Drug-loading gelatin-fibroin composite micron-sized particles, preparation method therefor, and use thereof

Abstract

Drug-loading gelatin-fibroin composite micron-sized particles, a preparation method therefor, and use thereof. The present invention relates to the field of biomedical materials. The provided drug-loading gelatin-fibroin composite micron-sized particles have large compactness, uniform and controllable particle size, good dispersibility, a wide drug loading spectrum, and MMP9 responsiveness, which improve the safety of drug administration, facilitate the stabilization of drug efficacy, improve targeted drug delivery efficiency, and reduce toxicity to other tissues. The particles can be used for preparing a drug having a pore-free compact structure and MMP9 responsiveness and constructing a universal drug delivery system. The provided preparation method for the drug-loading gelatin-fibroin composite micron-sized particles has a highly controllable preparation process, high stability across different batches, a high yield, and a simple and easy preparation process, which facilitate the stabilization of drug efficacy and industrial scale-up production.

Description

Drug-loaded gelatin-silk fibroin composite microparticles, preparation method thereof and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials, and relates to a preparation method of composite microparticles, in particular to a drug-loaded gelatin-silk fibroin composite microparticle, a preparation method thereof and application thereof. BACKGROUND

[0002] Silk fibroin, as a natural biomacromolecule material with abundant source, has the characteristics of good biocompatibility, biodegradability, easy processing and chemical modification, and has the potential as a drug delivery carrier material. Silk fibroin is orderly arranged by hydrophilic amino acid residue segments and hydrophobic amino acid residue segments. The spacing of the hydrophilic and hydrophilic segments and the existence of the hydrophilic long-chain amino acid residues provide the possibility for the self-assembly of silk fibroin in aqueous solution. The silk fibroin molecules in the random coiled state can be aggregated to form stable beta crystals under the induction of external conditions, which is the basic principle for the preparation of all silk fibroin-based materials. Self-assembly refers to the process that certain macromolecules spontaneously form stable structures under certain conditions, and through self-assembly, supermolecules or molecular aggregates with controllable properties can be formed. Due to the construction of hydrophobic and hydrophilic segments of the molecular chain and the kinetic characteristics of easy beta folding, silk fibroin has potential self-assembly characteristics.

[0003] Up to now, there are various methods to induce the preparation of silk fibroin microparticles, such as heat treatment, electrospray, ion induction, supercritical fluid induction, dialysis method and desolvation method. Among them, the desolvation method is the most commonly used method, which mixes the silk fibroin solution with polar protic solvents such as methanol, ethanol, propanol, isopropanol and acetone, and then collects the precipitated product. The precipitate is washed and freeze-dried to obtain microparticles with a size range of 100 nm to 20 μm. Nanoparticles and microparticles can be further screened by differential centrifugation. However, the traditional method represented by desolvation has great defects, which are as follows: (1) the preparation process is uncontrollable, the prepared particles have wide size distribution, large difference in particle morphology, poor dispersibility and easy aggregation in solution; (2) low batch stability, not suitable for mass production; (3) low yield, easy to cause waste of raw materials; (4) narrow drug loading spectrum, the drug loading amount of different drugs with different physicochemical properties is greatly different; (5) the particles are not compact enough, and drug loss, burst release and other situations are prone to occur.

[0004] SUMMARY

[0005] The technical problem solved by the present application is to provide a drug-loaded gelatin-silk fibroin composite microparticle to overcome the shortcomings of the microparticles prepared by only using desolvation method, such as wide particle size distribution, narrow drug loading spectrum, and easy loss or burst release of the loaded drug.

[0006] Another object of the present application is to provide a preparation method of the drug-loaded gelatin-silk fibroin composite microparticle to overcome the shortcomings of the prior art, such as uncontrollable preparation process, low yield, and poor batch stability.

[0007] Still another object of the present application is to provide an application of the drug-loaded gelatin-silk fibroin composite microparticle, which can be widely used for preparing a non-porous compact structure with MMP9 responsiveness.

[0008] To achieve the above objects, the technical solution adopted by the present application is as follows:

[0009] A drug-loaded gelatin-silk fibroin composite microparticle, the raw materials for preparing the effective components thereof include gelatin, silk fibroin, and a loaded drug, the mass ratio of the gelatin to the silk fibroin is 1:20-1, and the mass ratio of the loaded drug to the silk fibroin is 1:10-50; the loaded drug includes a water-soluble drug and a hydrophobic drug.

[0010] As a limitation of the drug-loaded gelatin-silk fibroin composite microparticle, the drug encapsulation efficiency thereof is 81.4-98.8%, and the drug loading amount is 1.55-10.01%.

[0011] As a further limitation of the drug-loaded gelatin-silk fibroin composite microparticle, the surface thereof is smooth and non-porous, the particle size is 0.1-20 μm, and the microparticle has MMP9 responsiveness.

[0012] The present application further provides a preparation method of the drug-loaded gelatin-silk fibroin composite microparticle, which comprises the following steps performed in sequence:

[0013] S1. Preparation of a silk fibroin solution and a gelatin solution

[0014] Take regenerated silk fibroin, prepare an initial regenerated silk fibroin solution by adding water, dissolve and hydrate, and remove the insoluble aggregates to obtain a silk fibroin solution;

[0015] Take gelatin, prepare an initial gelatin solution by adding water, and then perform water bath to obtain a gelatin solution;

[0016] S2. Preparation of a gelatin-silk fibroin mixed system

[0017] The gelatin solution and the silk fibroin solution are mixed in proportion, the mass ratio of gelatin to silk fibroin is 1:20-1, and the gelatin-silk fibroin mixed system is prepared after uniform mixing.

