Organic nano particle with high osteogenic performance as well as preparation method and application of organic nano particle

Abstract

The invention provides organic nanoparticles with high osteogenic performance as well as a preparation method and application thereof, and belongs to the technical field of osteogenic drug preparation. The preparation method of the organic nanoparticles with high osteogenic performance comprises the following steps: (1) preparing fluorinated polyethyleneimine, preparing double bonds from the fluorinated polyethyleneimine, and then grafting a targeting peptide E7 to obtain a carrier F-PEI-E7; and (2) entrapping an osteogenesis promoting drug in the carrier F-PEI-E7 to obtain the organic nanoparticles with high osteogenesis performance. The invention provides an innovative strategy: F-PEI is used as a carrier, an osteogenesis promoting drug (beta-GP) is entrapped, and through E7 targeting peptide (EPLQLKM) modification, precise targeting delivery of the drug is realized. The organic nanoparticles are high in stability, the in-vitro osteogenesis promoting effect is equivalent to that of a classical formula for in-vitro osteogenesis induction, and the organic nanoparticles have an excellent in-vivo osteogenesis promoting effect.

Description

Technical Field

[0001] The present invention relates to the technical field of osteogenic drug preparation, and in particular to an organic nanoparticle with high osteogenic performance and a preparation method and application thereof. Background Art

[0002] Spatially speaking, bone loss includes systemic osteoporosis and local bone defects. Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass, microstructural destruction, decreased bone strength and increased susceptibility to fractures. Bone defects, especially large bone defects, are a type of local severe bone loss. Both types of diseases seriously threaten the health and quality of life of patients. For osteoporosis, drug therapy is currently the main treatment, but its poor long-term efficacy, high treatment costs and adverse reactions limit its widespread application. For bone defects, bone transplantation is mainly used to treat large bone defects. However, both autologous bone transplantation and allogeneic bone transplantation have many shortcomings. Therefore, it is particularly important to develop a new, efficient and safe anti-bone loss therapy.

[0003] Bone marrow mesenchymal stem cells (BMMSCs) play a key role in bone tissue repair because of their multidirectional differentiation potential, such as osteogenic differentiation and chondrogenic differentiation. They have been widely studied in the prevention and treatment of osteoporosis and the repair of large bone defects, and have achieved good results. Among them, the "classic formula for in vitro osteogenic induction" consists of β-glycerophosphate (β-GP), vitamin C (Vc) and dexamethasone (Dex), which is a classic solution for inducing MSCs to differentiate into osteoblasts. However, when used in vivo, problems such as insufficient drug stability and uneven distribution limit its efficacy.

[0004] To overcome these challenges, previous studies have explored the use of drug delivery carriers to improve the stability and targeting of drugs. Cationic polymers such as polyethyleneimine (PEI) have attracted much attention due to their efficient transmembrane transport capabilities. However, the cytotoxicity of PEI limits its clinical application. To improve this problem, researchers have fluorinated PEI to obtain fluorinated PEI (F-PEI). F-PEI not only improves the biocompatibility of drugs, but also enhances the stability and delivery efficiency of drug molecules in cells.

[0005] However, current bone loss treatments still have problems such as poor long-term efficacy and severe adverse reactions, which may be related to high doses, single targets or off-target effects. Based on this, the present invention is proposed. Summary of the invention

[0006] The purpose of this invention is to provide new strategies and ideas for the treatment of bone loss. This study will evaluate the therapeutic effect, targeting and sustained release of this system by treating osteoporosis or large bone defect animal models through systemic or local administration, laying a solid foundation for future clinical applications.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides a method for preparing organic nanoparticles with high osteogenic performance, comprising the following steps:

[0009] (1) preparing fluorinated polyethyleneimine, preparing double bonds in the fluorinated polyethyleneimine, and then grafting the targeting peptide E7 to obtain a carrier F-PEI-E7;

[0010] (2) The osteogenic drug is encapsulated in the carrier F-PEI-E7 to obtain organic nanoparticles with high osteogenic properties.

