Preparation method of targetable temperature-sensitive drug-loaded composite microspheres based on attapulgite

Abstract

The embodiment of the application discloses a preparation method of a target temperature-sensitive drug-loaded composite microsphere based on attapulgite, which comprises the following steps: carrying out purification treatment on original attapulgite based on an aqueous solution of inorganic strong acid to obtain purified attapulgite; mixing the purified attapulgite, gypenoside, inulin and folic acid to obtain a mixture, and carrying out mixing treatment on the mixture based on deionized water to obtain a composite microsphere suspension; conveying the composite microsphere suspension into a spray dryer to carry out granulation to obtain primary drug-loaded composite microspheres; adopting a melt impregnation method to coat lauric acid on the surface and pores of the primary drug-loaded composite microspheres, and cooling the primary drug-loaded composite microspheres coated with lauric acid to room temperature to obtain the target temperature-sensitive drug-loaded composite microspheres. The application can not only realize temperature change-based on-off type control release of drugs, significantly improve the targeting and treatment accuracy of drugs, but also reduce the systemic toxic side effects of chemotherapy from the source, and realize efficient and low-toxicity synergistic treatment.

Description

Technical Field

[0001] This invention belongs to the field of drug delivery system technology, specifically relating to a method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite. Background Technology

[0002] Malignant tumors are a major disease posing a serious threat to human health, and chemotherapy plays a vital role in clinical treatment. However, traditional chemotherapy drugs generally suffer from poor targeting, wide systemic distribution, and significant toxic side effects, severely impacting their therapeutic efficacy and application prospects. To overcome these limitations, intelligent responsive nanomedicine delivery systems have become a research hotspot. Among them, systems that utilize tumor microenvironment characteristics (such as pH value and specific enzymes) or external stimuli (such as light and heat) to achieve controlled drug release show great potential. In particular, near-infrared light, due to its advantages such as large tissue penetration depth and excellent spatiotemporal controllability, is widely used in photothermal therapy and its synergistic treatment with chemotherapy.

[0003] Among numerous nanocarriers, attapulgite, a natural nanoclay mineral, is considered a highly promising drug carrier matrix due to its high specific surface area, excellent biocompatibility, and superior adsorption properties. However, pure attapulgite drug delivery systems have significant drawbacks: firstly, drug release behavior is difficult to control precisely, lacking an effective "on / off" mechanism, which may lead to premature drug release before reaching the target or insufficient release when needed; secondly, it lacks active targeting capabilities, exhibiting low selectivity for tumor tissues, limiting its efficacy and potentially increasing systemic toxicity. On the other hand, phase change materials (PCMs), represented by natural fatty acids (such as lauric acid), are being explored as thermally responsive drug release "gating" materials due to their reversible solid-liquid phase transition properties and high phase transition enthalpy. However, direct use of PCMs in practical applications faces two major challenges: firstly, molten materials are prone to leakage, leading to uncontrollable drug release and decreased carrier stability; secondly, the solid-liquid phase transition process involves phenomena such as supercooling, making precise control of the release temperature difficult.

[0004] Furthermore, although existing studies have used manganese-based materials and metal-organic frameworks to construct photothermal-chemotherapy synergistic systems, these synthetic materials often face issues such as unclear in vivo metabolism and questionable long-term biosafety. Meanwhile, many existing systems have limited functionality, making it difficult to simultaneously achieve multiple functions such as colon-specific delivery, active tumor targeting, and precise controlled release triggered by near-infrared light. Therefore, there is an urgent need in this field for a new technological solution that can construct a multifunctional integrated drug delivery system based on biocompatible natural materials. This system must effectively address multiple challenges, including carrier stability, precise drug release, target specificity, and biosafety, to achieve highly efficient and low-toxicity synergistic tumor therapy. Summary of the Invention

[0005] To address the aforementioned issues, this application proposes a method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite. These microspheres not only enable intelligent controlled release of drugs, improving therapeutic efficacy, but also reduce the systemic toxic side effects of chemotherapy, achieving highly efficient and low-toxicity synergistic therapy.

[0006] A method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite, the method comprising:

[0007] Step 1: Purify the raw attapulgite using an aqueous solution of an inorganic strong acid and deionized water to obtain purified attapulgite;

[0008] Step 2: The purified attapulgite, gypenosides, inulin and folic acid are mixed according to a preset mass percentage to obtain a mixture. The mixture is then mixed with deionized water to obtain a composite microsphere suspension.

[0009] Step 3: The composite microsphere suspension is transported to a spray dryer by a peristaltic pump for granulation to obtain primary drug-loaded composite microspheres;

[0010] Step 4: Use a melt impregnation method to coat lauric acid onto the surface and pores of the primary drug-loaded composite microspheres, and cool the primary drug-loaded composite microspheres coated with lauric acid to room temperature to obtain targeted thermosensitive drug-loaded composite microspheres.

[0011] In an optional implementation, step one includes:

[0012] The original attapulgite mineral is crushed and sieved to obtain attapulgite powder within the preset particle size range.

[0013] The attapulgite powder was activated by an aqueous solution of an inorganic strong acid, and the acid-activated mixture was naturally cooled to room temperature. The cooled mixture was then separated to obtain a solid product.

[0014] The solid product was repeatedly washed with deionized water until the filtrate was neutral. The washed solid product was then dried, ground, and sieved to obtain purified attapulgite.

[0015] In an optional embodiment, the acid activation of the attapulgite powder using an aqueous solution of an inorganic strong acid includes:

[0016] The attapulgite powder is mixed with an aqueous solution of an inorganic strong acid, such that the H⁺ concentration of the acid solution is in the range of 1~4 mol / L, and the mixture is stirred continuously for 2~6 hours under heating conditions of 50℃~80℃.

[0017] In an optional embodiment, the mixture consists of raw materials in the preset mass percentage:

[0018] Purified attapulgite: 50-85 parts; Gynostemma pentaphyllum saponins: 10-30 parts; Inulin: 10-25 parts; Folic acid: 0.1-1 parts.

