A polypeptide-modified long-afterglow nanocomposite, and a preparation method and application thereof

Abstract

The application discloses a kind of polypeptide modified long afterglow nano-complex and its preparation method and application, nano-complex, for core-shell structure, core-shell structure is composed of the core body in inside and the shell that surrounds core body, core body is long afterglow nanomaterial, shell is by polypeptide modification and modified molecule modification, and drug loaded dendritic polymer;The nano-complex of the application can realize multiscale imaging to osteoclast, through in vivo afterglow imaging and in vivo two-photon microscopic imaging, the morphology, migration and distribution of osteoclast can be more accurately analyzed, avoid photo toxicity and background interference, improve imaging sensitivity;The nano-complex of the application is loaded with osteoporosis treatment drug alendronate ALN, and treatment drug can be replaced according to actual demand, and treatment scheme is adjusted, so that the drug loading system has universality.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and in particular relates to a polypeptide-modified long-afterglow nanocomposite and a preparation method and application thereof. Background Art

[0002] Osteoporosis is a systemic skeletal disease characterized by decreased bone mass and deterioration of bone microarchitecture, caused by an imbalance between osteoclastic bone resorption and osteoblastic bone formation. This leads to increased bone fragility and fracture risk, placing a significant economic burden on patients and significantly impacting their quality of life. Osteoclasts, which resorb bone by releasing protons and enzymes, have been used as target cells for molecular research and drug development in osteoporosis. Currently, clinical treatments for osteoporosis primarily use drugs such as bisphosphonates and hormones. For example, bisphosphonates can bind to the bone matrix and induce osteoclast apoptosis. However, these drugs have systemic receptors and short metabolic cycles, resulting in relatively significant side effects and suboptimal therapeutic efficacy. Therefore, the development of more effective targeted therapies and osteoclast imaging nanocomposites presents significant challenges and opportunities in biomedicine, materials science, and analytical chemistry.

[0003] Compared with commonly used histological analysis or computed tomography (CT), optical imaging has outstanding advantages such as high sensitivity, non-invasiveness, non-radioactivity, and real-time. With the development of optical imaging technology and optical probes, it is possible to image the migration, positioning, and bone resorption function of osteoclasts. However, traditional imaging probes require continuous excitation light sources during in vivo imaging, resulting in high background signal interference and inability to achieve more sensitive osteoclast imaging. Moreover, it is impossible to simultaneously achieve real-time and long-term imaging of osteoclasts at the microscopic level. Summary of the Invention

[0004] The purpose of the present invention is to provide a polypeptide-modified long-afterglow nanocomposite and its preparation method and application, which can perform multi-scale imaging of the morphology, migration and distribution of osteoclasts; and can deliver ALN to osteoclasts in a targeted manner to achieve targeted treatment of osteoporosis.

[0005] The present invention adopts the following technical solution: a polypeptide-modified long-lasting glow nanocomposite, which is a core-shell structure. The core-shell structure consists of an internal core and a shell surrounding the core. The core is a long-lasting glow nanomaterial, and the shell is a dendrimer modified with polypeptides and modifying molecules and loaded with drugs.

[0006] Further, the dendrimer is an ethylenediamine core, generation 3.0;

[0007] The polypeptide is D-Asp8-SH, i.e., a polypeptide composed of 8 D-aspartic acids connected together, and the C-terminus is connected to a sulfhydryl group;

[0008] The modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS,

[0009] The medication is alendronate or a bisphosphonate for treating osteoporosis.

[0010] Furthermore, the long afterglow nanomaterial is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 .

[0011] Furthermore, the particle size of the core is 12±5.7 nm, and the thickness of the shell is 2.4 nm.

[0012] A method for preparing a polypeptide-modified long-lasting glow nanocomposite comprises the following steps:

[0013] Step 1: Weigh the long afterglow nanomaterial and disperse it in NaOH solution, stir, centrifuge, and freeze-dry to obtain PLNP-OH;

[0014] Step 2: dissolve PLNP-OH in N,N-dimethylformamide, add 3-iodopropyltrimethoxysilane, stir and centrifuge to obtain iodinated long-lasting nanomaterial PLNP-I;

[0015] Step 3: adding the dendrimer and triethylamine to the PLNP-I methanol dispersion, ultrasonicating, stirring, centrifuging, and washing to obtain the dendrimer PLNP-PAMAM whose core is a long-lasting nanomaterial;