[0011] Preferably, the step of preparing fluorinated polyethyleneimine is: dissolving polyethyleneimine and tridecafluoroheptylethylene oxide in a methanol solution respectively, mixing and reacting, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain fluorinated polyethyleneimine;

[0012] The mass ratio of the polyethyleneimine to tridecafluoroheptylethylene oxide is 1:0.18-0.72;

[0013] The molecular weight of the polyethyleneimine is 18 to 25 KDa;

[0014] The reaction time is 24 to 72 hours, and the dialysis washing solution is methanol.

[0015] Preferably, the step of preparing the double bond is: mixing the fluorinated polyethyleneimine and 4-dimethylaminopyridine in a dimethylformamide solvent, reacting overnight in the dark, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain the fluorinated polyethyleneimine containing the double bond;

[0016] The mass ratio of the fluorinated polyethyleneimine to 4-dimethylaminopyridine is 6:1 to 6.5:1;

[0017] The dialysis detergent is double distilled water.

[0018] Preferably, the step of grafting the targeting peptide E7 is: dissolving the thiol-modified targeting peptide E7 in acetonitrile, dissolving the double-bond-containing fluorinated polyethyleneimine in PBS, mixing the two for reaction, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain the carrier F-PEI-E7;

[0019] The mass volume ratio of the thiol-modified targeting peptide E7 to acetonitrile is 3-5 mg:1 mL;

[0020] The mass volume ratio of the double-bond-containing fluorinated polyethyleneimine to PBS is 4 mg:1 mL to 4 mg:3 mL;

[0021] The mass ratio of the thiol-modified targeting peptide E7 to the double-bond-containing fluorinated polyethyleneimine is 1:4 to 2:3;

[0022] The mixing reaction time is 24 to 72 hours.

[0023] Preferably, when the osteogenic drug is β-GP, the step of encapsulating β-GP in the carrier F-PEI-E7 is: dissolving β-GP and F-PEI-E7 in PBS for reaction, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain organic nanoparticles β-GP@F-PEI-E7;

[0024] The mass volume ratio of β-GP, F-PEI-E7 and PBS is 10-15 mg: 3-10 μg: 50 mL;

[0025] The reaction time is 24 to 72 hours.

[0026] Preferably, the bone-promoting drugs also include nucleic acid, protein or small molecule compound drugs that have the effect of treating bone loss.

[0027] Preferably, the molecular weight cutoff of the dialysis bag for dialysis is 3000-4000 KDa, and the dialysis time is 24-72 hours;

[0028] The freeze drying is carried out by freezing at -80°C overnight and then placing at 20 Pa and -40°C for freeze drying for 72 to 120 hours.

[0029] The present invention also provides organic nanoparticles with high osteogenic performance obtained by the preparation method.

[0030] The present invention also provides an application of the organic nanoparticles in preparing a medicine for treating systemic osteoporosis and bone defects.

[0031] Preferably, the drug is β-GP@F-PEI-E7 gel, which is obtained by mixing β-GP@F-PEI-E7 and HAMA gel.

[0032] The present invention proposes an innovative strategy: using F-PEI as a carrier to encapsulate osteogenic drugs (β-GP), and modifying it with the E7 targeting peptide (EPLQLKM) to achieve precise targeted delivery of the drug. The E7 targeting peptide can specifically recognize and target MSCs, allowing the drug to concentrate in the bone area that needs to be repaired, thereby improving the efficiency and quality of regenerative therapy. By optimizing the fluorination degree of F-PEI, the uptake efficiency of β-GP, and the targeting conditions, this study aims to achieve rapid, efficient, and low-toxic drug delivery, thereby promoting bone tissue regeneration.

