A targeted magnetic nano-composite of palygorskite, a preparation method thereof and application thereof in preparation of an in-vitro medicine controlled-release medicine

Abstract

The application discloses a targeted magnetic nano-composite of palygorskite, a preparation method and application thereof in preparation of an in-vitro drug controlled release medicine. In the application, an anti-tumor drug DOX is loaded through an acid-cleavable acylhydrazone bond, and the drug release rate is lower in a normal physiological environment and higher in a tumor acid environment compared with electrostatic adsorption and pore storage of the DOX with HPal, so that the drug controlled release performance is obviously improved. In addition, loading of magnetic Fe3O4 nanoparticles and grafting of FA enrich the means of transporting the DOX to a designated area by the carrier, that is, the tumor cells can be positioned by external magnetic field control or receptor-ligand interaction targeting. MTT analysis and cell uptake research prove the targeting effect of the FA, and the therapeutic effect of the DOX can be effectively improved.

Description

Technical Field

[0001] This invention relates to a targeted magnetic nanocomposite of palygorskite, its preparation method, and its application in the preparation of controlled-release drugs in vitro, belonging to the field of drug delivery. Background Technology

[0002] Palygorskite (Pal) is a natural silicate mineral with outstanding characteristics such as good chemical stability, large specific surface area, biocompatibility, and high surface activity, thus attracting extensive research in fields such as catalysts, adsorbents, and biomedicine. Pal can be combined with other inorganic nanoparticles or functional molecules to achieve multifunctionality. For example, Zhu et al. designed a Pal nanocomposite (atta@Fe3O4@[Ru(bpy)2(fmp)]Cl2) for dual-modal fluorescence / magnetic resonance imaging [Non-Patent Literature 1]. He et al. synthesized LaF3:Yb using Pal as a support. 3+ ;Tm 3+ / Pal upconversion complexes are used for photocatalytic nitrogen fixation [Non-Patent Literature 2]. Xu et al. designed visible light-sensitized Pal-based lanthanide complexes with high quantum yield, emission design, and long luminescence time, which can be applied to cell imaging and biolabeling [Non-Patent Literature 3]. Wu et al. used Pal as a shape-controlled template to assemble gold nanospheres on their surface through electrostatic force to form rod-shaped gold-Pal nanocomposites for near-infrared photothermal therapy [Non-Patent Literature 4].

[0003] Pallasic acid (Pal) has active hydroxyl groups on its surface, enabling it to bind target molecules through electrostatic interactions and hydrogen bonding. Its pores and interlayer voids provide ample storage space for target molecules, making it commonly used as an adsorbent or additive in composite materials to remove molecules such as dyes, thus finding applications in environmental treatment. For example, Tian et al. prepared a carbon / paleite composite adsorbent using starch as the carbon source via in-situ carbonization, which showed superior decolorization of crude palm oil compared to acid-activated Pallasic acid, and also effectively removed dyes such as methylene blue, methyl violet, and malachite green [Non-Patent Literature 5]. Due to its adsorption properties, Pallasic acid is also considered an ideal drug carrier. However, the weak interaction between Pallasic acid and drug molecules makes it difficult to prevent premature drug release, clearly failing to meet the requirements of a drug carrier and limiting its practical application value.

[0004] According to literature reports, the interaction between nanomaterials and cells is closely related to particle size, shape, and surface chemistry. Compared with cubic, cylindrical, and spherical nanoparticles, rod-shaped nanomaterials have significant advantages in terms of cell internalization rate and intracellular transport [Non-Patent Literature 6 and 7]. Given the abundant domestic reserves of Pal, it is meaningful to design Pal-based nanocomposites with optimized drug release properties.

[0005] Polydopamine (PDA) is a major component of proteins secreted by marine mussels. Under weakly alkaline conditions, it can spontaneously form a robust hydrophilic coating on the surface of various materials. For example, Wang et al. coated MoSe2 with PDA to improve its in vivo / in vitro chemical stability [Non-Patent Literature 8]. Simultaneously, as a biocompatible and biodegradable macromolecule with abundant surface functional amino and catechol groups, PDA can react with amine- or thiol-containing molecules through Michael addition or Schiff base reactions, serving as a secondary reaction platform to enrich material properties.

[0006] To expand the application of PAL in the field of drug delivery, this article aims to use PDA as a secondary reaction platform to provide PAL with abundant active functional groups for further functionalization, and to enhance its application value in drug transport by combining the magnetic targeting effect of iron tetroxide nanoparticles (Fe3O4).

[0007] Considering the pH differences between different microenvironments such as normal tissues (pH 7.0-7.4) and tumor cell intracellular bodies (pH 5.0-6.0), the anti-tumor drug DOX is bound to an acidic, cleavable hydrazone bond (Hz), and then, combined with magnetic guidance and the overexpression of folate receptors in 4T1 cells, DOX is targeted to the tumor region.