[0019] In an optional embodiment, the mixing treatment based on the mixture and deionized water to obtain the composite microsphere suspension includes:

[0020] The mixture is added to a container containing deionized water to obtain a suspension. The container containing the suspension is placed in an ultrasonic processor for ultrasonic treatment to obtain a pre-mixed suspension.

[0021] The initially mixed suspension was transferred to a mechanical stirrer for stirring to obtain a composite microsphere suspension.

[0022] In an optional embodiment, the ultrasonic treatment is performed under the following conditions: ultrasonic dispersion at a power of 300-600W for 10-30 minutes.

[0023] In an optional embodiment, the stirring conditions are: continuous stirring at a speed of 500~1000 rpm for 0.5~2 hours.

[0024] In an optional implementation, step three includes:

[0025] The composite microsphere suspension was transported to a spray dryer by a peristaltic pump for granulation to obtain primary drug-loaded composite microspheres;

[0026] The primary drug-loaded composite microspheres are recovered using a collection device.

[0027] In an optional embodiment, the peristaltic pump rotates at a speed of 1 to 5 RPM, and its feed flow rate can be set to 1 to 5 mL / min.

[0028] In an optional implementation, step four includes:

[0029] Lauric acid is heated in a constant temperature water bath until it is completely melted into a liquid state, thus obtaining molten lauric acid.

[0030] The primary drug-loaded composite microspheres are mixed with molten lauric acid at a specific mass ratio, and the mixture is continuously mechanically stirred under constant temperature conditions. The molten lauric acid is adsorbed onto the pores and surface of the primary drug-loaded composite microspheres by impregnation to form a coating layer, thus obtaining a mixture.

[0031] After coating, the mixture was removed from the water bath and allowed to cool naturally to room temperature to obtain targeted thermosensitive drug-loaded composite microspheres.

[0032] The embodiments of this application have the following beneficial effects:

[0033] This application discloses a method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite. The method includes purifying raw attapulgite using an aqueous solution of an inorganic strong acid and deionized water to obtain purified attapulgite; mixing the purified attapulgite, gypenosides, inulin, and folic acid according to a preset mass percentage to obtain a mixture; mixing the mixture with deionized water to obtain a composite microsphere suspension; granulating the composite microsphere suspension using a peristaltic pump in a spray dryer to obtain primary drug-loaded composite microspheres; coating the surface and pores of the primary drug-loaded composite microspheres using a melt impregnation method; and cooling the lauric acid-coated primary drug-loaded composite microspheres to room temperature to obtain targeted thermosensitive drug-loaded composite microspheres. The method proposed in this invention combines the drug-carrying capacity of attapulgite, the chemotherapy effect of drugs, the active targeting of folic acid, the colonic enzyme response of inulin, and the photothermal controlled release function of lauric acid. It successfully constructs a high-performance, multifunctional synergistic drug delivery system integrating "colon targeting, active target acquisition, and near-infrared photothermal triggered drug release," ultimately achieving a synergistic anti-tumor effect far exceeding that of single therapies. It not only enables temperature-based "on-off" controlled drug release, significantly improving drug targeting and therapeutic precision, but also reduces the systemic toxicity of chemotherapy at its source, achieving highly efficient and low-toxicity synergistic treatment. Attached Figure Description

[0034] To more clearly illustrate the technical solution of the present invention, the accompanying drawings required in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and therefore should not be regarded as a limitation on the scope of protection of the present invention. In the various drawings, similar components are numbered similarly.

[0035] Figure 1 The image shows scanning electron microscope images of the targeted thermosensitive drug-loaded composite microspheres and various intermediate products prepared according to the embodiments of this application.

[0036] Figure 2 The DSC curve of the targeted thermosensitive drug-loaded composite microspheres according to an embodiment of this application is shown.

[0037] Figure 3 The infrared spectrum of the targeted thermosensitive drug-loaded composite microspheres according to an embodiment of this application is shown.

[0038] Figure 4 The thermogravimetric analysis curves of the targeted temperature-sensitive drug-loaded composite microspheres prepared according to the embodiments of this application are shown.

[0039] Figure 5 The X-ray diffraction pattern of the targeted temperature-sensitive drug-loaded composite microspheres prepared according to the embodiments of this application is shown;

[0040] Figure 6 The cell viability of HeLa cells under different concentration gradients of the targeted thermosensitive drug-loaded composite microspheres prepared in the embodiments of this application is shown.

[0041] Figure 7 The cell survival rates of HeLa cells under different conditions are shown for the targeted thermosensitive drug-loaded composite microspheres prepared in the embodiments of this application. Detailed Implementation

[0042] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0043] To more clearly demonstrate the implementation steps and advantages of this invention, the specific implementation methods are described below with reference to the illustrations.

[0044] This application proposes a method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite. The prepared targeted thermosensitive drug-loaded composite microspheres can not only achieve intelligent controlled drug release and improve therapeutic efficacy, but also reduce the systemic toxic side effects of chemotherapy, achieving highly efficient and low-toxicity synergistic therapy. Exemplarily, the specific implementation steps of this method are as follows:

[0045] (1) Purification of attapulgite:

[0046] The raw attapulgite mineral is crushed and sieved, for example, using a jaw crusher or gyratory crusher, or further crushed using a cone crusher or impact crusher. A vibrating screen can also be used, with particle size classification achieved by adjusting the screen mesh size to obtain attapulgite powder within a preset particle size range. The preset particle size range can be set according to actual conditions, such as 100~400 mesh. An aqueous solution of an inorganic strong acid is used for acid activation. Specifically, the attapulgite powder and acid solution are mixed at a certain solid-liquid ratio, ensuring the H⁺ concentration of the acid solution is in the range of 1~4 mol / L. The mixture is stirred continuously for 2~6 hours under heating conditions of 50℃~80℃. The inorganic strong acid can be any one of hydrochloric acid, sulfuric acid, or nitric acid. The preferred acid activation temperature is 60~70℃, and the preferred reaction time is 3~4 hours.

