CN114426554A - Organic fluorescent small molecular compound, organic fluorescent nano-carrier and preparation method and application thereof - Google Patents

Abstract

The present invention provides an organic fluorescent small molecule compound, an organic fluorescent nano-carrier and a preparation method and application thereof. The structural formula of the organic fluorescent small molecule compound is shown in formula 1:

The organic fluorescent small molecule compound modifies polypeptide, protein, polyethylene glycol, nucleic acid aptamer or folic acid and its derivatives at its controllable site to obtain a fluorophore, and the fluorophore can be used as an anti-tumor drug carrier; the present invention Also provided is an anti-tumor drug, which has the characteristics of tumor targeting, high sustained release rate, good therapeutic effect, and low cardiotoxicity compared with free anti-tumor drugs; The route is simple, the reaction efficiency is high, the yield is high, and the method has high industrial application prospect.

Description

Organic fluorescent small molecule compound, organic fluorescent nanocarrier and preparation method and application thereof

technical field

The patent of the present invention relates to the technical field of biomedical materials, in particular to an organic fluorescent small molecule compound, an organic fluorescent nanocarrier and a preparation method and application thereof.

Background technique

In general, the longer the circulation time in the body, the more time the nanocarriers have to accumulate to the lesion site, and thus the better to exert their diagnostic or therapeutic effects. However, in the prior art, traditional nanocarriers are still insufficient to meet the requirements for long-cycle performance. In recent years, nanocarriers, based on the characteristics of high curvature and small size, which can improve the distribution of drugs in vivo and have the effect of high permeability and retention (EPR effect) on tumor sites, have received extensive attention. Once the nanocarriers are applied in vivo, the long-circulation property becomes an important factor for their application.

The currently widely used chemotherapeutic drugs are widely distributed in the body, especially in some normal tissues, which not only reduces the bioavailability of the drugs, but also causes significant toxic and side effects to normal tissues. In addition, the circulation time of the drug in the blood is too short, and it is difficult to reach the lesion site. Therefore, improving the selectivity of chemotherapeutic drugs to cancer cells, reducing their distribution in non-targeted sites, and improving drug utilization are the keys to improving the efficacy of cancer treatment drugs. For example, as a member of the anthracycline antibiotics, doxorubicin has strong antitumor activity. However, due to its poor tissue distribution, large toxic and side effects, and long-term use easily lead to multidrug resistance and other factors that affect its efficacy, these adverse factors severely limit its clinical application.

In conclusion, it is very necessary to provide a drug carrier with long-circulation properties and improving the utilization rate of drugs, especially tumor drugs.

SUMMARY OF THE INVENTION

As a member of the nano-drug delivery system, polymer micelles have attracted much attention in recent years. Compared with other carriers, they show obvious advantages: (1) low critical micelle concentration and strong anti-blood dilution; (2) ) Small particle size, narrow distribution range, can use ERP effect (high permeability and retention effect of solid tumors) to achieve passive targeting; (3) With a special core-shell structure, the hydrophilic shell can avoid RES (net (4) The surface has modified functional ligands to achieve active targeting. Based on this, researches on prodrug micelles are also emerging one after another.

HLA4P@DOX is composed of small-molecule near-infrared second-region fluorophores and anthraquinone antibiotics. And NIR-II imaging for continuous real-time tracking of drug delivery and treatment in vivo. Compared with free DOX, it has the characteristics of high sustained release rate, good therapeutic effect, and low cardiotoxicity.

