A multi-barrier drug release preparation and preparation method thereof

Abstract

The present invention proposes a multi-barrier drug-release preparation and a preparation method thereof, which belongs to the technical field of sustained-release drug preparations. It includes drug-loaded nano-microspheres and drug-loaded hydrogels, wherein the drug-loaded nano-microspheres and the drug-loaded hydrogels form a composite structure through physical cross-linking; the particle size range of the drug-loaded nano-microspheres is 50nm-500μm; the drug loading in the drug-loaded nano-microspheres is greater than the drug loading in the drug-loaded hydrogel; the drug includes a hydrophilic drug or a hydrophobic drug. The multi-barrier drug release preparation of the present invention can make up for the problem of burst release or delayed release of a single barrier carrier, and finally achieve zero-order long-acting release of the drug, and stably maintain the effective concentration of the drug in the body for a long time.

Description

Technical Field

[0001] The present invention relates to the technical field of sustained-release pharmaceutical preparations, and in particular to a multi-barrier drug step-by-step release preparation and a preparation method thereof. Background Art

[0002] Drug sustained-release preparations can be released at a certain release rate within a specific time, effectively controlling the blood drug concentration in the body and exerting the best therapeutic effect of the drug. Single barrier is a drug sustained-release preparation that is currently being studied more. The drug is directly adsorbed, embedded or grafted into the carrier material by physical or chemical methods, mainly including membranes, stents, gels, nano-microspheres, microneedles, etc., and the spatial hindrance of the carrier material is used to delay the release rate of the drug to achieve the purpose of slow release. There are a series of problems with single barrier carriers. First, the conditions for material molding and processing are severe and harsh, and it is difficult to maintain the stability of bioactive drugs; second, the material degradation rate is uncontrollable, affecting the sustained and stable release of the drug; finally, the immunogenicity of the material is high when applied in the body, especially for fragile and sensitive tissues in the body. It is easy to cause inflammatory reactions and has poor biosafety. Natural polymer materials have been proven to have high biocompatibility and safety, can stabilize the activity of the drug itself, and have become one of the popular materials in the field of drug delivery systems. Although the safety risk problem of synthetic polymer materials during use has been solved to a certain extent, there are still problems such as sudden release, delayed release, and incomplete release in the entire release process, which reduces the efficacy of the drug while also increasing the cost of treatment. Therefore, materials with high biocompatibility are selected to prepare multi-barrier carrier drug step-by-step release preparations. The typical feature of step-by-step release is that it has a nearly linear (zero-order) release, in which the drug release rate is closely related to the physical and chemical properties and spatial distribution of the carrier material. Therefore, selecting natural polymer materials with high biocompatibility, developing mild processing methods for carrier materials (maintaining drug activity and no organic solvent residue) to prepare multi-barrier drug step-by-step release preparations, accurately controlling the drug release rate and cycle, delaying the retention and degradation time of the carrier in the body while ensuring the effective therapeutic concentration of the drug in the lesion, and improving the safety of in vivo use will be the key strategy for the efficient and safe treatment of chronic diseases.

[0003] Silk fibroin has been proven to have not only excellent biocompatibility and controllable in vivo degradability, but also other unique physical and chemical properties. First, silk fibroin molecules are composed of hydrophilic regions and hydrophobic crystalline regions. The hydrophobic crystalline region is mainly composed of β-folds composed of repeated amino acid sequences such as alanine-glycine-alanine, or glycine-alanine-serine. While stabilizing the structure of silk fibroin itself, it can load and stabilize bioactive molecules such as enzymes and growth factors. Secondly, the level of silk fibroin β-fold content is related to the preparation and treatment of the solution, and the precise release of drugs can be achieved by controlling the degradation rate of the material. Finally, it has excellent plasticity and can be processed into electrostatic nanofibers, membranes, hydrogels, porous sponges, and nano-microspheres, which can provide a rich material form for the effective loading and delivery of drugs. At present, there are many studies on silk fibroin materials as single-barrier carriers, but there are few studies on silk fibroin materials as multi-barrier carriers. Some researchers have embedded silk protein nanoparticles in a gel matrix and found that the release time of the drug can be effectively delayed by controlling the particle size of the nanoparticles, the β-folding content, and the spatial distribution of the gel, and verified that the material has no cytotoxicity through human bone marrow mesenchymal stem cells. Some researchers have also loaded exenatide into a multi-barrier carrier (PLGA ball-PLGA-PEG-PLGA gel combination) for the first time and injected it once in vivo, and found that the blood sugar of mice can be maintained stably for up to 20 days, confirming that the multi-barrier carrier can achieve the effect of zero-order long-term sustained release of exenatide, providing a new treatment idea for the treatment of diabetic patients.

[0004] Silk fibroin gel and nano-microspheres are excellent drug delivery carriers. Silk fibroin gel with porous network is conducive to drug diffusion (burst release), and drugs loaded in silk fibroin nano-microspheres can effectively delay release (delayed release). Therefore, silk fibroin gel or silk fibroin nano-microspheres with single barrier carriers cannot achieve the expected effect of zero-order long-term drug release. Summary of the invention

[0005] The purpose of the present invention is to propose a multi-barrier drug release preparation and a preparation method thereof, wherein drug-loaded silk fibroin nano-microspheres are compounded with drug-loaded silk fibroin gels, specifically, the drug is first loaded into the silk fibroin nano-microspheres, and then mixed with the silk fibroin-drug solution contained therein, and a multi-barrier drug delivery carrier is formed through physical cross-linking. At the beginning, the drug is released from the gel with a low drug loading, accompanied by the release of part of the drug from the nano-microspheres with a high drug loading to the gel, and then released from the gel, forming a step-by-step release effect. The drug loading in the multi-barrier carrier requires that the silk fibroin nano-microspheres>silk fibroin gel, which is conducive to the smooth progress of the step-by-step release, makes up for the problem of sudden release or delayed release of the single-barrier carrier, and finally achieves zero-order long-term release of the drug, and maintains the effective concentration of the drug in the body for a long time.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides a multi-barrier drug-release preparation, comprising drug-loaded nano-microspheres and drug-loaded hydrogels, wherein the drug-loaded nano-microspheres and the drug-loaded hydrogels are physically cross-linked to form a composite structure;

[0008] The particle size of the drug-loaded nano-microspheres ranges from 50 nm to 500 μm;

[0009] The drug loading amount in the drug-loaded nano-microspheres is greater than the drug loading amount in the drug-loaded hydrogel; the drug includes a hydrophilic drug or a hydrophobic drug.

[0010] Preferably, the drug-loaded nano-microspheres are uniformly dispersed in the drug-loaded hydrogel; the drug molecules of the drug are bound to the drug-loaded nano-microspheres through non-covalent interactions / covalent bonds; the drug molecules of the drug are distributed in the voids in the gel structure of the drug-loaded hydrogel by diffusion, forming physical adsorption or chemical coupling to interact with the silk protein molecules in the drug-loaded hydrogel.