[0047] After the reaction is complete, allow the mixture to cool naturally to room temperature. Separate the solid product by centrifugation or filtration. For example, a high-speed centrifuge at 8000 rpm can be used for centrifugation. Discard the supernatant and collect the solid product. Then, repeatedly wash the solid product with deionized water until the resulting filtrate is neutral to thoroughly remove acid and dissolved impurities. Finally, dry the washed solid product in an oven at 60℃~100℃ for 6~12 hours, then grind and sieve to obtain purified attapulgite, i.e., purified attapulgite. The specific surface area of ​​the purified attapulgite is preferably 50~300 m² / g. This purification step effectively removes impurities such as carbonates and iron oxides from the original attapulgite mineral, improving its specific surface area and reactivity.

[0048] This application utilizes the unique advantages of natural attapulgite at the microstructure level. Unlike the artificially synthesized, uniformly structured gold nanocages in the prior art, attapulgite is a hydrous magnesium aluminum silicate clay mineral with a unique one-dimensional nanorod crystal structure. Its microstructure is composed of countless rod crystal bundles with nanometer-scale diameters and micrometer-scale lengths. After acid activation and purification, it can be effectively unbound and expose a huge specific surface area and abundant mesoporous structure.

[0049] (2) Preparation of composite microsphere suspension:

[0050] The purified attapulgite obtained above is mixed with the active pharmaceutical ingredients gypenosides, inulin, and the target agent folic acid in a specific mass ratio to obtain the corresponding mixture. Folic acid will serve as the target agent. The specific mass ratio consists of the following raw materials in the following mass percentages: purified attapulgite: 50-85 parts; gypenosides: 10-30 parts; inulin: 10-25 parts; folic acid: 0.1-1 parts. The molecular weight of inulin is 2,000-5,000 Da.

[0051] The above mixture is added together to a container filled with deionized water to obtain a suspension with a solid content between 2% and 10%. Preferably, the solid content of the suspension is 3% to 5%. Subsequently, the container containing the suspension is placed in an ultrasonic processor for ultrasonic treatment at a power of 300-600W for 10-30 minutes to obtain a pre-mixed suspension, ensuring that the nano-sized purified attapulgite is fully disintegrated and initially mixed with other components. This pre-mixed suspension can be used as a composite microsphere suspension for later use, or it can be stirred again to further form a uniform and stable composite microsphere suspension. Preferably, the ultrasonic dispersion time is 5-20 minutes.

[0052] After ultrasonic treatment, the preliminarily mixed suspension is transferred to a mechanical stirrer for stirring at a speed of 500-1000 rpm for 0.5-2 hours; preferably, the mechanical stirrer speed is 300-800 rpm for 0.5-1.5 hours, allowing the preliminarily mixed suspension to further form a uniform and stable composite microsphere suspension for later use. If the mechanical stirrer speed is too low, the materials in the suspension will not mix sufficiently, while if the speed is too high, the materials are prone to splashing and agglomeration.

[0053] In this application, inulin is used to achieve specific enzymatic hydrolysis in the colon, thereby releasing drug-loaded composite microspheres and realizing colon-targeted release. Furthermore, folic acid binds to the surface of the composite microspheres through physical adsorption and electrostatic interactions, enabling active targeting of tumor cells highly expressing folic acid receptors.

[0054] (3) Preparation of primary drug-loaded composite microspheres by spray drying:

[0055] The prepared composite microsphere suspension is transported to a spray dryer for granulation using a peristaltic pump. The peristaltic pump speed can be set to 1-5 RPM, and the feed flow rate can be set to 1-5 mL / min, preferably 1-4 mL / min. The inlet temperature of the spray dryer can be set to 150℃-200℃, and the outlet temperature can be maintained within the range of 70℃-110℃. In this application, the high-pressure airflow of the spray dryer atomizes the composite microsphere suspension into micron-sized droplets. The droplets are rapidly dried under the action of hot air, and after the moisture evaporates, solid composite microspheres with good sphericity are formed, thus obtaining primary drug-loaded composite microspheres based on attapulgite. The primary drug-loaded composite microspheres can also be screened to ensure that their particle size is mainly distributed between 1-50 μm. The dried product, the solid primary drug-loaded composite microspheres, is recovered by a collection device, which can be a cyclone separator. By precisely controlling the process parameters of the spray dryer, such as the inlet temperature and feed rate, primary drug-loaded composite microspheres with good sphericity, uniform particle size, and well-developed mesoporous structure can be obtained. This creates conditions for efficiently loading lauric acid and preventing its leakage.

[0056] This application utilizes attapulgite (ATP), a natural nano-clay mineral, as a rigid porous framework. Acid activation and purification significantly enhance its specific surface area and adsorption activity. Building upon this, inulin is innovatively introduced as a "bioswitch" for colonic enzyme responses, and folic acid as a "navigation head" for tumor targeting. These four components are integrated using spray drying technology to construct structurally stable primary drug-loaded composite microspheres. Firstly, this rigid nanofiber network structure provides a robust, highly porosity three-dimensional framework for loading chemotherapeutic drugs (Gynostemma pentaphyllum saponins) and subsequently encapsulating phase change materials (lauric acid). Its physical stability and drug loading capacity are far superior to hollow gold nanocages, effectively preventing leakage of the phase change material in the molten state. Secondly, the surface of attapulgite contains numerous silanol groups (Si-OH) and unsaturated charge sites, enabling it to efficiently load drug molecules and the target agent folic acid not only through physical adsorption and electrostatic interactions, but also to form strong hydrogen bonds with the hydroxyl groups of inulin. This allows the quaternary components (ATP / drug / inulin / folic acid) to tightly cross-link through various intermolecular forces during spray drying, forming structurally stable primary drug-loaded composite microspheres, rather than a simple physical mixture. This functional design based on the microstructure and surface chemistry of natural minerals is the material basis for achieving high-performance, multifunctional synergistic drug delivery systems.