[0008] The pH-dependent drug release characteristics of the samples were investigated by simulating normal blood and tumor cell environments with PBS buffers of different pH values. The biocompatibility and targeted recognition properties of the materials were verified through MTT assays and cellular uptake experiments.

[0009] Non-patent document 1: Zhu T, Ma

[0010] Non-patent literature 2: He C, Li X, Chen

[0011] Non-patent Document 3: Xu J, Sun Z, Jia L, et al. Visible light sensitized attapulgite-based lanthanide composites: microstructure, photophysical behaviour and biological application[J]. Dalto Transactions, 2011, 40(48): 12909-12916;

[0012] Non-patent Document 4: Wu P, Deng D, Gao J, et al. Tubelike Gold Sphere-Attapulgite Nanocomposites with a High Photothermal Conversion Ability in the Near-Infrared Region for Enhanced Cancer Photothermal Therapy[J]. ACS Applied Materials&Interfaces[J]. 2016, 8(16): 10243-10252;

[0013] Non-patent Document 5: Tian G, Wang W, Zhu Y, et al. Carbon / Attapulgite Composites as Recycled Palm Oil-Decoloring and Dye Adsorbents[J]. Materials, 2018, 11(1): 86-101;

[0014] Non-patent Document 6: Luo X, Zhang J, Wu Y P, et al. Multifunctional HNT@Fe3O4@PPy@DOX Nanoplatform for Effective Chemo-Photothermal Combination Therapy of Breast Cancer with MR Imaging[J]. ACS Biomaterials Science&Engineering, 2020, 6(6): 3361-3374;

[0015] Non-patent document 7: Gratton SEA, Ropp PA, Pohlhaus PD, et al. The effect of particle design on cellular internalization pathways [J]. Proceedings of the National Academy of Sciences of the United States of America, 2008, 105(33): 11613-11618;

[0016] Non-patent document 8: Wang C, Bai J, Liu Y, et al. Polydopamine Coated SelenideMolybdenum: A New Photothermal Nanocarrier for Highly Effective Chemo-photothermal Synergistic Therapy [J]. ACS Biomaterials Science & Engineering, 2016, 2(11): 2011-2017. Summary of the Invention

[0017] This invention designs an FA-grafted palygorskite@ferric oxide@polydopamine nanocomposite, namely FA@PMPal-Hz-DOX, as a smart drug carrier, loading the antitumor drug DOX via acidic, breakable acylhydrazone bonds. Addressing the low drug loading rate and premature drug leakage caused by the weak interaction between HCl-acidified palygorskite (HPal) and DOX, this invention provides HPal with numerous active sites by adding a PDA coating layer. This facilitates the carboxylation of the nanocomposite by thiomalic acid (MSA), thereby enabling the binding of the antitumor drug DOX via acylhydrazone bonds, significantly improving drug loading and encapsulation efficiency. Simultaneously, it facilitates the grafting of the targeting molecule FA onto the material surface. After loading DOX via acylhydrazone bonds, the cumulative drug release rate of the nanocomposite increases to 70.1% at pH 5.5 and 37°C, while decreasing to 10.1% at pH 7.4 and 37°C. Furthermore, after magnetic functionalization with iron oxide, HPal exhibits a saturation magnetic intensity of 4.25 emu / g, and the poor stability resulting from this magnetic functionalization is mitigated by the PDA coating. In addition, MTT assays and cellular uptake studies evaluated FA@PMPal-Hz-DOX at the cellular level, confirming its promising application prospects in drug delivery.

[0018] The technical solution adopted in this invention is as follows:

[0019] A targeted magnetic nanocomposite of palygorskite has a core-shell structure, with the core being palygorskite (Pal) and Fe3O4 nanoparticles loaded on the surface of the palygorskite, and a polydopamine layer coating the core.

[0020] The polydopamine layer also has the structures shown in formula (1) and / or formula (2), and has the structure shown in formula (3):

[0021]

[0022]

[0023]

[0024] Preferably, the preparation method of the nanocomposite includes the following steps:

[0025] S1: Pallas's palygorskite (Pal) is acidified with hydrochloric acid, and then the acidified Pal is reacted with organic iron salts by a solvothermal method to load Fe3O4 nanoparticles, thus obtaining Pal@Fe3O4, i.e. MPal.

[0026] S2: Disperse MPal in an alkaline buffer solution and add dopamine hydrochloride (DA·HCl). Under alkaline conditions, DA·HCl will coat PDA onto the surface of MPal to obtain MPal@PDA, i.e., PMPal.

[0027] S3: Disperse PMPal in alkaline buffer solution, then add folic acid (FA) and thiomalic acid (MSA) sequentially, stir the reaction, and after washing and magnetic separation, obtain the FA@PMPal-COOH nanocomposite.