[0011] Furthermore, the drug-loaded nano-microspheres and the drug-loaded hydrogel form a composite structure through physical cross-linking, including: forming a multi-barrier structure between the nano-microspheres and the gel through temperature change, pH adjustment or ionic cross-linking.

[0012] As a further improvement of the present invention, the hydrophilic drug is selected from at least one of amino acids, polypeptides, insulin, interferon or small molecule drugs with biological activity; the hydrophobic drug is selected from at least one of curcumin, berberine, dexamethasone, rifampicin, camptothecin, olanzapine or paclitaxel.

[0013] As a further improvement of the present invention, the matrix materials of the drug-loaded nano-microspheres and the drug-loaded hydrogel are silk fibroin, and the mass ratio of the nano-microspheres to the silk fibroin content in the hydrogel is in the range of 10:1-1:10.

[0014] The present invention further protects a method for preparing the above-mentioned multi-barrier drug graded release preparation, comprising the following steps:

[0015] Step (1.1), preparing a silk fibroin aqueous solution with a concentration of 1-40% wt / v;

[0016] Step (1.2), preparing a hydrophilic drug solution with a concentration range of 0.1-100 mg / mL;

[0017] Step (1.3), mixing the hydrophilic drug with the silk fibroin solution in step (1.1) to obtain a mixed solution, wherein the concentration of the hydrophilic drug is 0.5-50 mg / mL;

[0018] Step (1.4), preparing polyethylene glycol with a molecular weight of 4000-20000 into a polyethylene glycol solution with a weight percentage of 10%-60%, mixing the polyethylene glycol solution with part of the mixed solution of step (1.3), incubating at 37° C. for 24 hours, centrifuging and washing, and obtaining a suspension of silk fibroin nanoparticles loaded with hydrophilic drugs;

[0019] Step (1.5), subjecting the remaining mixed solution in step (1.3) to ultrasonic induction to form a turbid solution;

[0020] Step (1.6), measuring a certain concentration of the silk fibroin nano-microsphere suspension in step (1.4);

[0021] Step (1.7), mixing the solutions of step (1.5) and step (1.6), wherein the mass ratio of the silk fibroin content in the silk fibroin nano-microspheres to the silk fibroin content in the silk fibroin gel is in the range of 10:1-1:10, ensuring that the drug loading in the silk fibroin microspheres is greater than the drug loading in the silk fibroin gel, and ultrasonically dispersing uniformly until a gel is formed, wherein the gel is a silk fibroin multi-barrier hydrophilic drug release preparation, which is stored at 4°C.

[0022] The present invention further protects a method for preparing the above-mentioned multi-barrier drug graded release preparation, comprising the following steps:

[0023] Step (2.1), preparing a silk fibroin aqueous solution with a concentration of 1-40% wt / v;

[0024] Step (2.2), preparing polyethylene glycol with a molecular weight of 200-600 into a polyethylene glycol solution with a weight percentage of 40%-100%;

[0025] Step (2.3), dissolving the hydrophobic drug in the polyethylene glycol solution of step (2.2) to obtain a polyethylene glycol solution containing the hydrophobic drug, to obtain a first hydrophobic drug solution, wherein the concentration of the first hydrophobic drug solution is 0.1-100 mg / mL;

[0026] Step (2.4), mixing the above-mentioned part of the hydrophobic drug solution with the silk fibroin solution in step (2.1) to obtain a second hydrophobic drug solution, wherein the concentration of the second hydrophobic drug solution is 0.5-50 mg / mL;

[0027] Step (2.5), preparing a saturated ammonium sulfate solution;

[0028] Step (2.6), transferring the second hydrophobic drug solution in step (2.4) into a dialysis bag, and soaking it in the saturated ammonium sulfate solution in step (2.5) at a bath ratio of 1:80-120, dialyzing and incubating for 1-3 hours, and centrifuging and washing to obtain a suspension of silk fibroin nanoparticles loaded with hydrophobic drugs;

[0029] Step (2.7), subjecting the first hydrophobic drug solution in step (2.3) to ultrasonic induction to form a turbid solution;

[0030] Step (2.8), weighing a certain mass of the silk fibroin nano-microsphere suspension in step (2.6);

[0031] Step (2.9), mixing the turbid liquid in step (2.7) with the silk fibroin nano-microsphere suspension in step (2.8), wherein the mass ratio of the silk fibroin content in the silk fibroin nano-microsphere to the silk fibroin content in the silk fibroin gel is in the range of 10:1-1:10, ensuring that the drug loading in the silk fibroin microsphere is greater than the drug loading in the silk fibroin gel, and ultrasonically dispersing evenly until a gel is formed, wherein the gel is a silk fibroin multi-barrier hydrophilic drug release preparation, which is stored at 4°C.

[0032] As a further improvement of the present invention, the drug-loaded nano-microspheres are drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres, and the preparation method is as follows:

[0033] S1, preparing a silk fibroin aqueous solution;

[0034] S2, preparing a polyethylene glycol solution, mixing the polyethylene glycol solution with the silk fibroin aqueous solution in step S1, incubating, centrifuging and washing, and freeze-drying to obtain silk fibroin nano-microspheres;

[0035] S3, adding the silk fibroin nano-microspheres prepared in step S2 into water, adding dopamine hydrochloride and a catalyst, heating and stirring to react, centrifuging, washing, and freeze-drying to obtain polydopamine-modified silk fibroin nano-microspheres;

[0036] S4, adding the polydopamine-modified silk fibroin nano-microspheres in step S3 to the graphene oxide aqueous dispersion, adding drugs, heating and stirring to react, centrifuging, washing, and freeze-drying to obtain graphene oxide / polydopamine-modified silk fibroin microspheres loaded with drugs.

[0037] As a further improvement of the present invention, the concentration of the silk fibroin aqueous solution in step S1 is 1-40% wt / v; the molecular weight of the polyethylene glycol in step S2 is 4000-20000, the weight percentage of the polyethylene glycol solution is 10%-60%, the incubation temperature is 37-40°C, and the time is 18-24h; the mass ratio of the silk fibroin nano-microspheres, dopamine hydrochloride and the catalyst in step S3 is 100:12-15:0.5-1 The catalyst is a Tris-HCl solution with a pH of 8.5-9, the temperature of the heating and stirring reaction is 40-50°C, and the time is 2-4h; the mass ratio of the polydopamine-modified silk fibroin nano-microspheres, the drug, and the graphene oxide aqueous dispersion in step S4 is 15-20:3-5:100, the concentration of the graphene oxide aqueous dispersion is 0.5-1mg / mL, the temperature of the heating and stirring reaction is 35-45°C, and the time is 0.5-1h.

[0038] As a further improvement of the present invention, the drug-loaded hydrogel is a drug-loaded collagen / chitosan-modified silk fibroin hydrogel, and the preparation method is as follows:

[0039] T1, preparing silk fibroin aqueous solution;

[0040] T2, dissolving chitosan in acid solution to prepare a chitosan aqueous solution;

[0041] T3. Mix the silk fibroin aqueous solution in step T1 and the chitosan aqueous solution in step T2, add citric acid, EDC, and NHS, heat and stir to react, add collagen and drugs, stir to react, and obtain collagen / chitosan modified silk fibroin hydrogel loaded with drugs.