[0057] (4) Construction of a temperature-sensitive controlled-release system by encapsulating phase change materials:

[0058] To further endow the composite microspheres with temperature-responsive drug release characteristics, the physical properties of lauric acid in its molten state will be utilized to control the drug release rate. Specifically, a melt impregnation method will be used to coat the phase change material (PCM) lauric acid (LA) onto the surface and pores of the primary drug-loaded composite microspheres obtained in the above steps. The specific process is as follows: lauric acid is placed in a water bath and heated to above its melting point, allowing it to completely melt into a liquid state. Preferably, the water bath temperature is 60-95°C. The primary drug-loaded composite microspheres and molten lauric acid are mixed in a specific ratio, with a mass ratio of 1:2 to 1:3. This range represents the loading amount of the phase change material lauric acid onto the primary drug-loaded composite microspheres, allowing for better encapsulation of the phase change material within the rigid structure of the microspheres. Primary drug-loaded composite microspheres were continuously mechanically stirred and mixed with molten lauric acid under constant temperature conditions. The molten lauric acid was then adsorbed onto the pores and surface of the primary drug-loaded composite microspheres via an impregnation method for 20-60 minutes to ensure complete wetting and adsorption of the molten lauric acid into the pores and surface of the primary drug-loaded composite microspheres, simultaneously forming a coating layer on the surface of the primary drug-loaded composite microspheres. After coating, the mixture was removed from the water bath and allowed to cool naturally to room temperature. This allowed the lauric acid to completely solidify, forming a stable solid-solid phase change composite material. After slight grinding, the final product—targeted thermosensitive drug-loaded composite microspheres—was obtained.

[0059] The phase change material, lauric acid, has a phase change temperature of 40-45℃ and a solidification temperature of 35-40℃. Under near-infrared photothermal stimulation, the targeted thermosensitive drug-loaded composite microspheres rapidly heat up, melting the lauric acid and opening the drug release channels, resulting in the rapid release of the contained drug. After stimulation ceases, the temperature of the targeted thermosensitive drug-loaded composite microspheres drops to body temperature, the lauric acid solidifies, the drug release channels close, and the drug release rate significantly decreases, thus achieving precise "on-off" control of drug release. The near-infrared light can be 808nm with a power density of 1.0W / cm². The composite material targeted thermosensitive drug-loaded microspheres utilize a melt impregnation method to encapsulate lauric acid as a thermosensitive "gating" material within the pores of the primary drug-loaded microspheres. The lauric acid and the primary drug-loaded microspheres are physically bonded together, not only maintaining their inherent phase transition enthalpy and achieving efficient photothermal energy conversion and storage, but also utilizing their solid-liquid phase transition properties to achieve precise "on-off" control of drug release.

[0060] This application utilizes the unique microscopic confinement and nucleation effects generated when attapulgite and lauric acid are combined to achieve precise temperature-sensitive controlled-release performance that surpasses conventional phase change materials. In existing technologies, lauric acid merely acts as a "plug" to physically seal the pores of gold nanocages. This application is entirely different. In this application, molten lauric acid, under capillary force, can penetrate and fill the well-developed mesoporous network (mesoporous structure) formed by purified attapulgite nanorod crystals. On one hand, the large specific surface area and nanopores of purified attapulgite exert a strong spatial confinement effect and surface interaction on lauric acid molecules. This significantly reduces the "supercooling" phenomenon that easily occurs during the phase change of lauric acid, making its solidification temperature closer to its melting point. This results in a faster "off" response for drug release and more precise and reliable controlled-release behavior. On the other hand, the rigid framework of purified attapulgite provides a large number of heterogeneous nucleation sites for the solidification of lauric acid, promoting its uniform and rapid crystallization during cooling. This further ensures the effectiveness of the "off" state and avoids drug leakage caused by incomplete local crystallization of the phase change material. This microscopic confinement and nucleation effect mediated by natural minerals has fundamental differences and significant advantages over the simple surface coating of gold nanocages in terms of the precision of controlled release and the long-term stability of the system, and is the key to achieving efficient, low-toxicity synergistic therapy.

[0061] (5) Evaluation of the in vitro killing effect of targeted thermosensitive drug-loaded composite microspheres on HeLa cells (human cervical cancer cells).

[0062] Targeted thermosensitive drug-loaded composite microspheres were prepared into working solutions with multiple concentration gradients. Single-cell suspensions of target tumor cells (HeLa cells) were prepared and seeded in multi-well plates, and cultured to allow HeLa cells to adhere. Groups were set up, including a control group and multiple concentration experimental groups. Different concentrations of the targeted thermosensitive drug-loaded composite microsphere working solution were added to each of the multiple concentration experimental groups, and the cells were cultured for a certain period. Then, the cytotoxicity of the control group and the multiple concentration experimental groups after culture was detected using the CCK-8 assay. Specifically, CCK-8 reagent was added to each well, and after incubation in the dark, the absorbance at a specific wavelength was measured using a microplate reader. This was used to evaluate the effect of different concentrations of the targeted thermosensitive drug-loaded composite microsphere working solution on the survival rate of tumor cells, i.e., to evaluate the cytotoxicity of the targeted thermosensitive drug-loaded composite microspheres themselves. The use of the CCK-8 assay for cytotoxicity detection is well known to those skilled in the art and will not be described in detail here.

[0063] (6) Evaluation of the killing effect of antitumor drugs on tumor cells under photothermal stimulation.

[0064] Furthermore, this embodiment will also verify the synergistic therapeutic effect of targeted thermosensitive drug-loaded composite microspheres under near-infrared light stimulation. The test material is configured to include a blank control and multiple gradient working solutions around the optimal concentration determined in the preliminary experiment. The preliminary experiment is the experimental results of the effect of the working solutions of the targeted thermosensitive drug-loaded composite microspheres at different concentrations on the survival rate of tumor cells. The test material is the working solutions of the targeted thermosensitive drug-loaded composite microspheres at different concentrations. In other words, the drug-loaded composite microsphere working solutions within the optimal concentration range among the different concentrations of targeted thermosensitive drug-loaded composite microspheres are selected for the experiment, and a blank control group will also be set up for the experiment.

