Antibiotic slow-release system based on hierarchical porous silicon dioxide and preparation method of antibiotic slow-release system

Abstract

The invention discloses an antibiotic slow-release system based on hierarchical porous silicon dioxide and a preparation method of the antibiotic slow-release system, and belongs to the technical field of biological medicines. The antibiotic slow-release system comprises a spherical hierarchical-pore silicon dioxide microsphere carrier, amino functional groups covalently bonded to the outer surface and the inner surface of a pore channel of the spherical hierarchical-pore silicon dioxide microsphere carrier, antibiotic molecules loaded in the pore channel of the spherical hierarchical-pore silicon dioxide microsphere carrier, and a pH-responsive chitosan gating layer. The preparation method comprises the following steps: (1) preparing a hierarchical pore silicon dioxide microsphere carrier; (2) aminating the surface of the carrier; (3) loading antibiotics; and (4) constructing the pH responsive chitosan gating layer. According to the invention, the silicon dioxide carrier with a macroporous-mesoporous hierarchical structure is constructed to realize high drug loading capacity, and the pH-responsive chitosan gating layer is covalently bonded at the orifice of the silicon dioxide carrier to realize intelligent controlled release, so that the problem that high drug loading capacity, low burst release rate and biological safety are difficult to achieve at the same time in the prior art is solved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an antibiotic sustained-release system based on multi-level porous silica and a preparation method thereof. Background Art

[0002] In modern medical practice, the effective use of antibiotics is a key cornerstone for controlling bacterial infections and safeguarding human health. Traditional drug delivery methods, such as oral preparations or intravenous injections, can rapidly deliver drugs into the body, but they often suffer from the inherent flaw of "peak-and-valley" fluctuations in blood drug concentrations. This fluctuation can not only cause drug concentrations to exceed the safe range at peak values, leading to toxic side effects, but can also cause concentrations to fall below the minimum inhibitory concentration (MIC) at trough values, thereby weakening the therapeutic effect and even inducing bacterial resistance. To overcome these limitations, the development of efficient and safe sustained-release drug systems to achieve long-term, constant-rate release of drugs in the body, thereby maintaining stable and effective blood drug concentrations, has become a research focus of ongoing concern in the fields of pharmacy and materials science.

[0003] Against this backdrop, organic polymer carriers, represented by polymer microspheres and liposomes, emerged. These carriers encapsulate drug molecules within their three-dimensional network structures or internal cavities, leveraging the swelling and degradation of the polymer matrix or the passive diffusion of drug molecules to achieve sustained drug release. During certain historical periods, designing dosing regimens based on the biological half-life of drugs effectively addressed the problem of short drug half-lives, significantly extending the duration of drug action and, to a certain extent, improving therapeutic efficacy and patient compliance. Specifically, by regulating the molecular weight, degree of cross-linking, and hydrophilicity of the polymer, it is possible to initially control the drug release rate.

[0004] However, with the in-depth exploration of the concept of precision treatment and the deepening understanding of the complex infection microenvironment, some inherent characteristics of this type of polymer matrix-based carrier at the principle level have gradually revealed insurmountable limitations when responding to new challenges. The reason is that its drug loading mechanism is essentially a physical encapsulation. The drug molecules are unevenly distributed in the heterogeneous polymer matrix. The drug molecules close to the carrier surface can easily detach rapidly in the initial stage, forming a significant "burst effect", which not only causes drug waste, but may also cause local or systemic toxic reactions in the early stages of medication. In order to alleviate the burst release phenomenon, increasing the density of the polymer matrix has become a common method. However, this will inevitably reduce the internal space for drug loading, resulting in a significant drop in drug loading, which is usually difficult to exceed the 20% limit. This negative correlation between drug loading and burst release control constitutes a pair of core contradictions in the performance improvement of polymer carriers.

[0005] To overcome drug loading limitations, researchers have turned their attention to metal-organic frameworks (MOFs). These materials, with their highly ordered pore structures and enormous surface areas, can efficiently accommodate large numbers of drug molecules through physical adsorption or chemical bonding. The theoretical drug loading capacity of MOFs far exceeds that of traditional polymer carriers, thanks to their unique crystal structure and customizable properties. This development appears to offer an ideal solution to the problem of low drug loading. However, this development presents new challenges. For carriers like MOFs, their degradation behavior in physiological environments is often difficult to accurately predict and control. The coordination bonds between the metal ions and organic ligands that comprise their frameworks can break uncontrollably in the complex environment of body fluids, leading to premature disintegration of the carrier structure. This not only results in an instantaneous "dumping" release of the loaded drug, recreating a more dramatic burst release, but also presents new biosafety risks, including potential cytotoxicity or immunogenicity from degradation products, particularly free metal ions. Although metal-organic frameworks (MOFs) have shown great potential in drug delivery systems, such as achieving targeted delivery and controlled release of drugs through their highly tunable pore structure, the challenges of MOFs in drug release controllability and biocompatibility, such as the regulation of MOFs structure and properties and the evaluation of long-term effects on organisms, remain key obstacles to overcome in their clinical translation.

[0006] It can be seen that the existing technology has fallen into the following dilemma: 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 capacity and burst release control.

