Plant-derived component-based bone-targeting nanoparticles, and methods of making and using the same

Abstract

The application discloses a kind of bone-targeted nanoparticles based on plant source components and its preparation method and application, it is related to the technical field of biomedical nanomaterials.Preparation method includes: apple, ginseng and yam are mixed, boiling is boiled with water, after cooling, remove large particle residue by filtration, obtain clear mixed extract;Calcium salt solution is added to the mixed extract, mix evenly, obtain mixed solution;The mixed solution is frozen at-20 DEG C for 2h-12h, then thawed at 2 DEG C-8 DEG C for 1h-4h;The mixed solution after processing is purified, and the precipitate is collected, i.e.bone-targeted nanoparticles based on plant source components are obtained.The application uses three common plants of apple, ginseng and yam as raw materials, through the core process of "co-boiling extraction" and "calcium ion synergistic freeze-thaw method", successfully prepares the nanoparticles with significant bone targeting function, which can be used for preventing and / or treating osteoporosis.

Description

Technical Field

[0001] This invention relates to the field of biomedical nanomaterials technology, specifically to a bone-targeting nanoparticle based on plant-derived components, its preparation method, and its application. Background Technology

[0002] Osteoporosis, especially postmenopausal osteoporosis, is a systemic skeletal disease characterized by decreased bone mass and destruction of bone microstructure. The key to its treatment lies in effectively inhibiting bone resorption and promoting bone formation. Currently, chemical drugs dominate clinical treatment, but long-term use often results in serious side effects such as osteonecrosis of the mandible and cardiovascular events. Therefore, developing safe and effective alternative therapies derived from natural products has become a research hotspot.

[0003] Nanotechnology has provided a revolutionary tool for drug delivery, especially bone-targeting nanoparticles, which can improve drug accumulation at bone lesion sites, enhance efficacy, and reduce systemic side effects. Currently, strategies for constructing such nanoparticles largely rely on synthetic materials or traditional Chinese medicine ingredients with known efficacy (such as Eucommia ulmoides). However, the biocompatibility and safety of synthetic materials are questionable, while the active ingredients and mechanisms of action of traditional Chinese medicine raw materials with well-defined efficacy have often been thoroughly studied, potentially leading to insufficient inventiveness and difficulty in obtaining effective patent protection for related products. Summary of the Invention

[0004] The purpose of this invention is to at least solve one of the technical problems existing in the prior art, and to provide a bone-targeting nanoparticle based on plant-derived components, its preparation method and application. The nanoparticles utilize three common plant raw materials, apple, Panax notoginseng and yam, and through an innovative preparation process, they achieve excellent bone targeting properties and can effectively improve bone density and bone quality in osteoporosis models.

[0005] This invention utilizes three raw materials—apple, Panax notoginseng, and yam—that are not known for treating osteoporosis, and through an innovative process, prepares nanoparticles with bone-targeting functions. Specifically, this invention uses a "calcium ion synergistic freeze-thaw method" to induce the active ingredients of the three raw materials to self-assemble into nuclei and mineralize in situ, unexpectedly obtaining nanoparticles with excellent bone-targeting properties and bone-promoting activity, providing a novel solution to the aforementioned problems.

[0006] The technical solution of the present invention is as follows: In a first aspect, the present invention provides a method for preparing bone-targeting nanoparticles based on plant-derived components, comprising the following steps: S1. Mix apples, Panax notoginseng and yam, add water and boil together. After cooling, filter to remove large particles of residue and obtain a clear mixed extract. S2. Add calcium salt solution to the mixed extract, mix well to obtain a mixed solution; S3. Freeze the mixture at -20°C for 2-12 hours, and then thaw it at 2°C-8°C for 1-4 hours; S4. Purify the mixture after S3 treatment, collect the precipitate, and obtain bone-targeting nanoparticles based on plant-derived components.