[0033] At present, the "classic formula for in vitro osteogenic induction" consists of β-glycerophosphate (β-GP), vitamin C (Vc) and dexamethasone (Dex). When this method is used to induce cell osteogenic in vitro, due to the instability of Vc, it needs to be prepared on the spot, and the three ingredients are also relatively cumbersome. The present invention uses fluorinated polyethyleneimine (F-PEI) as a carrier to encapsulate β-glycerophosphate (β-GP) to form organic nanoparticles. The organic nanoparticles have high stability, and the in vitro osteogenic effect is comparable to the "classic formula for in vitro osteogenic induction", and has excellent in vivo osteogenic effect.

[0034] The present invention provides an organic nanoparticle β-GP@F-PEI- E7 , which can achieve efficient, low-toxic, stable cell uptake of the drug β-GP in vitro and precise targeting of mesenchymal stem cells in the bone microenvironment. The organic nanoparticles have the following advantages:

[0035] (1) Precision treatment: β-GP@F-PEI- E7 The system precisely targets mesenchymal stem cells in the bone microenvironment, increases drug utilization, and significantly improves treatment outcomes.

[0036] (2) Reduce side effects: By reducing off-target effects, the system significantly reduces the adverse reactions of traditional osteoporosis treatment drugs and improves treatment safety.

[0037] (3) Cost-effectiveness: Optimize drug uptake and release, reduce drug dosage, simplify treatment process, and save treatment costs for patients.

[0038] (4) Scientific research innovation: Provide new ideas for the treatment of osteoporosis, promote interdisciplinary cooperation, and promote scientific research progress in related fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is the identification result of β-GP@F-PEI-E7 in Example 1. The transmission electron microscope image and EDX image of β-GP@F-PEI-E7 show the distribution of elements such as C, N, O, S, P, and F.

[0040] Figure 2These are the cytotoxicity test results of PEIβ-GP@F-PEI-E7 with different degrees of fluorination in Example 2.

[0041] Figure 3 Schematic diagram of ALP staining of each group in Example 3, scale bar = 100 μm.

[0042] Figure 4 Schematic diagram of ARS staining of each group in Example 3, scale bar = 100 μm.

[0043] Figure 5 This is a statistical chart showing that the ALP protein and ARS content of each group in Example 3 increased significantly (p<0.05).

[0044] Figure 6 The statistical chart shows that the relative expression levels of Alp and Runx2 in each group increased significantly (p<0.05).

[0045] Figure 7 These are in vivo images of the small animals 12 hours after the different drugs in Example 4 were injected into the mice.

[0046] Figure 8 The healing of skull defects in rats after 4 weeks of treatment in Example 5, A. New bone parameters of skull defects in rats, *p<0.05; B. MicroCT stereogram of skulls of rats in each group, the green box represents the skull defect regeneration area.

[0047] Fig. 9 This is a micro-CT cross-sectional image of the femur of mice after 4 weeks of treatment with osteoporosis small β-GP@F-PEI-E7 in Example 6.

[0048] Fig.10 These are the bone mass, bone density, and trabecular regeneration parameters of the proximal femur of mice treated with osteoporosis small β-GP@F-PEI-E7 for 4 weeks in Example 6, **p<0.05. DETAILED DESCRIPTION

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1 Preparation and screening of β-GP@FPEI-E7

[0051] a) Preparation of fluorinated PEI derivatives. First, weigh 1 g of PEI (25 KDa) and 0.18 g, 0.36 g, 0.72 g, and 1.44 g of tridecafluoroheptyl oxirane (C9H5F 13 O) in a glass bottle and dissolved in 10 mL of methanol; then, C9H5F 13The O solution was slowly added dropwise to the methanol solution of PEI, a stirrer was placed in it, and it was slowly stirred at room temperature (25°C) on a magnetic stirrer for 48 hours; a ready-to-use dialysis bag (3500KDa) was used to collect the mixture after the stirring reaction was completed, and it was sealed with a dialysis bag clamp and dialyzed in methanol for 48 hours, during which the washing liquid was continuously replaced. Finally, the liquid in the dialysis bag was placed in a centrifuge tube and frozen in a -80°C refrigerator overnight, and dried in a freeze dryer (20Pa, -40°C) for 48 hours to collect the polymer (F 12 -PEI (0.18 g), F 24 -PEI (0.36 g), F 36 -PEI (0.72 g), F 48 -PEI (1.44 g, hereinafter referred to as F-PEI) was stored in a refrigerator at 4°C.

