A bone-targeted oral micro-nano hydrogel microsphere and its preparation method and application

Abstract

The present invention provides a bone-targeted oral micro-nano hydrogel microsphere, which adopts a click chemistry method to graft deferoxamine, Asp8 peptide and polyethylene glycol onto a POSS nano-platform to obtain organic-inorganic hybrid nanoparticles, and then encapsulate the organic-inorganic hybrid nanoparticles in a pH-responsive hydrogel microsphere formed by alginate and chitosan. The bone-targeted oral micro-nano hydrogel microsphere can realize functions such as oral delivery, intragastric protection, intestinal sustained / controlled release and active targeting of bone tissue, can significantly improve the intestinal absorption rate and bioavailability of deferoxamine drugs, and can activate the HIF-1α / VEGF signaling pathway to promote H-type angiogenesis, while promoting heme oxygenase-1 expression, inhibiting bone absorption, and effectively alleviating bone loss. The micro-nano hydrogel microsphere system provided by the present invention can synergistically promote intrabone vascularization and inhibit bone absorption, and can be well used in the prevention and treatment of postmenopausal osteoporosis.

Description

Technical Field

[0001] The invention belongs to the technical field of drug-loaded hydrogel preparation, and specifically relates to bone-targeted oral micro-nano hydrogel microspheres and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a systemic bone metabolic disease characterized by decreased bone mass, destruction of trabecular microstructure, increased bone brittleness, and susceptibility to fractures. Postmenopausal osteoporosis (PMOP) is caused by estrogen deficiency and is one of the most common bone diseases in women. Its main pathological change is the imbalance between bone vascularization and bone metabolism. PMOP patients can suffer fractures with slight external forces. At the same time, patients with fractures are bedridden for a long time, which can easily cause complications such as deep vein thrombosis, pneumonia, and urinary tract infection, causing great pain to patients.

[0003] Recent studies have shown that there is a specific coupling relationship between bone angiogenesis, especially H-type blood vessels, and bone metabolism, which plays an important role in the development and prevention of postmenopausal osteoporosis. The development of corresponding drugs to regulate bone angiogenesis and bone metabolism has become a new research direction for the treatment of postmenopausal osteoporosis.

[0004] As early as 2006, some scholars proposed that iron overload is one of the risk factors for osteoporosis. Existing studies have shown that iron accumulation and estrogen deficiency have an important impact on postmenopausal osteoporosis.

[0005] Deferoxamine (DFO) is an FDA-approved 3+ Chelating agents are widely used in the clinical treatment of iron overload caused by various reasons. Literature reports that DFO activates the expression of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) and promotes angiogenesis in bones, including H-type blood vessels. Therefore, DFO has broad application prospects in the treatment of postmenopausal osteoporosis.

[0006] Although deferoxamine has shown great potential in the treatment of osteoporosis, it has a short half-life in vivo (only 5 to 15 minutes in rodents), poor drug availability, inconvenient administration, continuous administration time, and high concentrations caused by rapid drug absorption. The current clinical administration method for deferoxamine to treat iron overload is mainly to inject DFO around the wound surface through intravenous or intramuscular injection, but it often requires repeated injections, and there is no consensus on the optimal injection dose and course of treatment. The many adverse reactions caused by these problems have greatly reduced patient compliance and quality of life, and seriously hindered the clinical transformation of DFO.

[0007] Zheng Nina[1] In a study published in 2019 by et al., "Feasibility and Safety Study of Deferoxamine Mesylate as Adjuvant Treatment for Postmenopausal Osteoporosis", the dosage method adopted was: 10% deferoxamine mesylate 25-60 mg / kg continuous intravenous infusion, once a day, generally 1 week as a course of treatment, and the treatment cycle is up to 6 months.

[0008] Yu Chen and Xu Youjia, Department of Orthopedics, The Second Affiliated Hospital of Soochow University [2] A paper published in the 2011 Chinese Medical Association's Sixth National Osteoporosis and Bone Mineral Disease Academic Conference and the 10th Anniversary of the Establishment of the Chinese Medical Association's Osteoporosis and Bone Mineral Disease Branch Paper Collection used deferoxamine treatment on female SD rats 3 months after surgical castration. The treatment method was 3 times a week, 100 mg / kg each time, and the treatment lasted for 12 weeks.

[0009] Ge Yan [3] In the article "The Efficacy of Deferioxamine in Patients with Transfusion-Related Iron Overload and Its Effect on Bone Density" published in 2020, 55 clinical patients were treated with intravenous infusion of deferoxamine mesylate, requiring continuous medication for 5 days, with a 2-day interval, and continuous treatment for up to 3 months.

[0010] Liao Qinghui from the Department of Endocrinology, Longgang District People's Hospital of Shenzhen, and Tan Qin from the Department of Nephrology, Longgang District Traditional Chinese Medicine Hospital of Shenzhen [4] The aim of this study was to explore the effect of deferoxamine mesylate on the treatment of osteoporosis and its effect on the bone metabolism markers of patients. 70 elderly female patients with osteoporosis who visited our hospital were given deferoxamine mesylate by injection. The administration method was continuous subcutaneous infusion via intravenous or infusion pump, 8 to 12 hours each time, once a day, 5 to 7 times a week, and a total course of 12 weeks.

[0011] Although the above studies have shown that deferoxamine has a good therapeutic effect on iron overload and osteoporosis, the existing administration method has problems such as high administration frequency, long injection time, and long treatment cycle. Long-term infusion can cause local skin reactions such as local swelling, infection, and rash, and even lead to growth retardation, bone diseases, and auditory neurotoxicity. These problems greatly limit the clinical transformation of DFO.

[0012] In order to solve the problems of short vascular retention half-life and poor drug availability of local injection of DFO, tissue engineering DFO drug-loaded stents were developed. Many researchers have used different stent materials to achieve DFO loading and drug delivery.

[0013] JiaP et al. [5]DFO was added dropwise to the surface of a porous polylactic-co-glycolic acid (PLGA) scaffold and freeze-dried to synthesize a PLGA / DFO drug-loaded scaffold. The results showed that about 80% of DFO was released within 7 days and was almost completely released within 28 days.

[0014] Yan Y et al. [6] A 3D printed biodegradable scaffold was prepared by surface aminolysis and layer-by-layer assembly technology, which can control the effective release of DFO. The results showed that the DFO-loaded scaffold had a rapid release characteristic within the first 20 hours, and then the release was relatively slow, with a release of nearly 90% in 120 hours.

[0015] Yao Q et al [7] A novel 3D composite scaffold composed of mesoporous silicate nanoparticles (MSNs) and nanofibrous gelatin (GF) was developed to load bone morphogenetic protein 2 (BMP2) and DFO to form a new dual drug delivery system. The results showed that DFO could be continuously released in the scaffold for 10 days, and the release of DFO significantly promoted BMP2-induced osteogenic differentiation.

[0016] Ran Q et al [8] Titanium dioxide nanotubes (TNT) were used as drug nanoreservoirs to load DFO, and then a multilayer covering structure of chitosan (CHI) and gelatin (Gel) was deposited by layer-by-layer assembly (LBL) technology to obtain a TNT-DFO-LBL scaffold. The DFO release rate of the TNT-DFOLBL scaffold within 12 hours was relatively low, about 47.6%, and the release reached about 90% after 7 days.

