Compounds Containing Bis(isatin anhydride) Functional Groups, Polymers with Dual pH and Temperature Stimuli Responsiveness, and Their Preparation Methods and Applications

Abstract

The present invention relates to a compound containing a bisisatoic anhydride functional group, a polymer with dual pH and temperature stimuli-responsive properties, and a preparation method and application thereof. The compound containing the bisisatoic anhydride functional group has a structure shown in formula (1). The polymer with dual pH and temperature stimuli-responsive properties is obtained by a polymerization reaction of the compound containing the bisisatoic anhydride functional group described in the present invention with oligoethylene glycol diamine. The aqueous solution of the polymer has dual pH and temperature stimuli-responsive properties and has LCST-type phase transition characteristics. The amino groups on its main chain make it positively charged in an aqueous solution, and it can be conjugated with negatively charged siRNA through electrostatic interaction, and can be used to load siRNA and deliver siRNA drugs, so it has potential application value in the fields of intelligent responsive materials and pharmaceutical biology. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the technical fields of intelligent polymers and biomedicine, and particularly relates to a compound containing a bisisatoic anhydride functional group, a polymer with dual pH and temperature stimuli-responsive properties, a preparation method thereof, and an application in loading siRNA. Background Art

[0002] Stimuli-responsive polymers refer to a class of materials whose physical or chemical properties can change in response to changes in the external environment (light, temperature, pH, ionic strength, etc.), and are also known as intelligent materials and intelligent polymers. In recent years, they have broad application prospects in the fields of medicine, life science, and nanomaterial science.

[0003] Due to the fact that the environment in actual application scenarios is often complex and variable, single-stimuli-responsive polymer materials are often difficult to meet the actual usage requirements. Therefore, polymers with multiple stimuli-responsive properties have attracted much attention from researchers in recent years. This type of polymer material can respond to two or more stimuli, and there is a relationship of mutual promotion and restriction between multiple stimuli responses, which has very important application value. Summary of the Invention

[0004] Based on this, the purpose of the present invention is to provide a novel polymer with dual pH and temperature stimuli-responsive properties and a preparation method thereof.

[0005] In order to achieve the above invention purpose, the present invention includes the following technical solutions.

[0006] In the first aspect, the present invention provides a compound containing a bisisatoic anhydride functional group, having the structure shown in formula (1):

[0007]

[0008] In the second aspect, the present invention provides a preparation method of the compound containing a bisisatoic anhydride functional group, including the following steps: reacting isatoic anhydride and triethylene glycol under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the product.

[0009] Preferably, the preparation method of the compound containing a bisisatoic anhydride functional group includes the following steps:

[0010] Dissolve isatoic anhydride, triphenylphosphine, and triethylene glycol in tetrahydrofuran, cool in an ice bath, dropwise add a tetrahydrofuran solution of diisopropyl azodicarboxylate to the obtained solution, and react at 15°C to 40°C for 12h to 48h to obtain the product.

[0011] In the third aspect, the present invention provides a polymer with dual pH and temperature stimuli-responsive properties, having the structure shown in formula (2):

[0012]

[0013] Wherein x is selected from 2, 3, 4, 5.

[0014] The pH- and temperature-dual stimulus-responsive polymer described in the present invention is obtained by the polymerization reaction of the compound containing bisisatoic anhydride functional groups described in the present invention and oligoethylene glycol diamine.

[0015] Wherein, the oligoethylene glycol diamine can be selected from at least one of 1,2-bis(2-aminoethoxy)ethane, 1,11-diamino-3,6,9-trioxaundecane, 3,6,9,12-tetraoxatetradecane-1,14-diamine, and 3,6,9,12,15-pentaoxaheptadecane-1,17-diamine.

[0016] In a fourth aspect, the present invention provides a preparation method of the pH- and temperature-dual stimulus-responsive polymer described above, comprising the following steps:

[0017] Adding the compound containing bisisatoic anhydride functional groups described in the present invention into a mixed solvent of N,N-dimethylformamide and water, heating to dissolve and then cooling to 15°C to 30°C, and then adding oligoethylene glycol diamine, and reacting at a temperature of 15°C to 30°C to obtain the product.

[0018] In a fifth aspect, the present invention provides the application of the pH- and temperature-dual stimulus-responsive polymer described above in the preparation of a carrier for loading siRNA or delivering siRNA.

