CN117683372A - A kind of nanophotothermal reagent and its preparation method and application - Google Patents

Abstract

The invention discloses a heptamethine cyanine dye and a near infrared absorption nanometer photo-thermal reagent, wherein the micromolecular cyanine dye is designed and synthesized on the basis of a commercial dye IR780, and has higher photo-thermal conversion efficiency. The nanometer photothermal reagent of the invention is a small molecule cyanine dye Cy-H which passes through DSPE-PEG ₂₀₀₀ Encapsulation of the formed nanoparticles, which improves the solubility of the cyanine dye in waterAggregation in the liquid promotes the water solubility and biocompatibility of the cyanine dye molecules, so that the reagent has higher molar extinction coefficient in water, and compared with single cyanine dye, the absorption spectrum has the promotion of 70 nm. At the same time, the nano preparation can isolate ¹ O ₂ To reduce photobleaching and enhance molecular stability. The nano photothermal reagent provided by the invention is applied to three tumor cells of A549, MCF-7 and 4T1, and has excellent phototoxicity, safer dark toxicity and excellent photothermal performance.

Description

A kind of nanophotothermal reagent and its preparation method and application

Technical field

The invention relates to the technical fields of fine chemicals and nanomaterials, and in particular to a nanophotothermal reagent and its preparation method and application.

Background technique

Surgery, chemotherapy, and radiotherapy are still the most commonly used cancer treatments. However, these traditional treatments still have many limitations. For example, it is difficult to remove small tumors with surgery, resulting in easy recurrence of cancer and incomplete treatment; the selection of chemotherapy drugs. Radiotherapy is very poor and will cause damage to healthy tissues and cells during treatment, resulting in huge side effects; radiotherapy will cause functional disorders in the body and cause considerable pain to cancer patients. Therefore, scientific researchers have been looking for treatment methods that are thorough and have minimal side effects. PTT is a non-invasive, real-time controllable and mild tumor treatment method, and the selection of PTA is particularly important as the biggest influence factor on the effect of PTT.

In recent years, many PTAs have been reported. Cyanine dyes have received widespread attention due to their high molar extinction coefficient and good biocompatibility. Among them, the heptamethacyanine dye has strong near-infrared absorption, and its molecules are easy to modify, making it very suitable for use as photothermal reagents. However, heptamethocyanine dyes usually have poor tumor targeting properties and are easily metabolized during treatment. Moreover, most heptamethocyanine dyes have poor photostability and are easily photobleached. At the same time, the photothermal conversion efficiency is not high, and high drug concentrations and light doses are required for treatment, which severely limits the use of heptamethocyanine dyes. Application in photothermal therapy. Therefore, it is of great significance to develop heptamethacyanine dyes with high photothermal conversion efficiency and high stability.

Contents of the invention

According to the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a photothermal reagent of heptamethocyanine dye with high photothermal conversion efficiency and high stability and a preparation method thereof, aiming to solve the problem of photothermal use of heptamethocyanine dye in tumor treatment. Problems include poor stability, easy bleaching and low photothermal conversion efficiency.

The technical solution of the present invention is as follows:

A small molecule cyanine dye, characterized by having the structure of formula I:

Wherein, R is a linear alkyl group having 8 to 20 carbon atoms, and X ^- is an anion. The number of positive charges carried by the anion is equal to the number of positive charges carried by the cation in Formula I.

For some specific small molecule cyanine dyes, R is a linear alkyl group with 8-16 carbon atoms, and X ^is selected from BF4 ^- , Cl ^- , Br ^- , I ^- , NO ^3- , SO ₄ ^2- , ClO ₄ ^- , CH ₃ COO ^- , CH ₃ SO ₃ ^- and CF ₃ SO ₃ ^- .

For some specific small molecule cyanine dyes, R is a straight-chain alkyl group with 10-15 carbon atoms.

For some specific small molecule cyanine dyes, R is a straight-chain alkyl group with 11-13 carbon atoms.

A nanoparticle includes at least one of the small molecule cyanine dyes described in Formula I.

The nanoparticles also include DSPE-PEG ₂₀₀₀ (distearyl phosphatidylethanolamine-polyethylene glycol 2000) used to encapsulate the small molecule cyanine dye.

The diameter of the nanoparticles is 10-200nm.

