Small biological molecule cluster type nano coating agent material and preparation method thereof

Abstract

The invention relates to the technical field of nano drug delivery, in particular to a small biological molecule cluster type nano coating agent material and a preparation method thereof, the small biological molecule cluster type nano coating agent material takes dopamine hydrochloride as a main body, and a long carbon chain is grafted on a side chain and amino acid and saccharide molecules are connected in series. According to the invention, through the high demand and high uptake characteristic of tumor cells on amino acids and carbohydrates in the proliferation process, active targeting on tumors is realized, drugs can be accurately delivered to lesion areas of the tumors, the targeting accuracy is significantly improved, and the problem of inaccurate targeting of traditional nano-carriers is effectively overcome.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drug delivery, and particularly to a biomolecular cluster-type nano-coating agent material and a preparation method thereof. Background Art

[0002] In tumor treatment, the delivery of nano-drugs by nano-carriers has become a cutting-edge and highly potential treatment method. Due to their unique small size, usually between 1 and 1000 nm, nano-carriers can smoothly penetrate physiological barriers, such as the abnormal blood vessel walls around tumor tissues, to precisely target cancer cells and solve the problem of tumors.

[0003] Traditional nano-carriers, such as distearoyl phosphatidylethanolamine-polyethylene glycol (DSP-PEG) and Pluronic F127, mainly rely on the "enhanced permeability and retention effect" (EPR effect) to guide the loaded drugs to accumulate in the tumor area, optimize the distribution of drugs in the body, and increase the drug concentration at the tumor site. Compared with traditional drug dosage forms, the treatment effect has been significantly improved. However, with the in-depth research and clinical practice, the EPR effect also has defects in the tumor targeting process, such as low targeting accuracy and large potential toxic and side effects.

[0004] In contrast, active targeting has shown unique advantages in tumor treatment. During the abnormal proliferation and metabolism of tumor cells, the demand for amino acids and sugars increases significantly. This is because amino acids are important raw materials for tumor cells to synthesize biological macromolecules such as proteins and nucleic acids, while sugars provide energy and the material basis for the rapid proliferation of tumor cells to build biological membranes. Based on this characteristic, amino acid cluster-type nano-coating agents have emerged. By integrating amino acids and sugar molecules into nano-carriers and using the high-demand uptake mechanism of tumor cells for these substances, the active enrichment of nano-carriers in tumor cells is achieved, thereby achieving the purpose of actively targeting tumors, providing new ideas and methods for improving the tumor treatment effect, and having broad application prospects. Summary of the Invention

[0005] The purpose of the present invention is to provide a biomolecular cluster-type nano-coating agent material and a preparation method thereof. The provided nano-coating agent utilizes the high-demand uptake of amino acids and sugars during the abnormal process of tumors to achieve the targeting effect, and at the same time enhances its water solubility to achieve good drug coating and delivery effects, solving the problems of low targeting accuracy and large toxic and side effects of traditional nano-carriers.

[0006] To achieve the above object, the present invention provides a biomolecular cluster-type nano-coating agent material, with dopamine hydrochloride as the main body, grafting long carbon chains and connecting amino acids and sugar molecules in series on the side chain. The structural formula is as follows,

[0007]

[0008] In the formula, R1 is an alkyl group with C1 to C 30 , and R2 is one or more of the following structural formulas,

[0009]

[0010] n is the degree of polymerization, and n is 50 to 100.

[0011] The present invention also provides a preparation method of the above-mentioned bio-small molecule cluster-type nano-coating agent material, including the following steps,

[0012] S1. Dissolve dopamine hydrochloride, potassium carbonate, and di-tert-butyl dicarbonate (BOC anhydride) in N,N-dimethylformamide, stir and react to obtain Compound 1, and then add potassium carbonate and octadecyl bromide to Compound 1, and carry out a condensation reflux reaction to obtain Compound 2;

[0013] S2. Dissolve Compound 2 and trifluoroacetic acid in dichloromethane, react at room temperature to obtain Reaction Solution 1, add saturated sodium bicarbonate aqueous solution to Reaction Solution 1, and stir and react to obtain Compound 3;