[0007] On the other hand, novel porous materials, such as MOFs, have significantly increased drug loading, but this has also introduced new challenges: uncontrollable release behavior and potential biotoxicity. While gating mechanisms can achieve controlled drug release to a certain extent, such as patent publication number CN106267230A, which utilizes a drug molecule "self-gating" mechanism, the drug molecules themselves may be cytotoxic (e.g., the cardiotoxicity of DOX), and residual groups after cleavage may trigger nonspecific reactions, thus still failing to address the potential biotoxicity issue.

[0008] In summary, while polymer carriers exhibit excellent biocompatibility, their physical encapsulation mechanisms limit drug loading and reduce their ability to suppress burst release. While porous materials such as metal-organic frameworks (MOFs) offer high drug loading and, combined with gating mechanisms, can achieve controlled drug release, they pose potential biosafety risks. Therefore, how to design a new drug sustained-release system that can effectively load large-capacity drug molecules in its structure and integrate a precisely controllable release mechanism to respond to specific pathological microenvironments (such as the acidic characteristics of the infection site), thereby overcoming the inherent contradictions in the existing technology between drug loading, burst release effect and sustained-release period and potential biological toxicity, has become one of the main problems that need to be solved in this field. Summary of the Invention

[0009] The purpose of the present invention is to provide an antibiotic sustained-release system based on multi-level porous silica and a preparation method thereof, so as to overcome the inherent contradictions among drug sustained-release carriers in the prior art in terms of drug loading capacity, burst release control, controllability of release behavior and biosafety.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is as follows: The antibiotic sustained-release system based on hierarchically porous silica includes: Spherical multi-level porous silica microsphere carrier, the spherical multi-level porous silica microsphere carrier has a dual-mode pore structure composed of a macroporous network that penetrates each other and mesopores extending from the inner wall of the macroporous network; amino functional groups covalently bonded to the outer surface and inner surface of the pores of the spherical hierarchically porous silica microsphere carrier; Antibiotic molecules loaded inside the pores of spherical hierarchically porous silica microsphere carriers; The pH-responsive chitosan gating layer is a covalently bonded coating that is coated on the outer surface of the spherical multi-level porous silica microsphere carrier. It is in a dense contracted state under a physiological neutral environment to close the pores and prevent the leakage of antibiotic molecules; it changes to a swollen and loose state under a slightly acidic environment to open the pores and release antibiotic molecules.

[0011] Specifically, the diameter of the spherical multi-level 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 mesopore specific surface area is greater than 600m 2 / g; the total pore volume of the spherical multi-level porous silica microsphere carrier is greater than 1.2cm 3 / g.

[0012] Furthermore, 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 a 3-aminopropyltriethoxysilane molecule as a silane coupling agent, through a hydrolysis condensation reaction between its triethoxysilane group and the silanol group on the surface of the spherical multi-level porous silica microsphere carrier.

[0013] Preferably, the antibiotic molecule is levofloxacin or ciprofloxacin.

[0014] Specifically, the pH-responsive chitosan gating layer is constructed using a glutaraldehyde crosslinker; the aldehyde groups of the glutaraldehyde crosslinker 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 themselves to form imine bonds that covalently bond the chitosan network; the pH-responsive chitosan gating layer has a swelling ratio of ≤6 in an environment of pH=7.4 and a swelling ratio of ≥15 in an environment of pH=5.5.

[0015] Preferably, the chitosan has a weight average molecular weight of 50 kilodaltons to 100 kilodaltons and a degree of deacetylation of 85%.

[0016] Furthermore, the molar ratio of the glutaraldehyde crosslinking agent to the amino groups in the chitosan molecule 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.

[0017] At the same time, the present invention also provides a method for preparing the above-mentioned antibiotic sustained-release system based on multi-level porous silica, comprising the following steps: (1) Preparation of hierarchically porous silica microsphere carriers: Using a dual-template method, in a mixed solvent system with ethanol and deionized water as the main components and ammonia as the catalyst, polymethyl methacrylate nanospheres are used as macroporous templates and triblock copolymers are used as mesoporous templates. A silicon source is added to carry out hydrolysis and condensation reactions to form composite microspheres. The two organic templates are removed by high-temperature calcination to obtain spherical hierarchically porous silica microsphere carriers with a dual-mode pore structure. (2) Amination of the carrier surface: After the spherical multi-level porous silica microsphere carrier is dried and activated under vacuum conditions at 100°C to 150°C, it is refluxed with a silane coupling agent in an organic solvent at 80°C to 120°C for 12 to 24 hours to covalently bond the amino functional groups to the outer surface of the spherical multi-level porous silica microsphere carrier and the inner surface of the pores; (3) Antibiotic loading: The spherical multi-level porous silica microsphere carrier after amino treatment is dispersed in a buffer solution with a pH of 7.0 to 7.4 and a concentration of 3-5 mg / mL antibiotic molecules. After stirring and adsorption, separation, washing, and drying are performed to obtain a drug-loaded intermediate; (4) Construction of pH-responsive chitosan gating layer: The drug-loaded intermediate is dispersed in an acidic buffer solution, chitosan solution is added and mixed, and glutaraldehyde solution as a cross-linking agent is added to carry out an in situ cross-linking reaction, so that chitosan forms a covalently bonded gating layer on the outer surface of the pores of the spherical multi-level porous silica microsphere carrier. After washing and drying, an antibiotic sustained-release system is obtained.