[0007] Optionally, in step S1, the mass ratio of apples, Panax notoginseng, and yam is 5-7:2-5:2-5, the amount of water used is 5-15 times the total weight of the raw materials, and the boiling time is 20-40 minutes. Preferably, the mass ratio of apples, Panax notoginseng, and yam is 6:3:4, the amount of water used is 10 times the total weight of the raw materials, and the boiling time is 30 minutes.

[0008] Optionally, in S2, the calcium salt solution includes at least one of calcium chloride solution, calcium gluconate solution, and calcium citrate solution. Preferred is calcium chloride solution.

[0009] Optionally, in S2, the final concentration of calcium ions in the mixture is 0.5 mM-5 mM.

[0010] Optionally, in S3, the freezing and thawing process is repeated 2 to 4 times.

[0011] Optionally, in S4, the purification method includes at least one of ultracentrifugation, size exclusion chromatography, and tangential flow ultrafiltration, wherein the ultracentrifugation method includes centrifugation at 100,000g - 150,000g for 60 minutes - 120 minutes.

[0012] Secondly, the present invention provides bone-targeting nanoparticles based on plant-derived components obtained by the preparation method described above.

[0013] Optionally, the plant-derived bone-targeting nanoparticles have an average particle size of 50 nm to 200 nm and a zeta potential of -10 mV to -30 mV.

[0014] Thirdly, the present invention provides a pharmaceutical composition comprising the aforementioned plant-derived bone-targeting nanoparticles and a pharmaceutically acceptable carrier.

[0015] Optionally, the dosage form of the pharmaceutical composition is an oral preparation, an injection, or a spray.

[0016] Fourthly, the present invention provides the use of the aforementioned plant-derived bone-targeting nanoparticles or the aforementioned pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

[0017] The bone-targeting nanoparticles of the present invention achieve targeted enrichment of bone sites through their surface properties and the calcium and phosphorus compounds they contain, thereby enabling their use in the prevention and / or treatment of osteoporosis.

[0018] This invention has at least one of the following beneficial effects: (1) This invention creatively uses three common plants—apple, Panax notoginseng, and yam—as raw materials, and successfully prepares nanoparticles with significant bone-targeting function through a core process combining "azeotropic extraction" and "calcium ion synergistic freeze-thaw method." In this invention, the three plant raw materials have a synergistic effect: apple provides pectin and other polysaccharide components as natural carriers and contributes to initial targeting; Panax notoginseng is rich in flavonoids, which exert antioxidant and osteopromoting effects; and the polysaccharide components in yam effectively stabilize the nanostructure and promote the absorption of active ingredients. Furthermore, through the "bridging" effect of calcium ions and the unique physical stress generated by freeze-thaw cycles, the three plant components are strongly driven to achieve tight self-assembly and structural solidification at the molecular level, forming bone-like apatite microcrystals in situ. Ultimately, this results in nanoparticles with excellent bone targeting and the potential to treat osteoporosis.

[0019] (2) The raw materials of the present invention are safe and readily available, the process is green and innovative, and the prepared bone-targeting nanoparticles are highly safe and have no toxic side effects, providing a brand-new solution for the targeted treatment of osteoporosis. Attached Figure Description

[0020] Figure 1 Transmission electron microscopy images of plant-derived bone-targeting nanoparticles prepared under different preparation conditions.

[0021] Figure 2 Potential maps of bone-targeting nanoparticles based on plant-derived components prepared under different preparation conditions.

[0022] Figure 3 Bone microstructure diagrams of bone-targeting nanoparticles based on plant-derived components prepared under different preparation conditions. Detailed Implementation

[0023] To make the technical problems solved, the technical solutions, and the beneficial effects of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0024] Unless otherwise specified in the examples, the conditions were performed according to standard conditions or the manufacturer's recommendations. All reagents and instruments used were commercially available products unless otherwise specified.

[0025] Example 1 Example 1 provides a bone-targeting nanoparticle, which comprises the following raw materials in parts by weight: 30 parts apple, 15 parts Panax notoginseng, and 20 parts yam.