[0028] Preferably, in step S1, the hydrochloric acid acidification uses 0.5-2 mol / L hydrochloric acid, and the acidification conditions are 55-65℃ for 2-10 hours; the organic iron salt is acetylacetone iron; and the hot melt method conditions are 200-350℃ for 3-8 hours.

[0029] Preferably, in step S2, the weight ratio of MPal to dopamine hydrochloride is 1:2-8, and the reaction time is 5-20 hours.

[0030] Preferably, in step S3, the weight ratio of folic acid (FA) and thiomalic acid (MSA) and PMal is 15:50-150:10-35, and the reaction time is 5-20 hours.

[0031] The above-mentioned palygorskite-targeted magnetic nanocomposites are used in the preparation of controlled-release drugs in vitro.

[0032] Preferably, the palygorskite-targeted magnetic nanocomposite is bonded to the antitumor drug doxorubicin (DOX) via acidic, cleavable acylhydrazone bonds.

[0033] Preferably, the specific method for bonding the antitumor drug DOX to the palygorskite-targeted magnetic nanocomposite is as follows:

[0034] The FA@PMPal-COOH nanocomposite was dispersed in an acidic buffer solution, and activated with EDC and NHS. After activation, hydrazine hydrate and DOX solution were added, the reaction was stirred, and after washing and magnetic separation, the mixture was freeze-dried to obtain the palygorskite-targeted magnetic nanocomposite FA@PMPal-Hz-DOX bonded to the antitumor drug DOX.

[0035] The mass ratio of FA@PMPal-COOH nanocomposite to EDC and NHS was 20:130-170:60-100, and the reaction time was 31-33 h.

[0036] Preferably, the palygorskite-targeting magnetic nanocomposite FA@PMPal-Hz-DOX, which is bonded to the antitumor drug DOX, targets and localizes tumor cells through magnetic guidance or receptor-ligand interaction.

[0037] A method for improving the stability of Fe3O4 nanoparticle-loaded palygorskite by magnetic enhancement involves coating the surface of Fe3O4 nanoparticle-loaded palygorskite with PDA.

[0038] The beneficial effects of this invention are as follows:

[0039] 1. By acidifying Pal with hydrochloric acid, appropriate acid activation can loosen the pores of Pal, increase its specific surface area, and release more hydroxyl functional groups to facilitate subsequent modification. Fe3O4 nanoparticles can be firmly fixed on the surface of HPal by solvothermal method. Moreover, the preparation of MPal by solvothermal method can not only retain the magnetic properties of Fe3O4, but also avoid the problem of easy agglomeration of Fe3O4 when used alone.

[0040] 2. After PDA is coated on the surface of MPal, the introduction of non-magnetic organic components reduces the magnetization of MPal. However, its abundant functional groups not only improve the stability of the material in solution, but also provide a large number of functional groups for modification, which is convenient for subsequent grafting of FA, MSA, etc.

[0041] 3. The loading of Fe3O4 nanoparticles and the grafting of FA enrich the means of transporting DOX to the designated area, which can be achieved by controlling the external magnetic field or by receptor-ligand interaction to target and locate tumor cells.

[0042] 4. FA@PMPal-Hz-DOX, through its surface carboxylhydrazide hydratylation, can bind DOX via acylhydrazone bonds, effectively improving drug loading efficiency and significantly enhancing encapsulation efficiency. In simulated tumor cell environments, its cumulative drug release rate increased to 70.1%, confirming that FA@PMPal-Hz-DOX has the ability to stably transport drugs and release large quantities of drug molecules under predetermined conditions.

[0043] 5. In the MTT assay, FA@PMPal-Hz-DOX clearly demonstrated the targeting effect of FA, and its cytotoxicity at all concentrations was higher than that of free DOX, confirming the excellent performance of FA@PMPal-Hz-DOX as a drug carrier.

[0044] 6. Cellular uptake studies confirmed that FA@PMPal-Hz-DOX enhanced the uptake behavior of Hep G2 cells, meeting the performance requirements of drug delivery carriers. Attached Figure Description

[0045] Figure 1 A schematic diagram of the synthesis of FA@PMPal-Hz-DOX;

[0046] Figure 2 The reaction mechanism of FA@PMPal-Hz-DOX;

[0047] Figure 3 Infrared spectra of (a) Pal; (b) HPal; (c) MPal; (d) PMPal; (e) PDA; (f) PMPal-COOH; (g) FA@PMPal-COOH; (h) FA@PMPal-Hz-DOX and (i) MSA;

[0048] Figure 4 The ultraviolet spectra of MPal; FA@PMPal-COOH; FA; DOX and FA@PMPal-Hz-DOX;

[0049] Figure 5 XRD patterns of (a) HPal, (b) MPal and (c) PMPal;

[0050] Figure 6 The magnetization curves of MPal and PMPal are shown. The built-in figure is a photograph of MPal(d) and PMPal(a) dispersed in water and their response behavior to an external magnetic field over one minute.