[0052] b) Preparation of double bonds. Weigh 200 mg of F-PEI and 32 mg of 4-dimethylaminopyridine (DMAP) in a bottle, add 10 mL of dimethylformamide (DMF) to dissolve, stir for 24 h in dark, collect the stirred mixture using a ready-to-use dialysis bag (3500 KDa), seal it with a dialysis bag clip and dialyze it in double distilled water for 48 h, during which the washing liquid is constantly replaced. Then freeze-dry (same as a) to obtain a fluorinated PEI polymer containing double bonds, which is weighed.

[0053] c) Grafting of targeting peptide. Weigh 4 mg of thiol-modified E7 targeting peptide (EPLQLKM) and 16 mg of double-bond-containing fluorinated PEI polymer into a 15 ml centrifuge tube, dissolve the targeting peptide with 1 ml of acetonitrile, dissolve the double-bond-containing F-PEI polymer with 5 ml of PBS, then add the targeting peptide solution dropwise to the double-bond-containing F-PEI polymer solution, place on a shaker to react for 48 hours, then perform dialysis (same as b) purification, freeze-dry (same as a) and weigh to obtain the product F-PEI-E7.

[0054] d) Drug encapsulation: 10 mg of β-GP and 5 μg of F-PEI-E7 were weighed and mixed in 50 ml PBS at room temperature (25°C) for 48 hours, and then freeze-dried (same as a) and weighed to obtain β-GP@F-PEI-E7, which was stored at 4°C.

[0055] β-GP@F-PEI-E7 identification. Transmission electron microscopy (TEM) and elemental energy dispersive X-ray spectroscopy (EDX) were used to determine the morphology and elemental composition of β-GP@F-PEI-E7. The results showed that β-GP@F-PEI-E7 was round and contained characteristic elements such as C, N, O, S, P, and F ( Figure 1 ).

[0056] Example 2 Cytotoxicity detection of PEI carriers with different fluorination degrees.

[0057] The nanoparticles with different fluorination degrees prepared in Example 1 (β-GP@F-PEI-E7) and the nanoparticles prepared with non-fluorinated PEI (β-GP@PEI-E7) were prepared into solutions with different concentrations (see Figure 2 ) were co-cultured with hBMMSCs, specifically: 2000 cells were seeded in each well of a 96-well plate, 6 wells were seeded in each group, 100 μl / well of culture medium with nanoparticle concentrations of 0 μg / mL, 0.01 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 10 μg / mL, 20 μg / mL, 30 μg / mL, and 40 μg / mL were added, and cell proliferation was detected using CCK8 after co-culture at 37°C for 24 hours. Figure 2 As shown, the F with the least cytotoxicity was screened out 24 -PEI-E7 as carrier. (β-GP@F 48 -PEI-E7 is not considered because of its poor water solubility) The organic nanoparticles used in the following experiments are all F 24 -PEI-E7 was prepared as a carrier.

[0058] Example 3 Osteogenesis Detection of β-GP@F-PEI-E7

[0059] Bone marrow mesenchymal stem cells (hBMMSCs) were co-cultured with β-GP@F-PEI-E7 (same as Example 2), and their osteogenic markers alkaline phosphatase (ALP) and calcium nodules were detected. Figures 3 to 6 As shown, Figures 3 to 6 PM represents normal culture medium (α-MEM+10% FBS+1% double antibody); OM represents (10mM β-GP+10mM Vitamin C+1mM Dex) added to PM; β-GP represents 10mM β-GP added to PM; FP represents 0.1ug / ml F-PEI-E7 vector added to PM; FPB represents 10mM β-GP@F-PEI added to PM; FPBE represents 10mM β-GP@F-PEI-E7 added to PM.

