A biological small molecule cluster type nano-coating agent material and a preparation method thereof

Abstract

The present application relates to the technical field of nanomedicine delivery, in particular to a biological small molecule cluster type nanocoating agent material and a preparation method thereof, wherein the biological small molecule cluster type nanocoating agent material takes dopamine hydrochloride as a main body, and is side-linked with long carbon chains, amino acids and saccharide molecules. The present application realizes active targeting of tumors through high demand and high uptake characteristics of amino acids and saccharide substances in the proliferation process of tumor cells, can accurately deliver drugs to the lesion area of tumors, significantly improves the accuracy of targeting, and effectively overcomes the problem of inaccurate targeting of traditional nanocarriers.

Description

Technical Field

[0001] This invention relates to the field of nanomedicine delivery technology, and in particular to a biomolecule cluster-type nanocoating material and its preparation method. Background Technology

[0002] In cancer treatment, nanocarrier delivery of nanomedicines has become a cutting-edge and highly promising therapeutic approach. Due to their uniquely small size, typically between 1 and 1000 nm, nanocarriers can successfully penetrate physiological barriers, such as the abnormal blood vessel walls surrounding tumor tissue, enabling precise targeting of cancer cells and overcoming the challenge of cancer treatment.

[0003] Traditional nanocarriers, such as distearylphosphatidylethanolamine-polyethylene glycol (DSP-PEG) and Pluronic F127, primarily rely on the enhanced penetration and retention (EPR) effect to guide the enrichment of loaded drugs in the tumor region, optimize drug distribution in vivo, and increase drug concentration at the tumor site, significantly improving therapeutic efficacy compared to traditional drug formulations. However, with further research and clinical practice, the EPR effect also has limitations in tumor targeting, such as low targeting precision and potentially significant toxic side effects.

[0004] In contrast, active targeting has demonstrated unique advantages in cancer treatment. Tumor cells exhibit a significantly increased demand for amino acids and carbohydrates during abnormal proliferation and metabolism. This is because amino acids are essential raw materials for tumor cells to synthesize proteins, nucleic acids, and other biomolecules, while carbohydrates provide energy for rapid tumor cell proliferation and form biomembranes. Based on this characteristic, amino acid cluster-type nanocarriers have emerged. By integrating amino acid and carbohydrate molecules into nanocarriers and utilizing the high uptake mechanism of these substances by tumor cells, the nanocarriers are actively enriched within tumor cells, thereby achieving active targeting of the tumor. This provides a new approach and method for improving cancer treatment efficacy and has broad application prospects. Summary of the Invention

[0005] The purpose of this invention is to provide a biological small molecule cluster-type nano-coating material and its preparation method. The provided nano-coating material utilizes the high demand for amino acids and sugars in the abnormal process of tumors to achieve a targeting effect, while enhancing its water solubility to achieve good drug encapsulation and delivery effects, thus solving the problems of low targeting accuracy and large toxic side effects of traditional nanocarriers.

[0006] To achieve the above objectives, this invention provides a bio-small molecule cluster-type nano-coating material, with dopamine hydrochloride as the main component, and long carbon chains branched on the sides and amino acids and sugar molecules linked in series, as shown in the following structural formula.

[0007]

[0008] In the formula, R1 represents C1 to C2. 30 The alkyl group, R2 is one or more of the following structural formulas.

[0009]

[0010] n represents the degree of polymerization, which ranges from 50 to 100.

[0011] This invention also provides a method for preparing the above-mentioned biomolecule cluster-type nano-coating material, comprising the following steps:

[0012] S1. Dopamine hydrochloride, potassium carbonate and ditert-butyl dicarbonate (BOC anhydride) are dissolved in N,N-dimethylformamide and stirred to react to obtain compound 1. Potassium carbonate and bromooctadecane are then added to compound 1 and refluxed to obtain compound 2.