The present invention aims to solve one of the technical problems existing in the prior art at least to a certain extent. Therefore, in the first aspect of the present invention, the present invention provides an application of an organic fluorescent small molecule compound in the preparation of a drug carrier, The structural formula of the organic fluorescent small molecule compound is shown in formula 1:

和H中的一种，n取自0～18中的整数，m取自0～20中的整数；Wherein, R ₁ is selected from one of S and Se, R ₀ and R ₂ are independently selected from one of O, S, Se and NR ₁₁ , and R ₁₁ is selected from one of H, methyl and ethyl. species; R ₃ , R ₄ , R ₅ , R ₆ are each independently selected from

and one of H, n is taken from an integer from 0 to 18, and m is taken from an integer from 0 to 20;

中的一种，n取自0～18中的整数，m取自0～20中的整数，X选自F、Cl、Br、I和N₃中的一种。R ₇ , R ₈ , R ₉ , R ₁₀ are each independently selected from

One of , where n is an integer from 0 to 18, m is an integer from 0 to 20, and X is selected from one of F, Cl, Br, I and N ₃ .

In one or more embodiments of the present invention, the structural formula of the organic fluorescent small molecule compound is as follows:

In one or more embodiments of the present invention, the application of the above-mentioned organic fluorescent small molecule compound in the preparation of a drug carrier includes: modifying polypeptides, proteins, polymers at the controllable site of the organic fluorescent small molecule compound Ethylene glycol, nucleic acid aptamer or folic acid and derivatives thereof obtain a fluorophore, and the fluorophore is used as an antitumor drug carrier.

Preferably, the fluorophore is used as an anthraquinone antibiotic antitumor drug carrier.

Further, the anthraquinone antibiotic antitumor drugs include one or more of doxorubicin, daunorubicin, doxorubicin, epirubicin, idarubicin and mitoxantrone.

In a second aspect of the present invention, the present invention provides an anti-tumor drug, comprising the organic fluorescent small molecule compound described in the first aspect of the present invention or a polypeptide or protein modified at a controllable site of the organic fluorescent small molecule compound , polyethylene glycol, nucleic acid aptamers or fluorophores obtained from folic acid and its derivatives.

In one or more embodiments of the present invention, the anti-tumor drug targets tumors.

In the third aspect of the present invention, the present invention provides a preparation method of the antitumor drug described in the second aspect of the present invention, comprising:

Step 1): remove the acid in the active ingredient salt of the antitumor drug to obtain a free drug, and modify the polypeptide, protein, polyethylene glycol, nucleic acid aptamer or folic acid and The fluorophore obtained by its derivative and the free drug are added to DMSO to obtain a mixed solution;

Step 2): adding deionized water or PBS into the container, then dropping the mixture obtained in step 1) into the bottom of the container, and ultrasonically treating it to obtain a mixture;

Step 3): dialyze the mixture obtained in step 2) to remove the free drug that is not successfully encapsulated.

In one or more embodiments of the present invention, in the step 1), in the mixed solution, the mass ratio of the free drug to the fluorophore is 1:5; preferably, the antitumor drug active ingredients are selected from One or more of daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone. Preferably, the active ingredient salt of the antitumor drug is doxorubicin hydrochloride.

In one or more embodiments of the present invention, in the step 2), the dropping speed of the mixed droplets into the container is 60 to 110 drops per minute; an ultrasonic cleaning machine is used for ultrasonic treatment, and the ultrasonic power is controlled to be 230～250W, the working frequency is controlled at 40KHz, and the ultrasonic time is controlled at 5～20 seconds.

In one or more embodiments of the present invention, in step 3), the molecular weight cut-off of the dialysis bag used in the dialysis is 10kDa, deionized water is used as the external fluid, and the dialysis time is controlled to be 24-60 hours, during which time Change the external fluid more than 6 times.

In the fourth aspect of the present invention, the present invention provides an organic fluorescent nanocarrier, characterized in that, the organic fluorescent nanocarrier comprises the organic fluorescent small molecule compound described in the first aspect of the present invention or the organic fluorescent small molecule The fluorophore obtained by modifying the controllable site of the compound with polypeptide, protein, polyethylene glycol, nucleic acid aptamer or folic acid and its derivatives.