[0060] The results showed that ALP staining in β-GP@F-PEI-E7 group ( Figure 3 ) and Alizarin red staining ( Figure 4 ) (showing calcium nodules) were all darker, and the Alp and Runx2 genes ( Figure 6 ) relative expression and ALP content and ARS content ( Figure 5 ) were significantly increased (p<0.05).

[0061] Example 4 Targeting evaluation of β-GP@F-PEI-E7

[0062] 200ul of β-GP, β-GP@F-PEI and β-GP@F-PEI-E7 (100mg / ml) with Cy5 fluorescence were injected into mice through the tail vein, and the in vivo imaging of the small animals was performed after 12h. Figure 7 As shown, the results showed that the fluorescence intensity of the femur in the β-GP@F-PEI-E7 group was significantly increased compared with the other two groups (p<0.05).

[0063] Example 5 Detection of the osteogenic ability of β-GP@F-PEI-E7 in skull defect repair

[0064] A rat skull defect model (diameter = 5 mm) was created, and the defect was filled with methacryloyl hyaluronic acid (HAMA) gel containing β-GP@F-PEI-E7. After 4 weeks, the rats were killed by cervical dislocation, and the skulls were collected for micro-CT examination of bone regeneration. Figure 8 As shown, Figure 8 CTL represents the blank group without any treatment; ZOL represents HAMA mixed with zoledronic acid (0.5 mg / ml) and then photocured; HAMA represents HAMA gel filling; PB (or β-GP) represents HAMA mixed with β-GP (10 mM) and then photocured; FPB represents HAMA mixed with β-GP@F-PEI (10 mM) and then photocured; FPBE represents HAMA mixed with β-GP@F-PEI-E7 (10 mM) and then photocured; the concentration of HAMA is: HAMA-400K concentration is 3% (w / v), the volume ratio of HAMA to drug is 1:1, and 20 ul of gel is used in each well.

[0065] The results showed that large areas of new bone were formed in the HAMA gel group containing β-GP@F-PEI-E7, and the bone mineral content and bone volume fraction of the β-GP@F-PEI-E7 group increased significantly (p<0.05).

[0066] Example 6 Anti-osteoporosis ability detection

[0067] The ovariectomized mouse model (OVX) was successfully induced after 8 weeks of treatment, and then the saline containing β-GP@F-PEI-E7 was intraperitoneally injected. After 4 weeks, the mice were killed by cervical dislocation, and the femurs of the mice were obtained and micro-CT scanning was used to detect bone regeneration. Fig. 9 , 10As shown in the figure, Sham group represents the sham operation group; OVX group represents the ovariectomy and saline injection group; ZOL represents the ovariectomy and zoledronate injection group (1 mg / kg); β-GP represents the ovariectomy and β-GP injection group (250 mg / kg); FPB represents the ovariectomy and β-GP@F-PEI injection (250 mg / kg); FPBE represents the ovariectomy and β-GP@F-PEI-E7 injection (250 mg / kg); all the above drugs were weighed according to body weight and injected with 500ul saline as the solvent per mouse.

[0068] The results showed that the bone density and the number and thickness of trabeculae in the β-GP@F-PEI-E7 group increased significantly (p<0.05).

