Biodegradable nanohydrogel microspheres, their preparation methods, drug-loaded hydrogels and their preparation methods

Abstract

This invention provides a method for preparing biodegradable nanohydrogel microspheres, comprising the following steps: A) irradiating an aqueous solution of polyvinylpyrrolidone with high-energy rays to obtain a micro-crosslinked prepolymer; B) mixing polyvinylpyrrolidone, hyaluronic acid, and water, mixing the resulting initial mixture with the micro-crosslinked prepolymer, irradiating with high-energy rays, and freeze-drying to obtain biodegradable nanohydrogel microspheres. This application also provides drug-loaded hydrogels and their preparation methods. The biodegradable nanohydrogel microspheres prepared by the method provided by this invention have a particle size of 100–500 nm and cytotoxicity level 0; furthermore, the nanohydrogel microspheres prepared using this application can effectively load drugs and have high biosafety. Simultaneously, the method is simple and can be mass-produced.

Description

Technical Field

[0001] This invention relates to the field of new material processing technology, and in particular to biodegradable nanohydrogel microspheres, their preparation methods, drug-loaded hydrogels, and their preparation methods. Background Technology

[0002] Biodegradable materials, as a novel type of intelligent drug carrier, can control the release process of the encapsulated substance through their own degradation. Hydrogels, as a type of polymer material with good biocompatibility, also play a unique role in controlled drug release systems.

[0003] Existing methods for preparing hydrogel microspheres include emulsion polymerization, reverse emulsion polymerization, suspension polymerization, and precipitation polymerization. For example, emulsion polymerization involves selecting a water-in-oil or oil-in-water system based on the polymer structure, and polymerizing the polymer in microdroplets through high-speed emulsification. The particle size of the nanohydrogels prepared by this method can be controlled by adjusting the amount of surfactant and crosslinking agent, as well as the stirring speed. However, these methods introduce excessive amounts of organic solvents, small-molecule emulsifiers, and catalysts, leading to complex post-processing, high preparation costs, and hindering large-scale production.

[0004] Chinese patent application CN201810958392.0 discloses a method for preparing and applying highly biocompatible hydrogel microspheres. It uses polyaspartic acid-based polymers as raw materials to ensure good biodegradability and biocompatibility. The hydrogel microspheres are obtained through a "suspension-like polymerization" process in an organic phase containing a dispersant, ultimately yielding highly biocompatible hydrogel microspheres for applications such as blood purification, drug sustained release, and skincare cosmetics. However, the hydrogel microspheres prepared by this method have a complex reaction system and preparation process, making it difficult to determine the final composition and achieve large-scale production.

[0005] For polymeric compounds used as drug carriers, the most basic requirements are good biocompatibility and good biodegradability. However, due to the complexity of the reaction system and process, these basic requirements are often difficult to achieve. How to achieve a pure reaction system and a simple process to serve as an effective drug carrier has become an urgent problem to be solved in the development of intelligent hydrogel materials. Summary of the Invention

[0006] The technical problem solved by this invention is to provide a method for preparing biodegradable nanohydrogel microspheres. The biodegradable nanohydrogel microspheres prepared by this method can effectively encapsulate small molecule drugs and achieve drug sustained release.

[0007] In view of this, this application provides a method for preparing biodegradable nanohydrogel microspheres, comprising the following steps:

[0008] A) Irradiate an aqueous solution of polyvinylpyrrolidone with high-energy rays to obtain a micro-crosslinked prepolymer;

[0009] B) Polyvinylpyrrolidone, hyaluronic acid and water are mixed to obtain an initial mixture. The initial mixture and the micro-crosslinked prepolymer are mixed and irradiated with high-energy rays. After freeze-drying, biodegradable nanohydrogel microspheres are obtained.

[0010] Preferably, in step A), the concentration of the polyvinylpyrrolidone aqueous solution is 0.3-0.5 wt%, the electron beam energy of the high-energy irradiation is 2-5 MeV, and the irradiation dose is 5-10 kGy.

