Group controllable and adjustable zwitterionic polymer, preparation method and application thereof

Abstract

A preparation method of a group controllable and adjustable zwitterionic polymer comprises the following specific steps: step 1, preparing PCBMA by using CBMA as a raw material, dissolving the PCBMA in deionized water, adding an EDC solution and an NHS solution, adjusting the pH value of the solution to 6.0-6.5, and reacting and activating at room temperature for 1-3 hours to obtain an activated PCBMA solution; and 2, dissolving an amide drug monomer in DMSO, adding the activated PCBMA solution, dropwise adding a NaOH solution, carrying out a stirring reaction at room temperature for 24-48 h, dialyzing the obtained product through a dialysis bag for 24-48 h, and carrying out freeze drying to obtain the amide zwitterionic polymer modified by the amide drug monomer. The pH value transition point of the surface of the nanoparticle interface material is regulated and controlled through a group regulation and control method, so that the targeting application requirements in different tumor phenotypes, different tumor growth stages and the interiors of tumors are met.

Description

Technical Field

[0001] The invention relates to the field of biomedical materials, and in particular to a group-controllable adjustable zwitterionic polymer, a preparation method and application thereof. Background Art

[0002] Nanoparticles have great potential in the treatment of diseases, inflammation and cancer, especially in the diagnosis and treatment of cancer. The acidic microenvironment pH range of the tumor site is about 6.0-6.8, and it has the characteristics of acid response, which provides an effective technical solution for the design of targeted nanomaterials. However, the phenotype, growth stage and internal acidity of different tumor cells are different and have the characteristics of dynamic change. Therefore, the development of interface materials that are responsive to different acidic environments of tumors plays an important role in improving the application ability of nanoparticles.

[0003] The current acidic environment responsive interface materials have only one transition point. In addition, the method of adjusting the pH transition point of the material has not yet been established. In order to meet the diagnosis and treatment needs of different tumor cells, we designed and modified groups to regulate the pH transition point on the surface of nanoparticle interface materials. We invented a method for group regulation of the transition point to adapt to the targeted application needs of different tumor cell phenotypes, growth stages and internal acidity. Summary of the invention

[0004] In order to overcome the above shortcomings, the present invention provides a preparation method of a group-controllable zwitterionic polymer and its application in the charge reversal pH transition point, and the group modification of PCBMA (polymer of zwitterionic monomer carboxylic acid betaine) is carried out through amide drug monomers to achieve the adjustment of pH response transition point and meet the functional requirements of slightly acidic environment with different pH levels in various pathological sites.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: A method for preparing a group-controllable zwitterionic polymer, the specific steps comprising: Step 1: Prepare PCBMA using CBMA as raw material, dissolve it in deionized water and add EDC solution and NHS solution, adjust the solution pH to 6.0-6.5, react and activate at room temperature for 1-3 hours to obtain an activated PCBMA solution; Step 2: dissolve the amide drug monomer in DMSO, add the activated PCBMA solution, dropwise add NaOH solution, stir and react at room temperature for 24-48 hours, dialyze the obtained product through a dialysis bag for 24-48 hours, and freeze-dry to obtain an amide drug monomer-modified amide zwitterionic polymer.

[0006] Further optimized, the preparation method of PCBMA in step 1 is: a. Ultrasonic dissolution of AIBN and DMF to obtain an AIBN solution, and ultrasonic dissolution of BSPA and DMF to obtain a BSPA solution; b. After CBMA is dissolved in deionized water, add the BSPA solution, AIBN solution and anhydrous DMF in step a and mix well. Vacuum at 0-5°C and introduce nitrogen for 5-7 times. After deoxygenation, react at 70-75°C for 24-48h. c. The solution after the deoxygenation reaction in step b is dialyzed against deionized water for 24-36 hours and freeze-dried to obtain PCBMA.

[0007] Further optimization, the volume ratio of deionized water to DMF is 1:1-1:5, the weight ratio of AIBN to BSPA is 1:2-1:4, and the weight ratio of CBMA to BSPA is 1:20-1:40.

[0008] Further optimized, the amide drug monomer is one of benzenesulfonamide, sulfacetamide, and sulfadoxine.

[0009] Further optimization, during the PCBMA activation process, the amounts of PCBMA, EDC solution, and NHS solution used were 0.05-0.1 mmol, 0.1-0.2 mmol, and 0.1-0.2 mmol, respectively.