[0013] S2. Compound 2 and trifluoroacetic acid are dissolved in dichloromethane and reacted at room temperature to obtain reaction solution one. Saturated sodium hydrocarbon aqueous solution is added to reaction solution one and the reaction is stirred to obtain compound 3.

[0014] S3. Compound 3 was dissolved in dichloromethane, triethylamine was added first and the mixture was stirred and reacted, and then dibromoisobutyryl bromide was added and reacted at room temperature to obtain compound 4.

[0015] S4. Compound 4 was dissolved in anisole, and CuBr and N-acryloyloxysuccinimide were added to react and give reaction solution II. N,N,N',N”,N”-pentamethyldivinyltriamine was added to reaction solution II to give compound 5.

[0016] S5. Compound 5 is dissolved in N,N-dimethylformamide, and triethylamine, amino acids and sugar molecules are added to react and obtain compound 6, which is a bio-small molecule cluster type nano-coating material.

[0017] Preferably, the molar ratio of dopamine hydrochloride, potassium carbonate and ditert-butyl dicarbonate in S1 is 1:1:1.5 to 2, and the molar ratio of compound 1 to potassium carbonate and octadecane bromo is 1:1:2 to 3.

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

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

[0020] The reaction time in S2 at room temperature is 12 hours, and the reaction time with stirring is 2 hours.

[0021] Preferably, the molar ratio of compound 3 in S3 to triethylamine and dibromoisobutyryl bromide is 1:1:2-3;

[0022] The reaction temperature for the stirred reaction in S3 is room temperature, and the reaction time is 0.5 h. The reaction time for the room temperature reaction is 4 to 6 h.

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

[0024] Preferably, in step S4, reaction liquid two is cooled to a solid state using liquid nitrogen, the gas in the reaction atmosphere is replaced with nitrogen, and then placed at room temperature until the solid melts. This process is repeated three times.

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

[0026] Preferably, the amino acids in S5 include one or more of arginine, cysteine, leucine, isoleucine, and valine; and the carbohydrate molecule D-(+)-glucosamine hydrochloride.

[0027] The beneficial effects of this invention are:

[0028] (1) This invention achieves active targeting of tumors by taking advantage of the high demand and high uptake of amino acids and sugars by tumor cells during proliferation. It can deliver drugs precisely to the lesion area of ​​the tumor, significantly improving the accuracy of targeting and effectively overcoming the problem of inaccurate targeting of traditional nanocarriers.

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

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

[0031] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0032] Figure 1 This is the 1H NMR spectrum of compound 2 obtained in Example 1 of this invention;

[0033] Figure 2 This is the 1H NMR spectrum of compound 3 obtained in Example 1 of this invention;

[0034] Figure 3 This is the 1H NMR spectrum of compound 4 obtained in Example 1 of this invention;

[0035] Figure 4 This is the 1H NMR spectrum of compound 5 obtained in Example 1 of this invention;

[0036] Figure 5 This is the 1H NMR spectrum of compound 6-leucine from Example 3 of this invention;

[0037] Figure 6 This is the 1H NMR spectrum of compound 6-valine from Example 5 of this invention;

[0038] Figure 7 This is a CPC data graph of the compound 6-leucine obtained in Example 3 of the present invention;

[0039] Figure 8 These are confocal images of the products obtained in Examples 1-5 of the present invention at different time points in cells and in vivo. Detailed Implementation

[0040] The present invention will be further described below with reference to the accompanying drawings and embodiments. Unless otherwise defined, the technical or scientific terms used in this invention should be understood in their ordinary sense by those skilled in the art. The features mentioned above or in the specific examples mentioned in this invention can be combined arbitrarily, and these specific embodiments are only used to illustrate the invention and are not intended to limit the scope of the invention.

[0041] This invention provides a bio-based small molecule cluster-type nano-coating material, with dopamine hydrochloride as the main component, and long carbon chains branched on the sides and amino acids and sugar molecules linked in series, as shown in the following structural formula.