[0011] Preferably, in step B), the mass ratio of polyvinylpyrrolidone to hyaluronic acid is 1:(0.1-1), and the concentration of polyvinylpyrrolidone in the initial mixture is 0.1-0.5 wt%.

[0012] Preferably, in step B), the electron beam energy of the high-energy irradiation is 2-5 MeV, and the irradiation dose is 10-20 kGy.

[0013] This application also provides biodegradable nanohydrogel microspheres prepared by the aforementioned preparation method, which are block copolymers formed by crosslinking polyvinylpyrrolidone and hyaluronic acid.

[0014] This application also provides a drug-loaded hydrogel, which is composed of hydrogel microspheres and an amino-containing small molecule drug loaded in the hydrogel microspheres, wherein the hydrogel microspheres are biodegradable nano-hydrogel microspheres prepared by the preparation method described above or the biodegradable nano-hydrogel microspheres described above.

[0015] Preferably, the small molecule drug is doxorubicin hydrochloride, methotrexate, or cytarabine.

[0016] This application also provides a method for preparing the drug-loaded hydrogel, including:

[0017] Hydrogel microspheres, amino-containing small molecule drugs, and solvents were mixed in the dark to obtain drug-loaded hydrogels;

[0018] The hydrogel microspheres are the biodegradable nanohydrogel microspheres prepared by the preparation method described above or the biodegradable nanohydrogel microspheres as described in claim 5.

[0019] Preferably, the solvent is a PBS buffer solution with pH=7.4, and the mixing time is 24-48h.

[0020] Preferably, the small molecule drug is 6 to 25 wt% of the total mass of the hydrogel microspheres, the small molecule drug, and the solvent.

[0021] This application provides a method for preparing biodegradable hydrogel microspheres. First, an aqueous solution of polyvinylpyrrolidone (PVP) is irradiated with high-energy rays to cause weak intramolecular cross-linking of PPVP monomolecules, forming a core for microsphere growth. Then, a mixed solution of PPVP and hyaluronic acid is added to the reaction system, allowing the microspheres to continue growing on the cross-linked cores of the PPVP monomolecules. Under irradiation conditions, PPVP and hyaluronic acid undergo intramolecular and intermolecular cross-linking, and hyaluronic acid simultaneously degrades with the irradiation. The resulting chain ends continue to bond with PPVP, thus forming block copolymer microspheres primarily composed of PPVP and hyaluronic acid.

[0022] The nano-hydrogel microspheres provided in this application can encapsulate small molecule drugs through electrostatic interactions between the carboxyl groups on hyaluronic acid and small molecules with amino groups, thereby achieving drug sustained release. When the hydrogel microspheres carrying small molecule drugs enter the human body, the hyaluronic acid segments in the chain are degraded by hyaluronidase in the body, causing the microspheres to disintegrate. As the degradation continues, the small molecule drugs encapsulated in them are gradually released, thereby achieving drug sustained release. Attached Figure Description

[0023] Figure 1 The hydrodynamic radii of PHA-Nanogel and PHA-Dox-Nanogel in Example 1;

[0024] Figure 2 The hydrodynamic radii of PHA-Nanogel and PHA-Nanogel after 3h, 5h and 8h of hyaluronidase treatment in Example 1;

[0025] Figure 3 The hydrodynamic diameter of PHA-Nanogel after 5 hours of hyaluronidase treatment in Examples 1, 4, 5, and 6;

[0026] Figure 4 The results are the cell compatibility test results of the PHA-Nanogel materials in Examples 1, 5, and 6.

[0027] Figure 5 The results of cell proliferation inhibition experiments of PHA-Dox-Nanogel, the drug-loaded hydrogel microspheres in Examples 1, 7, and 8, after treatment with hyaluronidase for 5 hours. Detailed Implementation

[0028] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and not for limiting the scope of the claims of the present invention.

