Drug sustained-release polypeptide hydrogel and preparation method thereof

Abstract

The invention belongs to the field of hydrogel, and particularly discloses drug sustained-release polypeptide hydrogel and a preparation method thereof, the drug sustained-release polypeptide hydrogel comprises a polypeptide polymer, a cross-linking agent, a drug and a solvent; a main chain of the polypeptide polymer is a glycine-alanine-serine repetitive unit, and side chains of the polypeptide polymer are grafted with carboxyl and sulfydryl bifunctional groups; the cross-linking agent is prepared from oxidized hyaluronic acid and thiolated chitosan; the solvent is a phosphate buffer solution containing 5 to 10 mM of reduced glutathione; according to the preparation method, a pH, reduction and enzyme triple responsive cross-linked network is constructed, a surface large-aperture-internal small-aperture gradient pore structure is designed, hyaluronic acid, chitosan, polypeptide and other natural polymer base materials are adopted, and HA-CD44 targeting peptide modification is introduced, so that the hydrogel is prepared. Accurate trigger release of the drug in a pathological microenvironment, a two-phase slow release mode, biocompatibility improvement of non-toxic degradation products, and active targeting ability of increase of drug enrichment amount at a tumor site are correspondingly realized.

Description

Technical Field

[0001] The invention belongs to the field of hydrogels, and particularly relates to a drug sustained-release polypeptide hydrogel and a preparation method thereof. Background Art

[0002] As a new drug delivery system, sustained-release hydrogel has the advantages of controlling drug release rate, improving drug bioavailability, and reducing drug administration frequency. Currently, the application of hydrogel in drug sustained release has received widespread attention. Hydrogel is a three-dimensional cross-linked polymer network structure with high water content, good biocompatibility and controllable degradation properties. It can embed various drugs, including proteins, peptides, small molecule drugs, etc., to achieve continuous and stable drug release.

[0003] Traditional hydrogels mostly use biodegradable polymers such as gelatin, chitosan, and polylactic acid to achieve sustained drug release by regulating cross-linking density or drug loading methods. However, a single preparation technology can easily lead to uneven drug release or imprecise control. There is an urgent need to develop new sustained-release hydrogels to improve the controllability and effectiveness of drug release. In addition, in recent years, sustained-release hydrogels prepared by combining nanotechnology, microencapsulation technology, and other methods have shown broader application prospects and are expected to achieve important breakthroughs in drug controlled release, tissue engineering, wound dressings, and other fields.

[0004] However, the single response mechanism of traditional drug sustained-release hydrogels makes it impossible to accurately match the pathological microenvironment, the drug release is insufficiently controllable, and the uniform pore structure causes a sudden release of the initial drug dose, resulting in a significant burst release effect. Summary of the Invention

[0005] The purpose of the present invention is to provide a drug sustained-release polypeptide hydrogel and a preparation method thereof, so as to solve the problems of insufficient controllability of drug release and significant burst release effect of traditional drug sustained-release hydrogels mentioned in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A drug sustained-release polypeptide hydrogel comprising a polypeptide polymer, a cross-linking agent, a drug and a solvent;

[0008] The main chain of the polypeptide polymer is a repeating unit of glycine-alanine-serine, with a side chain grafted with carboxyl and thiol bifunctional groups, and a molecular mass of 8000-12000Da;

[0009] The cross-linking agent includes oxidized hyaluronic acid and thiolated chitosan in a mass ratio of 1:0.8-1.2;

[0010] The drug loading amount is 10%-40% of the total mass of the hydrogel;

[0011] The solvent is a phosphate buffer solution containing 5-10 mM reduced glutathione, with a pH value of 7.0-7.6.

[0012] Preferably, the side chain active group grafting rate of the polypeptide polymer is 20%-35%, the molar ratio of the carboxyl group to the thiol group is 2-3:1, the mass fraction of the polypeptide polymer in the hydrogel is 18%-30%, and a gradient pore structure is formed with a surface pore diameter of 50-100 nm and an internal pore diameter of 10-30 nm.

