Preparation method and application of flaxseed cyclic peptide bone-targeted nano-delivery system

Abstract

The present invention belongs to the technical field of drug carriers, and discloses a preparation method and application of a flaxseed cyclic peptide bone-targeted nano-delivery system. The present invention prepares nano-sized spherical calcium carbonate particles by an emulsion method, and then selects calcium carbonate particles as nano-carriers of the delivery system, and utilizes its unique porous structure and large specific surface area to load flaxseed cyclic peptide, showing a high drug loading rate. At the same time, the bone-targeted substance selected for the delivery system is asparagine peptide, which has good biocompatibility. The flaxseed cyclic peptide bone-targeted nano-delivery system obtained by the present invention can greatly reduce the biosafety issues such as obvious side effects and systemic toxicity caused by traditional drugs; and has shown significant therapeutic effects on osteoporosis in animal experiments, greatly reversing bone problems such as trabecular loss caused by osteoporosis, and maintaining bone homeostasis.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug carriers, and particularly relates to a preparation method and application of a flaxseed cyclic peptide bone-targeted nano-delivery system. Background Art

[0002] Osteoporosis is a global public health issue facing an aging society. Osteoporosis causes systemic bone loss, generalized pain, height loss, kyphosis, and susceptibility to brittle fractures. The resulting fractures and complications bring significant pain to patients, shorten their lifespan, and severely impact their quality of life. Currently, osteoporosis treatment relies primarily on long-term medication. Commonly used anti-osteoporosis drugs include bisphosphonates, estrogens, calcium supplements, and vitamin D. Bisphosphonates have low oral bioavailability and must be taken alone on an empty stomach. They are commonly associated with gastrointestinal adverse reactions such as nausea, vomiting, abdominal distension, and abdominal pain. Long-term use can cause numerous side effects and even increase the risk of gastrointestinal tumors. Long-term use of synthetic hormones, such as estrogen, increases the risk of breast cancer, uterine hyperplasia, and carcinoma in women, as well as stroke, cardiovascular disease, and cerebrovascular disease. Calcium supplements have low bioavailability, making their therapeutic effect suboptimal even at high doses. Therefore, finding new natural active substances, improving the bioavailability of drugs, reducing side effects and the number of medications, and increasing the targeting and efficacy of drugs are issues that need to be urgently addressed in the clinical treatment of osteoporosis, and are of great significance for improving the quality of life of the elderly.

[0003] Flaxseed cyclopeptide (CLs) is a homocyclic peptide formed by the cyclization of 8 to 9 amino acids with a molecular weight of approximately 960 to 1100 Da. It is present in flax seeds and flax rhizomes and is a lipid accompaniment unique to flax seeds. Kaneda et al. reported that flaxseed cyclopeptide AI can inhibit the differentiation of osteoclasts by inhibiting the expression of the c-fos gene in cells and reducing the expression of the RANKL receptor RANK in M-CSF-induced mouse bone marrow mononuclear macrophages, i.e., osteoclast precursor cells (Kaneda T, Nakajima Y, Koshikawa S, Nugroho AE, Morita H. Cyclolinopeptide F, a cyclic peptide from flaxseed inhibited RANKL-induced osteoclastogenesis via downergulation of RANK expression. J Nat Med. 2019 Jun; 73(3): 504-512.). However, the surface structure of flaxseed cyclic peptides is composed of hydrophobic groups, resulting in poor water solubility and low bioavailability. Therefore, this factor limits its further clinical application. How to solve the solubility problem of flaxseed and improve its bioavailability to better play its role in the treatment and prevention of osteoporosis has great practical significance and broad market prospects.

[0004] Bone tissue is characterized by high hardness, poor permeability, and specialized physiological and biochemical processes. Non-targeted drug delivery routes make it difficult to achieve drug accumulation in bone tissue. Systemic drug delivery for the treatment of bone diseases often suffers from poor absorption by target tissues, necessitating increased drug doses to achieve effective therapeutic concentrations in bone tissue. This not only reduces therapeutic efficacy but also can easily cause severe adverse reactions in non-bone tissues or organs, increasing the risk of potential systemic toxicity and drug-resistant infections, as well as severe side effects such as hepatotoxicity and nephrotoxicity. Nanodrug delivery systems (NDDS) utilize nanoparticles as drug carriers, including nanoliposomes, nanospheres, nanocapsules, solid nanoparticles, and gel nanoparticles. Nanoparticles are easily surface-modified to alter their distribution and achieve targeted delivery. They also offer excellent stability, biocompatibility, and minimal toxic side effects. The small size of NDDS allows them to reach the smallest capillaries. Blood macrophages have limited clearance of nanoparticles, similar to their stealth properties, resulting in a circulation time in the body several times longer than traditional drugs. NDDS also facilitates drug permeation through biological membranes and is suitable for intracellular targeting, making it a novel drug delivery system with excellent performance and broad application prospects. Furthermore, nanoparticles modified with targeting agents for active targeting have great potential for transforming the future of disease treatment. Nanoparticles preferentially aggregate in target tissues and cells, reducing drug toxicity to normal tissues. These, including responsive nanoparticle drug carriers, are promising future development directions, providing new strategies for future disease treatment.

