Compound containing double isatoic anhydride functional groups, polymer with pH and temperature dual stimulation response as well as preparation method and application of polymer

Abstract

The invention relates to a compound containing double isatoic anhydride functional groups, a polymer with pH and temperature dual stimulation response as well as a preparation method and application of the polymer. The compound containing the bis-isatoic anhydride functional group has a structure as shown in a formula (1). The pH and temperature dual-stimulus-responsive polymer is obtained by carrying out polymerization reaction on the compound containing the double isatoic anhydride functional group and oligomeric ethylene glycol diamine. The aqueous solution of the polymer has pH and temperature dual stimulation response and has LCST type phase transition characteristics, the amino group on the main chain of the polymer makes the polymer show positive electricity in the aqueous solution, the polymer can be conjugated with electronegative siRNA through electrostatic interaction, and the polymer can be used for loading siRNA and delivering siRNA drugs, and can be used for preparing siRNA drugs. Therefore, the method has potential application value in the fields of intelligent response materials and medical biology. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of intelligent polymer and biomedicine technology, and specifically relates to a compound containing a bis-isatoic anhydride functional group, a polymer having dual stimulation responses of pH and temperature, a preparation method thereof, and an application thereof 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.). They are also called smart materials and smart polymers. In recent years, they have broad application prospects in the fields of medicine, life sciences, and nanomaterials science.

[0003] Since the actual application scenarios are often complex and changeable, polymer materials that respond to a single stimulus often fail to meet the actual use requirements. Therefore, polymers with multiple stimulus responses have attracted much attention from researchers in recent years. This type of polymer material can respond to two or more stimulus sources, and there is a relationship of mutual promotion and restriction between multiple stimulus 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 new type of polymer that responds to dual stimulations of pH and temperature and a preparation method thereof.

[0005] In order to achieve the above-mentioned object of the invention, the present invention includes the following technical solutions.

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

[0007]

[0008] In a second aspect, the present invention provides a method for preparing the compound containing a bis-isatoic anhydride functional group, comprising the following steps: isatoic anhydride and triethylene glycol react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain.

[0009] Preferably, the method for preparing the compound containing a bis-isatoic anhydride functional group comprises the following steps:

[0010] Dissolve isocyanic anhydride, triphenylphosphine and triethylene glycol in tetrahydrofuran, place in an ice bath, dropwise add a tetrahydrofuran solution of diisopropyl azodicarboxylate into the resulting solution, raise the temperature to 15°C to 40°C and react for 12h to 48h to obtain the product.

[0011] In a third aspect, the present invention provides a polymer that is dual responsive to pH and temperature stimulation, having a structure shown in formula (2):

[0012]

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

[0014] The polymer responsive to dual stimulation of pH and temperature described in the present invention is obtained by polymerization reaction of the compound containing bis-isatoic anhydride functional group described in the present invention and oligoethylene glycol diamine.

[0015] The oligoethylene glycol diamine may 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 method for preparing the polymer responsive to both pH and temperature stimuli, comprising the following steps:

[0017] The compound containing the bis-isatoic anhydride functional group of the present invention is added to a mixed solvent of N,N-dimethylformamide and water, heated to dissolve and then cooled to 15°C to 30°C, and then oligoethylene glycol diamine is added and reacted at a temperature of 15°C to 30°C to obtain the product.

[0018] In a fifth aspect, the present invention provides the use of the polymer responsive to dual stimulation of pH and temperature in the preparation of a carrier for loading 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 a bis-isatoic anhydride functional group. The compound has high reactivity, and its reactivity with primary amines is much stronger than that of isatoic anhydride, thus overcoming the defect that the reaction efficiency of isatoic anhydride and primary amines is low. The new compound can be used as a polymerization monomer to polymerize with oligoethylene glycol diamine to obtain a polymer that responds to both pH and temperature stimulations.

[0021] The present invention uses a new compound containing a bis-isatoic anhydride functional group and oligoethylene glycol diamine as reaction monomers, and prepares a new polymer through a polymerization reaction. The aqueous solution of the polymer has a dual stimulus response of pH and temperature, and has an LCST-type phase transition characteristic, 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 has an amine group on its main chain that makes it positively charged in an aqueous solution, and can be conjugated with negatively charged siRNA through electrostatic action, and can be used to load siRNA and deliver siRNA drugs, so it has potential application value in the fields of intelligent response materials and medical biology.

[0022] The pH and temperature dual stimulus responsive polymer of the present invention is synthesized by isatoic anhydride-primary amine reaction, and the reaction conditions are mild, the operation is simple, and the post-treatment process is very simple, which is conducive to industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0024] Figure 2 The GPC spectra of polymers P1, P2, P3 and P4 in the examples are shown in FIG.

