Drug loading system, responsive long-acting drug release apatite type bone cement, and preparation method and application of responsive long-acting drug release apatite type bone cement

Abstract

The invention relates to a drug loading system, responsive long-acting drug release apatite type bone cement as well as a preparation method and application of the responsive long-acting drug release apatite type bone cement. The drug loading system RES (at) LDH comprises a layered double hydroxide carrier LDH and a drug resveratrol RES loaded on the layered double hydroxide carrier LDH through electrostatic adsorption.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical material preparation, and specifically relates to a drug-carrying system, a responsive long-acting drug-releasing apatite-type bone cement, and a preparation method and application thereof. Background Art

[0002] Affected by the aging population and the lifestyle of modern people, the incidence of metabolic bone diseases such as osteoporosis continues to rise and has become an important health issue of global concern. There is an urgent need to develop more effective bone repair and regeneration materials. Among them, calcium phosphate cement (CPC), as a new type of bone repair material, is similar to the composition of natural bone tissue. It has the advantages of injectability, plasticity, good biocompatibility, low curing temperature, porous structure that allows cell growth, and degradability compatible with bone formation. When the solid phase and liquid phase are mixed to form a slurry and injected into the bone defect site, the bone cement solidifies in situ (hydration reaction) and assists in the formation of new bone. In practical applications, it also shows excellent osteoinductive activity. For example, the addition of biphasic calcium phosphate to PMMA can improve the osteogenic effect of bone cement; the PO4 released by CPC 3- It can reduce the expression of RANK, thereby hindering the signal cascade and downregulating osteoclast-targeted genes, inhibiting osteoclast differentiation.

[0003] However, when used to fill bone defects in patients with metabolic bone diseases, higher requirements are placed on the mechanical properties and targeted therapeutic effects of bone cement. Bone cement loaded with a certain drug or a specific bioactive ingredient can deliver the drug preparation directly to the bone defect site, achieving sustained release of local high concentrations. This not only improves the therapeutic effect of the drug, but also achieves precise release of the drug, reducing the side effects of systemic medication. It plays an important role in promoting bone regeneration, inhibiting infection, and reducing complications. For example, the drug loading system composed of teriparatide, a synthetic analog of parathyroid hormone, is loaded into bone cement, which has a direct therapeutic effect on osteoporotic lesions. Vitamin D and calcium agents are loaded into bone cement for sustained release to maintain local drug concentrations, promoting bone mineralization and bone repair.

[0004] In drug-loaded bone cement, drugs can be loaded in various ways, including direct mixing, liquid phase mixing, carrier embedding, additive-assisted, special process, etc. From the advantages and disadvantages of these methods, the carrier embedding method can fully maintain the activity and stability of the drug, control the drug release rate, and avoid the "burst release" phenomenon caused by too fast early release, which will lead to excessive local drug concentration and increase the risk of toxic side effects, while the later release is insufficient and cannot maintain effective therapeutic concentration. Using substances that are responsive to external environmental stimuli as drug carriers can also achieve responsive drug controlled release. Such as pH responsiveness, physical stimulus responsiveness, and physiological signal responsiveness. In this research, the main problems are the precision control of drug release and the biocompatibility and safety of the carrier itself. Affected by the degradation of the carrier and bone cement, the drug release kinetics are unstable. If the carrier is not sensitive enough to the conditioned stimulus, the local drug concentration is too low to have a therapeutic effect, and the drug release triggered by the stimulus is too intense, which will lead to too fast drug release and cannot exert a long-term therapeutic effect. At the same time, the choice of carrier is also very important. In addition to considering the ability to effectively load the drug, it is also necessary to pay attention to the chemical composition of the drug carrier and the adverse effects such as cytotoxicity caused by structural changes during the degradation process. Summary of the invention

[0005] In view of the above technical problems, the object of the present invention is to provide a drug loading system, a responsive long-acting drug-releasing apatite-type bone cement, and a preparation method and application thereof.

[0006] In a first aspect, the present invention provides a drug loading system RES@LDH, wherein the drug loading system RES@LDH comprises: a layered double hydroxide carrier LDH, and a drug resveratrol RES loaded on the layered double hydroxide carrier LDH by electrostatic adsorption.

[0007] Preferably, the layered double hydroxide carrier LDH is a two-dimensional nanosheet composed of a hydroxide layer containing metal cations and anions between the layers, wherein the metal cations include Mg 2+ 、Al 3+ At least one of; The drug-carrying system RES@LDH is a hexagonal nanosheet with a size of 30-200 nm, a thickness of 5-20 nm, preferably 10 nm, and a specific surface area of ​​49.49-50.04 m 2 / g; In the drug-carrying system RES@LDH, the loading amount of the drug resveratrol RES is 10-15%.