[0005] Therefore, developing a flaxseed cyclic peptide drug that improves flaxseed cyclic peptide absorption, has high bioavailability, has good bone tissue targeting, and can significantly improve osteoporosis has important research significance and application value. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the above-mentioned prior art, the primary purpose of the present invention is to provide a method for preparing a nano-bone-targeted delivery system that is efficient, simple in process, non-toxic and non-irritating, environmentally friendly, biodegradable, biocompatible, and highly stable, which is an expansion of the existing targeted nano-delivery system. The present invention, from the perspective of naturalness, innovation, and health and safety, prepares nano-sized spherical calcium carbonate particles by an emulsion method. The obtained calcium carbonate crystal type is vaterite, and it has high biocompatibility and biodegradability. The preparation process is green, safe, and simple in process, and the calcium carbonate yield is high. Calcium carbonate particles are selected as nano-carriers of the delivery system, and their unique porous structure and large specific surface area are utilized to load drugs, showing a high drug loading rate. At the same time, the bone-targeting substance selected for the delivery system is asparagine peptide, which has good biocompatibility. From the raw materials to the preparation process, a green and environmentally friendly concept is demonstrated, avoiding the potential toxic and harmful risks brought by other chemical substances.

[0007] Another object of the present invention is to overcome the defects of the prior art of flaxseed cyclic peptide, such as poor absorption, rapid elimination, unsuitability for long-term administration and lack of targeting, and to provide a bone-targeted nano-delivery system loaded with flaxseed cyclic peptide to improve the absorption and digestion of flaxseed cyclic peptide in the body. At the same time, this nano-delivery system can greatly reduce the dosage of the drug, significantly improve the bioavailability, extend the dosing time interval, have a certain sustained-release effect and better targeting of bone tissue.

[0008] Another object of the present invention is to provide the aforementioned bone-targeted flaxseed cyclic peptide-loaded nanoparticle delivery system and its use in the preparation of anti-osteoporosis drugs. The flaxseed cyclic peptide bone-targeted nanoparticle delivery system obtained by the present invention can significantly reduce biosafety issues such as significant side effects and systemic toxicity caused by traditional drugs. In vivo animal experiments have demonstrated significant therapeutic effects on osteoporosis, significantly reversing trabecular bone loss and other bone problems caused by osteoporosis, and maintaining bone homeostasis.

[0009] The purpose of the present invention is achieved through the following solutions:

[0010] A method for preparing a novel bone-targeted flaxseed cyclic peptide-loaded nano-delivery system comprises the following steps:

[0011] (1) Preparation of porous calcium carbonate nanoparticles (CA)

[0012] An emulsifier, an oil phase, and a co-emulsifier are weighed and placed in a container, stirred evenly, and then a calcium chloride solution and a sodium carbonate solution are added to the container. The mixture is stirred at room temperature overnight. After the reaction is completed, the mixture is centrifuged. The resulting precipitate is washed with the oil phase and ethanol in sequence, and then dried to obtain porous calcium carbonate nanoparticles.

[0013] (2) Preparation of flaxseed cyclic peptide-loaded calcium carbonate nanoparticles (JCA)

[0014] Weigh the flaxseed cyclic peptide in a container, add the alcohol dispersion containing the porous calcium carbonate nanoparticles of step (1), mix well, shake on a shaker at room temperature for 12 to 48 hours, remove, centrifuge, precipitate and dry to obtain calcium carbonate nanoparticles loaded with the flaxseed cyclic peptide;

[0015] (3) Preparation of bone-targeting modifier (CSD8)

[0016] Carboxymethyl chitosan or hyaluronic acid is weighed and dissolved in water, stirred to fully dissolve, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added, and stirred for activation under nitrogen protection. Subsequently, a bone targeting agent is weighed and dissolved in water. The aqueous solution of the bone targeting agent is added to the activated carboxymethyl chitosan or hyaluronic acid aqueous solution under a nitrogen atmosphere, the pH value is adjusted, and the reaction is stirred at room temperature in the dark with nitrogen. The reacted sample is placed in a dialysis bag, dialyzed with water, and freeze-dried after dialysis to obtain a coupled product of hyaluronic acid and bone targeting agent or carboxymethyl chitosan and bone targeting agent, i.e., a bone-targeted surface modifier.

[0017] (4) Preparation of bone-targeted flaxseed cyclic peptide-loaded nanoparticles (JCA / CSD8)

[0018] The bone-targeted surface modifier of step (3) is weighed and dissolved in water, and fully stirred to dissolve. Dithiothreitol (DTT) is added to the aqueous solution of the bone-targeted surface modifier, and stirred in a nitrogen atmosphere in the dark. The aqueous dispersion of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide of step (2) is weighed and added to the aqueous solution of the bone-targeted surface modifier, and stirred. Finally, oxygen is introduced for cross-linking, followed by centrifugation, and the supernatant is discarded to obtain bone-targeted nanoparticles loaded with flaxseed cyclic peptide.

[0019] The emulsifier described in step (1) is a quaternary ammonium salt type cationic emulsifier, preferably cetyltrimethylammonium bromide (CTAB).

[0020] The oil phase described in step (1) is an oil-soluble organic substance, preferably n-hexane.

[0021] The co-emulsifier described in step (1) is an alcohol-soluble organic substance, preferably n-butanol.

[0022] The stirring in step (1) is preferably carried out by heating to 35-45°C, more preferably by heating to 40°C.

[0023] The concentration of the calcium chloride solution described in step (1) is the same as that of the calcium carbonate solution, and the concentration is 1 to 3 mol / L, preferably 2 mol / L.

[0024] The amounts of the emulsifier, oil phase, co-emulsifier, calcium chloride solution, and sodium carbonate solution described in step (1) satisfy: the amount of calcium chloride solution added accounts for 0.3% to 1.5% of the overall reaction solution, preferably 0.78%; the amount of calcium carbonate solution added accounts for 0.3% to 0.5% of the overall reaction solution, preferably 0.78%; the volume ratio of the oil phase to the aqueous phase is 160:1 to 17:1, preferably 54:1, wherein the volume of the aqueous phase refers to the sum of the volumes of the calcium chloride solution and the sodium carbonate solution; the volume of the co-emulsifier is 17 to 25% of the volume of the oil phase, preferably 18.5%; the amount of the emulsifier satisfies that 1.8 to 2.2 g of emulsifier is included per 27 ml of the oil phase, preferably 2.046 g of emulsifier is included per 27 ml of the oil phase.