[0018] S3. Preparation of drug-gelatin-silk fibroin mixed system

[0019] The loaded drug is dispersed into the gelatin-silk fibroin mixed system, and the drug-gelatin-silk fibroin mixed system is obtained after ultrasonic treatment.

[0020] S4. Preparation of drug-loaded gelatin-silk fibroin composite microparticles

[0021] The drug-gelatin-silk fibroin mixed system is added dropwise into an acetone solution, uniformly mixed, centrifuged, and freeze-dried to prepare the drug-loaded gelatin-silk fibroin composite microparticles.

[0022] It has been reported that rigid and fixed orientation molecules can induce the formation of silk fibroin secondary structure and accelerate the solidification process. Inspired by this, the present application explores a new method for preparing microparticles by controllable induction of silk fibroin β-sheet formation. The present application finds that gelatin molecules with a triple helix structure can induce silk fibroin to produce microphase separation, and the size of the separated microphase is closely related to the ratio of gelatin / silk fibroin. Specifically, there is a balance in the whole process of inducing phase separation, that is, the formation of silk fibroin β-sheet induced by gelatin molecules and the limitation of β-sheet formation by the steric hindrance effect of gelatin. Low concentration of gelatin is more conducive to the formation of β-sheet, so the phase separation is mainly of the crystal type with anti-parallel β-sheet, and due to the rapid effect of β-sheet formation, the final separated microphase is small in size. When the concentration of gelatin increases, the steric hindrance effect is significantly enhanced, and the β-sheet is significantly reduced, which instead induces the formation of Silk I crystal type. The Silk I crystal type is formed by random coiling between and within silk fibroin (SF) molecules, so the range of microphase separation becomes larger. Through the pre-assembly process of microphase separation and the subsequent desolvation-mediated "densification" process, the finally prepared drug-loaded gelatin-silk fibroin composite microparticles have a compact and few-pore internal structure, thus having excellent drug loading, activity protection and controlled release capabilities.

[0023] As the first limitation to the above preparation method of drug-loaded gelatin-silk fibroin composite microparticles, in step S1, the preparation method of regenerated silk fibroin comprises the following steps: cocoon degumming with weak alkali, dissolution with a ternary solution composed of calcium chloride, ethanol and water, water dialysis, and freeze-drying to prepare regenerated silk fibroin.

[0024] As a second limitation of the above preparation method of the drug-loaded gelatin-silk fibroin composite microparticles, the water bath in step S1 has a water bath temperature of 40-90 DEG C and a water bath time of 0.5-2 h.

[0025] As a third limitation of the above preparation method of the drug-loaded gelatin-silk fibroin composite microparticles, the drug loaded in step S3 includes a chemical drug, a small molecule interfering RNA, a protein drug, or a plasmid nucleic acid drug.

[0026] As a fourth limitation of the above preparation method of the drug-loaded gelatin-silk fibroin composite microparticles, the ultrasonic treatment in step S3 is probe ultrasonic treatment, which has an ultrasonic power of 0.2-5 W, an amplitude of 2-20%, and an ultrasonic time of 90-120 s.

[0027] As a further limitation of the above preparation method of the drug-loaded gelatin-silk fibroin composite microparticles, the volume ratio of the drug-gelatin-silk fibroin mixed system to acetone in step S4 is 1:2-10.

[0028] The centrifugal speed is 10,000-20,000 rpm, and the centrifugal time is 10-40 min.

[0029] The application also provides use of the above drug-loaded gelatin-silk fibroin composite microparticles in preparation of a non-porous compact structure and MMP9-responsive drug delivery system.

[0030] Compared with the prior art, the application has the following technical progress:

[0031] ①Compared with the prior art, the drug-loaded gelatin-silk fibroin composite microparticles provided by the application have high compactness, which can completely coat and protect the loaded drug, and the drug is not easy to be lost or released in the storage and in-vivo treatment, and the safety is higher. In addition, the compact microparticles have better mucus penetration ability, and the appropriate particle size and surface charge endow the gelatin-silk fibroin mixed system with the ability of mucosal penetration.

[0032] ②The drug-loaded gelatin-silk fibroin composite microparticles provided by the application have excellent MMP-9-responsive drug release ability, and can realize drug controlled release, especially the controlled release of tumor-specific drugs, which can significantly reduce the toxic and side effects on other tissues.

[0033] ③The self-assembly process induced by microphase separation includes a pre-assembly process between the drug and the carrier, which eliminates the influence of the physicochemical properties such as hydrophobicity and charge characteristics of the drug on the loading process, greatly reduces the "threshold" of drug loading, and thus makes the drug-loaded gelatin-silk fibroin composite microparticles provided by the application have a wider drug loading spectrum.

[0034] IV. The drug-loaded gelatin-silk fibroin composite microparticles provided by the application are prepared by using silk fibroin as a base material. Silk fibroin has obvious advantages in biocompatibility, biodegradability, bioactivity and mechanical properties compared with synthetic polymer materials, and is widely available and low in price. In addition, silk fibroin has multiple properties in drug loading, delivery and targeting, and thus is selected as a base material for constructing a broad-spectrum drug delivery platform.

