WO2022134862A1 - Organic conjugated polymer photo-thermal reagent for treating malignant melanoma, nanoparticle, and preparation method therefor and use thereof - Google Patents

Abstract

An organic conjugated polymer photo-thermal reagent for treating malignant melanoma, a nanoparticle, and a preparation method therefor and the use thereof. The structure of the organic conjugated polymer photo-thermal reagent for treating malignant melanoma is as represented by formula (I). The organic conjugated polymer photo-thermal reagent has the advantages of using easily available raw materials, having mild synthesis conditions, having a simple preparation method, being convenient in terms of purification and easy in terms of realization. The organic conjugated polymer for treating malignant melanoma and an amphiphilic compound are prepared into nanoparticles by means of a co-precipitation method, and the nanoparticles have a good solubility in a water environment and have excellent biocompatibility. The nano-sized particles can enter cells easily, have an excellent photo-stability, chemical properties and photo-thermal conversion efficiency, have a good photo-thermal treatment effect on melanoma (B16) cells, and have few side effects.

Description

Organic conjugated polymer photothermal agent, nanoparticle, preparation method and application for treating malignant melanoma technical field

The invention belongs to the technical field of anti-tumor drugs, and in particular relates to an organic conjugated polymer photothermal reagent for treating malignant melanoma, nanoparticles, and a preparation method and application.

Background technique

Malignant melanoma is a highly malignant tumor derived from melanocytes, which mostly occurs in the skin, but also in the mucosa and internal organs, accounting for about 3% of all tumors. Skin malignant melanoma is the third most common skin cancer (accounting for 6.8%-20%). In recent years, the incidence and mortality of malignant melanoma have increased year by year. In light-skinned people, the incidence of malignant melanoma has increased by about 3% to 7% per year in recent years, and it is the malignant tumor with the fastest increasing incidence. one of the tumors. It has a lower age of death than other solid tumors. Except for early surgical resection, malignant melanoma lacks effective treatment and has a poor prognosis. Therefore, early diagnosis and treatment of malignant melanoma is extremely important.

Photothermal therapy selectively heats the tumor site through photothermal materials under the action of light, and excessive heat is generated inside the cancer cells, thereby killing the cancer cells and inhibiting the growth of the tumor. Because the proliferation rate of cancer cells is much faster than that of normal tissue, the blood vessels in tumor tissue are not fully developed, the blood vessel wall is defective, and the tolerance to heat is lower than that of normal cell tissue. When the intracellular temperature reaches 40 °C, the proteins in the cell begin to deform, and 50 °C will cause irreversible damage. Taking advantage of this, photothermal therapy can cause damage to cancer cells and destroy tumor tissue without affecting normal cells and tissues. Photothermal therapy has the advantages of being minimally invasive, having little effect on normal cells and tissues, and having few side effects. Photothermal therapy for melanoma using organic photothermal materials is a new treatment method.

Through the continuous efforts of scientific researchers, the current photothermal materials are mainly divided into noble metal materials, carbon-based materials, transition metal compound nanomaterials and organic photothermal materials. 1) Although noble metal materials (such as Au, Ag, etc.) have high photothermal conversion efficiency, they have poor metabolism in the body, high cost, and some disadvantages of toxic and side effects; 2) Although carbon-based materials are non-toxic and have high photothermal conversion efficiency, However, the weak absorption in the near-infrared band also limits its further application; 3) The two-dimensional materials of transition metal compounds have high photothermal conversion efficiency in the near-infrared region, but are complicated to prepare, large in size, not easily absorbed by cells, and metabolized slowly, etc. It is also the bottleneck of its development. 4) Organic photothermal materials have the advantages of strong near-infrared absorption, good biocompatibility, easy functionalization of structure, and short metabolism time in vivo. Therefore, compared with other types of photothermal materials, organic photothermal materials provide a new material system for tumor photothermal therapy.