[0065] Tumor cell suspensions were prepared and seeded into multi-well plates. After cell adhesion, multiple experimental groups were set up, including a blank control group, a simple photothermal stimulation group, a simple material treatment group, and a combined material and photothermal treatment group, each receiving corresponding treatment. The blank control group received no treatment. The simple photothermal stimulation group received photothermal stimulation by near-infrared laser irradiation with the temperature controlled within a preset range, followed by continued culturing of the material. The simple material treatment group only underwent culturing of the material. The combined material and photothermal treatment group received photothermal stimulation by near-infrared laser irradiation after incubation for a certain period, with the temperature controlled within a preset range, followed by continued culturing for a period after photothermal stimulation. The preset temperature range was 40-55℃. Near-infrared laser irradiation was used in the simple photothermal stimulation group and the combined material and photothermal treatment group, with temperature controlled by an infrared imager. The cell viability of each experimental group was then detected using the CCK-8 assay. The absorbance at a specific wavelength was measured to evaluate the killing effect of the test material on tumor cells under single treatment, no treatment, simple photothermal stimulation, and combined photothermal stimulation.

[0066] The wavelength of the aforementioned near-infrared light can be 808 nm, and the power density is 1.0 W / cm². Under near-infrared light irradiation, the simple photothermal stimulation group and the material combined with photothermal treatment group can raise the local temperature to above 40°C within 5 minutes, thereby triggering the melting of the phase change material and releasing the drug loaded on the drug-loaded composite microspheres. Therefore, the targeted thermosensitive drug-loaded composite microspheres prepared by the above method have a phase change enthalpy (ΔHm) of not less than 60 J / g and possess good thermal energy storage and release capabilities.

[0067] Example

[0068] 100 grams of raw attapulgite mineral was weighed and preliminarily pulverized using a pulverizer, then passed through a 200-mesh standard sieve to obtain attapulgite powder within a predetermined particle size range. The attapulgite powder was placed in a flask containing an aqueous solution of an inorganic strong acid with a H+ concentration of 4 mol / L, and placed in a 60°C constant temperature water bath. The mixture was mechanically stirred continuously at 60°C for 4 hours. After the reaction, the mixture of the inorganic strong acid aqueous solution and the attapulgite powder was allowed to cool naturally to room temperature, and then centrifuged at 8000 rpm using a high-speed centrifuge. The supernatant was discarded, and the collected solid precipitate was collected. The solid precipitate was repeatedly washed with deionized water and centrifuged until the supernatant tested neutral using pH paper to ensure complete removal of residual acid and soluble impurities. The washed solid product was a wet solid. The wet solid was transferred to a forced-air drying oven and dried at 80°C for 10 hours to obtain a dried block product. Finally, the dried block product is ground and sieved again to obtain purified attapulgite powder, i.e., purified attapulgite.

[0069] Accurately weigh 5.5 g of purified attapulgite, 2.5 g of gypenosides, 1.95 g of inulin, and 0.05 g of folic acid (as a targeting agent). Add all four materials together to a 500 mL beaker, and then add 200 mL of deionized water to obtain a suspension. The solid content of the suspension is approximately 4.8% (w / v). Furthermore, in some embodiments, the mass ratio of purified attapulgite to the active pharmaceutical ingredients gypenosides, inulin, and the targeting agent folic acid can also be as shown in Table 1.

[0070] Attapulgite (g) Gynostemma pentaphyllum saponins (g) Inulin (g) Folic acid (g) Deionized water (ml) 5.5 2.5 1.95 0.05 200 2.5 2.5 1.95 0.05 200 7.5 2.5 1.95 0.05 200

[0071] Table 1

[0072] The beaker was placed in an ultrasonic cell disruptor and ultrasonically dispersed for 10 minutes at 400W to obtain a preliminary mixed suspension. This process fully disintegrated the nano-sized attapulgite and uniformly dispersed it in the liquid phase, while also promoting the initial mixing of the components. After ultrasonication, the beaker was transferred to a magnetic stirrer and stirred continuously at 600rpm for 0.5 hours, so that the preliminary mixed suspension eventually formed a uniform, stable, and easily spray-dried composite microsphere suspension for later use.

[0073] The prepared composite microsphere suspension was poured into the feed bottle of a spray dryer. The main process parameters of the spray dryer were set as follows: inlet air temperature was 180℃, outlet air temperature was automatically maintained at approximately 85℃; the feed flow rate of the peristaltic pump was set to 3 mL / min. Under negative pressure, the composite microsphere suspension was atomized into micron-sized droplets, which were instantly dried in hot air, and the moisture evaporated rapidly to form solid composite microspheres. The dried product was collected by a cyclone separator to obtain light brown, free-flowing primary drug-loaded composite microsphere powder. Laser particle size analysis showed that the particle size of the primary drug-loaded composite microspheres was mainly distributed in the range of 5-25 μm, and scanning electron microscopy (SEM) observation showed that it had good sphericity and a rich micro-nano pore structure on the surface.

[0074] 1.0 g of the prepared primary drug-loaded composite microspheres and 2.0 g of lauric acid (LA) were accurately weighed. The lauric acid was placed in a 50 mL round-bottom flask and heated in a 90°C constant-temperature water bath until it was completely melted into a transparent liquid. Under continuous mechanical stirring, the primary drug-loaded composite microspheres were slowly and evenly added to the molten lauric acid, maintaining the water bath temperature at 90°C. Stirring and impregnation continued for 40 minutes to ensure the molten lauric acid fully wetted and penetrated into the pores of the composite microspheres via capillary action, forming a coating layer on their surface. After coating, the mixture of primary drug-loaded composite microspheres and molten lauric acid was removed from the water bath, allowed to cool naturally at room temperature, and allowed to stand for 2 hours to allow the lauric acid to completely solidify. The resulting solidified block product, after slight grinding, yielded attapulgite-targeted thermosensitive drug-loaded composite microspheres. This composite material combines the chemotherapy of drugs, the active targeting of folic acid, the colonic enzyme response of inulin, and the photothermal controlled release function of lauric acid.