[0029] In view of the performance requirements of polymeric compounds used as drug carriers in the prior art, this application provides a method for preparing biodegradable nanohydrogel microspheres. The prepared biodegradable nanohydrogel microspheres can effectively load drugs and achieve sustained drug release. Specifically, this application provides a method for preparing biodegradable nanohydrogel microspheres, including the following steps:

[0030] A. High-energy radiation was applied to an aqueous solution of polyvinylpyrrolidone to obtain a micro-crosslinked prepolymer;

[0031] B) Polyvinylpyrrolidone, hyaluronic acid and water are mixed to obtain an initial mixture. The initial mixture and the micro-crosslinked prepolymer are mixed and irradiated with high-energy rays. After freeze-drying, degradable drug-loaded nanohydrogel microspheres are obtained.

[0032] In the preparation of biodegradable hydrogel microspheres, this application first irradiates an aqueous solution of polyvinylpyrrolidone (PVP) with high-energy rays to obtain a micro-crosslinked prepolymer. During this process, PPVP undergoes weak internal crosslinking of individual molecules through high-energy ray irradiation, forming the nucleus for microsphere growth. The aqueous solution of PPVP is obtained by mixing PPVP with water, and the concentration of the aqueous solution is 0.3–0.5 wt%, specifically, 0.3 wt%, 0.4 wt%, or 0.5 wt%. In the high-energy ray irradiation, the electron beam energy is 2–5 MeV, and the irradiation dose is 5–10 kGy; specifically, the electron beam energy is 2 MeV, 3 MeV, 4 MeV, or 5 MeV, and the irradiation dose is 5 kGy, 6 kGy, 7 kGy, 8 kGy, 9 kGy, or 10 kGy. In this application, if this step is omitted, microspheres cannot be formed because the radiation degradation rate of hyaluronic acid is greater than the cross-linking rate of polyvinylpyrrolidone and hyaluronic acid. Therefore, if the subsequent steps are carried out directly, only a mixed solution of polyvinylpyrrolidone and hyaluronic acid can be obtained after irradiation.

[0033] This application then mixes polyvinylpyrrolidone, hyaluronic acid, and water to obtain an initial mixture. This initial mixture is then mixed with the micro-crosslinked prepolymer and subjected to high-energy irradiation. After freeze-drying, degradable drug-loaded hydrogel nanospheres are obtained. During this process, a mixed solution of polyvinylpyrrolidone and hyaluronic acid is added to the micro-crosslinked prepolymer, allowing it to continue growing on the micro-crosslinked cores of the single-molecule polyvinylpyrrolidone. Under irradiation conditions, polyvinylpyrrolidone and hyaluronic acid undergo intramolecular and intermolecular crosslinking, and hyaluronic acid simultaneously degrades with the irradiation. The resulting chain ends continue to bond with polyvinylpyrrolidone, thereby forming block copolymer microspheres primarily composed of polyvinylpyrrolidone and hyaluronic acid. In the above process, the mass ratio of polyvinylpyrrolidone to hyaluronic acid is 1:(0.1-1), and the concentration of polyvinylpyrrolidone is 0.1-0.5 wt%. Specifically, the mass ratio of polyvinylpyrrolidone to hyaluronic acid is 1:0.1, 1:0.2, 1:0.3, 1:0.4, 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, or 1:1, and the concentration of polyvinylpyrrolidone is 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, or 0.5 wt%. The high-energy ray irradiation uses an electron beam energy of 2–5 MeV and an irradiation dose of 10–20 kGy; specifically, the high-energy ray irradiation uses an electron beam energy of 2 MeV, 3 MeV, 4 MeV, or 5 MeV, and the irradiation dose is 10 kGy, 11 kGy, 12 kGy, 13 kGy, 14 kGy, 15 kGy, 16 kGy, 17 kGy, 18 kGy, 19 kGy, or 20 kGy. With increasing irradiation dose, the microsphere density gradually increases, and the porosity gradually decreases. In this step, if polyvinylpyrrolidone is not added, and only hyaluronic acid is added, the hyaluronic acid can only be grafted onto the outside of the micro-crosslinked prepolymer microspheres and cannot provide a crosslinked block structure, resulting in very small microsphere size and specific surface area, essentially losing its encapsulation capacity.

