Preparation method and application of a p(nipam-co-am) loaded hypericin hydrogel

Abstract

The present application relates to a preparation method and application of a hypericin composite hydrogel with P(NIPAM-co-AM) as a carrier, and belongs to the technical field of medical materials. N-isopropyl acrylamide monomers and acrylamide are dissolved in deionized water, a crosslinking agent and an initiator are added to the above solution to form a hydrogel precursor solution; hypericin is dissolved in an organic solvent, added to the hydrogel precursor solution, and a promoter is added, the solution is mixed thoroughly, poured into a glass mold, and an ice water bath is used to obtain a hydrogel, and the organic solvent and unreacted monomers are removed. The advantage is that the LCST of NIPAM is increased to 38.49 DEG C by modification with AM, which is closer to the normal human body temperature; real-time intelligent control release of the hypericin drug is realized by temperature stimulation, which significantly improves the solubility and bioavailability of the hypericin drug; it can be used for treating trichinella infection in tissues and organs, and it is proved that the hypericin has a good treatment effect on tissue and organ parasites.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical materials technology, and specifically relates to a method for preparing hypericin composite hydrogel with P(NIPAM-co-AM) as a carrier and its application. Background Technology

[0002] Hydrogels are a class of highly hydrophilic three-dimensional network gels with numerous advantages such as good biocompatibility, softness, and biodegradability. They can provide an environment similar to natural tissues and have attracted widespread attention in biomedical and other fields. Poly(N-isopropylacrylamide) (PNIPAM) is a typical thermosensitive hydrogel, polymerized from the monomer N-isopropylacrylamide (NIPAM). The chemical formula of NIPAM is C6H. 11 NO has the structure shown in formula (Ⅰ).

[0003]

[0004] PNIPAM exhibits excellent temperature sensitivity, displaying different pore sizes at varying temperatures, thus making it widely used in drug delivery and cell encapsulation. PNIPAM possesses a low critical solution temperature (LCST) close to human body temperature, undergoing a rapid hydrophilic-to-hydrophobic transition around 32°C. Below the LCST, the hydrogel absorbs water and swells. Above the LCST, the hydrogel shrinks, reducing the drug-carrying space provided by the carrier and allowing drug release, thus achieving intelligent controlled drug release. Modification of PNIPAM can improve its mechanical properties, regulate its LCST value, and enhance its temperature response rate, enabling better application of its temperature-sensitive properties in tissue engineering and sustained drug release.

[0005] Acrylamide (AM) is an organic compound with the chemical formula C3H5NO and has the structure shown in formula (II).

[0006]

[0007] Ammonium acetate (AM) has a wide range of applications, commonly used as a raw material in organic synthesis and polymer materials. Its polymers are water-soluble, making it suitable for producing flocculants for water treatment, particularly effective at flocculating proteins and starches in water. Besides flocculation, it also possesses excellent thickening, shear resistance, drag reduction, and dispersing properties. As a soil conditioner, it increases soil water permeability and moisture retention; as a paper filler, it increases paper strength, replacing starch and water-soluble ammonia resins; as a chemical grouting agent, it is used for leak sealing in civil engineering projects such as tunnel excavation, oil well drilling, mines, and dams; as a fiber modifier, it improves the properties of synthetic fibers; as a preservative, it can be used for corrosion protection of underground components; and it can also be used as an additive in the food industry, a dispersant for pigments, and a printing and dyeing paste. Combined with phenolic resin solutions, it can be used to make adhesives for glass fibers, and with rubber, it can be used to make pressure-sensitive adhesives. Polymerization with monomers such as vinyl acetate, styrene, vinyl chloride, and acrylonitrile can produce many synthetic materials. AM can also be used as a raw material for pharmaceuticals, pesticides, dyes, and coatings.

[0008] Hypericin is a dianthrone compound, specifically 4,4',5,5',7,7'-hexahydroxy-2,2'-dimethyl-meso-naphthodianthrone, with the chemical formula C. 30 H 16 O8 has the structure shown in formula (Ⅲ).