[0026] Example 1 also provides a method for preparing the above-mentioned bone-targeting nanoparticles, as follows: (1) Azeotropic extraction and primary filtration: Weigh apples, Panax notoginseng and yam according to the weight ratio, mix them, add water at a ratio of 10 times the total weight of raw materials, boil together for 30 minutes; after cooling, filter to remove large particles of residue and obtain a clear mixed extract. (2) Calcium ion addition and mixing: Add calcium chloride solution to the mixed extract obtained in step (1), mix evenly, and make the final calcium ion concentration 2.5 mM; (3) Freeze-thaw cycle induced assembly: The mixture obtained in step (2) was frozen at -20°C for 8 hours and then thawed at 4°C for 2 hours. This process is one freeze-thaw cycle, and the process is repeated for 3 cycles. (4) Purification of nanoparticles: The mixture after step (3) is centrifuged at 4°C and 120,000g for 90 minutes, the supernatant is discarded, the precipitate is collected and resuspended in sterile water to obtain the bone-targeting nanoparticles.

[0027] The average particle size of the bone-targeting nanoparticles prepared in Example 1 was measured to be 301 nm.

[0028] like Figure 1 The image shown is a transmission electron microscope (TEM) image of the prepared bone-targeting nanoparticles. As can be seen from the image, the bone-targeting nanoparticles prepared in Example 1 formed a bone-like apatite microcrystalline structure.

[0029] Zeta potential is a key parameter characterizing the surface charge of particles and the stability of a dispersion system. A higher absolute value indicates stronger electrostatic repulsion between particles and a more stable system. For example... Figure 2 As shown, the Zeta potential of the bone-targeting nanoparticles prepared in Example 1 was determined by Malvern nanoparticle size potential analysis. The results showed that the Zeta potential of the bone-targeting nanoparticles prepared in Example 1 was -12.4 mV.

[0030] Example 2 Example 2 provides a bone-targeting nanoparticle, which comprises the following raw materials in parts by weight: 40 parts apple, 20 parts Panax notoginseng, and 25 parts yam.

[0031] Example 2 also provides a method for preparing the above-mentioned bone-targeting nanoparticles, as follows: (1) Azeotropic extraction and primary filtration: Weigh apples, Panax notoginseng and yam according to the weight ratio, mix them, add water at a ratio of 10 times the total weight of raw materials, boil them together for 40 minutes; after cooling, filter to remove large particles of residue and obtain a clear mixed extract. (2) Calcium ion addition and mixing: Add calcium gluconate solution to the mixed extract obtained in step (1), mix evenly, and make the final calcium ion concentration 5 mM. (3) Freeze-thaw cycle induced assembly: The mixture obtained in step (2) is frozen at -20°C for 12 hours, and then thawed at 8°C for 4 hours. This process is one freeze-thaw cycle, and is repeated for 4 cycles. (4) Purification of nanoparticles: The mixture after step (3) is centrifuged at 4°C and 150,000g for 60 minutes, the supernatant is discarded, the precipitate is collected and resuspended in sterile water to obtain the bone-targeting nanoparticles.

[0032] Example 3 Example 3 provides a bone-targeting nanoparticle, which comprises the following raw materials in parts by weight: 20 parts apple, 10 parts Panax notoginseng, and 15 parts yam.

[0033] Example 3 also provides a method for preparing the above-mentioned bone-targeting nanoparticles, as follows: (1) Azeotropic extraction and primary filtration: Weigh apples, Panax notoginseng and yam according to the weight ratio, mix them, add water at a ratio of 10 times the total weight of raw materials, boil together for 20 minutes; after cooling, filter to remove large particles of residue and obtain a clear mixed extract. (2) Calcium ion addition and mixing: Add calcium citrate solution to the mixed extract obtained in step (1), mix evenly, and make the final calcium ion concentration 0.5 mM; (3) Freeze-thaw cycle induced assembly: The mixture obtained in step (2) is frozen at -20°C for 2 hours, and then thawed at 2°C for 1 hour. This process is one freeze-thaw cycle, and two cycles are repeated. (4) Purification of nanoparticles: The mixture after step (3) is centrifuged at 4°C and 100,000g for 120 minutes, the supernatant is discarded, the precipitate is collected and resuspended in sterile water to obtain the bone-targeting nanoparticles.