[0017] Park et al. [9] An injectable thermosensitive hydrogel loaded with DFO nanoparticles was prepared, which greatly prolonged the renal clearance time of the drug after subcutaneous injection. Compared with simple DFO nanoparticles, the half-life was extended by 47 times.

[0018] The above research results show that the effective release of DFO can be controlled by using tissue engineering drug-loaded stents, but the existing stents generally have the characteristics of rapid release in the early stage and slow release in the later stage. They still cannot effectively solve the side effects caused by the rapid release of DFO and the problem of determining a reasonable method of medication. How to achieve long-term, stable and slow release of DFO on stent materials still needs further development.

[0019] Oral administration is convenient, painless and low-cost, and patients have high compliance. However, the complex gastrointestinal physiological environment greatly limits the delivery effect of oral drugs, such as the acidic environment in the stomach, a large number of digestive enzymes, and the mucus / mucosal barrier in the intestine, which leads to drug degradation and inactivation, short intestinal retention time and low bioavailability.

[0020] To improve the therapeutic effect of oral drugs, although researchers have tried many methods for efficient oral delivery, such as nanoparticle carriers, ionic liquids, hydrogel microspheres, microneedle delivery systems, and micro-injectors, most methods are limited to overcoming a single oral barrier, and their bioavailability is still low. TM The results of a phase III clinical trial of oral salmon calcitonin prepared by nasal spray technology for the treatment of postmenopausal osteoporosis showed that oral delivery of salmon calcitonin was more effective in increasing lumbar spine and hip bone density than nasal spray administration, but its bioavailability was only 1%.

[0021] Zhang Ru

[10] In the master's thesis "Preparation, Physicochemical Properties and Cell Viability of Deferoxamine Mesylate Nanoliposomes" published in 2019, deferoxamine mesylate nanoliposomes were prepared, which improved the bioavailability compared with free deferoxamine mesylate. However, the research results further showed that the release of deferoxamine mesylate nanoliposomes in simulated gastric fluid for 12 hours reached 63.5%, and the release in simulated intestinal fluid for 10 hours was 51.5%. Due to the defect of poor gastrointestinal absorption of DFO, its oral bioavailability is still not high even if nanoliposomes are used for administration.

[0022] Although DFO has achieved good therapeutic effects in the treatment of osteoporosis, and people have been working hard to develop new drug carriers to overcome the disadvantages of direct intravenous injection, the existing technology still has the following shortcomings: 1) DFO is poorly absorbed by the gastrointestinal tract, with an absolute bioavailability of less than 2%, and it is difficult to significantly improve its bioavailability through tissue engineering drug delivery; 2) The existing drug carriers have not yet conducted relevant research on how to avoid the toxic and side effects caused by DFO, and no corresponding solutions have been found.

[0023] Therefore, how to develop a new drug delivery system to solve the obvious side effects of DFO during long-term injection; and the low bioavailability of existing controlled-release preparations for oral administration and the inability to avoid drug-induced side effects have become technical problems that need to be solved urgently.

[0024] The references cited are as follows:

[0025] [1] Zheng Nina, Lu Junling, Sheng Hui. Feasibility and safety of deferoxamine mesylate as an adjuvant therapy for postmenopausal osteoporosis[J]. Chinese Journal of Osteoporosis, 2019, 25(7):4.

[0026] [2] Yu Chen, Xu Youjia. Experimental study on the therapeutic effect of deferoxamine on osteoporotic rats after castration [C] / / The 6th National Osteoporosis and Bone Mineral Disease Academic Conference of the Chinese Medical Association and the 10th Anniversary of the Establishment of the Osteoporosis and Bone Mineral Disease Branch of the Chinese Medical Association. 0.

[0027] [3] Ge Yan. Efficacy of deferoxamine in patients with transfusion-related iron overload and its effect on bone density[J]. Heilongjiang Traditional Chinese Medicine, 2020(2):2.

[0028] [4] Liao Qinghui, Tan Qin. Efficacy of deferoxamine mesylate in the treatment of osteoporosis and its effect on patients' bone metabolism indicators[J]. Hainan Medicine, 2016(20):4.

[0029] [5]Peng J, Hao C, Hui K, et al. Deferoxamine released frompoly(lacticco-glycolic acid)promotes healing of osteoporotic bone defect via enhancedangiogenesis and osteogenesis[J].J BiomedMater ResA, 2016,104(10):2515-2527.

[0030] [6] YanY, Chen H, Zhang H, et al. Vascularized 3D printed scaffolds for promoting bone regeneration[J]. Biomaterials, 2019, 190-191:97-110.

[0031] [7]Yao QQ,Liu YX,Selvaratnam B,et al.Mesoporous silicatenanoparticles / 3D nano fi brous scaffold-mediated dual-drug delivery for bonetissue engineering[J].J Control Release,2018,279:69-78.

[0032] [8]Ran QC, Yu YL, Chen WZ, et al. Deferoxamine loaded titaniananotubes substrates regulate osteogenic and angiogenic differentiation ofMSCs via activation of HIF-1αsignaling[J].Mater Sci Eng C Mater BiolAppl,2018,91:44-54.

[0033] [9]Park SH,Kim RS,Stiles WR,et al.Injectable ThermosensitiveHydrogels for a Sustained Release of Iron Nanochelators[J].Adv Sci(Weinh),2022,9(15):e2200872.DOI:10.1002 / advs.202200872.

[0034]

[10] Zhang Ru. Preparation, physicochemical properties and cell viability of deferoxamine mesylate nanoliposomes[D]. Hebei Normal University. Summary of the invention

[0035] The present invention is to solve the above technical problems, thereby providing a bone-targeted oral micro-nano hydrogel microsphere and its preparation method and application. The technical purpose of the present invention is to provide a micro-nano hydrogel microsphere that can be orally administered, which, on the one hand, solves the problem that the existing DFO administration method has many side effects, on the other hand, solves the problem that the existing DFO administration system has low bioavailability and poor gastrointestinal absorption, and thirdly, can further improve the efficacy of DFO in the treatment of postmenopausal osteoporosis.

[0036] In order to achieve the above technical objectives, the present invention adopts the following technical solutions:

[0037] The present invention first provides a bone-targeted oral micro-nano hydrogel microsphere, which adopts a click chemistry method to co-graft deferoxamine, Asp8 peptide and polyethylene glycol onto a POSS nanoplatform to obtain organic-inorganic hybrid nanoparticles, and then encapsulates the organic-inorganic hybrid nanoparticles in pH-responsive hydrogel microspheres formed by alginate and chitosan.

[0038] Furthermore, the polyethylene glycol is polyethylene glycol 400.

[0039] Furthermore, the POSS is a polyhedral oligomeric silsesquioxane.