The beneficial effects of the present invention are:

1. The present invention provides an application of an organic fluorescent small molecule compound in the preparation of a drug carrier, wherein the organic fluorescent small molecule compound modifies polypeptides, proteins, polyethylene glycol, nucleic acid aptamers or folic acid and Its derivatives obtain fluorophores, and the fluorophores can be used as anti-tumor drug carriers;

2. The present invention provides an anti-tumor drug, which, compared with free anti-tumor drugs, has the characteristics of tumor targeting, high sustained release rate, good therapeutic effect, and low cardiotoxicity;

3. The present invention provides a method for preparing the above antitumor drug, which has a simple synthetic route, high reaction efficiency and high yield, and has a high industrial application prospect;

4. The present invention provides a drug carrier. The anti-tumor drug has the characteristics of tumor targeting, high sustained release rate, good therapeutic effect and low cardiotoxicity under the encapsulation of the drug carrier.

Description of drawings

Fig. 1 is the hydrogen nuclear magnetic spectrum of HLA4;

Fig. 2 is the carbon nuclear magnetic spectrum of HLA4;

Fig. 3 is the absorption and emission spectra of HLA4;

Fig. 4 is the preparation process of converting compound HLA4 into HLA4P which can be used for bioimaging;

Fig. 5 is the characterization of compound HLA4P by hydrogen nuclear magnetic spectrum;

Figure 6 is a transmission electron microscope image of the compound HLA4 that can self-assemble to form nanoparticles after being connected to polyethylene glycol;

7 is a schematic diagram of the HLA4P@DOX self-assembly prepared in Example 2;

Fig. 8 is the cell uptake imaging of the confocal microscope of Example 3;

Fig. 9 is the TEM and DLS images of HLA4P@DOX prepared in Example 2;

Figure 10 is the biodistribution diagram of DOX and HLA4P@DOX at different time nodes after tail vein injection in Example 5;

Figure 11 is a biodistribution diagram of different organs at different time points after tail vein injection of DOX in Example 5;

12 is a biodistribution diagram of different organs at different time points after tail vein injection of HLA4P@DOX in Example 5;

Figure 13 is a graph showing the tumor size of mice given various treatments in Example 6;

Figure 14 is a near-infrared second region imaging diagram of mice under various treatments in Example 7;

15 is a graph of H&E staining of different organs of mice administered various treatments in Example 8. FIG.

Detailed ways

The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be construed as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, the technique or condition described in the literature in the field or the product specification is used. If the specific conditions are not indicated in the following examples, follow the conventional conditions or the conditions suggested by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art. The consumables and reagents used are not specified. Bought for the market. Unless otherwise defined, professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described can also be used in the present invention.

Example 1

The structural formula of the organic fluorescent small molecule compound is shown in formula 1:

和H中的一种，n取自0～18中的整数，m取自0～20中的整数；Wherein, Y, Z are independently selected from one of O, S, Se and NR ₉ , R ₉ is selected from one of H, methyl and ethyl; R ₁ , R ₂ , R ₃ , R ₄ individually selected from

and one of H, n is taken from an integer from 0 to 18, and m is taken from an integer from 0 to 20;

R ₅ , R ₆ , R ₇ , R ₈ are each independently selected from

中的一种，n取自0～18中的整数，m取自0～20中的整数，X选自F、Cl、Br、I和N₃中的一种。

One of , where n is an integer from 0 to 18, m is an integer from 0 to 20, and X is selected from one of F, Cl, Br, I and N ₃ .

The preparation route of the above-mentioned organic fluorescent small molecule compound (compound shown in formula 1) is as follows:

The following experimental group 1 takes the compound HLA4 as an example to illustrate the preparation of the organic fluorescent small molecule compound (the compound represented by formula 1).