[0042] As a further improvement of the present invention, the concentration of the silk fibroin aqueous solution in step T1 is 1-40% wt / v; the acid solution in step T2 is an acetic acid or phosphoric acid solution with a concentration of 1-2wt%, and the concentration of the chitosan aqueous solution is 1-20wt%; the mass ratio of the silk fibroin aqueous solution, chitosan aqueous solution, citric acid, EDC, NHS, collagen and the drug in step T3 is 100:30-50:2-3:10-15:10-15:2-4:5-7, the temperature of the heating and stirring reaction is 80-90°C, the time is 5-7h, and the stirring reaction time is 1-3h.

[0043] The present invention further protects a method for preparing the above-mentioned multiple-barrier drug-release preparation, comprising the following steps: uniformly mixing drug-loaded nano-microspheres and drug-loaded hydrogels to ensure that the drug loading amount in the microspheres is greater than the drug loading amount in the hydrogels, uniformly dispersing by ultrasonication, and storing at 4° C. to obtain the multiple-barrier drug-release preparation.

[0044] The present invention achieves a multi-barrier carrier (linear release) with step-by-step release by regulating the different gradients of the drug loading of silk fibroin microspheres and silk fibroin gel carrier matrix. The silk fibroin microspheres containing high-concentration drugs can release the drugs into the silk fibroin gel matrix with low-concentration drugs through a diffusion mechanism, and then release them through diffusion, which focuses on the effect of step-by-step release. The specific particle size range of the microspheres, the mass ratio between the microspheres and the gel, and the drug loading in the microspheres and the gel are all key indicators for realizing the technology.

[0045] The microspheres prepared by the present invention are obtained by physical crosslinking. No crosslinking agent or organic solvent is used in the preparation process. The silk fibroin microspheres are prepared only by inducing agents such as ammonium sulfate or polyethylene glycol. No chemical crosslinking agent (glutaraldehyde) needs to be added, and the microspheres have biosafety and biocompatibility.

[0046] The process of preparing the multi-barrier drug-released preparation by the method of the present invention is simple, wherein the preparation of the silk fibroin microspheres does not require the introduction of an oil phase, and only requires stirring, chemical reaction cross-linking, one-step blending or salting out to obtain the microspheres.

[0047] The present invention has the following beneficial effects:

[0048] The present invention compounds the drug-loaded silk fibroin nano-microspheres with the drug-loaded silk fibroin gel, specifically, the drug is first loaded into the silk fibroin nano-microspheres, and then blended with the silk fibroin-drug solution contained therein, and a multi-barrier drug delivery carrier is formed through physical cross-linking. At the beginning, the drug is released from the gel with a low drug loading, accompanied by the release of part of the drug from the nano-microspheres with a high drug loading to the gel, and then released from the gel, forming a step-by-step release effect. The drug loading in the multi-barrier carrier requires that the silk fibroin nano-microspheres> the silk fibroin gel, which is conducive to the smooth progress of the step-by-step release, makes up for the problem of sudden release or delayed release of the single barrier carrier, and finally achieves zero-order long-acting release of the drug, and stably maintains the effective concentration of the drug in the body for a long time.

[0049] The silk fibroin multi-barrier drug release preparation of the present invention adopts polyethylene glycol and salt ions of different molecular weights and silk fibroin, has no organic solvent added, has high biological safety, and can be directly used in clinical practice.

[0050] The silk fibroin multi-barrier drug release preparation of the present invention can be directly injected without using a solubilizer, thereby improving the safety of the preparation.

[0051] The release results of the silk fibroin multi-barrier drug release preparation of the present invention show that the preparation can make up for the burst release or delayed release problem of a single barrier carrier, ultimately achieving zero-order long-acting drug release and stably maintaining the effective concentration of the drug in the body for a long time.

[0052] The present invention prepares drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres, and after the surface of the silk fibroin microspheres is modified by polydopamine, the active groups on the surface of the microspheres are further enriched, and the complexing ability to drug molecules can be further improved through the rich active groups such as hydroxyl, amino, carboxyl, etc., so as to help load more drug molecules and better achieve sustained and controlled release. In addition, it is also helpful to combine with graphene oxide through van der Waals force, so that the surface of the microspheres is loaded with graphene oxide, and graphene oxide has a two-dimensional nanomaterial with a honeycomb single-layer structure, and there are a large number of active groups on the surface, such as hydroxyl, carboxyl, epoxy, etc., and a large specific surface area can increase the drug loading, and can also increase the drug loading of poorly soluble drugs through π-π stacking and electrostatic attraction.

[0053] The present invention further prepares collagen / chitosan modified silk fibroin hydrogel loaded with drugs. By modifying chitosan and silk fibroin hydrogel, the solubility of poorly soluble drugs such as antibiotics and hormones can be improved, and the slow and sustained release of drugs can be achieved without causing immune inflammatory reactions. Furthermore, the introduction of collagen into the prepared hydrogel can shorten the gelation time and improve the cross-linking degree, mechanical strength and biocompatibility of the hydrogel. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0055] Attached Figure 1 Scanning electron micrograph of the silk fibroin nano-microspheres loaded with rhodamine B in Example 1 of the present invention.

[0056] Attached Figure 2 This is a scanning electron micrograph of the freeze-dried silk fibroin multi-barrier rhodamine B (hydrophilic) release preparation in Example 1 of the present invention.

[0057] Attached Figure 3 This is the in vitro release curve of the Rhodamine B (hydrophilic) release preparation with multiple barriers of silk fibroin in Example 2 of the present invention.

[0058] Attached Figure 4 This is the cumulative release curve of Rhodamine B (hydrophilic) of the silk fibroin multi-barrier in Example 4 of the present invention.

[0059] Attached Figure 5 Scanning electron micrograph of the silk fibroin nanospheres loaded with calcein in Example 5 of the present invention.

[0060] Attached Figure 6 This is a scanning electron micrograph of the calcein-releasing preparation of the lyophilized silk fibroin multi-barrier in Example 5 of the present invention.

[0061] Attached Figure 7 This is the in vitro release curve of the calcitonin rate-release preparation with silk fibroin multiple barriers in Example 6 of the present invention.

[0062] Attached Figure 8 This is the in vitro release curve of the calcein release preparation with multiple barriers of silk fibroin in Example 8 of the present invention.

[0063] Attached Fig. 9 This is a scanning electron microscope image of the silk fibroin nano-microspheres loaded with curcumin in Example 9 of the present invention.

[0064] Attached Fig.10 This is a scanning electron micrograph of the lyophilized silk fibroin multi-barrier curcumin release preparation in Example 9 of the present invention.