[0042]

[0043] In the formula, R1 represents C1 to C2. 30 The alkyl group, R2 is one or more of the following structural formulas.

[0044]

[0045] n represents the degree of polymerization, which ranges from 50 to 100.

[0046] This invention also provides a method for preparing the above-mentioned biomolecule cluster-type nano-coating material, comprising the following steps:

[0047] S1. Dopamine hydrochloride, potassium carbonate and ditert-butyl dicarbonate are dissolved in N,N-dimethylformamide and stirred to react to obtain compound 1. Potassium carbonate and bromooctadecane are then added to compound 1 and refluxed to obtain compound 2.

[0048] S2. Compound 2 and trifluoroacetic acid are dissolved in dichloromethane and reacted at room temperature to obtain reaction solution one. Saturated sodium hydrocarbon aqueous solution is added to reaction solution one and the reaction is stirred to obtain compound 3.

[0049] S3. Compound 3 was dissolved in dichloromethane, triethylamine was added first and the mixture was stirred and reacted, and then dibromoisobutyryl bromide was added and reacted at room temperature to obtain compound 4.

[0050] S4. Compound 4 was dissolved in anisole, and CuBr and N-acryloyloxysuccinimide were added to react and give reaction solution II. N,N,N',N”,N”-pentamethyldivinyltriamine was added to reaction solution II to give compound 5.

[0051] S5. Compound 5 is dissolved in N,N-dimethylformamide, and triethylamine, amino acids and sugar molecules are added to react and obtain compound 6, which is a bio-small molecule cluster type nano-coating material.

[0052] In some embodiments of the present invention, the synthetic route for preparing bio-small molecule cluster-type nano-coating agent materials is as follows:

[0053]

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

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

[0056] In some embodiments of the present invention, after the 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 a white solid compound 2. The present invention does not limit the amount of dichloromethane and anhydrous sodium sulfate; 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 in S2 at room temperature is 12 hours, and the reaction time with stirring is 2 hours.

[0059] In some embodiments of the present invention, S2 saturated sodium hydrocarbon aqueous solution is added dropwise to reaction solution one.

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

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

[0062] The reaction temperature for the stirred reaction in S3 is room temperature, and the reaction time is 0.5 h. The reaction time for the room temperature reaction is 4 to 6 h.

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

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

[0065] Preferably, in step S4, reaction liquid two is cooled to a solid state using liquid nitrogen, the gas in the reaction atmosphere is replaced with nitrogen, and then placed at room temperature until the solid melts. This process is repeated three times.

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

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

[0068] Preferably, the amino acids in S5 include one or more of arginine, cysteine, leucine, isoleucine, and valine; the carbohydrate molecules include D-(+)-glucosamine hydrochloride.

[0069] In some embodiments of the present invention, after the reaction in step S5 is completed, the supernatant is taken and dialyzed for 24 hours using a dialysis bag with a molecular weight of 2000 until N,N-dimethylformamide is removed. Then, all liquids are freeze-dried using a freeze dryer to obtain compound 6, namely, a bio-small molecule cluster type nano-coating material.

[0070] Example 1

[0071] This invention provides a method for preparing bio-small molecule cluster-type nano-coating materials, comprising the following steps:

[0072] S1. At room temperature, in a single-necked round-bottom flask equipped with a magnetic stirrup, 3 g of dopamine hydrochloride was dissolved in N,N-dimethylformamide. Then, 2.18 g of potassium carbonate and 3.5 g of BOC anhydride were added, and the mixture was reacted at room temperature for 4 h to obtain compound 1. 2.18 g of potassium carbonate and 15 g of bromooctadecane were added to compound 1, and the mixture was refluxed overnight. The resulting mixture was extracted with dichloromethane, and the organic layer was collected and dried over anhydrous sodium sulfate. The crude product was purified by silica gel chromatography to give a white solid, compound 2.

