Activatable nanoprobe for early diagnosis and reverse progress of diabetic osteoporosis and preparation method of activatable nanoprobe

Abstract

The invention relates to an activatable nanoprobe for early diagnosis and progress reversal of diabetic osteoporosis, which comprises a probe monomer, and the probe monomer is polymerized to form the nanoprobe. The invention provides an activatable nanoprobe RAFPNPs for early diagnosis and reverse progression of diabetic osteoporosis, the probe belongs to an activatable probe, and the probe can maintain the stability of a nanostructure in normal tissues and can be quickly cleared in a short time, so that the probe has excellent biological safety; when the nanoprobe enters the target tissue, the highly expressed biomarker in the target tissue can specifically activate the nanoprobe, so that the nanostructure of the nanoprobe is disintegrated, and the controllable release of the therapeutic drug is realized.

Description

Technical Field

[0001] The present invention belongs to the field of nanoprobes, and particularly relates to an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis and a preparation method thereof. Background Art

[0002] Osteoporosis (OP) is a systemic disease characterized by decreased bone mass and destruction of bone microstructure. Due to the reduction of bone strength, it is very easy to cause complications such as osteoporotic fractures. Statistics show that OP causes 8.9 million fractures worldwide, seriously threatening the life, health and safety of the elderly and causing a huge social burden.

[0003] However, unlike other diseases, OP lacks obvious clinical symptoms. Before serious complications such as fractures occur, most patients are not diagnosed with OP. Once OP enters the irreversible stage, the brittle fractures caused by it are fatal to patients. Therefore, OP is also called the "invisible cancer" of the elderly. At present, OP is clinically divided into primary and secondary according to the cause. The former is mainly related to factors such as aging and menopause in women, and the latter is secondary to endocrine and metabolic diseases. Because there is a "vicious cycle" between metabolic diseases and OP caused by them, that is, metabolic diseases lead to the occurrence of OP, and the biological factors produced in the process of bone loss in OP patients further aggravate the progression of the metabolic disease. Therefore, compared with primary OP, secondary OP is usually accompanied by more serious complications. At the same time, among all secondary OP, diabetic osteoporosis (DOP) has the highest prevalence, and about 1 / 2 to 2 / 3 of patients with diabetes mellitus (DM) are accompanied by decreased bone density. Compared with DM patients with normal bone density, the fracture rate of DOP patients is 2 to 3 times higher, and the risk of disability and mortality is also significantly increased.

[0004] At present, in terms of diagnosis, the commonly used clinical method is to diagnose diabetes by measuring fasting blood glucose and other methods, and to measure bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA) to evaluate the patient's bone loss to determine osteoporosis, and only after excluding factors such as aging and postmenopausal women can they be diagnosed as DOP. However, most OP patients with reduced bone density have obvious systemic bone loss. The DXA method of measuring BMD to diagnose DOP lacks early diagnosis capabilities and is difficult to intervene in treatment in a timely manner. In addition, the clinical diagnosis method is to diagnose DM and OP separately, which is difficult to differentiate from other types of OP (such as elderly OP, hormone-related OP in postmenopausal women, etc.), and therefore lacks diagnostic accuracy. In terms of treatment, conventional clinical treatment drugs are mainly divided into the following three categories: 1) Drugs that promote bone metabolism to regulate diabetes, such as calcium supplements, vitamin D, etc.; 2) Anti-DM drugs that regulate bone metabolism, such as metformin (Met), sulfonylurea insulin, thiazolidinediones, and glucagon-like peptide-1 (GLP-) antagonists, etc. ( Figure 2 ); 3) Anti-osteoporosis drugs regulate sugar metabolism, such as anti-bone resorption drugs bisphosphonates and their derivatives. Studies have shown that the above drugs have different degrees of efficacy in regulating diabetes and inhibiting the progression of DOP.