A nanophotothermal reagent prepared by the following method:

Dissolve the small molecule cyanine dye and DSPE-PEG ₂₀₀₀ according to claim 1 in an organic solvent and add it dropwise to the dispersion medium under the action of ultrasound; or,

The small molecule cyanine dye according to claim 1 is dissolved in an organic solvent and then added dropwise to the solution of DSPE-PEG ₂₀₀₀ and dispersion medium under the action of ultrasound;

The mixture is sonicated until no turbidity is observed or no turbidity is observed, and the organic solvent is removed by dialysis through a water-based filter membrane with a diameter of 0.45 μm and 0.22 μm in order to obtain the nanophotothermal reagent; or,

The dialyzed dispersion is concentrated to obtain the nanophotothermal reagent; or,

The concentrated dispersion is freeze-dried to obtain a powdered nanophotothermal reagent.

The organic solvent is a solvent miscible with water, and the dispersion medium is ultrapure water or PBS.

The mass ratio of the nanophotothermal reagent, small molecule cyanine dye, DSPE-PEG ₂₀₀₀ and dispersion medium is 1:5-15:5-15.

The mass ratio of the nanophotothermal reagent, small molecule cyanine dye, DSPE-PEG ₂₀₀₀ and dispersion medium is 1:9-11:9-11; the organic solvent is one or more of DMSO, THF and DMF. kind.

The power of ultrasound is 20-220W, and the ultrasound time is 3min-3h. As the power increases, the time becomes shorter.

The synthesis method of the small molecule cyanine dye adopts the traditional synthesis method of cyanine dye to synthesize the compound Cy-Cl. The compound Cy-Cl reacts with sodium methylmercaptide to remove the chlorine atom to obtain the small molecule cyanine dye. The specific synthesis reaction is as follows:

Application of the small molecule cyanine dye in the preparation of tumor treatment drugs.

Application of the nanoparticles in tumor treatment.

Application of the nanophotothermal reagent in tumor treatment.

Further, the specific steps of the preparation method of the nano-photothermal reagent:

Dissolve the cyanine dye in the organic solvent tetrahydrofuran, disperse DSPE-PEG ₂₀₀₀ in ultrapure water, and then slowly drop the organic solution of the small molecule cyanine dye into the DSPE-PEG ₂₀₀₀ ultrapure water under the action of ultrasound, and add The mixture was ultrasonicated at 180 W for 3 minutes, and then the resulting dispersion was filtered through water-based filters with diameters of 0.45 μm and 0.22 μm, added to the dialysis bag, and dialyzed in ultrapure water or PBS for 12-24 hours.

Further, the mass ratio of the cyanine dye to DSPE-PEG ₂₀₀₀ is 1:10

Further, the power of the ultrasonic action can be 20W-180W; the dialysis bag is an aqueous dialysis bag with a molecular weight of 3500. The outstanding advantages of the small molecule cyanine dye and nanophotothermal reagent of the present invention are:

(1) The aggregation of small molecule cyanine dyes in water is improved in the form of nanoparticles or nanoreagents, so that the photothermal drug has excellent water solubility and biocompatibility.

(2) The small molecule cyanine dye of the present invention has a higher molar extinction coefficient and a red shift of 70nm compared to a single cyanine dye. Traditional cyanine dyes with photothermal effects usually contain Cl atoms in their structure, but the present invention removes Cl atoms. The Cl atom on the traditional cyanine dye structure, combined with the long carbon chain alkyl groups modified on both sides of the molecule, significantly improves the high photothermal conversion efficiency of the entire small molecule cyanine dye.

(3) The small molecule cyanine dye wrapped in DSPE-PEG ₂₀₀₀ can isolate ¹ O ₂ to reduce photobleaching and give the molecule good stability.

(3) The nanophotothermal reagents or nanoparticles provided by the present invention have a high photothermal conversion efficiency of 71.2%, can be well enriched in tumor cells, and have good targeting properties.

Description of the drawings

Figure 1: The structural formula of the small molecule cyanine dye Cy-H and its hydrogen nuclear magnetic resonance spectrum.

Figure 2: The structural formula of the small molecule cyanine dye Cy-H and its carbon NMR spectrum.

Figure 3: The structural formula of the small molecule cyanine dye Cy-H and its high-resolution mass spectrum.

Figure 4: a is the dynamic light scattering (DLS) picture of the nano-photothermal reagent; b is the particle size change chart of the nano-photothermal reagent over seven days; c is the transmission electron microscope picture of the nano-photothermal reagent.