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

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

[0027] Figure 5 This is the gel electrophoresis diagram of polymer P2 prepared in Example 2 loaded with siRNA at different N / P levels. DETAILED DESCRIPTION

[0028] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0029] The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended 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 being based on other factors not described, unless the context clearly dictates otherwise. In addition, 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 involve a compound containing a bis-isatoic anhydride functional group, having a structure shown in formula (1):

[0033]

[0034] The compound containing the bis-isatoic anhydride functional group of the present invention has high reactivity with oligoethylene glycol diamine and can be polymerized with the oligoethylene glycol diamine to obtain a polymer that responds to both pH and temperature stimulations.

[0035] Some of the embodiments involve a method for preparing a compound containing a bis-isatoic anhydride functional group according to the present invention, comprising the following steps: isatoic anhydride and triethylene glycol react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the compound.

[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 reaction temperature 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 more preferably 24 hours.

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

[0040] In some preferred embodiments, the method for preparing the compound containing a bis-isatoic anhydride functional group comprises the following steps:

[0041] Dissolve isocyanic anhydride, triphenylphosphine and triethylene glycol in tetrahydrofuran, place in an ice bath, dropwise add a tetrahydrofuran solution of diisopropyl azodicarboxylate into the resulting solution, raise the temperature to 15°C to 40°C and react for 12h to 48h to obtain the product.

[0042] Some of the embodiments also involve a polymer that is responsive to both pH and temperature stimuli and has a structure shown in formula (2):

[0043]

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

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

[0046] The polymer capable of responding to dual stimulation of pH and temperature described in the present invention is obtained by polymerization reaction of the compound containing the bis-isatoic anhydride functional group described in the present invention and oligoethylene glycol diamine.

[0047] The oligoethylene glycol diamine may 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 bis-isatoic anhydride functional group to oligoethylene glycol diamine is 1:1.

[0049] Some of the embodiments also involve a method for preparing a polymer responsive to both pH and temperature stimuli according to the present invention, comprising the following steps:

[0050] The compound containing the bis-isatoic anhydride functional group of the present invention is added to a mixed solvent of N,N-dimethylformamide and water, heated to dissolve and then cooled to 15°C to 30°C, and then oligoethylene glycol diamine is added and reacted at a temperature of 15°C to 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 to 98:2, more preferably 99:1.

[0052] In some preferred embodiments, the temperature of the heating dissolution is 38°C to 42°C.

[0053] In some preferred embodiments, the reaction time is 6 h to 48 h, more preferably 20 h to 30 h, and 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 ether, the obtained precipitate is redissolved and reprecipitated, and this operation is repeated 2-4 times, and finally the obtained solid is dried to obtain the polymer that responds to both pH and temperature stimuli.

[0055] The polymer prepared by the present invention has a dual stimulus response of pH and temperature in its aqueous solution and has a LCST phase transition characteristic, and its phase transition temperature can be adjusted according to the type of oligoethylene glycol diamine and the pH of the system. The polymer prepared by the present invention has positive charge in the solution and can be used to load negatively charged siRNA for delivering siRNA drugs.

[0056] The present invention is further described in detail below with reference to specific embodiments.

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

[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 place in an ice bath. Slowly dropwise add a solution of 17.8 g (88.1 mmol) of diisopropyl azodicarboxylate in tetrahydrofuran (50 mL) to the resulting solution under ice bath conditions. After the addition is complete, heat the mixture to room temperature and react for 24 h. The reaction mixture is spin-dried and the crude product is separated by column chromatography (the eluent is a mixed solvent of ethyl acetate / petroleum ether in 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 H NMR spectroscopy. Figure 1 As shown, it is shown that the monomer has been synthesized, and its structural formula is:

[0062]

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

[0064]

[0065] 10 mL of DMF / water mixed solvent (mass ratio of DMF:water is 99:1) was added to 2.00 g of monomer (1) (4.5 mmol), stirred and heated to 40°C to completely dissolve it, 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 diethyl ether for precipitation, the precipitate was redissolved in tetrahydrofuran and then precipitated with diethyl ether, and this was repeated 3 times. The obtained solid was vacuum dried to obtain polymer P1.

[0066] Example 2

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

[0068] 2. The synthesis method of polymer P2 with dual stimulus response of temperature and pH is basically the same as that of Example 1, except that the oligoethylene glycol diamine used is 1,11-diamino-3,6,9-trioxaundecane, and the added amount is 0.84 g. The reaction formula is as follows:

[0069]

[0070] Example 3

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

[0072] 2. The synthesis method of polymer P3 with dual stimulus response of temperature and pH is basically the same as that of Example 1, except that the oligoethylene glycol diamine used is 3,6,9,12-tetraoxatetradecane-1,14-diamine, and the added amount is 1.08 g. The reaction formula is as follows:

[0073]

[0074] Example 4

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

[0076] 2. The synthesis method of polymer P4 with dual stimulus response to temperature and pH is basically the same as that of Example 1, except that the oligoethylene glycol diamine used is 3,6,9,12,15-pentaoxaheptadecan-1,17-diamine, and the added amount is 1.28 g. The reaction formula is as follows:

[0077]

[0078] Comparative Example 1

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

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

[0081] Example 5 Characterization and Testing

[0082] 1. The polymer was analyzed by gel permeation chromatography. 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 15700, 18300, 20100 and 22300, respectively.