[0014] S3. Dissolve Compound 3 in dichloromethane, first add triethylamine for stirring reaction, and then add dibromo isobutyryl bromide to react at room temperature to obtain Compound 4;

[0015] S4. Dissolve Compound 4 in anisole, add CuBr and N-acryloxysuccinimide to react to obtain Reaction Solution 2, and add N,N,N',N'',N''-pentamethyldiethylenetriamine to Reaction Solution 2 to obtain Compound 5;

[0016] S5. Dissolve Compound 5 in N,N-dimethylformamide, add triethylamine, amino acid, and sugar molecule to react to obtain Compound 6, that is, the bio-small molecule cluster-type nano-coating agent material.

[0017] Preferably, in S1, the molar ratio of dopamine hydrochloride, potassium carbonate, and di-tert-butyl dicarbonate is 1:1:1.5 to 2, and the molar ratio of Compound 1 to potassium carbonate and octadecyl bromide is 1:1:2 to 3.

[0018] Preferably, in S1, the stirring reaction is carried out at room temperature for 4 h, and the time of the condensation reflux reaction is 12 h.

[0019] Preferably, in S2, the molar ratio of Compound 2 to trifluoroacetic acid is 1:1.5;

[0020] In S2, the reaction time at room temperature is 12 h, and the stirring reaction time is 2 h.

[0021] Preferably, in S3, the molar ratio of Compound 3 to triethylamine and dibromo isobutyryl bromide is 1:1:2 to 3;

[0022] In S3, the reaction temperature of the stirring reaction is room temperature, the reaction time is 0.5 h, and the reaction time for the room temperature reaction is 4 - 6 h.

[0023] Preferably, in S4, the molar ratio of compound 4 to N - acryloxysuccinimide is 1:50 - 100.

[0024] Preferably, in S4, the reaction solution II is cooled to a solid using liquid nitrogen, the gas in the reaction atmosphere is replaced with nitrogen, and then it is placed at room temperature until the solid melts, and this is repeated three times.

[0025] Preferably, in S5, the molar ratio of compound 5 to triethylamine, amino acid, and sugar molecule is 1:50:50.

[0026] Preferably, the amino acid in S5 includes one or more of arginine, cysteine, leucine, isoleucine, valine; the sugar molecule is D-(+)-glucosamine hydrochloride.

[0027] Advantages of the present invention:

[0028] (1) Through the high demand and high uptake characteristics of tumor cells for amino acids and sugar substances during the proliferation process, the present invention achieves active targeting of tumors, can precisely deliver drugs to the diseased areas of tumors, significantly improves the targeting accuracy, and effectively overcomes the problem of inaccurate targeting of traditional nanocarriers.

[0029] (2) The precise targeting effect of the present invention enables the drug to act more effectively on tumor cells, enhances the therapeutic effect of the drug on tumors, while reducing the distribution of the drug in normal tissues, reducing the toxic and side effects on normal tissues, and improving the treatment safety and effectiveness of patients.

[0030] (3) The design of the materials of the present invention enhances their water solubility, is beneficial to the encapsulation of drugs and their transportation in the body, improves the stability and bioavailability of drugs, and further optimizes the drug delivery performance.

[0031] The technical solution of the present invention will be further described in detail below through the drawings and examples. Description of the Drawings

[0032] Figure 1 is the nuclear magnetic resonance hydrogen spectrum of compound 2 prepared in Example 1 of the present invention;

[0033] Figure 2 is the nuclear magnetic resonance hydrogen spectrum of compound 3 prepared in Example 1 of the present invention;

[0034] Figure 3 is the nuclear magnetic resonance hydrogen spectrum of compound 4 prepared in Example 1 of the present invention;

[0035] Figure 4 1H NMR spectrum of Compound 5 prepared in Example 1 of the present invention;

[0036] Figure 5 1H NMR spectrum of Compound 6-leucine in Example 3 of the present invention;

