-water bath, stirring for 15min, slowly dropping ethyl iodide, slowly heating the reaction system to room temperature, continuously stirring for 2h, adding water to quench the reaction, extracting, and separating and purifying by using column chromatography to obtain a yellow solid (compound 2);

s2, dissolving the compound 2 in ultra-dry tetrahydrofuran, stirring in an ice water bath for 5min, dropwise adding a methyl magnesium chloride/tetrahydrofuran solution, heating the reaction system to 60 ℃ for reaction for 2h, adding water for quenching reaction, adding a potassium iodide solution to precipitate an orange-red solid, and performing suction filtration to obtain a compound 3;

s3, dissolving the compound 3 and the squaric acid in n-butanol/toluene in a volume ratio of 1: 1, heating the mixture to reflux reaction for 3 hours by using a water separator, and performing column chromatography separation and purification (eluent: dichloromethane/methanol volume ratio 100: 1) after rotationally evaporating the reaction solvent in vacuum to obtain a brown product, namely SQ 1.

The reaction process is as follows:

further, the ratio of the amounts of the 1, 8-naphthalimide, sodium hydride and ethyl iodide substances in S1 is 5: 25: 6.

further, the ratio of the amount of the compound 2, the methyl magnesium chloride/tetrahydrofuran and the potassium iodide in S2 is 1: 3: 0.001.

further, the ratio of the amount of compound 3 to the amount of squaric acid substance in S3 is 2: 1.

further, the synthesis method of the PSQ in the step 2) comprises the following steps: dissolving SQ1 obtained in the step 1) in dichloromethane, slowly dropwise adding methyl trifluoromethanesulfonate, stirring at room temperature for 6h, adding a potassium iodide solution for extraction, performing vacuum rotary evaporation on the dichloromethane solution, and separating and purifying by column chromatography (eluent: dichloromethane/methanol volume ratio 100: 1) a brown product (MSQ) is obtained; mixing MSQ with NH₂-PEG₅₀₀₀Dissolving in dichloromethane, stirring at room temperature for 6h, spin-drying the reaction solvent, and separating and purifying by column chromatography (eluent: dichloromethane/methanol volume ratio 20: 1) to obtain brown oily liquid, namely PSQ.

The reaction process is as follows:

further, the ratio of SQ1 to the amount of methyl trifluoromethanesulfonate species is 1: 3; the MSQ and NH₂-PEG₅₀₀₀The ratio of the amounts of substances is 1: 1.

further, the specific operation of step 3) self-assembly is as follows: dissolving the PSQ obtained in the step 2) in dimethyl sulfoxide, slowly dropping deionized water under vigorous stirring, then stirring overnight at room temperature, and dialyzing to obtain a nanoparticle solution.

The reaction process is as follows:

further, the mass-to-volume ratio of the PSQ to the dropped deionized water is 20: 1.8, mg: mL.

Further, deionized water is used for dialysis, the molecular weight cutoff is 3500, and the deionized water is replaced every 6 hours; the obtained nanoparticle solution was stored at 4 ℃ for further use.

Compared with the prior art, the invention has the beneficial effects that:

the invention firstly synthesizes traditional 1, 3-type squaraine photo-thermal agents SQ1 and SQ1 which have high molar extinction coefficient, and because of poor water solubility and chemical instability, the invention connects PEG modified by amino at 2' position of central four-membered ring of SQ1 through covalent bond₅₀₀₀And (4) obtaining a 1,2, 3-type squaraine macromolecular photo-thermal agent through long chain. The photo-thermal agent has high light stability, chemical stability and good water solubility in a monomolecular state; meanwhile, the PSQ macromolecule is an amphiphilic molecule, can be self-assembled in water to form nanoparticles with uniform particle size and morphology, and the nanoparticles show excellent photo-thermal property in-vitro photo-thermal performance research.