[0035] V. In the preparation process of the drug-loaded gelatin-silk fibroin composite microparticles, gelatin is used as a phase separation inducer, and a phase separation system is successfully obtained under the promotion of low-energy ultrasound. The phase separation process has the following four characteristics: (a) the separated phase is a mixed system of silk fibroin and gelatin; (b) under the action of mild ultrasound, the silk fibroin / gelatin separated phase is in the form of micro-separated phase with uniform size dispersed in the solvent, and thus the process is referred to as a gelatin-induced micro-phase separation process; (c) the size of the micro-separated phase is strictly positively correlated with the silk fibroin / gelatin mass ratio (SF / GEL), that is, by changing the silk fibroin / gelatin mass ratio, micro-separated phases with different dispersion volumes can be obtained, which is a strategy for regulating the particle size of the drug-loaded silk fibroin / gelatin composite microparticles; (d) the micro-phase separation process is accompanied by a self-assembly process, in which silk fibroin changes from an amorphous structure to a crystal structure, and the self-assembly process can promote the pre-loading of drugs and the change of the micro-separated phase to a compact and stable structure.

[0036] Therefore, the preparation process provided by the application is highly controllable. By adjusting the gelatin / silk fibroin (GEL / SF) mass ratio, microparticles with different particle sizes and surface charges can be obtained, and the size can be adjusted in the range of 100 nm to 20 μm. Due to the micro-phase separation self-assembly induced by gelatin molecules, the product particle size is uniform, the morphology is stable, the dispersion is excellent, and the stability of products between different batches can be guaranteed by precisely adjusting the gelatin / silk fibroin ratio.

[0037] The "loose" system obtained by the pre-assembled stable silk fibroin / gelatin microphase separation is made into a more compact and non-porous structure of compact particles by a traditional desolvation method, and a non-porous compact structure is constructed. The particles obtained by the preparation method provided by the application have the characteristics of compact and non-porous structure, which is mainly due to the pre-assembly process mediated by microphase separation and the subsequent desolvation of the particle structure. Compared with the silk fibroin microparticles alone, the content of Silk I crystal structure in the drug-loaded gelatin-silk fibroin composite microparticles is significantly increased. Because compared with the anti-parallel beta-sheet of Silk II, Silk I is mainly composed of random coil and alpha-helix, and the crystal structure is more "plastic", and the adaptability of space filling is realized in the process of densification of the drug-loaded gelatin-silk fibroin composite microparticles. Therefore, the significant increase of Silk I structure is not only the symbol of the compact and non-porous structure of the drug-loaded gelatin-silk fibroin composite microparticles, but also the basis for its generation.

[0038] The preparation method of the drug-loaded gelatin-silk fibroin composite microparticles provided by the application has high yield, mainly because the phase separation process has synergistic effect, thereby promoting the phase separation of the whole system, so that the loss rate of the material is low and the drug loading capacity is high. At the same time, the increase of the feeding amount will not cause the increase of the loss rate, but the yield will also increase with the increase of the feeding amount. In addition, the preparation process is simple and easy to operate, the raw material cost is relatively low, and it is convenient for large-scale production.

[0039] In summary, the drug-loaded gelatin-silk fibroin composite microparticles provided by the application have high compactness, uniform and controllable particle size, good dispersibility, wide drug loading spectrum and MMP9 responsiveness. The drug-loaded gelatin-silk fibroin composite microparticles not only can improve the safety of drug use, but also can improve the stability of drug efficacy, improve the targeting delivery efficiency of drugs and reduce the toxicity to other tissues. The preparation method of the drug-loaded gelatin-silk fibroin composite microparticles provided by the application has high controllability, high stability between different batches, high yield and simple preparation process, which is beneficial to the stability of drug efficacy and industrialized production, and has wide application prospect.

[0040] The application can be used for preparing drugs with non-porous compact structure and MMP9 responsiveness, constructing a universal drug delivery system, and especially constructing a tumor drug delivery platform. BRIEF DESCRIPTION OF DRAWINGS

[0041] The application will be further described in detail below in combination with the drawings and specific embodiments.

[0042] Figure 1 is a scanning electron microscope image of the doxorubicin hydrochloride-loaded gelatin / silk fibroin composite microparticles and silk fibroin microparticles in Example 1 of the present application, wherein Figure (i) is the surface of the gelatin / silk fibroin composite microparticles, Figure (ii) is the cross-section of the gelatin / silk fibroin composite microparticles, Figure (iii) is the surface of the silk fibroin microparticles, and Figure (iv) is the cross-section of the silk fibroin microparticles;

[0043] Figure 2 is a particle size distribution histogram of the doxorubicin hydrochloride-loaded gelatin / silk fibroin composite microparticles and silk fibroin microparticles in Example 1 of the present application;

[0044] Figure 3 is a distribution profile of doxorubicin hydrochloride in the gelatin / silk fibroin microseparation phase in Example 1 of the present application;

[0045] Figure 4 is a drug release curve of the doxorubicin hydrochloride-loaded gelatin / silk fibroin composite microparticles in Example 1 of the present application;

[0046] Figure 5 is a particle size change graph of the doxorubicin hydrochloride-loaded gelatin / silk fibroin composite microparticles in different release buffers in Example 1 of the present application;

[0047] Figure 6 is a particle size distribution histogram of four kinds of drug-loaded gelatin / silk fibroin composite microparticles prepared in Examples 2 to 5 of the present application;