[0025] The stirring speed for overnight stirring in step (1) is 700 r / min to 1000 r / min.

[0026] The particle size of the porous calcium carbonate nanoparticles obtained in step (1) is 50 to 70 nm.

[0027] The flaxseed cyclic peptide described in step (2) is at least one of flaxseed cyclic peptide A, flaxseed cyclic peptide B, flaxseed cyclic peptide C, flaxseed cyclic peptide D, flaxseed cyclic peptide E, flaxseed cyclic peptide F, flaxseed cyclic peptide G, flaxseed cyclic peptide H, flaxseed cyclic peptide I, and flaxseed cyclic peptide J, preferably flaxseed cyclic peptide J.

[0028] The mass ratio of the porous calcium carbonate nanoparticles to the flaxseed cyclic peptide in step (2) is 2:1 to 1:4, preferably 1:1;

[0029] The concentration of the alcohol dispersion containing the porous calcium carbonate nanoparticles of step (1) in step (2) is 20-50%, wherein the alcohol is preferably at least one of methanol and ethanol.

[0030] The present invention utilizes the unique porous structure and large specific surface area of ​​spherical porous vaterite calcium carbonate to efficiently load the poorly soluble substance flaxseed cyclic peptide, providing spherical porous vaterite calcium carbonate nanoparticles with a high flaxseed cyclic peptide load. By controlling the composition and particle size of the nanoparticles, the solubility and absorption of the flaxseed cyclic peptide can be improved while also controlling the in vivo process of the loaded flaxseed cyclic peptide, resulting in sustained, controlled, and pH-responsive release. Experiments have shown that the ratio of flaxseed cyclic peptide to calcium carbonate has a certain influence on the loading rate. A higher loading rate can reduce the drug dosage of the nanodelivery system, improving therapeutic efficiency. The present invention controls the drug loading rate to exceed 45%.

[0031] The molar ratio of carboxymethyl chitosan or hyaluronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) described in step (3) is 1:4:4 to 1:6:6, preferably 1:5:5.

[0032] The stirring activation in step (3) is preferably magnetic stirring for 40 min to 90 min;

[0033] The bone targeting substance in step (3) is aspartic acid octapeptide (Asp8);

[0034] In step (3), the molar ratio of the carboxymethyl chitosan or hyaluronic acid to the bone targeting agent Asp8 is 1:1 to 1:3, with the preferred ratio being 1:1.

[0035] The pH value adjustment in step (3) refers to adjusting to pH=7;

[0036] The time for the light-proof nitrogen-filled stirring reaction in step (3) is 5 to 8 hours, preferably 6 hours.

[0037] The molecular weight cut-off of the dialysis bag into which the post-reaction solution in step (3) is loaded is 8000 to 14000 Da.

[0038] The dialysis time in step (3) is 24 hours to 72 hours, preferably 48 hours.

[0039] Bone-targeted drug delivery systems can specifically transport drugs to bone tissue, reducing their distribution and binding in non-bone tissues. The inorganic matrix of bones is basically composed entirely of hydroxyapatite (HAP). Molecules that have a specific affinity for hydroxyapatite, such as bisphosphonates, tetracyclines, acidic oligopeptides, etc., can be used as targets or carriers for bone-targeted drugs, so that the drugs can selectively act on bone tissue, that is, active bone-targeted drug delivery. The principle is: the anionic groups of the bone-targeting molecules bind to the Ca2+ molecules on the surface of HAP. 2+ Binding is through chelation. Generally, high-crystalline hydroxyapatite has a higher concentration of Ca on its surface. 2+ The optimal calcium spacing provides a favorable environment for bone targeting. Furthermore, the bone-targeting agent aspartic acid offers potential advantages over other targeting agents. Amino acids can be enzymatically degraded and cleared from the body after binding to enzymes, demonstrating good biodegradability. Furthermore, to impart long-circulating properties and invisibility or steric stability to nanoparticles, thereby broadening their application, surface modifiers such as carboxymethyl chitosan, hyaluronic acid, or polyethylene glycol are also being used.

[0040] The concentration of the aqueous solution of the bone-targeted surface modifier in step (4) is the same as that of the aqueous dispersion of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide, which is 0.5 mg / mL-2 mg / mL, preferably 1 mg / mL.

[0041] The mass ratio of dithiothreitol added in step (4) to the bone-targeted surface modifier is 8 to 10:1; the mass ratio of the bone-targeted surface modifier to the calcium carbonate nanoparticles loaded with flaxseed cyclic peptide is 2:1 to 1:1, preferably 1:1.

[0042] After adding DTT in step (4), the stirring time in the dark is 18 to 30 hours, preferably 24 hours.

[0043] In step (4), the aqueous dispersion of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide is added to the aqueous solution of the bone-targeted surface modifier and the stirring time is 10 hours to 18 hours, preferably 12 hours.

[0044] The cross-linking time after the introduction of oxygen in step (4) is 1 h to 3 h, preferably 2 h.

[0045] The centrifugal speed in step (4) is 3000 r / min to 5000 r / min, and the centrifugal time is 20 min.

[0046] In the above preparation steps, if no temperature is clearly stated, the steps are carried out at room temperature.

[0047] A bone-targeted flaxseed cyclic peptide-loaded nano-delivery system prepared by the above method.

[0048] The above-mentioned bone-targeted flaxseed cyclic peptide-loaded nano-delivery system and the application of flaxseed cyclic peptide J in the preparation of anti-osteoporosis drugs.