[0008] In a second aspect, the present invention provides a method for preparing the above-mentioned drug delivery system RES@LDH, the preparation method comprising the following steps: (1) adding a mixed solution containing MgCl2 and AlCl3 dropwise into a NaOH solution for precipitation reaction, filtering and washing to obtain a precipitated product, and then adding deionized water to the precipitated product for hydrothermal reaction to obtain a layered double hydroxide carrier LDH; (2) The layered double hydroxide carrier LDH aqueous solution is mixed with the resveratrol RES ethanol solution, and the mixture is shaken at room temperature in the dark to allow the drug resveratrol RES to be fully loaded on the layered double hydroxide carrier LDH. The mixture is washed again and dried to obtain the drug loading system RES@LDH.

[0009] Preferably, the mixed solution containing MgCl2 and AlCl3 is prepared by the following process: weighing MgCl2·6H2O and AlCl3·6H2O, adding them to deionized water in turn, and vigorously stirring at room temperature for 10 to 30 minutes to obtain; the molar concentrations of MgCl2·6H2O and AlCl3·6H2O in the mixed solution containing MgCl2 and AlCl3 are 0.30 to 1.00 mol / L and 0.10 to 0.50 mol / L, respectively, preferably 0.7 mol / L and 0.3 mol / L; The molar concentration of the NaOH solution is 0.15 to 0.45 mol / L, preferably 0.45 mol / L.

[0010] Preferably, the precipitation reaction time is no more than 1 hour, preferably 0.5 to 1 hour, more preferably 0.5 hour; The temperature of the hydrothermal reaction is 80-110° C., preferably 100° C., and the reaction time is 12-20 hours, preferably 16 hours.

[0011] Preferably, the concentration ranges of the layered double hydroxide carrier LDH aqueous solution and the resveratrol RES ethanol solution are 0.5-2.0 mg / mL and 0.1-4.0 mg / mL, respectively, preferably 1 mg / mL and 0.25 mg / mL; The shaking time at room temperature and away from light is 12 to 36 hours, preferably 24 hours.

[0012] In the third aspect, the present invention provides a responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH, wherein the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH comprises a liquid phase component and a solid phase component; the liquid phase component comprises deionized water, and the solid phase component comprises the above-mentioned drug loading system RES@LDH, white calcium phosphate WH powder, tetracalcium phosphate TTCP powder and dihydrated calcium phosphate DCPD powder.

[0013] Preferably, the liquid-to-solid ratio of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH is 0.25 to 0.35 mL / g, preferably 0.3 mL / g; The mass ratio of the solid phase components is TTCP large particles: TTCP small particles: DCPD: WH: RES@LDH = 7-13: 2-6: 3.5-4.5: 1.5-2.5: 0.5-2, preferably 10: 3: 4: 2: 1; wherein the particle size of the TTCP small particles is less than 25 microns, and the particle size range of the TTCP large particles is 200-400 microns, preferably 250-400 microns.

[0014] In a fourth aspect, the present invention provides a method for preparing the above-mentioned responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH, the preparation method comprising the following steps: weighing solid phase components and liquid phase components according to the mass ratio of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH, and fully solidifying them through stirring and hydration reaction to obtain the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH.

[0015] In a fifth aspect, the present invention provides an application of the above-mentioned responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH in the preparation of a material with dual effects of repairing and treating bone defects in metabolic bone diseases.

[0016] Beneficial Effects (1) The drug-carrying system provided by the present invention not only has a high drug loading rate and encapsulation rate by loading the anti-oxidative stress drug RES on the drug carrier LDH, and realizes uniform effective loading of the drug, but also has good biocompatibility and degradability, and can be used as one of the solid phase components of drug-loaded calcium phosphate bone cement; (2) The apatite-type bone cement with responsive long-acting drug release provided by the present invention has the characteristics of long-acting responsive drug release and the function of dissolving bioactive ions, so that the bone cement can be injected into the bone defect site in a weakly acidic microenvironment (H + ions) and replenish the Ca required for the bone integration process 2+ Mg 2+ Bioactive ions can achieve long-lasting therapeutic effects and promote bone healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The SEM images and element distribution diagrams of the drug carrier LDH and the drug delivery system RES@LDH in Example 1; Figure 2 , 3 They are the XRD patterns and FTIR spectra of the drug carrier LDH and the drug delivery system RES@LDH in Example 1, respectively; Figure 4-6 Schematic diagrams of average particle size, specific surface area and surface Zeta potential of drug carrier LDH and drug delivery system RES@LDH in Example 1 respectively; Figure 7 The SEM image of the drug-loaded bone cement CPC-WH-RES@LDH in Example 1 and the element surface scanning image of the 2000 times local magnification; Figure 8 The XRD pattern of the drug-loaded bone cement CPC-WH-RES@LDH in Example 1; Fig. 9 The drug release curve of the drug-loaded bone cement CPC-WH-RES@LDH in Example 1, and the drug release amount, release concentration and drug release rate of 500 mg sample in 5 mL PBS are also marked; Fig.10 The topographic images of the interior and surface of the drug-loaded bone cement CPC-WH-RES@LDH after drug release in Example 1; Fig.11 This is a comparison chart of the compressive strength of the bone cement prepared in Example 1 and Comparative Examples 1-3 after curing for 3 days. DETAILED DESCRIPTION

[0018] The present invention is further described below by the following embodiments. It should be understood that the following embodiments are only used to illustrate the present invention, but not to limit the present invention.