[0034] Comparative Example 1 Compared with Example 1, the only difference is that no calcium salt solution is added, while the other steps and conditions are the same as in Example 1.

[0035] like Figure 1The image shown is a TEM image of the prepared bone-targeting nanoparticles. As can be seen from the image, the bone-targeting nanoparticles prepared in Comparative Example 1 did not form a bone-like apatite microcrystalline structure, which indicates that the addition of calcium ions affects the formation of the bone-like apatite microcrystalline structure.

[0036] like Figure 2 As shown, the zeta potential of the bone-targeting nanoparticles prepared in Comparative Example 1 is -14.3 mV.

[0037] Comparative Example 2 Compared with Example 1, the only difference is that: no freeze-thaw cycle treatment is performed, and after mixing evenly in step (2), step (4) is directly subjected to ultracentrifugation. The remaining steps and conditions are the same as in Example 1.

[0038] like Figure 1 The image shown is a TEM image of the prepared bone-targeting nanoparticles. As can be seen from the image, the bone-targeting nanoparticles prepared in Comparative Example 2 did not form a bone-like apatite microcrystalline structure, which indicates that whether or not freeze-thaw cycle treatment is performed will affect the formation of the bone-like apatite microcrystalline structure.

[0039] like Figure 2 As shown, the zeta potential of the bone-targeting nanoparticles prepared in Comparative Example 2 is -10.4 mV, therefore the stability of the bone-targeting nanoparticles prepared in Comparative Example 2 is lower than that in Example 1.

[0040] Comparative Example 3 Compared with Example 1, the only difference is that apples are not added, while the other steps and conditions are the same as in Example 1.

[0041] like Figure 1 The image shown is a TEM image of the prepared bone-targeting nanoparticles. As can be seen from the image, the bone-targeting nanoparticles prepared in Comparative Example 3 form an unstable bone-like apatite microcrystalline structure. This indicates that the addition of apples affects the structural stability of the bone-like apatite microcrystalline structure.

[0042] like Figure 2 As shown, the zeta potential of the bone-targeting nanoparticles prepared in Comparative Example 3 is -3.1 mV, therefore the stability of the bone-targeting nanoparticles prepared in Comparative Example 3 is lower than that in Example 1.

[0043] Comparative Example 4 Compared with Example 1, the only difference is that Panax notoginseng is not added, while the other steps and conditions are the same as in Example 1.

[0044] like Figure 1The image shown is a TEM image of the prepared bone-targeting nanoparticles. As can be seen from the image, the bone-targeting nanoparticles prepared in Comparative Example 4 form an unstable bone-like apatite microcrystalline structure. This indicates that whether or not Panax notoginseng is added will affect the structural stability of the bone-like apatite microcrystalline structure.

[0045] like Figure 2 As shown, the zeta potential of the bone-targeting nanoparticles prepared in Comparative Example 4 is -2.8 mV, therefore the stability of the bone-targeting nanoparticles prepared in Comparative Example 4 is lower than that in Example 1.

[0046] Comparative Example 5 Compared with Example 1, the only difference is that yam is not added, while the other steps and conditions are the same as in Example 1.

[0047] like Figure 1 The image shown is a TEM image of the prepared bone-targeting nanoparticles. As can be seen from the image, the bone-targeting nanoparticles prepared in Comparative Example 5 did not form a bone-like apatite microcrystalline structure. This indicates that whether or not yam is added will affect the formation of the bone-like apatite microcrystalline structure.