[0019] The present invention has the following beneficial effects:

[0020] The present invention uses isatoic anhydride and triethylene glycol as raw materials to react to obtain a new compound containing bisisatoic anhydride functional groups. This compound has high reactivity, and its reactivity with primary amines is much stronger than that of isatoic anhydride, overcoming the defect of low reaction efficiency between isatoic anhydride and primary amines. This new compound can be used as a polymerization monomer to polymerize with oligoethylene glycol diamine to obtain a pH- and temperature-dual stimulus-responsive polymer.

[0021] The present invention uses a new compound containing bisisatoic anhydride functional groups and oligoethylene glycol diamine as reaction monomers, and prepares a new polymer through a polymerization reaction. The aqueous solution of this polymer has pH- and temperature-dual stimulus response, and has LCST-type phase transition characteristics, and its phase transition temperature can be adjusted according to the type of oligoethylene glycol diamine and the system pH. For the polymer prepared in the present invention, the amino groups on its main chain make it positively charged in aqueous solution, and can be conjugated with negatively charged siRNA through electrostatic interaction, and can be used for loading siRNA and delivering siRNA drugs, so it has potential application value in the fields of intelligent response materials and medicine and biology.

[0022] The pH- and temperature-responsive polymer of the present invention is synthesized by the reaction of isatoic anhydride and primary amine. The reaction conditions are mild, the operation is simple, and the post-treatment process is very convenient, which is conducive to industrial production. Description of the Drawings

[0023] Figure 1 It is the NMR spectrum of monomer (1) in the examples.

[0024] Figure 2 It is the GPC spectra of polymers P1, P2, P3 and P4 in the examples.

[0025] Figure 3 It is the temperature-transmittance curves of polymers P1, P2, P3 and P4 in water (concentration: 0.5 wt%) in the examples.

[0026] Figure 4 It is the temperature-transmittance curves of polymer P2 prepared in Example 2 in different pH solutions (concentration: 0.5 wt%).

[0027] Figure 5 It is the gel electrophoresis diagrams of polymer P2 prepared in Example 2 loaded with siRNA at different N / P ratios. Detailed Description of the Invention

[0028] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosed content of the present invention more thorough and comprehensive.

[0029] For the experimental methods without specific conditions indicated in the following examples, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturers. All kinds of common chemical reagents used in the examples are commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those skilled in the technical field to which the present invention belongs. The terms used in the description of the present invention are only for the purpose of describing specific embodiments and are not used to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0031] In addition, as used in the present invention, the term "or" is an inclusive "or" symbol and is equivalent to the term "and / or", unless the context clearly dictates otherwise. The term "based on" is not exclusive and allows for other factors not described, unless the context clearly dictates otherwise. Further, throughout the specification, the meanings of "a", "an", and "the" include plural referents. The meaning of "in" includes "in" and "on".

[0032] Some of the embodiments thereof relate to a compound containing a bisisatoic anhydride functional group, having the structure shown in formula (1):

[0033]

[0034] The compound containing a bisisatoic anhydride functional group of the present invention has a high reactivity with oligoethylene glycol diamine and can polymerize with oligoethylene glycol diamine to obtain a polymer with dual pH and temperature stimuli responsiveness.

[0035] Some of the embodiments thereof relate to a preparation method of the compound containing a bisisatoic anhydride functional group of the present invention, comprising the following steps: reacting isatoic anhydride and triethylene glycol under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the product.

[0036] In some preferred embodiments, the reaction is carried out in a solvent, and the solvent is preferably tetrahydrofuran.

[0037] In some preferred embodiments, the temperature of the reaction is preferably 15°C to 40°C, more preferably 20°C to 30°C.

[0038] In some preferred embodiments, the reaction time is 12 h to 48 h, more preferably 20 h to 30 h, and even more preferably 24 hours.

[0039] In some preferred embodiments, the molar ratio of isatoic anhydride, triphenylphosphine, triethylene glycol, and diisopropyl azodicarboxylate is 2.0 to 3.0: 2.0 to 3.0: 1: 2.0 to 3.0, more preferably 2.1 to 2.3: 2.1 to 2.3: 1: 2.1 to 2.3.

[0040] In some preferred embodiments, the preparation method of the compound containing a bisisatoic anhydride functional group comprises the following steps:

[0041] Dissolve isatoic anhydride, triphenylphosphine, and triethylene glycol in tetrahydrofuran, cool in an ice bath, dropwise add a tetrahydrofuran solution of diisopropyl azodicarboxylate to the resulting solution, and heat to 15°C to 40°C for reaction for 12 h to 48 h to obtain the product.