[0083] 2. Dissolve P1, P2, P3, P4 and P5 polymers in water (concentration of 0.5wt%), test the transmittance of the solution using an ultraviolet spectrophotometer at a constant heating rate (1°C / min), and obtain the polymer temperature sensitivity curve. Figure 3 As shown, it can be clearly observed that the polymer of the present invention has very obvious LCST 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 increases from 22.0°C to 67.5°C. For P5, it cannot be dissolved even when the temperature drops to near freezing point.

[0084] 3. Take polymer P2 as a representative, dissolve it in water (concentration is 0.5wt%), adjust the pH of the system and measure its phase transition behavior at different pH. Figure 4 As shown, 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 amine group and better hydrophilicity.

[0085] 4. Dissolve siRNA and polymer P2 in PBS buffer solution. According to the N / P ratio (N / P = amino content in polymer / phosphate content in siRNA), add the polymer solution to a certain amount of siRNA solution (siRNA concentration is 0.2 mM) through a pipette and react at room temperature for 30 minutes to ensure complete conjugation. The resulting conjugate was analyzed by gel electrophoresis. The results are as follows: Figure 5 As shown, as the N / P ratio gradually increases, the electrophoretic ability of negatively charged siRNA gradually decreases. When N / P≥16, there is no free siRNA, indicating that polymer P2 can conjugate siRNA through electrostatic interaction.

[0086] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A compound containing a bis-isatoic anhydride functional group, characterized in that: It has the structure shown in formula (1):

2. A method for preparing a compound containing a bis-isatoic anhydride functional group according to claim 1, characterized in that: The method comprises the following steps: isatoic anhydride and triethylene glycol react under the action of triphenylphosphine and diisopropyl azodicarboxylate to obtain the product.

3. The method for preparing a compound containing a bis-isatoic anhydride functional group according to claim 2, characterized in that: The reaction is carried out in a solvent, and the solvent is preferably tetrahydrofuran; and / or, The reaction temperature is 15°C to 40°C, preferably 20°C to 30°C; and / or, The reaction time is 12h to 48h, preferably 20h to 30h; and / or, The molar ratio of isatoic anhydride, triphenylphosphine, triethylene glycol and diisopropyl azodicarboxylate is 2.0-3.0:2.0-3.0:1:2.0-3.0, preferably 2.1-2.3:2.1-2.3:1:2.1-2.

3.

4. The method for preparing a compound containing a bis-isatoic anhydride functional group according to claim 2 or 3, characterized in that: The steps include: Dissolve isocyanic anhydride, triphenylphosphine and triethylene glycol in tetrahydrofuran, place in an ice bath, dropwise add a tetrahydrofuran solution of diisopropyl azodicarboxylate into the resulting solution, raise the temperature to 15°C to 40°C and react for 12h to 48h to obtain the product.

5. A polymer that responds to both pH and temperature stimuli, characterized in that: It has the structure shown in formula (2): Wherein x is selected from 2, 3, 4, 5; Preferably, the number average molecular weight of the polymer is 15,000-23,000.

6. A polymer that responds to both pH and temperature stimuli, characterized in that: The compound is obtained by polymerization reaction of the compound containing bis-isatoic anhydride functional group as claimed in claim 1 with oligoethylene glycol diamine.

7. The pH and temperature dual stimulus responsive polymer according to claim 6, characterized in that: The 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; and / or, The molar ratio of the compound containing the bis-isatoic anhydride functional group to the oligoethylene glycol diamine is 1:

1.

8. A method for preparing a polymer responsive to both pH and temperature dual stimulation according to any one of claims 5 to 7, characterized in that: The steps include: The compound containing the bis-isatoic anhydride functional group as claimed in claim 1 is added to a mixed solvent of N,N-dimethylformamide and water, heated to dissolve and then cooled to 15°C to 30°C, and then oligoethylene glycol diamine is added and reacted at a temperature of 15°C to 30°C to obtain the product.

9. The method for preparing a polymer responsive to both pH and temperature stimulation according to claim 8, characterized in that: The mass ratio of N,N-dimethylformamide to water in the mixed solvent of N,N-dimethylformamide and water is 99:0.5 to 98:2, preferably 99:1; and / or, The temperature of the heating dissolution is 38°C to 42°C; and / or, The reaction time is 6 h to 48 h, preferably 20 h to 30 h; and / or, After the reaction is completed, the following post-treatment steps are also included: after the reaction is completed, the reaction solution is precipitated in ether, the obtained precipitate is redissolved and reprecipitated, and this operation is repeated 2-4 times, and finally the obtained solid is dried to obtain the polymer that responds to both pH and temperature stimulations.

10. Use of the pH and temperature dual stimulus responsive polymer according to any one of claims 5 to 7 in the preparation of a carrier for loading or delivering siRNA.

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