[0037] Figure 6 1H NMR spectrum of Compound 6-valine in Example 5 of the present invention;

[0038] Figure 7 CPC data graph of Compound 6-leucine prepared in Example 3 of the present invention;

[0039] Figure 8 Confocal imaging graphs of the products prepared in Examples 1-5 of the present invention at different time points in cells and in vivo. Detailed implementation manners

[0040] The present invention will be further described below in conjunction with the drawings and examples. Unless otherwise defined, the technical terms or scientific terms used in the present invention should have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs. The features mentioned above in the present invention or the features mentioned in the specific examples can be combined arbitrarily. These specific examples are only used to illustrate the present invention and not to limit the scope of the present invention.

[0041] The present invention provides a biological small molecule cluster type nano-coating agent material, with dopamine hydrochloride as the main body, grafting long carbon chains and connecting amino acids and sugar molecules in the side chain. The structural formula is as follows.

[0042]

[0043] In the formula, R1 is an alkyl group with C1-C 30 and R2 is one or more of the following structural formulas.

[0044]

[0045] n is the degree of polymerization, and n is 50-100.

[0046] The present invention also provides a preparation method of the above biological small molecule cluster type nano-coating agent material, including the following steps.

[0047] S1. Dissolve dopamine hydrochloride, potassium carbonate and di-tert-butyl dicarbonate in N,N-dimethylformamide, stir and react to obtain Compound 1, and then add potassium carbonate and octadecyl bromide to Compound 1, and carry out a condensation reflux reaction to obtain Compound 2;

[0048] S2. Dissolve compound 2 and trifluoroacetic acid in dichloromethane, react at room temperature to obtain reaction solution 1, add saturated aqueous sodium bicarbonate solution to reaction solution 1, and stir to react to obtain compound 3;

[0049] S3. Dissolve compound 3 in dichloromethane, first add triethylamine and stir to react, then add dibromo isobutyryl bromide and react at room temperature to obtain compound 4;

[0050] S4. Dissolve compound 4 in anisole, add CuBr and N-acryloxysuccinimide to react to obtain reaction solution 2, add N,N,N',N'',N''-pentamethyldiethylenetriamine to reaction solution 2 to react to obtain compound 5;

[0051] S5. Dissolve compound 5 in N,N-dimethylformamide, add triethylamine, amino acid and sugar molecule to react to obtain compound 6, namely the biomolecular cluster-type nano-coating agent material.

[0052] In some embodiments of the present invention, the synthesis route of the preparation method of the biomolecular cluster-type nano-coating agent material is as follows:

[0053]

[0054] Preferably, the molar ratio of dopamine hydrochloride, potassium carbonate and di-tert-butyl dicarbonate in S1 is 1:1:1.5-2, and the molar ratio of compound 1 to potassium carbonate and octadecyl bromide is 1:1:2-3.

[0055] Preferably, the stirring reaction in S1 is carried out at room temperature for 4 h, and the time of the condensation reflux reaction is 12 h.

[0056] In some embodiments of the present invention, after the condensation reflux reaction in step S1 is completed, the mixture is extracted with dichloromethane, the organic layer is collected and dried with anhydrous sodium sulfate, and the crude product is purified by silica gel chromatography to obtain white solid compound 2. The present invention does not limit the amounts of dichloromethane and anhydrous sodium sulfate, and the amounts well-known to those skilled in the art can be used.

[0057] Preferably, the molar ratio of compound 2 to trifluoroacetic acid in S2 is 1:1.5;

[0058] The reaction time at room temperature in S2 is 12 h, and the stirring reaction time is 2 h.

[0059] In some embodiments of the present invention, the saturated aqueous sodium bicarbonate solution in S2 is dropped into reaction solution 1.

[0060] In some embodiments of the present invention, after the stirring reaction in step S2 is completed, the product is filtered, extracted with dichloromethane solution, and then rotary evaporated to obtain white solid compound 3.