[0065] Attached Fig.11 This is the in vitro release curve of the silk fibroin multi-barrier curcumin release preparation in Example 10 of the present invention. DETAILED DESCRIPTION

[0066] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0067] Example 1: Preparation of a silk fibroin multi-barrier rhodamine B (hydrophilic) release formulation

[0068] (1) Prepare 10% wt / v silk fibroin solution;

[0069] (2) Use deionized water to directly dissolve rhodamine to prepare a 5 mg / mL rhodamine B solution;

[0070] (3) mixing the solutions of step (1) and step (2) in equal volumes to obtain a mixed solution having a hydrophilic drug solution of rhodamine B of 2.5 mg / mL and a silk fibroin concentration of 5%;

[0071] (4) preparing a polyethylene glycol solution with a molecular weight of 10,000 into a polyethylene glycol solution with a weight percentage of 50%, mixing the polyethylene glycol solution and the solution of step (3) in equal volumes, incubating at 37° C. for 24 h, and washing by centrifugation to obtain a suspension of silk fibroin nanoparticles loaded with rhodamine B;

[0072] (5) subjecting the silk fibroin solution containing different concentrations of rhodamine B in (3) to ultrasonic induction to form a red turbid solution;

[0073] (6) measuring a certain concentration of the silk fibroin nanoparticle sustained-release preparation loaded with rhodamine B in step (4);

[0074] (7) Blending the solutions of step (5) and step (6), wherein the mass ratio of the silk fibroin content in the silk fibroin nano-microspheres to the silk fibroin content in the silk fibroin gel is 1:3, and uniformly dispersing by ultrasonication until a gel is formed, which is the silk fibroin multi-barrier rhodamine B release preparation, and storing at 4°C.

[0075] Scanning electron microscopy observation (attached Figure 1 ), drug-loaded microspheres can be prepared in one step using a silk fibroin solution of high molecular weight polyethylene glycol and rhodamine B.

[0076] 0% SHMs refers to a silk fibroin multi-barrier Rhodamine B release preparation formed by mixing a 10 mg / mL Rhodamine B silk fibroin microsphere suspension and a 0% wt / v silk fibroin solution in an equal volume ratio; 1% SHMs refers to a silk fibroin multi-barrier Rhodamine B release preparation formed by mixing a 10 mg / mL Rhodamine B silk fibroin microsphere suspension and a 1% wt / v silk fibroin solution in an equal volume ratio by ultrasound; 3% SHMs refers to a silk fibroin multi-barrier Rhodamine B release preparation formed by mixing a 10 mg / mL Rhodamine B silk fibroin microsphere suspension and a 1% wt / v silk fibroin solution in an equal volume ratio; 5% SHMs refers to a silk fibroin multi-barrier Rhodamine B release preparation formed by mixing a 10 mg / mL Rhodamine B silk fibroin microsphere suspension and a 5% wt / v silk fibroin solution in an equal volume ratio.

[0077] Scanning electron microscopy observation (attached Figure 2 ), silk fibroin nanospheres loaded with rhodamine B were embedded in the surrounding porous silk fibroin scaffold to form a multi-barrier carrier (3% SHMs freeze-dried sample).

[0078] Example 2: In vitro release assay of rhodamine B (hydrophilic) release formulations with multiple barriers of silk fibroin

[0079] 0.2 g of the silk fibroin multi-barrier rhodamine B release preparation prepared according to the method of Example 1 was weighed into a 2 mL centrifuge tube, 1 mL of PBS release solution was added to the centrifuge tube, and the tube was sealed and placed on a shaker at 37° C. At the specified time point, 0.5 mL of the release solution was taken out for testing, and then 0.5 mL of fresh release solution was added to the centrifuge tube, which was sealed and continued to be placed on a shaker at 37° C.

[0080] The fluorescence value of the test solution was measured under the conditions of excitation wavelength of 546nm and emission wavelength of 568nm. The concentration of rhodamine B in the test solution was calculated based on the concentration-fluorescence value, and then the cumulative release rate of rhodamine B within a specified time was calculated (see Appendix Figure 3 ), compared with the sample without silk fibroin gel (0% SHMs), the silk fibroin gel multi-barrier rhodamine B release preparation showed good sustained release performance, and as the silk fibroin concentration increased from 1% to 5%, the sustained release performance of rhodamine became more prominent, the cumulative release rate in 24 hours decreased from 100% to 50%, and the sustained release time lasted for 120 hours. The data show that the silk fibroin multi-barrier carrier has a sustained release effect on hydrophilic drugs, which can solve the problem of too fast sudden release from a single barrier carrier.

[0081] Example 3: Preparation of a silk fibroin multi-barrier rhodamine B (hydrophilic) release formulation

[0082] According to the method of Example 1, silk fibroin nano-microspheres loaded with rhodamine B were first prepared, and then the silk fibroin nano-microspheres with different contents were blended with rhodamine-silk fibroin solution by ultrasound to prepare silk fibroin multi-barrier rhodamine B (hydrophilic) release preparations. 3%-SHMs-5 refers to a silk fibroin multi-barrier rhodamine B release preparation formed by blending 5 mg / mL of rhodamine B silk fibroin microsphere suspension and 3% of rhodamine-containing silk fibroin solution in an equal volume ratio, in which the drug loading amount in the silk fibroin microspheres is equal to the drug loading amount in the silk fibroin gel; 3%-SHMs-10 refers to a silk fibroin microsphere suspension of 10 mg / mL of rhodamine B and a silk fibroin solution containing rhodamine in an equal volume ratio. In the silk fibroin multiple-barrier Rhodamine B release preparation formed by mixing, the drug loading in the silk fibroin microspheres is twice the drug loading in the silk fibroin gel; 3%-SHMs-30 refers to a silk fibroin multiple-barrier Rhodamine B release preparation formed by mixing 30 mg / mL Rhodamine B silk fibroin microsphere suspension and 3% Rhodamine-containing silk fibroin solution in an equal volume ratio, in which the drug loading in the silk fibroin microspheres is 6 times the drug loading in the silk fibroin gel.

[0083] Example 4: In vitro release assay of rhodamine B (hydrophilic) release formulations with multiple barriers of silk fibroin

[0084] 0.2 g of the silk fibroin multi-barrier rhodamine B release preparation prepared according to the method of Example 3 was weighed into a 2 mL centrifuge tube, 1 mL of PBS release solution was added to the centrifuge tube, and the tube was sealed and placed on a shaker at 37° C. At the specified time point, 0.5 mL of the release solution was taken out for testing, and then 0.5 mL of fresh release solution was added to the centrifuge tube, which was sealed and continued to be placed on a shaker at 37° C.

[0085] The fluorescence value of the test solution was measured under the conditions of excitation wavelength of 546nm and emission wavelength of 568nm. The concentration of rhodamine B in the test solution was calculated based on the concentration-fluorescence value, and then the cumulative release rate of rhodamine B within a specified time was calculated (see Appendix Figure 4 ), compared with the low-content silk fibroin nano-microsphere sample (3%-SHMs-5), the high-content silk fibroin nano-microsphere multi-barrier rhodamine B release preparation showed better sustained release performance, and the release curve was close to zero-order release. This is because there is a gradient release of rhodamine B in the silk fibroin nano-microspheres and the surrounding gel. At the beginning, the drug was released from the gel with low drug loading, accompanied by part of the drug released from the nano-microspheres with high drug loading into the gel, and then released through the gel, forming a step-by-step release effect. The greater the gradient difference in drug loading between the gel and the microspheres, the closer it is to linear release.