Figure 5: a is the UV-visible absorption spectrum of the small molecule cyanine dye Cy-H; b is the fluorescence absorption spectrum of Cy-H.

Figure 6: Temperature-raising curves of Cy-H (a) and nano-photothermal reagents (b, c, d) under 760nm laser; e is the heating and cooling cycle diagram of nano-photothermal reagents.

Figure 7: Fluorescence images of nanophotothermal reagents incubated with MCF-7 cells for different times, scale bar: 20 μm.

Figure 8: a shows the survival rate of A549, 4T1 and MCF-7 cells incubated with different concentrations of nanophotothermal reagents under dark and light conditions; b shows the live/dead cell staining of MCF-7 cells under different conditions. Scale bar: 200 μm.

Detailed ways

The present invention provides a nanophotothermal reagent and its preparation method and application. In order to make the purpose and technical solution of the present invention clearer, the present invention will be further explained in detail below. It should be clear that the examples of specific implementations described here are only for further explanation of the present invention, but not for limiting the present invention.

The distearyl phosphatidylethanolamine-polyethylene glycol 2000 (DSPE-PEG ₂₀₀₀ ) used in the examples of the present invention was purchased from Shanghai Pengshuo Biotechnology Co., Ltd., and the PBS buffer was purchased from Beijing Solebao Technology Co., Ltd. In the examples The names and units are commonly used in this field, for example, μM is the concentration unit and the unit is micromolar/ml.

Example 1: Preparation of a series of small molecule cyanine dyes I1-I5 and nanophotothermal reagents

The small molecule cyanine dyes I1-I5 of the present invention are prepared in the following manner (R is a linear alkyl group with different carbon numbers):

(1) Anhydrous DMF (17.5 mL, 136.5 mmol) and anhydrous DCM (17.5 mL) were stirred in an ice bath for 30 min. POCl ₃ (17.5 mL, 115 mmol) and anhydrous DCM (5 mL) were mixed within 0.5 h. Slowly drip, and then slowly inject cyclohexanone (5g, 50mmol) into the above solution. The resulting mixture was stirred at 80°C for 5 h under nitrogen protection. After the reaction was completed, it was poured into ice water with stirring to obtain a light yellow precipitate. The solid was filtered off, washed with water, and dried in vacuo to give product 1a as a pale yellow solid (7.1 g, 84.3%). It was used directly in the next step without further purification.

(2) Combine 2,3,3-trimethyl-3H-indole with 1-iodooctane, 1-iododecane, 1-iodododecane, 1-iodotetradecane, and 1-iododecane respectively. Dissolve hexane in 10 mL toluene and stir under reflux overnight. After the reaction is cooled, it is washed with diethyl ether and dried under vacuum to obtain a purple-red solid product with different alkyl substitutions.

Next, take R as a twelve-carbon straight-chain alkyl group as an example, and the operations for the remaining carbon numbers are the same.

(3) Dissolve compound 1a (0.378g, 2.2mmol), compound 2a (2g, 4.39mmol) and sodium acetate (0.18g, 2.2mmol) in 5mL acetic anhydride under nitrogen protection, and react at 60°C for 1 hour. After the reaction, 20 mL of diethyl ether was added and refrigerated at -20°C for 3 hours. A golden solid was obtained by suction filtration, which was then purified by column chromatography to obtain a golden compound Cy-Cl (1.5 g, 74.2%).

¹ H NMR (500MHz, CDCl ₃ ) δ8.33 (d, J = 14.1Hz, 2H), 7.43–7.35 (m, 4H), 7.30–7.23 (m, 2H), 7.15 (d, J = 7.9Hz, 2H),6.23(d,J＝14.1Hz,2H),4.20(t,J＝7.3Hz,4H),2.74(t,J＝5.9Hz,4H),2.03–1.92(m,2H),1.72( s,12H),1.50–1.12(m,40H),0.87(t,J＝6.8Hz,6H).ESI-MS:m/z calcd for[MI ^- ] ⁺ :791.6005,measured:791.6016.

(4) Under _N2 atmosphere, dissolve Cy-Cl (1g, 1.1mmol) in 100mL DMF, then add sodium methylmercaptide (20% hydrate) (7.71g, 22mmol), and then add the mixture in The reaction was carried out overnight at 100°C. After the reaction was cooled, a large amount of saturated brine and ethyl acetate were used for extraction. The organic phase was collected and dried with anhydrous sodium sulfate. After drying, the organic phase was concentrated and separated by column chromatography to obtain the final product Cy- H (0.2g, 20.5%).