[0009]

[0010] Hypericin is found in St. John's wort and related plants, and is the most bioactive substance in St. John's wort. Hypericin possesses various pathological effects, including antiviral, antibacterial, and antidepressant properties. Simultaneously, hypericin is also a good natural photosensitizer; upon photoactivation, it produces singlet oxygen, which can be used to treat various cancers. Our research has found that hypericin is an immune agonist, capable of producing type I interferon and inflammatory cytokines, and can be applied to the immunotherapy of Trichinella spiralis. Hypericin has high medicinal value, but its insolubility in water greatly limits its clinical application. Common drug delivery carriers include liposomes, micelles, and nanoparticles, but they cannot simultaneously achieve high drug loading capacity, high drug delivery efficiency, excellent biocompatibility and degradability, and controlled drug release. Summary of the Invention

[0011] This invention provides a method for preparing P(NIPAM-co-AM) loaded hypericin hydrogel and its application, in order to solve the problems of poor water solubility and low bioavailability of hypericin. The method achieves real-time intelligent controlled release of hypericin drug through temperature response and applies it to anti-trichinosis, demonstrating that hypericin has a good therapeutic effect on tissue and organ parasites.

[0012] The technical solution adopted in this invention is a poly(N-isopropylacrylamide-co-acrylamide)

[0013] The preparation method of P(NIPAM-co-AM) loaded hypericin hydrogel includes the following steps:

[0014] First, dissolve 1000–2000 mg of N-isopropylacrylamide monomer and 50–100 mg of acrylamide in 8 mL of deionized water;

[0015] The second step involves adding 5–10 mg of crosslinking agent and 20–40 mg of initiator to the above solution to form a hydrogel precursor solution.

[0016] The third step is to dissolve 20-30 mg of hypericin in 2 mL of organic solvent, resulting in a concentration of 10-15 mg / mL.

[0017] The fourth step is to add the prepared hypericin solution to the hydrogel precursor solution, and add 20-30 μL of accelerator, and stir vigorously with a magnetic stirrer.

[0018] Fifth step: Pour the thoroughly mixed solution into a glass mold and place it in an ice water bath to obtain a hydrogel;

[0019] Step 6: Place the hydrogel in deionized water for 2-4 hours to remove organic solvents and unreacted monomers.

[0020] In the first step of this invention, the content of NIPAM is 125-250 mg / mL and the content of acrylamide is 12.5-25 mg / mL.

[0021] The crosslinking agent used in the second step of this invention is N,N'-methylenebisacrylamide.

[0022] In the second step of this invention, the initiator is potassium persulfate.

[0023] The organic solvent mentioned in the third step of this invention is one of Tween 80 and DMSO.

[0024] The accelerator used in the fourth step of this invention is tetramethylethylenediamine.

[0025] A P(NIPAM-co-AM)-loaded hypericin hydrogel prepared using the method of the present invention.

[0026] Application of P(NIPAM-co-AM) loaded hypericin hydrogel in the preparation of drugs for treating parasitic infections of tissues and organs.

[0027] Application of P(NIPAM-co-AM) loaded hypericin hydrogel in the preparation of drugs for treating trichinosis infection of tissues and organs.

[0028] The beneficial effects of this invention are:

[0029] The P(NIPAM-co-AM) hypericin-loaded hydrogel obtained by the method of this invention, through AM modification of NIPAM, raises its LCST to 38.49℃, which is closer to normal human body temperature; real-time intelligent controlled release of hypericin drug is achieved through temperature stimulation, significantly improving the solubility and bioavailability of hypericin drug; it can be used to treat trichinosis infection in tissues and organs, proving that hypericin has a good therapeutic effect on parasites in tissues and organs. Attached Figure Description

[0030] Figure 1 This is the Fourier transform infrared (FTIR) spectrum of the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in this invention;

[0031] Figure 2 This is the LCST measurement diagram of the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in this invention;

[0032] Figure 3 This is a scanning electron microscope (SEM) image of the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in this invention.

[0033] Figure 4 This is a diagram showing the effect of P(NIPAM-co-AM) loaded hypericin hydrogel on trichinella larvae under a microscope at 4×10 magnification.

[0034] Figure 5 This is a pathological histochemical staining image of the diaphragm of mice used in an in vivo drug experiment. Detailed Implementation

[0035] Example 1

[0036] First, weigh 1000 mg of N-isopropylacrylamide and 50 mg of acrylamide, add them to 8 mL of deionized water, and stir at room temperature until completely dissolved.

[0037] The second step involves adding 5 mg of cross-linking agent N,N'-methylenebisacrylamide and 20 mg of initiator potassium persulfate to the above solution to form a hydrogel precursor solution.

[0038] Third step: Weigh 20mg hypericin and add it to 2mL of organic solvent DMSO or Tween-80, and sonicate for 5-10 minutes to completely dissolve it.

[0039] The fourth step involves adding the prepared hypericin solution to the hydrogel precursor solution, along with 20 μL of the accelerator tetramethylethylenediamine, and stirring vigorously with a magnetic stirrer.