[0019] First, the present invention provides a drug-carrying system RES@LDH, wherein the drug-carrying system RES@LDH comprises: a layered double hydroxide carrier LDH, and a drug resveratrol RES loaded on the layered double hydroxide carrier LDH by electrostatic adsorption.

[0020] Wherein, the resveratrol (RES) is a natural plant polyphenol with good antioxidant, anti-inflammatory and anti-cancer properties.

[0021] Wherein, the layered double hydroxide carrier LDH is a two-dimensional nanosheet composed of a hydroxide layer containing metal cations and an interlayer anion; in some embodiments, the metal cation may include Mg 2+ 、Al 3+ Mg / Al-LDH layered double hydroxide has good drug loading performance, does not destroy the ion microenvironment in the osteogenic microenvironment, and has good biocompatibility.

[0022] Layered double hydroxides (LDHs) are a class of two-dimensional nanosheets consisting of hydroxide layers containing metal cations and interlayer anions. -It forms an octahedron and coordinates with the metal cation at the center. - It is also shared by three octahedral units (or three metal ions), presenting a hexagonal nanosheet.

[0023] LDH itself has good biocompatibility, and its elastic modulus (9-35GPa) is similar to that of cortical bone (5-23GPa), which can avoid stress shielding and provide sufficient mechanical support. The bioactive metal ions in the LDH structure play a role in different stages of the bone defect repair process. 2 / g), variable interlayer spacing (0.73-2.28nm) and positive charge and other surface characteristics, it has a strong loading capacity for functional substrates (drugs, bioactive molecules, genes and photosensitizers). The loaded functional substrates can be protected by LDH and remain stable in body fluids, thereby improving the therapeutic efficiency. In addition, the LDH structure has pH-responsive drug release characteristics. With the change of pH, the binding of interlayer anions to drugs is weakened, and the drugs are released from the interlayers.

[0024] At the same time, LDH tends to interact with negatively charged cell surface proteins or bacterial membranes, thereby being internalized and quickly escaping through lysosomes, diffusing into the cytoplasm and mitochondria, which can achieve subcellular organelle targeted drug delivery and enhance the therapeutic effect. In the treatment of tumors and inflammation, a weakly acidic and H2O2-rich microenvironment can accelerate the degradation of LDH and release more drugs, which has the effect of responsive drug delivery.

[0025] In some embodiments, the drug delivery system RES@LDH is a hexagonal nanosheet with a size of 30-200 nm, a thickness of 5-20 nm, preferably 10 nm, and a specific surface area of ​​49.49-50.04 m 2 / g.

[0026] Among them, the morphology of the drug-carrying system RES@LDH is mainly affected by factors such as the crystal structure of the LDH carrier, interlayer interaction and growth mode, and steric hindrance. This structural feature is conducive to the adsorption of drug molecules between the LDH layers and on the surface. In addition, the size and thickness of LDH will affect its drug loading and release performance: LDH with relatively large size can provide a larger surface area and more drug loading sites, thereby having a higher drug loading capacity; while LDH with relatively small size tends to have a relatively large specific surface area, and the contact area between drug molecules and the external environment is also larger, resulting in a relatively fast drug release rate. Moreover, layered double hydroxide nanoparticles with smaller sizes are more likely to pass through various barriers in the body, such as blood vessel walls, cell membranes, etc., so that targeted drug delivery can be better achieved. Smaller particles may also have a longer circulation time in the body, increasing the chance of contact with target cells or tissues, and improving the targeting and therapeutic effects of drugs. In terms of thickness, the interlayer spacing of relatively thin LDH is relatively large, which makes it easier for drug molecules to be inserted into the interlayers to achieve intercalation drug loading. Relatively thick LDH may have a stronger wrapping and protection effect on drug molecules, and the release of drugs is more dependent on the slow degradation of the layer or the diffusion of drug molecules. The release rate is relatively slow and more sustained. Therefore, in order to achieve long-term and responsive drug release behavior, the size and thickness of LDH should not be too large or too small, but should be kept within a uniform range.

[0027] In some embodiments, in the drug loading system RES@LDH, the loading amount of the drug resveratrol RES can be 10-15%.

[0028] The following is an exemplary description of the preparation method of the drug delivery system RES@LDH provided by the present invention. The preparation method may include the following steps: (1) adding a mixed solution containing MgCl2 and AlCl3 dropwise into a NaOH solution for precipitation reaction, filtering, centrifuging and washing with deionized water until the supernatant is neutral to obtain a precipitated product, and then adding deionized water to the precipitated product for hydrothermal reaction to obtain a layered double hydroxide carrier LDH; (2) The layered double hydroxide carrier LDH aqueous solution is mixed with the resveratrol RES ethanol solution, and the mixture is shaken at room temperature in the dark to allow the drug resveratrol RES to be fully loaded on the layered double hydroxide carrier LDH. The mixture is centrifuged again, washed, and dried to obtain the drug loading system RES@LDH.