¹ H NMR (500MHz, CDCl ₃ ) δ7.65 (d, J = 12.6Hz, 2H), 7.35 (m, 5H), 7.20 (t, J = 7.4Hz, 2H), 7.07 (d, J = 8.0Hz ,2H),6.05(d,J＝13.1Hz,2H),4.04(t,4H),2.52(t,4H),1.95(m,2H),1.72(s,12H),1.52–1.17(m, 40H), 0.87 (t, J＝6.9Hz, 6H).ESI-MS: m/z calcd for [MI ^- ] ⁺ :757.6394, measured:757.6387.

Preparation method of photothermal nanoparticle reagent of the present invention:

Preparation of Cy-H NPs

First, dissolve 1 mg Cy-H in 1 mL THF and 10 mg DSPE-PEG ₂₀₀₀ in 5 mL ultrapure water. Then slowly add the THF solution containing Cy-H to the ultrapure solution containing DSPE-PEG ₂₀₀₀ under 180W ultrasonic action. water, and the mixed solution was sonicated for 3 minutes, and the resulting dispersion was filtered through water-based membranes with diameters of 0.45 μm and 0.22 μm, and then the resulting dispersion was put into a dialysis bag and dialyzed in ultrapure water for 12 hours. The dialysis bag is an aqueous phase dialysis bag with a molecular weight of 3500.

Preparation of Cy-H NPs-1

First, dissolve 1 mg Cy-H in 1 mL DMSO and 10 mg DSPE-PEG ₂₀₀₀ in 5 mL PBS, and then add the THF solution containing Cy-H dropwise to the ultrapure water containing DSPE-PEG ₂₀₀₀ under 40W ultrasonic action. The mixed solution was sonicated for 60 minutes, and the resulting dispersion was filtered through water-based membranes with diameters of 0.45 μm and 0.22 μm, and then the resulting dispersion was put into a dialysis bag and dialyzed in PBS for 12 hours. The dialysis bag used had a molecular weight of 3500 aqueous phase dialysis bag.

Preparation of Cy-H NPs-2

First, dissolve 1 mg Cy-H in 1 mL THF and 15 mg DSPE-PEG ₂₀₀₀ in 10 mL ultrapure water. Then slowly add the THF solution containing Cy-H to the ultrapure solution containing DSPE-PEG ₂₀₀₀ under 180W ultrasonic action. water, and the mixed solution was sonicated for 3 minutes, and the resulting dispersion was filtered through water-based filters with diameters of 0.45 μm and 0.22 μm, and then the resulting dispersion was put into a dialysis bag and dialyzed in ultrapure water for 1 day. The dialysis bag used is an aqueous phase dialysis bag with a molecular weight of 3500.

Preparation of Cy-H NPs-3

First, dissolve 1 mg Cy-H in 1 mL THF and 10 mg DSPE-PEG ₂₀₀₀ in 10 mL PBS. Then slowly add the THF solution containing Cy-H to the ultrapure water containing DSPE-PEG ₂₀₀₀ under 100W ultrasound. , and the mixed solution was sonicated for 20 minutes, and the obtained dispersion was filtered through a water-based filter membrane with a diameter of 0.45 μm and 0.22 μm, and then the obtained dispersion was put into a dialysis bag and dialyzed in PBS for 12 hours. The dialysis bag used was molecular weight 3500 aqueous dialysis bag.

Preparation of Cy-H NPs-4

First, dissolve 1 mg I2 in 1 mL DMF and 5 mg DSPE-PEG ₂₀₀₀ in 5 mL ultrapure water. Then slowly add the THF solution containing Cy-H to the ultrapure water containing DSPE-PEG ₂₀₀₀ under 180W ultrasonic action. , and the mixed solution was sonicated for 3 minutes, and the obtained dispersion was filtered through water-based membranes with diameters of 0.45 μm and 0.22 μm, and then the obtained dispersion was put into a dialysis bag and dialyzed in PBS for 12 hours. The dialysis bag used was molecular weight 3500 aqueous dialysis bag.

