Doxorubicin molecular imprinting hydrogel and preparation method thereof

Abstract

The invention relates to the technical field of molecular imprinting and biological medicine, in particular to doxorubicin molecular imprinting hydrogel and a preparation method thereof, and the doxorubicin molecular imprinting hydrogel is prepared by taking DOX as a template and adopting N-isopropylacrylamide and N-[3-(dimethylamino) propyl] methacrylamide as bifunctional monomers through free radical polymerization. According to the method, efficient and specific separation of DOX in a complex biological system is achieved through the functional monomer ratio, the cross-linking agent concentration and the solution pH value, and the defects in the prior art are overcome.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of molecular imprinting and biological medicine, in particular to a doxorubicin molecular imprinting hydrogel and a preparation method thereof. BACKGROUND

[0002] Doxorubicin (DOX) is an important broad-spectrum antitumor drug, but in complex biological systems, its separation and purification face challenges such as complex environment, low drug concentration, and many interfering substances. Traditional separation methods cannot meet the needs of high efficiency and specificity, which restricts the efficacy and safety of the drug.

[0003] Although molecular imprinting technology (MIT) can construct specific recognition sites, it has low imprinting efficiency and poor stability of recognition sites when dealing with water-soluble molecules such as DOX, making it difficult to achieve efficient separation in complex biological systems.

[0004] Molecular imprinting hydrogels (MIHs) improve separation performance through three-dimensional network structure and stimulus response characteristics, but existing research has not fully considered the adsorption efficiency and specificity of DOX in the selection of functional monomers and cross-linking design, especially in complex interference environments.

[0005] Currently, there is no research in the prior art that applies both temperature-sensitive and pH-responsive functional monomers to a DOX molecular imprinting hydrogel system. SUMMARY

[0006] The technical problem to be solved by the present application is to provide a doxorubicin molecular imprinting hydrogel and a preparation method thereof, which realizes efficient and specific separation of DOX in complex biological systems through the ratio of functional monomers, the concentration of cross-linking agent, and the pH value of the solution, filling the gap in the prior art.

[0007] To solve the above technical problems, the present application adopts the following technical solutions: A doxorubicin molecular imprinting hydrogel, using DOX as a template, using N-isopropyl acrylamide and N-[3-(dimethylamino)propyl]methacrylamide as bifunctional monomers, and being prepared by free radical polymerization.

[0008] Preferably, the free radical polymerization reaction also includes a cross-linking agent, and the cross-linking agent is N,N'-methylenebisacrylamide.

[0009] The preparation method of the doxorubicin molecular imprinting hydrogel according to any one of the preceding items, comprising the following steps: S1, dissolving N-isopropyl acrylamide and N,N'-methylenebisacrylamide in pure water, then adding N-[3-(dimethylamino)propyl]methacrylamide to obtain a mixed solution; S2, to the mixed solution obtained in S1, 1 mol / L hydrochloric acid was added to adjust the pH of the mixed solution to 7.0, and then a DOX solution was added; S3, nitrogen was bubbled into the solution of S2 for 10 minutes to deoxygenate the mixture, and then ammonium persulfate and tetramethyl ethylenediamine were immediately added, and the reaction was carried out under nitrogen sealing at room temperature for 2 hours to obtain a DOX molecularly imprinted hydrogel crude body; S4, DOX removal: the DOX molecularly imprinted hydrogel crude body prepared in step S3 was first eluted with 10% (v / v) acetic acid multiple times, and then repeatedly washed with pure water to obtain a DOX molecularly imprinted hydrogel.

[0010] Preferably, the molar ratio of N-isopropyl acrylamide, N-[3-(dimethylamino) propyl] methyl acrylamide and N,N'-methylene bisacrylamide in step S1 is 200:20~1:10.

[0011] Preferably, the molar ratio of N-isopropyl acrylamide, N-[3-(dimethylamino) propyl] methyl acrylamide and N,N'-methylene bisacrylamide in step S1 is 200:20~1:100.

[0012] Preferably, the concentration of the DOX solution in step S2 is 1.6 mg / mL.

[0013] Preferably, the amount of ammonium persulfate used in step S3 is 500 μL at a concentration of 6% (w / v).

[0014] Preferably, the amount of tetramethyl ethylenediamine added in step S3 is 15 μL at a concentration of 99%.

[0015] The doxorubicin molecularly imprinted hydrogel according to any one of the above is applied in a drug controlled release system.