Drawings

FIG. 1 is a scheme for the synthesis of PSQ, a macromolecular photo-thermal agent.

FIG. 2 is an absorption spectrum of SQ1 with PSQ in phosphate buffered saline and methylene chloride, respectively.

FIG. 3 shows the maximum absorption intensity of SQ1, PSQ, and a commercial photothermal agent ICG after 12 hours of natural light.

FIG. 4 is a graph of SQ1 and PSQ as the change in their maximum absorption intensity after 35 minutes of reaction with GSH at 37 ℃.

In fig. 5: (A) calculated absorbance spectra for SQ1 and PSQ; (B) reaction energy curves for SQ1/PSQ and GSH; (C) the front molecular orbital of SQ1 and PSQ.

FIG. 6 is a schematic diagram of the molecular structure and simplified model of PSQ, self-assembled in aqueous solution to form nanoparticles.

Fig. 7 is a graph of the dynamic light scattering particle size of the nanoparticles, and the inset is an environmental scanning electron microscope image of the nanoparticles.

FIG. 8 shows the power density of 0.1 W.cm for aqueous solutions of nanoparticles of different concentrations^-2The temperature rise profile of the aqueous solution after 4 minutes of laser irradiation.

FIG. 9 shows 500. mu.g/mL^-1The nano particle aqueous solution is subjected to a temperature curve of 6 cycles of laser irradiation temperature rise and natural cooling.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Example 1

(1) Preparation of SQ 1: the first step is as follows: dissolving 1, 8-naphthalimide (compound 1, 5mmol) in N, N-dimethylformamide (20mL), slowly adding sodium hydride (25mmol) under the condition of ice-water bath, stirring for 15min, slowly dropping ethyl iodide (6mmol), slowly heating the reaction system to room temperature, then continuing stirring for 2h, adding water to quench the reaction, extracting, separating and purifying by using column chromatography (eluent petroleum ether, ethyl acetate volume ratio of 15: 1) to obtain compound 2, wherein the yellow solid is 827mg, and the yield is 84%. The second step is that: dissolving a compound 2(2mmol) in ultra-dry tetrahydrofuran (20mL), stirring in an ice-water bath for 5min, dropwise adding a methyl magnesium chloride/tetrahydrofuran solution (2mL,3mol/L) through a syringe, heating a reaction system to 60 ℃, reacting for 2h, adding water, quenching, adding a potassium iodide solution (2mL,1mmol/L) to precipitate an orange-red solid, and performing suction filtration to obtain a compound 3, wherein the next reaction can be directly performed without treatment. The third step: compound 3(2mmol) and squaric acid (1mmol) were dissolved in n-butanol/toluene 1: 1, heated to reflux for 3h by using a water separator, and subjected to separation and purification by column chromatography (eluent: dichloromethane/methanol volume ratio 100: 1) after the reaction solvent is rotationally evaporated in vacuum to obtain 249mg of SQ1 brown product with the yield of 53.2%.

(2) Preparation of PSQ: the first step is as follows: dissolving SQ1(1mmol) in dichloromethane, slowly dropwise adding methyl trifluoromethanesulfonate (3mmol), stirring at room temperature for 6h, adding a KI solution for extraction, carrying out vacuum rotary evaporation on the dichloromethane solution, and then carrying out column chromatography separation and purification (eluent: dichloromethane/methanol volume ratio 100: 1) to obtain a brown product (MSQ); the second step is that: mixing MSQ (0.1mmol) with NH₂-PEG₅₀₀₀(0.1mmol) was dissolved in 20mL of dichloromethane, stirred at room temperature for 6 hours to spin-dry the reaction solvent, and then purified by column chromatography (eluent: dichloromethane/methanol volume ratio 20: 1) to obtain 296mg of PSQ brown oily liquid in a yield of 53.3%. The specific process is shown in fig. 1.