Preparation of Cy-H NPs-5

First, dissolve 1 mg I4 in 1.5 mL DMF and 10 mg DSPE-PEG ₂₀₀₀ in 15 mL ultrapure water. Then slowly add the THF solution containing Cy-H to the ultrapure water containing DSPE-PEG ₂₀₀₀ under 180W ultrasonic action. in, and the mixed solution was sonicated for another 3 minutes, and the obtained dispersion was filtered through water-based filters with diameters of 0.45 μm and 0.22 μm, and then the obtained dispersion was put into a dialysis bag and dialyzed in PBS for 24 hours. The dialysis bag used was Aqueous dialysis bag with a molecular weight of 3500.

Using Cy-H as the standard solution, a UV-vis spectrometer was used to calibrate the concentration of the prepared Cy-H NPs. It was determined that the concentration of Cy-H in the Cy-H NPs reagent prepared in the example was 20 μM, using an ultrafiltration tube (outside Tube 15ml; pore size: 3kd) Concentrate the nanoreagent to the required concentration or add solvent to dilute it to the required concentration for subsequent testing.

The different concentrations of photothermal nanoreagents mentioned in this article are all prepared by this method.

Example 2: Stability and DLS and SEM analysis of nanophotothermal reagents

Zetasizer Nano-ZS90 was used to conduct dynamic light scattering (DLS) experiments on nanophotothermal reagents. As shown in Figure 4a. The measured hydrodynamic diameter of the nanophotothermal reagent is 174.2nm, and the PDI is 0.238. The lower PDI indicates that the nanoparticles have good dispersion. Then the hydrated particle size of the nanoparticles was tested within seven days, as shown in Figure 4b. The hydrated particle size of the nanophotothermal reagent had almost no change within seven days, indicating that the prepared nanoparticles have good stability and can be stored for a long time. Then, a transmission electron microscope (TEM) was used to test the morphology of the nanophotothermal reagent, as shown in Figure 4c. The image shows that Cy-H and DSPE-PEG ₂₀₀₀ are co-assembled into uniform nanoparticles. The particle size distribution of the nanophotothermal reagent is Between 10 and 200nm, it can be well enriched and retained at the tumor site through the EPR effect. This property makes it very suitable for photothermal treatment of tumors.

Example 3: UV-visible spectrum characterization of small molecule cyanine dye Cy-H and nano-photothermal reagent

In this example, the preparation method of small molecule cyanine dye mother liquor and different concentration solutions:

Use a six-digit electronic balance to weigh the small molecule cyanine dye Cy-H solid, prepare it with DMSO into a mother solution with a concentration of 10 μM, and then dilute it with dichloromethane, methanol and other organic solvents (the specific solvent is shown in Figure 5a) to a concentration of 2 μM. Spectral testing.

As shown in Figure 5a, the absorption peaks of Cy-H in various organic solvents are around 750nm, but the maximum absorption peak in water is at 680nm, which shows that the small molecule cyanine dye Cy-H will aggregate in water, and The blue shift of the wavelength will prevent Cy-H from using longer wavelength lasers for subsequent photothermal treatments, which will limit its application in photothermal treatments. As shown in Figure 5b, the absorption spectrum of nanophotothermal reagents in water is similar to the absorption of Cy-H in organic solvents, indicating that the wrapping of DSPE-PEG ₂₀₀₀ can reduce the aggregation of Cy-H and can be used to achieve higher tissue penetration depth. Near-infrared light is used to excite nanophotothermal reagents to produce photothermal effects. In addition, the fluorescence of nanophotothermal reagents in water is significantly weaker than the fluorescence of Cy-H in the organic phase, which means that the energy of the excited state can be converted more into thermal energy, resulting in higher photothermal conversion efficiency.

Example 4: In vitro photothermal performance evaluation of nanophotothermal reagents

In order to test the photothermal capabilities of the photothermal reagent Cy-H and the encapsulated nanoparticles Cy-H NPs, different concentrations (0, 10 μM, 30 μM, 50 μM) of the small molecule photothermal reagent Cy-H were tested at an optical power density of 500 mW. /cm ² temperature rise under 760nm laser. Small molecule photothermal reagents of different concentrations were irradiated under a 760nm laser, and the infrared camera FLIR-1910582 was used to monitor the temperature changes every 30 seconds for a total of 10 minutes. Afterwards, the temperature changes of nanoparticle Cy-H NPs with different concentrations (0μM, 10μM, 30μM, 50μM) were tested under the irradiation of 760nm 500mW/cm ² laser. Next, the effects of optical power density and Cy-H NPs concentration on temperature changes were explored. To determine the impact of the situation, 30μM nanoparticles Cy-HNPs were configured to be irradiated with different optical power densities (200mW/cm ² , 300mW/cm ² , 400mW/cm ² ) under a 760nm laser, and an infrared camera was used to monitor every 30s. After that, the optical power density of the 760nm laser was kept at 400mW/cm ² , Cy-H NPs of different concentrations (20μM, 30μM, 40μM) were configured, and an infrared camera was used to monitor every 30s.