[0016] The beneficial effects of the present application are: The present application fills the gap in the prior art by using DOX as a template, using N-isopropyl acrylamide (NIPAM) and N-[3-(dimethylamino) propyl] methyl acrylamide (DMAPMA) as bifunctional monomers, and preparing a molecularly imprinted hydrogel by free radical polymerization. Through the ratio of functional monomers, the concentration of crosslinking agent and the pH value of the solution, efficient and specific separation of DOX in complex biological systems is achieved, filling the gap in the prior art.

[0017] The present application adopts N-isopropyl acrylamide (NIPAM) as a temperature-sensitive functional monomer, N-[3-(dimethylamino)propyl] methacrylamide (DMAPMA) as a pH-responsive functional monomer, and N,N'-methylene bisacrylamide (BIS) as a crosslinking agent to prepare a double-stimulus-responsive molecular imprinting hydrogel. NIPAM has temperature-sensitive properties and can undergo reversible phase transition at a specific temperature, so that the hydrogel exhibits different swelling states in different temperature environments, thereby providing the hydrogel with intelligent response characteristics and helping to realize selective adsorption and release of doxorubicin under different temperature conditions; DMAPMA contains an amino group and can produce hydrogen bonding, electrostatic interaction and the like with specific functional groups in the doxorubicin molecule, thereby enhancing the specific recognition ability of the hydrogel to doxorubicin; the two functional monomers provide specific recognition sites for the doxorubicin molecule and synergize in a specific molar ratio, and the imprinting factor is as high as 2.26, indicating that the molecular imprinting hydrogel has high specific recognition ability to doxorubicin.

[0018] The present application overcomes the defects of low molecular imprinting efficiency and unstable recognition sites of water-soluble molecular imprinting in the prior art. In the present application, the molar ratio of each component is optimized to significantly improve the stability of the material. The molar ratio of N-isopropyl acrylamide, N-[3-(dimethylamino)propyl] methacrylamide and N,N'-methylene bisacrylamide plays a key role in the stability of the final material. The present application selects the molar ratio of N-isopropyl acrylamide, N-[3-(dimethylamino)propyl] methacrylamide and N,N'-methylene bisacrylamide in the range of 200:20-1:10 and 200:20-1:100. Under this specific molar ratio condition, the tertiary amine group of DMAPMA and the hydroxyl group / amino group of doxorubicin can form directional hydrogen bonding, and the isopropyl group of NIPAM can enhance the binding force through hydrophobic interaction, and the two can synergistically construct a three-dimensional complementary cavity. In addition, the amount of the crosslinking agent N,N'-methylene bisacrylamide is adjusted to ensure the specific shape of the imprinting cavity and the structural stability.

[0019] In the present application, N-[3-(dimethylamino)propyl] methacrylamide is used as a pH-responsive functional monomer, and its charge state is easily affected by the pH environment and changes, thereby affecting the interaction with DOX. In order to further improve the selectivity of the molecular imprinting hydrogel, the present application has carried out research and test on the pH condition. The results show that for epirubicin which has extremely similar structure, the selectivity factor of the molecular imprinting hydrogel prepared by the present application can reach 1.97, thereby significantly improving the selective recognition ability to DOX.

[0020] According to actual test data, the hydrogel has good temperature-sensitive performance in the temperature range of 25-90 DEG C, and the swelling rate changes significantly with temperature, which provides the possibility for its application in drug controlled release system. In addition, the prepared doxorubicin molecular imprinting hydrogel has good mechanical stability and biocompatibility, and is expected to have wide application prospect in the field of biomedicine, especially in drug delivery and separation technology. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The scanning electron microscope image of the cross section of the molecular imprinting hydrogel of example 2 of the present application; Figure 2 The infrared spectrum of the molecular imprinting hydrogel of example 7 of the present application and the non-molecular imprinting hydrogel of comparative example 1; wherein DOX-MIH7 is the molecular imprinting hydrogel of example 7 which is not completely eluted; Figure 3 The thermogravimetric analysis curve of the molecular imprinting hydrogel of example 7 of the present application and the non-molecular imprinting hydrogel of comparative example 1; Figure 4 The swelling ratio of the molecular imprinting hydrogels of examples 1-10 of the present application under different proportions; Figure 5 The influence of pH on the selective adsorption of the molecular imprinting hydrogels of examples 2 and 7 of the present application; Figure 6 The imprinting factor evaluation of the molecular imprinting hydrogels of examples 1-10 of the present application. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of those skilled in the art, the present application is further described below in combination with examples and drawings, and the content mentioned in the embodiments is not a limitation of the present application. EXAMPLE