[0029] In some embodiments, the mixed solution containing MgCl2 and AlCl3 can be prepared by the following process: weigh MgCl2·6H2O and AlCl3·6H2O, add them into deionized water in turn, and stir vigorously at room temperature for 10 to 30 minutes; the molar concentrations of MgCl2·6H2O and AlCl3·6H2O in the mixed solution containing MgCl2 and AlCl3 can be 0.30 to 1.00 mol / L and 0.10 to 0.50 mol / L, respectively, preferably 0.7 mol / L and 0.3 mol / L.

[0030] In some embodiments, the molar concentration of the NaOH solution may be 0.15 to 0.45 mol / L, preferably 0.45 mol / L.

[0031] An appropriate amount of sodium hydroxide can promote the orderly growth of LDH and make the structure more regular. During the reaction, metal ions first react with NaOH to form hydroxide precipitates, which are gradually converted into LDH during subsequent hydrothermal crystallization and other treatments. When the NaOH concentration is too low, the pH value of the reaction system cannot reach the alkaline conditions required to form LDH; too high a NaOH concentration may cause the hydroxide precipitates to form too quickly and disorderly, unable to form a regular layered structure, resulting in agglomeration. The final product may be irregular large particles, or cause excessive exchange or removal of anions between LDH layers, destroying the crystal structure of LDH.

[0032] In some embodiments, the mixed solution containing MgCl2 and AlCl3 can be added dropwise to the NaOH solution at a rate of 5-12 mL / min, and the mixed solution is always stirred vigorously during the mixing process.

[0033] In some embodiments, the precipitation reaction time is no more than 1 hour, preferably 0.5 to 1 hour, and more preferably 0.5 hour. The formation of LDH involves a complex process of multiple steps such as metal hydroxide precipitation, layered structure assembly, and interlayer anion exchange. Sufficient reaction time can ensure the full completion of the entire process, but too long a reaction time will cause the already formed LDH particles to agglomerate or grow, affecting the adsorption properties of their surface; at the same time, too long a reaction time is also likely to induce side reactions to generate impurities, affecting the purity of LDH.

[0034] The hydrothermal reaction can optimize the LDH crystal structure and rearrange the LDH to form a LDH with higher crystallinity and more regular layered structure. In some embodiments, the temperature of the hydrothermal reaction can be 80 to 110° C., preferably 100° C., and the reaction time can be 12 to 20 hours, preferably 16 hours.

[0035] The crystal growth of LDH requires a certain amount of time to complete the orderly arrangement of the structure. If the hydrothermal reaction time is too short, the crystals will not have enough time to fully grow and rearrange, resulting in low crystallinity and imperfect crystal structure; long-term hydrothermal reaction will enhance the interaction between LDH particles and easily cause agglomeration. The agglomerated particles become larger in size and have a smaller specific surface area, which may affect their dispersibility and interaction with other substances in applications; excessive hydrothermal reaction may cause the crystal phase of LDH to change from the desired crystal phase to other unstable or undesirable crystal phases, thereby changing its performance.

[0036] In some embodiments, the concentrations of the layered double hydroxide carrier LDH aqueous solution and the resveratrol RES ethanol solution may be in the range of 0.5-2.0 mg / mL and 0.1-4.0 mg / mL, respectively, preferably 1 mg / mL and 0.25 mg / mL.

[0037] In some embodiments, the shaking time at room temperature and away from light can be 12 to 36 hours, preferably 24 hours. Sufficient shaking time can ensure sufficient adsorption of the drug, while too long shaking time will affect the stability of the carrier.

[0038] In some embodiments, the second centrifugal washing can be washed with anhydrous ethanol for no less than two times; the drying temperature can be 40-60° C., preferably 55° C., and the drying time can be 2-10 hours, preferably 5 hours.

[0039] In addition, the present invention also provides a responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH. The responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH includes a liquid component and a solid component; the liquid component may include deionized water, and the solid component may include the above-mentioned drug-carrying system RES@LDH, white calcium phosphate WH powder, tetracalcium phosphate TTCP powder, and dihydrated calcium hydrogen phosphate DCPD powder (CaHPO4·2H2O).

[0040] The present invention introduces the second most abundant bone mineral in human bone tissue, white calcium phosphate, into the solid phase composition of calcium phosphate bone cement. By utilizing its high compressive strength and fast degradation in physiological environment, the mechanical properties of CPC can be enhanced, and CPC can also be introduced into pores as WH degrades to adjust its degradation rate. The Ca, Mg, and P substances released by WH can also promote the growth of new bones. WH can improve the osteogenic effect of PMMA bone cement, and the Mg released by WH can also promote the growth of new bones. 2+ It can promote the upregulation of nerve growth factor (NGF) and vascular endothelial growth factor (VEGF).