[0049] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0050] (1) This invention, for the first time, uses spherical calcium carbonate nanoparticles prepared by an emulsion method as a carrier to deliver active substances and synergistically promote osteogenesis for the treatment of osteoporosis. The raw materials used in the preparation process have the advantages of being natural and healthy, highly biocompatible and degradable, low-cost, and having strong physiological activity. The successful construction of these bone-targeted nanoparticles addresses the potential problems of significant side effects and systemic toxicity associated with traditional emulsion drugs.

[0051] (2) The present invention is the first to use bone-targeted nanoparticles to efficiently load flaxseed cyclic peptide, improve its solubility and absorption problems, and can control the release of the loaded flaxseed cyclic peptide, with the characteristics of sustained release, controlled release and pH responsiveness.

[0052] (3) The calcium carbonate nanoparticles provided by the present invention can be loaded with a large amount of flaxseed cyclic peptide, controlling the particle size of the calcium carbonate to less than 80 nm. Furthermore, through bone-targeted modification, they have active bone targeting, improving the therapeutic efficiency of the nanoparticles and reducing drug dosage. Furthermore, the nanoparticles, after surface modification with a bone-targeting agent, are approximately 120 nm in size, which facilitates stable transport and long-term circulation in the body, solving the problems of traditional drugs accumulating in non-targeted tissues and requiring large drug dosages.

[0053] (4) The bone-targeted nanoparticles loaded with flaxseed cyclic peptide provided by the present invention have a significant therapeutic effect on osteoporosis model mice, significantly reversing the problems of trabecular bone loss and thinning in the model mice, while showing no significant toxic side effects. This is a new green, environmentally friendly, highly biocompatible, and therapeutically effective nano-delivery system. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a diagram showing the cytotoxicity of the flaxseed cyclic peptide J of the present invention.

[0055] Figure 2 represents the expression levels of different genes in the MC3T3-E1 cells of the present invention.

[0056] Figure 3 This is a schematic diagram of the process of Example 2 of the present invention.

[0057] Figure 4 Fourier transform infrared spectra of different nanoparticles in Example 2

[0058] Figure 5 This is a scanning electron microscope image of the calcium carbonate nanoparticles prepared in Example 2.

[0059] Figure 6 TEM images of different nanoparticles prepared in Example 2.

[0060] Figure 7 These are the in vitro drug release curves of different nanoparticles prepared in Example 2, A is the release curve of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide, and B is the release curve of bone-targeted flaxseed cyclic peptide-loaded nanoparticles.

[0061] Figure 8 These are femoral scans of the animals in the different dosing groups prepared in Example 2. DETAILED DESCRIPTION

[0062] The present invention will be described in further detail below with reference to the Examples and accompanying drawings, but the embodiments of the present invention are not limited thereto. Where specific conditions are not specified in the Examples, conventional conditions or conditions recommended by the manufacturer were followed. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0063] The present invention aims to prepare a novel flaxseed cyclic peptide-loaded bone-targeted nano-delivery system and explore its application. Natural flaxseed cyclic peptide, vaterite nano-calcium carbonate, carboxymethyl chitosan, and aspartic acid are used as raw materials. The preparation process and application evaluation of the novel flaxseed cyclic peptide bone-targeted nano-delivery system are studied in the following examples.

[0064] The structural formula of the flaxseed cyclic peptide J in the embodiment is shown below:

[0065]

[0066] Example 1

[0067] The present invention weighed different concentrations of flaxseed cyclic peptide J (CLJ), namely 5 μM, 10 μM and 20 μM, to explore its effect on promoting osteogenic differentiation.

[0068] (1) MC3T3-E1 cells were seeded in a 96-well plate (1 × 104 cells / well) and cultured with 10% fetal bovine serum (FBS) and minimal essential medium (α-MEM) for 24 h. After cell attachment, different concentrations of CLJ were added to the 96-well plate and incubated for 48 h. Then, 10 μL of CCK8 solution was added to each well and incubated for 2 h. Finally, cell viability was calculated based on the optical density at a wavelength of 450 nm.

[0069] (2) Total RNA was collected on ice and extracted using TRIzol reagent. The PrimeScript RT Reagent kit was used to obtain cDNA obtained by reverse transcription of mRNA, and the transcription levels of genes related to osteogenic differentiation, including alkaline phosphatase (ALP), nuclear transcription factor (Runx2), and bone non-specific gene (COL-1), were detected.

[0070] According to the research results, it was found that flaxseed cyclic peptide J has low cytotoxicity to MC3T3-E1, and the cell survival rate is still above 100% when the concentration is 100μM. And for the first time, it was found that flaxseed cyclic peptide J not only inhibits the differentiation and maturation of osteoclasts, but also promotes the osteogenic differentiation of osteoblast precursor cells MC3T3-E1, activates osteoblast-related signaling pathways in cells, increases the expression levels of related genes such as Runx2, promotes cell mineralization nodules and upregulates the expression of osteogenic differentiation-related genes such as type I collagen (Col-I) and alkaline phosphatase (ALP). Therefore, due to its good biocompatibility and biological activity, flaxseed cyclic peptide J can be used not only as an inhibitor of osteoclast differentiation, but also as an inducer of osteoblast differentiation, and has the potential to be used as a natural health product.