[0023] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropylacrylamide (NIPAM) and 0.02 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, then 32 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, then 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes, oxygen was removed, 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove the residual acid, and doxorubicin (DOX) molecularly imprinted hydrogel, namely MIH1, was obtained. Example

[0024] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropylacrylamide (NIPAM) and 0.02 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, then 32 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, then 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes, oxygen was removed, 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove the residual acid, and doxorubicin (DOX) molecularly imprinted hydrogel, namely MIH1, was obtained. Example

[0025] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropylacrylamide (NIPAM) and 0.02 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, then 32 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, then 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes, oxygen was removed, 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove the residual acid, and doxorubicin (DOX) molecularly imprinted hydrogel, namely MIH1, was obtained. Example

[0026] Synthesis of the molecularly imprinted hydrogel: 0.2260 g of N-isopropylacrylamide (NIPAM) and 0.02 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water to obtain a monomer solution of 9 mL. The pH was adjusted to 7 using hydrochloric acid, and 1 mL of a 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes to remove oxygen, and 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out under nitrogen at room temperature for 2 hours. The prepared molecularly imprinted hydrogel was eluted several times with 10% (v / v) acetic acid. Then, it was repeatedly washed with pure water to remove residual acid, and a doxorubicin molecularly imprinted hydrogel, MIH4, was obtained. Example

[0027] Synthesis of the molecularly imprinted hydrogel: 0.2260 g of N-isopropylacrylamide (NIPAM) and 0.02 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water to obtain a monomer solution of 9 mL. The pH was adjusted to 7 using hydrochloric acid, and 1 mL of a 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes to remove oxygen, and 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out under nitrogen at room temperature for 2 hours. The prepared molecularly imprinted hydrogel was eluted several times with 10% (v / v) acetic acid. Then, it was repeatedly washed with pure water to remove residual acid, and a doxorubicin molecularly imprinted hydrogel, MIH4, was obtained. Example

[0028] Synthesis of the molecularly imprinted hydrogel: 0.2260 g of N-isopropylacrylamide (NIPAM) and 0.02 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water to obtain a monomer solution of 9 mL. The pH was adjusted to 7 using hydrochloric acid, and 1 mL of a 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes to remove oxygen, and 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out under nitrogen at room temperature for 2 hours. The prepared molecularly imprinted hydrogel was eluted several times with 10% (v / v) acetic acid. Then, it was repeatedly washed with pure water to remove residual acid, and a doxorubicin molecularly imprinted hydrogel, MIH4, was obtained. Example

[0029] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropyl acrylamide (NIPAM) and 0.2 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, then 16 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, then 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes, oxygen was removed, 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove residual acid, and doxorubicin molecularly imprinted hydrogel, MIH7, was obtained. Example

[0030] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropyl acrylamide (NIPAM) and 0.2 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, then 16 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, then 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes, oxygen was removed, 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove residual acid, and doxorubicin molecularly imprinted hydrogel, MIH7, was obtained. Example

[0031] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropyl acrylamide (NIPAM) and 0.2 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, then 16 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, then 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes, oxygen was removed, 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added, and the reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove residual acid, and doxorubicin molecularly imprinted hydrogel, MIH7, was obtained. Example

[0032] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropyl acrylamide (NIPAM) and 0.2 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, and 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes to remove oxygen, and 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added. The reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove residual acid, and doxorubicin molecularly imprinted hydrogel, namely MIH10, was obtained.

[0033] Comparative Example 1 The same as Example 7, except that 1 mL of 1.6 mg / mL DOX solution was not added.

[0034] Synthesis of molecularly imprinted hydrogel: 0.2260 g of N-isopropyl acrylamide (NIPAM) and 0.2 g of N,N'-methylenebisacrylamide (BIS) were dissolved in pure water, and then 16 μL of N-[3-(dimethylamino)propyl]methacrylamide (DMAPMA) was added to obtain 9 mL of monomer solution. The pH was adjusted to 7 with hydrochloric acid, and 1 mL of 1.6 mg / mL DOX solution was added and mixed well. Nitrogen was bubbled for 10 minutes to remove oxygen, and 500 μL of 6% ammonium persulfate (APS) and 15 μL of tetramethyl ethylenediamine (TEMED) were quickly added. The reaction was carried out at room temperature under nitrogen for 2 hours. The prepared molecularly imprinted hydrogel was eluted with 10% (v / v) acetic acid for several times. Then, it was repeatedly washed with pure water to remove residual acid, and doxorubicin molecularly imprinted hydrogel, namely NIH7, was obtained.