[0016] Step 4: reacting PLNP-PAMAM with the modified molecule in an N,N-dimethylformamide solution containing triethylamine;

[0017] Step 5: Add the polypeptide dissolved in PBS, stir, wash, and freeze-dry in a dark environment at room temperature to obtain a dendrimer PLNP-Asp whose core is a long-lasting nanomaterial and is modified with the polypeptide and the modifying molecule;

[0018] Step 6: Add PLNP-Asp to the drug saline solution, incubate at room temperature, centrifuge, and wash to obtain a dendrimer PLNP-Asp / ALN whose core is a long-lasting nanomaterial, modified with a polypeptide and a modifying molecule, and loaded with a drug.

[0019] Furthermore,

[0020] The dendrimer is ethylenediamine,

[0021] The polypeptide is D-Asp8-SH, i.e., a polypeptide composed of 8 D-aspartic acids connected together, and the C-terminus is connected to a sulfhydryl group;

[0022] The modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS,

[0023] The drug is alendronate or bisphosphonates for treating osteoporosis;

[0024] Long afterglow nanomaterials are Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 .

[0025] A peptide-modified long-afterglow nanocomplex is used to target osteoclasts for in vivo afterglow imaging and microscopic two-photon imaging.

[0026] A polypeptide-modified long-lasting nanocomposite is used to prepare a drug for treating osteoporosis.

[0027] The beneficial effects of the present invention are:

[0028] 1. The nanocomplex of the present invention can achieve multi-scale imaging of osteoclasts. Through in vivo afterglow imaging and in vivo two-photon microscopy, the morphology, migration and distribution of osteoclasts can be analyzed more accurately, avoiding phototoxicity and background interference, and improving imaging sensitivity.

[0029] 2. The nanocomposite of the present invention is loaded with alendronate (ALN), a drug for treating osteoporosis. The drug can be replaced and the treatment plan adjusted according to actual needs, so the drug loading system has universal applicability.

[0030] 3. The nanocomplex of the present invention can effectively improve the targeting of osteoclasts, thereby achieving targeted drug release, greatly reducing the dosage, reducing toxic side effects, and avoiding long-term injections. Therefore, the nanocomplex of the present invention has both targeted imaging and therapeutic functions;

[0031] 4. The present invention first loads the ethylenediamine core to provide a carrier for loading osteoporosis therapeutic drugs, which can achieve the effect of sustained drug release, improve drug utilization efficiency, and reduce toxic side effects; at the same time, it provides the amino group required for subsequent targeted modification, which can increase the biocompatibility of the nanomaterial after active PEG modification, and can be used for subsequent connection of targeted polypeptides. Modifying the polypeptide on the surface of the nanomaterial can achieve targeting of the target osteoclasts, providing a basis for subsequent targeted imaging and treatment; the step-by-step modification method has simple synthesis conditions and can give the nanomaterial multiple functions, making the nanocomplex suitable for osteoporosis-related imaging and treatment research. The related modification method can also be extended to the surface modification of other functional materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The morphological characterization of PLNP-Asp under transmission electron microscopy;

[0033] Figure 2 Fourier transform infrared spectra of long afterglow nanocomposites PLNP-OH, PLNP-I, PLNP-PAMAM, and PLNP-Asp;

[0034] Figure 3 Zeta potential analysis diagram of long afterglow nanocomposites PLNP-OH, PLNP-I, PLNP-PAMAM, PLNP-Asp, and PLNP-Asp / ALN;

[0035] Figure 4 The particle size distribution diagrams of long afterglow nanocomposites PLNP-OH, PLNP-I, PLNP-PAMAM, PLNP-Asp, and PLNP-Asp / ALN;

[0036] Figure 5 is the ALN concentration standard curve;

[0037] Figure 6 is the excitation-emission spectrum of PLNP-Asp, where λ em =700nm;

[0038] Figure 7 This is the long afterglow decay curve of PLNP-Asp / ALN at 700 nm after 1 minute of UV light irradiation;

[0039] Figure 8 Comparison of afterglow excitation, emission, and fluorescence intensities of PLNP-OH, PLNP-Asp, and PLNP-Asp / ALN;

[0040] Figure 9 This is a near-infrared afterglow image of a PLNP-Asp / ALN aqueous solution (100 μL, 1 mg / mL). PLNP-Asp / ALN was irradiated with UV light or LED light for 1 min before imaging.

