Antibiotic sustained release system based on hierarchical porous silica and preparation method thereof

Abstract

The application discloses an antibiotic sustained-release system based on a multi-stage pore silica and a preparation method thereof, and belongs to the technical field of biological medicines, wherein the antibiotic sustained-release system comprises: a spherical multi-stage pore silica microsphere carrier, amino functional groups covalently bonded to the outer surface and the inner surface of pores of the spherical multi-stage pore silica microsphere carrier, antibiotic molecules loaded in the interior of the pores of the spherical multi-stage pore silica microsphere carrier, and a pH-responsive chitosan gating layer. The preparation method comprises the following steps: (1) preparing the multi-stage pore silica microsphere carrier; (2) aminoating the surface of the carrier; (3) loading the antibiotics; and (4) constructing the pH-responsive chitosan gating layer. The application constructs a silica carrier with a macropore-mesopore hierarchical structure to realize high drug loading, and covalently bonds a pH-responsive chitosan gating layer to the pore opening to realize intelligent controlled release, thereby solving the problem that high drug loading and low burst release rate and biological safety are difficult to be achieved simultaneously in the prior art.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an antibiotic sustained-release system based on a multi-level pore silica and a preparation method thereof. BACKGROUND

[0002] In modern medical practice, the effective application of antibiotics is a key cornerstone to control bacterial infection and protect human health. Traditional drug delivery methods, such as oral preparations or intravenous injection, can quickly deliver drugs into the body, but they have the inherent defect of blood drug concentration fluctuating in a "peak-valley" manner. This fluctuation not only may cause drug concentration to exceed the safe range at the peak value and induce toxic side effects, but also may be lower than the minimum inhibitory concentration (MIC) at the valley value, thereby weakening the therapeutic effect and even inducing bacterial drug resistance. In order to overcome these limitations, the development of efficient and safe drug sustained-release systems to achieve long-term and constant release of drugs in the body, thereby maintaining a stable and effective blood drug concentration, has become a research focus in the fields of pharmacy and material science.

[0003] Under this background, organic polymer carriers represented by polymer microspheres and liposomes have emerged as the times require. These carriers encapsulate drug molecules in their three-dimensional network structure or internal cavity, and use the swelling and degradation of the polymer matrix or the passive diffusion of drug molecules to achieve sustained release of drugs. In a certain historical period, the drug delivery scheme was designed according to the biological half-life of the drug, effectively solving the problem of short drug half-life, significantly prolonging the drug action time, and thus improving the therapeutic effect and patient medication compliance to some extent. Specifically, by adjusting the molecular weight, crosslinking degree and hydrophilicity / hydrophobicity of the polymer, the release rate of the drug can be preliminarily controlled.

[0004] However, with the in-depth exploration of the concept of precision therapy and the deepening understanding of the complex infection microenvironment, some inherent characteristics of such polymer matrix-based carriers in principle have gradually shown insurmountable limitations in coping with new challenges. The reason lies in that the drug loading mechanism is essentially a physical embedding, and the drug molecules are unevenly distributed in the heterogeneous polymer matrix. The drug molecules near the surface of the carrier are easily released rapidly in the initial stage, forming a significant "burst effect", which not only causes waste of drugs, but also may induce local or systemic toxicity in the early stage of medication. In order to alleviate the burst phenomenon and increase the density of the polymer matrix, it has become a common means to increase the density of the polymer matrix, however, this will inevitably reduce the internal space for drug loading, and the drug loading capacity is usually difficult to exceed 20%. This negative correlation between drug loading capacity and burst control constitutes a pair of core contradictions in the performance improvement of polymer carriers.

[0005] To overcome the limitation of drug loading, researchers have begun to focus on metal-organic frameworks (MOFs) materials, which can efficiently accommodate a large number of drug molecules through physical adsorption or chemical bonding due to their highly regular pore structure and large specific surface area. The theoretical drug loading of MOFs materials far exceeds that of traditional polymer carriers, which benefits from their unique crystal structure and designable properties. This progress seems to provide an ideal way to solve the problem of low drug loading. However, new problems have followed. For MOFs carriers, the degradation behavior in the physiological environment is often difficult to accurately predict and control. The coordination bond between the metal ions and organic ligands that make up the framework may break uncontrollably in the complex body fluid environment, leading to premature disintegration of the carrier structure, not only causing the instantaneous "dumping" release of the loaded drugs, which reappears the more severe burst problem, but also its degradation products, especially free metal ions, may exhibit potential cytotoxicity or immunogenicity, bringing new biological safety risks. Although metal-organic frameworks (MOFs) show great potential for drug delivery systems, such as achieving targeted delivery and controlled release of drugs through their highly adjustable pore structure, the challenges of MOFs in drug release controllability and biocompatibility, such as the regulation of MOFs structure and properties and the long-term impact assessment on the biological body, are still key obstacles that need to be overcome in the clinical translation process.

[0006] It can be seen that the prior art is in the following dilemma:

[0007] On the one hand, traditional carriers represented by polymer microspheres have good biocompatibility, but their material structure itself determines that there is an irreconcilable contradiction between drug loading and burst control.

[0008] On the other hand, new porous materials represented by MOFs greatly improve the drug loading, but introduce new problems of uncontrollable release behavior and potential biological toxicity. Although the use of a gating mechanism can achieve control of drug release to some extent, for example, patent publication number CN106267230A uses a "self-gating" mechanism of drug molecules, but the drug molecules themselves may have cytotoxicity (such as the cardiotoxicity of DOX), and the residual groups after breaking may trigger non-specific reactions, so the potential biological toxicity problem has not been solved.

[0009] In summary, although the polymer carrier performs excellently in biocompatibility, its physical embedding mechanism limits the improvement of drug loading and leads to the decrease of burst suppression ability. Porous materials such as metal-organic frameworks (MOFs) have high drug loading, and the combination of gating mechanism can achieve control of drug release, but may bring potential biological safety risks.