[0069] As can be seen from the above examples, the present invention has designed a new type of therapy, using fluorinated polyethyleneimine (F-PEI) modified with mesenchymal cell targeting peptide E7 as a carrier to encapsulate β-glycerophosphate (β-GP) and construct a β-GP@F-PEI-E7 targeted delivery system. By optimizing the degree of F-PEI fluorination and β-GP uptake conditions, high-efficiency, low-toxicity, stable uptake and precise targeting of mesenchymal stem cells in the bone microenvironment are achieved. It has been verified that this method promotes the cellular uptake of β-glycerophosphate sodium (β-GP) in vitro, improves the in vitro osteogenic induction ability, and can effectively treat menopausal osteoporosis in mice and repair skull defects in rats in vivo.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for preparing organic nanoparticles with high osteogenic properties, characterized in that: The steps include: (1) preparing fluorinated polyethyleneimine, preparing double bonds in the fluorinated polyethyleneimine, and then grafting the targeting peptide E7 to obtain a carrier F-PEI-E7; (2) The osteogenic drug is encapsulated in the carrier F-PEI-E7 to obtain organic nanoparticles with high osteogenic properties.

2. The preparation method according to claim 1, characterized in that The steps of preparing fluorinated polyethyleneimine are: dissolving polyethyleneimine and tridecafluoroheptylethylene oxide in a methanol solution respectively, mixing and reacting, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain fluorinated polyethyleneimine; The mass ratio of the polyethyleneimine to tridecafluoroheptylethylene oxide is 1:0.18-0.72; The molecular weight of the polyethyleneimine is 18 to 25 KDa; The reaction time is 24 to 72 hours, and the dialysis washing solution is methanol.

3. The preparation method according to claim 2, characterized in that: The step of preparing the double bond is: mixing the fluorinated polyethyleneimine and 4-dimethylaminopyridine in a dimethylformamide solvent, reacting overnight in a dark environment, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain the fluorinated polyethyleneimine containing the double bond; The mass ratio of the fluorinated polyethyleneimine to 4-dimethylaminopyridine is 6:1 to 6.5:1; The dialysis detergent is double distilled water.

4. The preparation method according to claim 3, characterized in that: The step of grafting the targeting peptide E7 is as follows: dissolving the thiol-modified targeting peptide E7 in acetonitrile, dissolving the double-bond-containing fluorinated polyethyleneimine in PBS, mixing the two for reaction, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain the carrier F-PEI-E7; The mass volume ratio of the thiol-modified targeting peptide E7 to acetonitrile is 3-5 mg:1 mL; The mass volume ratio of the double-bond-containing fluorinated polyethyleneimine to PBS is 4 mg:1 mL to 4 mg:3 mL; The mass ratio of the thiol-modified targeting peptide E7 to the double-bond-containing fluorinated polyethyleneimine is 1:4 to 2:3; The mixing reaction time is 24 to 72 hours.

5. The preparation method according to claim 4, characterized in that: When the osteogenic drug is β-GP, the step of encapsulating β-GP in the carrier F-PEI-E7 is as follows: dissolving β-GP and F-PEI-E7 in PBS for reaction, dialyzing the reaction mixture, collecting the trapped solution in the bag, and freeze-drying to obtain organic nanoparticles β-GP@F-PEI-E7; The mass volume ratio of β-GP, F-PEI-E7 and PBS is 10-15 mg: 3-10 μg: 50 mL; The reaction time is 24 to 72 hours.

6. The preparation method according to claim 5, characterized in that: The osteogenic drugs also include nucleic acid, protein or small molecule compound drugs that have the effect of treating bone loss.

7. The preparation method according to any one of claims 1 to 6, characterized in that: The molecular weight cut-off of the dialysis bag for dialysis is 3000-4000 KDa, and the dialysis time is 24-72 hours; The freeze drying is carried out by freezing at -80°C overnight and then placing at 20 Pa and -40°C for freeze drying for 72 to 120 hours.

8. Organic nanoparticles with high osteogenic properties obtained by the preparation method according to any one of claims 1 to 7.

9. Use of the organic nanoparticles according to claim 8 in the preparation of a drug for treating systemic osteoporosis and bone defects.

10. The use according to claim 9, characterized in that The drug is β-GP@F-PEI-E7 gel, which is obtained by mixing β-GP@F-PEI-E7 and HAMA gel.

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