[0010] Further optimization, the molecular weight MWCO of the dialysis bag used for PCBMA in step 2 is 2000-3500, and the volume ratio of DMSO to deionized water is 1:1-1:4.

[0011] A group-controllable and adjustable zwitterionic polymer prepared by the preparation method.

[0012] The invention discloses an application of group-controllable regulation of the charge reversal pH transition point of zwitterionic polymers.

[0013] Further optimization includes the following steps: reacting NaBH4 at room temperature for 2-4 hours under an inert gas atmosphere, adding QDs uniformly dispersed by ultrasound, continuing to react at room temperature for 24-36 hours under an inert gas atmosphere, then dialyzing in deionized water in the dark for 3-6 hours through a dialysis membrane, dissolving the dialyzwitterionic polymer-modified QDs in a buffer solution with a pH gradient of 7.2-6.0, and detecting the changes in surface Zeta potential under various pH environments using a laser particle size analyzer.

[0014] The beneficial effects of the present invention are: (if possible, the effect of realizing controllable charge reversal pH transition point based on group mechanism is supplemented) 1. The amide zwitterionic polymer synthesized by the present invention is an interface material, which plays the role of functionalized nanoparticles. The amide zwitterionic polymer performs surface modification on fluorescent nanomaterials. In the cell endocytosis experiment, the surface modified quantum dots achieve charge reversal in the pH range of 6.4-6.8, thereby improving cell uptake. 2. Ischemia, inflammation and cancer sites all present an acidic microenvironment with acid response in the range of pH 6.8-pH 6.0. With different pH microenvironments, their charge reversal pH response transition points can be adjusted by changing the groups to meet the multifunctional requirements of the above pathological sites; In summary, the present invention regulates the pH transition point on the surface of the nanoparticle interface material by means of a group regulation method to adapt to the targeted application requirements in different tumor phenotypes, different tumor growth stages and inside the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the reaction formula for the preparation of amide zwitterionic polymer PCBSs; Figure 2 This is the H NMR spectrum of the carboxyl zwitterionic polymer PCBMA; Figure 3 is the H NMR spectrum of carboxyl zwitterionic polymer PCBSA; Figure 4 This is the H-NMR spectrum of the amide zwitterionic polymer PCBSAC; Figure 5 This is the H-NMR spectrum of the amide zwitterionic polymer PCBSDOX; Figure 6 Zeta potential spectra of PCBSA, PCBSAC, and PCBSDOX surface-modified quantum dots QDs under different pH conditions; Figure 7 These are the images of liver cancer cells HepG2 taking red QDs modified on the surface of PCBSA, yellow QDs modified on the surface of PCBSAC, and green QDs modified on the surface of PCBSDOX. DETAILED DESCRIPTION

[0016] In order to more clearly understand the above-mentioned purposes, features and advantages of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The following embodiments are implemented based on the technical solutions of the present invention, and detailed implementation methods and specific operating procedures are given. However, the present invention can also be implemented in other ways different from those described herein. Therefore, the protection scope of the present invention is not limited to the following embodiments.

[0017] Example 1 A method for preparing a group-controllable zwitterionic polymer, the specific steps are as follows.

[0018] Step 1: Preparation of carboxyl zwitterionic polymer PCBMA: First, weigh 0.12mmol AIBN (azobisisobutyronitrile), take 4mL anhydrous DMF (dimethylformamide) and sonicate for 3-5min to completely dissolve it, and prepare the AIBN solution with a concentration of 5mg / mL; Weigh 0.018 mmol BSPA (chain transfer agent 3-[[(benzylthio)thiocarbonyl]thio]propionic acid), draw 1 mL DMF and add it to a centrifuge tube, use ultrasonic sonication for 3 min to completely dissolve it, and the concentration of the prepared BSPA solution is 5 mg / mL; Weigh 1.3 mol CBMA (zwitterionic monomer carboxylic acid betaine), transfer it to a Schlenk bottle, add 0.5 mL of deionized water to the Schlenk bottle to completely dissolve the CBMA, transfer 1.426 mL of BSPA solution and 0.215 mL of AIBN solution to the Schlenk bottle, then add 1.4 mL of redistilled anhydrous DMF to the bottle to make the volume ratio of deionized water to anhydrous DMF be 1:5, the solution is clear and transparent and light yellow, place it in a -5°C circulating refrigerator, cycle the vacuum-filling nitrogen for 5-7 times to remove all oxygen in the Schlenk bottle and the solution, seal the mouth of the Schlenk bottle with a sealing film, which is conducive to better reaction under anaerobic conditions, place the Schlenk bottle in a magnetic stirring oil bath at 72°C for 36 hours, use a dialysis membrane with a molecular weight (MWCO) of 2000, and dialyze with deionized water for 24 h, freeze-drying to obtain the dry product PCBMA; the synthesized carboxyl zwitterionic polymer PCBMA was subjected to 1HNMR (nuclear magnetic hydrogen spectrum) determination. As shown in the figure, there are multiple characteristic peaks marked as a, b, c, d, e, f, g, and m in the spectrum, belonging to different groups on the PCBMA chain segment.

