A supramolecular chiral photothermal agent based on cysteine ​​and its application

Abstract

This case involves a supramolecular chiral photothermal agent based on cysteine ​​and its application. Equimolar amounts of NaOH and chiral cysteine ​​are dissolved in deionized water, mixed and then added dropwise to a liquid containing metal ions M. 2+ The solution was stirred evenly, and then the precipitate was collected by centrifugation and washed with water and ethanol to obtain D / L-Cys-M 2+ ; Then, indocyanine green was loaded to obtain the supramolecular chiral photothermal agent D / L-Cys-ICG-M 2+ The present invention uses chiral cysteine, Cu 2+ / Zn 2+ A chiral supramolecular photothermal agent was constructed using indocyanine green as the assembly unit, and the preparation steps are simple and easy to operate; the supramolecular photothermal agents of different chirality have different interactions with cells and different photothermal therapeutic effects; it shows that chiral characteristics can be used as an important regulatory factor in the design of photothermal agents and an innovative strategy for improving tumor photothermal therapy agents.

Description

Technical Field

[0001] The present invention relates to the field of supramolecular assembly and biomedical technology, and in particular to a cysteine-based supramolecular chiral photothermal agent and applications thereof. Background Art

[0002] Photothermal therapy is an emerging cancer treatment method that uses active or passive targeting to cause photothermal agents with photothermal conversion capabilities to accumulate at the tumor site. Through in vitro near-infrared light irradiation, the absorbed light energy is converted into heat energy, thereby causing the temperature of the tumor site to rise and activating the apoptosis pathway of tumor cells. As a non-invasive tumor treatment strategy, near-infrared light has the advantages of strong tissue penetration, minimal tissue damage, and the ability to achieve spatiotemporally and temporally controllable in vitro selective irradiation, which is in line with the development direction of targeted tumor treatment. Therefore, given the current status of long tumor treatment cycles and painful procedures, the development of high-efficiency, low-toxicity photothermal therapy methods is of great significance.

[0003] Improving the photothermal conversion efficiency of photothermal agents is currently the main strategy for improving the efficacy of photothermal therapy. Methods for improving photothermal conversion efficiency mainly focus on the following two aspects: (1) enhancing the photothermal conversion efficiency of organic photothermal agents by expanding the conjugated structure; (2) improving the photothermal conversion efficiency of inorganic photothermal agents by regulating the composition or structure. Although the above strategies have effectively promoted the development of photothermal therapy to a certain extent, there are disadvantages in the implementation process, such as cumbersome synthesis steps and reduced biocompatibility, which have hindered the practical application of photothermal therapy.

[0004] In addition to the photothermal conversion efficiency of the photothermal reagent, the strength of the interaction between the reagent and tumor cells is also a key factor influencing the effectiveness of photothermal therapy. Given the chiral characteristics of the phospholipid bilayer and integral membrane proteins that constitute the cell membrane, differences in the interactions between different chiral nanomaterials and cells are inevitable. However, there are few reports on the regulation of the interaction between supramolecular chiral nanomaterials and cells, and even fewer studies on the regulation of the efficacy of photothermal therapy by supramolecular chiral nanomaterials. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the present invention uses chiral cysteine ​​(D-Cys or L-Cys), copper ions or zinc ions (Cu 2+ or Zn 2+ ) and indocyanine green (ICG) as assembly units to construct a chiral supramolecular photothermal agent. This material has a significant effect on photothermal therapy and provides theoretical and technical support for the development of efficient photothermal agents for tumor treatment.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A supramolecular chiral photothermal agent based on cysteine ​​is prepared by the following steps:

[0008] 1) Dissolve equimolar amounts of NaOH and D-cysteine ​​or L-cysteine ​​in deionized water, mix and add dropwise to the mixture containing metal ions M 2+ The solution was stirred evenly, and then the precipitate was collected by centrifugation and washed with water and ethanol to obtain the corresponding D-Cys-M 2+ or L-Cys-M 2+ ;

[0009] 2) D-Cys-M 2+ or L-Cys-M 2+ Disperse in deionized water, add indocyanine green and stir for 10 hours, then wash three times to obtain the supramolecular chiral photothermal agent D-Cys-ICG-M. 2+ or L-Cys-ICG-M 2+ .