[0051] Figure 7 TEM images of (A)MPal and (B)PMPal;

[0052] Figure 8SEM images of (A)HPal, (B)MPal, and (C)PMPal;

[0053] Figure 9 EDS spectra of (A)MPal and (B)PMPal, and SEM images of (C)MPal and (D)PMPal corresponding to C, O, and Fe elements;

[0054] Figure 10 The cumulative drug release curves for FA@PMPal-Hz-DOX at (a) pH 7.4, 37℃; (b) pH 5.5, 37℃ and HPal at (c) pH 5.5, 37℃; (d) pH 7.4, 37℃ are shown.

[0055] Figure 11 To (a) cell viability of Hep G2 cells exposed to various concentrations of FA@PMPal-COOH; and (b) MTT assay of free DOX and FA@PMPal-Hz-DOX (n=3);

[0056] Figure 12 Fluorescence microscopy images of Hep G2 liver cancer cells incubated with the material. Detailed Implementation

[0057] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0058] Example

[0059] This embodiment describes a method for preparing a targeted magnetic nanocomposite of palygorskite. The nanocomposite is used for the delivery of the antitumor drug DOX. The preparation method of the nanocomposite includes the following steps:

[0060] S1: Preparation of MPal: Pallasia galena (Pal) is acidified with hydrochloric acid, and then Fe3O4 nanoparticles are loaded onto the acidified Pal using a solvothermal method to obtain magnetic properties, resulting in Pal@Fe3O4, i.e., MPal. The specific steps are as follows:

[0061] 1. Preparation of HPal by Pal acidification: Pre-ground Pal was ultrasonically dispersed in a solution with a concentration of 1 mol / L. -1 After being evenly dispersed in hydrochloric acid solution, the mixture was magnetically stirred at 60°C for 4 hours. After cooling to room temperature, the upper yellow liquid was removed by centrifugation for 3 minutes at a speed of 2000 rpm. The mixture was then washed with deionized water until the solution was neutral. The solution was then freeze-dried to obtain acidified white Pal sample powder, i.e., HPal.

[0062] 2. Preparation of MPal by solvothermal method: 0.2g HPal and 1g acetylacetone iron were ultrasonically dispersed in 100mL triethylene glycol. The mixture was mechanically stirred at 270℃ for 6h. After the reaction was completed, the mixture was washed twice with ethanol and deionized water, and MPal was obtained by freeze drying to remove the solution.

[0063] S2: Preparation of PMPal: MPal is ultrasonically dispersed in an alkaline buffer solution, and dopamine hydrochloride (DA·HCl) is added. The PDA is coated onto the MPal surface by the self-polymerization properties of DA·HCl under alkaline conditions, yielding MPal@PDA, i.e., PMPal. The specific method is as follows:

[0064] Take 22 mg of MPal and sonicate it in a 0.01 M Tris-HCl buffer solution with a pH of 8.5. Add 0.1 g of DA·HCl and stir mechanically overnight at room temperature. The solution will gradually change from brown to dark black. After the reaction is complete, remove the upper layer solution by magnetic separation. Then wash the remaining solution with deionized water until the upper layer solution is clear. The final precipitate is PMPal.

[0065] S3: Preparation of FA@PMPal-COOH nanocomposite: PMPal was dispersed in alkaline buffer, and then folic acid (FA) and thiomalic acid (MSA) were added sequentially. The mixture was stirred overnight at room temperature, and then the FA@PMPal-COOH nanocomposite was obtained by washing with deionized water and magnetic separation multiple times. The specific method is as follows:

[0066] The PMPal obtained in step S2 was ultrasonically dispersed in 50 mL of Tris-HCl buffer with pH 8.5 and a concentration of 0.01 M. 15 mg FA and 0.1 g MSA were added sequentially, and the mixture was stirred overnight at room temperature. The FA@PMPal-COOH nanocomposite was obtained by washing with deionized water and magnetic separation multiple times.

[0067] Drug loading

[0068] 20 mg of FA@PMPal-COOH nanocomposite was ultrasonically dispersed in 30 mL of 0.2 M PBS buffer (pH 6.0). Then, 158 mg of EDC and 88 mg of NHS were added to activate the carboxyl functional groups on the material surface. After 15 min, 80 μL of hydrazine hydrate was added and the reaction proceeded for 6 h. Finally, 3 mL of 1.8 mg / mL PBS buffer was added. -1 The DOX solution was placed in the above solution and stirred for 24 hours in the dark at room temperature. Finally, the palygorskite-targeted magnetic nanocomposite FA@PMPal-Hz-DOX bonded to the antitumor drug DOX was obtained by magnetic separation and then washed three times with deionized water and freeze-dried.

