Folic acid heparin-iron compound as well as preparation method and application thereof

Abstract

The invention discloses a folic acid heparin-iron compound and a preparation method and application thereof.The folic acid heparin-iron compound is of a microsphere structure, the size is 20-100 nm, the folic acid heparin-iron compound is composed of folic acid, heparin and iron, and the folic acid heparin-iron compound is used for tumor targeted therapy through the emerging treatment strategy of ferroptosis. The ferroptosis tumor treatment material folic acid heparin-iron compound provided by the invention shows concentration-dependent cell inhibition when being co-cultured with tumor cells for different times, which indicates that the material has good tumor cell inhibition ability.

Description

Technical Field

[0001] The invention belongs to the field of biomedical materials, and in particular relates to a heparin folate-iron complex and a preparation method and application thereof. Background Art

[0002] Heparin is a natural polysaccharide composed of highly sulfated uronic acid and glucosamine in alternating arrangement. It can affect the growth of transplanted tumors and is an endogenous polysaccharide widely present in human cells. It has good biocompatibility and safety, and the probability of immune rejection when administered is low. In addition, there are a large number of chemical groups such as carboxyl, amino and hydroxyl groups in heparin molecules, which provide the possibility for the modification of heparin. Therefore, heparin and its derivatives are widely used as drug carriers. The active targeted delivery strategy aims to modify specific targeting molecules on the surface of the drug delivery system, guide the modified nanodrugs to cross the physiological barrier and increase the distribution of drugs in the lesion site by identifying the corresponding targets. Folic acid can achieve active targeting by identifying folate receptors highly expressed in tumor tissues.

[0003] However, although the existing technologies have shown good biocompatibility and certain targeting potential when using heparin as a drug carrier for anti-tumor treatment, most of them focus on the delivery of traditional chemotherapy drugs, and the research on the use of emerging ferroptosis mechanisms for anti-tumor treatment is still insufficient. In particular, there is still a gap in how to effectively bind iron ions to heparin-based carriers and increase the iron ion concentration in the tumor site through specific targeting strategies such as folate receptor-mediated active targeting, thereby inducing ferroptosis. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a folate heparin-iron complex with good tumor inhibition ability; another purpose of the present invention is to provide a preparation method and application of the folate heparin-iron complex.

[0005] Technical solution: The folic acid heparin-iron complex described in the present invention is a microsphere structure with a size of 20 to 100 nm, and is composed of folic acid, heparin and iron; the mass ratio of folic acid, heparin and iron is 1:4 to 6:0.1 to 0.5.

[0006] Preferably, the size of the heparin folate-iron complex is 20 to 50 nm.

[0007] The method for preparing the heparin folate-iron complex of the present invention comprises the following steps:

[0008] (1) cation exchange resin pretreatment;

[0009] (2) dissolving heparin in deionized water, passing it through the cation exchange resin pretreated in step (1), collecting the acidic part of the solution, adjusting the pH of the solution to 8-9, and then freeze-drying the solution. Dissolving the freeze-dried solid in a solvent, wherein the solvent is anhydrous DMF or anhydrous DMSO; adding excess succinic anhydride, reacting, dialyzing the resulting solution in deionized water, and then freeze-drying to obtain a white powder solid;

[0010] (3) dissolving the white powder solid obtained in step (2) in a solvent, wherein the solvent is anhydrous DMSO or anhydrous DMF; adding a coupling agent, reacting, adding folic acid, stirring the reaction, dialyzing the reaction solution, and lyophilizing to obtain a yellow solid; dissolving the yellow solid, passing it through a cation exchange resin, collecting the acidic liquid, adjusting the pH value to 8 to 9, and then lyophilizing to obtain a light yellow solid;

[0011] (4) dissolving the light yellow solid obtained in step (3) to obtain a heparin folate solution, adding FeCl3 solution, stirring to react, dialyzing and then freeze-drying to obtain a heparin folate-iron complex.

[0012] Preferably, the mass ratio of the white powder solid to folic acid in step (3) is 4 to 6:1; and the mass ratio of folic acid heparin to FeCl3 in step (4) is 8 to 40:1.

[0013] Preferably, in step (1), the specific method of pretreatment is to load the cation exchange resin into a silica gel glass column and rinse it with deionized water until it is colorless, then acidify the ion resin with hydrochloric acid, stop acidification when the collected solution is acidic, and then rinse the silica gel column with deionized water, and stop rinsing when the pH of the outflowing solution is close to neutral for later use.