[0040] It has been verified by the examples that the micro-nano hydrogel microsphere system provided by the present invention can achieve functions such as oral delivery, intragastric protection, intestinal slow / controlled release and active targeting of bone tissue, thereby negatively affecting bone angiogenesis and bone resorption. In vivo imaging results show that oral delivery of the micro-nano hydrogel microspheres can effectively improve the intestinal absorption rate and bioavailability of the DFO drug through intestinal adhesion and bone-targeted delivery. In vivo and in vitro experiments have shown that the micro-nano hydrogel microspheres can activate the HIF-1α / VEGF signaling pathway to promote H-type angiogenesis, while promoting the expression of heme oxygenase-1 (HO-1), inhibiting bone resorption, and effectively alleviating bone loss. Therefore, the micro-nano hydrogel microsphere system provided by the present invention can synergistically promote bone vascularization and inhibit bone resorption, providing a new approach for the prevention and treatment of postmenopausal osteoporosis.

[0041] However, the existing DFO administration method has the problems of high administration frequency, long injection time, and long treatment cycle. The side effects caused by long-term infusion are difficult to solve, such as local swelling, infection, and rash, and even growth retardation, bone diseases, and auditory neurotoxicity. In addition, the administration method using tissue engineering scaffolds to load DFO still has the problems of rapid drug release, poor drug absorption, and low bioavailability. In particular, the scaffold material is easily degraded in gastric juice and intestines, which further leads to the inability of the drug to target the bone lesion site, resulting in low bioavailability of DFO and the inability to achieve oral administration.

[0042] The Asp8 peptide selected in the present invention is an oligopeptide (Asp8) composed of eight repeated aspartic acid sequences, which can specifically bind to highly crystallized hydroxyapatite, thereby targeting the bone resorption surface covered by osteoclasts. As a new type of nanomaterial, POSS has a unique Si-O-Si three-dimensional cage structure, and organic groups with different functions or structures can be modified on the eight arms, so that the original molecule obtains higher thermal stability and stronger deaggregation effect, becoming an ideal platform for constructing intelligent single-molecule organic-inorganic nanocomposite sensing materials. The present invention uses a click chemistry method to graft DFO, Asp8 and polyethylene glycol 400 onto the POSS nanoplatform, synthesizes organic-inorganic hybrid nanoparticles (PDAPNPs), and further uses pH-responsive polymer hydrogel microspheres formed by alginate (Alg) and chitosan (Cs) to encapsulate PDAPNPs, achieving only a small amount of release in simulated gastric acid, and the drug release in simulated small intestinal fluid reaches 80%, which can be well used for oral administration, and can well achieve the effects of oral delivery, gastric protection, and intestinal slow / controlled release.

[0043] The second object of the present invention is to provide a method for preparing the bone-targeted oral micro-nano hydrogel microspheres as described above, comprising the following steps:

[0044] (1) POSS-SH was prepared by hydrolysis and condensation of 3-mercaptopropyltriethoxysilane, and then deferoxamine, Asp8 peptide, and polyethylene glycol were grafted onto POSS-SH by click chemistry to synthesize organic-inorganic hybrid nanoparticles in one pot.

[0045] (2) dissolving the organic-inorganic hybrid nanoparticles obtained in step (1) in a sodium alginate solution, ultrasonically dispersing the nanoparticles into a uniform solution, and then rapidly gelling the sheared microdroplets in a calcium chloride solution by gas microfluidics technology to prepare calcium ion cross-linked calcium alginate microspheres;

[0046] (3) The calcium alginate microspheres cross-linked with calcium ions obtained in step (2) are mixed with a chitosan solution to construct micro-nano hydrogel microspheres with a core-shell structure through electrostatic interaction.

[0047] Furthermore, the molar ratio of POSS-SH, deferoxamine, Asp8 peptide and polyethylene glycol in step (1) is 2:1:1:8.

[0048] Furthermore, the method for synthesizing organic-inorganic hybrid nanoparticles in one pot in step (1) is as follows: POSS-SH prepared by condensation reaction is dissolved together with polyethylene glycol and a photoinitiator in tetrahydrofuran, and pre-reacted under ultraviolet light for 2.5 hours, and then a tetrahydrofuran solution containing deferoxamine, a photoinitiator and an Asp8 peptide is added thereto, and the reaction is continued under ultraviolet light for 8 hours, and the solvent is removed by rotary evaporation to obtain the target product, organic-inorganic hybrid nanoparticles.

[0049] Furthermore, in step (2), the concentration of the sodium alginate solution is 1% w / v, and the concentration of the CaCl2 solution is 100 mmol / L.

[0050] Furthermore, the concentration of the chitosan solution in step (3) is 1% w / v, and the mixed solution is shaken at 37° C. and 100 rpm for 30 min.

[0051] The third object of the present invention is to provide the use of the bone-targeted oral micro-nano hydrogel microspheres as described above, which is to prepare the micro-nano hydrogel microspheres into drugs for regulating vascularization and bone metabolism.

[0052] On the other hand, the micro-nano hydrogel microspheres of the present invention can also be prepared into oral medicines for treating postmenopausal osteoporosis.

[0053] The beneficial effects of the present invention are as follows:

[0054] (1) The present invention has developed a bone-targeted micro / nano hydrogel microsphere system through gas microfluidics and ion crosslinking technology. The micro / nano hydrogel microsphere system can well achieve functions such as oral delivery, gastric protection, intestinal sustained / controlled release, and active targeting of bone tissue.

[0055] (2) The bone-targeted oral micro-nano hydrogel microspheres provided by the present invention release only a small amount of drug in simulated gastric acid, and release up to 80% of the drug in simulated small intestinal fluid, which can greatly improve its bioavailability in the gastrointestinal tract during oral administration.

[0056] (3) The bone-targeted oral micro-nano hydrogel microspheres provided by the present invention have the properties of intestinal adhesion and bone-targeted delivery. In vivo and in vitro experiments have confirmed that the micro-nano hydrogel microspheres can promote H-type angiogenesis by activating the HIF-1α / VEGF pathway, inhibit the expression of osteoclast-related genes by promoting HO-1 expression, thereby negatively affecting intra-bone angiogenesis and bone resorption, and effectively alleviating bone loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Characterization of POSS-SH; (A) POSS-SH 1 HNMR spectrum; (B) FTIR spectrum of POSS-SH.

[0058] Figure 2 Synthesis and characterization results of PDAPNPs.

[0059] Figure 3 The molecular weight of PDAPNPs was determined by MALDI-TOF method.

[0060] Figure 4 FTIR spectrum of PDAPNPs.

[0061] Figure 5 Research on the process of preparing PDAP@Alg / Cs microspheres using gas microfluidics and ion cross-linking technology. Scale bar: 100μm.

[0062] Figure 6 for the synthesis and characterization of microspheres.

[0063] Figure 7 Optical microscopy images of the morphological changes of PDAP@Alg / Cs microspheres in SGF, SIF, and SCF. Scale bar: 200 μm.

[0064] Figure 8 For the biocompatibility and targeting of microspheres.

[0065] Fig. 9 Live / dead staining results for microspheres.

[0066] Fig.10The CCK-8 method was used to detect the effects of different concentrations of PDAPNPs on the activity of HUVECs (n=5).

[0067] Fig.11 In vitro vascularization experiments of mammospheres.

[0068] Fig.12 The CCK-8 method was used to detect the effects of different concentrations of PDAPNPs on the vitality of BMMs (n=5).

[0069] Fig.13 This is an in vitro experiment on the inhibition of bone resorption by microspheres.

[0070] Fig.14 The therapeutic effect of microspheres.

[0071] Fig.15 Representative micro-CT images of cortical bone in the middle of the femur.