[0018] Specifically, 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 Pluronic P123 was dissolved as a triblock copolymer; (102) Tetraethyl orthosilicate was added dropwise as a silicon source under stirring, 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 h to form composite microspheres; (103) The composite microspheres were calcined at 550 °C for 6 h to remove the template and solidify the silica skeleton to obtain spherical hierarchically porous silica microsphere carriers.

[0019] Specifically, step (4) includes the following steps: (401) The drug-loaded intermediate was dispersed in an acetic acid-sodium acetate buffer solution at pH 5.0 to obtain a suspension of drug-loaded microspheres; (402) Chitosan of the same mass as the drug-loaded intermediate was dissolved in another portion of acetic acid-sodium acetate buffer solution of the same volume and pH value to obtain a chitosan solution; (403) The chitosan solution was added to the suspension of drug-loaded microspheres, and a 25% w / w aqueous solution of glutaraldehyde was slowly added dropwise under magnetic stirring to control the final molar ratio of glutaraldehyde to amino groups in the chitosan molecule to be in 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) The product was washed twice with acetic acid-sodium acetate buffer solution at pH 5.0 and then washed with deionized water to remove unreacted chitosan, glutaraldehyde, and reaction by-products. The final product was then freeze-dried to obtain the antibiotic sustained-release system.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention constructs a macropore-mesopore through-channel structure (macropore diameter 150-250nm, mesopore diameter 10-15nm, mesopore specific surface area>600 m 2 / g, total pore volume>1.2cm 3 / g), the carrier has both macroporous high-speed transport channels and mesoporous high specific surface area storage units, and the antibiotic loading capacity reaches 32% to 33%, which is significantly higher than that of traditional polymer carriers and pure mesoporous silica carriers. Combined with the physical barrier effect of the covalently bonded chitosan gating layer, it solves the technical problem of the existing technology that it is difficult to achieve both drug loading and burst release rate.

[0021] (2) The present invention introduces amino functional groups on the surface through covalent bonding of 3-aminopropyltriethoxysilane, and utilizes 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.

[0022] (3) The present invention uses glutaraldehyde cross-linking agent to construct a covalently bonded chitosan gating layer at the pore mouth, which belongs to the external material gating structure. Under a physiological neutral environment (pH = 7.4), chitosan deprotonates and shrinks, and the 2-hour burst release rate is <5%, which is more than 90% lower than that of a carrier without a gating layer; the burst release rate is more than 75% lower than that of existing gating mechanisms (such as self-gating mechanisms); under a slightly acidic environment (pH = 5.5), chitosan protonates 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.

[0023] (4) Compared with the drug molecule self-gating mechanism adopted in the prior art, the external material gating mechanism adopted in the present invention and chitosan as the gating material have better biocompatibility potential, better reduce the potential risk of toxic side effects, and realize the safety design of "carrier stability-gating degradability". In addition, the chitosan gating layer forms a covalent bonding network through glutaraldehyde cross-linking, firmly anchored to the carrier surface, and can still maintain structural integrity and switching function under multiple pH cycle stimulation (neutral / acidic alternation), avoiding the risk of shedding of the physical adsorption coating; further, in addition to maintaining structural integrity and switching function, the present invention controls the cross-linking density of the gating layer by regulating the molar ratio of glutaraldehyde to chitosan amino groups (0.5:1 to 1.5:1), and also realizes the controllable design of release time and rate. For example, the release is slow at low cross-linking density (0.5:1) and accelerated at high cross-linking density (1.5:1), thereby matching the needs of different scenarios. The self-gating mechanism of drug molecules used in existing technologies relies on the dynamic breakage of the benzimide bond. Once the bond breaks and the drug is released in an acidic environment, the gating structure becomes ineffective and repeated responses or dosage regulation cannot be achieved.

[0024] (5) The present invention adopts a dual-template system of polymethyl methacrylate nanospheres (macroporous template) and Pluronic P123 (mesoporous template). By precisely controlling the template ratio, hydrolysis and condensation conditions, and calcination parameters, a spherical multi-level porous silica carrier with a dual-mode pore structure can be stably prepared to ensure uniform pore size distribution and good pore connectivity, providing a reliable structural basis for high drug loading and controlled release. The process has high repeatability and is suitable for mass production.

[0025] (6) The present invention adopts acetic acid-sodium acetate buffer solution (pH=5.0) to disperse the drug-loaded intermediate and dissolve chitosan of specific molecular weight in a step-by-step manner, and combines the in-situ reaction process of slowly adding glutaraldehyde cross-linking agent under magnetic stirring to achieve uniform coating of the chitosan gating layer on the surface of the drug-loaded microspheres; subsequent graded washing with buffer solution and deionized water and freeze-drying treatment effectively removes unreacted free chitosan and glutaraldehyde, avoids the interference of impurities on the release performance, and at the same time maintains the integrity of the carrier pore structure, further improving the biosafety and storage stability of the sustained-release system. DETAILED DESCRIPTION

[0026] The present invention provides a hierarchically porous silica-based sustained-release antibiotic system. Its core concept is to construct a composite microsphere system that integrates a high-capacity drug storage unit, an efficient internal transport channel, and a precise external environment-responsive gating unit. The structural foundation of this system is a spherical, hierarchically porous silica microsphere carrier. The carrier's diameter is controlled within the range of 1 to 2 microns, effectively preventing rapid clearance by the mononuclear phagocyte system (RES) while providing an optimal physical dimension for retention in the target area and subsequent drug release. The carrier's interior is not a homogeneous solid, but rather exhibits an interconnected dual-mode pore network. This dual-mode pore network is composed of a macroporous framework with pore sizes ranging from 150 to 250 nanometers and mesopores extending from the inner walls of the macroporous framework. The macropores form high-speed transport channels for rapid loading and release of drug molecules within the carrier, while the mesopores, with pore sizes ranging from 8 to 15 nanometers, serve as the primary storage unit for drug molecules due to their large total internal surface area. This synergistic structural design provides a solid physical foundation for achieving high drug loading.