[0075] In this embodiment, Figure 1 Scanning electron microscope images of the targeted thermosensitive drug-loaded composite microspheres (LA-composite pharmaceutical materials) and various intermediate products prepared by the above preparation method. Figure 1 (a) A scanning electron microscope image of purified attapulgite mineral, produced by... Figure 1(a) It can be seen that after acid activation treatment, the aggregates on the attapulgite decrease, and the attapulgite is unbound to a certain extent, forming purified attapulgite. The purified attapulgite rod crystals constitute a well-developed mesoporous structure. These changes are helpful for the subsequent construction of primary drug-loaded composite microspheres based on purified attapulgite and other pharmaceutical materials. Specifically, it helps gypenosides, inulin, and folic acid to be adsorbed onto the mesoporous structure formed by the purified attapulgite rod crystals, and also helps in the further encapsulation of phase change materials. Primary drug-loaded composite microspheres (composite pharmaceutical materials) are shown below. Figure 1 (b) and Figure 1 As shown in (c), the primary drug-loaded composite microspheres constructed in this example have a size of approximately 20 µm and a good spherical structure. The primary drug-loaded composite microspheres exhibit many intersecting channels and have a large specific surface area, providing a large number of adsorption sites for the phase change material. Figure 1 (d) is a scanning electron microscope image of targeted thermosensitive drug-loaded composite microspheres (LA-composite medical materials). Figure 1 (d) It can be seen that the constructed composite pharmaceutical material encapsulates the phase change material lauric acid in its rigid structure.

[0076] See appendix Figure 2 The image shows the DSC (Differential Scanning Calorimetry) curve of the targeted thermosensitive drug-loaded composite microspheres (LA-composite medical material). The melting and solidification phase transition enthalpies of the targeted thermosensitive composite microspheres reached 86.4 J / g and 80.6 J / g, respectively. In this embodiment, the melting and solidification phase transition temperatures of lauric acid were 42.9℃ and 40.0℃, respectively. During photothermal therapy, the temperature can rise above the melting point of lauric acid, causing the drug to be released from the targeted thermosensitive drug-loaded composite microspheres. After irradiation is stopped, the temperature needs to drop to a level far below the solidification temperature of lauric acid for the drug release channel to close. Furthermore, the purified attapulgite used in this embodiment, as a porous medium, provides nucleation sites for lauric acid due to its large specific surface area and pore structure, which helps to reduce supercooling and make the solidification temperature closer to the melting temperature, thereby allowing the targeted thermosensitive drug-loaded composite microspheres to respond more precisely.

[0077] As shown in Figure 3, the infrared spectrum of the targeted thermosensitive drug-loaded composite microspheres (LA-composite pharmaceutical material) is included, comprising the LA-composite pharmaceutical material, the composite pharmaceutical material, and LA (lauric acid). The infrared spectrum of lauric acid shows a wavenumber of 2918 cm⁻¹. -1 and 2850cm -1 The adsorption peaks are caused by aliphatic CH vibrations, corresponding to the stretching vibrations of CH2 and CH3, respectively, at 1701 cm⁻¹. -The strong absorption peak at ¹ is attributed to the C=O stretching vibration. When lauric acid is combined with pharmaceutical materials, no new absorption peaks can be observed in the spectrum of the prepared targeted temperature-sensitive drug-loaded composite microspheres (LA-composite pharmaceutical materials, i.e., targeted temperature-sensitive drug-loaded composite microspheres), indicating that there is no chemical interaction between lauric acid and the composite pharmaceutical materials, and that they have good compatibility.

[0078] See appendix Figure 4 The thermogravimetric analysis curves of the targeted temperature-sensitive drug-loaded composite microspheres are shown. Figure 4 It is known that the maximum loading (weight) of lauric acid in the composite pharmaceutical material is approximately 51%. The porous structure and large specific surface area of ​​the composite pharmaceutical material provide a large number of adsorption sites for lauric acid. Encapsulating lauric acid in the rigid structure of the composite pharmaceutical material gives the LA-composite pharmaceutical material (targeted thermosensitive drug-loaded composite microspheres) excellent thermal energy storage capacity. Furthermore, from Figure 4 It can be seen that the decomposition temperature of the composite pharmaceutical material is approximately 200℃, indicating high thermal stability.

[0079] Appendix Figure 5 The infrared spectrum and X-ray diffraction (XRD) pattern of the targeted thermosensitive drug-loaded composite microspheres are shown. The sharp and intense diffraction peaks with diffraction angles (2θ) around 9.7°, 21.6°, and 24.0° in the XRD pattern are LA peaks. The relative LA intensity of the characteristic diffraction peaks of the LA-composite pharmaceutical material decreases, but no new diffraction peaks appear, indicating that the LA-composite pharmaceutical material retains its original crystal structure, exhibits excellent chemical compatibility, and that no chemical reaction or crystal structure change occurs during the composite process of LA and the pharmaceutical material.

[0080] In addition, multiple working solutions of targeted thermo-sensitive drug-loaded composite microspheres with different concentration gradients were prepared, and their in vitro killing effect on HeLa cells was evaluated. In other words, this embodiment will evaluate the cytotoxicity of the targeted thermo-sensitive drug-loaded composite microspheres themselves. The targeted thermo-sensitive drug-loaded composite microspheres prepared according to the above method were prepared into working solutions with five concentration gradients of 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL using PBS buffer (phosphate buffer saline). Logarithmic growth phase HeLa cells were seeded into 96-well plates. After 24 hours of culture and adhesion, the original culture medium in the wells was replaced with the prepared culture medium containing different concentrations of working solution. A blank group and a control group were set up. Culture was continued for another 24 hours. In the blank group, the original culture medium was replaced with culture medium with a working solution concentration of 0, and in the control group, the original culture medium was replaced with the prepared culture medium containing different concentrations of working solution. After the drug treatment ended, cell viability was detected using the CCK-8 assay. The experiment evaluated the antitumor activity of the targeted thermo-sensitive drug-loaded composite microspheres by detecting cell viability using the CCK-8 assay.