[0073] The reaction formula is as follows:

[0074]

[0075] S2. In a single-necked round-bottom flask equipped with a magnetic stirrup, 1 g of compound 2 obtained in S1 was dissolved in 10 mL of dichloromethane. 0.3 g of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 12 h to obtain reaction solution one. Then, 200 mL of saturated sodium hydrochloride aqueous solution was added dropwise to reaction solution one, and the mixture was stirred for 2 h. After filtration and extraction with dichloromethane solution, the mixture was finally rotary evaporated to obtain a white solid compound 3. The reaction formula is as follows:

[0076]

[0077] S3. In a single-necked round-bottom flask equipped with a magnetic stir bar, 0.5 g of compound 3 obtained in S2 was dissolved in 5 mL of dichloromethane. First, 0.12 g of triethylamine was added, and the mixture was stirred at room temperature for 0.5 h. Then, 0.5 g of dibromoisobutyryl bromide was added, and the reaction was carried out at room temperature for 4-6 h. The product was extracted with dichloromethane and water, dried with anhydrous sodium sulfate, purified by silica gel chromatography, and finally dried and concentrated the organic layer to obtain a pale yellow oily compound 4. The reaction formula is as follows:

[0078]

[0079] S4. In a polymerization reaction tube equipped with a magnetic stir bar, 100 mg of compound 4 obtained in S3 was dissolved in anisole. 2 mg of cuprous bromide and 1 g of N-acryloyloxysuccinimide were added to the solution to obtain reaction solution II. Reaction solution II was cooled to a solid state using liquid nitrogen. The gas in the reaction tube was replaced with nitrogen, and the reaction tube was placed at room temperature until the reaction solution melted. This process was repeated three times. N,N,N',N”,N”-pentamethyldivinyltriamine was added to the reaction tube using a syringe, and the reaction was allowed to proceed for 12 h. The product was then dissolved in dichloromethane, filtered using a needle filter, and added dropwise to an ether solution. The mixture was stirred at room temperature for 1 h, the upper layer of ether solution was removed, the precipitate was collected, and dried in a vacuum drying oven to obtain compound 5.

[0080] The reaction formula is as follows:

[0081]

[0082] S5. In a single-necked round-bottom flask equipped with a magnetic flask, compound 5 was dissolved in N,N-dimethylformamide. Triethylamine, arginine (Arg), and D-(+)-glucosamine hydrochloride were added to the solution in a molar ratio of 1:50:50. The reaction was allowed to proceed for 48 h. The supernatant was collected and dialyzed against a dialysis bag with a molecular weight of 2000 for 24 h until N,N-dimethylformamide was removed. All liquids were then lyophilized 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), otherwise the same as in Example 1, yielding 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), otherwise the same as in Example 1, yielding 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), otherwise it is the same as in Example 1, yielding 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), otherwise the same as in Example 1, yielding compound 6-valine. The reaction formula is as follows:

[0095]

[0096] Characterization experiment

[0097] The compounds 2, 3, 4, and 5 obtained in Example 1, compound 6-leucine from Example 3, and compound 6-valine from Example 5 were subjected to nuclear magnetic resonance (NMR) detection. The results are as follows: Figures 1-6 As shown, from Figure 1 -to Figure 6 The proton NMR spectra show characteristic proton signals that are identical to those of compounds 2, 3, 4, and 5 in Example 1, compound 6-leucine in Example 3, and compound 6-valine in Example 5. Therefore, it can be concluded that the above compounds were successfully prepared in Examples 1, 3, and 5.

[0098] Figure 7 This is a CPC data graph of the compound 6-leucine obtained in Example 3 of the present invention. Figure 7 It can be seen that the molecular weight of the obtained polymer is MW = 4917 g / mol, which further demonstrates that the compound 6-leucine was successfully obtained in Example 3.