Experimental Group 1: Preparation of Compound HLA4

Step 1): Preparation of Compound 3a:

Take compound 2a (2g, 5.9mmol), zinc powder (13.8g, 212.4mmol) and ammonium chloride (18.8g, 354mmol) into a 500mL round-bottom flask, add methanol-water (v/v, 9:1) 100 mL and 100 mL of dichloromethane, bubbling argon into the reaction solution for 5 min to remove oxygen in the system, and react at room temperature for 2 hours under argon protection. After the reaction, it was cooled to room temperature, and the methanol was removed by rotary evaporation. The residue was redissolved in 150 mL of dichloromethane, washed with water (30 mL×3) three times, and saturated brine (30 mL×3) three times. The organic phase was dried over anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to obtain the intermediate. The above intermediate, N-sulfanilide (2.47g, 17.8mmol) and trimethylchlorosilane (2.57g, 23.7mmol) were taken into a 50mL round bottom flask, 20mL of pyridine was added under argon protection, and the reaction solution was added. Bubbling argon gas for 5 min to remove oxygen in the system, and reacting at room temperature for 2 hours under the protection of argon gas. After the reaction, it was cooled to room temperature, and the pyridine was removed by rotary evaporation. The residue was redissolved in 150 mL of dichloromethane, washed with water (30 mL×3) three times, and saturated brine (30 mL×3) three times. The organic phase was dried with anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to obtain 1.62 g of compound 3a, yield: 90%.

The structural determination data of compound 3a are as follows:

¹ H NMR (400MHz, CDCl ₃ )δ7.43(s,1H),6.93(s,1H),2.65(t,J=7.7Hz,2H),1.93-1.62(m,2H),1.46-1.14( m, 19H), 0.90 (t, J=6.8Hz, 3H). ¹³ C NMR (101MHz, CDCl ₃ )δ156.18, 144.66, 134.65, 125.64, 120.24, 112.46, 31.94, 30.49, 30.43, 29.69, 29.62, 29.48, 29.38, 29.35, 22.71, 14.14.

Step 2): Preparation of Compound 4a:

The above blue compound 3a (840 mg, 1.31 mmol) and N-bromosuccinimide (NBS) (780 mg, 3.93 mmol) were added to a 50 mL round-bottomed flask, and 20 mL of pyridine was added under argon protection. Bubbling argon gas for 5 min to remove oxygen in the system, and reacting at room temperature for 2 hours under the protection of argon gas. After the reaction, it was cooled to room temperature, and the pyridine was removed by rotary evaporation. The residue was redissolved in 150 mL of dichloromethane, washed with water (30 mL×3) three times, and saturated brine (30 mL×3) three times. The organic phase was dried over anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to obtain 953 mg of compound 3a. Yield: 91%.

The structural determination data of compound 4a are as follows:

HRMS(ESI)Calcd for:C ₅₂ H ₄₁ N ₆ O ₈ S ₄₊ ([M+H]+):800.8769,found:800.8743.

Step 3): Preparation of Compound 6a:

Take compound 4a (720mg, 0.904mmol), 15% by mass of sodium bicarbonate, compound 5a (1.23g, 2.26mmol) and tetrakistriphenylphosphine palladium (10mg, 0.008mmol) into a 50mL round bottom flask , 20 mL of tetrahydrofuran was added under argon protection, argon was bubbled into the reaction solution for 5 min to remove oxygen in the system, and the reaction was carried out at room temperature for 2 hours under argon protection. After the reaction, it was cooled to room temperature, and the tetrahydrofuran was removed by rotary evaporation. The residue was redissolved in 150 mL of dichloromethane, washed three times with water (30 mL×3) and three times with saturated brine (30 mL×3). The organic phase was dried with anhydrous magnesium sulfate for 3 hours, filtered, and the filtrate was spin-dried to obtain 1.03 g of 6a, yield: 80%.