As shown in Figure 6a, infrared camera FLIR-1910582 was used to test the temperature rise of the small molecule cyanine dye Cy-H at different concentrations (0, 10 μM, 30 μM, 50 μM) under a 760nm laser with an optical power density of 500mW/ ^cm2 in an ultrapure water system. In this case, as the concentration of Cy-H increases, the heating ability becomes stronger and stronger. 0, 10μM, 30μM and 50μM increased the temperature by 2.1℃, 18.2℃, 36.9℃ and 50.6℃ respectively within 5min. However, the 10 μM and 30 μM Cy-H aqueous solutions almost no longer heated up at 3 or 4 minutes because Cy-H was photobleached and lost its photothermal generation ability. As shown in Figure 6b, after being wrapped into nanoparticles, the nano-photothermal reagent shows a stronger temperature-raising ability. Under the same optical power density, 0, 10 μM, 30 μM and 50 μM nano-photothermal reagents respectively increased the temperature by 2.1% in 5 minutes. ℃, 35.8℃, 50.6℃ and 53.7℃, the temperature-raising ability of nanoparticles is significantly better than that of small molecules. This is because the wrapping of DSPE-PEG ₂₀₀₀ isolates part of ¹ O ₂ , thereby reducing the photobleaching of Cy-H. As shown in Figure 6c, the temperature rise of 0, 10μM, 20μM and 30μM nano-photothermal reagents within 5 minutes at 400mW/cm ² was also tested, and in Figure 6d, 30μM nano-photothermal reagent was tested at 200mW/cm ² and 300mW/cm ² As well as the temperature rise at 400mW/ ^cm2 , it can be seen that the temperature rise ability of the nanophotothermal reagent is proportional to the concentration and optical power density. Therefore, controllable temperature increase can be achieved by regulating its concentration and optical power density.

After that, Zetasizer Nano-ZS90 was used to conduct a dynamic light scattering (DLS) experiment on the nano-photothermal reagent to test the stability of the nano-photothermal reagent at a concentration of 10 μM. Figure 6e shows the nano-photothermal reagent under 760 nm, 300 mW/cm ² laser irradiation. Temperature changes under four consecutive heating-cooling cycles. It can be seen from the figure that even after four consecutive heating and cooling processes, the nano-photothermal reagent can still maintain its original heating ability, indicating that the nano-photothermal reagent has excellent photostability and excellent thermal stability and can last of play a role in photothermal therapy.

Example 5: Cellular uptake of nanophotothermal agents

The specific implementation method of cell culture and co-incubation of cells and nanoreagents in this example:

The cells used were: MCF-7 cells (mouse breast cancer cells), A549 cells (human lung cancer cells), and 4T1 cells (mouse breast cancer cells). All the cells used above were cultured in DMEM medium containing 1% double antibody (penicillin-streptomycin mixture) and 10% fetal calf serum at 37°C in a cell culture incubator containing 5% CO ₂ and 21% O ₂ nourish. When the cells grow to the logarithmic growth phase, use trypsin containing EDTA to digest the cells, and then transfer them to a 96-well plate or confocal culture dish for subsequent cell experiments. When cells need to be incubated with a certain concentration of nanoreagents, the culture medium is used as a solvent to prepare the nanoreagents for incubation, and the culture medium used for cultivation is replaced with a culture medium containing a corresponding concentration of nanoreagents, leaving the rest unchanged.

In order to determine when is the optimal time for cell therapy, cellular uptake experiments are needed to determine the time of maximum enrichment of Cy-H NPs within cells. Although the fluorescence of Cy-H has been quenched a lot after being encapsulated into nanoparticles, it is enough to monitor the enrichment of nanoparticles in cells. We used human breast cancer cells MCF-7 to conduct subsequent experiments. First, culture the MCF-7 cells in a 35 mm confocal culture dish and incubate them in a 37°C cell culture incubator for 24 hours. When the cells are completely attached, they can be used for cellular uptake experiments. At this time, the Cy-H NPs nanoreagent with a concentration of 20 μM was incubated with MCF-7 cells for different times, and an FV-3000 laser confocal scanning microscope (Olympus) was used to observe the fluorescence changes in the cells at different times. Fluorescence intensity is used to determine the maximum enrichment time of nanoparticles in cells. Wash 3 times with phosphate buffered saline (PBS) before imaging. The excitation wavelength of nanoparticles is 640nm, and the emission wavelength is selected within the range of 700-800nm for detection.