[0013] Preferably, the drug is a protein drug with polyethylene glycol modified on its surface, the molecular weight of the polyethylene glycol is 2000-5000 Da, and the end of the polyethylene glycol is connected to the drug via a hydrazone bond.

[0014] Preferably, the oxidized hyaluronic acid has an aldehyde content of 1.2-1.8 mmol / g, and the thiol degree of the thiolated chitosan is 15%-25%.

[0015] According to the above-mentioned method for preparing a drug sustained-release polypeptide hydrogel, the method comprises the following steps:

[0016] S1. Dissolve the polypeptide polymer in a solvent to prepare a solution with a concentration of 20-30 mg / mL, and ultrasonically treat the solution at 32-37°C for 40 minutes under inert gas protection. The ultrasonic treatment power is 150 W and the frequency is 40 kHz to obtain a polypeptide solution.

[0017] S2. The drug and the mesoporous silica nanocarrier with a pore size of 4-6 nm were mixed in a solvent and shaken at room temperature for 20 minutes to form a drug-nanocomplex. The peptide solution was then added and stirred at 200 rpm for 15 minutes to obtain a drug-loaded peptide mixture.

[0018] S3, dissolve the crosslinker in the solvent to obtain a crosslinker solution, and input the crosslinker solution and the drug-loaded polypeptide mixture into the Y-shaped microfluidic chip at a volume ratio of 1:5. 2 The mixed solution was irradiated with 365 nm ultraviolet light for 1 hour to obtain a photocurable gel precursor;

[0019] S4. Inject the photocurable gel precursor into the mold and cure it for 1.5 hours at a temperature of 37° C., a 5% concentration of CO 2 , and a humidity of 90% to form a primary hydrogel, which is then subjected to gradient freeze-drying to obtain the final hydrogel.

[0020] Preferably, after the gradient freeze-drying in step S4, the hydrogel is immersed in a 1.5% chitosan solution containing 0.8% by mass of HA-CD44 targeting peptide and self-assembled at 4° C. for 2 hours.

[0021] Preferably, the mold pore size gradient in step S4 is 100 to 20 μm.

[0022] Preferably, the gradient freeze-drying in step S4 is 4°C for 2 hours, -20°C for 6 hours, and -80°C for 12 hours.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention constructs a triple responsive cross-linking network of pH, reduction and enzyme, designs a gradient pore structure with large surface pores and small internal pores, adopts natural polymer substrates such as hyaluronic acid, chitosan and polypeptides, and introduces HA-CD44 targeting peptide modification. These achieve the corresponding precise triggered release of drugs in pathological microenvironments, a biphasic sustained-release mode, improved biocompatibility of non-toxic degradation products, and active targeting capabilities with increased drug enrichment in tumor sites. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort. Among them:

[0026] Figure 1 It is a flowchart of the method steps of the present invention. DETAILED DESCRIPTION

[0027] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0028] In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention may also be implemented in other ways different from those described herein. Those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in various places throughout this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive of other embodiments.

[0030] As attached Figure 1 As shown:

[0031] Example 1: This example provides a drug sustained-release polypeptide hydrogel, comprising a polypeptide polymer, a cross-linking agent, a drug, and a solvent;

[0032] The main chain of the polypeptide polymer is a repeating unit of glycine-alanine-serine, with carboxyl and thiol bifunctional groups grafted on the side chains. The molecular weight is 10,000 Da, the grafting rate of the side chain active groups of the polypeptide polymer is 25%, the molar ratio of carboxyl to thiol is 2.5:1, and the mass fraction of the polypeptide polymer in the hydrogel is 25%.

[0033] The cross-linking agent includes oxidized hyaluronic acid and thiolated chitosan in a mass ratio of 1:1, the oxidized hyaluronic acid has an aldehyde content of 1.5 mmol / g, and the thiol degree of thiolated chitosan is 20%;

[0034] The drug loading was 20% of the total mass of the hydrogel, and PEG2000 modified insulin (PEG terminal hydrazone bond connection);

[0035] The solvent was phosphate buffer solution containing 8 mM reduced glutathione, with a pH of 7.4.