[0019] Preparation of activated carboxyl zwitterionic polymers: Weigh 0.01 mol of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide), take 1 mL of deionized water and ultrasonicate to completely dissolve EDC to obtain a 0.01 mol / mL EDC solution; weigh 0.005 mol of NHS (N-hydroxysuccinimide), add 1 mL of deionized water and ultrasonicate to completely dissolve it to obtain a 0.005 mol / mL NHS solution; weigh 0.025 mmol of PCBMA, transfer it to a small flask, add 1 mL of deionized water to the flask, stir lightly on a magnetic stirrer to completely dissolve PCBMA, then use a pipette to draw 0.1 mL of 0.01 mol / mL EDC solution and 0.005 mol / mL NHS solution and add them to the round-bottom flask, react and activate for 2 hours at room temperature to obtain an activated PCBMA solution.

[0020] Step 2: Preparation of amide zwitterionic polymer PCBSA: Weigh 0.088mmol benzenesulfonamide and transfer it to a 5mL centrifuge tube. Add 0.5mL DMSO (dimethyl sulfoxide) to the centrifuge tube and use an ultrasonic cleaner to completely dissolve it. Add the activated PCBMA and benzenesulfonamide DMSO mixed solution to a 10mL round-bottom flask, add a 10mol / L NaOH solution to adjust the pH of the solution, and use precision test paper to make the pH of the solution 8.5. React at room temperature for 48h. The reaction product is dialyzed with a dialysis bag with a molecular weight of MWCO of 1000 for 24h to remove impurities, and then freeze-dried to obtain the amide zwitterionic polymer PCBSA. The polymer PCBSA is subjected to 1H NMR measurement. As shown in the figure, the 1H NMR spectrum of the amide zwitterionic polymer PCBSA generated by modifying the sulfonamide drug monomer benzenesulfonamide has a, b, c, d, e, f, g, m, and multiple characteristic peaks belonging to different groups on the PCBSA segment, and also has a characteristic peak belonging to the benzene ring on the modified sulfonamide drug, marked as n.

[0021] Example 2 The difference from Example 1 is that the amide drug monomer used is sulfacetamide: 0.088mmol sulfacetamide was weighed, dissolved in 0.5mL anhydrous DMSO, mixed with the activated PCBMA solution, and a 10mol / L NaOH solution was added dropwise to adjust the pH of the mixed solution to 8.5. The reaction was carried out at room temperature for 48h. After the reaction was completed, the amide zwitterionic polymer PBCSAC was obtained by dialysis with a dialysis bag with a molecular weight of 1000 and freeze-dried. The polymer PBCSAC was measured by 1H NMR. As shown in the figure, the 1H NMR spectrum of the amide zwitterionic polymer PCBSAC generated by modifying the sulfonamide drug monomer sulfacetamide not only has a, b, c, d, e, f, g, m, and multiple characteristic peaks belonging to different groups on the PBCSAC chain segment, but also after modification, a characteristic peak n belonging to the benzene ring on the modified sulfonamide drug also appears.

[0022] Example 3 The difference from Example 1 is that the amide drug monomer uses sulfadoxine: 0.088mmol sulfadoxine is dissolved in 0.5mL anhydrous DMSO, added to the activated PCBMA solution, stirred in an ice-water bath, and a 10mol / L NaOH solution is added dropwise to adjust the pH of the solution to 8.5, so that sulfadoxine is completely dissolved, reacted in an ice-water bath for 30min, and then the ice-water bath reaction is removed, and the reaction is carried out at room temperature for 48h, dialyzed with a dialysis bag with a molecular weight of 100 for 24h, and then freeze-dried to obtain an amide zwitterionic polymer PCBSDOX. The polymer PCBSDOX is subjected to 1H NMR measurement. As shown in the figure, the 1H NMR spectrum of the amide zwitterionic polymer PCBSDOX generated by modifying the sulfonamide drug monomer sulfacetamide has a, b, c, d, e, f, g, m, and multiple characteristic peaks belonging to different groups on the PCBSDOX segment, and after modification, a characteristic peak n belonging to the benzene ring on the modified sulfonamide drug also appears.