The reported compound TPA-T-TQ (from the American Chemical Society Nano, Vol. 11, No. 7, 2017, pp. 7177-7188) has a photothermal conversion efficiency of up to 74% at 808 nm, but the compound has a complex structure and is synthesized The steps are cumbersome. Therefore, photothermal reagents with simple structures and easy synthesis routes are more conducive to the development of industrialization; and polymers have synergistic amplification effects, and their photothermal properties are superior to those of small molecular compounds. Therefore, polymers with simple structures can promote the development of photothermal agents for photothermal therapy.

technical solutions

In order to treat the above-mentioned malignant melanoma, the primary purpose of the present invention is to provide a class of organic conjugated polymer photothermal reagents for the treatment of malignant melanoma. The photothermal reagents and amphiphilic compounds are prepared into nanoparticles by co-precipitation method, which have excellent water solubility, photostability, biocompatibility and photothermal conversion performance, and have great potential in the field of photothermal therapy of malignant melanoma. application potential.

The photothermal agent of the present invention is an organic conjugated polymer for treating malignant melanoma, and has high light capture ability and photothermal conversion efficiency. Polyethylene glycol (DSPE-PEG2000, DSPE-PEG5000) forms nanoparticles by co-precipitation method, which have excellent solubility and biocompatibility in aqueous solution, and are a class of photothermal reagents with excellent performance and potential.

Another object of the present invention is to provide the above-mentioned photothermal agent for treating malignant melanoma and the preparation method of nanoparticles.

Another object of the present invention is to provide the application of the above-mentioned photothermal agent for treating malignant melanoma, and the application of the organic photothermal agent in photothermal therapy. Especially as a photothermal agent for photothermal therapy of malignant melanoma. The present invention obtains an organic photothermal reagent with high photothermal conversion efficiency through rational molecular structure design, and has excellent curative effect in photothermal treatment of malignant melanoma. The cancer cells are melanoma (B16) cells.

The object of the present invention is achieved through the following technical solutions:

The organic conjugated polymer photothermal agent for treating malignant melanoma has the following chemical structural formula:

(I)

In the formula, R is a linear or branched alkyl group with 1-20 carbon atoms, R ₁ is a linear or branched alkyl group with 1-20 carbon atoms,

,

, R ₂ is hydrogen, straight-chain, branched or cyclic alkyl or alkoxy with 1 to 20 carbon atoms.

Ar has the following structure:

wherein R3 is a straight-chain, branched or cyclic alkyl group having 1 to 20 carbon atoms;

The degree of polymerization n is any integer from 2 to 300. 

The preparation method of the above-mentioned organic conjugated polymer photothermal reagent for treating malignant melanoma is:

In an inert atmosphere, the polymerized monomers M1 and M2 are dissolved in the organic solvent, then the catalyst tetrakistriphenylphosphine palladium is added, and the reaction is performed for 4 to 24 hours. After the reaction is stopped, the reaction solution is purified to obtain the target polymer;

The structural formula of the polymerized monomer M1 is

, the structural formula of the polymerized monomer M2 is

.

Further, the mol ratio of polymerized monomers M1 and M2 is 1:1; the organic solvent is tetrahydrofuran, chlorobenzene, o-dichlorobenzene, preferably o-dichlorobenzene; temperature of reaction is 90～160 ℃ Celsius, the reaction time is 4 to 12 hours, preferably 6 to 8 hours.

Further, the inert atmosphere is nitrogen or rare gas atmosphere; the purification refers to cooling the obtained reaction solution to room temperature, adding methanol to precipitate, filtering to obtain a crude product, and then dissolving the crude product in toluene and using silica gel. As the stationary phase, column chromatography was performed with acetone as the eluent, the solvent was concentrated, precipitated in methanol again, stirred, filtered, and dried under vacuum to obtain a polymer solid; , remove small molecules; then the solid is dissolved in deionized water, added to methanol for precipitation, and the target product is obtained after vacuum drying.