[0010] Therefore, how to design a new drug sustained-release system, which can effectively load large capacity drug molecules in structure, and at the same time integrate a precise controllable release mechanism to respond to a specific pathological microenvironment (such as the acidic characteristics of the infection site), so as to overcome the inherent contradictions between the drug loading capacity, burst effect, sustained-release period and potential biological toxicity in the prior art, has become one of the main problems to be solved in the field. SUMMARY

[0011] The purpose of the present application is to provide an antibiotic sustained-release system based on hierarchical porous silica and a preparation method thereof, which overcomes the inherent contradictions between drug loading capacity, burst control, release behavior controllability and biological safety of the drug sustained-release carrier in the prior art.

[0012] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows:

[0013] The antibiotic sustained-release system based on hierarchical porous silica comprises:

[0014] The spherical hierarchical porous silica microsphere carrier has a dual-mode pore structure composed of an interpenetrating macroporous network and mesopores extending from the inner wall of the macroporous network inside the spherical hierarchical porous silica microsphere carrier;

[0015] The amino functional groups are covalently bonded to the outer surface and the inner surface of the pores of the spherical hierarchical porous silica microsphere carrier;

[0016] The antibiotic molecules are loaded inside the pores of the spherical hierarchical porous silica microsphere carrier;

[0017] The pH-responsive chitosan gating layer is a covalently bonded coating layer covering the outer surface of the spherical hierarchical porous silica microsphere carrier, and is in a dense and contracted state under physiological neutral environment to close the pores and prevent leakage of antibiotic molecules; and is transformed into a swollen and loose state under a slightly acidic environment to open the pores and release the antibiotic molecules.

[0018] Specifically, the diameter of the spherical hierarchical porous silica microsphere carrier is 1um to 2um; the pore size of the macroporous network is 150nm to 250nm; the pore size of the mesopore is 8nm to 15nm, and the specific surface area of the mesopore is greater than 600m 2 / g; the total pore volume of the spherical hierarchical porous silica microsphere carrier is greater than 1.2cm 3 / g.

[0019] Further, the amino functional groups covalently bonded to the outer surface and inner surface of the pores of the spherical multi-level porous silica microsphere carrier are formed by hydrolytic condensation reaction between the triethoxysilane group of 3-aminopropyl triethoxysilane molecules as silane coupling agents and the silicon hydroxyl groups on the surface of the spherical multi-level porous silica microsphere carrier.

[0020] As a preference, the antibiotic molecules are levofloxacin or ciprofloxacin.

[0021] Specifically, the pH-responsive chitosan gating layer is constructed by glutaraldehyde crosslinking agent; the aldehyde groups of the glutaraldehyde crosslinking agent react with the amino functional groups on the surface of the spherical multi-level porous silica microsphere carrier and the amino groups of the chitosan molecules to form imine bonds of a covalently bonded chitosan network; the swelling ratio of the pH-responsive chitosan gating layer in a pH=7.4 environment is ≤6, and the swelling ratio in a pH=5.5 environment is ≥15.

[0022] As a preference, the weight average molecular weight of the chitosan is 50 kilodaltons to 100 kilodaltons, and the degree of deacetylation is 85%.

[0023] Further, the molar ratio of the glutaraldehyde crosslinking agent to the amino groups in the chitosan molecules is 0.5:1 to 1.5:1, so as to adjust the crosslinking density and response performance of the pH-responsive chitosan gating layer.

[0024] Meanwhile, the application also provides a preparation method of the above-mentioned antibiotic sustained-release system based on multi-level porous silica, comprising the following steps:

[0025] (1) Preparation of the multi-level porous silica microsphere carrier: using a double-template method, in a mixed solvent system with ethanol and deionized water as main components and ammonia as catalyst, poly(methyl methacrylate) nanospheres as macroporous template agent and triblock copolymer as mesoporous template agent, a silicon source is added to form composite microspheres through hydrolytic condensation reaction, and the two organic template agents are removed by high-temperature calcination to obtain spherical multi-level porous silica microsphere carriers with double-mode pore structure;

[0026] (2) Amination of the surface of the carrier: after drying and activating the spherical multi-level porous silica microsphere carrier under vacuum conditions at 100°C to 150°C, the spherical multi-level porous silica microsphere carrier is refluxed with a silane coupling agent in an organic solvent at 80°C to 120°C for 12 to 24 hours, so that the amino functional groups are covalently bonded to the outer surface and inner surface of the pores of the spherical multi-level porous silica microsphere carrier;

[0027] (3) Loading of antibiotics: the aminated spherical multi-level porous silica microsphere carrier is dispersed in a buffer solution with a pH of 7.0 to 7.4 and containing antibiotic molecules with a concentration of 3-5 mg / mL, and after stirring and adsorption, the carrier is separated, washed, and dried to obtain a drug-loaded intermediate;

[0028]

[0028] (4) Construction of pH-responsive chitosan gate layer: the drug-loaded intermediate is dispersed in an acidic buffer solution, chitosan solution is added after mixing, and glutaraldehyde solution as a crosslinking agent is added for in-situ crosslinking reaction, so that the chitosan forms a covalently bonded gate layer on the outer surface of the pore of the spherical multi-level porous silica microsphere carrier, and after washing and drying, an antibiotic sustained-release system is obtained.

[0029] Specifically, the step (1) comprises the following steps:

[0030] (101) Disperse polymethyl methacrylate nanospheres with a particle size of 200 nm in a mixed solvent composed of 100 mL of ethanol, 25 mL of deionized water, and 5 mL of 28% w / w concentrated ammonia water, and dissolve pluronic P123 as a triblock copolymer;

[0031] (102) Under stirring conditions, dropwise add tetraethyl orthosilicate as a silicon source, wherein the mass ratio of polymethyl methacrylate nanospheres to tetraethyl orthosilicate is 1:5, and the mass ratio of pluronic P123 to tetraethyl orthosilicate is 1:4; carry out hydrolysis and condensation reaction at 30°C for 24 hours to form composite microspheres;

[0032] (103) Calcine the composite microspheres at 550°C for 6 hours to remove the template agent and solidify the silica framework, and obtain a spherical multi-level porous silica microsphere carrier.