[0036] A method for preparing a drug-sustaining polypeptide hydrogel comprises the following steps:

[0037] S1. Weigh 2.5 g of polypeptide polymer and dissolve it in 100 mL of solvent. Ultrasonicate at 35°C for 40 minutes under N2 protection to obtain a polypeptide solution. The ultrasonic treatment power is 150 W and the frequency is 40 kHz.

[0038] S2: 1 g insulin and 0.5 g mesoporous silica nanocarriers were mixed in 50 mL of solvent and shaken at room temperature for 20 minutes to form a drug-nanocomplex. The peptide solution was then added and stirred at 200 rpm for 15 minutes to obtain a drug-loaded peptide mixture.

[0039] S3, 1.5g of oxidized hyaluronic acid aldehyde content and 1.5g of thiolated chitosan thiol content were dissolved in 50mL of solvent to obtain a crosslinker solution, and the crosslinker solution and drug-loaded polypeptide mixture were input into the Y-shaped microfluidic chip at a volume ratio of 1:5, and the chip outlet was heated at 10mW / cm 2 The mixed solution was irradiated with 365 nm ultraviolet light for 1 hour to obtain a photocurable gel precursor;

[0040] S4. Inject the photocurable gel precursor into a pore gradient mold (100 nm on the surface → 20 nm inside), and cure it at 37°C, 5% CO2, and 90% humidity for 1.5 hours to form a primary hydrogel. Treat it at 4°C for 2 hours → -20°C for 6 hours → -80°C for 12 hours, and freeze-dry it. Immerse it in a 1.5% chitosan solution containing 0.8% HA-CD44 targeting peptide by mass, and self-assemble at 4°C for 2 hours to obtain the final hydrogel.

[0041] Performance testing:

[0042] Release curve: In the control group in pH 7.4 PBS, 30% ± 0.5% insulin was released in 24 hours, and 75% ± 1.8% was released cumulatively in 72 hours; under pH 5.5 (simulating tumor microenvironment) + 10mM GSH conditions, 65% ± 1.2% was released in 24 hours, and 90% ± 2.5% was released cumulatively in 72 hours.

[0043] Mechanical strength: Compression modulus 15-20kPa, meeting the gel strength requirements for subcutaneous injection.

[0044] Conclusion: Acidic pH causes carboxyl groups to protonate → gel swelling + GSH cuts disulfide bonds → burst release; initial surface large pores rapidly release the drug (24h), while internal small pores maintain long-term release (>72h).

[0045] Example 2: This example provides a drug sustained-release polypeptide hydrogel, comprising a polypeptide polymer, a cross-linking agent, a drug, and a solvent;

[0046] The main chain of the polypeptide polymer is a repeating unit of glycine-alanine-serine, with carboxyl and thiol bifunctional groups grafted on the side chains. The molecular weight is 8000Da, the grafting rate of the side chain active groups of the polypeptide polymer is 30%, the molar ratio of carboxyl and thiol is 2:1, and the mass fraction of the polypeptide polymer in the hydrogel is 18%.

[0047] The cross-linking agent includes oxidized hyaluronic acid and thiolated chitosan in a mass ratio of 1:0.8, the oxidized hyaluronic acid has an aldehyde content of 1.2 mmol / g, and the thiol degree of thiolated chitosan is 15%;

[0048] The drug loading was 30% of the total mass of the hydrogel, and PEG5000 modified doxorubicin (PEG terminal hydrazone bond connection);

[0049] The solvent was a phosphate buffer solution containing 5 mM reduced glutathione, with a pH of 7.0.

[0050] The preparation method is basically the same as that of Example 1, except that:

[0051] S2. During the preparation of drug-nanocomplexes, the oscillation time was extended to 25 min to ensure that doxorubicin was fully adsorbed on the mesoporous silica;

[0052] S4. The mold pore size gradient was adjusted to 80 nm on the surface → 30 nm inside. After freeze-drying, the targeting modification step was omitted (if active targeting is required, it can be retained).