[0034] This application also provides biodegradable nanohydrogel microspheres prepared by the above method, which are block copolymers formed by crosslinking polyvinylpyrrolidone and hyaluronic acid.

[0035] In this application, the radius of the biodegradable nanohydrogel microspheres is 100–500 nm.

[0036] This application also provides a drug-loaded hydrogel, which is composed of hydrogel microspheres and a small molecule drug with an amino group encapsulated in the hydrogel microspheres, wherein the hydrogel microspheres are the biodegradable nanohydrogel microspheres described in the above-mentioned scheme.

[0037] In this application, the amino-containing small molecule drug is doxorubicin hydrochloride. The radius of the drug-loaded hydrogel is 160–600 nm.

[0038] This application also provides a method for preparing drug-loaded hydrogels, including:

[0039] Hydrogel microspheres, amino-containing small molecule drugs, and solvents were mixed in the dark to obtain drug-loaded hydrogels;

[0040] The hydrogel microspheres are the biodegradable nanohydrogel microspheres described in the above scheme.

[0041] In the drug-loaded hydrogel provided in this application, small molecule drugs are encapsulated on hydrogel microspheres through self-assembly. The solvent is specifically a PBS buffer solution with a pH of 7.4, and the small molecule drug comprises 6–25 wt% of the total mass of the hydrogel microspheres, the small molecule drug, and the solvent; more specifically, the small molecule drug comprises 8–15 wt% of the total mass of the hydrogel microspheres, the small molecule drug, and the solvent. The mixing time is 24–48 h, specifically 24 h, during which the hydrogel microspheres encapsulate the small molecule drug through self-assembly.

[0042] The nanogel microspheres (PHA-Nanogel) prepared in this invention form a block structure between polyvinylpyrrolidone and hyaluronic acid. Under the action of hyaluronidase, the hyaluronic acid segments can be degraded, thus allowing the nanogel microspheres to degrade after entering the body. These nanogel microspheres can effectively encapsulate specific small molecule drugs through self-assembly. After entering the body, the degradation of the microspheres under the action of in vivo enzymes can lead to the sustained release of the encapsulated drug. The hydrogel microspheres synthesized by this application via radiation method have a pure reaction system, requiring no addition of emulsifiers, initiators, or other small molecule additives, and eliminating the need for cumbersome post-processing. Furthermore, the high-energy irradiation results in high reaction efficiency. The preparation method of the nanogel microspheres has the advantages of simple process and ease of operation, enabling large-scale production.

[0043] To further understand the present invention, the following detailed description of the biodegradable nanohydrogel microspheres, their preparation method, and drug-loaded hydrogels provided by the present invention is provided in conjunction with the embodiments. The scope of protection of the present invention is not limited by the following embodiments.

[0044] Example 1

[0045] 1) Polyvinylpyrrolidone was prepared into a 0.5 wt% aqueous solution and irradiated with high-energy X-rays-electron beams at an energy of 2 MeV and an irradiation dose of 10 kGy to form a micro-crosslinked prepolymer;

[0046] 2) Polyvinylpyrrolidone and hyaluronic acid were dissolved in purified water at a ratio of 1:1, wherein the concentration of polyvinylpyrrolidone was 0.5wt%. The mixture was stirred at 25°C until it was completely dissolved. The mixture was then blended with the micro-crosslinked prepolymer obtained in step 1) at a ratio of 1:1. The mixture was then irradiated again with high-energy X-rays-electron beams at an electron beam energy of 2MeV and an irradiation dose of 20kGy to obtain the block copolymerized polyvinylpyrrolidone and hyaluronic acid nanogel microspheres (PHA-Nanogel).

[0047] 3) The prepared PHA-Nanogel stock solution was freeze-dried to obtain PHA-Nanogel freeze-dried powder;

[0048] 4) PHA-Nanogel lyophilized powder, doxorubicin hydrochloride, and PBS solution (pH=7.4) were mixed at a mass ratio of 8:1:6 and stirred at room temperature in the dark for 24 hours to prepare nano-hydrogel microspheres (PHA-Dox-Nanogel) loaded with doxorubicin.