(3) As can be seen from fig. 2, the maximum absorption wavelengths of SQ1 and PSQ in dichloromethane are 883 and 875nm, respectively, while in aqueous solution both show a significant blue shift, with SQ1 blue-shifted to 710nm and PSQ blue-shifted to 740 nm. Next, the light stability of SQ1, PSQ, and commercial photothermal agent ICG was investigated, and their absorbances at the maximum absorption wavelength were measured every 1h after the natural light continuous irradiation for 12h, as can be seen from fig. 3, after SQ1 and PSQ were continuously irradiated for 12h, the absorbances at the maximum absorption wavelength did not change significantly, but were always decreased compared to ICG, and it was found that SQ1 and PSQ had better light stability than ICG. Chemical stability of PSQ versus SQ1 was next investigated, treating SQ1 and PSQ with excess GSH under 37 ℃ water bath conditions, respectively, with time resolution of the absorption change as shown in fig. 4, with the absorption spectrum of PSQ remaining almost unchanged after 30 minutes, with regard to SQ1, the absorption intensity decreases sharply with time, and the solution fades almost completely after 10 minutes. This indicates that the chemical stability of PSQ is clearly superior to SQ 1. To further understand the optical behavior and stability of SQ1 and PSQ, the absorption spectra of PSQ and SQ1 and the mechanism of reaction with GSH were calculated by DFT and TDDFT methods. As shown in fig. 5(a), the absorption wavelengths of SQ1 and PSQ molecules from the ground state to the excited state in DCM were 881 and 862nm, respectively, and all calculated absorptions were in good agreement with the experimental values. As shown in figure 5(B), the reaction energy curves for SQ1 and PSQ with GSH were calculated, the first step being the nucleophilic addition of GSH to the SQ1 central four-membered ring, forming the adduct PSQ1 via the transition state TSSQ 1. The energy barrier and reaction energy of this process were 15.83 kcal/mol and-3.99 kcal/mol, respectively. However, the reaction between PSQ and GSH is very different. The nucleophilic addition of GSH to the PSQ central four-membered ring results in an energy barrier of 22.76 kcal/mole for the transition state TSPSQ, followed by the formation of the product PPSQ. The energy of the reaction between PSQ and GSH was 4.02 kcal/mole. As an endothermic reaction and possess a high energy barrier and mean that the latter is difficult to perform at physiological conditions. However, the reaction between SQ1 and GSH proceeds easily under the same conditions, since it is an exothermic reaction and possesses a lower reaction energy barrier.

(4) As shown in fig. 6, PSQ is an amphiphilic molecule, squaraine moiety is hydrophobic, PEG long-chain moiety is hydrophilic, and the molecular configuration is schematically shown in the figure, and it can self-assemble to form nanoparticles with uniform particle size after stirring aqueous solution, and then dialyze in deionized water for 72h, and characterize the nanoparticles with an environmental scanning electron microscope and a dynamic light scattering particle size analyzer, respectively, and the results show that the average particle size of the nanoparticles is 105nm spherical particles. This indicates that the particles can be enriched in tumor tissues by the enhanced permeability and retention Effect (EPR) of solid tumors, thereby achieving good photothermal treatment efficiency. Next, the photothermal effect of the nanoparticles was studied, and firstly, different concentrations of nanoparticle aqueous solutions exhibited different temperature-raising effects under the excitation of laser (0.1W/cm, 808nm), wherein 500 μ g/mL of nanoparticles were raised from 28 ℃ to 98 ℃ after 4 minutes of irradiation, and the result is shown in fig. 8, while in fig. 9, a set of temperature curves of laser heating temperature-raising and natural cooling temperature-lowering at 6 cycles, which are the maximum concentration, the nanoparticles exhibited good photothermal stability, and still could maintain good temperature-raising effect at 6 th cycle.

The above-described embodiments are only preferred embodiments of the present invention and are not intended to limit the present invention. Various changes and modifications can be made by one skilled in the art, and any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.