[0042] In some of these embodiments, there is also involved a polymer with dual pH and temperature stimuli responses, having the structure shown in formula (2):

[0043]

[0044] wherein x is selected from 2, 3, 4, 5.

[0045] In some of these embodiments, the number average molecular weight of the polymer is 15,000 - 23,000.

[0046] The polymer with dual pH and temperature stimuli responses of the present invention is obtained by the polymerization reaction of the compound containing bisisatoic anhydride functional groups of the present invention and oligoethylene glycol diamine.

[0047] Among them, the oligoethylene glycol diamine can be selected from at least one of 1,2-bis(2-aminoethoxy)ethane, 1,11-diamino-3,6,9-trioxaundecane, 3,6,9,12-tetraoxatetradecane-1,14-diamine, and 3,6,9,12,15-pentaoxaheptadecane-1,17-diamine.

[0048] In some preferred embodiments, the molar ratio of the compound containing bisisatoic anhydride functional groups to oligoethylene glycol diamine is 1:1.

[0049] In some of these embodiments, there is also involved a preparation method of the polymer with dual pH and temperature stimuli responses of the present invention, including the following steps:

[0050] Adding the compound containing bisisatoic anhydride functional groups of the present invention into a mixed solvent of N,N-dimethylformamide and water, heating to dissolve and then cooling to 15°C - 30°C, and then adding oligoethylene glycol diamine, and reacting at a temperature of 15°C - 30°C to obtain the product.

[0051] In some preferred embodiments, the mass ratio of N,N-dimethylformamide to water in the mixed solvent of N,N-dimethylformamide and water is 99:0.5 - 98:2, more preferably 99:1.

[0052] In some preferred embodiments, the temperature for heating to dissolve is 38°C - 42°C.

[0053] In some preferred embodiments, the reaction time is 6h - 48h, more preferably 20h - 30h, and even more preferably 24 hours.

[0054] In some embodiments, after the reaction is completed, the following post-treatment steps are further included: after the reaction is completed, the reaction solution is precipitated in diethyl ether, and the obtained precipitate is redissolved and reprecipitated, and this operation is repeated 2-4 times. Finally, the obtained solid is dried to obtain the polymer with dual pH and temperature stimuli responses.

[0055] The polymer prepared by the present invention has dual pH and temperature stimuli responses in its aqueous solution, and has LCST-type phase transition characteristics, and its phase transition temperature can be adjusted according to the type of oligoethylene glycol diamine and the system pH. The polymer prepared by the present invention is positively charged in solution and can be used to load negatively charged siRNA for delivering siRNA drugs.

[0056] The present invention will be further described in detail below with specific embodiments.

[0057] In the following examples, unless otherwise specified, the room temperature refers to the indoor temperature of 20°C - 30°C.

[0058] Example 1

[0059] 1. Synthesis of monomer (1)

[0060] Dissolve 14.3 g (87.7 mmol) of isatoic anhydride, 23.0 g (87.7 mmol) of triphenylphosphine, and 6.0 g (40.0 mmol) of triethylene glycol in 200 mL of tetrahydrofuran, and cool in an ice bath; under the ice bath condition, slowly dropwise add a solution of 17.8 g (88.1 mmol) of diisopropyl azodicarboxylate in tetrahydrofuran (50 mL) to the obtained solution. After the addition is completed, warm up to room temperature and react for 24 h; spin-dry the reaction mixture, and separate the crude product by column chromatography (the eluent is a mixed solvent of ethyl acetate / petroleum ether with a volume ratio of 3:1) to obtain 10.9 g of monomer (1) with a yield of 62%.

[0061] The structure of monomer (1) was characterized by 1H NMR, and the spectral results are as Figure 1 shown, indicating that the monomer has been synthesized, and its structural formula is:

[0062]

[0063] 2. Synthesis of polymer P1 with dual temperature and pH stimuli responses

[0064]

[0065] To 2.00 g of monomer (1) (4.5 mmol), 10 mL of a DMF / water mixed solvent (mass ratio of DMF:water is 99:1) was added. The mixture was stirred and heated to 40 °C until completely dissolved, and then the solution was cooled to room temperature. 0.67 g (4.5 mmol) of 1,2-bis(2-aminoethoxy)ethane was added to the solution, and the reaction was carried out at room temperature for 24 h; the reaction solution was poured into ether for precipitation, and the precipitate was redissolved in tetrahydrofuran and then precipitated with ether again. This was repeated 3 times, and the resulting solid was dried under vacuum to obtain polymer P1.