[0049] Example 2

[0050] The preparation method is the same as in Example 1, except that the concentration of polyvinylpyrrolidone in step 1) is 0.4 wt% and the concentration of polyvinylpyrrolidone in step 2) is 0.4 wt%.

[0051] Example 3

[0052] The preparation method is the same as in Example 1, except that the concentration of polyvinylpyrrolidone in step 1) is 0.4 wt% and the concentration of polyvinylpyrrolidone in step 2) is 0.2 wt%.

[0053] Example 4

[0054] The preparation method is the same as in Example 1, except that the mass ratio of polyvinylpyrrolidone to hyaluronic acid in step 2) is 1:0.8.

[0055] Example 5

[0056] The preparation method is the same as in Example 1, except that the mass ratio of polyvinylpyrrolidone to hyaluronic acid in step 2) is 1:0.5.

[0057] Example 6

[0058] The preparation method is the same as in Example 1, except that the mass ratio of polyvinylpyrrolidone to hyaluronic acid in step 2) is 1:0.1.

[0059] Example 7

[0060] The preparation method is the same as in Example 1, except that the mass ratio of each component in step 4) is 2:1:4.

[0061] Example 8

[0062] The preparation method is the same as in Example 1, except that the mass ratio of each component in step 4) is 1:1:2.

[0063] Comparative Example 1

[0064] The preparation method is the same as in Example 1, except that only 0.5 wt% of hyaluronic acid is added in step 2).

[0065] The testing methods for the above embodiments are as follows:

[0066] 1) Microscopic morphology analysis of PHA-Nanogel: The hydrodynamic diameter (Rh) was determined by dynamic light scattering method, and the microscopic morphology was observed by transmission electron microscopy;

[0067] 2) Cell compatibility test of hydrogel microsphere material: L929 fibroblasts were used to test the cell compatibility of the dressing;

[0068] 3) Drug loading and encapsulation efficiency of PHA-Dox-Nanogel: The absorption peak of the drug-loaded microspheres at 480 nm in the ultraviolet spectrum was tested;

[0069] 4) Cell proliferation inhibition experiment of drug-loaded hydrogel microspheres PHA-Dox-Nanogel after hyaluronidase treatment at 24h, 48h, and 72h: MTT assay was performed using an enzyme-linked immunosorbent assay (ELISA) reader. The MTT assay was performed on HeLa Cell Line (cervical cancer cell line), with Free Dox as the control group.

[0070] The test results are as follows:

[0071] Figure 1 The graph shows the hydrodynamic radius curves of the PHA-Dox-Nanogel prepared in Example 1. Figure 1 It can be seen that the radius of PHA-Nanogel is 105 nm, and the radius of PHA-Dox-Nanogel after encapsulation with doxorubicin is 186 nm.

[0072] Figure 2 This is a graph showing the hydrodynamic radius curves of PHA-Nanogel after 3h, 5h, and 8h of hyaluronidase treatment in Example 1. Figure 2 It can be seen that under the action of hyaluronidase, the radius of the nanohydrogel microspheres gradually increases and the peak shape becomes wider, indicating that the hyaluronic acid segments in the microspheres undergo a degradation reaction, causing the microspheres to gradually disintegrate and the particle size to increase.

[0073] Figure 3The graphs show the hydrodynamic radius curves of PHA-Nanogel after 5 hours of hyaluronidase treatment in Examples 1, 4, 5, 6, and Comparative Example 1. Figure 3 It can be seen that as the hyaluronic acid segments in the nano-hydrogel microspheres gradually increase, the hydrodynamic radius of the microspheres shows an upward trend, and the peak shape becomes wider, indicating that its degradation rate is faster.

[0074] Figure 4 The graphs show the cell compatibility of different concentrations of PHA-Nanogel material in Examples 1, 5, and 6. In each group of bars, from left to right, the bars represent Example 1, Example 5, Example 6, and PEI25K. Figure 4 It can be seen that the cytotoxicity of the nano-hydrogel microspheres prepared in the examples is all grade 0, indicating that the prepared nano-hydrogel microsphere materials have good biological safety.