[0061] Preferably, in S3, the molar ratio of compound 3, triethylamine, and dibromoisobutyryl bromide is 1:1:2 - 3;

[0062] In S3, the reaction temperature for the stirring reaction is room temperature, the reaction time is 0.5 h, and the reaction time for the room temperature reaction is 4 - 6 h.

[0063] In some embodiments of the present invention, after the room temperature reaction in step S3 is completed, the prepared mixture is extracted with dichloromethane and water, then dried with anhydrous sodium sulfate, purified by silica gel chromatography, and after purification, the organic layer is dried and concentrated to obtain a light yellow oily compound 4.

[0064] Preferably, in S4, the molar ratio of compound 4 to N - acryloxysuccinimide is 1:50 - 100.

[0065] Preferably, in S4, the reaction solution II is cooled to a solid with liquid nitrogen, the gas in the reaction atmosphere is replaced with nitrogen, and then placed at room temperature until the solid melts, repeating three times.

[0066] In some embodiments of the present invention, after the reaction with N,N,N',N”,N”-pentamethyldiethylenetriamine in step S4 is completed, the product is dissolved in dichloromethane, filtered with a needle filter head, dropped into an ether solution, stirred at room temperature for 1 h, the upper ether solution is removed, the precipitate is collected and dried in a vacuum drying oven to obtain compound 5.

[0067] Preferably, in S5, the molar ratio of compound 5, triethylamine, amino acid, and sugar molecule is 1:50:50.

[0068] Preferably, the amino acid in S5 includes one or more of arginine, cysteine, leucine, isoleucine, and valine; the sugar molecule includes D-(+)-glucosamine hydrochloride.

[0069] In some embodiments of the present invention, after the reaction in step S5 is completed, the upper clear liquid is taken, dialyzed with a dialysis bag with a molecular weight of 2000 for 24 h until N,N - dimethylformamide is removed, and then all the liquid is freeze - dried with a freeze dryer to obtain compound 6, which is the bio - small - molecule - cluster - type nano - coating agent material.

[0070] Example 1

[0071] The present invention provides a preparation method of a bio - small - molecule - cluster - type nano - coating agent material, including the following steps:

[0072] S1. At room temperature, in a single-neck round-bottom flask equipped with a magnetic stir bar, dissolve 3 g of dopamine hydrochloride in N,N-dimethylformamide. Then add 2.18 g of potassium carbonate and 3.5 g of BOC anhydride thereto, and react at room temperature for 4 h to obtain Compound 1. Add 2.18 g of potassium carbonate and 15 g of octadecyl bromide to Compound 1, and reflux overnight. Extract the resulting mixture with dichloromethane, collect the organic layer and dry it with anhydrous sodium sulfate. Purify the crude product by silica gel chromatography to obtain white solid Compound 2.

[0073] The reaction formula is as follows:

[0074]

[0075] S2. In a single-neck round-bottom flask equipped with a magnetic stir bar, dissolve 1 g of Compound 2 obtained in S1 in 10 mL of dichloromethane, add 0.3 g of trifluoroacetic acid, and react at room temperature for 12 h to obtain Reaction Solution 1. Then, drop 200 mL of saturated sodium bicarbonate aqueous solution into Reaction Solution 1, stir for 2 h, filter and extract with dichloromethane solution, and finally rotary evaporate to obtain white solid Compound 3. The reaction formula is as follows:

[0076]

[0077] S3. In a single-neck round-bottom flask equipped with a magnetic stir bar, dissolve 0.5 g of Compound 3 obtained in S2 in 5 mL of dichloromethane. First, add 0.12 g of triethylamine, stir at room temperature for 0.5 h, then add 0.5 g of dibromo isobutyryl bromide, and react at room temperature for 4 - 6 h. Then, extract the product with dichloromethane and water, dry it with anhydrous sodium sulfate, purify it by silica gel chromatography, and finally dry and concentrate the organic layer to obtain light yellow oily Compound 4. The reaction formula is as follows:

[0078]