The structural determination data of compound 6a are as follows: ¹ H NMR (400MHz, CDCl ₃ )δ7.47(s, 2H), 7.36(d, J=8.5Hz, 4H), 7.33-7.28(m, 5H), 7.16(d, J=9.7Hz, 8H), 7.08(dd, J=15.1, 7.9Hz, 10H), 4.22(dd, J=10.8, 6.1Hz, 4H), 2.96(t, J=7.8Hz, 4H), 2.71( dd, J=16.0, 8.0Hz, 4H), 2.65 (t, J=7.8Hz, 4H), 1.71 (dt, J=15.1, 7.6Hz, 4H), 1.38–1.22 (m, 36H), 1.06–0.98 (m, 4H), 0.90 (t, J=6.7Hz, 6H), 0.08 (s, 18H). ¹³ C NMR (101 MHz, CDCl ₃ ) δ 173.11, 147.49, 145.59, 135.77, 129.63, 129.33, 129.27, 125.07, 124.61,123.14,122.64,62.70,36.11,31.94,30.96,30.42,29.72,29.70,29.67,29.64,29.60,29.50,29.38,28.99,22.71,17.35,14.15,-1.44.

MALDI-TOF-MS Calcd for: C ₇₄ H ₈₀ N ₆ O ₄ S ₄ ([M+H]+): 1474.51, found: 1474.9806.

Step 4): Preparation of Compound HLA4:

Compound 6a (100 mg, 0.068 mmol) and trifluoroacetic acid (5 mL) were added to a 50 mL round-bottomed flask, 20 mL of dichloromethane was added under argon protection, and argon was bubbled into the reaction solution for 5 min to remove oxygen in the system, The reaction was carried out at room temperature for 2 hours under argon protection. After the reaction was completed, it was cooled to room temperature, and the dichloromethane was removed by rotary evaporation to obtain 85 mg of HLA4. Yield: 98%.

The structural determination data of compound HLA4 are as follows:

MALDI-TOF-MS Calcd for: C ₇₄ H ₈₀ N ₆ O ₄ S ₄ ([M+H]+): 1272.57., found: 1272.4865.

The HLA4 NMR spectrum of the compound prepared in the experimental group 1 of Example 1 is shown in Figure 1; the characterization of the compound HLA4 prepared in the experimental group 1 in Example 1 is shown in Figure 2; The absorption and emission spectra of the obtained compound HLA4 are shown in FIG. 3 .

The following experimental group 2 is the preparation of the compound HLA4 prepared in the above experimental group 1, which can be used for bioimaging probe HLA4P.

Experimental group 2: Preparation of fluorophore HLA4P

Take compound HLA4 (123 mg, 0.226 mmol), MPEG2000NH ₂ (1.23 g, 0.565 mmol), 100 μL DIPEA, N-(2-aminoethyl) maleimide trifluoroacetate (0.7611 mg, 0.030 mmol) and HATU (11.410 mg, 0.030 mmol) was added to a 50 mL round-bottomed flask, 20 mL of N,N dimethylformamide was added under argon protection, argon was bubbled into the reaction solution for 5 min to remove oxygen in the system, and under argon protection at room temperature The reaction was continued for 2 hours. After the reaction was completed, it was cooled to room temperature, and the N,N dimethylformamide was removed by rotary evaporation to obtain 1.1 g of HLA4P. Yield: 90%.

The HLA4P structure determination data of the compound H NMR characterization is shown in Figure 5.

Figure 6 is a transmission electron microscope image of the compound HLA4 that can self-assemble to form nanoparticles after being linked to polyethylene glycol.

Example 2

Preparation of Fluorophore-Encapsulated Antitumor Drugs (HLA4P@DOX)

First, the hydrochloric acid in DOX.HCl (doxorubicin hydrochloride) was removed. 5 mg of the fluorophore HLA4P prepared in Example 1 and 1 mg of the hydrophobic free drug DOX (doxorubicin) were added to the DMSO solution to obtain a mixed solution. Add deionized water or PBS to the round-bottom flask, and slowly drip the mixture of HLA4P and DOX into the bottom. The dropping speed of the mixed droplets into the container is 60-110 drops per minute, and after the addition, ultrasonic treatment is performed for 10s. The ultrasonic power is controlled to 240W, and the operating frequency is controlled to 40KHz. The mixture was obtained, the mixture was dialyzed to remove free DOX for about 48 hours, the molecular weight cut-off of the dialysis bag used in the dialysis was 10kDa, deionized water was used as the outer fluid, the dialysis time was controlled to be 48 hours, and the outer fluid was replaced more than 6 times during the period, That is, the HLA4P@DOX is obtained.