[0048] like Figure 2 As shown, the zeta potential of the bone-targeting nanoparticles prepared in Comparative Example 5 is -3.2 mV, therefore the stability of the bone-targeting nanoparticles prepared in Comparative Example 5 is lower than that in Example 1.

[0049] Tests and Results The bone-targeting nanoparticles obtained in Examples 1-3 and Comparative Examples 1-5 were tested as follows: (a) Safety evaluation experiment The safety of the bone-targeting nanoparticles obtained in Examples 1-3 and Comparative Examples 1-5 was tested, and the specific testing process is as follows: Ninety 16-month-old male C57BL / 6J naturally aging mice were selected, with 10 mice in each group, and were acclimatized for one week. Before the experiment, each group was fasted for 12 hours but allowed free access to water. The mice were then administered 10 ml / kg of nanoparticle suspensions from Examples 1-3 and Comparative Examples 1-6 via gavage. One group was administered 10 ml / kg of physiological saline (blank control group) via gavage. All mice were housed separately and observed for 15 consecutive days. Changes in body weight, behavioral activity, food and water intake, and mortality were recorded during the observation period. The results are shown in Table 1.

[0050] Table 1 Safety Evaluation Results As can be seen from the above, no abnormal behavior or death was observed in any of the groups of mice during the experiment, and their weight steadily increased, indicating that the bone-targeting nanoparticles obtained in this invention are safe to use.

[0051] (II) Pharmacodynamic evaluation of osteoporosis treatment The bone-targeting nanoparticles obtained in Examples 1-3 and Comparative Examples 1-5 were used to treat age-related osteoporosis, and their efficacy was tested. The specific testing process is as follows: 1. Laboratory animals Seventy naturally aged male C57BL / 6J mice aged 18 months were selected, and another 10 young male C57BL / 6J mice aged 3 months were selected as the young control group.

[0052] 2. Experimental Grouping Naturally aging mice were randomly divided into a model group, an example group, a control group 1, and a control group 2, with 10 mice in each group. A separate young control group of 10 mice was also included.

[0053] 3. Administration method Model group and young control group: 10 mL / kg of normal saline was administered by gavage, 3 times a week for 12 consecutive weeks; Example 1 group: 10 mL / kg of the nanoparticles from Example 1 was administered by gavage 3 times a week for 12 consecutive weeks; Comparative Example 1: 10 mL / kg of nanoparticles from Comparative Example 1 were administered by gavage three times a week for 12 consecutive weeks. Comparative Example 2: 10 mL / kg of nanoparticles from Comparative Example 2 were administered by gavage 3 times a week for 12 consecutive weeks.

[0054] Comparative Example 3: 10 mL / kg of nanoparticles from Comparative Example 3 were administered by gavage three times a week for 12 consecutive weeks.

[0055] Comparative Example 4: 10 mL / kg of nanoparticles from Comparative Example 4 were administered by gavage three times a week for 12 consecutive weeks.

[0056] Comparative Example 5: 10 mL / kg of nanoparticles from Comparative Example 5 were administered by gavage three times a week for 12 consecutive weeks.

[0057] 4. Detection Indicators Twenty-four hours after the last administration, the experimental animals in each group were anesthetized and euthanized. The bilateral femurs and tibias were then separated for the following tests: 4.1 Bone Microstructure Analysis Micro-computed tomography (μ-CT) was used to scan and analyze the distal right femur. Key parameters included bone volume fraction (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), and trabecular bone separation (Tb.Sp). Results are shown below. Figure 3 As shown in Table 2.

[0058] Table 2 Comparison of bone microstructure parameters ( ± s, n=10) From Table 2 and Figure 3 It can be seen that, compared with the model group, the bone-targeting nanoparticles obtained in Example 1 of the present invention can significantly improve the bone microstructure of naturally aging osteoporotic mice and increase bone density and bone quality.