[0027] To further enhance the carrier's affinity for specific drug molecules and provide chemical anchors for subsequent functionalization, a layer of amino groups was covalently bonded to the carrier's surface via a chemical reaction. These amino groups were introduced via a hydrolysis-condensation reaction of 3-aminopropyltriethoxysilane (APTES) in an organic solvent. The triethoxysilane groups in the APTES molecule reacted with the inherent silanol (Si-OH) groups on the silica surface, which increased in density after thermal activation, to form stable silanol-silicon (Si-O-Si) covalent bonds, firmly anchoring the aminopropyl segments to the inner and outer pore walls of the carrier. This modification not only transforms the silica surface from hydrophilic to potentially positively charged, significantly enhancing electrostatic adsorption and hydrogen bonding for negatively charged or lone-paired drug molecules, but also exposes the primary amino groups (-NH2) at the ends, which serve as key reaction sites for constructing subsequent gating structures.

[0028] Within the pores of the amino-functionalized hierarchical porous carrier, an antibiotic molecule, such as levofloxacin, is loaded as a model drug. Due to the carboxyl groups and nitrogen-containing heterocycles in its structure, the levofloxacin molecule is able to generate strong electrostatic interactions with protonated amino groups at physiological pH. Its abundant 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 molecule to be efficiently captured and stably stored within the carrier's mesopores. The design of the present invention allows the final levofloxacin loading to consistently reach 32% to 33% (mass percentage) of the total mass of the sustained-release system, significantly exceeding that of traditional drug carriers.

[0029] Furthermore, the functional core of the sustained-release system, the gating unit, is a chitosan hydrogel network constructed on the outer surface of the pore of the drug-loaded carrier through an in situ polymerization and cross-linking reaction after drug loading. This chitosan gating layer is achieved using a glutaraldehyde cross-linker. The aldehyde groups at both ends of the glutaraldehyde cross-linker undergo nucleophilic addition-dehydration reactions with amino groups protruding from the carrier surface and amino groups on the chitosan molecular chain, respectively, to form stable imine bonds. This covalent bonding firmly "rivets" the chitosan network to the microspheres, forming a physical barrier. The chitosan used in the present invention has a weight-average molecular weight of 50 to 100 kilodaltons and a degree of deacetylation of 85%. This moderate molecular weight ensures that the chitosan chains are of sufficient length to form effective network coverage, while also preventing excessive viscosity from hindering uniform dispersion in the reaction system and effective contact with the carrier surface. An 85% degree of deacetylation indicates a high density of amino groups on the chitosan chains, which is crucial for effective cross-linking and imparting sharp pH responsiveness to the gating layer, as quantified by the swelling ratio (wet weight / dry weight): ≤6 (tightly closed) at pH 7.4 and ≥15 (fully open) at pH 5.5. The molar ratio of glutaraldehyde to amino groups in the reaction system was controlled within a range of 0.5:1 to 1.5:1. By manipulating this ratio, the cross-link density of the chitosan network can be adjusted, thereby fine-tuning the compactness of the gating layer and its swelling / contraction behavior under different pH conditions.

[0030] The response mechanism of the chitosan gating layer originates from the reversible protonation-deprotonation transformation of its amino groups. In a normal physiological environment (pH ≈ 7.4), the amino groups of chitosan (pKa ≈ 6.5) mainly exist in the form of deprotonated -NH2. The chain segments attract each other due to hydrogen bonding and hydrophobic interactions, causing the entire hydrogel network to be in a shrunken and dense state, thereby effectively closing the pores of the silica carrier and minimizing the premature leakage of the internally loaded antibiotic molecules. However, when the system enters the local slightly acidic environment (pH < 6.5) caused by bacterial infection, the high concentration of protons in the environment causes a large number of amino groups on the chitosan chain to be protonated and converted into positively charged -NH3 + The strong electrostatic repulsion between the chain segments forces the molecular chains to stretch and relax each other, causing the entire hydrogel network to swell significantly and the network pore size to increase, thereby "opening" the channel for drug release, allowing the antibiotics stored inside the carrier to diffuse to the infected lesion in a controlled and continuous manner.

[0031] The present invention also provides a method for preparing the aforementioned hierarchically porous silica-based antibiotic sustained-release system. This method is conducted entirely 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 broken down into the following sequential steps.

[0032] The first step is to prepare the hierarchically porous silica microsphere carrier. This step uses a dual-template method. Pre-synthesized, uniformly sized polymethyl methacrylate (PMMA) nanospheres are dispersed in a mixed solvent system consisting of ethanol, deionized water, and concentrated ammonia. These serve as hard templates, and their size directly determines the pore size of the final macropores in the carrier.