[0040] Fifth step: Pour the above thoroughly mixed solution into a glass mold and place it in an ice-water bath to obtain P(NIPAM-co-AM) loaded hypericin hydrogel;

[0041] Step 6: Place the prepared hydrogel in deionized water for 2 hours to remove organic solvents and unreacted monomers.

[0042] Example 2

[0043] First, weigh 1500 mg of N-isopropylacrylamide and 75 mg of acrylamide, add them to 8 mL of deionized water, and stir at room temperature until completely dissolved.

[0044] The second step involves adding 7.5 mg of crosslinking agent N,N'-methylenebisacrylamide and 30 mg of initiator potassium persulfate to the above solution to form a hydrogel precursor solution.

[0045] Third step: Weigh 25mg hypericin and add it to 2mL of organic solvent DMSO or Tween-80, and sonicate for 5 to 10 minutes to completely dissolve it.

[0046] The fourth step involves adding the prepared hypericin solution to the hydrogel precursor solution, along with 25 μL of the accelerator tetramethylethylenediamine, and stirring vigorously with a magnetic stirrer.

[0047] Fifth step: Pour the above thoroughly mixed solution into a glass mold and place it in an ice-water bath to obtain P(NIPAM-co-AM) loaded hypericin hydrogel;

[0048] Step 6: Place the prepared hydrogel in deionized water for 3 hours to remove organic solvents and unreacted monomers.

[0049] Example 3

[0050] First, weigh 2000 mg N-isopropylacrylamide and 100 mg acrylamide, add them to 8 mL of deionized water, and stir at room temperature until completely dissolved;

[0051] The second step involves adding 10 mg of cross-linking agent N,N'-methylenebisacrylamide and 40 mg of initiator potassium persulfate to the above solution to form a hydrogel precursor solution.

[0052] Third step: Weigh 30mg hypericin and add it to 2mL of organic solvent DMSO or Tween-80, and sonicate for 5 to 10 minutes to completely dissolve it.

[0053] The fourth step involves adding the prepared hypericin solution to the hydrogel precursor solution, along with 30 μL of the accelerator tetramethylethylenediamine, and stirring vigorously with a magnetic stirrer.

[0054] Fifth step: Pour the above thoroughly mixed solution into a glass mold and place it in an ice-water bath to obtain P(NIPAM-co-AM) loaded hypericin hydrogel;

[0055] Step 6: Place the prepared hydrogel in deionized water for 4 hours to remove organic solvents and unreacted monomers.

[0056] Figure 1 The image shows the Fourier transform infrared (FTIR) spectrum of the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in Example 3 of this invention. As can be seen from the image, in the P(NIPAM-co-AM)-loaded hypericin hydrogel, at 3441.34 cm⁻¹... -1 The peak at 1032.69 cm⁻¹ represents the stretching vibration of the hydroxyl group (-OH). -1 The peaks at 1644.02 cm⁻¹ represent stretching vibrations of the CO bond and are characteristic absorption peaks of hypericin. -1 and 1525.42cm -1 The absorption peaks correspond to the stretching vibration peaks of the amide-I band (C=O) and amide-II band (NH) of the amide group (-CONH), respectively, and are characteristic absorption peaks of P(NIPAM-co-AM), proving that P(NIPAM-co-AM) loaded hypericin hydrogel was successfully prepared.

[0057] Figure 2 The image shows the LCST measurement of the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in Example 3 of this invention. As can be seen from the image, by modifying PNIPAM with AM, the LCST of the P(NIPAM-co-AM)-loaded hypericin hydrogel increased to 38.49℃, which is closer to normal human body temperature.

[0058] Figure 3 The image shows a scanning electron microscope (SEM) image of the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in Example 3 of this invention. As can be seen from the image, the P(NIPAM-co-AM)-loaded hypericin hydrogel has an irregular porous structure.

[0059] The effects of the present invention will be illustrated below through pharmacodynamic experiments.

[0060] Experimental Example 1: In vitro pharmacodynamic experiments demonstrated the inhibitory effect of the hydrogel prepared in this invention on the muscle larvae of Trichinella spiralis.