[0041] Therefore, the present invention introduces a drug-carrying system of "active bone mineral" white patite (WH) and magnesium aluminum layered double hydroxide (LDH) loaded with antioxidant drug resveratrol (RES) into the solid phase component of apatite-type bone cement to prepare drug-carrying bone cement. The drug RES is loaded onto the surface of the carrier LDH through hydrogen bonding and electrostatic adsorption, thereby achieving RES on the H + Moreover, by adding the drug-carrying system (RES@LDH) and the Mg-containing bone mineral component white phosphate (WH) into the solid phase raw material, the bone cement can achieve the purpose of long-term responsive drug release and dissolution of bioactive ions.

[0042] Furthermore, the hydration process of the apatite-based bone cement provided by the present invention involves a series of complex chemical reactions, including a dissolution stage, a nucleation stage, a crystal growth stage, and a solidification stage. In the dissolution stage, after the solid phase powder contacts the liquid phase, Ca is released. 2+ and PO4 3- ; Then comes the nucleation stage, when Ca in the solution 2+ and PO4 3- When the concentration reaches the supersaturated state, they begin to recombine to form the crystal nucleus of the hydration product hydroxyapatite; after the crystal nucleus is formed, it enters the crystal growth stage, the ions in the solution continue to deposit on the surface of the crystal nucleus, the HAp crystal gradually grows, and with the growth and interweaving of the crystal, the CPC gradually hardens; the last is the solidification stage, the CPC is completely solidified, the internal structure is continuously optimized, the porosity is reduced, and a solid material with a certain mechanical strength is formed. Therefore, in theory, in the dissolution stage, WH, TTCP and DCPD can all release Ca 2+ and PO4 3- , participate in the hydration reaction of bone cement to form a new calcium phosphate bone cement system.

[0043] The drug-loaded bone cement provided by the present invention not only has the hydrogen ion responsive graded drug controlled release performance for long-term drug administration, but also can continuously release Ca 2+ Mg 2+ Bioactive ions such as these can solve the problem of low drug release rate in existing drug-loaded bone cements and their inability to exert long-term drug release effects as required.

[0044] The present invention incorporates "active bone mineral" white calcium phosphate WH into the solid phase component of bone cement, and utilizes its high compressive strength and fast degradation in a physiological environment to improve the mechanical strength of bone cement. At the same time, more pores can be formed as WH degrades, thereby increasing the degradation rate of bone cement. At the same time, WH can release biologically active ions such as Ca, Mg, and P, which enter the hydration layer on the surface of bone apatite crystals through blood and extracellular fluid and are absorbed, thereby accelerating the bone integration process on the surface of bone cement.

[0045] The present invention uses layered double hydroxide LDH as a drug carrier of bone cement, and its application in patients with metabolic bone diseases has significant advantages, including excellent drug loading and delivery capabilities, multifunctional bioactive properties, promoting the reversal of osteoporosis, improving the interface stability of bone cement, and antibacterial and anti-inflammatory effects. These advantages make LDH a promising new drug carrier material. At the same time, using white calcium phosphate WH with high compressive strength as one of the solid phase components of bone cement has significant advantages in the application of patients with metabolic bone diseases, including promoting angiogenesis, promoting bone differentiation, bioabsorbability, and good biocompatibility and osteoconduction. These advantages make WH the key to improving bone integration behavior and mechanical strength in drug-loaded bone cement.

[0046] As an example, the white calcium phosphate WH powder can be prepared by the following process: add 0.37M Ca(OH)2 and 0.13M Mg(OH)2 solid powders in small amounts and multiple times to 500mL of 85°C deionized water, and stir for at least 1h until the solid powders are evenly dispersed in the water; then, add 500mL of 0.5M H3PO4 dropwise at a rate of 12mL / min, continue stirring for 30min and then cool to room temperature; then, age for 24h, discard the supernatant, wash the precipitate to neutrality, and dry it in an oven at 100°C for 12h; grind and sieve the agglomerated powder after drying to obtain the white calcium phosphate WH powder.

[0047] As an example, the tetracalcium phosphate TTCP powder used can be prepared by the following solid phase reaction process: equimolar CaCO3 and CaHPO4·2H2O are planetarily ball milled for 6 hours, then calcined in air (1450°C, 12h), ground and sieved to obtain the tetracalcium phosphate TTCP powder.

[0048] In some embodiments, the liquid-to-solid ratio (L / S) of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH can be 0.25 to 0.35 mL / g, preferably 0.3 mL / g.

[0049] When the maximum solid volume fraction SVFmax of the fine powder and the coarse powder of the solid phase component of the bone cement is equal, the injectability is optimal, and the loosening effect and the wall effect of CPC during injection can be reduced. In some embodiments, the mass ratio of the solid phase components can be TTCP large particles: TTCP small particles: DCPD: WH: RES@LDH = 7-13: 2-6: 3.5-4.5: 1.5-2.5: 0.5-2, preferably 10: 3: 4: 2: 1; wherein the particle size of the TTCP small particles is less than 25 microns, preferably 788-878 nanometers, and the particle size range of the TTCP large particles is 200-400 microns, preferably 250-400 microns, so that its particle size is much larger than the TTCP small particles, DCPD powder (particle size 744-870 nanometers), WH powder (particle size 762-826 nanometers) and RES@LDH nanosheets.