[0005] However, most drugs require a one-time oral or injection of a large dose to achieve clinical efficacy due to their lack of specificity, poor in vivo stability, and short in vivo half-life. The safety of large-dose administration needs further consideration. Research results show that some patients suffer from side effects such as hypercalcemia and bone hyperplasia due to overtreatment. Among them, patients who take bisphosphonates for a long time to treat DOP have serious adverse reactions such as atypical femoral fractures (Atypical femoral fractures, AFF). Therefore, it is urgent to develop a treatment strategy with high specificity, high in vivo stability and long-term treatment needs for the precise, efficient, safe and long-term treatment of DOP. Summary of the invention

[0006] The present invention proposes an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis and a preparation method thereof, wherein the probe is an activatable probe that can maintain the stability of its nanostructure in normal tissues and be quickly cleared in a short period of time, thus having excellent biosafety; and when it enters the target tissue, the highly expressed biomarkers in the target tissue can specifically activate the nanoprobe, causing its nanostructure to disintegrate, thereby achieving controlled release of therapeutic drugs.

[0007] An activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis comprises a probe monomer which forms a nanoprobe after polymerization.

[0008] Preferably, the probe monomer is a probe monomer RAFPNPs, and the structural formula is:

[0009]

[0010] A method for preparing an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis, comprising the following steps:

[0011] Step 1: Synthesis of compound A: Compound thioketone acid is subjected to amide condensation with lysine, phenylalanine and 4-pentynoic acid respectively by solid phase synthesis method, and 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine condensation agent are added to catalyze the reaction during the condensation. After the reaction is completed, the product is purified to obtain compound A;

[0012] Step 2: Synthesis of compound B: dissolving compound A and N-tert-butyloxycarbonylethylenediamine in anhydrous N'N-dimethylformamide, fully mixing, adding 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N'-diisopropylethylamine condensation agent to catalyze the reaction; after the reaction is completed, purifying by high performance liquid chromatography, and then drying by freeze drying to obtain compound B;

[0013] Step 3: Synthesis of compound C: dissolve compound B in a trifluoroacetic acid dichloromethane solution with a volume ratio of 1:1, and react at room temperature; after the reaction is completed, evaporate and concentrate the reaction solution, and then purify the product to obtain compound C;

[0014] Step 4: Synthesis of compound D: Compound C and activated poly(lactic acid-co-glycolic acid) copolymer are dissolved in an N,N-dimethylformamide solution containing piperidine to react, and then N,N'-diisopropylethylamine is added to catalyze the reaction; after the reaction is completed, the product is dialyzed to purify the product to obtain compound D;

[0015] Step 5: Synthesizing the probe monomer: dissolving compound D, copper sulfate pentahydrate, sodium ascorbate, and compound E in a mixed solution of dimethyl sulfoxide and water, and stirring the mixture under nitrogen protection; after the reaction, purifying the product, and freeze-drying the product to obtain the probe monomer;

[0016] Step 6: Polymerize the probe monomers to form nanoprobes.

[0017] Preferably, in step 4, when the product is dialyzed for purification, the molecular weight cut-off of the dialysis bag is 3000Da.

[0018] Preferably, in step 5, the volume ratio of dimethyl sulfoxide to water is 1:1.

[0019] Preferably, in step six, the specific steps of polymerizing the probe monomer to form a nanoprobe are as follows: dissolve the probe monomer, cyanine dye Cy5 and therapeutic drug Fer-1 in 500 μl of dimethyl sulfoxide, mix thoroughly by ultrasonication, shake at 37°C overnight, then add the mixture dropwise into 5 ml of water that is stirring at a uniform speed, stirring while adding, continue stirring for 1 hour after the addition is completed, and dialyze for 24 hours to obtain the nanoprobe.

[0020] The technical effects and advantages of the present invention are as follows: 1. The present invention provides an activatable nanoprobe RAFPNPs for early diagnosis and reversal of progression of diabetic osteoporosis. The probe is an activatable probe that can maintain the stability of the nanostructure in normal tissues and be quickly cleared in a short time, thus having excellent biosafety; and when it enters the target tissue, the highly expressed biomarkers in the target tissue can specifically activate the nanoprobe, causing its nanostructure to disintegrate, thereby achieving controlled release of therapeutic drugs.