As shown in Figure 7, before adding nanoparticles, there was no fluorescence signal in the cells. After adding nanoparticles, the fluorescence signal in the cells gradually increased during 0-5h and reached the maximum value at 5h, indicating that the cells were The most nanoparticles were enriched at this time, so in subsequent cytotoxicity experiments, the nanophotothermal reagent was incubated with the cells for 5 hours and then illuminated. And there is still strong fluorescence in the cells at 24 hours, indicating that the nanoparticles can stay in the cells for a long time, and photothermal treatment of cells can be carried out during this time period.

Example 6: Evaluation of cytotoxicity and photothermal effect of nanophotothermal reagents

The cytotoxicity of nanophotothermal reagents was tested through MTT experiment. A549 cells, MCF-7 cells and 4T1 cells were cultured in a 96-well plate (approximately 1×10 ⁴ cells per well, 100 μL DMEM medium was added). When the cells adhered and the density reached 80%, culture medium containing Cy-H NPs nanoreagents at different concentrations (specific concentrations are shown in Figure 8a) was added and incubated for 5 h, and then used with a 760nm laser (600mW/cm ² ) Irradiate for 15 minutes. At the same time, another control group with the same experimental conditions but without laser irradiation was also prepared for the study of cell dark toxicity. After incubating again at 37°C for 24 h, 100 μL of DMEM solution with 0.5 mg/mL MTT was added to each well and incubation continued for 4 h. Subsequently, the culture medium in the 96-well plate was removed, and 100 μL of DMSO was added to each well to dissolve the generated formazan crystals. Use a Bio-Rad microplate reader to measure the absorbance of the solution at 490nm and calculate the cell viability:

Among them, the experimental group represents the cell culture group treated with different concentrations of nanoparticles Cy-H NPs; the blank group represents the cell culture group that only adds culture medium: the control group represents the cell culture group that does not add Cy-H NPs: OD It is the absorbance value measured at 490nm of DMSO solution with dissolved formazan crystals. Each set of experiments was performed in parallel 4 times.

As shown in Figure 8a, under dark conditions, even at high concentrations, nanophotothermal reagents can only produce weak toxicity to the three types of cells. However, when the 760nm laser and nanophotothermal reagents act simultaneously, the three types of cells are showed great cytotoxicity, which effectively proves that nano-photothermal reagents can effectively kill cancer cells after irradiation with 600mW/cm ² and 760nm laser for 15 minutes. They have good phototoxicity and can effectively kill cancer cells under dark conditions. is safe.

In order to more intuitively observe the photothermal effect of nanophotothermal reagents, a living and dead cell staining experiment was performed.

The alcein AM/PI kit has the function of labeling live cells and dead cells simultaneously. After Calcein-AM enters the cell, it will be spliced by the esterase in living cells, causing Calcein-AM, which itself is non-fluorescent, to become a non-permeable polar molecule Calcein with strong green fluorescence (Ex=490nm, Em =515nm), so living cells can be observed by observing the fluorescence of Calcein retained in the molecule. It is difficult for PI (propidium iodide) to pass through the cell membrane of living cells. It can only pass through the cell membrane of dead cells and enter dead cells, and enters the nucleus and embeds DNA to produce red fluorescence. Therefore, PI can only stain dead cells. Among them, Calcein (Ex=490nm, Em=515nm) and PI (Ex=535nm, Em=617nm), so the 488nm laser can be used to excite Calcein and PI at the same time, and a confocal microscope can be used to observe the live/dead state of the cells.