[0050] The following is an exemplary description of the preparation method of the responsive long-acting drug-releasing apatite-based bone cement CPC-WH-RES@LDH provided by the present invention. The preparation method may include the following steps: The solid phase component and the liquid phase component are weighed according to the mass ratio of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH, and the mixture is fully solidified by stirring and hydrating reaction to obtain the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH.

[0051] The present invention cleverly integrates the anti-oxidative stress drug RES into the drug carrier LDH by designing an innovative drug delivery system, significantly improving the drug loading efficiency and encapsulation effect, and ensuring the uniformity of drug distribution and the effectiveness of release. In addition, the system also exhibits excellent biocompatibility and biodegradability, making it safer and harmless when used in vivo. It is particularly worth mentioning that this drug delivery system can be used as an important component of calcium phosphate bone cement, showing great application potential in the fields of orthopedic repair and drug sustained release, providing a more accurate and efficient solution for treatment.

[0052] Therefore, the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH provided by the present invention can be applied to the preparation of materials with dual effects of repairing and treating bone defects in metabolic bone diseases.

[0053] The following further examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to further illustrate the present invention and cannot be construed as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention all belong to the scope of protection of the present invention. The specific process parameters of the following examples are also only examples within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description of this article, and are not limited to the specific values ​​​​exemplified below. If not specifically stated, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0054] Example 1

[0055] The preparation method of the responsive long-acting drug-releasing apatite-type bone cement CPC-WH-RES@LDH provided in this embodiment comprises the following steps: (1) Preparation of RES@LDH drug delivery system. An aqueous solution (10 mL) containing 7 mmol of MgCl2·6H2O and 3 mmol of AlCl3·6H2O was added dropwise to an aqueous solution (40 mL) containing 18 mmol of NaOH, and stirred vigorously at room temperature for 30 min; then centrifuged and washed until neutral, and 40 mL of deionized water was added to perform a hydrothermal reaction (100°C, 16 h) to obtain a 10 mg / mL layered double hydroxide (LDH) solution; 1 mg / mL of LDH solution was mixed with 0.5 mg / mL of RES ethanol solution, and the mixture was shaken for 24 h to fully load the drug on the carrier. After drug loading, the solution was centrifuged and washed with anhydrous ethanol for at least twice, and then dried in an oven at 55°C for 5 hours to obtain the RES@LDH drug delivery system; (2) Preparation of CPC-WH-RES@LDH drug-loaded apatite calcium phosphate bone cement. The liquid phase is deionized water, the solid phase is RES@LDH drug-loaded system, white calcium phosphate WH small particles, tetracalcium phosphate TTCP powder and dihydrated calcium hydrogen phosphate (CaHPO4·2H2O, DCPD) powder, the liquid-solid ratio (L / S) is 0.3mL / g; the mass ratio of the solid phase components of the bone cement is TTCP large particles: TTCP small particles: DCPD: WH: RES@LDH = 10:3:4:2:1; the particle size of the TTCP small particles is less than 25 microns, and the particle size range of the TTCP large particles is 250-400 microns; the solid phase components are mixed evenly according to the above ratio to obtain a solid phase for bone cement, the solid phase components are mixed evenly according to the ratio, and then the liquid phase is added, and the mixture is stirred thoroughly until it solidifies to obtain the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH.

[0056] Figure 1The SEM images and element distribution diagrams of the drug carrier LDH and the drug delivery system RES@LDH in Example 1. As can be seen from the figure, LDH is a regular hexagonal nanosheet with a thickness of about 10 nanometers, and the Mg, Al, and O elements are evenly distributed; after loading RES, the morphology hardly changes; the distribution of C elements is almost the same as that of Mg, Al, and O elements, indicating that the drug is evenly loaded on LDH.

[0057] Figure 2 , 3 They are the XRD spectra and FTIR spectra of the drug carrier LDH and the drug-carrying system RES@LDH in Example 1. From the XRD spectra, we can see that compared with LDH nanoparticles, the XRD diffraction peak intensity decreases after loading RES; -1 The vibration peaks at 1028 and 1163 cm -1 The vibration peak at is the C-OH stretching vibration peak in RES, indicating the successful encapsulation of the drug.

[0058] Figure 4-6 Schematic diagrams of the average particle size, specific surface area and surface Zeta potential of the drug carrier LDH and the drug delivery system RES@LDH in Example 1. Figure 4 It can be seen that the particle size after loading the drug is slightly increased compared with LDH nanoparticles; Figure 5 The N2-isothermal adsorption-desorption curve shows that the specific surface area of ​​RES@LDH increases from 49.7 m 2 / g increased to 68.2m 2 / g, indicating that RES is loaded on the surface of LDH; from the Zeta potential, it can be seen that the negatively charged RES is loaded by the positively charged LDH through electrostatic adsorption, and the surface charge of the formed RES@LDH drug delivery system is reduced.