[0048] Figure 7 is a particle size distribution histogram of the doxorubicin hydrochloride-loaded gelatin / silk fibroin composite microparticles in Example 6 of the present application;

[0049] Figure 8 is a particle size distribution histogram of the drug-loaded gelatin / silk fibroin composite microparticles prepared in Examples 7 to 10 of the present application;

[0050] Figure 9 is a H&E staining image of the lung tissue sections of four kinds of tumor-bearing mice in Example 11 of the present application;

[0051] Figure 10 is a histogram of the number of breast cancer metastatic nodules of four kinds of tumor-bearing mice in Example 11 of the present application;

[0052] Figure 11 is a survival time histogram of four kinds of tumor-bearing mice in Example 11 of the present application. DETAILED DESCRIPTION

[0053] The present application will be further described in detail by specific examples. It should be understood that the described examples are the preferred examples of the present application and are only used to explain the present application and do not limit the present application.

[0054] The materials, reagents, etc. used in the examples of the present application can be obtained from commercial channels unless otherwise specified.

[0055] Example 1: A preparation method of doxorubicin hydrochloride-loaded gelatin / silk fibroin composite microparticles

[0056] One, the embodiment is a preparation method of doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles, which comprises the following steps performed in sequence:

[0057] S1. Preparation of silk fibroin solution and gelatin solution

[0058] Take 15g of cocoon, after degumming with 1000mL of 0.5M sodium carbonate, dissolve completely with 300mL of ternary solution, after dialysis with deionized water, freeze-drying to obtain regenerated silk fibroin; wherein the ternary solution is prepared by mixing calcium chloride, ethanol and deionized water in a molar ratio of 1:2:8;

[0059] Take 5g of regenerated silk fibroin, add 100mL of deionized water to prepare an initial regenerated silk fibroin solution, dissolve completely and hydrate for 2h, remove the insoluble aggregates, and prepare a silk fibroin solution with a mass concentration of 2%;

[0060] Take 1g of type A gelatin, prepare an initial gelatin solution with a mass concentration of 0.2% by adding deionized water, and dissolve completely in a 60°C water bath for 2h to prepare a gelatin solution;

[0061] S2. Preparation of gelatin-silk fibroin mixed system

[0062] Mix the silk fibroin solution and the gelatin solution in proportion, so that the mass ratio of gelatin to silk fibroin is 1:5, mix uniformly, and prepare a gelatin-silk fibroin mixed system; note that the mixing process should be gentle and slow to avoid generating a large amount of bubbles;

[0063] S3. Preparation of drug-gelatin-silk fibroin mixed system

[0064] Disperse 8mg of the loaded drug, which is a hydrophilic drug, doxorubicin hydrochloride (DOX), uniformly into 20mL of the gelatin-silk fibroin mixed system, and gently ultrasonic in a ultrasonic cell disruptor for 90s to promote the occurrence of microphase separation, to obtain a drug-gelatin-silk fibroin mixed system;

[0065] In this step, the mass ratio of doxorubicin hydrochloride to silk fibroin is 1:20;

[0066] The ultrasonic conditions are: ultrasonic power 1W, amplitude 10%;

[0067] S4. Preparation of doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles

[0068] The 20 mL drug-gelatin-silk fibroin mixed system was added dropwise into 100 mL acetone solution while vortexing, at which time the solution became turbid emulsion. After vortexing for 1 min, the precipitate containing the composite microparticles was collected by centrifugation at 10,000 rpm for 20 min, and then freeze-dried at -40 for 72 h to obtain the gelatin-silk fibroin composite microparticles 1 loaded with doxorubicin hydrochloride (denoted as DOX@GSC1), which was stored at 4°C.

[0069] Secondly, the structure, particle size, drug distribution, drug loading and encapsulation efficiency, MMP-9 responsiveness and other related indicators of the gelatin-silk fibroin composite microparticles loaded with doxorubicin hydrochloride prepared in the above were tested, as follows:

[0070] (1) Structure determination of the gelatin-silk fibroin composite microparticles 1 loaded with doxorubicin hydrochloride

[0071] In this embodiment, the differences in surface structure and internal structure between the gelatin-silk fibroin composite microparticles 1 loaded with doxorubicin hydrochloride (DOX@GSC1) and silk fibroin microparticles (SFP) were investigated by scanning electron microscopy, and the scanning images are shown in Figure 1.

[0072] As can be seen from Figure 1, the surface structure of SFP particles is uneven and not smooth, and the internal structure of the particles is porous with varying hole sizes. Compared with SFP particles, the DOX@GSC1 prepared by the pre-assembly process of microphase separation and the subsequent desolvation-mediated "densification" process has a smooth surface, uniform particle size, compact and less porous internal structure. This structure can completely cover and protect the loaded drug, effectively avoiding the loss and burst release of the drug during storage and in vivo treatment, and thus has the potential to have excellent drug loading, activity protection and controlled release capabilities.

[0073] (2) Particle size determination of the gelatin-silk fibroin composite microparticles 1 loaded with doxorubicin hydrochloride

[0074] The particle size of the gelatin-silk fibroin composite microparticles 1 loaded with doxorubicin hydrochloride prepared when the mass ratio of gelatin / silk fibroin (GEL / SF) was 1:5 was determined by dynamic light scattering, and the particle size statistical graph is shown in Figure 2.