[0033] Subsequently, the triblock copolymer Pluronic P123 is dissolved in the suspension. It acts as a soft template, 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 evenly dispersed, tetraethyl orthosilicate (TEOS) as a silicon source is added dropwise to the reaction system at a constant rate under vigorous mechanical stirring. Ammonia acts as a catalyst here, catalyzing the hydrolysis and condensation reaction of TEOS. The resulting silica precursor is deposited around the PMMA hard template and the P123 soft template. The entire reaction is carried out at a constant temperature of 30°C for 24 hours to ensure the full progress of the hydrolysis and condensation reaction and the formation of structurally complete composite microspheres.

[0034] After the reaction, the resulting white solid product was collected by centrifugation and washed multiple times with ethanol to remove residual reactants and uncoated template. Finally, the resulting composite microspheres were calcined in a high-temperature muffle furnace, completely burning away both the PMMA and P123 organic templates simultaneously while further crosslinking and solidifying the amorphous silica skeleton. The result was a spherical silica support with a macroporous-mesoporous through-channel structure, designated MS-SiO2.

[0035] Next, the support surface was amino-modified. The MS-SiO2 support prepared in the previous step was first dried under vacuum at 120°C for 12 hours to remove physically adsorbed water molecules and fully activate the silanol groups on the silica surface, providing ample reactive sites for the subsequent grafting reaction. The dried support powder was then dispersed in anhydrous toluene and handled under an inert atmosphere (e.g., nitrogen) to prevent airborne moisture from interfering with the reaction. 3-Aminopropyltriethoxysilane (APTES) was added to this suspension, and the reaction system was heated to 110°C and refluxed for 24 hours. Under high temperature and anhydrous conditions, the APTES molecules efficiently covalently bonded to the silanol groups inside and outside the pores of the MS-SiO2 support. After the reaction, the solid product was collected by centrifugation and repeatedly washed with anhydrous toluene and then anhydrous ethanol to completely remove any unreacted, physically adsorbed APTES molecules. Finally, the product was dried under vacuum conditions to obtain a functionalized carrier with amino functional groups uniformly covered on the surface, which was recorded as MS-SiO2-NH2.

[0036] Next comes the antibiotic loading process. Taking levofloxacin as an example, a phosphate-buffered solution of levofloxacin is first prepared. The amino-modified carrier MS-SiO2-NH2, prepared in the previous step, is then dispersed into the drug solution at a specific mass ratio (e.g., a 2:1 ratio of carrier to levofloxacin). The entire loading process is performed at 25°C, protected from light, with continuous magnetic stirring for 48 hours. This long loading time ensures that the drug molecules have ample time to diffuse through the macroporous channels into the deep layers of the carrier and be effectively captured by the high-surface-area mesoporous network. After loading, the drug-loaded microspheres are collected by high-speed centrifugation and repeatedly washed with deionized water. This step removes free drug adsorbed on the microsphere surface that has not entered the pores, 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, designated LVFX@MS-SiO2-NH2. During the drug loading process, the amino functional groups inside the pores stably bind to the antibiotic molecules through electrostatic interactions and hydrogen bonds, while the amino groups on the outermost surface of the carrier are not completely occupied by the drugs due to steric hindrance, and some free amino groups are still retained, providing reaction sites for the subsequent covalent cross-linking of the gating layer.

[0037] The final step is the construction of the chitosan gating layer. The drug-loaded intermediate LVFX@MS-SiO2-NH2 obtained in the previous step is redispersed in acetate buffer at pH 5.0. This acidic buffer is chosen because chitosan dissolves well when protonated at this pH. Separately, chitosan of a specific molecular weight (e.g., 50 kilodaltons) and degree of deacetylation (e.g., 85%) is dissolved in acetate buffer at pH 5.0. This chitosan solution is then mixed with the suspension of the drug-loaded intermediate. Under continuous stirring, the chitosan molecules are fully adsorbed and coated on the microsphere surface. Subsequently, an aqueous solution of glutaraldehyde (e.g., 25% w / w) is slowly added dropwise as a crosslinker. The reaction continues in a gentle water bath. During this time, the glutaraldehyde reacts with amino groups on the carrier surface and on the chitosan chains, forming a covalently crosslinked network that stably locks the chitosan layer into the pore region of the microsphere. After the reaction, the product was collected by centrifugation and thoroughly washed with acetate buffer (pH 5.0) and deionized water to remove unreacted chitosan, glutaraldehyde, and reaction byproducts. Finally, the purified product was freeze-dried to obtain the final antibiotic sustained-release system with pH-responsive release properties, designated CS-LVFX@MS-SiO2-NH2.

[0038] The present invention will be further described below with reference to the following examples. The embodiments of the present invention include but are not limited to the following examples.

[0039] Example 1 1. Preparation of hierarchically porous silica microsphere carrier (MS-SiO2) 1.0 g of 200 nm PMMA nanospheres were dispersed in a mixture of 100 ml of ethanol, 25 ml of deionized water, and 5 ml of concentrated ammonia (28% w / w). Ultrasonic treatment was performed for 15 minutes to achieve uniform dispersion. 1.25 g of Pluronic P123 was added and magnetically stirred until completely dissolved. Under vigorous stirring, 5.0 g of tetraethyl orthosilicate was added dropwise at a rate of 1 ml / min using a constant flow pump. After the addition was complete, stirring was continued at 30°C for 24 hours. The white precipitate was collected by centrifugation, washed three times with ethanol, and then dried in a 60°C oven for 12 hours. The dried white powder was placed in a muffle furnace, heated to 550°C at a rate of 2°C / min, and calcined at this temperature for 6 hours. After cooling to room temperature, a white MS-SiO2 powder was obtained.