[0059] Figure 7 The SEM image and the element surface scanning image of the 2000 times magnified local image of the drug-loaded bone cement CPC-WH-RES@LDH in Example 1. It can be seen from the figure that the internal structure of CPC-WH-RES@LDH is dense, and there are no obvious large pores, indicating that the small particles effectively fill the pores between the large particles, and the microscopic morphology is a regularly staggered lamellar structure, indicating that the hydration reaction is fully carried out and RES@LDH is evenly dispersed in the bone cement.

[0060] Figure 8It is the XRD spectrum of the drug-loaded bone cement CPC-WH-RES@LDH in Example 1. As can be seen from the figure, the phase composition of the drug-loaded bone cement is hydroxyapatite (the corresponding PDF card is No.74-0565), white calcium phosphate (the corresponding PDF card is No.70-2064), calcium phosphate (the corresponding PDF card is No.72-0713) and monetite (the corresponding PDF card is No.70-0360); among them, calcium phosphate and monetite have better degradation characteristics than hydroxyapatite, which can form through macropores after the internal degradation of bone cement to promote the release of drugs and the ingrowth of new bone.

[0061] The present invention tests the responsive drug release behavior of drug-loaded bone cement in the first 28 days in a PBS solution without H2O2 at pH=7, a PBS solution without H2O2 at pH=6, a PBS solution containing H2O2 at pH=7, and a PBS solution containing H2O2 at pH=6; the concentration of H2O2 is 0.3 mmol / L.

[0062] Fig. 9 The drug release curve of the drug-loaded bone cement CPC-WH-RES@LDH in Example 1 is shown, and the drug release amount, release concentration and drug release rate of 500 mg sample in 5 mL PBS are also marked. It can be seen from the figure that the drug can be divided into three stages in the overall drug release process: the first stage is about the first day, which is the rapid release stage of weakly adsorbed drugs on the surface. This is because the rapid release of weakly adsorbed drugs on the RES@LDH on the surface of the bone cement also conforms to the principle of "double the first dose", so that the local area quickly reaches an effective therapeutic concentration. In this stage, it is mainly the responsive drug release of the RES@LDH exposed on the surface of the bone cement. The H + and H2O2 ionization to produce H + The stability of the RES@LDH drug delivery system (the electrostatic adsorption between RES and LDH) was destroyed, and the drug release rate was accelerated. The second stage refers to the degradation of the bone cement body. The drug will be slowly released with the degradation of the bone cement. At the same time, the drug inside the bone cement can also be released faster through the connected pores formed after degradation. At this stage, H + This may be due to the faster degradation of bone cement in a weakly acidic environment. This stage ends at about the 16th day, which is due to the limited total amount of drugs in bone cement. After 16 days, it will enter the third stage. In this stage, the release rate of each group of drugs has a downward trend, but the drug release rate of the effective concentration is still maintained. The drug release cycle is expected to be about three months, and it is expected to achieve long-term therapeutic effects.

[0063] Fig.10The topographic images of the inside and surface of the drug-loaded bone cement CPC-WH-RES@LDH after drug release in Example 1. As can be seen from the figure, compared with the freshly prepared samples, more pore structures are formed inside the bone cement of each group, and the drug-loaded system can be observed. However, a large number of granular microscopic morphologies are formed on the surface due to the flushing of the PBS solution, and no drug-loaded nanoparticles are observed, which also verifies the conjecture about the drug release behavior. On the third day, the drugs on the surface have been completely released, and the drug enters the stage of uniform release as the bone cement degrades.

[0064] Comparative Example 1

[0065] The preparation method of the apatite bone cement CPC-WH-RES@LDH-A provided in this comparative example refers to Example 1, with the main difference being that in step (2), the liquid-to-solid ratio (L / S) is 0.2 mL / g.

[0066] Comparative Example 2

[0067] The preparation method of the apatite bone cement CPC-WH-RES@LDH-B provided in this comparative example refers to Example 1, with the main difference being that in step (2), all TTCP particles are large particles with a particle size range of 250 to 400 microns.

[0068] Comparative Example 3

[0069] The preparation method of apatite bone cement CPC provided in this comparative example refers to Example 1, with the main difference that: in step (2), the solid phase is only TTCP powder and DCPD powder, and the mass ratio of the solid phase components of the bone cement is TTCP large particles: TTCP small particles: DCPD = 10:3:4.