[0075] As can be seen from Figure 2, when the mass ratio of GEL / SF was 1:5, the particle size of the microparticles loaded with doxorubicin hydrochloride was 3 μm.

[0076] (3) Distribution of hydrophilic drugs in the gelatin-silk fibroin microseparation phase

[0077] The distribution of the water-soluble chemical drug doxorubicin hydrochloride in the solvent phase and the gelatin-silk fibroin separation phase during the phase separation was detected by fluorescence tracing method. The specific method was as follows:

[0078] Since DOX itself has red fluorescence, no fluorescence agent needs to be added in this embodiment. After obtaining the doxorubicin hydrochloride-gelatin-silk fibroin mixed system, the distribution of DOX in the solvent phase and the microseparation phase was observed by fluorescence microscopy, and the specific method was shown in FIG. 3.

[0079] As can be clearly seen from FIG. 3, after phase separation, the hydrophilic drug doxorubicin hydrochloride was significantly dispersed into the gelatin-silk fibroin microseparation phase, which was called the preloading process of the drug, and the threshold of drug loading was significantly reduced.

[0080] (4) Determination of drug loading and encapsulation efficiency

[0081] In this embodiment, the drug loading and encapsulation efficiency of doxorubicin hydrochloride in gelatin-silk fibroin composite microparticles (GSC) were investigated, and compared with the drug loading and encapsulation efficiency of poly(lactic-co-glycolic acid) (PLGA) commonly used in the art.

[0082] The drug loading and encapsulation efficiency in the present application were determined according to the method described in the literature of Liu Man et al., entitled "Research Progress of Influencing Factors of Drug Loading in Polymer Micelles", published in China Medical Industry Journal, 2017, Vol. 48, No. 9, pp. 1257-1263.

[0083] The determination results of drug loading and encapsulation efficiency are shown in Table 1.

[0084] Table 1: Statistical table of drug loading and encapsulation efficiency of doxorubicin hydrochloride in GSC and PLGA

[0085] As shown in Table 1, for the hydrophilic doxorubicin hydrochloride, the drug loading of the GSC mixed system was high (8.911%), and the encapsulation efficiency was more than 90%. As a control, under the same feeding amount, the drug loading and encapsulation efficiency of PLGA were significantly lower than those of gelatin-silk fibroin composite microparticles.

[0086] (5) MMP-9 responsiveness of doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1

[0087] Since the gelatin molecular chain has the recognition cleavage site of MMP-9, the gelatin molecules in the doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1 can be cleaved by MMP-9 to cause the dissociation of the particles, so that the doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1 have the MMP-9-responsive drug release performance. The doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1 were respectively placed in the ordinary release buffer and the ordinary release buffer added with MMP-9, and the drug release curves were determined and plotted, as shown in Fig. 4, and the particle size change graphs in different release buffers are shown in Fig. 5.

[0088] As can be seen from Fig. 4, the doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1 have very low drug release rate in the ordinary release buffer, and the cumulative release amount of the drug is 18% within 24 h, which also illustrates the advantages of the non-porous compact structure of the doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1. When MMP-9 is added in the release buffer, the drug release rate is significantly improved, and the cumulative release amount of the drug within 24 h reaches 72%, indicating the excellent MMP-9-responsive drug release ability of the composite microparticles.

[0089] As can be seen from Fig. 5, after incubation in the ordinary release buffer for 24 h, the particle size of the doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1 has little change. However, in the release buffer containing MMP-9, the particle size of the composite microparticles rapidly decreases in a short time, indicating that the composite microparticles are obviously dissociated, indicating that the MMP-9-responsive drug release is caused by the rapid dissociation of the particles, and the result also proves the rapid response of the doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles 1 to MMP-9.

[0090] Preparation methods of doxorubicin hydrochloride-loaded gelatin-silk fibroin composite microparticles with different particle sizes

[0091] Examples 2-5 are each a preparation method of a drug-loaded gelatin-silk fibroin composite microparticle, and the steps thereof are basically the same as those of Example 1, except that the mass ratio of gelatin to silk fibroin in the step "S2. Preparation of gelatin-silk fibroin mixed system" is different. The mass ratio of gelatin to silk fibroin in different examples is shown in Table 2.

[0092] Table 2 Statistics table of mass ratio of gelatin to silk fibroin in Examples 2-5

[0093] The particle sizes of DOX@GSC2, DOX@GSC3, DOX@GSC4 and DOX@GSC5 were determined in turn according to the method described in "(2) Measurement of the particle size of the gelatin-silk fibroin composite microparticle loaded with doxorubicin hydrochloride" in Example 1. The particle size histogram of the four gelatin-silk fibroin composite microparticles loaded with doxorubicin hydrochloride is shown in Figure 6.

[0094] As can be seen from Figure 6, when the mass ratio of gelatin to silk fibroin is 1:20, 1:10, 1:2 and 1:1, the particle sizes of the microparticles loaded with doxorubicin hydrochloride are 92 nm, 100 nm, 10 μm and 20 μm, respectively. This is because microphase separation can induce the pre-assembly of the gelatin-silk fibroin system, and thus the particle size of the obtained microparticles can be regulated by adjusting the mass ratio of gelatin to silk fibroin.