[0071] Example 2

[0072] (1) The present invention weighs 2.046 g of CTAB as an emulsifier, measures 27 mL of n-hexane and 5 mL of n-butanol into a 250 mL beaker as the oil phase and the co-emulsifier, respectively, and places the beaker in a 40°C water bath and stirs for 30 min. Then, 0.25 mL of calcium chloride solution (2 mol / L) and 0.25 mL of sodium carbonate solution (2 mol / L) are added to the above solution, and stirred at 700 r / min for 12 h at room temperature. After the reaction is completed, the solution is placed in a centrifuge and centrifuged at a speed of 15,000 r / min. The supernatant is discarded, and the precipitate is washed 2 to 3 times with an appropriate amount of n-hexane and then washed 2 to 3 times with anhydrous ethanol. Finally, the precipitate is dispersed in water, dried at low temperature, and used for standby use.

[0073] (2) Weigh 2 mg of flaxseed cyclopeptide J (purity > 85%) (preparation method reference: Liu X, Cai Z, Lee WJ, et al. A practical and fast isolation of 12-cyclolinopeptides (linusorbs) from flaxseed oil via preparative HPLC with phenyl-hexyl column [J]. Food Chemistry, 2021, 351: 129318.) into a 25 mL centrifuge tube, add 5 mL of nano-calcium carbonate dispersion alcohol solution containing 2 mg (drug / nanoparticles = 1:1, mass ratio), mix by ultrasonication, and shake on a shaker at room temperature for 24 h. Remove and centrifuge at 14000 r / min, and dry the precipitate.

[0074] (3) Weigh 24 mg of carboxymethyl chitosan and dissolve it in 20 mL of deionized water. Stir until fully dissolved. Add 57 mg of EDC and 35 mg of NHS to activate the carboxyl groups. Under N2 protection, stir magnetically for 1 hour. Weigh 65 mg of Asp8 and dissolve it in 20 mL of deionized water. Add the Asp8 solution to the activated carboxymethyl chitosan solution under N2 atmosphere. Adjust the pH to 7. Stir and react for 6 hours at room temperature in the dark with N2. The reacted sample is placed in a dialysis bag with a molecular weight cutoff of 8000-14000 Da and dialyzed with deionized water for 2 days. After dialysis, freeze-dry to obtain the coupling product of carboxymethyl chitosan and Asp8.

[0075] (4) Weigh 12 mg of the coupling substance obtained in step (3) and dissolve it in 24 mL of deionized water. Stir thoroughly to dissolve it. Add 100 mg of dithiothreitol (DTT) to the solution and stir it under a N2 atmosphere in the dark for 24 h. Take 24 mL of the 0.5 mg / mL nanoparticle dispersion obtained in step (2) and slowly add it to the coupling substance solution and stir for 12 h. Finally, introduce oxygen for cross-linking for 2 h, centrifuge at 3000 r / min, and discard the supernatant to obtain bone-targeted nanoparticles loaded with flaxseed cyclic peptide.

[0076] The resulting nanoparticles were then measured for size. Deionized water was added to prepare a 0.5 mg / mL nanosuspension. After 10 minutes of ultrasonic agitation, 1 mL of each sample was placed in a sample cell and placed in a Malvern nanoparticle size analyzer for particle size distribution and zeta potential. Electron micrographs of the particles and Table 1 indicate that the calcium carbonate nanoparticles had a particle size of approximately 60 nm, the flaxseed cyclic peptide-loaded calcium carbonate nanoparticles had a particle size of approximately 90 nm, and the bone-targeted flaxseed cyclic peptide nanoparticles had a particle size of approximately 120 nm. Zeta potential results showed that the zeta potential of the bone-targeted calcium carbonate nanoparticles before drug loading was +5.42 mV (Table 1). After drug loading, the zeta potential shifted to -17.71 mV, indicating a negative surface charge on the nanoparticles (Table 1). This is due to the negative charge of the drug. The surface zeta potential of the bone-targeted flaxseed cyclic peptide was also -15.54 (Table 1). At the same time, nanoparticles with negative surface charges tend to accumulate on the bone resorption surface of osteoporotic bones, which may be due to the presence of a gradient electric field at the osteoclast interface. 2+ , negatively charged OH - and PO4 3- They are released from the bone matrix into the surrounding environment. Due to the different diffusion coefficients of these charged ions, a gradient electric field is formed at the osteoclast resorption interface. This electric field can induce negatively charged nanoparticles to move toward the osteoclast interface.

[0077] Table 1 Particle size and charge values ​​of different nanoparticles in Example 2

[0078]

[0079] Example 3

[0080] Determination of the loading rate and encapsulation efficiency of flaxseed cyclic peptide by calcium carbonate nanoparticles by liquid chromatography

[0081] (1) Analytical HPLC conditions: Waters 2998-2695 (Waters Corporation, USA) chromatography system, using a reversed-phase column (Kinetex Phenyl Hexyl, 150 mm × 4.6 mm, 2.6), flow rate of 0.5 mL / min, column temperature of 25°C, elution program of acetonitrile:water initial volume ratio of 40:60, followed by gradient elution to acetonitrile:water volume ratio of 90:10, UV absorption wavelength of 214 nm. 10 μL of solution was injected each time for HPLC analysis and purity analysis.

[0082] (2) Concentration standard curve for flaxseed: Prepare cyclopeptide monomer standard solutions of different concentrations (10, 50, 100, 300, and 500 μg / mL) in acetonitrile and perform liquid chromatography analysis using the above-mentioned liquid chromatography conditions. With the monomer peak area as the Y-axis and the cyclopeptide monomer concentration as the X-axis, a linear regression equation was obtained, i.e., the equation for the cyclopeptide standard curve.

[0083] (3) The prepared flaxseed-loaded calcium carbonate nanoparticles were dispersed in an alcohol solution, shaken evenly, and then centrifuged to precipitate the particles at the bottom. The supernatant was removed by rotary evaporation to remove the solvent, and the weight (mg) was recorded. The supernatant was dissolved in a methanol solution at a concentration of 0.1 mg / mL and the purity was determined according to the chromatographic conditions of (1). The mass (mg) of the cyclopeptide in the supernatant was then obtained according to the cyclopeptide standard curve equation.