[0033] Specifically, the step (4) comprises the following steps:

[0034] (401) Disperse the drug-loaded intermediate in an acetic acid-sodium acetate buffer solution with pH=5.0 to obtain a suspension of drug-loaded microspheres;

[0035] (402) Dissolve chitosan with the same mass as the drug-loaded intermediate in another acetic acid-sodium acetate buffer solution with the same volume and pH value to obtain a chitosan solution;

[0036] (403) Add the chitosan solution to the suspension of drug-loaded microspheres, and slowly dropwise add 25% w / w glutaraldehyde aqueous solution under magnetic stirring, so that the final molar ratio of glutaraldehyde to amino groups in chitosan molecules is controlled in the range of 0.5:1 to 1.5:1;

[0037] (404) Perform water bath reaction on the mixed solution obtained in step (403), and after the reaction is completed, centrifuge to collect the product;

[0038] (405) Wash twice with an acetic acid-sodium acetate buffer solution with pH=5.0, and then wash with deionized water to remove unreacted chitosan, glutaraldehyde, and reaction byproducts, and then freeze-dry the final product to obtain an antibiotic sustained-release system.

[0039] Compared with the prior art, the present application has the following beneficial effects:

[0040] (1) The present application has a large pore-mesoporous through-pore structure (large pore diameter 150-250 nm, mesopore diameter 10-15 nm, mesopore specific surface area >600 m 2 / g, total pore volume >1.2 cm 3 / g), the carrier has a large pore high-speed transport channel and a mesopore high specific surface area storage unit, and the antibiotic loading capacity is 32%-33%, which is significantly higher than that of traditional polymer carriers and pure mesoporous silica carriers, and the physical barrier effect of the covalently bonded chitosan gating layer is combined to solve the technical problem that the drug loading capacity and burst release rate are difficult to achieve in the prior art.

[0041] (2) The present application covalently bonds 3-aminopropyltriethoxysilane on the surface to introduce amino functional groups, and uses the synergistic effect of electrostatic attraction and hydrogen bonding to enhance the adsorption of antibiotic molecules (such as levofloxacin and ciprofloxacin), thereby significantly improving the drug loading capacity and drug storage stability.

[0042] (3) The present application uses glutaraldehyde crosslinking agent to construct a covalently bonded chitosan gating layer at the pore mouth, which belongs to an external material gating structure. In a physiological neutral environment (pH=7.4), the chitosan is deprotonated and shrinks, and the burst release rate is less than 5% in 2 hours, which is reduced by more than 90% compared with carriers without a gating layer. Compared with existing gating mechanisms (such as self-gating mechanisms), the burst release rate is reduced by more than 75%. In a slightly acidic environment (pH=5.5), the chitosan is protonated and swells to open the release channel, and the 72-hour cumulative release rate is >83%, which can well achieve targeted on-demand drug delivery.

[0043] (4) Compared with the drug molecule self-gating mechanism used in the prior art, the external material gating mechanism used in the present application uses chitosan as the gating material, which has better biocompatibility potential and better reduces the risk of potential toxic side effects, and realizes the safe design of "stable carrier-gate degradable". Furthermore, the chitosan gating layer forms a covalently bonded network through glutaraldehyde crosslinking, which is firmly anchored on the surface of the carrier, and can still maintain the structure integrity and switching function under multiple pH cycle stimulation (neutral / acidic alternating), avoiding the risk of shedding of physically adsorbed coatings. Further, in addition to maintaining the structure integrity and switching function, the present application controls the crosslinking density of the gating layer by adjusting the molar ratio of glutaraldehyde to chitosan amino group (0.5:1 to 1.5:1), and also realizes the controllable design of release duration and rate, for example, slow release in low crosslinking density (0.5:1) and accelerated release in high crosslinking density (1.5:1), thereby matching different scene requirements. The drug molecule self-gating mechanism used in the prior art relies on the dynamic rupture of benzimidazole bond, and once the bond is broken to release the drug in an acidic environment, the gating structure is invalid, and cannot realize repeated response or dose regulation.

[0044] (5) The application adopts a double-template system of polymethyl methacrylate nanometer microspheres (macroporous template) and pluronic P123 (mesoporous template), and by accurately controlling the proportion of the template agent, the hydrolysis and condensation conditions and the calcination parameters, a spherical multi-level porous silica carrier with a double-mode pore structure can be stably prepared, the carrier has uniform pore size distribution and good pore connectivity, and the application provides a reliable structural basis for high drug loading and controlled release, and the process has high repeatability and is suitable for batch production.

[0045] (6) The application adopts an in-situ reaction process of dispersing the drug-loaded intermediate in acetic acid-sodium acetate buffer solution (pH=5.0), dissolving specific molecular weight chitosan, and slowly adding glutaraldehyde crosslinking agent under magnetic stirring, so that the chitosan gate layer is uniformly coated on the surface of the drug-loaded microspheres; and then the unreacted free chitosan and glutaraldehyde are effectively removed through the subsequent graded washing with buffer and deionized water and freeze-drying treatment, the interference of impurities on the release performance is avoided, the integrity of the carrier pore structure is maintained, and the biological safety and storage stability of the slow-release system are further improved. DETAILED DESCRIPTION

[0046] The application provides an antibiotic slow-release system based on a multi-level porous silica, and the core of the application is to construct a composite microsphere system integrating a high-capacity drug storage unit, an efficient internal transport channel and a precise external environment response gate unit. The structural basis of the system is a spherical multi-level porous silica microsphere carrier. The diameter of the carrier is controlled in the range of 1 micrometer to 2 micrometers, which can effectively avoid being quickly removed by the mononuclear phagocyte system (RES), and also provides a suitable physical size for the carrier to be retained in the target area and for subsequent drug release. The internal part of the carrier is not a homogeneous entity, but a double-mode pore network that is mutually connected. The double-mode pore network is composed of a macroporous framework with a pore size distribution of 150 nanometers to 250 nanometers and a mesopore extending from the inner wall of the macroporous framework. The macropore constitutes a high-speed transport channel for the rapid loading and release of drug molecules in the internal part of the carrier, and the mesopore with a pore size distribution of 8 nanometers to 15 nanometers serves as the main storage unit of the drug molecules by virtue of its large total internal surface area. The synergistic design of the structure lays a solid physical foundation for realizing high drug loading.