Operation steps: MCF-7 cells were inoculated into confocal culture dishes and cultured for 24 hours. They were divided into the following four groups: PBS group, light group (Light), drug-added group (Cy-H NPs), drug-added light group (Cy -H NPs+Light). PBS group: cells were incubated at 37°C after adding an equal amount of PBS to the drug; illumination group: cells were irradiated with 760nm laser (600mW/cm ² ) for 15 minutes and then incubated at 37°C; drug group: cells were added After adding 15 μM Cy-H NPs, the cells were incubated at 37°C; in the drug-added and illuminated group: 2 hours after adding 15 μM Cy-H NPs, the cells were irradiated with a 760nm laser (600mW/cm ² ) for 20 minutes and then incubated at 37°C. After all four groups of cells were incubated for 4 hours, the working solution for staining cells was prepared according to the requirements of the Calcein AM/PI double-staining kit, and then a FV-3000 laser confocal scanning microscope (Olympus) was used to image live/dead cells. Calcein and PI are both excited by 488nm laser. Calcein receives fluorescence signal at 500-540nm, and PI receives fluorescence signal at 650-690nm.

As shown in Figure 8b, the green fluorescence is the living cells stained by AM, and the red fluorescence is the dead cells stained by PI. Almost no red fluorescence was observed in the PBS group and the light group and nanoparticle group alone, indicating that light alone and nanophotothermal reagents will hardly cause damage to cells and have good biological safety. When the nanophotothermal reagent and the 760nm laser exist at the same time, almost only red fluorescence exists, indicating that the photothermal effect produced by the nanophotothermal reagent under light is enough to kill cancer cells and achieve therapeutic effects, and is expected to be applied in vivo.

Claims (12)

1. A small molecule cyanine dye having a structure according to formula I:

wherein R is a linear alkyl group having 8 to 20 carbon atoms, X ^- Is an anion, the number of positive charges of the anion is equal to the number of positive charges of the cation in the formula I.

2. The small molecule cyanine dye of claim 1, wherein R is a linear alkyl group having 8 to 16 carbon atoms, X ^- Selected from BF4 ^- 、Cl ^- 、Br ^- 、I ^- 、NO ^3- 、SO ₄ ^2- 、ClO ₄ ^- 、CH ₃ COO ^- 、CH ₃ SO ₃ ^- And CF (compact F) ₃ SO ₃ ^- 。

3. The small molecule cyanine dye of claim 1, wherein R is a linear alkyl group having 10 to 15 carbon atoms.

4. The small molecule cyanine dye of claim 1, wherein R is a linear alkyl group having 12 carbon atoms.

5. A nanoparticle comprising at least one of the small molecule cyanine dyes of claim 1.

6. The nanoparticle of claim 5, further comprising DSPE-PEG for encapsulating the small molecule cyanine dye ₂₀₀₀ 。

7. Nanoparticle according to claim 6, characterized in that the diameter of the nanoparticle is 10-200nm.

8. The nanometer photo-thermal reagent is characterized by being prepared by the following steps:

the method for preparing the dye of small molecule cyanine dye of claim 1, DSPE-PEG ₂₀₀₀ Dissolving in an organic solvent, and then dripping into a dispersion medium under the ultrasonic action; or,

the small molecule cyanine dye of claim 1 is added dropwise to DSPE-PEG under the action of ultrasound after being dissolved in an organic solvent ₂₀₀₀ In solution with a dispersion medium;

ultrasonic treating the mixture until no turbidity is observed, filtering with water-based filter membranes with diameters of 0.45 μm and 0.22 μm, and dialyzing to remove organic solvent to obtain the nano photothermal reagent; or,

concentrating the dialyzed dispersion liquid to obtain the nano photothermal reagent;

the organic solvent is a solvent which is mutually soluble with water, and the dispersion medium is ultrapure water or PBS.

9. The nano photothermal reagent of claim 8, wherein the small molecule cyanine dye, DSPE-PEG ₂₀₀₀ And the mass ratio of the dispersion medium is 1:5-15:5-15.

10. The nano photothermal reagent of claim 9, wherein the small molecule cyanine dye, DSPE-PEG ₂₀₀₀ And the mass ratio of the dispersion medium is 1:9-11:9-11;

the organic solvent is one or more of DMSO, THF, DMF.

11. The method for synthesizing the small-molecule cyanine dye according to claim 1, wherein the compound Cy-Cl reacts with sodium methyl mercaptide to remove chlorine atoms to obtain the small-molecule cyanine dye, and the specific synthesis reaction is as follows:

wherein R and X ^- Is as defined in claim 1.

12. Use of a small molecule cyanine dye according to any of claims 1-4, a nanoparticle according to any of claims 5-7, or a nano-photothermal agent according to any of claims 8-10 for the preparation of a medicament for the treatment of tumors.