[0072] Fig.16 The microspheres have the effects of promoting angiogenesis and inhibiting bone resorption.

[0073] Fig.17 (A) Immunofluorescence staining of NFATc1 in distal femur, scale bars for top and bottom images are 200 μm and 50 μm, respectively; (B) Quantification of mean fluorescence intensity of NFATc1 in different groups (n=6).

[0074] Fig.18 H&E staining of the heart, liver, spleen, lungs and kidneys. DETAILED DESCRIPTION

[0075] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with the embodiments. It is necessary to point out that the following embodiments are only used to explain and illustrate the present invention and are not used to limit the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above invention content still belong to the protection scope of the present invention.

[0076] Example 1

[0077] 1. Experimental Materials

[0078] 3-Mercaptopropyltriethoxysilane, acryloyl chloride, polyethylene glycol (PEG 400), tetrahydrofuran (THF), and potassium bromide (KBr) were purchased from Sinopharm Group (Shanghai, China); all reagents were of analytical grade. Desferrioxamine (DFO) and Asp8 peptide were purchased from Sigma-Aldrich (St. Louis, MO) and Chinapeptide (Shanghai, China), respectively. Caco-2 and HUVECs cells were from the National Authenticated Cell Culture Collection (Shanghai, China). POSS-SH and acryloyl chloride modified polyethylene glycol 400 (PEG-Ac) were synthesized according to the literature (Wei G, Zhang K, GuY, et al. Novel multifunctional nano-hybrid polyhedral oligomeric silsesquioxane-based molecules with high cell permeability: molecular design and application for diagnosis and treatment of tumors [J]. Nanoscale, 2021, 13 (5): 2982-2994. DOI: 10.1039 / d0nr07641d.) and the literature (Wei G, GuY, Lin N, et al. Autonomous Bionanorobots via a Cage-Shaped Silsesquioxane Vehicle for In Vivo Heavy Metal Detoxification [J]. ACS Appl Mater Interfaces, 2022, 14 (25): 29238-29249. DOI: 10.1021 / acsami.2c05736).

[0079] (II) Experimental methods

[0080] 1. Synthesis of organic-inorganic hybrid nanoparticles (PDAPNPs)

[0081] Take a 50.0mL three-necked flask, add 2.032g (0.2mmol) POSS-SH, 0.363mg (0.8mmol) PEG-Ac, 0.015g (0.06mmol) DMPA (dihydroxymethylpropionic acid) and dissolve in 20.0mL tetrahydrofuran, then react at room temperature using a 365nm portable ultraviolet lamp for 2.5h, then add 0.0320g (0.1mmol) DFO-Ac, 0.1200g (0.400mmol) DMPA and 0.0970g (0.1mmol) Asp8-Ac and dissolve in 10.00mL tetrahydrofuran, continue to use ultraviolet lamp to react for 8h, and remove the solvent by rotary evaporation to obtain the target product PDAPNPs with a yield of 91.3%.

[0082] FTIR (KBr): v = 3434 cm -1 (OH); 3350cm -1 ,3279cm -1 (NH); 1710cm -1 (C=O); 1659cm -1 (C=N); 3055cm -1 ,1689cm -1 ,1531cm -1 ,1492cm -1 (C6H6); 1276cm -1 (CO). 1 H NMR (CDCl3, 600MHz, ppm): δ = 0.71 (d, Si-CH2-), 4.19 (s, OH), 2.48 (s, -CH2-S), 1.48 (-S H),1.59(s,-CH2-),6.28,6.95-8.11(d,ArH),3.61(s,CH2-OH),10.83-10.29(d,-NH).

[0083] 2. Characteristics of PDAPNPs

[0084] Bruker AMX-600 NMR spectrometer detection 1 HNMR spectra (Bruker, Switzerland), scanning frequency of 600 MHz, solvents of D2O and DMSO-d6, tetramethylsilane (TMS) as internal standard. Thermo Nicolet 8700 infrared spectrometer was used to detect Fourier transform infrared (FTIR) spectra (Thermo Fisher, USA), samples were pressed into KBr powder, the number of scans was 32, and the test scanning wavelength was 4000-500 cm -1 , resolution 2cm -1. Lambda35 UV absorption spectrometer was used to test the UV-visible absorption spectrum (PerkinElmer, USA), with a wavelength range of 800-200 nm and a step length of 1 nm. LS-55 fluorescence spectrometer was used to test the fluorescence excitation spectrum and emission spectrum (PerkinElmer, USA), and the test solution was placed in a 1 cm quartz cuvette for testing. The morphology of PDAPNPs was observed using a transmission electron microscope (Hitachi, Japan). The molecular weight of PDAP NPs was determined using a MALDI-TOF mass spectrometer (Bruker, USA). Density functional theory (DFT) was used to calculate the HOMO-LUMO energy and band gap of the optimized structure at the B3LYP / 6-31G / LANL2DZ level of Gaussian 03 software. The B3LYP / 6-31g(d) model based on DFT is applicable to the molecular system of C, N, and O atoms.

[0085] 3. Synthesis of hydrogel microspheres

[0086] Hydrogel microspheres were prepared using gas microfluidics technology as follows:

[0087] Sodium alginate solution (1%, w / v) was injected through a coaxial needle, and the other end of the coaxial needle was connected to nitrogen. The shear force generated by the nitrogen flow (1.0L / min) cut it into homogeneous droplets, and the droplets fell into the calcium chloride aqueous solution (CaCl2, 100mmol / L) under the action of gravity. 2+ Cross-link Alg to form the core of the hydrogel microspheres. Collect the Alg hydrogel microspheres, wash them twice with ddH2O, and remove residual ions on the surface. Then soak them in a chitosan aqueous solution (1%, w / v) and shake them at 100rpm for 30 minutes at 37°C. Since the carboxyl group of Alg carries a negative charge and the amino group of Cs carries a positive charge, the two combine to form a water-insoluble polyelectrolyte complex, forming a Cs protective shell on the core surface of the Alg hydrogel microspheres. Collect the Alg / Cs hydrogel microspheres, wash them 3 times with ddH2O, remove excess Cs solution, and store them at 4°C in the dark.

[0088] When preparing PDAP@Alg / Cs, PDAP was dissolved in sodium alginate solution and ultrasonicated for 30 min to form a uniformly dispersed PDAP / Alg mixed solution. The remaining steps were the same.

[0089] 4. Characterization of hydrogel microspheres

[0090] The morphology of the microspheres was observed using an optical microscope (LSM800, ZEISS, Germany), and the diameters of 100 randomly selected microspheres were measured using Image J. Zetasizer Nano-S90 was used to measure the Zeta potential of the microspheres (ZetasizerNano S, Malvern, UK). Scanning electron microscopy was used to observe the surface morphology of freeze-dried microspheres (GeminiSEM 300, ZEISS, Germany). To facilitate the observation of the core-shell structure of the microspheres, we modified chitosan with FITC and alginate with Rho, and observed the core-shell structure of the microspheres under a confocal microscope (Carl Zeiss, Germany).