[0021] 2. The nanoprobe of the present invention has a stable optical signal, a long wavelength, and good penetration, and can perform imaging diagnosis of deep bone tissue.

[0022] 3. The nanoprobe of the present invention has good early imaging capability for diabetic osteoporosis, and can achieve specific imaging without obvious bone destruction. At the same time, the nanoprobe of the present invention can also realize differential diagnosis of diabetic osteoporosis and other types of osteoporosis.

[0023] 4. The nanoprobe of the present invention can effectively remove lipid peroxides in the diabetic osteoporosis area and inhibit the iron death of bone cells, thereby promoting bone formation and inhibiting bone resorption, achieving the therapeutic effect of reversing the progression of diabetic osteoporosis.

[0024] 5. The nanoprobe of the present invention has good biocompatibility and no obvious cytotoxicity. It is cleared by the kidney in a short time, reducing retention in normal tissues. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a synthetic route diagram of the probe monomer in the present invention;

[0026] Figure 2 The experimental results of the self-assembly of monomers encapsulating different concentrations of Cy5 dye into nanoprobes;

[0027] Figure 3 This is the result of cell imaging;

[0028] Figure 4 This is a diagram of cell therapy results;

[0029] Figure 5This is a schematic diagram of the diabetic osteoporosis mouse model process and experimental comparison diagram;

[0030] Figure 6 This is a schematic diagram of the treatment process and treatment results of the diabetic osteoporosis mouse model;

[0031] Figure 7 This is a diagram showing the mechanism of nanoprobes reversing the progression of diabetic osteoporosis.

[0032] Figure 8 Flow chart for nanoprobe preparation. DETAILED DESCRIPTION

[0033] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0034] Example 1

[0035] Synthesis of Compound A

[0036] 1200 mg of the compound acetal ketone acid was respectively subjected to amide condensation with 1650 mg of lysine, 985 mg of phenylalanine and 812 mg of 4-pentynoic acid by solid phase synthesis method, and 76 mg of 1-hydroxybenzotriazole, 1842 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 1240 μl of N,N'-diisopropylethylamine were added to catalyze the reaction, and each reaction lasted for 6 hours. After the reaction, the product was purified by preparative high performance liquid chromatography, evaporated and concentrated by rotary evaporator, and then freeze-dried to obtain compound A. The structural formula is as follows:

[0037]

[0038] Example 2

[0039] Synthesis of Compound B

[0040] Compound A (685 mg) and 324 mg of N-tert-butyloxycarbonylethylenediamine were dissolved in 5 ml of anhydrous N'N-dimethylformamide, and after thorough mixing, 351 mg of 1-hydroxybenzotriazole, 794 mg of benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and 278 μl of N,N'-diisopropylethylamine were added to catalyze the reaction, and stirred at room temperature overnight; after the reaction, the mixture was purified by preparative high performance liquid chromatography, evaporated and concentrated by rotary evaporator, and finally dried by freeze dryer to obtain compound B. The structural formula is shown below:

[0041]

[0042] Example 3

[0043] Synthesis of Compound C

[0044] Compound B (594 mg) was dissolved in a trifluoroacetic acid dichloromethane solution (3 ml in total) with a volume ratio of 1:1, stirred at room temperature, and the reaction progress was monitored by analytical high performance liquid chromatography. After the reaction was completed, the reaction solution was evaporated and concentrated, and then the product was directly purified by preparative high performance liquid chromatography, evaporated and concentrated by rotary evaporator, and then freeze-dried to obtain compound C. The structural formula is shown below:

[0045]

[0046] Example 4

[0047] Synthesis of Compound D

[0048] Compound C (527 mg) and activated poly(lactic acid-co-glycolic acid) (1534 mg) were dissolved in 5 ml of N,N-dimethylformamide solution to react, and then 218 μl of N,N'-diisopropylethylamine was added to catalyze the reaction. The reaction was carried out at room temperature overnight. After the reaction, the product was dialyzed for 48 hours, during which the water was changed several times. After the dialysis, the product was freeze-dried to obtain compound D, the structural formula of which is shown below:

[0049]