[0053] Performance testing:

[0054] Cell targeting: In tumor cells that highly express CD44 (such as HeLa cells), the hydrogel uptake efficiency is 40% higher than that of the non-targeted group.

[0055] In vitro degradation: In a solution containing MMP-2 protease, the degradation rate reached 60% within 48 hours, showing enzyme response characteristics.

[0056] The drug-releasing polypeptide hydrogel of the present invention achieves controlled release and targeted delivery of drugs through a multi-responsive cross-linking mechanism and gradient pore structure design. The specific principles are as follows:

[0057] 1. Construction of a dual-responsive cross-linking network

[0058] Dynamic imine bond cross-linking (pH response): The aldehyde group of oxidized hyaluronic acid (oHA) and the thiol group of thiolated chitosan (SH-CS) form a stable imine bond in a neutral environment (pH 7.0-7.6) to maintain the mechanical strength of the hydrogel; when in an acidic microenvironment (such as tumor tissue pH ≈ 5.5), the imine bond is hydrolyzed and broken, triggering the degradation of the hydrogel and releasing the drug.

[0059] Disulfide cross-linking (reduction response): The side chain thiol groups of the polypeptide polymer and the SH-CS thiol groups form disulfide bonds (-SS-) under physiological conditions, enhancing network stability; under the action of high concentrations of reduced glutathione (GSH, such as the intracellular environment), the disulfide bonds break, the hydrogel structure dissociates, and accelerates drug release.

[0060] Enzyme-responsive assisted degradation: If a polypeptide sequence (such as GPLGLAG) that can be cleaved by matrix metalloproteinase (MMP-2) is introduced into the cross-linker, the cross-linker will break under the action of the MMP-2 enzyme highly expressed in tumor tissue, further promoting the degradation of the hydrogel and achieving synergistic drug release of the "triple response to the pathological microenvironment" (pH + reduction + enzyme).

[0061] 2. Drug release regulation mechanism of gradient pore structure

[0062] Large surface pores (50-100nm): allow rapid drug diffusion and achieve initial burst release (such as 30%-65% release in the first 24 hours), suitable for scenarios requiring rapid onset of effect (such as blood sugar control).

[0063] Small internal pore size (10-30nm): Delays drug diffusion through pore sieving effect, combined with the physical restraint of the cross-linked network to achieve sustained release of drugs (such as cumulative release of 75%-90% in 72 hours), maintaining long-term therapeutic effects.

[0064] Pore ​​formation mechanism: The flow velocity gradient within the microfluidic chip leads to uneven distribution of the cross-linker (high cross-linker concentration in the outer layer → rapid formation of large pores; slow diffusion of cross-linker in the inner layer → slow formation of small pores). Combined with gradient freeze-drying (low-temperature gradient induces graded growth of ice crystals), a pore structure that gradually shrinks from the surface to the inside is eventually formed.

[0065] 3. Chemical coupling mechanism of drug loading and release

[0066] Synergy of physical adsorption and chemical coupling: drugs (such as proteins) are physically adsorbed and loaded through mesoporous silica nanocarriers, and at the same time, ionic bonds or covalent bonds (such as carbodiimide coupling) are formed between the side chain carboxyl groups of the polypeptide polymer and the amino groups of the drug to reduce the burst effect.

[0067] PEG-modified pH-responsive release: The drug surface is modified with polyethylene glycol (PEG) connected via a hydrazone bond. In an acidic environment, the hydrazone bond breaks, releasing the free drug and preventing the drug from dissociating prematurely in the blood circulation.

[0068] 4. Active recognition mechanism of targeted modification

[0069] The chitosan solution was used to self-assemble HA-CD44 targeting peptide on the hydrogel surface, which was specifically combined with hyaluronic acid (HA) by utilizing the overexpressed CD44 receptor on the surface of tumor cells (affinity constant ≈ 10 6 M-1), to achieve active targeting of tumor tissue, reduce exposure of normal tissues, and reduce toxic side effects.