[0084] Drug loading capacity (LC) = (total amount of flaxseed cyclic peptide added - mass of flaxseed cyclic peptide in the supernatant) / total weight of flaxseed cyclic peptide-loaded calcium carbonate nanoparticles × 100%.

[0085] Encapsulation efficiency (EE) = (total amount of flaxseed cyclic peptide added - mass of flaxseed cyclic peptide in supernatant) / total amount of flaxseed cyclic peptide initially added × 100%

[0086] The mass ratio of calcium carbonate particles to flaxseed cyclic peptide J in Example 2 was adjusted, and the other conditions remained unchanged to obtain calcium carbonate nanoparticles loaded with flaxseed cyclic peptide. The drug loading rate and encapsulation efficiency of calcium carbonate particles for flaxseed cyclic peptide at different mass ratios were calculated. The specific results are shown in Table 2:

[0087] Table 2 Drug loading rate and encapsulation efficiency of flaxseed cyclic peptide by calcium carbonate particles in Example 3

[0088]

[0089] As shown in Table 2, the ratio of nano-calcium carbonate to flaxseed cyclic peptide gradually increased from 2:1 to 1:4. Appropriately reducing the amount of nano-calcium carbonate particles can effectively increase the loading rate and encapsulation efficiency of the nanoparticles for the flaxseed cyclic peptide. As the flaxseed cyclic peptide content increases, the encapsulation efficiency also gradually increases, but the loading rate gradually decreases. Therefore, the present invention selects the optimal addition ratio of calcium carbonate nanoparticles to flaxseed cyclic peptide to be 1:1, resulting in a loading rate of 47.8% and an encapsulation efficiency of 95.32%.

[0090] Example 4

[0091] The intermolecular interaction mechanism of flaxseed cyclic peptide-loaded bone-targeting nanoparticles was analyzed by Fourier transform infrared spectroscopy.

[0092] Weigh 1-2 mg of sample, add 200 mg of KBr powder (200 mesh), grind evenly in an agate mortar under infrared light, and put into a tableting mold for tableting. The tableted sample was detected by Fourier infrared spectroscopy with a wavelength range of 400-4000 cm -1 , the number of scans was 16, and the interactions between the components were compared.

[0093] Figure 4 (a) is the infrared spectra of carboxymethyl chitosan (CMCS) and carboxymethyl chitosan coupled with a target (CSD8). The infrared spectrum of CMCS shows that 160 cm -1 and 1409cm -1 The characteristic absorption peaks of 2918cm are the asymmetric and symmetric stretching vibration peaks of carboxyl groups, indicating the presence of carboxyl groups. -1 and 1327cm -1 The absorption peaks on the left and right are the stretching vibration and bending vibration absorption peaks of CH, 3408 cm -1 The strong absorption peaks around 3408cm in CSD8 are the stretching vibration absorption peaks of OH and NH. -1 The peaks of -OH and -NH are broadened, which is caused by more -OH groups. The asymmetric stretching vibration of -COO is stronger and starts at 1601cm -1 Move to 1651cm -1 , indicating that CMCS in CSD8 formed an amide bond with Asp8, and also indicating that the coupling compound was successfully prepared.

[0094] Figure 4 (b) is the infrared spectra of calcium carbonate nanoparticles (CA), flaxseed cyclic peptide (CLs) and nanoparticles loaded with flaxseed cyclic peptide (JCA). It can be seen that the peak of calcium carbonate particles is located at 1090 cm -1 , 875cm -1 and 747cm -1The characteristic spectrum of vaterite calcium carbonate is shown at 1440 cm -1 The strong absorption peak at 1090 cm is the asymmetric stretching of carbonate ions; -1 and 875cm -1 The absorption peaks at are the out-of-plane bending and symmetric stretching in the non-centrosymmetric structure of calcium carbonate. Characteristic peaks of calcium carbonate and flaxseed cyclic peptide can be observed in the infrared spectrum of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide, respectively. In summary, this indicates that the calcium carbonate particles have been successfully loaded with flaxseed cyclic peptide.

[0095] Figure 4 (c) is the infrared spectrum of flaxseed cyclotide bone-targeted nanoparticles (JCA / CSD8), from which the characteristic absorption peaks of the surface targeting coupler CSD8 and the nanoparticle JCA can be seen respectively, indicating that CSD8 has been successfully coated on the surface of the JCA nanoparticles.

[0096] Example 5

[0097] The morphology of flaxseed cyclopeptide-loaded bone-targeting nanoparticles was observed using scanning electron microscopy and transmission electron microscopy.

[0098] A sample of prepared calcium carbonate nanoparticle powder was placed on a conductive adhesive and directly bonded to a copper stage, where it was sprayed with a layer of gold. The sample morphology was observed using a scanning electron microscope at an accelerating voltage of 20 kV.

[0099] The prepared bone-targeted nanoparticles were dispersed in deionized water to prepare a suspension with a concentration of 1 mg / mL, and then dropped onto a 200-mesh carbon-coated copper grid. After fully drying at room temperature, the morphology of the nanoparticles was observed using a field emission transmission electron microscope (TEM).

[0100] Scanning electron microscopy ( Figure 5 ), it can be found that the calcium carbonate obtained by the present invention has a unique porous structure, which is vaterite crystal type calcium carbonate. The porous structure on the surface can adsorb drugs through physical action. According to the transmission electron microscopy image ( Figure 6 ), it can be seen that the nanoparticles obtained in the present invention have a smooth spherical structure. After drug loading, the particle size increases slightly, but the spherical structure remains unchanged. The bone-targeted nanoparticles that have undergone bone-targeted surface modification have irregular shapes on the outer surface of the spherical particles, coated with a translucent organic layer, indicating that the targeting modifier has been successfully coated on the outer layer of the drug-loaded calcium carbonate nanoparticles.