[0041] Figure 10 is the normalized emission intensity of PLNP-Asp / ALN under different two-photon excitation wavelengths (emission wavelength is 700 nm);

[0042] Figure 11 The kinetic curves of ALN release from PLNP-Asp / ALN in phosphate buffers with different pH values ​​(pH = 6.0, 6.8, 7.4);

[0043] Figure 12Toxicity study of different concentrations of PLNP-Asp and PLNP-Asp / ALN on MC3T3-E1 (a) and RAW 264.7 (b) cells;

[0044] Figure 13 Cell imaging of PLNP-Asp / ALN;

[0045] Figure 14 The results of TRAP staining experiments after ALN, PLNP-Asp, PLNP-PAMAM / ALN and PLNP-Asp / ALN were co-incubated with cells;

[0046] Figure 15 Hemolysis analysis of different concentrations of PLNP-Asp / ALN;

[0047] Figure 16 The results of serum biochemical tests after tail vein injection of ALN, PLNP-Asp, PLNP-PAMAM / ALN, and PLNP-Asp / ALN in ovariectomized mice;

[0048] Figure 17 Histological studies were performed after tail vein injection of ALN, PLNP-Asp, PLNP-PAMAM / ALN, and PLNP-Asp / ALN in ovariectomized mice;

[0049] Figure 18 In vivo imaging of ovariectomized mice at 30 minutes and 4 hours after tail vein injection of PLNP-PAMAM, PLNP-Asp, and PLNP-Asp / ALN, respectively, and afterglow imaging and relative intensity of major organs and bones 6 hours later;

[0050] Figure 19 Immunofluorescence staining of femoral sections of ovariectomized mice after tail vein injection of PLNP-Asp / ALN;

[0051] Figure 20 This is a three-dimensional two-photon microscopic image of the femoral head of an ovariectomized mouse;

[0052] Figure 21 Schematic diagram of Micro-CT of the femur in mice after injection of different samples;

[0053] Figure 22 Statistical analysis of Micro-CT of the femur in mice after injection of different samples. DETAILED DESCRIPTION

[0054] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0055] It should be noted that the structures, proportions, sizes, etc. depicted in the drawings of this specification are only used to match the contents disclosed in the specification so that people familiar with this technology can understand and read them. They are not used to limit the conditions under which the present invention can be implemented. Therefore, they have no substantive technical significance. Any structural modification, change in proportional relationship or adjustment of size should still fall within the scope of the technical content disclosed in the present invention without affecting the efficacy and purpose that can be achieved by the present invention.

[0056] The present invention discloses a peptide-modified long-lasting glow nanocomposite with a core-shell structure. The core-shell structure consists of a long-lasting glow nanomaterial and a shell surrounding the core. The core is a dendrimer modified with peptides and modifying molecules and loaded with a drug. The core has a particle size of 12±5.7 nm, and the shell has a thickness of 2.4 nm.

[0057] The dendrimer is an ethylenediamine core, 3.0 generation; the polypeptide is D-Asp8-SH, which is a polypeptide composed of 8 D-aspartic acids connected together, and the C-terminus is connected to a sulfhydryl group; the modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS, and the drug is alendronate or a bisphosphonate drug for the treatment of osteoporosis; the long-lasting nanomaterial is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3 + 0.0075 .

[0058] The present invention also discloses a method for preparing a polypeptide-modified long-lasting glow nanocomposite, which comprises the following steps:

[0059] Step 1: Weigh the long afterglow nanomaterial and disperse it in NaOH solution, stir, centrifuge, and freeze-dry to obtain PLNP-OH;

[0060] Step 2: dissolve PLNP-OH in N,N-dimethylformamide, add 3-iodopropyltrimethoxysilane, stir and centrifuge to obtain iodinated long-lasting nanomaterial PLNP-I;

[0061] Step 3: adding the dendrimer and triethylamine to the PLNP-I methanol dispersion, ultrasonicating, stirring, centrifuging, and washing to obtain the dendrimer PLNP-PAMAM whose core is a long-lasting nanomaterial;

[0062] Step 4: reacting PLNP-PAMAM with the modified molecule in an N,N-dimethylformamide solution containing triethylamine;

[0063] Step 5: Add the polypeptide dissolved in PBS, stir, wash, and freeze-dry in a dark environment at room temperature to obtain a dendrimer PLNP-Asp whose core is a long-lasting nanomaterial and is modified with the polypeptide and the modifying molecule;

[0064] Step 6: Add PLNP-Asp to the drug saline solution, incubate at room temperature, centrifuge, and wash to obtain a dendrimer PLNP-Asp / ALN whose core is a long-lasting nanomaterial, modified with a polypeptide and a modifying molecule, and loaded with a drug.