[0081] Cell viability was determined using the CCK-8 assay: 10 μL of CCK-8 solution (Cell Counting Kit) was added to each well of a 96-well plate, and the plate was incubated in the dark for 2 hours. The absorbance of each well was then measured using a microplate reader at a wavelength of 450 nm. Cell viability (%) was calculated using the following formula: Cell viability (%) = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. OD value, or optical density, reflects the amount of cell metabolites. The experimental group included a control group and a blank group.

[0082] In this embodiment, Figure 6 To investigate the cytotoxic effects of different concentration gradients of temperature-sensitive drug-loaded composite microspheres on HeLa cells (human cervical cancer cells), the experiment used the CCK-8 assay to detect cell viability and evaluate the antitumor activity of the temperature-sensitive drug-loaded composite microspheres. The results of the experiments at different concentration gradients are shown below. Figure 6As shown, the killing effect of the targeted thermo-sensitive drug-loaded composite microspheres on HeLa cells exhibits a significant concentration-dependent effect: the cell viability of HeLa cells in the control group was 100%; at 0.2 mg / mL, the cell viability of HeLa cells remained close to that of the control group, at 95.02%; however, as the concentration of the working solution prepared with the targeted thermo-sensitive drug-loaded composite microspheres increased, the cell viability decreased significantly. At a working solution concentration of 0.6 mg / mL, the cell viability dropped to approximately 54%, close to the IC50 value; the lowest cell viability was reached at 1 mg / mL, approximately 15.50%. This indicates that the composite pharmaceutical material itself possesses significant antitumor activity, with its effective concentration mainly located in the range of 0.6–1 mg / mL. The experimental results of the killing effect of different concentration gradients of the targeted thermo-sensitive drug-loaded composite microspheres on HeLa cells provide crucial concentration data for subsequent synergistic killing experiments under photothermal stimulation.

[0083] This embodiment will also verify the synergistic therapeutic effect of targeted thermosensitive drug-loaded composite microspheres under near-infrared light stimulation. Based on the evaluation results of the in vitro anti-proliferative activity of the targeted thermosensitive drug-loaded composite microspheres on HeLa cells, three concentrations of 0.4, 0.6, and 0.8 mg / mL were selected for the experiment. HeLa cells in the logarithmic growth phase were seeded into 96-well plates and cultured for 24 hours until they adhered. Then, they were divided into four groups: ① blank control group, ② photothermal stimulation group alone, ③ material treatment group alone, and ④ material and photothermal combined treatment group. After grouping, ① the blank control group received no treatment; ③ the material-only treatment group treated HeLa cells with targeted thermosensitive drug-loaded composite microspheres at concentrations of 0.4, 0.6, and 0.8 mg / mL, respectively; ④ the combined material and photothermal treatment group treated HeLa cells with the optimal concentration of targeted thermosensitive drug-loaded composite microspheres (0.4, 0.6, and 0.8 mg / mL). Additionally, ② the photothermal stimulation group and ④ the combined material and photothermal treatment group were irradiated for 5 minutes with a near-infrared laser at a wavelength of 808 nm and a power density of 1.0 W / cm², with the temperature controlled within the range of 40-55℃ using an infrared thermal imager. After irradiation, all groups were cultured for another hour. Subsequently, cell viability was assessed using the CCK-8 assay to evaluate the antitumor activity of the targeted thermosensitive drug-loaded composite pharmaceutical material.

[0084] Figure 7The study investigated the cytotoxic effect of the prepared targeted thermosensitive drug-loaded composite pharmaceutical material on HeLa cells under different conditions. The experimental results clearly demonstrated the cytotoxic effect of the LA-composite pharmaceutical material on HeLa cells under different conditions: the cell survival rate in the blank control group was close to 100%, indicating the reliability of the experimental system; when using only the LA-composite pharmaceutical material, its cytotoxic effect showed a significant concentration-dependent variation. Figure 7 It can be seen that at concentrations of 0.4 mg / mL, 0.6 mg / mL, and 0.8 mg / mL, the cell viability rates were 83.27%, 74.30%, and 51.57%, respectively, demonstrating the efficacy of the LA-composite medical material based on the chemotherapy of Gynostemma pentaphyllum saponins. The most crucial comparison occurred in the LA-composite medical material + photothermal stimulation group. At this point, the optimal concentration of the LA-composite medical material, 0.6 mg / mL, showed a sharp drop in cell viability to 24.91%. This was not only far lower than the cell viability rate (74.30%) of the material alone at the same concentration (0.6 mg / mL) without photothermal stimulation, but also significantly lower than the higher concentration, specifically lower than the 51.57% cell viability rate of the material alone at a concentration of 0.8 mg / mL. The experimental results strongly demonstrate that there is a remarkable synergistic effect between near-infrared photothermal stimulation and LA-composite medical materials, rather than a simple additive effect. The mechanism is that the heat generated by photothermal stimulation melts the phase change material lauric acid, thereby triggering the "explosive" precise release of the drug from the attapulgite carrier, ultimately achieving a synergistic enhancement of the killing effect of chemotherapy and photothermal therapy.

[0085] Furthermore, the proposed solution achieves a synergistic effect between chemotherapy and photothermal therapy, demonstrating superior in vitro antitumor performance. In vitro cell experiments show that the targeted thermosensitive drug-loaded composite microspheres prepared in this application not only possess concentration-dependent antitumor activity (IC50 approximately 0.6 mg / mL), but more importantly, when combined with near-infrared photothermal stimulation, they produce a synergistic killing effect of "1+1>2". At the optimal concentration (0.6 mg / mL in this embodiment), the cell survival rate of the "material + photothermal" combined treatment group dropped sharply to 24.91%, far lower than that of single therapy, strongly demonstrating the superiority of this system in efficiently integrating chemotherapy and photothermal therapy. Simultaneously, the composite material has a high phase transition enthalpy (ΔHm>80 J / g), ensuring efficient photothermal conversion and energy storage. This provides a novel technical solution for precise, efficient, and low-toxicity synergistic tumor therapy, achieving integrated and optimized performance of "heat storage-drug loading-controlled release".