[0086] Example 5: Preparation of calcein-releasing preparation with silk fibroin multi-barrier

[0087] (1) Prepare 10% wt / v silk fibroin solution;

[0088] (2) Use deionized water to directly dissolve rhodamine to prepare a 1 mg / mL calcein solution;

[0089] (3) mixing equal volumes of the solutions of step (1) and step (2) to obtain a mixed solution having a calcein solution concentration of 0.5 mg / mL and a silk fibroin concentration of 1.5%;

[0090] (4) Prepare saturated ammonium sulfate solution: weigh 752 g of ammonium sulfate solid and stir with 1 L of water at 20 °C until completely dissolved;

[0091] (5) The mixed solution in step (3) is loaded into a semipermeable membrane dialysis bag with a molecular weight of 3,500 Da, fixed at both ends with rubber bands, and immersed in an ammonium sulfate solution at a bath ratio of 1:100 (silk fibroin solution: saturated ammonium sulfate solution), dialyzed and incubated for 2 h, and centrifuged and washed to obtain a suspension of silk fibroin nanoparticles loaded with calcein;

[0092] (6) subjecting the solution in (3) to ultrasonic induction to form a turbid solution;

[0093] (7) Weighing a certain mass of the silk fibroin nanoparticle sustained-release preparation in step (5);

[0094] (8) Blending the solutions of step (6) and step (7), wherein the mass ratio of the silk fibroin content in the silk fibroin nano-microspheres to the silk fibroin content in the silk fibroin gel is in the range of 5:1, ensuring that the loading amount of calcein in the silk fibroin microspheres is more than 4 times the loading amount of the drug in the silk fibroin gel, and uniformly dispersing by ultrasonic until a gel is formed, which is the silk fibroin multi-barrier calcein release preparation, and storing at 4°C.

[0095] Scanning electron microscopy observation (attached Figure 5 ), drug-loaded microspheres can be prepared by salting out method using salt solution and calcein silk protein solution. 0% SHMs refers to a silk protein multi-barrier calcein release preparation formed by mixing 10 mg / mL calcein silk protein microsphere suspension and 0% wt / v silk protein solution in equal volume ratio; 1% SHMs refers to a silk protein multi-barrier calcein release preparation formed by mixing 10 mg / mL calcein silk protein microsphere suspension and 1% wt / v silk protein solution in equal volume ratio by ultrasound; 3% SHMs refers to a silk protein multi-barrier calcein release preparation formed by mixing 10 mg / mL calcein silk protein microsphere suspension and 1% wt / v silk protein solution in equal volume ratio; 5% SHMs refers to a silk protein multi-barrier calcein release preparation formed by mixing 10 mg / mL calcein silk protein microsphere suspension and 5% wt / v silk protein solution in equal volume ratio. Scanning electron microscopy observation (attached Figure 6 ), calcein-loaded silk fibroin nanospheres were embedded in the surrounding porous silk fibroin scaffold to form a multi-barrier carrier (3% SHMs freeze-dried sample).

[0096] Example 6: In vitro release assay of calcein-releasing silk fibroin multi-barrier formulation

[0097] 0.2 g of the calcein release preparation of silk fibroin multi-barrier prepared according to the method of Example 5 was weighed into a 2 mL centrifuge tube, 1 mL of PBS release solution was added to the centrifuge tube, and the tube was sealed and placed on a shaker at 37° C. At the specified time point, 0.5 mL of the release solution was taken out for testing, and then 0.5 mL of fresh release solution was added to the centrifuge tube, which was sealed and continued to be placed on a shaker at 37° C.

[0098] The fluorescence value of the test solution was measured under the conditions of excitation wavelength 490nm and emission wavelength 515nm. The concentration of calcein in the test solution was calculated based on the concentration-fluorescence value, and then the cumulative release rate of calcein within a specified time was calculated (see Appendix Figure 7), compared with the sample without silk fibroin gel (0% SHMs), the multi-barrier calcein release preparation containing silk fibroin gel showed good sustained-release performance, and as the silk fibroin concentration increased from 1% to 5%, the sustained-release performance of calcein became more prominent.

[0099] Example 7: Preparation of calcein-releasing preparation with silk fibroin multi-barrier

[0100] According to the method of Example 5, calcein-loaded silk fibroin nano-microspheres were first prepared, and then different contents of silk fibroin nano-microspheres were blended with calcein-silk fibroin solution by ultrasound to prepare silk fibroin multi-barrier calcein release preparations. 3%-SHMs-5 refers to a silk fibroin multi-barrier calcein release preparation formed by blending 5 mg / mL calcein silk fibroin microsphere suspension and 3% calcein-containing silk fibroin solution in an equal volume ratio, and the drug loading amount in the silk fibroin microspheres is equal to the drug loading amount in the silk fibroin gel; 3%-SHMs-10 refers to a 10 mg / mL calcein silk fibroin microsphere suspension and 3% calcein-containing silk fibroin solution in an equal volume ratio. In the silk fibroin multi-barrier calcein releasing preparation formed by blending, the drug loading amount in the silk fibroin microspheres is twice the drug loading amount in the silk fibroin gel; 3%-SHMs-30 refers to a silk fibroin multi-barrier calcein releasing preparation formed by blending 30 mg / mL calcein silk fibroin microsphere suspension and 3% calcein-containing silk fibroin solution in an equal volume ratio, and the drug loading amount in the silk fibroin microspheres is 6 times the drug loading amount in the silk fibroin gel.

[0101] Example 8: In vitro release assay of calcein-releasing silk fibroin multi-barrier formulation

[0102] 0.2 g of the calcein release preparation of silk fibroin multi-barrier prepared according to the method of Example 7 was weighed into a 2 mL centrifuge tube, 1 mL of PBS release solution was added to the centrifuge tube, and the tube was sealed and placed on a shaker at 37° C. At the specified time point, 0.5 mL of the release solution was taken out for testing, and then 0.5 mL of fresh release solution was added to the centrifuge tube, which was sealed and continued to be placed on a shaker at 37° C.

[0103] The fluorescence value of the test solution was measured under the conditions of excitation wavelength 490nm and emission wavelength 515nm. The concentration of calcein in the test solution was calculated based on the concentration-fluorescence value, and then the cumulative release rate of calcein within a specified time was calculated (see Appendix Figure 8), compared with the low-content silk fibroin nano-microsphere sample (3%-SHMs-5), the high-content silk fibroin nano-microsphere multi-barrier calcein release preparation showed better sustained release performance. At the beginning, the drug was released from the gel with low drug loading, accompanied by part of the drug released from the nano-microsphere with high drug loading to the gel, and then released from the gel, forming a step-by-step release effect. The greater the gradient difference in drug loading between the gel and the microsphere, the more obvious the sustained release performance.