[0050] Example 5

[0051] Synthetic probe monomer

[0052] Compound D (854 mg), copper sulfate pentahydrate (67 mg), sodium ascorbate (49 mg), and compound E (764 mg) were dissolved in a mixed solution of dimethyl sulfoxide and water, and stirred for 24 hours under nitrogen protection; after the reaction, the product was dialyzed for 48 hours to purify it, and then freeze-dried to obtain a probe monomer. The structural formula is shown below, and the synthetic route of the probe monomer is shown below Figure 1 Said:

[0053]

[0054] Example 6

[0055] Synthetic nanoprobes

[0056] Compound monomer F (5 mg), cyanine dye Cy5 (0.5 mg) and therapeutic drug Fer-1 (0.35 mg) were dissolved in 500 μL of dimethyl sulfoxide, thoroughly mixed by ultrasound, and shaken at 37°C overnight. The mixture was then added dropwise to 5 ml of water that was stirring at a constant speed, stirring while adding, and stirring was continued for 1 hour after the addition was completed. After the stirring was completed, the nanoparticles were dialyzed for 24 hours to obtain. The process is as follows Figure 8 shown.

[0057] Example 7

[0058] Conduct in vitro experiments

[0059] The monomers were wrapped with different concentrations of Cy5 dye to self-assemble into nanoparticles; then the ultraviolet characteristic absorption peak was tested. Figure 2 (a, b) It can be seen that its ultraviolet characteristic absorption peak increases in equal proportion; the ultraviolet absorption experiment was carried out, and the results are as follows Figure 2 (c), its ultraviolet absorption shows a red shift; the fluorescence intensity is tested experimentally, and the results are as follows Figure 2 (d) Due to the aggregation-induced quenching (ACQ) effect, the fluorescence intensity is also significantly reduced after encapsulation; the monomers encapsulate different concentrations of Cy5 dye to self-assemble into nanoparticles and place them in PBS buffer and fetal bovine serum FBS. The stability test results of the nanostructures are as follows: Figure 2 (e) It can be seen that the stability of its nanostructure can be maintained in PBS buffer and fetal bovine serum FBS; the nanoparticles were co-incubated with hydrogen peroxide (H2O2), and the experimental results were Figure 2 (f, g), it can be seen that the UV absorption is restored to the level of free Cy5, and the fluorescence intensity is also significantly increased, indicating that it is effectively activated by H2O2. The fluorescence imaging system is used for testing, and the results are as follows Figure 2 (h), it can be seen that the fluorescence imaging system and its quantification are consistent with (g); after incubation, the particle size of the nanoparticles is tested, and the test results are as follows Figure 2 (i, j), it can be seen that after incubation, the particle size of the nanoparticles also decreases, indicating that the size becomes smaller; the fluorescence intensity is tested during the incubation time, and the results are as follows Figure 2 (k), it can be seen that the fluorescence intensity increases with the increase of incubation time; the nanoparticles were incubated with H2O2 and other proteases to verify their fluorescence activation. The results are shown in Figure 2 (l), it can be seen that the nanoprobe has a very high specificity for H2O2; the nanoparticles were tested in plasma, and the results were as follows Figure 2 (m) It can be seen that the proteins adsorbed on the surface of the nanoparticles in plasma form a protein corona.

[0060] In summary, the results show that ROS can specifically activate the nanoprobe and enhance its fluorescence signal. At the same time, the activated nanoprobe slowly degrades after deposition due to its reduced water solubility, achieving sustained release of the drug.

[0061] Example 8

[0062] Perform cell imaging

[0063] Induction of bone marrow-derived macrophages into osteoclasts in vitro and imaging of osteoclasts. The specific process is shown in the figure Figure 3 (a), cells were co-incubated with the probe monomer RAFPNPs, and the results were shown in Figure 3 (b, c), it can be seen that the uptake of nanoparticles increased significantly, showing excellent targeting to osteoclasts; flow cytometry analysis was performed on the same number of groups as confocal microscopy, and the results are as follows Figure 3 (d, e, f), we can see that the results are similar to Figure 3 (b, c) are consistent.

[0064] The above results indicate that RAFPNPs have excellent targeting ability to osteoclasts.