FIG. 6 is a schematic diagram of the self-assembly process of preparing HLA4P@DOX in Example 2. FIG. Using the anticancer drug doxorubicin hydrochloride (DOX) as a model drug, the drug loading and controlled release capacity of HLA4P were investigated. HLA4P@DOX was prepared by a typical nano-encapsulation method. The HLA4P@DOX was characterized by TEM and DLS, and its average size was 150 nm and 180 nm, respectively, which was slightly higher than the self-assembled size of a single HLA4P. According to the UV-vis absorbance of DOX, the encapsulation efficiency of HLA4P@DOX was determined to be 65%. Figure 9 shows the TEM and DLS images of HLA4P@DOX prepared in Example 2.

Example 3

Confocal Laser Scanning

CT-26 cells were grown in culture flasks. After 24 hours, 1 ml of culture medium was added to the confocal dish, followed by HLA4P@DOX. After 4 hours, the medium in the small dish was removed and 1 mL of PBS was added. A 4% paraformaldehyde solution (1 mL) was added to each plate and left for 15 minutes for cell fixation. After 15 min, the paraformaldehyde fixative was taken out, PBS buffer was added for 3 times, and 200 μL of DAPI staining solution was added. After 10 min incubation with DAPI staining solution, the staining solution was also removed and washed 3 times with PBS buffer. Cells were observed under a confocal microscope Leica-LCS-SP8-STED. CT-26 cells were imaged for cellular uptake using free DOX (CDOX=60 μM) and HLA4P@DOX (CDOX=60 μM) over 8 hours using confocal microscopy. FIG. 8 is the cellular uptake imaging of the confocal microscope of Example 3. FIG. The results showed that when CT26 cells were cultured with free DOX for 6 h, the drug uptake efficiency was about 50%. In contrast, after 6 h incubation with HLA4P@DOX, the drug uptake increased to about 80%, indicating that HLA4P@DOX promoted the cellular internalization and accumulation of DOX. Confocal laser scanning microscopy further demonstrated the enhanced uptake of HLA4P@DOX by CT-26 cells. The red fluorescence of HLA4P@DOX was much stronger than that of free DOX, indicating that CT-26 cells had higher uptake of it.

Example 4

Calculation of Encapsulation Efficiency and Drug Loading

Calibration curves were performed for different concentrations of DOX dissolved in DMSO. After dialysis, DOX was separated from the blood. HLA4P@DOX was dissolved in dimethyl sulfoxide and the absorbance was measured at 480 nm. The drug loading efficiency (DLE) and drug loading content (DLC) were analyzed by the following calculation formulas:

HLA4P can effectively encapsulate the antitumor drug doxorubicin (DOX, the encapsulation rate is about 65%), and achieve sustained release of DOX.