[0066] Example 2

[0067] 1. The synthesis of monomer (1) was the same as that in Example 1.

[0068] 2. The synthesis method of the temperature- and pH-dual-responsive polymer P2 was basically the same as that in Example 1, except that the oligomeric ethylene glycol diamine used was 1,11-diamino-3,6,9-trioxaundecane, and the addition amount was 0.84 g. The reaction formula is as follows:

[0069]

[0070] Example 3

[0071] 1. The synthesis of monomer (1) was the same as that in Example 1.

[0072] 2. The synthesis method of the temperature- and pH-dual-responsive polymer P3 was basically the same as that in Example 1, except that the oligomeric ethylene glycol diamine used was 3,6,9,12-tetraoxatetradecane-1,14-diamine, and the addition amount was 1.08 g. The reaction formula is as follows:

[0073]

[0074] Example 4

[0075] 1. The synthesis of monomer (1) was the same as that in Example 1.

[0076] 2. The synthesis method of the temperature- and pH-dual-responsive polymer P4 was basically the same as that in Example 1, except that the oligomeric ethylene glycol diamine used was 3,6,9,12,15-pentaoxaheptadecane-1,17-diamine, and the addition amount was 1.28 g. The reaction formula is as follows:

[0077]

[0078] Comparative Example 1

[0079] 1. The synthesis of monomer (1) was the same as that in Example 1.

[0080] 2. The synthesis method of the temperature and pH responsive polymer P5 is basically the same as that of Example 1, except that the oligoethylene glycol diamine used is 2,2-oxybis(ethylamine), and the addition amount is 0.47 g.

[0081] Characterization and Testing of Example 5

[0082] 1. The polymer was analyzed by gel permeation chromatography, and the results are shown in Figure 2 , indicating that the series of polymers P1, P2, P3 and P4 were successfully synthesized, and their number average molecular weights were 15,700, 18,300, 20,100 and 22,300 in turn.

[0083] 2. Polymers P1, P2, P3, P4 and P5 were respectively dissolved in water (concentration: 0.5 wt%), and at a constant heating rate (1 °C / min), the transmittance of the solution was measured using a UV spectrophotometer to obtain the thermosensitive curves of the polymers. As shown in Figure 3 , it can be clearly observed that the polymers of the present invention have very obvious LCST-type phase transition characteristics. As the ethylene glycol content in the oligoethylene glycol diamine unit gradually increases, the LCST (lower critical phase transition temperature) of the polymer shows an increasing trend, increasing from 22.0 °C to 67.5 °C. For P5, it cannot be dissolved even when the temperature drops to near the freezing point.

[0084] 3. Taking polymer P2 as a representative, it was dissolved in water (concentration: 0.5 wt%), the pH of the system was adjusted, and its phase transition behavior at different pH values was measured. As shown in Figure 4 , it can be found that as the pH gradually decreases, the LCST of the polymer gradually increases, mainly because the decrease in pH leads to a higher degree of protonation of the amino group and better hydrophilicity.

[0085] 4. siRNA and polymer P2 were respectively dissolved in PBS buffer solution. According to the N / P ratio (N / P = amino group content in the polymer / phosphate group content in siRNA), a certain amount of siRNA solution (siRNA concentration: 0.2 mM) was added to the polymer solution with a pipette gun, and the reaction was carried out at room temperature for 30 min to ensure complete conjugation. The obtained conjugate was analyzed by gel electrophoresis, and the results are shown in Figure 5 , as the N / P ratio gradually increases, the electrophoretic ability of negatively charged siRNA gradually decreases. When N / P ≥ 16, there will be no free siRNA, indicating that polymer P2 can conjugate siRNA through electrostatic interaction.

[0086] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the invention patent shall be subject to the appended claims.

Claims

1. A compound containing a bisisatoic anhydride functional group, characterized in that, It has the structure shown in formula (1): 。 2. A method for preparing a compound containing a bisisatoic anhydride functional group as described in claim 1, characterized in that, It includes the following steps: isatoic anhydride and triethylene glycol react under the action of triphenylphosphine and diisopropyl azodicarboxylate, and that's it.