[0079] S4. In a polymerization reaction tube equipped with a magnetic stir bar, dissolve 100 mg of Compound 4 obtained in S3 in anisole, add 2 mg of cuprous bromide and 1 g of N-acryloxysuccinimide thereto to obtain Reaction Solution 2. Cool Reaction Solution 2 to solid with liquid nitrogen, displace the gas in the reaction tube with nitrogen, place the reaction tube at room temperature until the reaction solution melts, and repeat three times. Use a syringe to add N,N,N',N'',N''-pentamethyldiethylenetriamine to the reaction tube, react for 12 h, then dissolve the product in dichloromethane, filter it with a needle filter head, drop it into an ether solution, stir at room temperature for 1 h, remove the upper ether solution, collect the precipitate and dry it in a vacuum drying oven to obtain Compound 5.

[0080] The reaction formula is as follows:

[0081]

[0082] S5. In a single-neck round-bottom flask with a magnetic stir bar, dissolve Compound 5 in N,N-dimethylformamide. Add triethylamine, arginine (Arg), and D-(+)-glucosamine hydrochloride to it. The molar ratio of triethylamine, arginine, and D-(+)-glucosamine hydrochloride is 1:50:50, and react for 48 h. Take the supernatant and dialyze it for 24 h using a dialysis bag with a molecular weight cut-off of 2000 until N,N-dimethylformamide is removed. Freeze-dry all the liquid using a freeze dryer to obtain Compound 6-arginine. The reaction formula is as follows:

[0083]

[0084] Example 2

[0085] The difference from Example 1 is that the amino acid in step S5 is cysteine (Cys), and the others are the same as in Example 1, to obtain Compound 6-cysteine. The reaction formula is as follows:

[0086]

[0087] Example 3

[0088] The difference from Example 1 is that the amino acid in step S5 is leucine (Leu), and the others are the same as in Example 1, to obtain Compound 6-leucine. The reaction formula is as follows:

[0089]

[0090] Example 4

[0091] The difference from Example 1 is that the amino acid in step S5 is isoleucine (Ile), and the others are the same as in Example 1, to obtain Compound 6-isoleucine. The reaction formula is as follows:

[0092]

[0093] Example 5

[0094] The difference from Example 1 is that the amino acid in step S5 is valine (Val), and the others are the same as in Example 1, to obtain Compound 6-valine. The reaction formula is as follows:

[0095]

[0096] Characterization experiment

[0097] Perform nuclear magnetic resonance detection on Compound 2, Compound 3, Compound 4, Compound 5 prepared in Example 1, Compound 6-leucine of Example 3, and Compound 6-valine of Example 5. The results are as Figures 1-6 shown. FromFigure 1 - to Figure 6 From the 1H NMR spectra, characteristic proton signals identical to those of Compound 2, Compound 3, Compound 4, Compound 5 of Example 1, Compound 6-leucine of Example 3, and Compound 6-valine of Example 5 can be seen. Thus, it can be known that the above compounds were successfully prepared in Example 1, Example 3, and Example 5.

[0098] Figure 7 This is the CPC data chart of Compound 6-leucine prepared in Example 3 of the present invention. From Figure 7 it can be seen that the molecular weight of the prepared polymer is MW = 4917 g / mol, further indicating that Compound 6-leucine was successfully prepared in Example 3.

[0099] Performance Test

[0100] 4T1, Hepg-2, and CT26 cells were seeded in cell culture dishes and incubated in complete DMEM (10% FBS) under standard conditions (37 °C, 5% CO2) for 24 h. Then, amino acid nanoparticles coated with a fluorescent dye were added to fresh DMEM and incubated in the culture dishes for 8 h. Hoechst was added for nuclear staining. The culture medium was removed, and the cells were washed twice with PBS buffer to remove residual nanoparticles. 1 mL of fresh DMEM was added. Finally, the cells were imaged using a fluorescence confocal microscope.

[0101] Through confocal imaging technology, the uptake of amino acid nanoparticles by 4T1, Hepg-2, and CT26 cells was detected. From Figure 8 it can be seen that red fluorescence can be observed in all cells, indicating that they can all be taken up by the cells. At the same time, compared with the F127 nanomaterial, the amino acid material has stronger fluorescence intensity and higher cell uptake rate.