[0095] Example 6 Preparation method of a gelatin-silk fibroin composite microparticle loaded with camptothecin

[0096] This example is a preparation method of a drug-loaded gelatin-silk fibroin composite microparticle, which comprises the following steps performed in turn:

[0097] S1. Preparation of a silk fibroin solution and a gelatin solution

[0098] Take 15 g of cocoon, degum it with 1000 mL of 0.5 M sodium carbonate, and then completely dissolve it in 300 mL of a ternary solution. After dialysis against deionized water, freeze-drying is performed to obtain regenerated silk fibroin; the ternary solution is prepared by mixing calcium chloride, ethanol and deionized water in a mass ratio of 1:2:8;

[0099] Take 5 g of regenerated silk fibroin, and prepare an initial regenerated silk fibroin solution by adding 100 mL of deionized water. After complete dissolution and hydration for 2 h, remove the insoluble aggregates, and prepare a silk fibroin solution with a mass concentration of 2%;

[0100] Take 1 g of gelatin type A, and prepare an initial gelatin solution by adding deionized water to obtain a gelatin mass concentration of 0.2%. After complete dissolution in a 60°C water bath for 2 h, prepare a gelatin solution;

[0101] S2. Preparation of a gelatin-silk fibroin mixed system

[0102] Mix the silk fibroin solution and the gelatin solution in a proportion such that the mass ratio of gelatin to silk fibroin is 1:5, and mix uniformly to obtain a gelatin-silk fibroin mixed system. Note that the mixing should be gentle and slow to avoid the generation of a large amount of bubbles;

[0103] S3. Preparation of a drug-gelatin-silk fibroin mixed system

[0104] The loaded drug, which is a hydrophobic drug, camptothecin 7.5 mg, is uniformly dispersed into 20 mL of the gelatin-silk fibroin mixed system, and is mildly ultrasonicated in an ultrasonic cell disruptor for 90 s to promote the occurrence of micro-phase separation, to obtain a drug-gelatin-silk fibroin mixed system;

[0105] In this step, the mass ratio of camptothecin to silk fibroin is 1:20.

[0106] The ultrasonic conditions are: ultrasonic power 1 W, amplitude 10%.

[0107] S4. Preparation of camptothecin-loaded gelatin-silk fibroin composite microparticles

[0108] The drug-gelatin-silk fibroin mixed system 20 mL is added dropwise into 100 mL of an acetone solution (i.e. the volume ratio of the drug-gelatin-silk fibroin mixed system to acetone is 1:5) while vortexing, at which time the solution becomes a turbid emulsion. After continuing to vortex for 1 min, the precipitate containing the composite microparticles is collected after centrifugation at 10,000 rpm for 20 min, and is freeze-dried at -40 for 72 h, to finally obtain camptothecin-loaded gelatin-silk fibroin composite microparticles, which are stored at 4°C.

[0109] The drug distribution, drug loading and encapsulation efficiency of the camptothecin-loaded gelatin-silk fibroin composite microparticles prepared in the above manner are also tested, as follows:

[0110] (1) Determination of the particle size of camptothecin-loaded gelatin-silk fibroin composite microparticles

[0111] The particle size of the camptothecin-loaded gelatin-silk fibroin composite microparticles prepared in this example, when the mass ratio of gelatin / silk fibroin (GEL / SF) is 1:5, is counted by a dynamic light scattering instrument, and the particle size histogram is shown in FIG. 7.

[0112] As can be seen from FIG. 7, when the mass ratio of GEL / SF is 1:5, the particle size of the camptothecin-loaded composite microparticles is 3 μm.

[0113] (2) Distribution of the hydrophobic drug in the gelatin-silk fibroin micro-phase separation system

[0114] In this example, the distribution of the hydrophobic chemical drug, camptothecin (CPT), in the two phases during the phase separation process is detected by a fluorescence tracing method. The specific method is as follows:

[0115] The raw materials and experimental parameters of steps S1-S2 of this example are not changed, only the "camptothecin" in step S3 is replaced by "fluorescein FITC conjugated camptothecin (FITC-CPT), and the remaining raw materials of step S3 are not changed, and the FITC-CPT-gelatin-silk fibroin mixed system is prepared through step S3. By taking FITC-CPT as a hydrophobic model drug, the distribution of FITC-CPT in the solvent phase and the microseparation phase is observed by fluorescence microscopy, and details are shown in FIG. 8.

[0116] As can be clearly seen from FIG. 8, after phase separation occurs, the hydrophobic drug FITC conjugated camptothecin is significantly dispersed into the gelatin-silk fibroin microseparation phase.

[0117] (3) Determination of drug loading and encapsulation efficiency

[0118] This example investigates the drug loading and encapsulation efficiency of the gelatin-silk fibroin (GSC) mixed system for camptothecin, and compares the drug loading and encapsulation efficiency with the commonly used drug-loaded polymer material poly(lactic-co-glycolic acid) (PLGA) in the art.

[0119] The determination of drug loading and encapsulation efficiency in this application is carried out by the method described in Reference Example 1.

[0120] The determination results of drug loading and encapsulation efficiency are shown in the following Table 3.

[0121] Table 3 Statistical table of drug loading and encapsulation efficiency of GSC and PLGA for camptothecin

[0122] As shown in Table 3, for the hydrophobic chemical drug camptothecin, the drug loading of the GSC mixed system is relatively high, which is 8.073%, and the encapsulation efficiency is more than 94%. As a control, under the same feeding amount, the drug loading and encapsulation efficiency of PLGA are significantly lower than those of the gelatin-silk fibroin composite microparticles.