The preparation method of organic conjugated polymer nanoparticles for treating malignant melanoma is as follows:

The polymer photothermal reagent and the amphiphilic compound are dissolved in an organic solvent, the mixed solution is added to an ultrapure aqueous solution under ultrasonic conditions, and the preparation is completed by ultrasonication at room temperature.

Further, the amphiphilic compound is F127, DSPE-PEG2000, DSPE-PEG5000, preferably F127; the mass ratio of the polymer and the amphiphilic compound is 1:1～50; further preferably, the mass ratio of the polymer:F127 is preferably 1 : 5～50, more preferably 1:10; Polymer: the mass ratio of DSPE-PEG2000 is preferably 1:1～20, more preferably 1:5; Polymer: the mass ratio of DSPE-PEG5000 is preferably 1:1～20 , more preferably 1:5.

The organic conjugated polymer nanoparticle for treating malignant melanoma prepared by the above preparation method.

Meanwhile, the present invention also provides the application of the organic conjugated polymer nanoparticles for treating malignant melanoma as a photothermal reagent in the field of photothermal therapy.

beneficial effect

Compared with the prior art, the beneficial effects of the present invention are embodied in:

1. The photothermal material of the present invention is an organic conjugated polymer, which is easy to modify in structure and has strong light capturing ability. Using near-infrared laser such as 808 nm as a light source for photothermal treatment of malignant melanoma, the penetration depth is strong and the therapeutic effect is strong. Better, less side effects, with clinical application prospects.

2. The photothermal material of the present invention has high photothermal conversion efficiency, good solubility in organic solvents, and is prepared into water-soluble nanoparticles, which can ensure the photothermal conversion efficiency of nanoparticles, and improve its water solubility and biological safety. sex. It has excellent biocompatibility to cells and can effectively kill cells under light conditions, especially melanoma (B16) cells.

3. The photothermal material of the present invention has easily available raw materials, mild synthesis conditions, simple preparation method and convenient purification.

Description of drawings

Fig. 1 is the absorption spectrum diagram of the organic conjugated polymer photothermal reagent P2 nanoparticle aqueous solution of embodiment 2;

Fig. 2 is the heating curve of the organic conjugated polymer photothermal reagent P2 nanoparticle aqueous solution under different powers of embodiment 2;

FIG. 3 is a bar graph of cell viability of photothermal reagent P2 nanoparticle aqueous solution co-cultured with B16 cells before and after irradiation of Example 2.

Embodiments of the present invention

The present invention will be described in further detail below with reference to the examples and accompanying drawings, but the embodiments and protection scope of the present invention are not limited thereto.

Example 1

Organic Conjugated Polymer Photothermal Reagents P1 Synthesis

Under an argon atmosphere, the polymerized monomer M1 (317.6 mg, 0.50 mmol) and the monomer M2 (233.9 mg, 0.50 mmol) were added into a 50 mL two-necked flask, and then 6 mL of purified o-dichlorobenzene was added, and then tetrakis Phenylphosphine palladium (2.80 mg, 12.45 μmol) was heated to 140 °C, reacted for 8 hours, and the reaction was stopped. After the temperature dropped to room temperature, the product was added dropwise to 300 mL of methanol for precipitation, filtered, and the crude product was dissolved in In 20 mL of toluene, use 200-300 mesh silica gel as the stationary phase, use acetone as the eluent to carry out column chromatography, concentrate the solvent, precipitate out in methanol again, stir, filter, and vacuum dry to obtain a polymer solid; Finally, the mixture was extracted with methanol, acetone and n-hexane for 24 hours each to remove small molecules; the solid was dissolved in deionized water, dropped into methanol for precipitation, and dried in vacuo to obtain polymer P1. The results of ¹ H NMR, GPC and elemental analysis show that the obtained compound is the target product, and the chemical reaction equation of the preparation process is as follows:

The obtained polymer P1 was detected, and the number average molecular weight Mn of the GPC test was 10500, the weight average molecular weight Mw was 154000, and the molecular weight distribution index PDI was 1.47.