[0040] 2. Amination (MS-SiO2-NH2) Dry 1.0 g of the 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. Add 1.0 ml of 3-aminopropyltriethoxysilane under a nitrogen atmosphere and magnetic stirring. Heat the mixture to 110°C and reflux for 24 hours. After the reaction is complete, cool to room temperature and collect the solid by centrifugation. Wash it twice with 20 ml of anhydrous toluene and then three times with 20 ml of anhydrous ethanol. Dry the resulting product in a vacuum oven at 60°C for 12 hours to obtain MS-SiO2-NH2.

[0041] 3. Levofloxacin loading (LVFX@MS-SiO2-NH2) Accurately weigh 200 mg of levofloxacin and dissolve it in 40 ml of PBS buffer (pH 7.0). Weigh 100 mg of MS-SiO2-NH2 powder and add it to the drug solution. Incubate the mixture in the dark at 25°C with magnetic stirring for 48 hours. After the reaction, centrifuge at 10,000 rpm for 10 minutes and collect the precipitate. Wash the precipitate three times with deionized water to remove any drug physically adsorbed on the particle surface. Freeze-dry the washed product for 24 hours to obtain LVFX@MS-SiO2-NH2 powder. The drug loading was calculated by measuring the levofloxacin concentrations in the supernatant and washing solutions.

[0042] 4. Construction of chitosan gating layer 50 mg of LVFX@MS-SiO2-NH2 was weighed and dispersed in 25 mL of acetic acid-sodium acetate buffer (pH 5.0). Another 50 mg of chitosan (molecular weight 50 kilodaltons, degree of deacetylation 85%) was dissolved in 25 mL of the same buffer. The chitosan solution was added to the suspension of drug-loaded microspheres and mixed thoroughly. Using a microinjector, 0.05 mL of a 25% w / w aqueous glutaraldehyde solution was slowly added dropwise at a rate of 5 μL / min under magnetic stirring. The mixture was reacted in a 40°C water bath for 6 hours. After cross-linking, the residual glutaraldehyde content was determined to be <0.01% by HPLC-MS. After the reaction, the product was collected by centrifugation and washed twice with acetic acid-sodium acetate buffer (pH 5.0) and then three times with deionized water. The final product was freeze-dried for 24 hours to obtain the target sustained-release system CS-LVFX@MS-SiO2-NH2.

[0043] Example 2 The preparation steps are exactly the same as those in Example 1, except that in step 4, chitosan with a weight average molecular weight of 100 kilodaltons (degree of deacetylation 85%) is used instead of chitosan with a weight average molecular weight of 50 kilodaltons.

[0044] Example 3 The preparation steps are exactly the same as those in Example 1, except that in step 4, the amount of glutaraldehyde aqueous solution added is increased to 0.14 ml.

[0045] Example 4 The preparation steps are basically the same as those in Example 1, except that in step 3, an equal molar amount of ciprofloxacin is used instead of levofloxacin for loading.

[0046] Comparative Example 1: Silica sustained-release system with only mesoporous structure The preparation steps were similar to those in Example 1, except that PMMA nanospheres were omitted as the macroporous template in Step 1. Instead, only Pluronic P123 was used as the mesoporous template, resulting in a pure mesoporous silica support (M-SiO2). The subsequent amination, drug loading, and chitosan gating layer construction steps were identical to those in Example 1.

[0047] Comparative Example 2: Sustained-release system without chitosan gating layer The preparation process was carried out according to steps 1 to 3 of Example 1 to obtain the drug-loaded intermediate LVFX@MS-SiO2-NH2, but the chitosan gating layer construction in step 4 was omitted. LVFX@MS-SiO2-NH2 was directly used as a control sample.

[0048] Comparative Example 3: Sustained-release system of physical adsorption chitosan The preparation process followed steps 1 to 3 of Example 1. In step 4, no glutaraldehyde crosslinking agent was added. The LVFX@MS-SiO2-NH2 solution was mixed and stirred with the chitosan solution for 4 hours, then directly centrifuged, washed, and dried, allowing the chitosan to be physically adsorbed onto the support surface solely through non-covalent bonding mechanisms such as electrostatic interactions.

[0049] The experimental results and analysis conclusions are as follows: Table 1 Physical structure parameter characterization of different carriers Conclusion: Observation shows that Example 1 prepared a silica support with a macroporous-mesoporous dual-mode pore structure. In contrast, Comparative Example 1, due to the lack of a macroporous template, only formed a pure mesoporous structure of worm-like mesopores. Although its specific surface area was higher, its pore volume was only 0.95 cm 3 / g, which is significantly lower than that in Example 1. This fundamental difference in structure directly leads to a huge disparity in drug loading capacity between the two.

[0050] Table 2 Drug loading and encapsulation efficiency of different sustained-release systems Conclusion: Thanks to the high mesoporous specific surface area provided by the macroporous-mesoporous synergistic structure, the drug loading and encapsulation efficiency of Examples 1-3 were significantly higher than those of Comparative Example 1, which only had a mesoporous structure, indicating a significant improvement in drug loading capacity. This demonstrates the superiority of the multi-level pore structure design of the present invention in increasing drug loading.