[0047] To further enhance the affinity of the carrier to specific drug molecules and provide chemical anchors for subsequent functionalization modification, the surface of the carrier is covalently bonded with a layer of amino functional groups through chemical reaction. The introduction of this amino functional group is achieved through the hydrolysis and condensation reaction of 3-aminopropyltriethoxysilane (APTES) in organic solvents. The triethoxysilane group in the APTES molecule reacts with the silicon hydroxyl (Si-OH) inherent to the silica surface, which increases in density after heat activation treatment, to form a stable silicon-oxygen-silicon (Si-O-Si) covalent bond, thereby firmly anchoring the aminopropyl segment in the inner wall and outer surface of the carrier's pores. This modification not only changes the silica surface from hydrophilic to a surface with positive charge potential, greatly enhancing the electrostatic adsorption and hydrogen bonding force of drug molecules with negative charge or lone pair electrons, but also its exposed primary amino group (-NH2) is a key reaction site for building subsequent gating structures.

[0048] Inside the amino-functionalized hierarchical pore carrier pores, antibiotic molecules such as levofloxacin are loaded as model drugs. The levofloxacin molecule can produce strong electrostatic interaction with protonated amino groups due to the presence of carboxyl and nitrogen-containing heterocyclic rings in its structure under physiological pH conditions, and its rich hydrogen bond donor and acceptor sites also enable it to form multiple hydrogen bonds with the silica surface and amino functional groups. These combined forces enable the drug molecules to be efficiently captured and stably stored in the mesopores of the carrier. The design of the present application enables the final loading of levofloxacin to reach a stable level of 32% to 33% (mass percentage) based on the total mass of the slow-release system, significantly surpassing traditional drug carriers.

[0049] Further, the functional core of the sustained-release system, namely the gating unit, is a layer of chitosan hydrogel network constructed on the outer surface of the pore of the drug-loaded carrier through in-situ polymerization and cross-linking reaction after the drug loading is completed. The chitosan gating layer is realized by glutaraldehyde cross-linking agent. The aldehyde groups at both ends of the glutaraldehyde cross-linking agent can respectively undergo nucleophilic addition-dehydration reaction with the amino functional groups protruding from the surface of the carrier and the amino groups on the chitosan molecular chain to form stable imine bonds. This covalent bonding mode firmly 'rivets' the chitosan network on the microspheres, forming a physical barrier. The chitosan used in the application has a weight average molecular weight of 50 to 100 kilodaltons and a deacetylation degree of 85%. The moderate molecular weight ensures that the chitosan chain has sufficient length to form an effective network coverage, and at the same time, it will not hinder the uniform dispersion of the chitosan in the reaction system and the effective contact with the surface of the carrier due to the high viscosity. And the deacetylation degree of 85% means that there is a high density of amino groups on the chitosan chain, which is crucial for effective cross-linking and imparting the gating layer with sharp pH responsiveness, which is quantified by the swelling ratio (wet weight / dry weight): ≤ 6 at pH = 7.4 (densely closed), ≥ 15 at pH = 5.5 (fully open). The molar ratio of glutaraldehyde to amino groups in the chitosan molecule in the reaction system is controlled in the range of 0.5:1 to 1.5:1, and by adjusting this ratio, the cross-linking density of the chitosan network can be adjusted, and in turn, the density of the gating layer and its swelling / shrinking behavior in different pH environments can be fine-tuned.

[0050] The response mechanism of the chitosan gating layer is derived from the reversible transformation of the protonation-deprotonation of its amino groups. In the normal physiological environment (pH ≈ 7.4), the amino groups of chitosan (pKa ≈ 6.5) mainly exist in the form of deprotonated -NH2, and the segments are attracted to each other due to hydrogen bonding and hydrophobic interaction, resulting in the whole hydrogel network being in a shrunk and dense state, thereby effectively closing the pores of the silica carrier and maximizing the inhibition of premature leakage of the internal loaded antibiotic molecules. However, when the system enters the local micro-acidic environment (pH < 6.5) caused by bacterial infection, the high concentration of protons in the environment causes the amino groups on the chitosan chain to be largely protonated, changing to positively charged -NH3 + . The strong electrostatic repulsion between the segments forces the molecular chains to stretch and relax, resulting in significant swelling of the whole hydrogel network, increasing the network pore size, thereby 'opening' the drug release channel, allowing the stored antibiotics in the carrier interior to be controlled and continuously diffused to the infection lesion.

[0051] The application also provides a preparation method of the above-mentioned antibiotic sustained-release system based on hierarchical pore silica, which is carried out in liquid phase under mild conditions, ensuring that the chemical structure and biological activity of the antibiotic are not destroyed. The preparation process can be specifically divided into the following continuous engineering steps.

[0052] First, the preparation of the hierarchical porous silica microsphere carrier. This step uses a double template method, in a mixed solvent system composed of ethanol, deionized water and concentrated ammonia water, first dispersed pre-synthesized, uniform particle size of polymethyl methacrylate (PMMA) nanospheres, which is used as a hard template agent, the size of which directly determines the pore size of the macropores in the final carrier.