[0091] 5. Microsphere swelling, release and degradation experiments

[0092] The water absorption of microspheres was measured by immersion method and their swelling behavior was studied. 5ml Add 10 mg of dried PDAP@Alg / Cs microspheres to the EP tube, weigh the weight (M1), then add 2 ml of simulated gastric juice, simulated small intestinal juice and simulated large intestinal juice respectively, shake at 100 rpm at 37 ° C, centrifuge at the corresponding time point, remove the supernatant, absorb the excess liquid with filter paper, and weigh the weight (Mt). Microsphere swelling rate calculation formula:

[0093] Swelling degree(%)=[(Mt-M1) / M0]×100

[0094] Where M0 is the initial mass of the microspheres.

[0095] To evaluate the release behavior of the microspheres, 100 mg of PDAP@Alg / Cs microspheres were immersed in a dialysis bag (molecular weight cutoff 3500 Da) containing 10 ml of SGF (containing 0.1% Tween80), and then the dialysis bag was immersed in 40 mL of the same release buffer, and the dialysis bag was constantly shaken at 100 rpm at 37 ° C. Every 0.5 h, 2 mL of external release buffer was collected, and the same volume of buffer was supplemented. After 2 h, the microspheres were transferred to SIF and SCF in turn, and the buffer was collected every 1 h. The concentration of PDAPNPs in each sample was determined using a UV spectrophotometer (Eppendorf, Germany) to draw the release curve.

[0096] To evaluate the degradation behavior of microspheres, PDAP@Alg / Cs microspheres were added to a 6-well plate, and 2 mL of simulated gastric fluid, simulated small intestinal fluid, and simulated large intestinal fluid were added respectively. The plate was kept at 37°C and oscillated at 100 rpm. The degradation process of the microspheres was observed under an optical microscope.

[0097] 6. Biocompatibility of hydrogel microspheres

[0098] The biocompatibility of PDAP@Alg / Cs microspheres was tested using a live / dead cell staining kit (Proteintech, China) and a Cell counting kit-8 (CCK-8) assay (DOJINDO, Japan). Briefly, sterile Alg / Cs and PDAP@Alg / Cs microspheres were immersed in DMEM medium and the extract was filtered after three days of immersion. Caco-2 cells were plated in 24-well plates (1×10 4 cells / well), and the corresponding leaching solution was added, and the cells were placed in an incubator at 37°C and 5% CO2. After incubation with CalceinAM / PI buffer for 15 min on days 1, 3, and 5, live cells (green) or dead cells (red) were observed under a fluorescence microscope. Caco-2 cells were plated in 96-well plates (1×10 4 cells / well), the corresponding leaching solution was added respectively, the cells were placed in an incubator at 37°C, 5% CO2, and CCK-8 reagent was incubated with the cells for 1 hour on the 1st, 3rd and 5th days, the supernatant was collected and placed in a new 96-well plate, and the absorbance was measured at 450nm with an enzyme reader (Molecular Devices, Japan).

[0099] 7. Cellular uptake studies

[0100] CLSM was used to observe the drug uptake by Caco-2 cells. In brief, Caco-2 cells were inoculated in a 24-well confocal plate. After 48 hours of culture, serum-free DMEM medium containing the same concentration of FITC and FITC-PDAP (40 μM) was added and incubated for 4 hours, with 3 replicates in each group. Then, the cells were washed 3 times with pre-cooled PBS, fixed with 4% paraformaldehyde for 20 minutes, and nuclear stain DAPI (life technologies, USA) was added and incubated for 5 minutes. Finally, Caco-2 cells were observed using CLSM.

[0101] 8. IVIS and tissue distribution

[0102] In order to facilitate the observation of drug distribution in vivo, FITC was used to modify the bone-targeted delivery system PDAPNPs and the non-bone-targeted delivery system PDPNPs. 8-week-old female C57BL / 6 mice were selected as animal models for tissue imaging. In brief, FITC-PDAPNPs (20 mg / kg DFO) were injected into the tail vein, FITC-PDAP NPs (20 mg / kg DFO) were gavaged, and FITC-PDAP@Alg / Cs (20 mg / kg DFO) were gavaged. At 1h, 2h, 4h and 8h, the small intestinal tissues of mice were collected after anesthesia, and the fluorescence signal intensity in the small intestine was observed using IVIS (PerkinElmer, USA). At the same time, FITC-PDP@Alg / Cs (20 mg / kg DFO) and FITC-PDAP@Alg / Cs (20 mg / kg DFO) were gavaged, and the femurs of mice were collected after anesthesia at 1h, 2h, 4h, 8h and 16h, and the hearts, livers, spleens, lungs and kidneys of mice were further collected at 4h to evaluate the distribution of drugs in vivo.

[0103] 9. Extraction of bone marrow-derived monocytes and macrophages

[0104] Primary BMMs were isolated from the long bones of 8-week-old female C57BL / 6 mice. Briefly, cells were isolated from the bone marrow of femurs and tibias and cultured in α-MEM (HyClone, USA) complete medium in 100 mm culture dishes for 24 h. Non-adherent cells were collected and centrifuged before adding α-MEM complete medium containing 30 ng / ml M-CSF (R&D, USA). After 3 days, adherent cells were taken as precursor osteoclasts.

[0105] 10. TRAP staining

[0106] The BMMs extracted above were inoculated into 24-well plates (1×10 5 cells / well), 50 μM PAP or PDAP was added to α-MEM complete medium containing M-CSF (30 ng / ml) and RANKL (100 ng / ml) (R&D Systems, USA) and cultured for 5 days until mature osteoclasts were formed. Then the cells were washed twice with PBS, fixed with 4% PFA for 20 min, and TRAP staining (Servicebio, China) was performed after membrane permeabilization with 0.2% TritonX-100, and the number of multinucleated giant cells with positive TRAP staining and 3 or more nuclei was counted.

[0107] 11. In vitro cell migration assay

[0108] HUVECs overexpressing GFP-HUVECs were seeded in 24-well plates (1×10 5When the cell confluence reached 100%, scratches were made on the monolayer cells with a 200 μL sterile pipette tip, and the floating cells were washed with PBS. The scratches were observed with a fluorescence microscope at 0 h and 24 h of culture, and statistical analysis was performed using Image J software.

[0109] 12. In vitro tube formation assessment

[0110] HUVECs were seeded in 24-well confocal plates (3 × 10 4 cells / well), added DMEM medium containing 200 μM PAP or PDAP, incubated the cells in an incubator for 4 h, fixed with 4% PFA for 20 min, and incubated with phalloidin (Proteintech, China) and DAPI in sequence to stain the cytoskeleton and cell nucleus. The tube formation was observed under a fluorescence microscope, and statistical analysis was performed using Image J software.

[0111] 13. Real-time fluorescence quantitative PCR

[0112] BMMs and HUVECs were seeded in 6-well plates (1×10 5 cells / well) and treated with complete medium containing 50 μM PAP or PDAP. Total RNA was extracted using TRIzol reagent (Invitrogen, USA) and reverse transcribed using PrimeScript reverse transcriptase kit (Takara, Japan) according to the manufacturer's protocol. Real-time PCR detection of cDNA was performed using SYBR Premix ExTaq (Takara, Japan). The primer sequences used are detailed in Table 1.