[0051] Table 3 In vitro cumulative release curve data in pH=7.4 buffer solution Conclusion: In an environment simulating normal body fluids at pH=7.4, the drug leakage of Examples 1, 2, and 3 within 72 hours was controlled within 13% due to the effective sealing effect of the chitosan gating layer, showing extremely low background release. Among them, Example 3 had the best sealing effect due to its higher degree of cross-linking. This result shows that under a physiologically 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 had a severe burst release of 42.5% within 2 hours; 75.3% of the drug was released within 12 hours. This is a completely uncontrollable explosive release that may lead to serious toxic side effects and drug waste. Although the comparative example 3 with physically adsorbed chitosan has a certain inhibitory effect, there is still significant leakage, indicating that the stability of the covalent bond is crucial.

[0052] Table 4 In vitro cumulative release curve data in pH=5.5 buffer solution Conclusion: In the pH=5.5 environment simulating the infection microenvironment, the chitosan gating layers of Examples 1-3 all responsively opened, achieving sustained and effective release of the drug. Among them, the release amount of Example 1 within 2 hours rose to 15.6%, and reached 68.9% and 88.1% at 24 hours and 72 hours, respectively, showing a sustained and efficient drug release pattern. This is in sharp contrast to the results in Table 3, proving the pH intelligent response characteristics of the system of the present invention. It can be seen that the chitosan gating layer can sensitively "sense" the drop in the pH value of the environment, and switch from a "closed" state to an "open" state, releasing antibiotics on demand. The release curve of Comparative Example 2 under this acidic condition is not much different from the release curve under its neutral condition. Both are rapid, uncontrolled releases, which contrasts that the drug release behavior of Examples 1, 2, and 3 is the result of precise regulation of the pH environment, rather than simple diffusion.

[0053] Furthermore, Example 2 (high molecular weight chitosan) released slowly, while Example 3 (high cross-linking degree) released quickly, indicating that the release kinetics can be precisely adjusted by regulating the gating layer parameters.

[0054] Table 5 Comparison of burst release rate within two hours Conclusion: The burst release rate of Example 1 at pH 7.4 was only 4.1%, far lower than that of Comparative Example 2 (42.5%) and Comparative Example 1 (12.5%). This demonstrates the synergistic effect of the multi-level pore structure and the covalently bonded gating layer, which provides a decisive advantage in effectively inhibiting burst drug release.

[0055] Table 6 Gating layer stability cycle test (pH = 7.4 / 5.5 alternating) Experimental description: Immerse the sample in a pH 7.4 buffer solution for 12 hours (valve closed), then transfer to a pH 5.5 buffer solution and test its release over 24 hours (valve open). Repeat this cycle six times, recording the cumulative release in each acidic environment.

[0056] Conclusion: In 6 pH stimulation cycles, the release behavior of Example 1 under acidic conditions was highly consistent (fluctuation range ≤1.4%), indicating that the low cross-linking density chitosan gating layer has excellent reversibility and structural stability, the swelling / contraction cycle does not cause fatigue damage, and the release rate is stable and controllable; the release amount of Example 3 gradually increased from 75.4% to 78.5%, indicating that the swelling rate of the highly cross-linked network is accelerated, which accelerates drug diffusion but does not destroy the switching function; while the release amount of Comparative Example 3 (physical adsorption) dropped sharply during the cycle (decreased by 86.4%), indicating that the physically adsorbed chitosan continued to fall off during the cycle and the gating function was basically lost in the 6th cycle.

[0057] Table 7 Comparison of release performance Conclusion: Under neutral conditions (pH = 7.4), the burst release rate of ciprofloxacin (5.2%) was slightly higher. This is because ciprofloxacin has a smaller molecular weight (approximately 331.3 g / mol) than levofloxacin (approximately 361.4 g / mol), resulting in a relatively higher diffusion rate within the gated layer network. Under acidic conditions (pH = 5.5), the overall release rate of ciprofloxacin was slower due to stronger electrostatic binding with the carrier amino group, requiring more proton competition for dissociation. In terms of drug loading, the ciprofloxacin loading (34.8%) (Example 4) was slightly higher than the levofloxacin loading (33.2%) (Example 1). This is due to the greater hydrophobicity of ciprofloxacin (logP = 0.8, levofloxacin logP = -0.4), which enhances the hydrophobic interaction with the amino-containing carrier. This indicates that the carrier and gated layer of the present invention are effective for another quinolone antibiotic, in addition to levofloxacin, and the difference in release behavior is consistent with the physicochemical properties of the drug.

[0058] Table 8 Swelling ratio of the gate layer Conclusion: Comparative Example 3, which physically adsorbs chitosan, opens the pores in both neutral and acidic environments, while Examples 1 and 3 are both in a sealed closed pore state in a neutral environment and open the pores in an acidic environment, which indicates that the gating mechanism designed in the present invention has a dynamic pH responsiveness function.

[0059] In summary, the present invention constructs an integrated system of "high-capacity storage-stable adsorption-intelligent release" through the three-level mechanism design of "multi-level pore structure-chemical anchoring-dynamic external gating", which solves the technical problem of high drug loading, low burst rate and biosafety in the existing technology, and provides a feasible and effective solution for the development of a new generation of intelligent drug delivery systems.

[0060] The above embodiments are only preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any changes that adopt the design principles of the present invention and any changes made through non-creative work on this basis should fall within the scope of protection of the present invention.