[0075] Table 1 shows the test results of drug loading and encapsulation efficiency of PHA-Dox-Nanogel material in Examples 1, 7 and 8.

[0076] Table 1. Test results of drug loading and encapsulation efficiency of PHA-Dox-Nanogel material in Examples 1, 7, and 8.

[0077] Sample Name PHA-Nanogel:Dox:PBS DLC (wt%) DLE (%) Example 1 8:1:6 5.76 86.36 Example 7 2:1:4 11.87 83.09 Example 8 1:1:2 20.12 80.48

[0078] As shown in Table 1, the drug loading gradually increases with the increase of doxorubicin hydrochloride addition. However, due to the limitation of microsphere encapsulation capacity, the encapsulation efficiency gradually decreases, but all remain above 80%, indicating that the hydrogel microsphere material prepared in this invention can effectively encapsulate small molecule drugs.

[0079] Figure 5 This is a bar graph showing the cell proliferation inhibition test results of different concentrations of drug-loaded hydrogel microspheres PHA-Dox-Nanogel in Examples 1, 7, and 8 after treatment with hyaluronidase for 24 h, 48 h, and 72 h. In each group of data, from left to right, the bars represent the data from Examples 1, 7, and 8, and doxorubicin, respectively. Figure 5 It is known that, compared with pure small molecule doxorubicin (Free Dox), drug-loaded hydrogel microspheres encapsulated in nano-hydrogel microspheres have a slow and sustained inhibitory effect on the proliferation of tumor cells over time. This is because, compared with single-dose administration, the gradual degradation of hydrogel microspheres enables slow and continuous drug delivery, thereby achieving sustained drug release.

[0080] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

[0081] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing biodegradable nanohydrogel microspheres, comprising the following steps: A) Irradiate an aqueous solution of polyvinylpyrrolidone with high-energy rays to obtain a micro-crosslinked prepolymer; B) Polyvinylpyrrolidone, hyaluronic acid and water are mixed to obtain an initial mixture. The initial mixture and the micro-crosslinked prepolymer are mixed and irradiated with high-energy rays. After freeze-drying, biodegradable nano-hydrogel microspheres are obtained. In step A), the concentration of the polyvinylpyrrolidone aqueous solution is 0.4~0.5wt%, the electron beam energy of the high-energy irradiation is 2~3MeV, and the irradiation dose is 8~10kGy; In step B), the mass ratio of polyvinylpyrrolidone to hyaluronic acid is 1:(0.1~1), the concentration of polyvinylpyrrolidone in the initial mixture is 0.1~0.5wt%, the electron beam energy of the high-energy irradiation is 2~5MeV, and the irradiation dose is 10~20kGy.

2. The biodegradable nanohydrogel microspheres prepared by the preparation method of claim 1 are formed by a block copolymer of polyvinylpyrrolidone and hyaluronic acid through crosslinking.

3. A drug-loaded hydrogel, comprising hydrogel microspheres and an amino-containing small molecule drug loaded in the hydrogel microspheres, wherein the hydrogel microspheres are biodegradable nano-hydrogel microspheres prepared by the preparation method of claim 1 or biodegradable nano-hydrogel microspheres of claim 2; and the small molecule drug is doxorubicin hydrochloride, methotrexate, or cytarabine.

4. The method for preparing the drug-loaded hydrogel according to claim 3, comprising: Hydrogel microspheres, amino-containing small molecule drugs, and solvents were mixed in the dark to obtain drug-loaded hydrogels; The hydrogel microspheres are the biodegradable nanohydrogel microspheres prepared by the preparation method of claim 1 or the biodegradable nanohydrogel microspheres of claim 2.

5. The preparation method according to claim 4, characterized in that, The solvent is a PBS buffer solution with pH=7.4, and the mixing time is 24~48h.

6. The preparation method according to claim 4, characterized in that, The small molecule drug comprises 6-25 wt% of the total mass of the hydrogel microspheres, the small molecule drug, and the solvent.

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