[0123] Examples 7-10 Preparation method of drug-loaded gelatin-silk fibroin composite microparticles

[0124] Examples 7-10 are respectively a preparation method of drug-loaded gelatin-silk fibroin composite microparticles, and their steps are basically the same as those of Example 1, except that the types and amounts of raw materials are different, and some process parameters in different steps. The types and amounts of raw materials, process parameters and product statistics of Examples 7-10 are shown in Table 4, and the index statistics of the products obtained in Examples 7-10 are shown in Table 5.

[0125] Table 4 Types and amounts of raw materials, process parameters and product statistics in Examples 7-10

[0126] The inventors measured the particle size, drug loading and encapsulation efficiency of the four kinds of drug-loaded gelatin-silk fibroin composite microparticles prepared in the above Examples 7-10 according to the method described in Example 1, and the statistical table of the measurement results is shown in Table 5.

[0127] Table 5 Statistical table of particle size, drug loading and encapsulation efficiency of the composite microparticles prepared in Examples 7-10

[0128] As can be seen from Table 5, the average particle size of the gelatin-silk fibroin microparticles loaded with different drugs is 3 μm, and the drug loading and encapsulation efficiency are high for small molecule chemical drugs, protein drugs and nucleic acid drugs, wherein the encapsulation efficiency of the four kinds of drug-loaded gelatin-silk fibroin microparticles is more than 94%, and the average encapsulation efficiency is 96.8%.

[0129] The inventors measured the particle size distribution of the four kinds of drug-loaded gelatin-silk fibroin composite microparticles prepared in the above Examples 7-10 according to the method described in Example 1, and the particle size distribution graph is shown in Figure 8.

[0130] As can be seen from Figure 8, the gelatin-silk fibroin composite microparticles loaded with small molecule chemical drugs, protein drugs and nucleic acid drugs have extremely similar particle size distribution, and the average particle size is 3 μm, indicating that the loading of drugs does not affect the self-assembly process of the particles.

[0131] From the particle size and particle size distribution graph of the drug-loaded gelatin-silk fibroin composite microparticles prepared in Examples 1, 6, 7-10, it can be seen that when the mass ratio of gelatin / silk fibroin (GEL / SF) is 1:5 and the loaded drugs are doxorubicin hydrochloride, camptothecin, anti-PD-L1 antibody, PD-L1 interfering RNA, IL-12 expressing plasmid drug and paclitaxel, respectively, the finally prepared drug-loaded gelatin-silk fibroin composite microparticles have almost the same particle size distribution, and the reason is that microphase separation can induce the pre-assembly of the gelatin-silk fibroin system, so by adjusting the mass ratio of gelatin / silk fibroin (GEL / SF), the particle size of the obtained particles can be adjusted; this experiment also shows that the loading of drugs does not have a significant effect on the overall structure of the particles.

[0132] As can also be seen from the above examples, the drug-loaded gelatin-silk fibroin composite microparticles have high compactness, uniform and controllable particle size, good dispersibility, wide drug loading spectrum and MMP9 responsiveness, and within the experimental range of the present application, the particle size is 0.1-20 μm, the encapsulation efficiency is 81.4-98.8%, and the drug loading is 1.55-10.01%.

[0133] Example 11 Application of drug-loaded gelatin-silk fibroin composite microparticles

[0134] In view of the drug-loaded gelatin-silk fibroin composite microparticles having the properties of compactness, uniform and controllable particle size, good dispersibility, wide drug loading spectrum and MMP9 responsiveness, and the potential advantages in improving the safety of drug use, the stability of drug efficacy, the efficiency of targeted drug delivery, etc., they can be used to prepare a non-porous compact structure and MMP9-responsive drug delivery system, and applied in clinical practice.

[0135] This example only takes the treatment of lung diseases by drug delivery through oral and nasal inhalation of PTX-loaded composite microparticles as an example for illustration. The in vivo anti-tumor activity of drug-loaded gelatin-silk fibroin composite microparticles is evaluated by constructing a mouse model of lung metastasis of breast cancer.

[0136] In this example, 24 male Balb / c mice of about 7 weeks old were selected, and a mouse model of lung metastasis of breast cancer was constructed by the method described in Yan Fei et al., Chinese Journal of Chinese Medicine, 2020, Vol. 45, No. 21, Study on Inhibition of Lung Metastasis of Breast Cancer by F127 Modified Lipid Polymer Nanoparticles Loaded with Fructus Schisandrae Chinensis B;

[0137] The above 48 mice were randomly divided into four groups according to their body weight, with 12 mice in each group. The four groups were PBS control group, free PTX treatment group, blank GSC treatment control group and PTX@GSC group. The tumor-bearing mice were administered by nebulization, and the types and doses of drugs administered to the mice in different groups during the experiment are shown in Table 6.

[0138] Table 6 Types and doses of drugs administered to mice in different groups

[0139] The PTX@GSC used in the PTX@GSC group of this example was prepared in Example 10.

[0140] After the breast cancer lung metastasis model was successfully constructed, the administration began, and the administration was performed every three days. Six mice in each group were randomly selected for H&E staining and counting of the number of breast cancer metastatic nodules, and the remaining six mice in each group were used to investigate the survival of tumor-bearing mice.