[0010] Furthermore, the metal ion M 2+ Cu 2+ or Zn 2+ ; Metal ion M 2+ The molar ratio of cysteine ​​to D-cysteine ​​or L-cysteine ​​is 1:2.

[0011] Furthermore, in step 2), D-Cys-M 2+ or L-Cys-M 2+ The mass ratio of indocyanine green to indocyanine green is 17:1.

[0012] The present invention further provides an application of the cysteine-based supramolecular chiral photothermal agent as described above in tumor photothermal therapy drugs, and the prepared supramolecular photothermal agents of different chirality have different interactions with cells and different photothermal treatment effects.

[0013] For the sake of convenience, “ / ” is used in this case to combine similar materials. In this article, “ / ” means “or”, for example, D / L-Cys means D-Cys or L-Cys, Cu 2+ / Zn 2+ Cu 2+ or Zn 2+ etc.

[0014] The beneficial effects of the present invention are: the present invention uses chiral cysteine ​​(D / L-Cys), Cu 2+ / Zn 2+ and indocyanine green (ICG) as the assembly unit to construct a chiral supramolecular photothermal agent, the preparation steps are simple and easy to operate; the prepared L / D-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) Supramolecular photothermal reagents have strong differential interactions with cells and differential photothermal therapeutic effects. Compared with L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) Supramolecular photothermal reagent, D-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) Supramolecular photothermal reagents have stronger interactions with cells and are more sensitive to L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) When the photothermal conversion efficiency of supramolecular photothermal reagents is the same, D-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ Supramolecular photothermal agents have a better photothermal killing effect on tumor cells. This suggests that chirality can be used as an important regulatory factor in the design of photothermal agents and an innovative strategy for improving tumor photothermal therapy agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0016] Figure 1 D / L-Cys-Cu 2+ Scanning electron microscopy (a), UV (b) and IR (c).

[0017] Figure 2 D / L-Cys-Cu 2+ UV absorption job plot (a), UV titration binding constant fitting plot (b).

[0018] Figure 3 D / L-Cys and D / L-Cys-Cu 2+ CD spectrum (a) and D / L-Cys-Zn 2+ CD spectrum of (b).

[0019] Figure 4 D / L-Cys-Cu 2+ 、D / L-Cys-Zn 2+Isothermal titration microcalorimetry (ITC) spectra (a, b, c, d), surface plasmon resonance (SPR) spectra (e, f) and D- / L-Cys-Cu interaction with liposomes 2+ The relationship between the fluorescence intensity of liposomes (fluorescein stained) at 522 nm and their concentration (g).

[0020] Figure 5 D / L-Cys, D / L-Cys-Cu 2+ , ICG and D / L-Cys-ICG-Cu 2+ UV-visible absorption spectrum.

[0021] Figure 6 D / L-Cys-ICG-Cu 2+ Under 808nm laser irradiation, different concentrations (1.0W / cm 2 ) (a) and the heating curves of different power densities (0.3 mg / mL) (b).

[0022] Figure 7 D / L-Cys-ICG-Cu 2+ Different concentrations (1W / cm 2 )(a) and thermal imaging images of different power densities (0.3 mg / mL) (b).

[0023] Figure 8 D-Cys-ICG-Cu 2+ After 808nm laser irradiation (1.0W / cm 2 ) six photothermal heating and cooling cycle curves (a) a single heating and cooling curve (b) and the corresponding fitting curve (c).

[0024] Figure 9 D / L-Cys-Cu 2+ Biological transmission electron microscopy image after incubation with human cervical cancer (Hela) cells for 24 hours.

[0025] Figure 10 Different concentrations of D / L-Cys-ICG-Cu 2+ (a) and D / L-Cys-ICG-Zn 2+ (b) With and without 808 nm laser (1.0 W / cm 2 ) shows the results of cytotoxicity experiments on human cervical cancer (Hela) cells after 10 minutes of irradiation.