[0053] Subsequently, the triblock copolymer Pluronic P123 is dissolved in the suspension, which is used as a soft template agent, and the micellar structure formed by self-assembly in the solution will define the size and morphology of the mesopores in the final carrier. After the template is uniformly dispersed, tetraethyl orthosilicate (TEOS) as the silicon source is added to the reaction system at a constant rate under vigorous mechanical stirring. Ammonia is used as a catalyst here to catalyze the hydrolysis and condensation of TEOS, and the generated silica precursor will be deposited around the PMMA hard template and P123 soft template. The entire reaction is carried out at a constant temperature of 30°C for 24 hours to ensure that the hydrolysis and condensation reactions proceed fully and form a structurally complete composite microsphere.

[0054] After the reaction is completed, the white solid product generated is collected by centrifugation and washed with ethanol several times to remove residual reactants and uncoated template agents. Finally, the obtained composite microspheres are calcined in a high-temperature muffle furnace to completely burn and remove the PMMA and P123 organic templates in one step, and the amorphous silica framework is further cross-linked and solidified, ultimately obtaining a spherical silica carrier with a macropore-mesopore through-pore structure, denoted as MS-SiO2.

[0055] Next, the amino modification of the carrier surface. The MS-SiO2 carrier prepared in the previous step is first dried at 120°C in a vacuum environment for 12 hours, and the purpose of this step is to remove physically adsorbed water molecules and fully activate the silicon hydroxyl groups on the surface of the silica to provide sufficient reaction sites for subsequent grafting reactions. Subsequently, the dried carrier powder is dispersed in anhydrous toluene and operated under an inert atmosphere such as nitrogen to prevent moisture in the air from interfering with the reaction. To the suspension, 3-aminopropyltriethoxysilane (APTES) is added, and the entire reaction system is heated to 110°C for reflux reaction for 24 hours. Under high temperature and anhydrous conditions, APTES molecules can covalently bond with the silicon hydroxyl groups inside and outside the MS-SiO2 carrier channels. After the reaction is completed, the solid product is collected by centrifugal separation and washed repeatedly with anhydrous toluene and anhydrous ethanol to completely remove any unreacted, physically adsorbed APTES molecules. Finally, the product is dried under vacuum to obtain a functionalized carrier with a uniform surface covered with amino functional groups, denoted as MS-SiO2-NH2.

[0056] The loading process of antibiotics follows. Take levofloxacin as an example. First, prepare a levofloxacin phosphate buffer solution. Then, disperse the amino-functionalized support MS-SiO2-NH2 prepared in the previous step in the above drug solution at a specific mass ratio (for example, the mass ratio of the support to levofloxacin is 2:1). The whole loading process is carried out at 25°C under light-avoiding conditions through continuous magnetic stirring for 48 hours. The long enough loading time ensures that the drug molecules have sufficient time to diffuse through the large-pore channels to the deep part of the support and be effectively captured by the mesoporous network with high specific surface area. After the loading is completed, the drug-loaded microspheres are collected by high-speed centrifugation and washed repeatedly with deionized water. This step aims to remove the free drugs adsorbed on the outer surface of the microspheres and not entered into the internal channels, which is crucial for controlling the burst release behavior of the final product. The washed product is freeze-dried to obtain a powdered drug-loaded intermediate, denoted as LVFX@MS-SiO2-NH2. During the drug loading process, the amino functional groups inside the channels are stably combined with the antibiotic molecules through electrostatic interaction and hydrogen bonding, while the amino groups on the outermost surface of the support are not completely occupied by drugs due to steric hindrance and still retain some free amino groups, which provide reaction sites for subsequent covalent cross-linking of the gating layer.

[0057] The last step is the construction of the chitosan gating layer. The drug-loaded intermediate LVFX@MS-SiO2-NH2 obtained in the previous step is re-dispersed in an acetate buffer solution with pH = 5.0. The reason for choosing an acidic buffer is that chitosan can be protonated and well dissolved under this pH condition. Another specific molecular weight (for example, 50 kilodaltons) and deacetylation degree (for example, 85%) of chitosan is also dissolved in the acetate buffer solution with pH = 5.0, and then the chitosan solution is mixed with the suspension of the drug-loaded intermediate. Under continuous stirring, the chitosan molecules are fully adsorbed and covered on the surface of the microspheres. Subsequently, a glutaraldehyde aqueous solution (for example, 25% w / w) as a cross-linking agent is slowly added dropwise. The reaction is continued under mild water bath conditions, during which the glutaraldehyde reacts with the amino groups on the surface of the support and the amino groups on the chitosan chains to form a covalent cross-linking network, thereby stably locking the chitosan layer in the pore region of the microspheres. After the reaction is completed, the product is collected by centrifugation and washed thoroughly with the acetate buffer solution with pH = 5.0 and deionized water in sequence to remove unreacted chitosan, glutaraldehyde, and reaction by-products. Finally, the purified product is freeze-dried to obtain the final antibiotic sustained-release system with pH intelligent release characteristics, denoted as CS-LVFX@MS-SiO2-NH2.

[0058] The application will be further described below with reference to the examples. The embodiments of the application include but are not limited to the following examples.

[0059] Example 1

[0060] 1. Preparation of mesoporous silica microsphere support (MS-SiO2)

[0061] Take 1.0 g of PMMA nanospheres with a particle size of 200 nm, disperse them in a mixture of 100 mL of ethanol, 25 mL of deionized water and 5 mL of concentrated ammonia water (28% w / w), and ultrasonically treat for 15 minutes to make them uniformly dispersed. Add 1.25 g of Pluronic P123 and magnetically stir until completely dissolved. Under vigorous stirring, add 5.0 g of tetraethyl orthosilicate at a rate of 1 mL / min through a constant flow pump. After the addition is complete, continue stirring at 30°C for 24 hours. Collect the white precipitate by centrifugation, wash it with ethanol three times, and then dry it in an oven at 60°C for 12 hours. Place the dried white powder in a muffle furnace, heat it at a rate of 2°C / min to 550°C, and calcine it at this temperature for 6 hours. After naturally cooling to room temperature, a white MS-SiO2 powder is obtained.