[0104] Example 9: Preparation of curcumin (hydrophobic) release formulation with multiple barriers of silk fibroin

[0105] Prepare a 3% wt / v silk fibroin solution;

[0106] Polyethylene glycol with a molecular weight of 400 is prepared into a polyethylene glycol solution with a weight percentage of 20%;

[0107] Dissolve curcumin with the polyethylene glycol solution of step (2) to prepare a 2 mg / mL curcumin solution;

[0108] (4) mixing equal volumes of the solutions of step (1) and step (3) to obtain a mixed solution having a curcumin solution concentration of 1 mg / mL and a silk fibroin concentration of 1.5%;

[0109] (5) Prepare saturated ammonium sulfate solution: weigh 752 g of ammonium sulfate solid and stir with 1 L of water at 20 °C until completely dissolved;

[0110] (6) The mixed solution in step (3) was loaded into a semipermeable membrane dialysis bag with a molecular weight of 3,500 Da, fixed at both ends with rubber bands, and immersed in an ammonium sulfate solution at a bath ratio of 1:100 (silk fibroin solution: saturated ammonium sulfate solution), dialyzed and incubated for 2 h, and centrifuged and washed to obtain a suspension of silk fibroin nanoparticles loaded with curcumin;

[0111] (7) subjecting the solution in (3) to ultrasonic induction to form a turbid solution;

[0112] (8) Weighing a certain mass of the silk fibroin nanoparticle sustained-release preparation in step (6);

[0113] (8) Blending the solutions of step (7) and step (8), wherein the mass ratio of the silk fibroin content in the silk fibroin nano-microspheres to the silk fibroin content in the silk fibroin gel is in the range of 5:1, ensuring that the loading amount of curcumin in the silk fibroin microspheres is more than 4 times the loading amount of the drug in the silk fibroin gel, and uniformly dispersing by ultrasonic until a gel is formed, which is a silk fibroin multi-barrier curcumin release preparation, and storing at a low temperature of 4°C.

[0114] Scanning electron microscopy observation (attached Fig. 9), drug-loaded nano-microspheres can be prepared by salting out using salt solution and curcumin silk protein solution. 0% SHMs refers to a silk protein multi-barrier curcumin release preparation formed by mixing 10 mg / mL curcumin silk protein microsphere suspension and 0% wt / v silk protein solution in an equal volume ratio; 1% SHMs refers to a silk protein multi-barrier curcumin release preparation formed by mixing 10 mg / mL curcumin silk protein microsphere suspension and 1% wt / v silk protein solution in an equal volume ratio by ultrasound; 3% SHMs refers to a silk protein multi-barrier curcumin release preparation formed by mixing 10 mg / mL curcumin silk protein microsphere suspension and 1% wt / v silk protein solution in an equal volume ratio; 5% SHMs refers to a silk protein multi-barrier curcumin release preparation formed by mixing 10 mg / mL curcumin silk protein microsphere suspension and 5% wt / v silk protein solution in an equal volume ratio. Scanning electron microscopy observation (attached Fig.10 ), curcumin-loaded silk fibroin nanospheres were embedded in the surrounding porous silk fibroin scaffold to form a multi-barrier carrier (3% SHMs freeze-dried sample).

[0115] Example 10: In vitro release assay of curcumin (hydrophobic) release formulation with silk fibroin multi-barrier

[0116] 0.2 g of the curcumin release preparation of silk fibroin multi-barrier prepared according to the method of Example 9 was weighed into a 2 mL centrifuge tube, 1 mL of PBS release solution was added to the centrifuge tube, and the tube was sealed and placed on a shaker at 37° C. At the specified time point, 0.5 mL of the release solution was taken out for testing, and then 0.5 mL of fresh release solution was added to the centrifuge tube, which was sealed and continued to be placed on a shaker at 37° C.

[0117] The absorbance of the test solution was measured under the condition of ultraviolet absorption wavelength of 425nm, and the concentration of curcumin in the test solution was calculated according to the concentration-absorbance value, and then the cumulative release rate of curcumin within a specified time was calculated (see Appendix Fig.11 ), compared with the sample without silk fibroin gel (0% SHMs), the multi-barrier curcumin release preparation containing silk fibroin gel showed good sustained-release performance, and as the silk fibroin concentration increased from 1% to 5%, the sustained-release performance of calcein became more prominent, showing a linear release trend.

[0118] Example 11: A method for preparing a multi-barrier drug-release formulation, comprising the following steps:

[0119] (1) Preparation of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres, as follows:

[0120] S1, preparing a 10% wt / v silk fibroin aqueous solution;

[0121] S2, preparing a 20% by weight polyethylene glycol (molecular weight 10000) solution, mixing the polyethylene glycol solution with the silk fibroin aqueous solution in step S1, incubating at 37° C. for 24 h, centrifuging and washing, and freeze-drying to obtain silk fibroin nano-microspheres;

[0122] S3, adding 100 parts by weight of the silk fibroin nano-microspheres prepared in step S2 to 500 parts by weight of water, adding 12 parts by weight of dopamine hydrochloride and 0.5 parts by weight of a catalyst, heating to 40° C., stirring for reaction for 2 hours, centrifuging, washing, and freeze-drying to obtain polydopamine-modified silk fibroin nano-microspheres;

[0123] The catalyst is a Tris-HCl solution with a pH of 8.5;

[0124] S4, adding 15 parts by weight of the polydopamine-modified silk fibroin nano-microspheres in step S3 to 100 parts by weight of a 0.5 mg / mL graphene oxide aqueous dispersion, adding 3 parts by weight of piperacillin sodium and tazobactam sodium, heating to 35° C., stirring for 0.5 h, centrifuging, washing, and freeze-drying to obtain drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres;

[0125] (2) Preparation of drug-loaded collagen / chitosan-modified silk fibroin hydrogel as follows:

[0126] T1, preparing a 10% wt / v silk fibroin aqueous solution;

[0127] T2, dissolving chitosan in 1wt% acetic acid solution to prepare a chitosan aqueous solution with a concentration of 10wt%;

[0128] T3, 100 parts by weight of the silk fibroin aqueous solution in step T1 and 30 parts by weight of the chitosan aqueous solution in step T2 were mixed, 2 parts by weight of citric acid, 10 parts by weight of EDC, and 10 parts by weight of NHS were added, heated to 80° C., stirred and reacted for 5 hours, 2 parts by weight of collagen and 5 parts by weight of piperacillin sodium tazobactam sodium were added, stirred and reacted for 1 hour, and a collagen / chitosan modified silk fibroin hydrogel loaded with drugs was obtained;

[0129] (3) 20 parts by weight of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres and 100 parts by weight of drug-loaded collagen / chitosan-modified silk fibroin hydrogel were mixed evenly, ultrasonically dispersed at 1000 W for 20 min, and stored at 4°C to prepare a multi-barrier drug-release preparation.