[0070] It will be appreciated that in the development of any actual embodiment, as in any engineering or design project, numerous implementation-specific decisions may be made. Such a development effort may be complex and time-consuming, but will, for those of ordinary skill having the benefit of this disclosure, be a routine undertaking of design, fabrication, and production without undue experimentation.

[0071] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A drug sustained-release polypeptide hydrogel, characterized in that: including polypeptide polymers, cross-linking agents, drugs, and solvents; The main chain of the polypeptide polymer is a repeating unit of glycine-alanine-serine, with a side chain grafted with carboxyl and thiol bifunctional groups, and a molecular mass of 8000-12000Da; The cross-linking agent includes oxidized hyaluronic acid and thiolated chitosan in a mass ratio of 1:0.8-1.2; The drug loading amount is 10%-40% of the total mass of the hydrogel; The solvent is a phosphate buffer solution containing 5-10 mM reduced glutathione, with a pH value of 7.0-7.

6.

2. The drug-releasing polypeptide hydrogel according to claim 1, characterized in that: The side chain active group grafting rate of the polypeptide polymer is 20%-35%, the molar ratio of the carboxyl group to the thiol group is 2-3:1, the mass fraction of the polypeptide polymer in the hydrogel is 18%-30%, and a gradient pore structure is formed with a surface pore diameter of 50-100nm and an internal pore diameter of 10-30nm.

3. The drug-releasing polypeptide hydrogel according to claim 1, characterized in that: The drug is a protein drug with polyethylene glycol modified on the surface. The molecular weight of the polyethylene glycol is 2000-5000 Da, and the end of the polyethylene glycol is connected to the drug through a hydrazone bond.

4. The drug-releasing polypeptide hydrogel according to claim 1, characterized in that: The oxidized hyaluronic acid has an aldehyde content of 1.2-1.8 mmol / g, and the thiol degree of the thiol-modified chitosan is 15%-25%.

5. The method for preparing a drug sustained-release polypeptide hydrogel according to any one of claims 1 to 4, characterized in that: The following steps are involved: S1. Dissolve the polypeptide polymer in a solvent to prepare a solution with a concentration of 20-30 mg / mL, and ultrasonically treat the solution at 32-37°C for 40 minutes under inert gas protection. The ultrasonic treatment power is 150 W and the frequency is 40 kHz to obtain a polypeptide solution. S2. The drug and the mesoporous silica nanocarrier with a pore size of 4-6 nm were mixed in a solvent and shaken at room temperature for 20 minutes to form a drug-nanocomplex. The peptide solution was then added and stirred at 200 rpm for 15 minutes to obtain a drug-loaded peptide mixture. S3, dissolve the crosslinker in the solvent to obtain a crosslinker solution, and input the crosslinker solution and the drug-loaded polypeptide mixture into the Y-shaped microfluidic chip at a volume ratio of 1:

5. 2 The mixed solution was irradiated with 365 nm ultraviolet light for 1 hour to obtain a photocurable gel precursor; S4. Inject the photocurable gel precursor into the mold and cure it for 1.5 hours at a temperature of 37° C., a 5% concentration of CO 2 , and a humidity of 90% to form a primary hydrogel, which is then subjected to gradient freeze-drying to obtain the final hydrogel.

6. The method for preparing a drug sustained-release polypeptide hydrogel according to claim 5, characterized in that: After the gradient freeze-drying in step S4, the hydrogel was immersed in a 1.5% chitosan solution containing 0.8% by mass of HA-CD44 targeting peptide and self-assembled at 4° C. for 2 hours.

7. The method for preparing a drug sustained-release polypeptide hydrogel according to claim 5, characterized in that: In step S4, the mold pore size gradient is 100 to 20 μm.

8. The method for preparing a drug sustained-release polypeptide hydrogel according to claim 5, characterized in that: The gradient freeze-drying in step S4 is 4°C for 2 hours, -20°C for 6 hours, and -80°C for 12 hours.

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