[0026] Figure 11 D / L-Cys-ICG-Cu 2+ (a) and D / L Cys-ICG-Zn 2+(b) With and without 808 nm laser (1.0 W / cm 2 ) Fluorescence microscopy of Hochest / PI staining of human cervical cancer cells (Hela) after 10 minutes of irradiation. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0028] In addition, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0029] Example 1: Preparation of supramolecular chiral nanomaterials

[0030] 2 mmol NaOH and 2 mmol D / L-Cys were dissolved in 10 mL deionized water. The mixtures were added dropwise to a CuCl2 aqueous solution (10 mL, 1 mmol) and stirred for 5 minutes. After the reaction, the precipitate was collected by centrifugation (6000 rpm, for 3 minutes) and then washed three times with water and ethanol to obtain supramolecular chiral nanomaterials, which were recorded as D / L-Cys-Cu 2+ , the supramolecular chiral nanomaterials are dispersed and preserved in aqueous solution.

[0031] like Figure 1 a SEM image shows that D / L-Cys-Cu with a size of 80 nm was obtained by supramolecular assembly. 2+ .like Figure 1 b UV-visible spectrum shows that cysteine ​​has an absorption peak at 209nm. The assembled D / L-Cys-Cu 2+ The absorption peak red-shifted to 215 nm, and a new absorption peak appeared at 250 nm. Figure 1 c Infrared spectrum shows that the thiol group at position 2552 disappears, indicating that cysteine ​​(Cys) combines with copper ions through the thiol group to assemble D / L-Cys-Cu 2+ .

[0032] like Figure 2 a, through Cu 2+ The relationship diagram of the UV absorption intensity of different stoichiometric ratios between Cu and cysteine ​​shows that 2+ A clear turning point was observed at a molar ratio of Cu / Cys of 1:2, which revealed that 2+ and cysteine ​​in a 1:2 stoichiometric ratio.2+ Coordination to form Cys-Cu 2+ -Cys, gradually self-assembled and formed supramolecular D / L-Cys-Cu 2+ .like Figure 2 b, UV titration experiment was carried out. 2+ Under the conditions of concentration, as the concentration of cysteine ​​gradually increased, the UV absorption intensity of the solution (monitored at 240 nm) gradually increased until saturation, which further confirmed that Cu 2+ The interaction between cysteine ​​and the cysteine ​​was further analyzed by analyzing the change of UV absorption intensity with cysteine ​​concentration, and the binding constant was calculated to be 272.88±48.03M using nonlinear curve fitting method. -1 and 291.39±48.87M -1 , which indicates that Cu 2+ Strong binding to cysteine.

[0033] like Figure 3 a Circular dichroism spectroscopy showed that D / L-Cys-Cu 2+ The supramolecular chiral material is mirror symmetrical, and cysteine ​​absorbs at 200nm. After assembly, D / L-Cys-Cu 2+ The absorption peak red-shifts to 209 nm, and new absorption peaks appear at 250 and 315 nm. Figure 3 b shows the D / L-Cys-Zn 2+ The supramolecular chiral material is mirror symmetrical, and after assembly, D / L-Cys-Zn 2+ A new absorption peak appeared at 235 nm; both indicated that D / L-Cys-M 2+ (Cu 2+ / Zn 2+ ) possesses the characteristics of supramolecular chirality.

[0034] Example 2: Investigating the Differences in the Effects of Chiral Supramolecular Nanomaterials on Cellular Interactions

[0035] The steps for simulating cell structure by homemade liposomes are as follows:

[0036] Dissolve 20 mg of lecithin and 2 mg of cholesterol in 20 mL of chloroform. Rotary evaporation removes the organic solvent to form a thin film. Add 10 mL of PBS buffer (pH 7.4) to the film and hydrate in a 52°C water bath for 5 minutes. After hydration, sonicate in an ice-water bath for 5 minutes to obtain a liposome solution. Store at 4°C.