Example 5

Biodistribution of HLA4P@DOX

ICR mice were randomly divided into different groups. HLA4P@DOX free DOX solution was injected intravenously into ICR mice. Blood samples were collected from various time points, and then whole blood samples were centrifuged at 13000 rpm for 15 minutes to collect plasma. We randomly divided CT-26 tumor-bearing Balb/c mice into different groups. HLA4P@DOX free DOX solution was injected intravenously into CT-26 mouse model. Mice were sacrificed at various time points after injection and hearts, spleens, livers, lungs, kidneys and tumors were harvested and weighed. These plasmas and tissues were suspended in a 70% ethanol solution containing 0.3N HCl, and the mixture was thoroughly homogenized. After a further centrifugation step, the absorbance of DOX in the supernatant was measured at a wavelength of 480 nm. According to the standard curve, the DOX content in mouse blood and various tissues was determined. Figure 10 is the biodistribution diagram of DOX and HLA4P@DOX at different time points after tail vein injection in Example 5; Figure 11 is the biodistribution diagram of different organs at different time points after tail vein injection of DOX in Example 5; Figure 12 is an example 5 Biodistribution maps of different organs at different time points after tail vein injection of HLA4P@DOX. It can be seen from the results in Figures 4, 5 and 6 that even though 72H HLA4P@DOX still has a certain concentration and is higher than free DOX, the side also reflects that HLA4P@DOX has an excellent potential for long-circulation therapy.

Example 6

Antitumor effect of HLA4P@DOX in vivo

CT-26-bearing mice (19-25 g) were randomized into 4 groups (n=5) and treated when the tumor volume of CT-26 mice increased with an average size ^of approximately 70 mm (recorded at this time point). as "Day 1"). The CT-26 tumor-bearing mice group was intravenously injected once. Free DOX (6 mg/kg), HLA4P, PBS and HLA4P@DOX (6 mg/kg) were injected intravenously into mice, respectively. Tumor volume was measured with an electronic vernier caliper. The analytical formula of CT-26 tumor volume is: V=e ² ×E/2, where E and e are the longest and shortest diameters of CT-26 tumors, respectively. Figure 13 is a graph of tumor size in mice given various treatments (PBS, DOX (6 mg/kg), HLA4P and HLA4P@DOX (6 mg/kg)). CT-26 mice were weighed every two days, and the survival rate of the mice was monitored all the time.

Tumor size and body weight were measured every 2 days. CT-26 tumors grew very fast after PBS and HLA4P treatment, and the tumor volume in the two control groups increased 13-fold and 12-fold, respectively, after 14 days of treatment. However, CT-26 tumors treated with free DOX grew slower in the first 6 days after injection. Mean tumor volume in the free DOX-treated group increased 6-fold after 14 days of treatment.

Example 7

Imaging of HLA4P@DOX Tracking Therapy

The mice in different treatment groups in Example 6 were subjected to near-infrared second-region fluorescence imaging, and FIG. 14 is the near-infrared second-region imaging of mice in different treatment groups (DOX (6mg/kg) and HLA4P@DOX (6mg/kg)) .

The results showed that HLA4P@DOX not only had bright NIR-II luminescence (~1055 nm) and tumor retention lasted for 14 days, but also had better therapeutic effect than free DOX in vitro and in vivo, with fewer side effects. HLA4P@DOX can be excited by 808 nm laser, showing the capability of real-time tracking and long-term continuous NIR-II imaging in vivo. Has a certain tracking effect.

The entire treatment process is precisely monitored by NIR-II fluorescence imaging. Significant NIR-II signal was obtained at tumor sites with high T/TN (>23), and the fluorescence signal could last for 14 days, indicating that HLA4P@DOX has superior tumor targeting ability and ultra-long tumor retention.

Example 8

Histological analysis

Tumors or major organs were obtained from mice in different treatment groups of Example 6 (free DOX (6 mg/kg), HLA4P, PBS and HLA4P@DOX (6 mg/kg)) and fixed with EDTA/formalin solution. After embedding, various tissue samples were stained with H&E staining. Figure 15 is a graph of H&E staining of different organs of mice in different treatment groups (PBS, DOX (6 mg/kg) and HLA4P@DOX (6 mg/kg)).

Although the embodiments of the present invention have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Changes, modifications, substitutions and modifications to the embodiments should all be included within the protection scope of the present invention.