[0035] Test Example 1 Study on temperature-sensitive properties of molecularly imprinted hydrogels with different ratios (i.e., molar ratios of N-isopropyl acrylamide, N-[3-(dimethylamino)propyl]methacrylamide, and N,N'-methylenebisacrylamide are 200:20~1:10 and 200:20~1:100): 0.02 g of molecularly imprinted hydrogel MIH7 was weighed, and the surface moisture was removed with filter paper, and then it was placed in a water bath at 25~90°C for heating for 5 minutes. The temperature was adjusted by a constant temperature water bath. After the precipitated water was removed, the mass of the hydrogel at this time was weighed. The swelling ratio (QT) changing with temperature was calculated by the formula as shown in the following formula (1). Figure 4

[0036] Test Example 2​ Adsorption of different proportions of molecularly imprinted hydrogels (Example 2 and Example 7) at different pH to doxorubicin (DOX) and epirubicin (EPI): 0.2 g of molecularly imprinted hydrogel was weighed in a 2 mL centrifuge tube. 1 mL of 2 μmol / L DOX solution or EPI solution with pH of 3, 4, 5, 6, 7 was added respectively, and adsorbed for 1 hour at 25 ℃, 150 rpm. Then, centrifuged at 10000 rpm for 5 minutes, and the supernatant was collected. The concentration of DOX in the supernatant was determined by fluorescence spectroscopy at λex=483 nm and λem=595 nm, and the adsorption of MIHs or NIHs to DOX was calculated according to Q=(C0-Ce)V) / m. As shown in Table 1. Figure 5

[0037] All the technical features in the present embodiment can be modified in appearance according to actual needs.

[0038] The above embodiments are the preferred implementation of the present application, in addition to this, the present application can be implemented in other ways, without departing from the technical solution concept of the present application, any obvious replacement within the protection scope of the present application.​

Claims

1. A doxorubicin molecularly imprinted hydrogel, characterized by: The nanostructured polymer was prepared by free radical polymerization using DOX as a template and N-isopropylacrylamide and N-[3-(dimethylamino)propyl]methacrylamide as bifunctional monomers.

2. The doxorubicin molecularly imprinted hydrogel according to claim 1, characterized in that: The free polymerization reaction also includes a cross-linking agent, which is N,N'-methylenebisacrylamide.

3. The method for preparing a doxorubicin molecularly imprinted hydrogel according to any one of claims 1 to 2, characterized in that: The following steps are involved: S1, dissolving N-isopropylacrylamide and N,N'-methylenebisacrylamide in pure water, and then adding N-[3-(dimethylamino)propyl]methacrylamide to obtain a mixed solution; S2, adding 1 mol / L hydrochloric acid to the mixed solution obtained in S1 to adjust the pH of the mixed solution to 7.0, and then adding the DOX solution; S3, nitrogen was bubbled into the solution of S2 for 10 minutes to deoxygenate the mixture, and then ammonium persulfate and tetramethylethylenediamine were immediately added. The mixture was sealed with nitrogen at room temperature for 2 hours to prepare a rough DOX molecularly imprinted hydrogel; S4, removal of DOX: The DOX molecularly imprinted hydrogel crude prepared in step S3 was first eluted with 10% (v / v) acetic acid multiple times, and then repeatedly washed with pure water to obtain the DOX molecularly imprinted hydrogel.

4. The method for preparing a doxorubicin molecularly imprinted hydrogel according to claim 3, wherein: In step S1, the molar ratio of N-isopropylacrylamide, N-[3-(dimethylamino)propyl]methacrylamide and N,N'-methylenebisacrylamide is 200:20 to 1:

10.

5. The method for preparing a doxorubicin molecularly imprinted hydrogel according to claim 3, wherein: In step S1, the molar ratio of N-isopropylacrylamide, N-[3-(dimethylamino)propyl]methacrylamide and N,N'-methylenebisacrylamide is 200:20 to 1:

100.

6. The method for preparing a doxorubicin molecularly imprinted hydrogel according to claim 3, wherein: The concentration of the DOX solution in step S2 was 1.6 mg / mL.

7. The method for preparing a doxorubicin molecularly imprinted hydrogel according to claim 3, wherein: In step S3, the ammonium persulfate is 6% (w / v) and the amount used is 500 μL.

8. The method for preparing a doxorubicin molecularly imprinted hydrogel according to claim 3, wherein: In step S3, the amount of tetramethylethylenediamine added is 15 μL, and the concentration is 99%.

9. Use of the doxorubicin molecularly imprinted hydrogel according to any one of claims 1-2 in a drug controlled release system.

Images