[0061] Hypericin dissolved in PBS, organic solvents, and P(NIPAM-co-AM)-loaded with hypericin hydrogels were added to serum-free RPMI 1640 medium at a concentration of 10 μg / ml. The activity of larvae co-cultured with the drugs in cell culture plates for 24 h was observed under a microscope. Figure 4 The effects of in vitro drugs on the muscle larvae of Trichinella spiralis were demonstrated. For example... Figure 4 As shown in (a), the trichinella muscle larvae in the negative control group with added PBS exhibited "snake-like movements" and good activity; Figure 4 (b) In the control group of hypericin drug dissolved in organic solvents, a large number of insoluble particles were suspended in the liquid, and most of the Trichinella spiralis muscle larvae showed good activity. Figure 4 (c) The P(NIPAM-co-AM)-loaded hypericin hydrogel was clear, with no insoluble particles, and most of the Trichinella spiralis muscle larvae exhibited a "spiral" shape, low activity, and a lack of movement. This demonstrates that the present invention significantly inhibits the activity of Trichinella spiralis muscle larvae in vitro, while significantly improving the solubility of hypericin and its bioavailability.

[0062] Experimental Example 2: In vivo pharmacodynamic experiments demonstrated that the hydrogel prepared in this invention has a good therapeutic effect on parasites in tissues and organs.

[0063] First, 300 trichinella muscle larvae were infected in 6-8 week old mice weighing 18-20g. Twenty-eight days post-infection, mice were administered PBS, hypericin solution dissolved in an organic solvent, or P(NIPAM-co-AM)-loaded hypericin hydrogel via gavage at a dose of 10 mg / kg. One week after administration, the infected mice were sacrificed, and the diaphragm was harvested. The samples were fixed in 4% formalin for 24 hours. Hematoxylin and eosin staining was then performed on the samples, including the preparation of paraffin blocks and 5 μm thick sections. The histopathological features of the HE-stained sections were examined under a microscope. Figure 5 (a) The diaphragm tissue section of the PBS negative control group showed a large number of Trichinella spiralis muscle cysts and extensive infiltration of eosinophils. Figure 5 (b) In the control group treated with hypericin dissolved in organic solvents, the number of Trichinella spiralis muscle larvae cysts decreased and eosinophil infiltration decreased. Figure 5 (c) The number of myolar cysts in the P(NIPAM-co-AM)-loaded hypericin hydrogel group was significantly reduced, accompanied by mild eosinophil infiltration. This demonstrates that the P(NIPAM-co-AM)-loaded hypericin hydrogel prepared in this invention has a good therapeutic effect on parasites in tissues and organs.

Claims

1. The application of a P(NIPAM-co-AM) hypericin-loaded hydrogel in the preparation of a drug for treating trichinosis infection of tissues and organs, wherein the P(NIPAM-co-AM) hypericin-loaded hydrogel is obtained by the following preparation method, comprising the following steps: First, dissolve 1000–2000 mg of N-isopropylacrylamide monomer and 50–100 mg of acrylamide in 8 mL of deionized water; The second step involves adding 5–10 mg of crosslinking agent and 20–40 mg of initiator to the above solution to form a hydrogel precursor solution. The third step is to dissolve 20-30 mg of hypericin in 2 mL of organic solvent, resulting in a concentration of 10-15 mg / mL. The fourth step is to add the prepared hypericin solution to the hydrogel precursor solution, and add 20-30 μL of accelerator, and stir vigorously with a magnetic stirrer. Fifth step: Pour the thoroughly mixed solution into a glass mold and place it in an ice water bath to obtain a hydrogel; Step 6: Place the hydrogel in deionized water for 2-4 hours to remove organic solvents and unreacted monomers.

2. The application of the P(NIPAM-co-AM) hypericin-loaded hydrogel according to claim 1 in the preparation of a medicament for treating trichinosis infection of tissues and organs, characterized in that: In the first step, the content of NIPAM is 125-250 mg / mL and the content of acrylamide is 12.5-25 mg / mL.

3. The application of the P(NIPAM-co-AM) hypericin-loaded hydrogel according to claim 1 in the preparation of a drug for treating trichinosis infection of tissues and organs, characterized in that: The crosslinking agent used in the second step is N,N'-methylenebisacrylamide.

4. The application of the P(NIPAM-co-AM) hypericin-loaded hydrogel according to claim 1 in the preparation of a medicament for treating trichinosis infection of tissues and organs, characterized in that: The initiator used in the second step is potassium persulfate.

5. The application of the P(NIPAM-co-AM) hypericin-loaded hydrogel according to claim 1 in the preparation of a medicament for treating trichinosis infection of tissues and organs, characterized in that: The organic solvent mentioned in the third step is one of Tween 80 and DMSO.

6. The application of the P(NIPAM-co-AM) hypericin-loaded hydrogel according to claim 1 in the preparation of a medicament for treating trichinosis infection of tissues and organs, characterized in that: The accelerator used in the fourth step is tetramethylethylenediamine.

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