[0101] Example 6

[0102] The in vitro drug release properties and pH responsiveness of bone-targeted nanoparticles were studied by liquid chromatography.

[0103] An appropriate amount of prepared bone-targeted nanoparticles was weighed and added to PBS with different pH values ​​(7.4, 6.8, and 4.5) to prepare a suspension with a concentration of 5.0 mg / mL. The suspension was shaken in a thermostatic shaker (37°C, 100 rpm) for 48 hours. At specific time points (1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 20 hours, 24 hours, 36 hours, 48 ​​hours, 60 hours, 72 hours, and 84 hours), the suspension was centrifuged, 5 mL of the supernatant was removed, and an equal volume of PBS solution was added (the PBS solution was the same as the PBS solution used to prepare the corresponding suspension). The concentration of flaxseed cyclic peptide in each release solution was determined by the liquid chromatography method described in Example 3.

[0104] Figure 7 A is the release curve of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide. Within 48 hours, the release amounts of flaxseed cyclic peptide were 22% and 24% at pH 7.4 and pH 6.8, respectively, while the cumulative release amount reached 86% at pH 4.5, and a burst release of 65% occurred within the first 12 hours. Figure 7 B is the release curve of bone-targeted flaxseed cyclic peptide loaded nanoparticles. Under pH 7.4 and pH 6.8 conditions, the release amount is about 8% to 10%. Under pH 4.5 conditions, the release curve is relatively gentle compared with that of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide, and there is no obvious burst release. Among them, the cumulative release amount of bone-targeted flaxseed cyclic peptide loaded nanoparticles reaches 58% at 48 hours.

[0105] The low release rate from the drug-loaded nanoparticles at pH 7.4 and 6.8 is attributed to the insolubility or very slow dissolution of the calcium carbonate nanoparticles, which results in slow diffusion and release of the drug within. However, at pH 4.5, the calcium carbonate is decomposed by acid, accelerating drug release, even experiencing an initial burst of release. This demonstrates that the drug-loaded nanoparticles prepared in this invention exhibit good pH responsiveness in drug release.

[0106] The release curve of bone-targeted flaxseed cyclic peptide loaded nanoparticles was gentler than that of flaxseed cyclic peptide loaded calcium carbonate nanoparticles, and there was no obvious burst release. The reason was that the coating of the outer layer of the surface targeting modifier reduced the direct contact between the calcium carbonate nanoparticles and the acidic solution. The surface modifier slowly swelled in the acidic solution, and the density was reduced. + The calcium carbonate particles that enter the interior of the nanoparticles cause them to slowly decompose, resulting in a good slow release of the drug. This example shows that the drug-loaded nanoparticles prepared by the present invention have a good sustained release of the drug.

[0107] Example 7

[0108] Effects of bone-targeted flaxseed cyclic peptide nanoparticles on rat bones.

[0109] The effects of bone-targeted flaxseed cyclic peptide nanoparticles on mouse bone metabolism were investigated using a classic ovariectomized (OVX) rat model. Three-month-old SPF C57BL / 6J mice underwent bilateral ovariectomy to simulate postmenopausal osteoporosis. Following recovery, the mice were divided into the following groups and administered intraperitoneally for 13 consecutive weeks.

[0110] Experimental groups: ① Sham group (i.e., mice underwent incision surgery at the same site without ovarian removal) (Sham, n=10, PBS 0.2 mL / day); ② Model group (OVX, n=10, PBS 0.2 mL / day); ③ Positive control estradiol (E2, n=10, 2 mg / kg / day); ④ Flaxseed cyclopeptide J (CLs, n=10, 10 mg / kg / day); ⑤ Flaxseed cyclopeptide-loaded calcium carbonate nanoparticles JCA provided in Example 2, 10 mg / kg / day); ⑥ Flaxseed cyclopeptide bone-targeted nanoparticles (JCA / CSD8, 10 mg / kg / day) provided in Example 2.

[0111] At the end of the experiment, the femurs of mice in each group were taken and placed in the small animal Micro-CT scanning imaging system for scanning to measure the femoral bone density of each group of animals. Figure 8 As shown in Table 3, (1) compared with the Sham group, the trabecular structure of the OVX group was significantly loose, the reticular structure degenerated, and a trabecular-free bone marrow area appeared. The bone microstructure was destroyed and the bone density decreased significantly. (2) The flaxseed cyclic peptide group increased the femoral bone density and bone mass of OVX rats, slightly better than the positive control estradiol level. (3) The calcium carbonate nanoparticles loaded with flaxseed cyclic peptide were better than the flaxseed cyclic peptide group, reflecting the synergistic therapeutic effect of calcium carbonate and flaxseed cyclic peptide. (4) Compared with the calcium carbonate nanoparticle group loaded with flaxseed cyclic peptide, the bone targeting of the nanoparticles was significantly improved after modification with the bone targeting agent aspartic acid, showing the best therapeutic effect. The bone morphology was similar to that of the Sham group. Nanoparticles with active bone targeting properties on the surface can effectively reduce the dosage and improve bioavailability, while solving the problems of low solubility and poor absorption of flaxseed cyclic peptide in application.