[0065] Among them, the dendrimer is ethylenediamine, the polypeptide is D-Asp8-SH, that is, a polypeptide composed of 8 D-aspartic acids connected, and the C-terminus is connected to a sulfhydryl group; the modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS, the drug is alendronate or a bisphosphonate drug for treating osteoporosis, and the long-lasting nanomaterial is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3 + 0.0075 .

[0066] The present invention also discloses a polypeptide-modified long afterglow nanocomposite for targeting osteoclasts to achieve in vivo afterglow imaging and microscopic two-photon imaging.

[0067] The present invention also discloses a polypeptide-modified long-afterglow nanocomposite for preparing a medicine for treating osteoporosis.

[0068] Example 1

[0069] The meaning of the symbols in this embodiment:

[0070] Long-lasting glow nanomaterials are designated as PLNPs. They offer the advantage of being free of in-situ excitation. After being excited by a red LED, they can emit a near-infrared 700nm afterglow and are also capable of femtosecond two-photon excitation. The dendrimer ethylenediamine is designated as PAMAM; the surface-modifying molecule maleimide polyethylene glycol active ester is designated as Mal-PEG-NHS; the targeting peptide polyaspartic acid is designated as D-Asp8-SH; the osteoporosis therapeutic drug is designated as ALN; and the long-lasting glow nanocomplex is designated as PLNP-Asp / ALN.

[0071] 1. Preparation of iodine-based long-lasting nanomaterial PLNP-I

[0072] 100 mg of long afterglow nanomaterial was weighed, dispersed in 5 mM NaOH solution, rapidly stirred overnight and then centrifuged, washed twice with ultrapure water, and freeze-dried to obtain PLNP-OH; then PLNP-OH was dissolved in 20 mL of N,N-dimethylformamide, 500 μL of 3-iodopropyltrimethoxysilane was added, and ultrasonic dispersion was performed; then stirred at 45 ° C for 12 h; centrifuged, and washed twice with methanol to obtain PLNP-I.

[0073] 2. Preparation of dendrimer PLNP-PAMAM with long-lasting nanomaterial as the core

[0074] 400 μL of PAMAM and 40 μL of triethylamine were added to 50 mg of PLNP-I methanol dispersion and sonicated; then stirred at 45°C overnight; centrifuged, and washed twice with methanol to obtain PLNP-PAMAM.

[0075] 3. Preparation of dendrimer PLNP-Asp with long-lasting nanomaterial as core, modified by peptide and modified molecules

[0076] 70 mg of PLNP-PAMAM and 140 mg of MAL-PEG-NHS were reacted in N, N-dimethylformamide solution containing 20 μL of triethylamine for 2 h; then 70 mg of D-Asp8-SH dissolved in 30 mL of PBS was added and stirred at room temperature in a dark environment for 12 h; then the solid was collected, washed twice with ultrapure water, and freeze-dried to obtain PLNP-Asp. The transmission electron microscopy image of PLNP-Asp is shown in the attached figure. Figure 1 shown.

[0077] 4. Preparation of a long-lasting nanomaterial core, modified with peptides and modified molecules, and loaded with drugs, a dendrimer PLNP-Asp / ALN

[0078] 5 mg of PLNP-Asp was added to 1 mg / mL, 1 mL of ALN aqueous solution and ultrasonically dispersed; then incubated overnight at room temperature; centrifuged, and washed with H2O three times to obtain PLNP-Asp / ALN.

[0079] 5. Characterization of PLNP-Asp / ALN

[0080] 5.1. Verification of PLNP surface modification and drug loading

[0081] As attached Figure 2 As shown in Figure 2, the infrared spectrum of the hydroxylated PLNP (PLNP-OH) has no obvious characteristic peaks. PLNP-I has strong -CH2- (2927 and 2851 cm -1) and O-Si-O (1101 and 1047 cm -1 ) stretching absorption peak, indicating that iodine has been successfully introduced into PLNP-OH. After coating with PAMAM, PLNP-PAMAM produced obvious C–N (1418cm -1 ) and N–H (1646 cm -1 ) extension band. The further synthesized PLNP-Asp has a new symmetrical COC stretching absorption peak (1026cm -1 ) and C=O stretching absorption peak (1738cm -1 ).