[0130] Example 12: A method for preparing a multi-barrier drug-release formulation, comprising the following steps:

[0131] (1) Preparation of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres, as follows:

[0132] S1, preparing a 40% wt / v silk fibroin aqueous solution;

[0133] S2, preparing a 60% by weight polyethylene glycol (molecular weight 20,000) solution, mixing the polyethylene glycol solution with the silk fibroin aqueous solution in step S1, incubating at 37° C. for 24 h, centrifuging and washing, and freeze-drying to obtain silk fibroin nano-microspheres;

[0134] S3, adding 100 parts by weight of the silk fibroin nano-microspheres prepared in step S2 to 500 parts by weight of water, adding 15 parts by weight of dopamine hydrochloride and 1 part by weight of a catalyst, heating to 50° C., stirring for reaction for 4 hours, centrifuging, washing, and freeze-drying to obtain polydopamine-modified silk fibroin nano-microspheres;

[0135] The catalyst is a Tris-HCl solution with a pH of 9;

[0136] S4, adding 20 parts by weight of the polydopamine-modified silk fibroin nano-microspheres in step S3 to 100 parts by weight of a 0.5 mg / mL graphene oxide aqueous dispersion, adding 5 parts by weight of piperacillin sodium and tazobactam sodium, heating to 45° C., stirring for 1 hour, centrifuging, washing, and freeze-drying to obtain drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres;

[0137] (2) Preparation of drug-loaded collagen / chitosan-modified silk fibroin hydrogel as follows:

[0138] T1, preparing a 40% wt / v silk fibroin aqueous solution;

[0139] T2, dissolving chitosan in 2wt% acetic acid solution to prepare a chitosan aqueous solution with a concentration of 20wt%;

[0140] T3, 100 parts by weight of the silk fibroin aqueous solution in step T1 and 50 parts by weight of the chitosan aqueous solution in step T2 were mixed, 3 parts by weight of citric acid, 15 parts by weight of EDC, and 15 parts by weight of NHS were added, heated to 90° C., stirred and reacted for 7 hours, 4 parts by weight of collagen and 7 parts by weight of piperacillin sodium tazobactam sodium were added, stirred and reacted for 3 hours, and a collagen / chitosan modified silk fibroin hydrogel loaded with drugs was obtained;

[0141] (3) 25 parts by weight of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres and 100 parts by weight of drug-loaded collagen / chitosan-modified silk fibroin hydrogel were mixed evenly, ultrasonically dispersed at 1000 W for 20 min, and stored at 4° C. to prepare a multi-barrier drug-release preparation.

[0142] Example 13: A method for preparing a multi-barrier drug-release formulation, comprising the following steps:

[0143] (1) Preparation of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres, as follows:

[0144] S1, preparing a 30% wt / v silk fibroin aqueous solution;

[0145] S2, preparing a 45% by weight polyethylene glycol (molecular weight 12000) solution, mixing the polyethylene glycol solution with the silk fibroin aqueous solution in step S1, incubating at 37° C. for 24 h, centrifuging and washing, and freeze-drying to obtain silk fibroin nano-microspheres;

[0146] S3, adding 100 parts by weight of the silk fibroin nano-microspheres prepared in step S2 to 500 parts by weight of water, adding 13 parts by weight of dopamine hydrochloride and 0.7 parts by weight of a catalyst, heating to 45° C., stirring for reaction for 3 hours, centrifuging, washing, and freeze-drying to obtain polydopamine-modified silk fibroin nano-microspheres;

[0147] The catalyst is a Tris-HCl solution with a pH of 8.7;

[0148] S4, adding 17 parts by weight of the polydopamine-modified silk fibroin nano-microspheres in step S3 to 100 parts by weight of a 0.5 mg / mL graphene oxide aqueous dispersion, adding 4 parts by weight of piperacillin sodium and tazobactam sodium, heating to 40° C., stirring for 1 hour, centrifuging, washing, and freeze-drying to obtain drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres;

[0149] (2) Preparation of drug-loaded collagen / chitosan-modified silk fibroin hydrogel as follows:

[0150] T1, preparing a 30% wt / v silk fibroin aqueous solution;

[0151] T2, dissolving chitosan in 1.5wt% acetic acid solution to prepare a chitosan aqueous solution with a concentration of 15wt%;

[0152] T3, 100 parts by weight of the silk fibroin aqueous solution in step T1 and 40 parts by weight of the chitosan aqueous solution in step T2 were mixed, 2.3 parts by weight of citric acid, 12 parts by weight of EDC, and 12 parts by weight of NHS were added, heated to 85° C., stirred and reacted for 6 hours, 3 parts by weight of collagen and 6 parts by weight of piperacillin sodium tazobactam sodium were added, stirred and reacted for 2 hours, and a collagen / chitosan modified silk fibroin hydrogel loaded with drugs was obtained;

[0153] (3) 22 parts by weight of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres and 100 parts by weight of drug-loaded collagen / chitosan-modified silk fibroin hydrogel were mixed evenly, ultrasonically dispersed at 1000 W for 20 min, and stored at 4° C. to prepare a multi-barrier drug-release preparation.

[0154] Comparative Example 1

[0155] Compared with Example 13, the difference is that step S3 is not performed in the preparation method of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres.

[0156] Preparation of drug-loaded graphene oxide-modified silk fibroin microspheres, as follows:

[0157] S1, preparing a 30% wt / v silk fibroin aqueous solution;

[0158] S2, preparing a 45% by weight polyethylene glycol (molecular weight 12000) solution, mixing the polyethylene glycol solution with the silk fibroin aqueous solution in step S1, incubating at 37° C. for 24 h, centrifuging and washing, and freeze-drying to obtain silk fibroin nano-microspheres;

[0159] S3. Add 17 parts by weight of the silk fibroin nano-microspheres in step S2 to 100 parts by weight of 0.5 mg / mL graphene oxide aqueous dispersion, add 4 parts by weight of piperacillin sodium and tazobactam sodium, heat to 40° C., stir to react for 1 hour, centrifuge, wash, and freeze-dry to obtain drug-loaded graphene oxide-modified silk fibroin microspheres.

[0160] Comparative Example 2

[0161] Compared with Example 13, the difference is that in the preparation method of drug-loaded graphene oxide / polydopamine-modified silk fibroin microspheres, step S4 is not performed, and the drug is directly mixed with the polydopamine-modified silk fibroin microspheres.