Claims

1. An antibiotic sustained-release system based on hierarchical porous silica, characterized in that: include: Spherical multi-level porous silica microsphere carrier, the spherical multi-level porous silica microsphere carrier has a dual-mode pore structure composed of a macroporous network that penetrates each other and mesopores extending from the inner wall of the macroporous network; amino functional groups covalently bonded to the outer surface and inner surface of the pores of the spherical hierarchically porous silica microsphere carrier; Antibiotic molecules loaded inside the pores of spherical hierarchically porous silica microsphere carriers; The pH-responsive chitosan gating layer is a covalently bonded coating that is coated on the outer surface of the spherical multi-level porous silica microsphere carrier. It is in a dense contracted state under a physiological neutral environment to close the pores and prevent the leakage of antibiotic molecules; it changes to a swollen and loose state under a slightly acidic environment to open the pores and release antibiotic molecules.

2. The antibiotic sustained-release system based on multi-level porous silica according to claim 1, characterized in that: The diameter of the spherical multi-level 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 mesopore specific surface area is greater than 600m 2 / g; the total pore volume of the spherical multi-level porous silica microsphere carrier is greater than 1.2cm 3 / g.

3. The antibiotic sustained-release system based on multi-level 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 multi-level porous silica microsphere carrier are formed by a 3-aminopropyltriethoxysilane molecule as a silane coupling agent, through a hydrolysis condensation reaction between its triethoxysilane group and the silanol group on the surface of the spherical multi-level porous silica microsphere carrier.

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

5. The antibiotic sustained-release system based on multi-level porous silica according to claim 3 or 4, characterized in that: The pH-responsive chitosan gating layer is constructed using a glutaraldehyde crosslinker; the aldehyde groups of the glutaraldehyde crosslinker react with amino functional groups on the surface of the spherical multi-level porous silica microsphere carrier and the amino groups of the chitosan molecules themselves to form imine bonds that covalently bond the chitosan network; the pH-responsive chitosan gating layer has a swelling ratio of ≤6 in an environment of pH=7.4 and a swelling ratio of ≥15 in an environment of pH=5.

5.

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

7. The antibiotic sustained-release system based on multi-level porous silica according to claim 6, characterized in that: The molar ratio of the glutaraldehyde cross-linking agent to the amino groups in the chitosan molecules is 0.5:1 to 1.5:

1.

8. A method for preparing the antibiotic sustained-release system according to any one of claims 1 to 7, characterized in that: The following steps are involved: (1) Preparation of hierarchically porous silica microsphere carriers: Using a dual-template method, in a mixed solvent system with ethanol and deionized water as the main components and ammonia as the catalyst, polymethyl methacrylate nanospheres are used as macroporous templates and triblock copolymers are used as mesoporous templates. A silicon source is added to carry out hydrolysis and condensation reactions to form composite microspheres. The two organic templates are removed by high-temperature calcination to obtain spherical hierarchically porous silica microsphere carriers with a dual-mode pore structure. (2) Amination of the carrier surface: After the spherical multi-level porous silica microsphere carrier is dried and activated under vacuum conditions at 100°C to 150°C, it is refluxed with a silane coupling agent in an organic solvent at 80°C to 120°C for 12 to 24 hours to covalently bond the amino functional groups to the outer surface of the spherical multi-level porous silica microsphere carrier and the inner surface of the pores; (3) Antibiotic loading: The spherical multi-level porous silica microsphere carrier after amino treatment is dispersed in a buffer solution with a pH of 7.0 to 7.4 and a concentration of 3-5 mg / mL antibiotic molecules. After stirring and adsorption, separation, washing, and drying are performed to obtain a drug-loaded intermediate; (4) Construction of pH-responsive chitosan gating layer: The drug-loaded intermediate is dispersed in an acidic buffer solution, chitosan solution is added and mixed, and glutaraldehyde solution as a cross-linking agent is added to carry out an in situ cross-linking reaction, so that chitosan forms a covalently bonded gating layer on the outer surface of the pores of the spherical multi-level porous silica microsphere carrier. After washing and drying, an antibiotic sustained-release system is obtained.

9. The method according to claim 8, characterized in that The step (1) comprises 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 Pluronic P123 was dissolved as a triblock copolymer; (102) Tetraethyl orthosilicate was added dropwise as a silicon source under stirring, 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 h to form composite microspheres; (103) The composite microspheres were calcined at 550 °C for 6 h to remove the template and solidify the silica skeleton to obtain spherical hierarchically porous silica microsphere carriers.

10. The method according to claim 8 or 9, characterized in that The step (4) comprises the following steps: (401) The drug-loaded intermediate was dispersed in an acetic acid-sodium acetate buffer solution at pH 5.0 to obtain a suspension of drug-loaded microspheres; (402) Chitosan of the same mass as the drug-loaded intermediate was dissolved in another portion of acetic acid-sodium acetate buffer solution of the same volume and pH value to obtain a chitosan solution; (403) The chitosan solution was added to the suspension of drug-loaded microspheres, and a 25% w / w aqueous solution of glutaraldehyde was slowly added dropwise under magnetic stirring to control the final molar ratio of glutaraldehyde to amino groups in the chitosan molecule to be in 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) The product was washed twice with acetic acid-sodium acetate buffer solution at pH 5.0 and then washed with deionized water to remove unreacted chitosan, glutaraldehyde, and reaction by-products. The final product was then freeze-dried to obtain the antibiotic sustained-release system.

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