[0141] Six mice were randomly selected from each of the four groups of mice, and a total of three administrations were performed. Fifteen days after the first administration, the mice were euthanized, and the lung tissue was dissected and subjected to H&E staining. The H&E staining images of lung tissue sections of the four tumor-bearing mice are shown in Figure 9. The number of tumor metastatic nodules in the lungs was also counted by H&E staining, and the number of breast cancer metastatic nodules in the four tumor-bearing mice is shown in Figure 10.

[0142] The remaining six mice in each of the four groups were used to investigate the survival of tumor-bearing mice, and the survival of the four tumor-bearing mice is shown in Figure 11.

[0143] As shown in FIGS. 9-10, the number of metastatic nodules in the PTX@GSC group was significantly lower than that in the free PTX group and the blank GSC group. Therefore, compared with the free PTX group and the blank GSC group, the PTX@GSC group significantly inhibited the growth of tumor nodules, reduced the number of tumor metastatic nodules, and prolonged the survival time. However, as shown in FIG. 11, the systemic toxic side effects caused by free PTX did not prolong the survival time of the mice, but rather accelerated the death of the mice after treatment. On the other hand, the blank GSC control group also showed a certain inhibitory effect on the growth of tumor metastatic nodules, which was caused by the immunoregulatory activity of the GSC itself. In summary, the gelatin-silk fibroin composite microparticles prepared by the present application have excellent drug delivery capacity.

[0144] Obviously, the above embodiments of the present application are only examples for clearly illustrating the present application, and are not intended to limit the embodiments of the present application. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. It is impossible to enumerate all the embodiments here. Any obvious changes or variations derived from the technical solutions of the present application are still within the protection scope of the present application.

Claims

1. Drug-loaded gelatin-silk fibroin composite microparticles, characterized by, The raw materials for preparing the effective component include gelatin, silk fibroin, and the loaded drug, wherein the mass ratio of the gelatin to the silk fibroin is 1:20-1, and the mass ratio of the loaded drug to the silk fibroin is 1:10-50.

2. The drug-loaded gelatin-silk fibroin composite microparticles according to claim 1, characterized by, The drug encapsulation rate of the drug-loaded gelatin-silk fibroin composite microparticles is 81.4-98.8%, and the drug loading amount is 1.55-10.01%.

3. The drug-loaded gelatin-silk fibroin composite microparticles according to claim 1 or 2, characterized by, The drug-loaded gelatin-silk fibroin composite microparticles have a smooth surface, no pores, a particle size of 0.1-20 μm, and MMP9 responsiveness.

4. The method for preparing drug-loaded gelatin-silk fibroin composite microparticles according to any one of claims 1 to 3, characterized by, The preparation method comprises the following steps performed in sequence: S1. Preparation of a silk fibroin solution and a gelatin solution Take regenerated silk fibroin, and prepare an initial regenerated silk fibroin solution by adding water, dissolve and hydrate, and remove insoluble aggregates to obtain a silk fibroin solution; Take gelatin, prepare an initial gelatin solution by adding water, and then perform water bath to obtain a gelatin solution; S2. Preparation of a gelatin-silk fibroin mixed system Mix the silk fibroin solution and the gelatin solution in a certain proportion, so that the mass ratio of the gelatin to the silk fibroin is 1:20-1, and mix uniformly to obtain a gelatin-silk fibroin mixed system; S3. Preparation of a drug-gelatin-silk fibroin mixed system Disperse the loaded drug into the gelatin-silk fibroin mixed system, and perform ultrasonic treatment to obtain a drug-gelatin-silk fibroin mixed system; S4. Preparation of drug-loaded gelatin-silk fibroin composite microparticles Drop the drug-gelatin-silk fibroin mixed system into an acetone solution, mix uniformly, centrifuge, and freeze-dry to obtain drug-loaded gelatin-silk fibroin composite microparticles.

5. The method of claim 4, wherein the drug-loaded gelatin-silk fibroin composite microparticles are prepared by the steps of: In step S1, The preparation method of the regenerated silk fibroin comprises the following steps: degumming of a cocoon by a weak alkali, dissolution by a ternary solution composed of calcium chloride, ethanol, and water, water dialysis, and freeze-drying to obtain regenerated silk fibroin.

6. The method of claim 4, wherein the drug-loaded gelatin-silk fibroin composite microparticles are prepared by the steps of: In step S1, the water bath temperature is 40-90 ℃, and the water bath time is 0.5-2 h.

7. The method of claim 4, wherein the drug-loaded gelatin-silk fibroin composite microparticles are prepared by the steps of: In step S3, the loaded drug includes a chemical drug, a nucleic acid drug, and a protein drug.

8. The method of claim 4 to 7, wherein the drug-loaded gelatin-silk fibroin composite microparticles are prepared by the steps of: In step S3, the ultrasonic treatment is probe ultrasonic treatment, the ultrasonic power is 0.2-5 W, the amplitude is 2-20%, and the ultrasonic time is 90-120 s.

9. The method of claim 8, wherein the drug-loaded gelatin-silk fibroin composite microparticles are prepared by the steps of: In step S4, the volume ratio of the drug-gelatin-silk fibroin mixed system to the acetone is 1:2-10. The centrifugal speed is 10,000-20,000 rpm, and the centrifugal time is 10-40 min.

10. Use of the drug-loaded gelatin-silk fibroin composite microparticles according to any one of claims 1-3 in the preparation of a drug delivery system having a non-porous compact structure and MMP9 responsiveness.

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