Claims (10)

1. an application of an organic fluorescent small molecule compound in the preparation of a drug carrier, is characterized in that, the structural formula of the organic fluorescent small molecule compound is as shown in formula 1:

Wherein, R ₁ is selected from one of S and Se, R ₀ and R ₂ are independently selected from one of O, S, Se and NR ₁₁ , and R ₁₁ is selected from one of H, methyl and ethyl. species; R ₃ , R ₄ , R ₅ , R ₆ are each independently selected from

and one of H, n is taken from an integer from 0 to 18, and m is taken from an integer from 0 to 20; R ₇ , R ₈ , R ₉ , R ₁₀ are each independently selected from

One of , where n is an integer from 0 to 18, m is an integer from 0 to 20, and X is selected from one of F, Cl, Br, I and N ₃ . 2. the application of a kind of organic fluorescent small molecular compound according to claim 1 in the preparation of drug carrier, it is characterized in that, described organic fluorescent small molecular compound structural formula is as follows:

3. The application of an organic fluorescent small molecule compound in the preparation of a drug carrier according to claim 1, characterized in that, comprising: modifying polypeptides, proteins, polyethylene Diol, nucleic acid aptamer or folic acid and derivatives thereof obtain a fluorophore, and the fluorophore is used as an antitumor drug carrier. 4 . The application of an organic fluorescent small molecule compound in the preparation of a drug carrier according to claim 3 , wherein the fluorophore is used as an anthraquinone antibiotic antitumor drug carrier. 5 . 5. the application of a kind of organic fluorescent small molecule compound in the preparation of drug carrier according to claim 4, is characterized in that, described anthraquinone antibiotic antitumor drug comprises doxorubicin, daunorubicin, doxorubicin One or more of star, epirubicin, idarubicin and mitoxantrone. 6. An anti-tumor drug, characterized in that it comprises the organic fluorescent small molecule compound of claim 1 or modified polypeptides, proteins, polyethylene glycol, nucleic acid suitable for the controllable site of the organic fluorescent small molecule compound. Fluorophores derived from ligands or folic acid and its derivatives. 7 . The anti-tumor drug according to claim 6 , wherein the anti-tumor drug targets tumors. 8 . 8. the preparation method of the antitumor drug of claim 6, is characterized in that, comprises: Step 1): remove the acid in the active ingredient salt of the antitumor drug to obtain a free drug, and modify the polypeptide, protein, polyethylene glycol, nucleic acid aptamer or folic acid and The fluorophore obtained by its derivative and the free drug are added to DMSO to obtain a mixed solution; Step 2): adding deionized water or PBS into the container, then dropping the mixture obtained in step 1) into the bottom of the container, and ultrasonically treating it to obtain a mixture; Step 3): dialyze the mixture obtained in step 2) to remove the free drug that is not encapsulated successfully; Preferably, in the step 1), in the mixture, the mass ratio of the free drug to the fluorophore is 1:5; preferably, the active ingredients of the antitumor drug are selected from daunorubicin, doxorubicin , one or more of epirubicin, idarubicin and mitoxantrone; Preferably, in the step 2), the dropping speed of the mixed droplets into the container is 60-110 drops per minute; ultrasonic treatment is performed using an ultrasonic cleaning machine, the ultrasonic power is controlled at 230-250W, and the working frequency is controlled at 40KHz , the ultrasonic time is controlled to be 5 to 20 seconds. 9 . The method for preparing an antitumor drug according to claim 8 , wherein in the step 3), the molecular weight cut-off of the dialysis bag used for the dialysis is 10 kDa, and deionized water is used as the external fluid, and the dialysis time is 10 kDa. 10 . Controlled for 24 to 60 hours, during which the external fluid was replaced more than 6 times. 10. An organic fluorescent nanocarrier, characterized in that the organic fluorescent nanocarrier comprises the organic fluorescent small molecule compound of claim 1 or modified polypeptides, proteins, Fluorophores obtained from polyethylene glycol, nucleic acid aptamers or folic acid and derivatives thereof.

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