[0069] For comparison, the same amount of HPal was used in drug loading experiments under the same conditions described above. To calculate the drug loading and encapsulation efficiency, the deionized water washing solution was collected, and the free DOX content was calculated using UV spectroscopy. The final drug loading and encapsulation efficiency are as follows:

[0070]

[0071]

[0072] In the formula, m0 and m1 are the initial DOX mass and the DOX mass removed by dialysis, respectively, and w0 is the carrier mass.

[0073] Drug controlled release research

[0074] To compare the DOX controlled-release performance of FA@PMPal-Hz-DOX and HPal-DOX, 3.8 mg of sample was ultrasonically dispersed in PBS buffer at pH 5.5 or 7.4, respectively. The solutions were then placed in a thermostatic shaker at 37°C and 150 rpm. At certain time intervals, the PBS supernatant was extracted by centrifugation, and the concentration of released DOX was measured to calculate the cumulative drug release rate. To minimize experimental errors, the drug release was repeated three times.

[0075] In vitro cell experiments

[0076] The cells selected for the experiment were Hep G2 liver cancer cells with high expression of folate receptors, purchased from the American Type Culture Collection (ATCC). Cell culture, cytotoxicity studies, and uptake studies are described below.

[0077] Cell culture

[0078] Hep G2 liver cancer cells were purchased from the American Type Culture Collection (ATCC) and grown in RPMI 1640 medium containing 1% penicillin-streptomycin and 10% fetal bovine serum in a humidified incubator (37°C and 5% CO2).

[0079] Cytotoxicity studies

[0080] MTT assay was performed using Hep G2 hepatocellular carcinoma cells as a model cell line to investigate the cytotoxicity of free DOX and DOX-loaded FA@PMPal-Hz-DOX. Cells were cultured at 2 × 10⁶ cells per well. 5 Cells were seeded at a density in 96-well plates and incubated overnight at 37°C with 5% CO2. Different concentrations of free DOX and DOX-loaded FA@PMPal-Hz-DOX were added to the cells (the calculated drug loading rates ensured that the concentrations of doxorubicin DOX were the same for both groups: 0.5, 1, 2, 4, and 8 μg / mL). -1Incubate for 24 hours. Add MTT (20 μL, 5 mg / mL) to the solution. -1 Add the solution to each well and continue culturing for 4 hours, then discard. Dissolve the generated MTT-formazan crystals in 100 μL DMSO, and record the absorbance of the resulting solution at 570 nm using a microplate reader. To minimize experimental error, the experiment was repeated three times. Furthermore, to confirm the biocompatibility of FA@PMPal-Hz-DOX, the same experiments were performed using FA@PMPal-Hz-DOX at concentrations of 25, 50, 75, 100, and 200 μg mL⁻¹. Cell viability was calculated using the following formula (untreated Hep G2 liver cancer cells in the cell culture medium served as the control group):

[0081]

[0082] Cellular uptake research

[0083] Hep G2 liver cancer cells were seeded in 6-well plates (density: 3 × 10⁶ cells / well). 5 Incubate for 16 hours in FA@PMPal-Hz-DOX or PMPal-Hz-DOX (2 μg / mL) loaded with DOX. -1 Cell treatment. Hep G2 liver cancer cells were fixed with 4% paraformaldehyde for 15 minutes, washed with PBS buffer, and then treated with 1 mL of 2 μg / mL [presumably a specific chemical formula]. -1 Phalloid peptide-FITC, after DPAI staining (1 mg mL) -1 Rinse again with PBS buffer (10 minutes), and observe cell images with a fluorescence microscope.

[0084] Synthesis mechanism

[0085] Synthesis of FA@PMPal-Hz-DOX as follows Figure 1 As shown, Pal increases its specific surface area through hydrochloric acid acidification and loads Fe3O4 nanoparticles via a solvothermal method. This not only retains the magnetic properties of Fe3O4 but also avoids the problem of easy agglomeration when Fe3O4 is used alone. To prevent the detachment of Fe3O4 nanoparticles and enhance the stability of the material in solution, PDA is coated onto the surface of MPal by utilizing the self-polymerization properties of dopamine hydrochloride under alkaline conditions. Due to the abundant functional groups on the PDA surface, MSA and FA can be grafted onto the material surface through a simple stirring process under alkaline conditions. FA is used for targeted delivery of DOX, and MSA provides carboxyl groups to facilitate subsequent DOX bonding via acylhydrazone bonds. The specific reaction mechanism is as follows: Figure 2As shown, under alkaline conditions, the catechol functional groups of the PDA matrix are oxidized to the corresponding quinones, which are then further grafted with MSA (containing a nucleophilic thiol group) and FA (containing a nucleophilic amino group) via a Schiff base reaction / Michael addition reaction. The carboxyl functional group obtained after MSA modification is then hydrazide-substituted, and finally bonded to the antitumor drug DOX via an acylhydrazone bond. The entire reaction process requires no complex reaction conditions or equipment, is simple to operate, and is easy to implement.