[0062] 2. Amination (MS-SiO2-NH2)

[0063] Dry 1.0 g of the above MS-SiO2 powder in a vacuum oven at 120°C for 12 hours. After cooling, transfer it to a three-necked flask containing 50 mL of anhydrous toluene. Under a nitrogen atmosphere and magnetic stirring, add 1.0 mL of 3-aminopropyltriethoxysilane. Heat the mixture to 110°C and reflux it for 24 hours. After the reaction is complete, cool it to room temperature and collect the solid by centrifugation. Wash it with 20 mL of anhydrous toluene twice and then with 20 mL of anhydrous ethanol three times. Dry the resulting product in a vacuum oven at 60°C for 12 hours to obtain MS-SiO2-NH2.

[0064] 3. Levofloxacin loading (LVFX@MS-SiO2-NH2)

[0065] Accurately weigh 200 mg of levofloxacin and dissolve it in 40 mL of PBS buffer solution with pH = 7.0. Weigh 100 mg of MS-SiO2-NH2 powder and add it to the above drug solution, then magnetically stir it in a dark environment at 25°C for 48 hours. After the reaction is complete, centrifuge it at 10,000 rpm for 10 minutes to collect the precipitate. Wash the precipitate with deionized water three times to remove the drug physically adsorbed on the outer surface of the particles. Freeze-dry the washed product for 24 hours to obtain LVFX@MS-SiO2-NH2 powder. Calculate the drug loading by measuring the concentration of levofloxacin in the supernatant and washing liquid.

[0066] 4. Construction of chitosan gating layer

[0067] Take 50 mg of LVFX@MS-SiO2-NH2 and disperse it in 25 mL of pH = 5.0 acetic acid-sodium acetate buffer solution. Take another 50 mg of chitosan (molecular weight 50 kilodaltons, deacetylation degree 85%) and dissolve it in 25 mL of the same buffer solution. Add the chitosan solution to the drug-loaded microsphere suspension and mix well. Use a microsyringe to take 0.05 mL of 25% w / w glutaraldehyde aqueous solution and slowly add it at a rate of 5 μL / min under magnetic stirring. Place the mixed system in a 40°C water bath for 6 hours. After crosslinking, use HPLC-MS to detect that the residual amount of glutaraldehyde is <0.01%. After the reaction is completed, centrifuge to collect the product, wash it twice with pH = 5.0 acetic acid-sodium acetate buffer solution, and then wash it three times with deionized water. Freeze-dry the final product for 24 hours to obtain the target sustained-release system CS-LVFX@MS-SiO2-NH2.

[0068] Example 2

[0069] The preparation steps are exactly the same as in Example 1, except that in Step 4, chitosan with a weight-average molecular weight of 100 kilodaltons (deacetylation degree 85%) is used instead of chitosan with a weight-average molecular weight of 50 kilodaltons.

[0070] Example 3

[0071] The preparation steps are exactly the same as in Example 1, except that in Step 4, the amount of glutaraldehyde aqueous solution added is increased to 0.14 mL.

[0072] Example 4

[0073] The preparation steps are basically the same as in Example 1, except that in Step 3, an equimolar amount of ciprofloxacin is used instead of levofloxacin for loading.

[0074] Comparative Example 1: Mesoporous silica sustained-release system without a chitosan gatekeeper layer

[0075] The preparation steps are similar to those in Example 1, except that in Step 1, no PMMA nanomicrosphere is added as a macropore template agent, and only Pluronic P123 is used as a mesopore template agent to prepare a pure mesoporous silica carrier (M-SiO2). The subsequent steps of amination, drug loading, and chitosan gatekeeper layer construction are exactly the same as in Example 1.

[0076] Comparative Example 2: Sustained-release system without a chitosan gatekeeper layer

[0077] The preparation process performs Steps 1 to 3 of Example 1 to obtain the drug-loaded intermediate LVFX@MS-SiO2-NH2, but does not perform Step 4 of constructing a chitosan gatekeeper layer. LVFX@MS-SiO2-NH2 is directly used as a control sample.

[0078] Comparative Example 3: Sustained-release system of physical adsorption of chitosan

[0079] The preparation process performed steps 1 to 3 of Example 1. In step 4, no glutaraldehyde crosslinking agent was added. After mixing LVFX@MS-SiO2-NH2 with chitosan solution for 4 hours, centrifugation, washing and drying were directly performed, so that chitosan was only physically adsorbed on the surface of the carrier by electrostatic interaction and other non-covalent bonds.

[0080] The experimental results and analysis conclusions are as follows:

[0081] Table 1: Physical structure parameter characterization of different carriers

[0082]

[0083] Conclusion: The observation shows that the silica carrier with a large pore-mesopore dual-mode pore structure is prepared in Example 1. In comparison, Comparative Example 1 lacks a large pore template and only forms a pure mesoporous structure of worm-like mesopores. Although its specific surface area is higher, its pore volume is only 0.95 cm 3 / g, which is significantly lower than that of Example 1. This fundamental difference in structure directly leads to a huge difference in drug loading capacity between the two.

[0084] Table 2: Drug loading and encapsulation efficiency of different sustained-release systems

[0085]

[0086] Conclusion: Due to the high mesopore specific surface area provided by the large pore-mesopore cooperative structure, the drug loading and encapsulation efficiency of Examples 1-3 are significantly higher than that of Comparative Example 1, which only has a mesoporous structure, indicating that the drug loading capacity has been greatly improved. It proves the superiority of the multi-level pore structure design of the present application in improving the drug loading capacity.