3. The preparation method of the compound containing a bisisatoic anhydride functional group according to claim 2, characterized in that, The said reaction is carried out in a solvent; and / or, The temperature of the said reaction is 15°C to 40°C; and / or, The time of the said reaction is 12 h to 48 h; and / or, The molar ratio of the said isatoic anhydride, triphenylphosphine, triethylene glycol and diisopropyl azodicarboxylate is 2.0 to 3.0: 2.0 to 3.0: 1: 2.0 to 3.

0.

4. The preparation method of the compound containing a bisisatoic anhydride functional group according to claim 3, characterized in that, The said solvent is tetrahydrofuran.

5. The preparation method of the compound containing a bisisatoic anhydride functional group according to claim 3, characterized in that, The temperature of the said reaction is 20°C to 30°C.

6. The preparation method of the compound containing a bisisatoic anhydride functional group according to claim 3, characterized in that, The time of the said reaction is 20 h to 30 h.

7. The preparation method of the compound containing a bisisatoic anhydride functional group according to claim 3, characterized in that, The molar ratio of the said isatoic anhydride, triphenylphosphine, triethylene glycol and diisopropyl azodicarboxylate is 2.1 to 2.3: 2.1 to 2.3: 1: 2.1 to 2.

3.

8. The preparation method of the compound containing a bisisatoic anhydride functional group according to any one of claims 2-7, characterized in that, It includes the following steps: Dissolve isatoic anhydride, triphenylphosphine and triethylene glycol in tetrahydrofuran, cool in an ice bath, dropwise add a tetrahydrofuran solution of diisopropyl azodicarboxylate to the obtained solution, and raise the temperature to 15°C to 40°C for reaction for 12 h to 48 h, and that's it.

9. A polymer with dual pH and temperature stimuli response, characterized in that, It has the structure shown in formula (2): Where x is selected from 2, 3, 4, 5; The number average molecular weight of the said polymer is 15000 to 23000.

10. A polymer with dual pH and temperature stimuli responsiveness, characterized in that, It is obtained by the polymerization reaction of the compound containing bis(isatoic anhydride) functional group described in claim 1 and oligoethylene glycol diamine; The said oligoethylene glycol diamine is selected from at least one of 1,2-bis(2-aminoethoxy)ethane, 1,11-diamino-3,6,9-trioxaundecane, 3,6,9,12-tetraoxatetradecane-1,14-diamine and 3,6,9,12,15-pentaoxaheptadecane-1,17-diamine; The molar ratio of the compound containing bis(isatoic anhydride) functional group to oligoethylene glycol diamine is 1:

1.

11. A method for preparing a pH- and temperature-dual-stimuli-responsive polymer according to claim 9 or 10, characterized in that, It includes the following steps: Add the compound containing bis(isatoic anhydride) functional group described in claim 1 into a mixed solvent of N,N-dimethylformamide and water, raise the temperature to dissolve and then cool to 15°C to 30°C, then add oligoethylene glycol diamine, and react at a temperature of 15°C to 30°C, and that's it.

12. The preparation method of the pH- and temperature-dual-stimulus-responsive polymer according to claim 11, wherein, In the said mixed solvent of N,N-dimethylformamide and water, the mass ratio of N,N-dimethylformamide to water is 99:0.5 to 98:2; and / or, The temperature for raising the temperature to dissolve is 38°C to 42°C; and / or, The time of the said reaction is 6 h to 48 h; and / or, After the reaction, it also includes the following post-treatment steps: after the reaction, precipitate the reaction solution in ether, redissolve the obtained precipitate and reprecipitate it, repeat this operation 2 - 4 times, and finally dry the obtained solid to obtain the pH and temperature dual-stimuli responsive polymer.

13. The preparation method of the pH- and temperature-dual stimulus-responsive polymer according to claim 12, wherein In the said mixed solvent of N,N-dimethylformamide and water, the mass ratio of N,N-dimethylformamide to water is 99:

1.

14. The preparation method of the pH- and temperature-dual stimulus-responsive polymer according to claim 12, characterized in that, The time of the said reaction is 20 h to 30 h.

15. Use of the pH and temperature dual-stimuli responsive polymer according to claim 9 or 10 in the preparation of a carrier for loading siRNA or delivering siRNA.

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