[0086] The PMPal nanocomposite was ultrasonically dispersed in 50 mL Tris-HCl buffer, and then 15 mg FA and 0.1 g MSA were added sequentially. After stirring overnight at room temperature, the nanocomposite was obtained by washing with deionized water and magnetic separation multiple times. For subsequent in vitro cell experiments, PMPal-COOH nanocomposite without FA grafting was prepared in the same manner.

[0087] Infrared spectroscopy analysis

[0088] Unprocessed Pal( Figure 3 (Region a) is at 3600-3700cm -1 1654cm -1 1028cm -1 The characteristic peaks at 1440 cm⁻¹ correspond to the stretching vibrations of coordinated water, the bending vibrations of adsorbed water, and the stretching vibrations of Si-O-Si, respectively. The FT-IR spectrum of HPal shows a peak at 1440 cm⁻¹. -1 The carbonate impurity peaks at the location disappeared. Figure 3 The region b indicates that Pal was purified by hydrochloric acid. Appropriate acid activation can loosen the pores of Pal, increase its specific surface area, and release more hydroxyl functional groups to facilitate subsequent modification. Fe3O4 nanoparticles can be firmly immobilized on the HPal surface via a solvothermal method, such as... Figure 3 As shown in region c, the infrared spectrum of MPal is at 585 cm⁻¹. -1 A strong absorption peak is observed at this location, which is attributed to the stretching vibration of the Fe-O group. The FT-IR spectrum of PDA is as follows: Figure 3 As shown in the e-region, these typical absorption peaks of PDA can also be found in... Figure 3 The presence of PDA in the d region confirms that the PDA was successfully coated onto the MPal surface. The infrared spectrum of the MSA ( Figure 3 The middle i region is displayed at 1735cm. -1 The carboxyl stretching vibration peak that appears at this location can also be found in Figure 3 As seen in region f, this confirms that MSA has successfully bonded to the surface of the PDA coating. FA can bond to the PDA coating layer via a Schiff base reaction, such as... Figure 3 The infrared spectrum of FA@PMPal-COOH in the middle g region is shown at 1194 cm⁻¹.-1 The absorption peak at this location corresponds to the CN stretching vibration peak of the aliphatic amine in FA. Therefore, the results indicate that FA has been coupled onto the PDA coating layer. Compared with FA@PMPal-COOH, the infrared spectrum of FA@PMPal-Hz-DOX ( Figure 3 (middle h region) at 1620cm -1 and 1208cm -1 The peak at 1458 cm⁻¹ corresponds to the skeletal vibration peaks of the C=N and NH bonds in the hydrazine bond. -1 The nearby characteristic peaks are the tensile vibration peaks of anthracene in conjugated DOX, and these results confirm that DOX is bonded to the FA@PMPal-COOH surface via acylhydrazone bonds.

[0089] Ultraviolet spectroscopy analysis

[0090] The ultraviolet spectrum of the material, such as Figure 4 As shown, FA exhibits characteristic peaks at 285 nm and 350 nm, while DOX shows characteristic absorption peaks at 233 nm, 250 nm, and 480 nm. In contrast, PMPal shows no obvious absorption peaks at these positions. Compared to PMPal, FA-PMPal-COOH displays characteristic absorption peaks similar to FA, further confirming FA grafting onto the material surface. Furthermore, FA-PMPal-Hz-DOX shows a characteristic peak similar to DOX at 480 nm, also confirming successful DOX loading onto its surface.

[0091] XRD analysis

[0092] like Figure 5 As shown, new peaks appearing at 2θ = 30.2°, 35.5°, 43.0°, 57.1°, and 62.8° in MPal correspond to the (200), (311), (400), (511), and (440) crystal planes of Fe3O4 nanoparticles, respectively, confirming the successful preparation of MPal. When a PDA layer is coated onto the surface of MPal, the peak shape does not change significantly, confirming that this modification process does not affect the crystal structure of the material.

[0093] VSM Analysis

[0094] The magnetic properties of MPal and PMpal were detected by VSM at room temperature. Figure 6The hysteresis regression curves shown indicate that both exhibit superparamagnetism. The saturation magnetization of MPal is 4.25 emu / g, attributed to the presence of Fe3O4 nanoparticles. After PDA layer coating, the saturation magnetization decreases to 2.37 emu / g due to the introduction of non-magnetic organic components. Although the PDA layer coating reduces the magnetization of MPal, its abundant functional groups not only improve the material's stability in solution but also provide a large number of functional groups suitable for subsequent grafting of FA, MSA, etc. Figure 6 The image inside shows MPal and PMPal in PBS buffer (pH=7.4, 0.2M). It is clear that MPal has poor stability and settles quickly, while PMPal exhibits good stability. Furthermore, under the influence of an external magnetic field, PMPal, like MPal, can adsorb onto the container wall within a short time.