[0113] Table 1. Primer sequences

[0114]

[0115] 14. Western blot analysis

[0116] BMMs and HUVECs were seeded in 6-well plates (1×10 5cells / well), treated with complete medium containing 50 μM PAP or PDAP, and then lysed on ice for 30 min using RIPA lysis buffer (Servicebio, China) containing protease inhibitors. The supernatant was collected after centrifugation. The collected supernatant was separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane (Millipore, USA). After blocking with 5% BSA (Servicebio, China) at room temperature for 1 h, it was incubated with the following specific primary antibodies at 4°C overnight: anti-HIF-1α (Servicebio, China), anti-VEGF (Servicebio, China), anti-TRAP (Abcam, USA), anti-CTSK (Abcam, USA), anti-NFATc1 (CST, USA), anti-HO-1 (Servicebio, China) and anti-β-actin (Servicebio, China), and then incubated with the corresponding secondary antibodies (Servicebio, China) for 1 h. After chemiluminescence, the blot was detected with a chemiluminescence system, and the optical density value was determined using Image J software.

[0117] 15. Osteoporosis mouse model

[0118] Animal experiments were approved by the Research Ethics Committee of the Second Affiliated Hospital of Soochow University. Thirty 8-week-old female C57BL / 6 mice were selected and randomly divided into 5 groups. After anesthesia, the hair on both sides of the back was cleaned, local disinfection and draping were performed, the skin and muscle tissue were incised, the abdominal cavity was opened to explore the ovaries, and the ovaries were removed after ligation and sutured layer by layer. In the Sham group, only the fat around the bilateral ovaries was removed. All mice were kept in a specific pathogen-free (SPF) environment. After resting for 1 week, the microspheres were immersed in 1% sodium carboxymethylcellulose and given by gavage. The Shan group and OVX group were given the same volume of 1% sodium carboxymethylcellulose (Aladdin, China), and the other groups were given Alg / Cs, PDAP (20 mg / kg DFO) and PDAP@Alg / Cs (20 mg / kg DFO) by gavage, respectively, once a day for 8 consecutive weeks. Mouse blood was collected and centrifuged at 3000 rpm for 20 min at 4°C to collect the supernatant. The bilateral femurs, tibias, heart, liver, spleen, kidney, and small intestine of the mice were collected for further analysis.

[0119] 16. Radiological evaluation

[0120] Micro-CT analysis of mouse femurs to evaluate the anti-osteoporosis effect of PDAP@Alg / Cs microspheres. Mouse femurs were collected and fixed with 4% paraformaldehyde solution, and then placed in a Micro-CT (Skyscan, Belgium) sample detection plate for fixation, scanning and three-dimensional reconstruction (resolution: 9μm, voltage: 50kV, source current: 500mA, rotation step: 0.5°). The regions of interest (ROI) of the cortical bone in the middle femur and the cancellous bone in the distal femur were selected to measure and compare the following bone morphological parameters, including: bone density (BMD, g / cm 3 ), bone volume / tissue volume (BV / TV,%), trabecular separation (Tb.Sp, mm), corticalbone density (Ct.BMD, g / cm 3 ) and cortical thickness (Ct.Th, mm).

[0121] 17. ELISA analysis

[0122] CTX-1 (Cloud-Clone, China) and P1NP (Cloud-Clone, China) ELISA kits were used to detect the levels of CTX-1 and P1NP in serum. All operations were performed according to the manufacturer's instructions.

[0123] 18. Histological sections and immunofluorescence analysis

[0124] The bone specimens were fixed in 4% PFA for 48 h, then transferred to EDTA decalcification solution for shaking decalcification for 4 weeks, and the solution was changed every other day. The specimens were dehydrated, paraffin-embedded, and sliced ​​(5 μm thick) for H&E staining and TRAP staining. For immunofluorescence staining, 5% BSA was used for blocking at room temperature for 30 min, and the specimens were incubated with the following specific primary antibodies at 4°C overnight: anti-EMCN (Santa Cruz, USA), anti-CD31 (Servicebio, China), anti-HIF-1α (Servicebio, China), anti-HO-1 (Servicebio, China), anti-NFATc1 (CST, USA), followed by incubation with the corresponding secondary antibodies (Servicebio, China) at room temperature for 1 h and observed under a fluorescence microscope. The images were analyzed using Image J software.

[0125] 19. Statistical analysis

[0126] GraphPad Prism 9.0 (GraphPad, USA) and SPSS software version 26.0 (IBM, USA) were used for statistical processing and analysis of the data. All data are expressed as mean ± SEM. Student's t test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for analysis of three or more experimental groups. Finally, in the legends of this study, *P<0.05, **P<0.01, ***P<0.001.

[0127] (III) Experimental results

[0128] 1. Synthesis and characterization of PDAPNPs

[0129] POSS-SH was prepared by hydrolysis condensation using 3-mercaptopropyltriethoxysilane as raw material. Its form is colorless, transparent and viscous liquid. 1 H NMR) and Fourier transform infrared (FTIR) spectroscopy showed that its structure was pure ( Figure 1 Then, DFO, Asp8 and PEG 400 were grafted onto POSS-SH via environmentally friendly click chemistry to synthesize bone-targeted organic-inorganic nanoparticles (PDAPNPs) in one pot ( Figure 2 ),pass 1 Its structure was characterized by H NMR, FTIR and MALDI-TOF mass spectrometry. Figure 2 As can be seen from the B-part figure, at 1.5ppm, it is the unreacted thiol peak, and between 6-8ppm is the characteristic peak of the benzene ring. According to MALDI-TOF mass spectrometry analysis, the molecular weight of PDAPNPs is about 2485.2 ( Figure 3 ). Figure 4 The FTIR in the figure can further prove the successful synthesis of PDAPNPs, and its structural stoichiometry can be precisely controlled according to the feed ratio. Transmission electron microscopy (TEM) images show that the prepared PDAPNPs have a uniform spherical morphology with an average particle size of 42nm ( Figure 2 In order to better characterize the tracer effect of the material in vivo, the fluorescence spectrum was quantitatively tested under 365nm ultraviolet light, and it was found that from 0μM to 200μM, as the concentration increased, the fluorescence intensity increased linearly ( Figure 2 Similarly, Figure 2The linear enhancement of UV intensity in the F-part is consistent with the fluorescence intensity. The change trends of UV-Vis spectrum and fluorescence spectrum are highly consistent, which not only indicates that the fluorescent group is successfully grafted onto PDAPNPs, but also that PDAPNPs have excellent fluorescence visualization effect, providing a good basis for tracking nanoparticles in vivo. Based on density functional theory (DFT) calculations, we found that when DFO is modified by POSS, the energy difference between HOMO and LUMO orbitals increases from 3.452 eV ( Figure 2 (G in the middle) is reduced to 2.018eV ( Figure 2 The molecular distance in its geometric chemical structure also increases ( Figure 2 These results suggest that POSS modification can form more stable compounds.