[0087] Table 3: In vitro cumulative release curve data in pH = 7.4 buffer solution

[0088]

[0089] Conclusion: In the simulated normal body fluid environment of pH = 7.4, the drug leakage of examples 1, 2 and 3 is controlled within 13% within 72 hours due to the effective blocking effect of the chitosan gating layer, and the background release is extremely low, among which example 3 has the best blocking effect due to the higher cross-linking degree. This result shows that in the physiological neutral environment, the covalently bonded chitosan gating layer is in a dense "closed" state, effectively blocking the drug molecules inside the carrier. In contrast, the non-gated comparative example 2 has a serious burst release of 42.5% within 2 hours; 75.3% of the drug is released within 12 hours, which is a completely uncontrollable burst release, which may cause serious toxic side effects and drug waste. Comparative example 3 with physically adsorbed chitosan still has significant leakage, indicating that the stability of covalent bonding is crucial.

[0090] Table 4 In vitro cumulative release curve data in pH = 5.5 buffer solution

[0091]

[0092] Conclusion: In the simulated infected microenvironment of pH = 5.5, the chitosan gating layer of examples 1-3 all responds to open, realizing the sustained and effective release of drugs, among which the release of example 1 increases to 15.6% within 2 hours, and reaches 68.9% and 88.1% at 24 hours and 72 hours, respectively, showing a sustained and efficient drug release mode. This is in sharp contrast to the results in Table 3, proving the pH intelligent response characteristics of the system, and it can be seen that the chitosan gating layer can sensitively "perceive" the decrease of the environmental pH value, and change from the "closed" state to the "open" state, releasing the antibiotic as needed. The release curve of comparative example 2 under this acidic condition is not much different from that under the neutral condition, both being rapid and uncontrollable release, which contrasts the drug release behavior of examples 1, 2 and 3, which is the result of precise regulation by pH environment, rather than simple diffusion.

[0093] In addition, example 2 (high molecular weight chitosan) releases slowly, and example 3 (high cross-linking degree) releases quickly, indicating that the release kinetics can be precisely adjusted by regulating the gating layer parameters.

[0094] Table 5 Burst release rate comparison within two hours

[0095]

[0096] Conclusion: The burst release rate of example 1 at pH = 7.4 is only 4.1%, which is much lower than that of comparative example 2 (42.5%) and comparative example 1 (12.5%). This confirms the synergistic effect of the multi-level pore structure and the covalently bonded gating layer, which has a decisive advantage in effectively inhibiting the burst release of drugs.

[0097] Table 6 Gated layer stability cycle test (pH = 7.4 / 5.5 alternation)

[0098]

[0099] Experimental description: The sample was soaked in pH = 7.4 buffer for 12 hours (valve closed), then transferred to pH = 5.5 buffer for 24 hours release test (valve open). Repeat this cycle 6 times, record the cumulative release amount in each acidic environment.

[0100] Conclusion: In 6 times of pH stimulation cycle, the release behavior of Example 1 under acidic conditions is highly consistent (fluctuation range ≤1.4%), indicating that the low cross-linking density of chitosan gated layer has excellent reversibility and structural stability, and the swelling / shrinking cycle does not cause fatigue damage, and the release rate is smooth and controllable; the release amount of Example 3 gradually increases from 75.4% to 78.5%, indicating that the swelling rate of the high cross-linking network is accelerated, which accelerates drug diffusion, but does not damage the switching function; while the release amount of Comparative Example 3 (physical adsorption) sharply decreases (decrease by 86.4%) in the cycle, indicating that the physically adsorbed chitosan continuously falls off in the cycle, and the gating function is basically lost in the 6th cycle.

[0101] Table 7 Release performance comparison

[0102]

[0103] Conclusion: Under neutral environment (pH = 7.4), the burst rate of ciprofloxacin (5.2%) is slightly higher, because the molecular weight of ciprofloxacin (about 331.3 g / mol) is smaller than that of levofloxacin (about 361.4 g / mol), and its molecular size is smaller, so it has a relatively higher diffusion rate in the gated layer network; under acidic environment (pH = 5.5), the release rate of ciprofloxacin is slowed down as a whole, because its electrostatic combination with the amino group of the carrier is stronger, and more protons are needed to compete for dissociation. In terms of drug loading, the loading amount of ciprofloxacin (Example 4) 34.8% is slightly higher than that of levofloxacin (Example 1) 33.2%, because ciprofloxacin is more hydrophobic (logP = 0.8, levofloxacin logP = -0.4), and the hydrophobic interaction with the amino group carrier is enhanced. This shows that in addition to levofloxacin, the carrier and gated layer of the present application are still effective for another quinolone antibiotic, and the release behavior difference conforms to the rules of drug physical and chemical properties.

[0104] Table 8 Gated layer swelling ratio

[0105]

[0106] Conclusion: The control sample 3 of physical adsorption of chitosan opens the pore channel in neutral and acidic environments, while the examples 1 and 3 are in the state of dense closed pore channel in neutral environment and open the pore channel in acidic environment, which shows that the designed gating mechanism has dynamic pH responsiveness.

[0107] In summary, the present application designs a three-level mechanism of "multi-level pore structure-chemical anchoring-dynamic external gating", and constructs an integrated system of "high capacity storage-stable adsorption-intelligent release", which solves the technical problems of high drug loading and low burst release and difficult to obtain biological safety in the prior art, and provides a feasible and effective solution for developing a new generation of intelligent drug delivery system.

[0108] The above examples are only preferred embodiments of the present application, and are not intended to limit the protection scope of the present application. Any changes made on the basis of the design principles of the present application and non-creative labor shall fall within the protection scope of the present application.