[0102] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A biological small molecule cluster type nano-coating agent material, characterized in that: Using dopamine hydrochloride as the main body, grafting long carbon chains, concatenating amino acids and sugar molecules on the side chain, the structural formula is as follows, In the formula, R1 is an alkyl group of C1 to C 30 , R2 is one or more of the following structural formulas, n is the degree of polymerization, and n is 50-100.

2. A preparation method of the biomacromolecule cluster-type nano-coating agent material as described in claim 1, characterized in that: It includes the following steps, S1. Dissolve dopamine hydrochloride, potassium carbonate and di-tert-butyl dicarbonate in N,N-dimethylformamide, stir and react to obtain Compound 1, then add potassium carbonate and octadecyl bromide to Compound 1, and carry out a condensation reflux reaction to obtain Compound 2; S2. Dissolve Compound 2 and trifluoroacetic acid in dichloromethane, react at room temperature to obtain Reaction Solution 1, add saturated sodium bicarbonate aqueous solution to Reaction Solution 1, and stir and react to obtain Compound 3; S3. Dissolve Compound 3 in dichloromethane, first add triethylamine for stirring reaction, and then add dibromo isobutyryl bromide to react at room temperature to obtain Compound 4; S4. Dissolve Compound 4 in anisole, add CuBr and N-acryloxysuccinimide to react to obtain Reaction Solution 2, and add N,N,N',N'',N''-pentamethyldiethylenetriamine to Reaction Solution 2 to obtain Compound 5; S5. Dissolve Compound 5 in N,N-dimethylformamide, add triethylamine, amino acids and sugar molecules to react to obtain Compound 6, that is, the biomolecular cluster-type nano-coating agent material.

3. The preparation method of the biomacromolecular cluster-type nano-coating agent material according to claim 2, characterized in that: In S1, the molar ratio of dopamine hydrochloride, potassium carbonate and di-tert-butyl dicarbonate is 1:1:1.5-2, and the molar ratio of Compound 1 to potassium carbonate and octadecyl bromide is 1:1:2-3.

4. The preparation method of the biomacromolecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S1, the stirring reaction is carried out at room temperature for 4 h, and the time of the condensation reflux reaction is 12 h.

5. The preparation method of the biomolecular cluster-type nano-coating agent material according to claim 2, wherein: In S2, the molar ratio of Compound 2 to trifluoroacetic acid is 1:1.5; In S2, the reaction time at room temperature is 12 h, and the stirring reaction time is 2 h.

6. The preparation method of the biomacromolecule cluster type nano-coating agent material according to claim 2, characterized in that: In S3, the molar ratio of Compound 3 to triethylamine and dibromo isobutyryl bromide is 1:1:2-3; In S3, the reaction temperature of the stirring reaction is room temperature, the reaction time is 0.5 h, and the reaction time at room temperature is 4-6 h.

7. The preparation method of the biomacromolecular cluster-type nano-coating agent material according to claim 2, characterized in that: In S4, the molar ratio of Compound 4 to N-acryloxysuccinimide is 1:50-100.

8. The preparation method of the biomolecular small cluster type nano encapsulant material according to claim 2, characterized in that: In S4, Reaction Solution 2 is cooled to a solid with liquid nitrogen, the gas in the reaction atmosphere is replaced with nitrogen, and then placed at room temperature until the solid melts, and this is repeated three times.

9. The preparation method of the biomacromolecular cluster-type nano-coating agent material according to claim 2, wherein: In S5, the molar ratio of Compound 5 to triethylamine, amino acids and sugar molecules is 1:50:

50.

10. The preparation method of the biomacromolecular cluster-type nano-coating agent material according to claim 2, wherein: The amino acids in S5 include one or more of arginine, cysteine, leucine, isoleucine, and valine; the sugar molecules include D-(+)-glucosamine hydrochloride.

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