[0130] 2. Synthesis and characterization of PDAP@Alg / Cs

[0131] In this study, sodium alginate hydrogel microspheres were prepared by gas shearing technology in high throughput. The microspheres quickly gelled in calcium chloride solution, and then chitosan ( Figure 5 and Figure 6 Figure A in the middle). It can be seen from an optical microscope that when the flow rate is as high as 20 ml / h, PDAP@Alg hydrogel microspheres and PDAP@Alg / Cs hydrogel microspheres with excellent morphology and uniform particle size can still be prepared. The average particle size of PDAP@Alg / Cs hydrogel microspheres is 255.1±12.0 μm ( Figure 6 The surface morphology of PDAP@Alg and PDAP@Alg / Cs hydrogel microspheres was observed by scanning electron microscopy (SEM). Figure 6 As shown in the middle D figure, the surface of PDAP@Alg hydrogel microspheres is relatively smooth, while the surface of PDAP@Alg / Cs hydrogel microspheres is rough. We labeled alginate and chitosan with rhodamine and fluorescein isothiocyanate, respectively, and under confocal laser scanning microscopy (CLSM), we can observe obvious core-shell structure hydrogel microspheres ( Figure 6 In addition, according to Zeta potential analysis, PDAP@Alg hydrogel microspheres are negatively charged, while PDAP@Alg / Cs hydrogel microspheres are positively charged ( Figure 6 The above results suggest that Cs was successfully encapsulated on the surface of PDAP@Alg microspheres to form hydrogel microspheres with a core-shell structure.

[0132] We simulated the retention time of drugs in the gastrointestinal tract after healthy people took the drugs. In simulated gastric fluid (SGF), the swelling rate of PDAP@Alg / Cs hydrogel microspheres was low, while in simulated small intestinal fluid (SIF) and simulated colon fluid (SCF), the microspheres expanded rapidly ( Figure 6 Since the swelling rate of PDAP@Alg / Cs hydrogel microspheres in acidic environment is extremely low, only 16% of PDAPNPs are released after 2 hours, and gradually released in SIF, reaching 80% after 4 hours ( Figure 6 In SGF, the PDAP@Alg / Cs hydrogel microspheres basically maintained their intact structure, gradually degraded in SIF, and completely degraded in SCF after 24 hours. It can be seen that the PDAP loaded by the Alg / Cs hydrogel microspheres was basically released in the small intestine, and the residual Alg / Cs hydrogel microspheres gradually degraded in the large intestine, and were almost completely degraded after 24 hours ( Figure 7 ).

[0133] 3. Biocompatibility and targeting performance

[0134] We evaluated the biocompatibility of hydrogel microspheres based on qualitative and quantitative methods, and co-cultured Caco-2 cell lines with hydrogel microspheres of different concentrations for 1, 3, and 5 days. Live / dead staining results showed that Alg / Cs, 1% PDAP@Alg / Cs, and 5% PDAP@Alg / Cs hydrogel microspheres had biocompatibility similar to that of the control group, while 25% PDAP@Alg / Cs had a significant inhibitory effect on cells on days 3 and 5 ( Figure 8 A-point chart and Fig. 9 ). The CCK-8 results also showed that 5% PDAP@Alg / Cs hydrogel microspheres still showed good biocompatibility on the 5th day ( Figure 8 (B). The cytotoxicity of 25% PDAP@Alg / Cs hydrogel microspheres may be related to the high drug concentration. Therefore, taking into account both drug concentration and biocompatibility, 5% PDAP@Alg / Cs hydrogel microspheres were used in subsequent experiments in this study.

[0135] Then, we used CLSM to observe the drug uptake by Caco-2 cells. Compared with free FITC, cells co-cultured with FITC-modified PDAPNPs (FITC-PDAPNPs) showed stronger green fluorescence signals at 4 h ( Figure 8(Figures C and D in the middle). Therefore, organic-inorganic nanoparticles can significantly improve the efficiency of drug entry into intestinal cells. In order to further observe the intestinal absorption of PDAP@Alg / Cs in mice, we administered FITC-PDAP@Alg / Cs hydrogel microspheres orally and used the InVivo Imaging System to visualize and track the FITC signal intensity in vivo. Compared with tail vein injection or oral administration of FITC-PDAP, the fluorescence signal intensity distribution in the small intestine of mice administered FITC-PDAP@Alg / Cs hydrogel microspheres was stronger and lasted longer, and could last up to 8h in the intestine ( Figure 8 The above results may be due to the increased intestinal absorption of PDAP and the enhanced retention of PDAPNPs in the intestine. On the other hand, in order to detect the effectiveness of the bone-targeted drug delivery system, the distribution of PDAPNPs in various tissues in mice was monitored by IVIS. The results showed that compared with the PDP@Alg / Cs hydrogel microspheres lacking the bone-targeting peptide Asp8, the fluorescence signal intensity in the femur of mice gavaged with PDAP@Alg / Cs hydrogel microspheres was stronger, reaching a peak at 4 hours, while the fluorescence signal intensity in other organs decreased ( Figure 8 (Fig. F and G).

[0136] 4. Promote vascularization in vitro

[0137] In order to determine whether PDAPNPs have an in vitro pro-angiogenic effect, we used PDAPNPs to intervene in human umbilical vein endothelial cells (HUVECs). Prior to this experiment, we used CCK-8 assay to evaluate the effect of PDAP NPs on HUVECs viability. The results showed that when the concentration of PDAPNPs reached 50 μM, there was no obvious toxicity to HUVECs cells ( Fig.10 ). Subsequently, we used PDAPNPs to intervene in HUVECs to determine whether PDAP NPs have the effect of promoting vascularization in vitro. Subsequently, we used cell migration experiments and tube formation experiments to clarify whether PDAPNPs have the effect of promoting angiogenesis in vitro. First, we synthesized PAPNPs, which lack DFO compared with PAPNPs. After PDAPNPs intervened in HUVECs for 24 hours, the cell migration experiment showed that the PDAPNPs group significantly promoted HUVECs cell migration compared with the PAP NPs group and the control group ( Fig.11 In vitro tube formation experiments also showed that the PDAP group significantly promoted the tube formation of HUVECs compared with the PAP group and the control group ( Fig.11 Similar conclusions were drawn from the analysis of the Number of meshes, Total length, and Number of Junctions using Image J software ( Fig.11 DF sub-figures). HIF-1α and VEGF are important regulatory factors in the process of intraosseous angiogenesis. The mRNA expression levels of Hif-1α and VEGF in the PDAP group were significantly higher than those in the PAP group and the control group, and the mRNA expression levels of Hif-1α and VEGF in the PAP group were higher than those in the control group ( Fig.11 Similar results were obtained for the protein expression of Hif-1α and VEGF ( Fig.11 The above in vitro experiments showed that PDAPNPs promoted angiogenesis by activating the HIF-1α / VEGF signaling pathway ( Fig.11 (L in the figure).