[0037] Take 5.8mM liposome solution and titrate D / L-Cys-M 2+ (Cu 2+ / Zn 2+) 6.57mM solution was subjected to ITC test:

[0038] like Figure 4 a and b, there are obvious heat changes during the liposome titration process, indicating that lipids and D / L-Cys-Cu 2+ There is a binding effect between the two and D-Cys-Cu 2+ The degree of interaction is greater, about L-Cys-Cu 2+ 3.3 times of . Similarly, Figure 4 c and d, lipid and D / L-Cys-Zn 2+ There is a binding effect between the two and D-Cys-Zn 2+ The degree of interaction is greater, about L-Cys-Zn 2+ 1.3 times of that.

[0039] Take 260μg / mL D / L-Cys-Cu 2+ 、D / L-Cys-Zn 2+ SPR test with 200 μg / mL liposome solution:

[0040] First, the buffer solution was passed through to rinse, and then the liposome solution was passed through, and then the PBS solution was passed through to rinse the excess liposome solution, and then the D / L-Cys-Cu 2+ 、D / L-Cys-Zn 2+ Solution, and finally rinse with PBS solution. Figure 4 The results of e and 4f show that under the interaction of the same dose of chiral supramolecular nanomaterials with liposomes, D-Cys-Cu 2+ Significantly higher than L-Cys-Cu 2+ More than ten times. At the same time, D-Cys-Zn 2+ Significantly higher than L-Cys-Zn 2+ The above results show that D-Cys-M 2+ Compared with L-Cys-M 2+ Stronger interaction with cells.

[0041] like Figure 4 g, Fluorescein-stained liposomes to D / L-Cys-Cu 2+ Fluorescence quenching test of D / L-Cys-Cu 2+ When liposome solution is added to the solution, the binding effect will produce quenching fluorescence. With the addition of liposome solution, the fluorescence intensity gradually increases. 2+ Compared with L-Cys-Cu 2+ Has a stronger interaction with liposomes. D / L-Cys-Cu 2+The concentration of liposomes was fixed at 9.7 μg / mL, and the concentration of liposomes ranged from 0 to 0.6 mg / mL.

[0042] Example 3: Preparation of supramolecular chiral photothermal agent

[0043] Take 20mL 8.525mg / mL of the chiral supramolecular material D / L-Cys-M prepared above and 2+ (Cu 2+ / Zn 2+ ), 10 mg ICG was added to aqueous solutions of different chiral materials, mechanically stirred for 10 h to load ICG, washed with water 3 times, and dispersed in aqueous solution for storage to obtain D / L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) solution.

[0044] from Figure 5 It can be seen that D / L-Cys-ICG-Cu 2+ With D / L-Cys (209nm), ICG (215nm and 780nm), D / L-Cys-Cu 2+ The same absorption peaks (215 nm and 258 nm) demonstrate the successful loading of ICG and the successful synthesis of the supramolecular chiral photothermal agent.

[0045] Since the interaction between the material and cells is mainly the surface interface, and ICG is coated inside the material, the D-Cys-ICG-M loaded with ICG 2+ The interaction with cells is also significantly higher than that of L-Cys-ICG-M 2+ .

[0046] Example 4: Study on the photothermal properties of supramolecular chiral photothermal reagents:

[0047] First, add 3 mL of the D / L-Cys-ICG-M prepared above into the cuvette. 2+ (Cu 2+ / Zn 2+ ) solution, and irradiate the cuvette with 808 nm laser at a laser power density of 1.0 W / cm 2 , use a thermocouple thermometer to measure the temperature change within 10 minutes.

[0048] like Figure 6 As shown in a, within 10 minutes of laser irradiation, different chiral materials have the same photothermal effect, and the solution temperature increases significantly with increasing concentration. Figure 6 b is D / L-Cys-ICG-Cu 2+ When the laser power density is 0.5, 1.0, 1.5, 2.0 W / cm 2, the solution temperature increases significantly with the increase of laser power density. Figure 7 As shown in a and b, different concentrations of different chiral supramolecular photothermal agents and different laser wattages are detected by 808 nm laser (1.0 W / cm 2 ) 10 minutes of irradiation infrared thermal image further confirmed Figure 6 Conclusions of experiments a and 6b.