[0095] Morphological analysis

[0096] MPal TEM image as follows Figure 7 As shown in region A, Pal exhibits a rod-like structure with black spherical Fe3O4 nanoparticles adhering to its surface, giving Pal magnetic properties. According to literature, under alkaline conditions, dopamine can adhere to the surface of materials with almost any properties. Through simple mechanical stirring, the surface of MPal is coated with a PDA coating, as shown... Figure 7 As shown in region B, a layer of PDA clearly self-polymerized on the MPal surface. SEM images are also used to analyze the morphological characteristics of the material, such as... Figure 8 As shown, the acidified HPal particles are approximately 0.4-1.0 μm long and about 10 nm in diameter. When spherical Fe3O4 nanoparticles are loaded onto the HPal surface, the surface becomes rough. When a PDA coating is applied to the MPal surface, its surface becomes smooth. The PDA coating process can also be verified by EDS analysis, such as... Figure 9 As shown in regions A and B, the C content of MPal is approximately 8.86%, and the Fe content is approximately 18.99%. After the PDA layer is applied, the C content increases to 70.13%, while the relative Fe content decreases to 0.73%. The C, O, and Fe elemental distributions of MPal and PMPal are shown below. Figure 9 As shown in regions C and D, the successful preparation of the material is further confirmed.

[0097] Drug loading and controlled release

[0098] The design purpose of FA@PMPal-Hz-DOX is to optimize the drug delivery performance of PAL (pal). Therefore, its drug loading and encapsulation efficiency were first compared with those of HPal. Equal amounts of FA@PMPal-Hz-DOX and HPal were subjected to the same drug loading process. The final drug loading efficiency of HPal was 13.8%, and its encapsulation efficiency was 53.7%; while that of FA@PMPal-Hz-DOX was 22.1%, and its encapsulation efficiency was 81.9%. HPal mainly loads DOX through electrostatic adsorption and its porous structure, hence its lower drug loading efficiency. FA@PMPal-Hz-DOX, on the other hand, achieves a higher drug loading efficiency through surface carboxylhydrazideization and subsequent acylhydrazone bonding of DOX. Furthermore, we compared the drug release behavior of HPal and FA@PMPal-Hz-DOX under different conditions. Figure 10 Region a represents the DOX release behavior of FA@PMPal-Hz-DOX at pH 7.4 and 37℃, with a cumulative drug release rate of only 10.1% after 30 hours. Acylhydrazone bonds are acidic, easily broken chemical bonds, relatively stable under normal physiological conditions, but become unstable at pH 5.5 and 37℃. Figure 10 In the simulated tumor cell environment (region b), the cumulative drug release rate increased to 70.1%. This significant pH-differential drug release confirms that FA@PMPal-Hz-DOX has the ability to stably transport drugs and release large quantities of drug molecules under predetermined conditions. In contrast, HPal at pH 7.4 and 37°C... Figure 10 The cumulative drug release rate in the middle d region was 34%, while at pH 5.5 and 37℃ ( Figure 10 The percentage of HPal released from the middle c region increased to 48.4%. The difference in HPal drug release under different pH conditions is mainly due to the difference in H+ release under acidic conditions. + Competing with functional groups that can form hydrogen bonds with DOX, and with enhanced DOX solubility under acidic conditions, HPal exhibits a higher release rate at pH 5.5. HPal also shows a significantly higher drug release rate at pH 7.4 compared to acylhydrazone-bonded conditions, primarily because drug molecules within the pores are difficult to stably preserve, and the hydrogen bonds with DOX are weak interactions. This result confirms that HPal is susceptible to loss during transport, limiting its application in drug delivery. In comparison, FA@PMPal-Hz-DOX significantly enhances the performance of HPal in controlled drug release.

[0099] In vitro cytotoxicity assay

[0100] Biocompatibility is a key performance characteristic for materials to be used as drug carriers. For example... Figure 11 As shown in region a, even when the FA@PMPal-COOH concentration increases to 200 μg / mL... -1The cell viability remained at 92%. Pal and PDA have been extensively reported to be biocompatible, therefore the biocompatibility of FA@PMPal-COOH after a series of modifications is also expected. When DOX is bonded to it via an acylhydrazone bond, an MTT assay is performed using DOX and FA@PMPal-Hz-DOX containing the same amount. Figure 11 As shown in region b, FA@PMPal-Hz-DOX clearly demonstrated the targeting effect of FA, and its cytotoxicity at all concentrations was higher than that of free DOX. These experiments confirm the excellent performance of FA@PMPal-Hz-DOX as a drug carrier.