[0059] Compared to Example 1, the nanoparticles obtained in Comparative Examples 1 (without calcium ion addition) and 2 (without freeze-thaw cycle treatment) showed some improvement, but the effect was significantly lower than that of Example 1, demonstrating that the calcium ion synergistic freeze-thaw method is crucial for the formation of nanoparticles with good bone targeting and therapeutic effects. Similarly, compared to Example 1, the nanoparticles obtained in Comparative Examples 3-5 showed some improvement, but the effect was significantly lower than that of Example 1, demonstrating that the type of plant-derived components is crucial for the formation of nanoparticles with good bone targeting and therapeutic effects. In this invention, apples provide pectin and other polysaccharide components as natural carriers and contribute to initial targeting; the flavonoids rich in Panax notoginseng exert antioxidant and osteogenic activity; and the polysaccharide components of Dioscorea opposita effectively stabilize the nanostructure and promote the absorption of active ingredients. That is, through the synergistic effect of three plant-derived components—apple, Panax notoginseng, and Dioscorea opposita—nanoparticles with good bone targeting and therapeutic effects are formed.

[0060] In summary, by comparing the results of Example 1 and Comparative Examples 1-5, it can be seen that without the addition of calcium salt solution, without freeze-thaw cycles, or with only two plant components, it is impossible to form a bone-like apatite microcrystalline structure, or the formed bone-like apatite microcrystalline structure is unstable. Therefore, the present invention uses three plant components with synergistic effects as raw materials, and through the "bridging" effect of calcium ions and the unique physical stress generated by freeze-thaw cycles, strongly drives the three plant components to achieve tight self-assembly and structural solidification at the molecular level, and form bone-like apatite microcrystalline structures in situ. Ultimately, the resulting nanoparticles possess excellent bone targeting and the potential to treat osteoporosis.

[0061] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing bone-targeting nanoparticles based on plant-derived components, characterized in that, Includes the following steps: S1. Mix apples, Panax notoginseng and yam, add water and boil together. After cooling, filter to remove large particles of residue and obtain a clear mixed extract. S2. Add calcium salt solution to the mixed extract, mix well to obtain a mixed solution; S3. Freeze the mixture at -80°C to -20°C for 2-12 hours, and then thaw it at 2°C to 8°C for 1-4 hours; S4. Purify the mixture after S3 treatment, collect the precipitate, and obtain bone-targeting nanoparticles based on plant-derived components.

2. The preparation method according to claim 1, characterized in that, In S1, the mass ratio of apple, Panax notoginseng and yam is 5-7:2-5:2-5, the amount of water used is 5 to 15 times the total weight of the raw materials, and the boiling time with water is 20 to 40 minutes.

3. The preparation method according to claim 1, characterized in that, In S2, the calcium salt solution includes at least one of calcium chloride solution, calcium gluconate solution, and calcium citrate solution.

4. The preparation method according to claim 1, characterized in that, In S2, the final concentration of calcium ions in the mixture is 0.5 mM-5 mM.

5. The preparation method according to claim 1, characterized in that, In S3, the freezing and thawing process is repeated 2 to 4 times.

6. The preparation method according to claim 1, characterized in that, In S4, the purification method includes at least one of ultracentrifugation, size exclusion chromatography, and tangential flow ultrafiltration, wherein the ultracentrifugation method includes centrifugation at 100,000g-150,000g for 60-120 minutes.

7. A bone-targeting nanoparticle based on plant-derived components obtained by the preparation method according to any one of claims 1 to 6.

8. The bone-targeting nanoparticles based on plant-derived components according to claim 7, characterized in that, The plant-derived bone-targeting nanoparticles have an average particle size of 50 nm to 200 nm and a zeta potential of -10 mV to -30 mV.

9. A pharmaceutical composition, characterized in that, It includes the plant-derived component-based bone-targeting nanoparticles as described in any one of claims 7 to 8, and a pharmaceutically acceptable carrier.

10. The use of the plant-derived bone-targeting nanoparticles of claim 7 or the pharmaceutical composition of claim 9 in the preparation of a medicament for the prevention and / or treatment of osteoporosis.

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