Preparation method of polymer nanoparticles:

5.0 mg of polymer P1 and 50 mg of amphiphilic triblock polymer F127 were dissolved in 2.0 ml of tetrahydrofuran solvent, and the dissolved mixture was quickly added to 10 ml of ultrapure water under ultrasonic conditions at room temperature to obtain Brown-black mixed solvent liquid. The obtained liquid was then placed in a fume hood, and the excess tetrahydrofuran solution was volatilized to obtain polymer nanoparticles with an apparent concentration of 500. ug/ml.

Example 2

Organic Conjugated Polymer Photothermal Reagents P2 Synthesis

The synthesis of organic conjugated polymer photothermal reagent P2 is similar to that of polymer P1, except that the monomer M2 is replaced by M3. The specific synthetic route is as follows:

The obtained polymer P2 was detected, and the number average molecular weight Mn of the GPC test was 10300, the weight average molecular weight Mw was 15400, and the molecular weight distribution index PDI was 1.50.

Preparation method of polymer P2 nanoparticles:

5.0 mg of polymer P2 and 50 mg of amphiphilic triblock polymer F127 were dissolved in 2.0 ml of tetrahydrofuran solvent, and the dissolved mixture was quickly added to 10 ml of ultrapure water under ultrasonic conditions at room temperature to obtain Brown-black mixed solvent liquid. The obtained liquid was then placed in a fume hood, and the excess tetrahydrofuran solution was volatilized to obtain polymer nanoparticles with an apparent concentration of 500. ug/ml.

The absorption spectrum of the organic conjugated polymer photothermal reagent P2 nanoparticle aqueous solution is shown in Figure 1. It can be seen from the figure that the polymer P2 has a strong absorption in the range of 400-1400 nm, and it is suitable to use a laser with a wavelength of 808 nm as the Light source for photothermal test;

The photothermal properties of the polymer nanoparticles P2 NPs were measured using an 808 nm laser. The polymer nanoparticles P2 NPs with a concentration of 50 μg/mL were placed under a laser light source with a wavelength of 808 nm, and the powers were 0.25, 0.50, 0.75, and 1.0 W/cm ² , and the polymer nanoparticles P2 NPs were recorded every 30 s temperature of the aqueous solution. 8 minutes of light each time. The heating and cooling curves of the polymer nanoparticles P2 NPs are shown in Fig. As the power increases, the maximum temperature of the aqueous solution of P2 NPs also increases. When the power is 1.0 W/cm ² , the maximum temperature is 78.5 degrees Celsius, and the photothermal conversion efficiency is 84.6%. This shows that the polymer P2 has excellent photothermal conversion efficiency and has excellent effects in the treatment of malignant melanoma, and is a promising photothermal material.

The cytotoxicity experiments of polymer nanoparticles P2 NPs were detected by CCK-8 method:

1) Dilute the polymer nanoparticle P2 NPs with complete medium (DMEM) to the concentration of 10 μg/mL, 20 μg/mL, 30 μg/mL, 40 μg/mL, 50 μg/mL.

2) The melanoma (B16) cells in logarithmic growth phase were digested with trypsin, and the cells were evenly diluted to a concentration of 5×10 ⁴ cells/mL.

3) Add the cell solution to a 96-well plate, 100 μL per well, shake it slightly, put it into an incubator at 37 °C and 5% CO ₂ , and incubate for 24 h.

4) Add the complete medium containing different concentrations of polymer nanoparticles P2 NPs into a 96-well plate, 100 μL per well, 10 wells for each concentration, each 5 wells as a group, a total of 2 groups, that is, illumination group and unlit group. 0 μg/mL was set as the control group. The 96-well plate was placed in an incubator for 12 h.