[0014] Further preferably, the cation exchange resin is 732 cation exchange resin.

[0015] More preferably, the hydrochloric acid concentration is 1 to 3 mol / L.

[0016] Preferably, in step (2), soluble heparin is dissolved in deionized water, passed through the cation exchange resin pretreated in step (1), and the acidic part of the solution is collected. After the collection is completed, the pH of the collected liquid is adjusted to 8-9 with an organic amine, and then the solution is freeze-dried. At room temperature, the freeze-dried solid is dissolved in a solvent, and the solvent is anhydrous DMF or anhydrous DMSO; excess succinic anhydride is added, and after reacting for 24h to 48h, the resulting solution is dialyzed in deionized water for 3-5 days to remove excess succinic anhydride raw material and solvent, and the dialyzed solution is freeze-dried to obtain a white powder solid.

[0017] More preferably, the organic amine is triethylamine or tri-n-butylamine.

[0018] More preferably, the concentration of the freeze-dried solid in the solvent is 0.05-2 g / ml.

[0019] Preferably, in step (3), the white powder solid obtained in step (2) is placed in a solvent, wherein the solvent is anhydrous DMSO or anhydrous DMF; N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) coupling agent are added, and the reaction is carried out at room temperature for 3 to 5 hours, and then the folic acid solution is added dropwise to the above solution. After stirring and reacting at room temperature overnight, the reaction solution is dialyzed with deionized water for 3 to 5 days, and the dialyzate is freeze-dried under vacuum conditions for 3 to 5 days to obtain a yellow solid. After the yellow solid is dissolved with a small amount of water, it is passed through a cation exchange resin, the acidic liquid is collected, a NaOH solution is added, and the pH value is adjusted to 8 to 9 and then freeze-dried to obtain a light yellow solid.

[0020] Further preferably, the solvent of the folic acid solution is anhydrous DMSO or anhydrous DMF.

[0021] Further preferably, the concentration of the NaOH solution is 1 mol / L.

[0022] Preferably, the concentration of the white powder solid obtained in step (2) in the solvent is 0.01 to 2 g / ml, the concentration of N-hydroxysuccinimide (NHS) in the solvent is 0.001 to 0.1 g / ml, and the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in the solvent is 0.001 to 0.1 g / ml.

[0023] Preferably, in step (4), the light yellow solid obtained in step (3) is dissolved in deionized water at room temperature by stirring to obtain a heparin folate solution, a FeCl3 solution is prepared, and the FeCl3 solution is added dropwise to the heparin folate solution at room temperature. After stirring overnight at room temperature, the reaction solution is dialyzed in deionized water at room temperature to remove uncoordinated FeCl3 solution, and the obtained solution is freeze-dried to obtain a heparin folate-iron complex.

[0024] Further preferably, the concentration of the FeCl3 solution is 10-20 mg / ml.

[0025] Preferably, the concentration of the light yellow solid in deionized water is 10 to 250 mg / ml.

[0026] Application of the folate heparin-iron complex of the present invention or the folate heparin-iron complex prepared by any preparation method of the present invention in tumor treatment.

[0027] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the present invention constructs a folate heparin-iron complex, increases and optimizes iron ions as an anti-tumor therapeutic agent, and uses it for tumor targeted therapy through the emerging therapeutic strategy of ferroptosis; the ferroptosis tumor treatment material folate heparin-iron complex provided by the present invention, when co-cultured with tumor cells for different time periods, all exhibits concentration-dependent cell inhibition, indicating that the material has good tumor cell inhibition ability. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a transmission electron micrograph of the folic acid heparin-iron complex microspheres of Example 1 of the present invention;

[0029] Figure 2 This is an infrared image of the folic acid heparin-iron complex of Example 1 of the present invention;

[0030] Figure 3 This is a diagram of cell activity at different times and concentrations of the folic acid heparin-iron complex of Example 1 of the present invention;

[0031] Figure 4 This is a diagram of the level of lipid peroxide generation of folic acid, heparin-iron complex according to Example 1 of the present invention. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0033] Embodiment 1:

[0034] ①Put 500g of 732 cationic resin into a silica glass column and rinse it with deionized water until it becomes colorless. Then, acidify the ion resin with 1mol / L hydrochloric acid. When the collected solution has a pH value of ~3, stop acidification. Rinse the silica gel column with 2L deionized water. When the pH value of the outflowing solution reaches ~7, stop rinsing and set aside.