[0023] The polymers PCBSA, PBCSAC and PCBSDOX prepared in Examples 1-3 were used to modify red fluorescent quantum dots QDs respectively.

[0024] Preparation of red fluorescent quantum dots QDs modified with amide zwitterionic polymer PCBSA: The concentration of QDs (fluorescent quantum dots) with a fluorescence emission wavelength of 620nm is 3mg / mL. 0.1mL of QDs solution is aspirated and sonicated for 30min in the dark to make the quantum dots evenly dispersed and easier to graft and modify. 15mg of NaBH4 (sodium borohydride) is weighed on a weighing balance. NaBH4 is in excess in the reaction. It is added to the Schlenk bottle and nitrogen is passed through. The reaction is carried out at room temperature for 2h to maximize the thiolation of PCBSA and obtain thiolated PCBSA. Then, the sonicated QDs solution is added to the Schlenk bottle. Nitrogen is passed through the bottle at room temperature and the reaction is stirred on a magnetic stirrer for 24h to replace the MPA (3-mercaptopropionic acid) on the surface of the quantum dots, so that the thiolated polymer PCBSA is modified and grafted onto the surface of the quantum dots. The solution obtained by the reaction is dialyzed for 6h in the dark with deionized water using a molecular weight WMCO8000 dialysis membrane.

[0025] Preparation of yellow fluorescent quantum dots QDs modified with amide zwitterionic polymer PCBSAC: The concentration of QDs with a fluorescence emission wavelength of 560 nm is 3 mg / mL. 0.1 mL of QDs solution is aspirated and sonicated for 30 minutes in the dark to make the quantum dots evenly dispersed and more easily grafted. 15 mg of NaBH4 is weighed on a weighing balance. NaBH4 is in excess in the reaction and is added to a Schlenk bottle. Nitrogen is passed through and the reaction is carried out at room temperature for 2 hours to maximize the thiolation of PCBSAC to obtain thiolated PCBSAC. Then, the sonicated QDs solution is added to the Schlenk bottle. Nitrogen is passed through and the reaction is carried out in the dark at room temperature. The reaction is stirred on a magnetic stirrer for 24 hours to replace the MPA on the surface of the quantum dots, so that the thiolated polymer PCBSAC is modified and grafted onto the surface of the quantum dots. The solution obtained by the reaction is dialyzed for 6 hours in the dark with deionized water using a molecular weight WMCO8000 dialysis membrane.

[0026] Preparation of green fluorescent quantum dots QDs modified with amide zwitterionic polymer PCBSDOX: The concentration of QDs with a fluorescence emission wavelength of 540 nm is 3 mg / mL. 0.1 mL of QDs solution is aspirated and sonicated for 30 minutes in the dark to make the quantum dots evenly dispersed and more easily grafted. 15 mg of sodium borohydride is weighed on a weighing balance. NaBH4 is in excess in the reaction. It is added to a Schlenk bottle and nitrogen is passed through. The reaction is carried out at room temperature for 2 hours to maximize the thiolation of PCBSDOX and obtain thiolated PCBSDOX. Then, the sonicated QDs solution is added to the Schlenk bottle. Nitrogen is passed through the bottle at room temperature and the reaction is carried out in the dark. The reaction is stirred on a magnetic stirrer for 24 hours to replace the MPA on the surface of the quantum dots, so that the thiolated polymer PCBSDOX is modified and grafted onto the surface of the quantum dots. The solution obtained by the reaction is dialyzed for 6 hours in the dark with deionized water using a molecular weight WMCO8000 dialysis membrane.

[0027] In order to determine the pH-responsive charge reversal range of quantum dots QDs modified with different amide zwitterionic polymers on the surface, PCBSA-@-QDs, PCBSAC-@-QDs, and PCBSDOX-@-QDs were dissolved in buffer solutions with a pH gradient of 7.2-6.0, and the changes in their surface Zeta potential under various pH environments were detected by a laser particle size analyzer. Figure 6 As shown in the figure, the Zeta potential of QDs modified by PCBSA, PCBSAC, and PCBSDOX in the environment of pH=6.8, pH=6.6, and pH=6.4 changed from negative charge value to positive charge value, showing cationic properties. Figure 6 It shows that the charge reversal point can be adjusted by changing the group and their pH response transition point.