5) Take out the 96-well plate in the light group, irradiate it with 808 nm laser (power 0.5 W/cm ² ) for 5.0 min, and then put it into the incubator for further incubation for 12 h. The 96-well plate in the unilluminated group does not need to receive light treatment. Incubate directly for 24 h.

6) Wash away the waste medium in the 96-well plate of the light group and the non-light group, add 100 μL of complete medium containing 10% CCK-8 to each well, and then put it back into the incubator for 1 h.

7) Put the 96-well plates of the light group and the non-light group into the microplate reader, and the absorption peak is 450 nm. Measure the absorbance of each well, calculate the average and standard deviation of the absorbance of 5 wells in each group, and calculate the melanin Tumor (B16) cell viability. Its CCK-8 test results are shown in Figure 3.

It can be seen from Figure 3 that the polymer nanoparticles P2 with different concentrations Under the condition of no light, the survival rate of B16 cells of NPs can be maintained above 90%. It shows that the polymer nanoparticles P2 NPs have low dark toxicity and excellent biocompatibility. While under light conditions, cell viability was significantly higher than that of polymer nanoparticles P2 The concentration of NPs was related, and the higher the concentration of P2 NPs, the lower the survival rate of melanoma (B16) cells. At 10 μg/mL, P2 NPs could kill 19% of melanoma (B16) cells; at 20 μg/mL, P2 NPs could kill 33% of melanoma (B16) cells; at 30 μg/mL At the concentration of mL, P2 NPs can kill 49% of melanoma (B16) cells; at the concentration of 40 μg/mL, P2 NPs can kill 62% of melanoma (B16) cells; at the concentration of 50 μg/mL, P2 NPs can kill 76% of melanoma (B16) cells; indicating that the polymer nanoparticles P2 NPs have excellent photothermal therapy effect on melanoma (B16) cells.

Example 3

Organic Conjugated Polymer Photothermal Reagents P3 Synthesis

The synthesis of organic conjugated polymer photothermal reagent P3 is similar to that of polymer P1, except that the monomer M2 is replaced by M4. The specific synthetic route is as follows:

The obtained polymer P3 was detected, and the number average molecular weight Mn of the GPC test was 12000, the weight average molecular weight Mw was 15900, and the molecular weight distribution index PDI was 1.32.

Preparation method of polymer P3 nanoparticles:

5.0 mg of polymer P3 and 25 mg of DSPE-PEG2000 were dissolved in 2.0 ml of tetrahydrofuran solvent. Under ultrasonic conditions at room temperature, the dissolved mixture was quickly added to 10 ml of ultrapure water to obtain a brown-black mixed solvent liquid. The obtained liquid was then placed in a fume hood, and the excess tetrahydrofuran solution was volatilized to obtain polymer nanoparticles with an apparent concentration of 500. ug/ml.

The photothermal conversion efficiency of the polymer nanoparticles P3 NPs was measured by an 808 nm laser, and the photothermal conversion efficiency was 78.6%.

Example 4

Organic Conjugated Polymer Photothermal Reagents P4 Synthesis

The synthesis of organic conjugated polymer photothermal reagent P4 is similar to that of polymer P1, except that the monomer M2 is replaced by M5. The specific synthetic route is as follows:

Preparation method of polymer P4 nanoparticles:

5.0 mg of polymer P4 and 25 mg of DSPE-PEG5000 were dissolved in 2.0 ml of tetrahydrofuran solvent. Under ultrasonic conditions at room temperature, the dissolved mixture was quickly added to 10 ml of ultrapure water to obtain a brown-black mixed solvent liquid. The obtained liquid was then placed in a fume hood, and the excess tetrahydrofuran solution was volatilized to obtain polymer nanoparticles with an apparent concentration of 500. ug/ml.