[0162] Preparation of drug-loaded polydopamine-modified silk fibroin microspheres, as follows:

[0163] S1, preparing a 30% wt / v silk fibroin aqueous solution;

[0164] S2, preparing a 45% by weight polyethylene glycol (molecular weight 12000) solution, mixing the polyethylene glycol solution with the silk fibroin aqueous solution in step S1, incubating at 37° C. for 24 h, centrifuging and washing, and freeze-drying to obtain silk fibroin nano-microspheres;

[0165] S3, adding 100 parts by weight of the silk fibroin nano-microspheres prepared in step S2 to 500 parts by weight of water, adding 13 parts by weight of dopamine hydrochloride and 0.7 parts by weight of a catalyst, heating to 45° C., stirring for reaction for 3 hours, centrifuging, washing, and freeze-drying to obtain polydopamine-modified silk fibroin nano-microspheres;

[0166] The catalyst is a Tris-HCl solution with a pH of 8.7;

[0167] S4. Add 17 parts by weight of the polydopamine-modified silk fibroin nanoparticles in step S3 to 100 parts by weight of water, add 4 parts by weight of piperacillin sodium and tazobactam sodium, heat to 40° C., stir to react for 1 hour, centrifuge, wash, and freeze-dry to obtain drug-loaded polydopamine-modified silk fibroin microspheres.

[0168] Comparative Example 3

[0169] Compared with Example 13, the difference is that no chitosan solution is added during the preparation of the drug-loaded collagen / chitosan modified silk fibroin hydrogel.

[0170] The preparation of drug-loaded hydrogels was performed as follows:

[0171] T1, preparing a 30% wt / v silk fibroin aqueous solution;

[0172] T2. Add 2.3 parts by weight of citric acid, 12 parts by weight of EDC, and 12 parts by weight of NHS to 100 parts by weight of the silk fibroin aqueous solution in step T1, heat to 85°C, stir and react for 6 hours, add 3 parts by weight of collagen and 6 parts by weight of piperacillin sodium and tazobactam sodium, stir and react for 2 hours, and obtain a drug-loaded hydrogel.

[0173] Comparative Example 4

[0174] Compared with Example 13, the difference is that no collagen is added during the preparation of the drug-loaded collagen / chitosan modified silk fibroin hydrogel.

[0175] Preparation of drug-loaded collagen / chitosan-modified silk fibroin hydrogels as follows:

[0176] T1, preparing a 30% wt / v silk fibroin aqueous solution;

[0177] T2, dissolving chitosan in 1.5wt% acetic acid solution to prepare a chitosan aqueous solution with a concentration of 15wt%;

[0178] T3. Mix 100 parts by weight of the silk fibroin aqueous solution in step T1 and 40 parts by weight of the chitosan aqueous solution in step T2, add 2.3 parts by weight of citric acid, 12 parts by weight of EDC, and 12 parts by weight of NHS, heat to 85°C, stir and react for 6 hours, add 6 parts by weight of piperacillin sodium and tazobactam sodium, stir and react for 2 hours, and obtain a drug-loaded collagen / chitosan modified silk fibroin hydrogel.

[0179] Embodiment 14

[0180] The performance of the multi-barrier drug progressive release preparations prepared in Examples 11-13 of the present invention and Comparative Examples 1-4 was tested. The results are shown in Table 1.

[0181] (1) In vitro cytotoxicity: The multi-barrier drug-release preparation was tested according to the ISO10993-5 standard test method.

[0182] (2) Tensile strength. The tensile properties of the multi-barrier drug-release formulations were tested at room temperature using a CMT4102 universal testing machine. The specimen used in the tensile test had a diameter of 5 mm, a length of 30 mm, and a tensile rate of 50 mm / min.

[0183] Table 1

[0184]

[0185] It can be seen from the above table that the multiple-barrier drug-release preparations prepared in Examples 11-13 of the present invention have low cytotoxicity and better mechanical strength.

[0186] Embodiment 15

[0187] The drug release performance of the multiple-barrier drug-gradual release preparations prepared in Examples 11-13 of the present invention and Comparative Examples 1-4 was tested. The results are shown in Table 2.

[0188] 50 mg of multi-barrier drug-released stepwise preparation was placed in the dialysis bag, 2 mL of phosphate buffer was added, and the dialysis bag was placed in a 50 mL phosphate buffer sealed bottle after sealing both ends. 5 mL of solution was drawn at 2, 8, 16, 24, 48, 72, and 96 h, and then phosphate buffer was added again; the absorbance of the solution drawn at each time point was measured with an ultraviolet spectrophotometer, the drug concentration was calculated, and the cumulative drug release rate was calculated according to the formula.

[0189] Cumulative drug release rate (%) =

[0190] Where V t is the total volume of the release medium (mL), C t is the drug mass concentration in the release medium at different time points (g / L), Vi is the sampling volume each time (mL), M is the total mass of the multiple-barrier drug progressive release preparation (mg), and A is the drug loading of the multiple-barrier drug progressive release preparation.

[0191] Table 2

[0192]

[0193] As can be seen from the above table, the multiple-barrier drug-release preparations prepared in Examples 11-13 of the present invention have a cumulative drug release of approximately 70.5-71.4% within 96 hours, and no obvious drug burst release occurs. The drug can be stably released within a specified time, and its sustained release effect is better than that of Comparative Examples 1-4.

[0194] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A multi-barrier drug release preparation, characterized in that: It comprises drug-loaded nano-microspheres and drug-loaded hydrogels, wherein the drug-loaded nano-microspheres and the drug-loaded hydrogels form a composite structure through physical cross-linking; The particle size of the drug-loaded nano-microspheres ranges from 50 nm to 500 μm; The drug loading amount in the drug-loaded nano-microspheres is greater than the drug loading amount in the drug-loaded hydrogel; the drug includes a hydrophilic drug; The method for preparing the multi-barrier drug-gradual release preparation comprises the following steps: Step (1.1), preparing a silk fibroin aqueous solution with a concentration of 1-40% wt / v; Step (1.2), preparing a hydrophilic drug solution with a concentration range of 0.1-100 mg / mL; Step (1.3), mixing the hydrophilic drug with the silk fibroin solution in step (1.1) to obtain a mixed solution, wherein the concentration of the hydrophilic drug is 0.5-50 mg / mL; Step (1.4), preparing polyethylene glycol with a molecular weight of 4000-20000 into a polyethylene glycol solution with a weight percentage of 10%-60%, mixing the polyethylene glycol solution with part of the mixed solution of step (1.3), incubating at 37° C. for 24 hours, centrifuging and washing, and obtaining a suspension of silk fibroin nanoparticles loaded with hydrophilic drugs; Step (1.5), subjecting the remaining mixed solution in step (1.3) to ultrasonic induction to form a turbid solution; Step (1.6), measuring a certain concentration of the silk fibroin nano-microsphere suspension in step (1.4); Step (1.7), mixing the suspensions of step (1.5) and step (1.6), wherein the mass ratio of the silk fibroin content in the silk fibroin nano-microspheres to the silk fibroin content in the silk fibroin gel is in the range of 10:1-1:10, the silk fibroin in the silk fibroin nano-microspheres is prepared by the step (1.4), the silk fibroin gel is subjected to ultrasonic induction treatment by the step (1.5), ensuring that the drug loading amount in the silk fibroin microspheres is greater than the drug loading amount in the silk fibroin gel, and ultrasonic dispersion is uniform until a gel is formed, the gel is a multi-barrier drug release preparation, and is stored at 4°C; the hydrophilic drug is a small molecule drug with biological activity.

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