[0028] In the acidic microenvironment of the tumor, the surface electrical properties of QDs modified with sulfonamide grafted polymers will flip to cationic properties. Based on electrostatic interactions, when the surface of the fluorescent imaging material is positively charged, it can attract the negatively charged cell membrane surface. In order to verify that the group can control the charge reversal performance of zwitterionic polymers, the cell uptake experiment of tumor liver cancer cells HepG2 was carried out to test the ability of the material to be taken up by cells. Through fluorescence microscopy, as shown in the figure, at pH=6.8, pH=6.6, and pH6.4, the fluorescence intensity of the quantum dots on the cells was different after the cells were incubated with different samples. Figure 7 It can be seen that the red fluorescent quantum dots modified by PCBSA, the yellow fluorescent quantum dots modified by PCBSAC, and the green fluorescent quantum dots modified by PCBSDOX showed significantly enhanced fluorescence intensity at cell pHe (pH=6.8), cell pHe (pH=6.6), and cell pHe (pH=6.4), confirming that their charge reversal points can adjust their pH response transition points by changing the groups.

[0029] The above shows and describes the main features, methods of use, basic principles and advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements according to actual conditions, and these changes and improvements fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A method for preparing a group-controllable zwitterionic polymer, characterized in that: The specific steps include: Step 1: Prepare PCBMA using CBMA as raw material, dissolve in deionized water and add EDC solution and NHS solution, adjust the solution pH to 6.0-6.5, and react and activate at room temperature for 1-3 hours to obtain an activated PCBMA solution; Step 2: dissolve the amide drug monomer in DMSO, add the activated PCBMA solution, dropwise add NaOH solution, stir and react at room temperature for 24-48 hours, dialyze the obtained product through a dialysis bag for 24-48 hours, and freeze-dry to obtain an amide drug monomer-modified amide zwitterionic polymer.

2. The method for preparing a group-controllable zwitterionic polymer according to claim 1, characterized in that: The preparation method of PCBMA in step 1 is: a. Dissolve AIBN and DMF by ultrasonic method to obtain AIBN solution, and dissolve BSPA and DMF by ultrasonic method to obtain BSPA solution; b. After CBMA is dissolved in deionized water, add the BSPA solution, AIBN solution and anhydrous DMF in step a and mix well. Vacuum at 0-5°C and introduce nitrogen for 5-7 times. After deoxygenation, react at 70-75°C for 24-48h. c. The solution after the deoxygenation reaction in step b is dialyzed against deionized water for 24-36 hours and freeze-dried to obtain PCBMA.

3. The method for preparing a group-controllable zwitterionic polymer according to claim 2, characterized in that: The volume ratio of deionized water to DMF is 1:1-1:5, the weight ratio of AIBN to BSPA is 1:2-1:4, and the weight ratio of CBMA to BSPA is 1:20-1:

40.

4. The method for preparing a group-controllable zwitterionic polymer according to claim 1, characterized in that: The amide drug monomer is one of benzenesulfonamide, sulfacetamide and sulfadoxine.

5. The method for preparing a group-controllable zwitterionic polymer according to claim 1, characterized in that: During the PCBMA activation process, the amounts of PCBMA, EDC solution and NHS solution used are 0.05-0.1 mmol, 0.1-0.2 mmol and 0.1-0.2 mmol respectively.

6. The method for preparing a group-controllable zwitterionic polymer according to claim 1, characterized in that: In the step 2, the molecular weight MWCO of the dialysis bag used for PCBMA is 2000-3500, and the volume ratio of DMSO to deionized water is 1:1-1:

4.

7. A group-controllable zwitterionic polymer prepared by the preparation method according to any one of claims 1 to 6.

8. Use of a zwitterionic polymer as claimed in claim 7, wherein the group can be controlled to adjust the charge reversal pH transition point.

9. The use of a group-controllable zwitterionic polymer for adjusting the charge reversal pH transition point as claimed in claim 8, characterized in that: The following steps are involved: NaBH4 is reacted at room temperature for 2-4 hours under an inert gas atmosphere, and ultrasonically dispersed QDs are added. The reaction is continued at room temperature for 24-36 hours under an inert gas atmosphere, and then dialyzed with deionized water in the dark for 3-6 hours through a dialysis membrane. The dialyzed zwitterionic polymer-modified QDs are dissolved in a buffer solution with a pH gradient of 7.2-6.0, and the changes in surface Zeta potential under various pH environments are detected using a laser particle size analyzer.

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