[0070] Fig.11 The compressive strength comparison chart of the bone cement prepared in Example 1 and Comparative Examples 1-3 after curing for 3 days. It can be seen from the figure that compared with CPC, the compressive strength of each drug-loaded bone cement is significantly improved, indicating that WH and the drug-loaded system are beneficial to the improvement of the density of the bone cement structure; among them, the compressive strength of CPC-WH-RES@LDH-A and B is significantly lower than that of CPC-WH-RES@LDH, which indicates that the solid-liquid ratio and the particle ratio of the solid phase component will affect the hydration reaction of the bone cement, and sufficient liquid phase content and the reasonable ratio of coarse powder and fine powder ensure sufficient contact between the solid and liquid phases, which is beneficial to the crystal formation, growth and curing of the bone cement.

[0071] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A drug delivery system RES@LDH, characterized in that: The drug-carrying system RES@LDH comprises: a layered double hydroxide carrier LDH, and a drug resveratrol RES loaded on the layered double hydroxide carrier LDH by electrostatic adsorption.

2. The drug delivery system RES@LDH according to claim 1, characterized in that: The layered double hydroxide carrier LDH is a two-dimensional nanosheet composed of a hydroxide layer containing metal cations and anions between the layers. The metal cations include Mg 2+ 、Al 3+ At least one of; The drug-carrying system RES@LDH is a hexagonal nanosheet with a size of 30-200 nm, a thickness of 5-20 nm, preferably 10 nm, and a specific surface area of ​​49.49-50.04 m 2 / g; In the drug-carrying system RES@LDH, the loading amount of the drug resveratrol RES is 10-15%.

3. A method for preparing the drug delivery system RES@LDH according to claim 1 or 2, characterized in that: The preparation method comprises the following steps: (1) adding a mixed solution containing MgCl2 and AlCl3 dropwise into a NaOH solution for precipitation reaction, filtering and washing to obtain a precipitated product, and then adding deionized water to the precipitated product for hydrothermal reaction to obtain a layered double hydroxide carrier LDH; (2) The layered double hydroxide carrier LDH aqueous solution is mixed with the resveratrol RES ethanol solution, and the mixture is shaken at room temperature in the dark to allow the drug resveratrol RES to be fully loaded on the layered double hydroxide carrier LDH. The mixture is washed again and dried to obtain the drug loading system RES@LDH.

4. The preparation method according to claim 3, characterized in that: The mixed solution containing MgCl2 and AlCl3 is prepared by the following process: weighing MgCl2·6H2O and AlCl3·6H2O, adding them to deionized water in turn, and vigorously stirring at room temperature for 10 to 30 minutes to obtain; the molar concentrations of MgCl2·6H2O and AlCl3·6H2O in the mixed solution containing MgCl2 and AlCl3 are 0.30 to 1.00 mol / L and 0.10 to 0.50 mol / L, respectively, preferably 0.7 mol / L and 0.3 mol / L; The molar concentration of the NaOH solution is 0.15 to 0.45 mol / L, preferably 0.45 mol / L.

5. The preparation method according to claim 3 or 4, characterized in that: The precipitation reaction time is no more than 1 hour, preferably 0.5 to 1 hour, more preferably 0.5 hour; The temperature of the hydrothermal reaction is 80-110° C., preferably 100° C., and the reaction time is 12-20 hours, preferably 16 hours.

6. The preparation method according to any one of claims 3 to 5, characterized in that: The concentration ranges of the layered double hydroxide carrier LDH aqueous solution and the resveratrol RES ethanol solution are 0.5-2.0 mg / mL and 0.1-4.0 mg / mL, respectively, preferably 1 mg / mL and 0.25 mg / mL; The shaking time at room temperature and away from light is 12 to 36 hours, preferably 24 hours.

7. A responsive long-acting drug-releasing apatite-based bone cement CPC-WH-RES@LDH, characterized in that: The responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH includes a liquid phase component and a solid phase component; the liquid phase component includes deionized water, and the solid phase component includes the drug-carrying system RES@LDH according to claim 1 or 2, white calcium phosphate WH powder, tetracalcium phosphate TTCP powder and dihydrated calcium phosphate DCPD powder.

8. The responsive long-acting drug-releasing apatite-based bone cement CPC-WH-RES@LDH according to claim 7, characterized in that: The liquid-to-solid ratio of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH is 0.25-0.35 mL / g, preferably 0.3 mL / g; The mass ratio of the solid phase components is TTCP large particles: TTCP small particles: DCPD: WH: RES@LDH = 7-13: 2-6: 3.5-4.5: 1.5-2.5: 0.5-2, preferably 10: 3: 4: 2: 1; wherein the particle size of the TTCP small particles is less than 25 microns, and the particle size range of the TTCP large particles is 200-400 microns, preferably 250-400 microns.

9. A method for preparing the responsive long-acting drug-releasing apatite-type bone cement CPC-WH-RES@LDH according to claim 7 or 8, characterized in that: The preparation method comprises the following steps: weighing solid phase components and liquid phase components according to the mass ratio of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH according to claim 7 or 8, and fully solidifying them through stirring and hydration reaction to obtain the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH.

10. Use of the responsive long-acting drug-releasing apatite bone cement CPC-WH-RES@LDH according to claim 7 or 8 in the preparation of a material with dual effects of repairing and treating bone defects in metabolic bone diseases.

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