[0138] 5. Inhibit bone resorption in vitro

[0139] In order to exclude the inhibitory effect of PDAPNPs toxicity on osteoclast formation, we used CCK-8 assay to detect the effect of PDAPNPs on BMMs cell viability. The results showed that when the PDAP concentration reached 50 μM, there was no obvious toxicity to BMMs cells ( Fig.12 ). Some genes, including tartrate-resistant acid phosphatase (Trap), cathepsin K (Ctsk), and nuclear factor of activated T cells 1 (Nfatc1), have been found to be involved in the differentiation and formation of osteoclasts. Subsequently, we detected the expression of these genes to determine whether PDAPNPs have an inhibitory effect on bone resorption in vitro. The mRNA expression levels of Acp5, Ctsk, and Nfatc1 in the PDAP group were significantly lower than those in the PAP group and the control group, among which the mRNA expression levels of Acp5, Ctsk, and Nfatc1 in the PAP group were lower than those in the control group ( Fig.13 Similar results were obtained for the protein expression of TRAP, CTSK and NFATC1 ( Fig.13 HO-1 is the rate-limiting enzyme in heme catabolism. Literature reports that DFO inhibits osteoclast formation by promoting HO-1 expression. qPCR and WB confirmed that the expression of HO-1 mRNA and protein in the PDAP group was significantly increased ( Fig.13 In vitro inhibition of osteoclastogenesis was further confirmed. After 30ng / ml M-CSF and 100ng / ml RANKL induced BMMs for 5 days, TRAP staining results showed that there were almost no mature osteoclasts in the PDAP group, and the number of TRAP-positive osteoclasts in the PAP group was reduced by 50% compared with the control group ( Fig.13 The above results indicate that PDAPNPs inhibit osteoclast function and generation by activating HO-1 ( Fig.13 (L in the figure).

[0140] 6. Anti-osteoporosis effect in ovariectomized mice

[0141] Eight-week-old female C57BL / 6 mice were selected and bilateral ovaries were removed to construct a classic postmenopausal osteoporosis model (OVX). All mice were randomly divided into five groups, namely Sham group, OVX group, Alg / Cs group, PDAP group and PDAP@Alg / Cs group. The first two groups were given normal saline by gavage once a day for 8 weeks and then killed for specimen collection ( Fig.14 (center A). Micro-CT and HE staining of the distal femur showed that the trabeculae in the OVX group were significantly lost, indicating that the postmenopausal osteoporosis model was successfully established ( Fig.14 (B and C in the figure). Compared with the OVX group and the PDAP group, the number of trabeculae in the PDAP@Alg / Cs group increased significantly. Bone microarchitecture parameters showed that compared with the control group, the bone mineral density (BMD) and bone volume fraction (BV / TV) in the PDAP group increased, and the number of trabeculae (Tb.Sp) decreased; while the BMD and BV / TV in the PDAP@Alg / Cs group increased more significantly, and Tb.Sp was more significant ( Fig.14 However, PDAP@Alg / Cs did not improve the cortical bone-related parameters ( Fig.15 ). The results of serum ELISA also showed that the CTX-1 content in the serum of the PDAP@Alg / Cs group was significantly decreased, and the P1NP content was significantly increased ( Fig.14 The above results suggest that after oral administration of PDAP@Alg / Cs, PDAPNPs exert a more significant bone protective effect because Alg / Cs protects PDAPNPs from being destroyed in the stomach and promotes absorption in the intestine.

[0142] To further verify the mechanism of improving osteoporosis in vivo, histological sections were used for detection. We used immunofluorescence to detect EMCN and CD31 expression. The number of H-type blood vessels (CD31hiEMCNhi) in the PDAP@Alg / Cs group increased significantly and was distributed in a stripe pattern, while the number of H-type blood vessels (CD31hiEMCNhi) in the OVX group decreased significantly and was distributed in a sparse dot pattern ( Fig.16 In order to verify whether oral administration of PDAP@Alg / Cs can activate the HIF-1α signaling pathway, we used immunofluorescence to detect the expression of HIF-1α. The data showed that, except for the Sham group, the HIF-1α fluorescence intensity in the PDAP@Alg / Cs group was the highest ( Fig.16 TRAP staining showed that the number of osteoclasts and the percentage of bone surface in the PDAP@Alg / Cs group were significantly reduced, almost returning to normal levels ( Fig.16 In the PDAP@Alg / Cs group, the fluorescence intensity of the osteoclast marker NFATc1 was also significantly lower ( Fig.17In addition, HO-1 immunofluorescence was also detected, and the results showed that HO-1 expression was significantly increased in the PDAP@Alg / Cs group ( Fig.16 These results are consistent with the in vitro experimental results.

[0143] To further verify the biocompatibility of PDAP@Alg / Cs after oral administration, HE staining was used to observe the main organs (heart, liver, spleen, kidney and small intestine), and no obvious pathological changes such as necrosis and inflammation were found in the sections of each organ. These results show that oral PDAP@Alg / Cs has good biocompatibility ( Fig.18 ).

Claims

1. A bone-targeted oral micro-nano hydrogel microsphere, characterized in that: The click chemistry method is used to graft deferoxamine, Asp8 peptide and polyethylene glycol onto polyhedral oligomeric silsesquioxane to obtain organic-inorganic hybrid nanoparticles, and then the organic-inorganic hybrid nanoparticles are encapsulated in pH-responsive hydrogel microspheres formed by alginate and chitosan.

2. The bone-targeted oral micro-nano hydrogel microspheres according to claim 1, characterized in that: The polyethylene glycol is polyethylene glycol 400.

3. A method for preparing bone-targeted oral micro-nano hydrogel microspheres as claimed in claim 1 or 2, characterized in that: The following steps are involved: (1) Polyhedral oligomeric silsesquioxane was prepared by hydrolysis and condensation using 3-mercaptopropyltriethoxysilane as the raw material. Then, deferoxamine, Asp8 peptide and polyethylene glycol were grafted onto the polyhedral oligomeric silsesquioxane by click chemistry to synthesize organic-inorganic hybrid nanoparticles in one pot. (2) dissolving the organic-inorganic hybrid nanoparticles obtained in step (1) in a sodium alginate solution, ultrasonically dispersing the nanoparticles into a uniform solution, and then rapidly gelling the sheared microdroplets in a calcium chloride solution by gas microfluidics technology to prepare calcium ion cross-linked calcium alginate microspheres; (3) The calcium ion cross-linked calcium alginate microspheres prepared in step (2) are mixed with chitosan solution to construct micro-nano hydrogel microspheres with a core-shell structure through electrostatic interaction.

4. The preparation method according to claim 3, characterized in that: The molar ratio of the polyhedral oligomeric silsesquioxane, deferoxamine, Asp8 peptide and polyethylene glycol in step (1) is 2:1:1:

8.

5. The preparation method according to claim 3, characterized in that: The method for synthesizing organic-inorganic hybrid nanoparticles in one pot in step (1) is as follows: polyhedral oligomeric silsesquioxane prepared by condensation reaction is dissolved together with polyethylene glycol and a photoinitiator in tetrahydrofuran, and a pre-reaction is carried out under ultraviolet light for 2.5 hours, and then a tetrahydrofuran solution containing deferoxamine, a photoinitiator and Asp8 peptide is added thereto, and the reaction is continued under ultraviolet light for 8 hours, and the solvent is removed by rotary evaporation to obtain the target product, organic-inorganic hybrid nanoparticles.

6. The preparation method according to claim 3, characterized in that: In step (2), the concentration of the sodium alginate solution is 1% w / v, and the concentration of the CaCl2 solution is 100 mmol / L.

7. The preparation method according to claim 3, characterized in that: The concentration of the chitosan solution in step (3) is 1% w / v, and the mixed solution is shaken at 37° C. and 100 rpm for 30 min.

8. Use of the bone-targeted oral micro-nano hydrogel microspheres according to claim 1 or 2 or the bone-targeted oral micro-nano hydrogel microspheres prepared by the method according to any one of claims 3 to 7 in the preparation of drugs for treating postmenopausal osteoporosis.

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