[0086] This invention utilizes a quaternary composite structure of attapulgite, gypenosides, inulin, and folic acid to achieve synergistic delivery of targeted and enzyme-responsive drugs, and employs lauric acid for thermosensitive coating to successfully prepare a multifunctional composite microsphere. This composite microsphere combines inulin-mediated colonic enzyme-responsive specific release with folic acid-mediated active tumor targeting, effectively reducing drug distribution in non-target tissues. More importantly, lauric acid, as a phase change material, possesses solid-liquid phase change properties (~43℃) that constitute a reliable thermosensitive "gating" switch, enabling rapid "explosive" drug release under near-infrared light irradiation, while release is significantly slowed at body temperature. This thermosensitive phase change gating mechanism coupled with photothermal effects achieves precise, on-demand drug release, fundamentally reducing the systemic toxicity of chemotherapy.

[0087] In summary, this application combines the drug-carrying capacity of attapulgite, the chemotherapy effect of drugs, the active targeting of folic acid, the colonic enzyme response of inulin, and the photothermal controlled release function of lauric acid to successfully construct a high-performance, multifunctional synergistic drug delivery system integrating "colon targeting, active target acquisition, and near-infrared photothermal triggered drug release," ultimately achieving a synergistic anti-tumor effect far exceeding that of single therapies. It not only enables temperature-based "on-off" controlled drug release, significantly improving drug targeting and therapeutic precision, but also reduces the systemic toxicity of chemotherapy at its source, achieving highly efficient and low-toxicity synergistic therapy.

[0088] The above are merely specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite, characterized in that, The method includes: Step 1: Purify the raw attapulgite using an aqueous solution of an inorganic strong acid and deionized water to obtain purified attapulgite; Step 2: The purified attapulgite, gypenosides, inulin and folic acid are mixed according to a preset mass percentage to obtain a mixture. The mixture is then mixed with deionized water to obtain a composite microsphere suspension. Step 3: The composite microsphere suspension is transported to a spray dryer by a peristaltic pump for granulation to obtain primary drug-loaded composite microspheres; Step 4: Use a melt impregnation method to coat lauric acid onto the surface and pores of the primary drug-loaded composite microspheres, and cool the primary drug-loaded composite microspheres coated with lauric acid to room temperature to obtain targeted thermosensitive drug-loaded composite microspheres.

2. The method for preparing targeted temperature-sensitive drug-loaded composite microspheres based on attapulgite according to claim 1, characterized in that, Step one includes: The original attapulgite mineral is crushed and sieved to obtain attapulgite powder within the preset particle size range. The attapulgite powder was activated by an aqueous solution of an inorganic strong acid, and the acid-activated mixture was naturally cooled to room temperature. The cooled mixture was then separated to obtain a solid product. The solid product was repeatedly washed with deionized water until the filtrate was neutral. The washed solid product was then dried, ground, and sieved to obtain purified attapulgite.

3. The method for preparing targeted temperature-sensitive drug-loaded composite microspheres based on attapulgite according to claim 2, characterized in that, The acid activation of the attapulgite powder using an inorganic strong acid aqueous solution includes: The attapulgite powder is mixed with an aqueous solution of an inorganic strong acid, such that the H⁺ concentration of the acid solution is in the range of 1~4 mol / L, and the mixture is stirred continuously for 2~6 hours under heating conditions of 50℃~80℃.

4. The method for preparing targeted temperature-sensitive drug-loaded composite microspheres based on attapulgite according to claim 1, characterized in that, The mixture is composed of the raw materials in the preset mass percentage: Purified attapulgite: 50-85 parts; Gynostemma pentaphyllum saponins: 10-30 parts; Inulin: 10-25 parts; Folic acid: 0.1-1 parts.

5. The method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite according to claim 1, characterized in that, The process of mixing the aforementioned materials with deionized water to obtain a composite microsphere suspension includes: The mixture is added to a container containing deionized water to obtain a suspension. The container containing the suspension is placed in an ultrasonic processor for ultrasonic treatment to obtain a pre-mixed suspension. The initially mixed suspension was transferred to a mechanical stirrer for stirring to obtain a composite microsphere suspension.

6. The method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite according to claim 5, characterized in that, The conditions for ultrasonic treatment are: ultrasonic dispersion for 10 to 30 minutes at a power of 300 to 600W.

7. The method for preparing targeted temperature-sensitive drug-loaded composite microspheres based on attapulgite according to claim 5, characterized in that, The stirring conditions are as follows: stirring continuously at a speed of 500~1000 rpm for 0.5~2 hours.

8. The method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite according to claim 1, characterized in that, Step three includes: The composite microsphere suspension was transported to a spray dryer by a peristaltic pump for granulation to obtain primary drug-loaded composite microspheres; The primary drug-loaded composite microspheres are recovered using a collection device.

9. The method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite according to claim 5, characterized in that, The peristaltic pump has a rotational speed of 1~5 RPM and its feed flow rate can be set to 1~5 mL / min.

10. The method for preparing targeted thermosensitive drug-loaded composite microspheres based on attapulgite according to claim 1, characterized in that, Step four includes: Lauric acid is heated in a constant temperature water bath until it is completely melted into a liquid state, thus obtaining molten lauric acid. The primary drug-loaded composite microspheres are mixed with molten lauric acid at a specific mass ratio, and the mixture is continuously mechanically stirred under constant temperature conditions. The molten lauric acid is adsorbed onto the pores and surface of the primary drug-loaded composite microspheres by impregnation to form a coating layer, thus obtaining a mixture. After coating, the mixture was removed from the water bath and allowed to cool naturally to room temperature to obtain targeted thermosensitive drug-loaded composite microspheres.

Images