[0112] Table 3 Bone density scan results of OVX mice in different drug groups in Example 7

[0113]

[0114] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a bone-targeted nano-delivery system loaded with flaxseed cyclic peptide, characterized in that The following steps are involved: (1) Preparation of porous calcium carbonate nanoparticles An emulsifier, an oil phase, and a co-emulsifier are weighed and placed in a container, stirred evenly, and then a calcium chloride solution and a sodium carbonate solution are added to the container. The mixture is stirred at room temperature overnight. After the reaction is completed, the mixture is centrifuged. The resulting precipitate is washed with the oil phase and ethanol in sequence, and then dried to obtain porous calcium carbonate nanoparticles. (2) Preparation of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide Weigh the flaxseed cyclic peptide in a container, add the alcohol dispersion containing the porous calcium carbonate nanoparticles of step (1), mix well, shake on a shaker at room temperature for 12 to 48 hours, remove, centrifuge, precipitate and dry to obtain calcium carbonate nanoparticles loaded with flaxseed cyclic peptide; (3) Preparation of bone-targeted modifiers Weigh carboxymethyl chitosan and dissolve it in water, stir to dissolve it, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and stir to activate it under nitrogen protection. Then weigh a bone targeting agent and dissolve it in water. Add the aqueous solution of the bone targeting agent to the activated carboxymethyl chitosan aqueous solution under a nitrogen atmosphere, adjust the pH value, stir and react at room temperature in the dark with nitrogen, place the reacted sample in a dialysis bag, dialyze with water, and freeze-dry to obtain a coupled product of carboxymethyl chitosan and the bone targeting agent, i.e., a bone-targeting surface modification product. (4) Preparation of bone-targeted flaxseed cyclic peptide-loaded nanoparticles The bone-targeted surface modifier of step (3) is weighed and dissolved in water, stirred and dissolved, dithiothreitol is added to the aqueous solution of the bone-targeted surface modifier, and stirred in a nitrogen atmosphere in the dark; the aqueous dispersion of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide of step (2) is weighed and added to the aqueous solution of the bone-targeted surface modifier, stirred, and finally oxygen is introduced for cross-linking, followed by centrifugation, and the supernatant is discarded to obtain bone-targeted nanoparticles loaded with flaxseed cyclic peptide; The flaxseed cyclic peptide described in step (2) is flaxseed cyclic peptide J; The bone targeting agent in step (3) is aspartic acid octapeptide.

2. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 1, characterized in that: The emulsifier described in step (1) is a quaternary ammonium salt type cationic emulsifier; The oil phase described in step (1) is an oil-soluble organic substance; The co-emulsifier described in step (1) is an alcohol-soluble organic substance; The concentration of the calcium chloride solution in step (1) is the same as that of the calcium carbonate solution, which is 1 to 3 mol / L; The amounts of the emulsifier, oil phase, co-emulsifier, calcium chloride solution, and sodium carbonate solution described in step (1) meet the following requirements: the amount of calcium chloride solution added accounts for 0.3% to 1.5% of the total reaction solution; the amount of calcium carbonate solution added accounts for 0.3% to 1.5% of the total reaction solution; the volume ratio of the oil phase to the aqueous phase is 160:1 to 17:1, wherein the volume of the aqueous phase refers to the sum of the volumes of the calcium chloride solution and the sodium carbonate solution; the volume of the co-emulsifier is 17 to 25% of the volume of the oil phase; and the amount of the emulsifier meets the requirement that 1.8 to 2.2 g of emulsifier is included in every 27 ml of the oil phase.

3. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 2, characterized in that: The emulsifier described in step (1) is hexadecyltrimethylammonium bromide; The oil phase in step (1) is n-hexane; The co-emulsifier described in step (1) is n-butanol.

4. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 1, characterized in that: The mass ratio of the porous calcium carbonate nanoparticles to the flaxseed cyclic peptide in step (2) is 2:1 to 1:

4.

5. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 4, characterized in that: The mass ratio of the porous calcium carbonate nanoparticles and the flaxseed cyclic peptide described in step (2) is 1:

1.

6. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 1, characterized in that: The molar ratio of carboxymethyl chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step (3) is 1:4:4 to 1:6:6; The stirring activation time in step (3) is 40 min to 90 min.

7. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 6, characterized in that: The molar ratio of carboxymethyl chitosan, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in step (3) is 1:5:

5.

8. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 1, characterized in that: The molar ratio of the carboxymethyl chitosan to the bone targeting agent in step (3) is 1:1 to 1:3; The pH value adjustment in step (3) refers to adjusting to pH=7; The time for the reaction in step (3) under nitrogen atmosphere and in the dark is 5 to 8 hours.

9. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 8, characterized in that: The molar ratio of the carboxymethyl chitosan to the bone targeting agent in step (3) is 1:

1.

10. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 1, characterized in that: The concentration of the aqueous solution of the bone-targeted surface modifier in step (4) is the same as the concentration of the aqueous dispersion of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide, which is 0.5 mg / mL to 2 mg / mL; The mass ratio of dithiothreitol added in step (4) to the bone-targeted surface modifier is 8 to 10:1; the mass ratio of the bone-targeted surface modifier to the calcium carbonate nanoparticles loaded with flaxseed cyclic peptide is 2:1 to 1:

1.

11. The method for preparing the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 1, characterized in that: After adding DTT in step (4), the stirring time in the dark is 18 to 30 hours; In step (4), the aqueous dispersion of calcium carbonate nanoparticles loaded with flaxseed cyclic peptide is added to the aqueous solution of the bone-targeted surface modifier and stirred for 10 to 18 hours; The cross-linking time after the introduction of oxygen in step (4) is 1 to 3 hours.

12. A bone-targeted nano-delivery system loaded with flaxseed cyclic peptide prepared according to the method of any one of claims 1 to 11.

13. Use of the bone-targeted flaxseed cyclic peptide-loaded nano-delivery system according to claim 12 in the preparation of anti-osteoporosis drugs.

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