[0065] Example 9

[0066] Conduct cell therapy

[0067] Inducing mesenchymal stem cells into osteoblasts in vitro, establishing an in vitro diabetic osteoporosis model, and treating its ferroptosis, the specific process is as follows Figure 4 (a), Confocal fluorescence imaging using ferroptosis detection agents after co-incubation with nanoprobe RAFPNPs. Figure 4 (b, c, d), it can be seen that both free small molecules and nanoprobes can effectively inhibit the ferroptosis of osteoblasts; the expression of ferroptosis-related proteins before and after nanoprobe treatment was analyzed by western blot. The results are as follows Figure 4 (e, f, g), it can be seen that both small molecules and nanoprobes can effectively inhibit the progression of ferroptosis; after cells were co-incubated with nanoprobe RAFPNPs, flow cytometry analysis and quantitative data were used, and the results were as follows Figure 4 (h~k), the results are consistent with the confocal imaging; the final product of intracellular ferroptosis metabolism, malondialdehyde, was quantitatively analyzed, and the results are as follows Figure 4 (l), it can be seen that both small molecule drugs and nanoprobes can effectively inhibit the progression of ferroptosis; the quantitative data of total iron ion concentration, trivalent iron ion concentration and dimethyl ferrous ion concentration in cells were detected, and the results are as follows Figure 4 (m~o), it can be seen that small molecule drugs and nanoprobes can effectively reduce the concentrations of the three iron ions, thereby inhibiting the progression of ferroptosis.

[0068] The above results indicate that at the cellular level, both small molecule drugs and nanoprobes can effectively inhibit the ferroptosis of osteoblasts, thereby having a therapeutic effect on osteoblasts.

[0069] Example 10

[0070] In vivo imaging of diabetic osteoporosis

[0071] like Figure 5 (a) A diabetic osteoporosis mouse model was established and the appearance, weight, blood sugar, bone microstructure and bone density of the model mice were compared with those of normal mice. Figure 5 (b~f), it can be seen that the diabetic osteoporosis model was successfully established; imaging and data measurement of diabetic osteoporosis mice, such as Figure 5 (g, h), it can be seen that the nanoprobe has excellent targeting and in vivo imaging capabilities for diabetic osteoporosis; the biodistribution of the nanoprobe in vivo after imaging is analyzed, and the results are as follows Figure 5 (i) It can be seen that the nanoprobes are efficiently enriched in the femur.

[0072] The above results indicate that the nanoprobe has excellent in vivo imaging capability for diabetic osteoporosis.

[0073] Embodiment 11

[0074] Reversing the progression of diabetic osteoporosis

[0075] like Figure 6 (a) A diabetic osteoporosis model was established. The model mice were divided into groups and CT, H&E and Masson staining were performed on the mice in each group to observe the changes in bone microstructure. The results are shown in Figure 2. Figure 6 (b, c, d), it can be seen that the nanoprobe can effectively inhibit the absorption of bone mass, almost returning to normal levels; the nanoprobe can effectively reduce the number of cavities in the bone, almost returning to normal levels; the nanoprobe can effectively promote the formation of new bone. The data of mice were verified and analyzed by bone CT. The results are as follows Figure 6 (e~i), we can see that the result is the same as Figure 6 (b) Consistent; data validation analysis was performed using H&E staining of bone mass. Figure 6 (j, k), we can see that the result is the same as Figure 6 (c) Consistent; data validation analysis was performed using Masson new bone. The results are shown in Figure 6 (d), we can see that the result is similar to Figure 6 (d)Consistency.