Claims

1. An antibiotic sustained-release system based on hierarchical porous silica, characterized in that, include: A spherical hierarchical porous silica microsphere carrier has an internal dual-mode pore structure consisting of an interconnected macroporous network and mesopores extending from the inner walls of the macroporous network. The macroporous network has a pore size of 150 nm to 250 nm, and the mesopores have a pore size of 8 nm to 15 nm, with a specific surface area greater than 600 m². 2 / g; The total pore volume of the spherical hierarchical porous silica microsphere carrier is greater than 1.2 cm³. 3 / g; Amino functional groups covalently bonded to the outer surface and inner surface of the pores of the spherical hierarchical porous silica microsphere carrier; Antibiotic molecules loaded inside the pores of a spherical hierarchical porous silica microsphere carrier; A pH-responsive chitosan-gated layer, a covalently bonded coating, is applied to the outer surface of a spherical hierarchical porous silica microsphere carrier. Under physiologically neutral conditions, it exhibits a dense, contracted state to seal the pores and prevent antibiotic molecule leakage; under slightly acidic conditions, it transforms into a swollen, porous state to open the pores and release antibiotic molecules. The pH-responsive chitosan-gated layer is constructed using a glutaraldehyde crosslinking agent. The aldehyde groups of the glutaraldehyde crosslinking agent react with the amino functional groups on the surface of the spherical hierarchical porous silica microsphere carrier and the amino groups of the chitosan molecules themselves to form imine bonds that covalently bond the chitosan network. The swelling ratio of the pH-responsive chitosan-gated layer is ≤6 at pH 7.4 and ≥15 at pH 5.

5. The molar ratio of the glutaraldehyde crosslinking agent to the amino groups in the chitosan molecules is 0.5:1 to 1.5:

1.

2. The antibiotic sustained-release system based on hierarchical porous silica according to claim 1, characterized in that, The diameter of the spherical hierarchical porous silica microsphere carrier is 1 μm to 2 μm.

3. The antibiotic sustained-release system based on hierarchical porous silica according to claim 2, characterized in that, The amino functional groups covalently bonded to the outer surface and inner surface of the pores of the spherical hierarchical porous silica microsphere carrier are formed by the hydrolysis and condensation reaction of 3-aminopropyltriethoxysilane molecules, which act as silane coupling agents, with the triethoxysilyl groups on the surface of the spherical hierarchical porous silica microsphere carrier.

4. The antibiotic sustained-release system based on hierarchical porous silica according to claim 3, characterized in that, The antibiotic molecule is levofloxacin or ciprofloxacin.

5. The antibiotic sustained-release system based on hierarchical porous silica according to claim 4, characterized in that, The chitosan has a weight-average molecular weight of 50 to 100 kilodaltons and a degree of deacetylation of 85%.

6. A method for preparing the antibiotic sustained-release system according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Preparation of hierarchical porous silica microsphere carrier: The dual-template method was adopted. In a mixed solvent system with ethanol and deionized water as the main components and ammonia as the catalyst, polymethyl methacrylate nanospheres were used as macroporous template agents and triblock copolymers were used as mesoporous template agents. A silicon source was added to carry out hydrolysis and condensation reaction to form composite microspheres. The two organic template agents were removed by high-temperature calcination to obtain spherical hierarchical porous silica microsphere carrier with dual-mode pore structure. (2) Aminoation of the carrier surface: After drying and activating the spherical hierarchical porous silica microsphere carrier under vacuum conditions at 100℃ to 150℃, it is refluxed with a silane coupling agent in an organic solvent at 80℃ to 120℃ for 12 to 24 hours, so that the amino functional groups are covalently bonded to the outer surface and the inner surface of the pores of the spherical hierarchical porous silica microsphere carrier. (3) Antibiotic loading: The aminated spherical hierarchical porous silica microsphere carrier was dispersed in a buffer solution with a pH of 7.0 to 7.4 and containing antibiotic molecules at a concentration of 3-5 mg / mL. After stirring and adsorption, the mixture was separated, washed, and dried to obtain the drug-loaded intermediate. (4) Construction of pH-responsive chitosan gated layer: The drug-loaded intermediate was dispersed in an acidic buffer solution, and chitosan solution was added and mixed. Then, glutaraldehyde solution as a crosslinking agent was added to carry out an in-situ crosslinking reaction, so that chitosan formed a covalently bonded gated layer on the outer surface of the pores of the spherical hierarchical porous silica microsphere carrier. After washing and drying, an antibiotic sustained-release system was obtained.

7. The method according to claim 6, characterized in that, Step (1) includes the following steps: (101) In a mixed solvent consisting of 100 mL of ethanol, 25 mL of deionized water and 5 mL of 28% w / w concentrated ammonia, polymethyl methacrylate nanospheres with a particle size of 200 nm were dispersed and Pranic P123 was dissolved as a triblock copolymer. (102) Tetraethyl orthosilicate was added dropwise under stirring conditions as a silicon source, wherein the mass ratio of polymethyl methacrylate nanospheres to tetraethyl orthosilicate was 1:5, and the mass ratio of Pluronic P123 to tetraethyl orthosilicate was 1:4; a hydrolysis-condensation reaction was carried out at 30°C for 24 hours to form composite microspheres. (103) The composite microspheres were calcined at 550°C for 6 hours to remove the template agent and solidify the silica skeleton, thereby obtaining a spherical multi-level porous silica microsphere carrier.

8. The method according to claim 6 or 7, characterized in that, Step (4) includes the following steps: (401) The drug-loaded intermediate was dispersed in an acetate-sodium acetate buffer solution with pH=5.0 to obtain a suspension of drug-loaded microspheres; (402) Dissolve chitosan of the same mass as the drug-loaded intermediate in another portion of the same volume and pH value of an acetate-sodium acetate buffer solution to obtain a chitosan solution. (403) Add the chitosan solution to the suspension of drug-loaded microspheres, and slowly add 25% w / w glutaraldehyde aqueous solution under magnetic stirring, so that the final molar ratio of glutaraldehyde to amino groups in chitosan molecules is controlled within the range of 0.5:1 to 1.5:

1. (404) The mixed solution obtained in step (403) is subjected to a water bath reaction. After the reaction is completed, the product is collected by centrifugation. (405) Wash twice with an acetate-sodium acetate buffer solution at pH 5.0, and then wash with deionized water to remove unreacted chitosan, glutaraldehyde and reaction byproducts. Then freeze-dry the final product to obtain the antibiotic sustained-release system.

Images