The photothermal conversion efficiency of the polymer nanoparticles P4 NPs was measured by an 808 nm laser, and the photothermal conversion efficiency was 66.4%.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, The simplification should be equivalent replacement manners, which are all included in the protection scope of the present invention.

Claims (10)

1. An organic conjugated polymer photothermal reagent for treating malignant melanoma, characterized in that its chemical structure is shown in formula (I):

   

   (I)

   In the formula, R is a straight-chain or branched alkyl group with 1-20 carbon atoms; R ₁ is a straight-chain or branched alkyl group with 1-20 carbon atoms,

   

   ,

   

   , R ₂ is hydrogen, straight-chain, branched or cyclic alkyl or alkoxy with 1 to 20 carbon atoms;

   Ar has the following structure:

   

    

   wherein R3 is a straight-chain, branched or cyclic alkyl group having 1 to 20 carbon atoms;

   The degree of polymerization n is any integer from 2 to 300.
2. The method for preparing an organic conjugated polymer photothermal reagent for treating malignant melanoma according to claim 1, characterized in that it comprises the following steps:

   In an inert atmosphere, the polymerized monomers M1 and M2 are dissolved in an organic solvent, then the catalyst tetrakistriphenylphosphine palladium is added, and the reaction is performed for 4 to 24 hours. After the reaction is stopped, the reaction solution is purified to obtain the target polymer;

   The structural formula of the polymerized monomer M1 is

   

   , the structural formula of the polymerized monomer M2 is

   

   .
3. The method for preparing an organic conjugated polymer photothermal reagent for treating malignant melanoma according to claim 2, wherein the molar ratio of the polymerized monomers M1 and M2 is 1:1; the organic solvent Be tetrahydrofuran, chlorobenzene, o-dichlorobenzene; The temperature of described reaction is 90～160 ℃, and the time of reaction is 4～12 hours.
4. The method for preparing an organic conjugated polymer photothermal reagent for treating malignant melanoma according to claim 3, wherein the reaction time is 6-8 hours.
5. The method for preparing an organic conjugated polymer photothermal reagent for treating malignant melanoma according to any one of claims 2-4, wherein the inert atmosphere is nitrogen or a rare gas atmosphere; the purification It refers to cooling the obtained reaction solution to room temperature, adding methanol for precipitation, filtering to obtain a crude product, and then dissolving the crude product in toluene. Precipitate in methanol again, stir, filter, and vacuum dry to obtain a polymer solid; finally, extract with methanol, acetone, and n-hexane in turn to remove small molecules; then dissolve the solid in deionized water, add methanol to precipitate The target product was obtained after vacuum drying.
6. A method for preparing organic conjugated polymer nanoparticles for treating malignant melanoma, comprising the following steps:

   Dissolving the organic conjugated polymer photothermal reagent for treating malignant melanoma and the amphiphilic compound according to claim 1 in an organic solvent, adding the mixed solution to an ultrapure aqueous solution under ultrasonic conditions, and ultrasonicating at room temperature Complete the preparation.
7. The method for preparing organic conjugated polymer nanoparticles for treating malignant melanoma according to claim 6, wherein the amphiphilic compound is F127, DSPE-PEG2000, DSPE-PEG5000; The mass ratio of the conjugated polymer photothermal reagent and the amphiphilic compound is 1:1~50.
8. The method for preparing organic conjugated polymer nanoparticles for treating malignant melanoma according to claim 7, wherein the organic conjugated polymer photothermal reagent: the mass ratio of F127 is 1:5～50, The mass ratio of organic conjugated polymer photothermal reagent: DSPE-PEG2000 is 1:1~20 or the mass ratio of organic conjugated polymer photothermal reagent: DSPE-PEG5000 is 1:1~20.
9. The organic conjugated polymer nanoparticle for treating malignant melanoma prepared by the preparation method of any one of claims 6-8.
10. Application of the organic conjugated polymer nanoparticles for treating malignant melanoma according to claim 9 in the field of photothermal therapy.