[0076] Example 12

[0077] Study on the mechanism of nanoprobes reversing the progression of diabetic osteoporosis

[0078] like Figure 7 (a~h), the proteomics and metabolomics of the femur of diabetic osteoporotic mice were studied, and it was found that the main mode of cell death in the femur of diabetic osteoporotic mice was ferroptosis; the diabetic osteoporotic mice were treated with nanoprobes, and the western blot analysis and quantitative data of ferroptosis-related proteins and osteogenic-related proteins in the femur of each group of mice after treatment were shown in Figure 2. Figure 7 (i~p), it can be seen that the nanoprobe can effectively inhibit the iron death of osteoblasts and promote bone formation, thereby reversing the progression of diabetic osteoporosis; the expression of osteoblast-related proteins in femoral tissue was quantitatively analyzed using an ELISA kit, and the results are as follows Figure 7 (q, r); ELISA kit was used to detect the quantitative analysis of malondialdehyde, the final product of ferroptosis, in femoral tissue and serum. The results are as follows Figure 7 (s, t); detect unsaturated fatty acids and palmitic acid, metabolites of ferroptosis in serum and conduct quantitative analysis. The results are as follows Figure 7 (u, w); detect osteogenic proteins in serum and conduct quantitative analysis. The results are as follows Figure 7 (v).

[0079] The above results indicate that the nanoprobes can effectively inhibit the ferroptosis of osteoblasts and promote bone formation, thereby effectively reversing the progression of osteoporosis.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. An activatable nanoprobe for early diagnosis and reversal of diabetic osteoporosis, characterized in that: The probe monomers are included, and the probe monomers are polymerized to form nanoprobes.

2. The activatable nanoprobe for early diagnosis and reversal of diabetic osteoporosis according to claim 1, characterized in that: The probe monomer is a probe monomer RAFPNPs, and the structural formula is:

3. A method for preparing an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis, comprising the following steps: Step 1: Synthesis of compound A: Compound thioketone acid is subjected to amide condensation with lysine, phenylalanine and 4-pentynoic acid respectively by solid phase synthesis method, and 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate and N,N'-diisopropylethylamine condensation agent are added to catalyze the reaction during the condensation. After the reaction is completed, the product is purified to obtain compound A; Step 2: Synthesis of compound B: dissolving compound A and N-tert-butyloxycarbonylethylenediamine in anhydrous N'N-dimethylformamide, fully mixing, adding 1-hydroxybenzotriazole, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate, and N,N'-diisopropylethylamine condensation agent to catalyze the reaction; after the reaction is completed, purifying by high performance liquid chromatography, and then drying by freeze drying to obtain compound B; Step 3: Synthesis of compound C: dissolve compound B in a trifluoroacetic acid dichloromethane solution with a volume ratio of 1:1, and react at room temperature; after the reaction is completed, evaporate and concentrate the reaction solution, and then purify the product to obtain compound C; Step 4: Synthesis of compound D: Compound C and activated poly(lactic acid-co-glycolic acid) copolymer are dissolved in an N,N-dimethylformamide solution containing piperidine to react, and then N,N'-diisopropylethylamine is added to catalyze the reaction; after the reaction is completed, the product is dialyzed to purify the product to obtain compound D; Step 5: Synthesizing the probe monomer: dissolving compound D, copper sulfate pentahydrate, sodium ascorbate, and compound E in a mixed solution of dimethyl sulfoxide and water, and stirring the mixture under nitrogen protection; after the reaction, purifying the product, and freeze-drying the product to obtain the probe monomer; Step 6: Polymerize the probe monomers to form nanoprobes.

4. The method for preparing an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis according to claim 3, characterized in that: In step 4, when the product is dialyzed for purification, the molecular weight cut-off of the dialysis bag is 3000Da.

5. The method for preparing an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis according to claim 3, characterized in that: In step 5, the volume ratio of dimethyl sulfoxide to water is 1:

1.

6. The method for preparing an activatable nanoprobe for early diagnosis and reversal of progression of diabetic osteoporosis according to claim 3, characterized in that: In step six, the specific steps of polymerizing the probe monomer to form a nanoprobe are as follows: dissolve the probe monomer, cyanine dye Cy5 and therapeutic drug Fer-1 in 500 μl of dimethyl sulfoxide, mix thoroughly by ultrasonication, and shake at 37°C overnight. Then, add the mixture dropwise into 5 ml of water that is stirring at a uniform speed, stirring while adding. After the addition is completed, continue stirring for 1 hour. After the stirring is completed, dialyze for 24 hours to obtain the nanoprobe.

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