[0049] like Figure 8 a is D-Cys-ICG-Cu 2+ After 808nm laser irradiation (1.0W / cm 2 ) of the four photothermal heating and cooling cycle curves. Figure 8 b shows a single heating and cooling curve. Figure 8 After fitting, the calculated photothermal conversion efficiency is 63.45%, which proves that the material has a good photothermal effect.

[0050] Example 5: Cytotoxicity and Photothermal Effect Analysis

[0051] The cytotoxicity of chiral materials was evaluated by CCK-8 assay. Hela (human cervical cancer cells) were plated at 1.5×10 4 The cells were seeded in 96-well plates at a density of 10 cells / well and cultured at 37°C, 5% CO2 for 24 hours. 2+ After 24 hours of incubation, biological transmission electron microscopy images were taken. Similarly, the cells were incubated with different concentrations (10, 15, 20, 25, 30, 40 μg / mL) of D / L-Cys-ICG-Cu 2+ Incubate for 24 h. Similarly, cells were treated with different concentrations (40, 60, 70, 80, 120 μg / mL) of D / L-Cys-ICG-Zn 2+ Incubate for 24 hours as the non-illumination group D / L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ )(-). Then the light-exposed group D / L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ )(+) Each well was exposed to 808 nm laser (1.0 W / cm 2 After 10 minutes of illumination, the cells were incubated for another 24 hours. Finally, CCK-8 solution was added to each well and the cells were incubated for another 0.5 hours. The absorbance at 450 nm was measured using a microplate reader.

[0052] like Figure 9 , D / L-Cys-Cu 2+ After co-incubation with cells, biological transmission electron microscopy images were taken. 2+Compared with L-Cys-Cu 2+ More D-Cys-Cu 2+ Compared with L-Cys-Cu 2+ High degree of interaction with cells.

[0053] D / L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) The results of cell toxicity and photothermal effect are shown in Figure 10 The analysis results showed that the materials did not cause obvious toxicity to cells under non-light conditions. What is more noteworthy is that D-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) were more cytotoxic than L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ). Under near-infrared laser irradiation conditions, the ability of the irradiation group to kill tumor cells was significantly higher than that of the non-irradiation group, and D-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ )PTT effect is significantly better than L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ).

[0054] The cells were stained with Hochest / PI and fluorescence microscopy images were taken. Figure 11 The analysis results showed that, consistent with the above conclusions, the ability of the light-irradiated group to kill tumor cells was significantly higher than that of the non-light-irradiated group, and D-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ) has a significantly better photothermal therapeutic effect than L-Cys-ICG-M 2+ (Cu 2+ / Zn 2+ ), which has good clinical application and prospects.

[0055] This indicates that chiral supramolecular nanomaterials have an important effect on regulating the efficacy of photothermal therapy. Furthermore, supramolecular photothermal agents of different chirality exhibit differential interactions with cells and exhibit different photothermal therapeutic effects, indicating that chirality can serve as an important regulatory factor in the design of photothermal agents and an innovative strategy for improving tumor photothermal therapy agents.

[0056] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the description and implementation methods. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A supramolecular chiral photothermal agent based on cysteine, characterized in that: It is prepared by the following steps: 1) Dissolve equimolar amounts of NaOH and D-cysteine ​​or L-cysteine ​​in deionized water, mix and add dropwise to the mixture containing metal ions M. 2+ The solution was stirred evenly, and then the precipitate was collected by centrifugation and washed with water and ethanol to obtain the corresponding D-Cys-M 2+ or L-Cys-M 2+ ; 2) D-Cys-M 2+ or L-Cys-M 2+ Disperse in deionized water, add indocyanine green and stir for 10 hours, then wash three times to obtain the supramolecular chiral photothermal agent D-Cys-ICG-M. 2+ or L-Cys-ICG-M 2+ ; The metal ion M 2+ Cu 2+ or Zn 2+ ; Metal ion M 2+ The molar ratio with D-cysteine ​​or L-cysteine ​​is 1:2; D-Cys-M in step 2) 2+ or L-Cys-M 2+ The mass ratio of indocyanine green to indocyanine green is 17:

1.

2. Use of the cysteine-based supramolecular chiral photothermal agent as claimed in claim 1 in tumor photothermal therapy.

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