[0099] Performance testing

[0100] 4T1, Hepg-2, and CT26 cells were seeded in cell culture dishes and incubated for 24 h in complete DMEM (10% FBS) under standard conditions (37°C, 5% CO2). Then, amino acid nanoparticles coated with fluorescent dye were added to fresh DMEM and incubated for 8 h. Hoschst was then 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 then added. Finally, the cells were imaged using a fluorescence confocal microscope.

[0101] The uptake of amino acid nanoparticles by 4T1, Hepg-2, and CT26 cells was detected using confocal imaging technology. Figure 8 As can be seen, red fluorescence was observed in all cells, indicating that the amino acid materials could be taken up by the cells. Furthermore, compared to F127 nanomaterials, the amino acid materials exhibited stronger fluorescence intensity and a higher cellular uptake rate.

[0102] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A bio-small molecule cluster-type nano-coating agent material, characterized in that: Based on dopamine hydrochloride, with side-linked carbon chains and linked amino acids or sugar molecules, the structural formula is as follows. In the formula, R1 represents C1~C 30 The alkyl group, R2 is one or more of the following structural formulas. ； n represents the degree of polymerization, which ranges from 50 to 100. Indicates the connection site.

2. A method for preparing a bio-small molecule cluster-type nano-coating agent material as described in claim 1, characterized in that: Includes the following steps, S1. Dopamine hydrochloride, potassium carbonate and ditert-butyl dicarbonate are dissolved in N,N-dimethylformamide and stirred to react to obtain compound 1. Potassium carbonate and bromooctadecane are then added to compound 1 and refluxed to obtain compound 2. S2. Compound 2 and trifluoroacetic acid are dissolved in dichloromethane and reacted at room temperature to obtain reaction solution one. Saturated sodium bicarbonate aqueous solution is added to reaction solution one and the reaction is stirred to obtain compound 3. S3. Compound 3 was dissolved in dichloromethane, triethylamine was added first and the mixture was stirred and reacted, and then dibromoisobutyryl bromide was added and reacted at room temperature to obtain compound 4. S4. Compound 4 was dissolved in anisole, and CuBr and N-acryloyloxysuccinimide were added to react and give reaction solution II. N,N,N',N'',N''-pentamethyldivinyltriamine was added to reaction solution II to give compound 5. S5. Compound 5 is dissolved in N,N-dimethylformamide, and triethylamine, amino acids or sugar molecules are added to react and obtain compound 6, which is a bio-small molecule cluster type nano-coating material.

3. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S1, the molar ratio of dopamine hydrochloride, potassium carbonate, and ditert-butyl dicarbonate is 1:1:1.5~2, and the molar ratio of compound 1 to potassium carbonate and octadecane bromo is 1:1:2~3.

4. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: The stirring reaction in S1 was carried out at room temperature for 4 hours, and the reflux reaction was carried out for 12 hours.

5. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S2, the molar ratio of compound 2 to trifluoroacetic acid is 1:1.5; The reaction time in S2 at room temperature is 12 hours, and the reaction time with stirring is 2 hours.

6. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S3, the molar ratio of compound 3 to triethylamine and dibromoisobutyryl bromide is 1:1:2~3; The reaction temperature for the stirred reaction in S3 is room temperature, and the reaction time is 0.5 h. The reaction time for the room temperature reaction is 4 to 6 h.

7. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S4, the molar ratio of compound 4 to N-acryloyloxysuccinimide is 1:50~100.

8. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S4, the reaction liquid 2 is cooled to solid using liquid nitrogen, the gas in the reaction atmosphere is replaced with nitrogen, and then placed at room temperature until the solid melts. This process is repeated three times.

9. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: In S5, the molar ratio of compound 5 to amino acid or sugar molecules and triethylamine is 1:50:

50.

10. The method for preparing the bio-small molecule cluster-type nano-coating agent material according to claim 2, characterized in that: The amino acids in S5 include one or more of arginine, cysteine, leucine, isoleucine, and valine; the carbohydrate molecules include D-(+)-glucosamine hydrochloride.

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