[0035] ② Dissolve 10 g of heparin in 20 mL of deionized water, add 10 mL of triethylamine to the solution of the acidic part collected in ①, adjust the pH of the collected liquid to 8.5, freeze-dry to obtain 9.5 g of white powder, dissolve it in 50 mL of anhydrous DMF, add 4.5 g of succinic anhydride, react at room temperature for 24 hours, dialyze the resulting solution in deionized water for 5 days, and freeze-dry to obtain 10.5 g of white powder solid.

[0036] ③ Take 8g of the white solid obtained in ②, add 50ml of anhydrous dimethyl sulfoxide to dissolve, add N-hydroxysuccinimide (NHS 0.25g) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI 0.30g) coupling agents, react at room temperature for 3 hours, add 2g of folic acid in dimethyl sulfoxide (DMSO) solution dropwise to the above solution, stir and react at room temperature for 24h, dialyze the reaction solution with deionized water for 5 days, freeze-dry for 3 days to obtain 4.8g of yellow solid, dissolve it with 5mL of water, pass it through 732 cation exchange resin, collect the acidic liquid, add 1mol / L NaOH solution, adjust the pH value to 8.2, and freeze-dry to obtain 8.5g of light yellow solid (HF).

[0037] ④ Preparation of folate heparin-iron complex: Take 200 mg of the powder prepared in ③, stir and dissolve it in 5 mL of deionized water at room temperature to obtain a folate heparin solution, prepare a 10 mg / mL FeCl3 solution, add 1 mL of the folate heparin solution at room temperature, stir at room temperature for 24 h, dialyze the reaction solution in deionized water at room temperature, and lyophilize to obtain a folate heparin-iron complex (HF-Fe).

[0038] Embodiment 2:

[0039] ①Put 500g of 732 cationic resin into a silica glass column and rinse it with deionized water until it is colorless. Then acidify the ion resin with 1mol / L hydrochloric acid. When the pH of the solution is ~3, stop acidification. Rinse the silica gel column with deionized water. When the pH of the outflowing solution is ~7, stop rinsing and set aside.

[0040] ② Dissolve 5 g of heparin in 10 mL of deionized water, pass through the cation exchange resin pretreated in ①, collect the acidic part of the solution, and after the collection is completed, add 10 mL of tri-n-butylamine to adjust the pH of the collected liquid to 8.6, and then freeze-dry the solution to obtain 4.5 g of white powder. At room temperature, take 4.5 g of white solid powder and dissolve it in 30 mL of anhydrous DMF, add 2.5 g of succinic anhydride, react at room temperature for 24 hours, and then dialyze the resulting solution in deionized water for 3 days to remove excess succinic anhydride raw material and solvent. The dialyzed solution is freeze-dried to obtain 4.8 g of white powder solid.

[0041] ③ Take 4.8 g of the white solid obtained in ②, add 50 mL of anhydrous dimethyl sulfoxide to dissolve, add N-hydroxysuccinimide (NHS 0.15 g) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI 0.22 g) coupling agents, react at room temperature for 3 hours, add 0.8 g of folic acid in dimethyl sulfoxide (DMSO) solution dropwise to the above solution, stir and react at room temperature for 24 hours, dialyze the reaction solution with deionized water for 5 days, and freeze-dry the dialyzate under vacuum conditions for 3 days to obtain 4.1 g of yellow solid, dissolve the yellow solid with 10 mL of water, pass it through 732 cation exchange resin, collect the acidic liquid, add 1 mol / L NaOH solution, adjust the pH value to 8.8, and freeze-dry to obtain 3.2 g of light yellow solid (HF).

[0042] ④ Take 200 mg of the powder prepared in ③, stir and dissolve it in 5 mL of deionized water at room temperature to obtain a heparin folate solution, add 2.5 mL of the prepared 10 mg / mL FeCl3 solution dropwise to the heparin folate solution at room temperature, stir at room temperature for 24 h, dialyze the reaction solution in deionized water at room temperature to remove the uncoordinated FeCl3 solution, and lyophilize the obtained solution to obtain the heparin folate-iron complex (HF-Fe).