[0101] Cellular uptake research

[0102] like Figure 12 The fluorescence microscopy images of FA@PMPal-Hz-DOX and PMPal-Hz-DOX shown indicate that weak red fluorescence of DOX was observed in Hep G2 cells incubated with untargeted PMPal-Hz-DOX, and even after 6 hours, the enrichment of PMPal-Hz-DOX in Hep G2 cells remained low. In contrast, FA@PMPal-Hz-DOX was highly enriched in Hep G2 cells, and this enrichment increased over time. Even after 3 hours, the red fluorescence intensity was higher than that of the untargeted DOX after 6 hours, confirming that FA@PMPal-Hz-DOX enhanced the uptake behavior of Hep G2 cells, meeting the performance requirements of a drug delivery vehicle.

[0103] in conclusion

[0104] As described above, we designed a pH-responsive drug delivery carrier, FA@PMPal-Hz-DOX, based on pal (parameters) and investigated its drug loading capacity, encapsulation efficiency, controlled drug release performance, biocompatibility, and cellular uptake. Compared to HPal (parameter-dependent anti-tumor drugs), its drug loading capacity and encapsulation efficiency are significantly improved. This study used acidic, cleavable acylhydrazone bonds to load the anti-tumor drug DOX. Compared to HPal's electrostatic adsorption and porosity for DOX storage, the release rate was lower under normal physiological conditions but higher in the acidic environment of tumors, significantly improving its controlled drug release performance. Furthermore, the loading of magnetic Fe3O4 nanoparticles and the grafting of FA enriched the means of transporting DOX to designated regions, enabling targeted localization of tumor cells via external magnetic field control or receptor-ligand interactions. MTT analysis and cellular uptake studies confirmed the targeting effect of FA, which can effectively improve the therapeutic efficacy of DOX. In conclusion, FA@PMPal-Hz-DOX improves the performance of pal and has good application potential in drug delivery.

[0105] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of a palygorskite-targeted magnetic nanocomposite in the preparation of in vitro controlled-release drugs, characterized in that, The targeted magnetic nanocomposite of palygorskite has a core-shell structure, with the core being palygorskite (Pal) and Fe3O4 nanoparticles loaded on the surface of the palygorskite, and a polydopamine layer coating the outside of the core. The polydopamine layer also has the structures shown in formula (1) and / or formula (2), and has the structure shown in formula (3): (1)， (2)， (3)； The targeted magnetic nanocomposite of palygorskite binds the antitumor drug doxorubicin (DOX) via acidic, cleavable acylhydrazone bonds. The specific method is as follows: The FA@PMPal-COOH nanocomposite was dispersed in an acidic buffer solution, and activated with EDC and NHS. After activation, hydrazine hydrate and DOX solution were added, the reaction was stirred, and after washing and magnetic separation, the mixture was freeze-dried to obtain the palygorskite-targeted magnetic nanocomposite FA@PMPal-Hz-DOX bonded to the antitumor drug DOX. The mass ratio of FA@PMPal-COOH nanocomposite to EDC and NHS was 20:130-170:60-100, and the reaction time was 31-33 h. The preparation method of FA@PMPal-COOH nanocomposite includes the following steps: S1: Pallas's palygorskite (Pal) is acidified with hydrochloric acid, and then the acidified Pal is reacted with organic iron salts by a solvothermal method to load Fe3O4 nanoparticles, thus obtaining Pal@Fe3O4, i.e. MPal. S2: Disperse MPal in an alkaline buffer solution and add dopamine hydrochloride (DA·HCl). Under alkaline conditions, DA·HCl will coat PDA onto the surface of MPal to obtain MPal@PDA, i.e., PMPal. S3: Disperse PMPal in alkaline buffer solution, then add folic acid (FA) and thiomalic acid (MSA) sequentially, stir the reaction, and after washing and magnetic separation, obtain the FA@PMPal-COOH nanocomposite.

2. The application according to claim 1, characterized in that, In step S1, the hydrochloric acid acidification uses 0.5-2 mol / L hydrochloric acid, and the acidification conditions are 55-65℃ for 2-10 h; the organic iron salt is acetylacetone iron; the solvothermal method conditions are 200-350℃ for 3-8 h.

3. The application according to claim 1, characterized in that, In step S2, the weight ratio of MPal to dopamine hydrochloride is 1:2-8, and the reaction time is 5-20 hours.

4. The application according to claim 1, characterized in that, In step S3, the weight ratio of folic acid (FA), thiomalic acid (MSA), and PMPal is 15:50-150:10-35, and the reaction time is 5-20 hours.

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