[0043] Embodiment 3:

[0044] ①Put 500g of cationic resin into a silica glass column and rinse it with deionized water until it becomes colorless. Then, acidify the ion resin with 1mol / L hydrochloric acid. When the pH of the solution is ~3, stop acidification. Rinse the silica gel column with deionized water. When the pH of the outflowing solution is ~7, stop rinsing and set aside.

[0045] ② Dissolve 5 g of heparin in 10 mL of deionized water, pass through the pretreated cation exchange resin in ①, collect the acidic part of the solution, and after the collection is completed, add 10 mL of tri-n-butylamine to adjust the pH of the collected liquid to 8.6, and then freeze-dry the solution to obtain 4.5 g of white powder. At room temperature, take 4.5 g of white solid powder and dissolve it in 30 mL of anhydrous DMF, add 2.5 g of succinic anhydride, react at room temperature for 24 hours, and then dialyze the resulting solution in deionized water for 5 days to remove excess succinic anhydride raw material and solvent. The dialyzed solution is freeze-dried to obtain 4.8 g of white powder solid.

[0046] ③ Take 4.8 g of the white solid obtained in ②, add 50 mL of anhydrous dimethyl sulfoxide to dissolve it, then add 0.25 g of 1-hydroxybenzotriazole, react at room temperature for 3 hours, then add 0.8 g of folic acid in dimethyl sulfoxide (DMSO) solution dropwise to the above solution, stir and react at room temperature for 24 hours, dialyze the reaction solution with deionized water for 5 days, and freeze-dry the dialyzate under vacuum conditions for 3 days to obtain 4.1 g of yellow solid, dissolve the yellow solid with a small amount of water, pass it through a cation exchange resin, collect the acidic liquid, add 1 mol / L NaOH solution, adjust the pH to 8.5, and then freeze-dry to obtain 3.2 g of light yellow solid (HF).

[0047] ④ Take 300 mg of the powder prepared in ③, stir and dissolve it in 5 mL of deionized water at room temperature to obtain a heparin folate solution, add 1.5 mL of the prepared 10 mg / mL FeCl3 solution dropwise to the heparin folate solution at room temperature, stir at room temperature for 24 h, dialyze the reaction solution in deionized water at room temperature to remove the uncoordinated FeCl3 solution, and lyophilize the obtained solution to obtain the heparin folate-iron complex (HF-Fe).

[0048] Figure 1 This is a transmission electron micrograph of the folate heparin-iron complex microspheres of Example 1 of the present invention. As can be seen from the figure, the size of the microspheres is less than 100 nm, and they can reach the tumor through the vascular system, and are suitable for tumor treatment; Figure 2 The infrared image of the heparin folate-iron complex of the present invention shows that the heparin folate compounded with Fe retains the main characteristic absorption peaks of its parent body, with the wave number of 1200-800cm -1 The fingerprint region corresponds to the coordination chelation between Fe and heparin, indicating that the folic acid heparin-iron complex of the present invention was successfully prepared; Figure 3 This is a cell activity diagram of the folic acid heparin-iron complex of the present invention at different times and concentrations. The material and tumor cells were co-cultured for different times, and concentration-dependent cell inhibition was shown. This shows that the material of the present invention has good tumor cell inhibition ability; Figure 4 The folate heparin-iron composite lipid peroxide generation level of the present invention is that when the material is co-incubated with cells, obvious green fluorescence is generated in the oxidized state, indicating that the material of the present invention has a significant tumor ferroptosis treatment effect.

Claims

1. A folic acid heparin-iron complex, characterized in that: The folic acid heparin-iron complex is a microsphere structure with a size of 20-100 nm, and is composed of folic acid, heparin and iron; the mass ratio of the folic acid, heparin and iron is 1:4-6:0.1-0.

5.

2. A method for preparing the folic acid heparin-iron complex according to claim 1, characterized in that: The following steps are involved: (1) cation exchange resin pretreatment; (2) dissolving heparin in deionized water, passing it through the cation exchange resin pretreated in step (1), collecting the acidic part of the solution, adjusting the pH of the solution to 8-9, and then freeze-drying the solution. Dissolving the freeze-dried solid in a solvent, wherein the solvent is anhydrous DMF or anhydrous DMSO; adding excess succinic anhydride, reacting, dialyzing the resulting solution in deionized water, and then freeze-drying to obtain a white powder solid; (3) dissolving the white powder solid obtained in step (2) in a solvent, wherein the solvent is anhydrous DMSO or anhydrous DMF; adding a coupling agent, reacting, adding folic acid, stirring the reaction, dialyzing the reaction solution, and lyophilizing to obtain a yellow solid; dissolving the yellow solid, passing it through a cation exchange resin, collecting the acidic liquid, adjusting the pH value to 8 to 9, and then lyophilizing to obtain a light yellow solid; (4) dissolving the light yellow solid obtained in step (3) to obtain a heparin folate solution, adding FeCl3 solution, stirring to react, dialyzing and then freeze-drying to obtain a heparin folate-iron complex.

3. The method for preparing the folic acid heparin-iron complex according to claim 2, characterized in that: The mass ratio of the white powder solid to folic acid in step (3) is 4 to 6:1; the mass ratio of folic acid heparin to FeCl3 in step (4) is 8 to 40:

1.

4. The method for preparing the folic acid heparin-iron complex according to claim 2, characterized in that: In step (1), the specific method of pretreatment is to load the cation exchange resin into a silica gel glass column and rinse it with deionized water until it is colorless, then acidify the ion resin with hydrochloric acid, stop acidification when the collected solution is acidic, and then rinse the silica gel column with deionized water. When the pH of the outflowing solution is close to neutral, stop rinsing and set aside.

5. The method for preparing the folic acid heparin-iron complex according to claim 2, characterized in that: In step (2), soluble heparin is dissolved in deionized water, passed through the cation exchange resin pretreated in step (1), and the acidic part of the solution is collected. After the collection is completed, the pH of the collected liquid is adjusted to 8-9 with an organic amine, and then the solution is freeze-dried. At room temperature, the freeze-dried solid is dissolved in a solvent, wherein the solvent is anhydrous DMF or anhydrous DMSO; excess succinic anhydride is added, and after reacting for 24h-48h, the obtained solution is dialyzed in deionized water for 3-5 days to remove excess succinic anhydride raw material and solvent, and the dialyzed solution is freeze-dried to obtain a white powder solid.

6. The method for preparing the folic acid heparin-iron complex according to claim 2, characterized in that: In step (3), the white powder solid obtained in step (2) is placed in a solvent, wherein the solvent is anhydrous DMSO or anhydrous DMF; N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) coupling agent are added, and the mixture is reacted at room temperature for 3 to 5 hours, and then the folic acid solution is added dropwise to the above solution. After stirring and reacting at room temperature overnight, the reaction solution is dialyzed with deionized water for 3 to 5 days, and the dialyzate is freeze-dried under vacuum conditions for 3 to 5 days to obtain a yellow solid. The yellow solid is dissolved with a small amount of water, and then passed through a cation exchange resin, the acidic liquid is collected, a NaOH solution is added, and the pH value is adjusted to 8 to 9 and then freeze-dried to obtain a light yellow solid.

7. The method for preparing the folic acid heparin-iron complex according to claim 6, characterized in that: The concentration of the white powder solid obtained in step (2) in the solvent is 0.01 to 2 g / ml, the concentration of N-hydroxysuccinimide (NHS) in the solvent is 0.001 to 0.1 g / ml, and the concentration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI) in the solvent is 0.001 to 0.1 g / ml.

8. The method for preparing the folic acid heparin-iron complex according to claim 2, characterized in that: In step (4), the light yellow solid obtained in step (3) is dissolved in deionized water at room temperature by stirring to obtain a heparin folate solution, a FeCl3 solution is prepared, and the FeCl3 solution is added dropwise to the heparin folate solution at room temperature. After stirring overnight at room temperature, the reaction solution is dialyzed in deionized water at room temperature to remove uncoordinated FeCl3 solution, and the obtained solution is freeze-dried to obtain a heparin folate-iron complex.

9. The method for preparing the folic acid heparin-iron complex according to claim 8, characterized in that: The concentration of the light yellow solid in deionized water is 10-250 mg / ml.

10. Use of the folate heparin-iron complex according to claim 1 or the folate heparin-iron complex prepared by any one of the preparation methods of claims 2 to 9 in tumor treatment.

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