[0082] As attached Figure 3 As shown in the zeta potential diagram, the zeta potentials of PLNP-OH and PLNP-I are -22.0±0.8mV and -14.8±0.6mV, respectively. PAMAM coating, however, significantly reverses this to +15.8±0.6mV. After PEGylation and surface modification with D-Asp8 peptide, the zeta potential is +5.5±1.0mV. Loading the drug, ALN, shifts the surface charge of PLNP-Asp / ALN to a negative charge of -3.1±0.7mV.

[0083] As attached Figure 4 Figure 2 shows the hydrated particle size of the long-lasting nanocomposite. After surface modification, the hydrated particle size increased from 18.0 nm to 28.1 nm. However, the hydrated particle size of PLNP-Asp / ALN did not increase significantly after drug loading.

[0084] As attached Figure 5 As shown, the ALN concentration standard curve can be used to calculate the ALN loading amount in the long afterglow nanocomposite to be 9.8±1% (mass percentage).

[0085] The above results showed that PLNP successfully coated the PAMAM layer and modified the D-Asp8 peptide, while completing the loading of the drug ALN.

[0086] 5.2 Optical properties of PLNPs after surface modification and drug loading

[0087] As attached Figure 6 As shown, this is the excitation-emission diagram of PLNP-Asp, which shows that PLNP-Asp can be excited by a wider excitation light and emit in the near-infrared region.

[0088] As attached Figure 7 As shown, it is the afterglow attenuation curve of PLNP-Asp / ALN measured at 700 nm after PLNP-Asp / ALN was irradiated with UV lamp for 1 min, indicating that PLNP-Asp / ALN has good afterglow performance.

[0089] As attached Figure 8 As shown in the Figure 3, the afterglow excitation and emission of PLNP-Asp and PLNP-Asp / ALN did not change significantly compared with PLNP-OH, indicating that surface modification and ALN loading had no effect on the afterglow performance.

[0090] As attached Figure 9 As shown, a PLNP-Asp / ALN aqueous solution was exposed to UV light for 1 minute, and the afterglow was observed to decay to 1 hour. It was then re-excited with a 650nm LED for 1 minute, and the afterglow observed after excitation revealed a strong afterglow after UV light exposure. The persistent luminescence imaging and reproducible excitation capabilities of PLNP-Asp / ALN in the near-infrared region facilitate in-situ excitation-free imaging, avoiding background fluorescence interference.

[0091] As attached Figure 10 Figure 2 shows the normalized emission intensity of the long-lasting fluorescence nanocomposite PLNP-Asp / ALN at different two-photon excitation wavelengths (emission wavelength is 700 nm). It can be seen that PLNP-Asp / ALN exhibits high fluorescence emission near 800-1100 nm. This indicates that PLNP-Asp / ALN can be applied to femtosecond two-photon microscopy for in vivo single-cell microscopic analysis.

[0092] These outstanding optical properties provide great potential for PLNP-Asp / ALN to target osteoclasts for multi-scale imaging in vivo and in vitro.

[0093] 5.3 Drug Release Capacity of PLNP-Asp / ALN

[0094] As attached Figure 11 Figure 2 shows the drug release kinetics of PLNP-Asp / ALN in phosphate buffer at different pH values ​​(pH = 6.0, 6.8, and 7.4). The amount of ALN released gradually increased over time. ALN initially showed a significant rapid release, reaching a steady state after 24 hours. The amount of ALN released under acidic pH conditions (6.0 and 6.8) was higher than that under pH 7.4. At pH 6.0 and 6.8, the 48-hour release rates reached 83.1% and 71.3%, respectively. These results indicate that PLNP-Asp loaded with ALN can protect ALN from the external environment, increase its blood circulation time, and thus increase the drug content in the target area, demonstrating the outstanding advantages of PLNP-Asp / ALN for targeted drug delivery in the treatment of osteoporosis.

[0095] 5.4 Toxicity Analysis of PLNP-Asp and PLNP-Asp / ALN

[0096] As attached Figure 12 As shown in the figure, the cytotoxicity of PLNP-Asp and PLNP-Asp / ALN against MC3T3-E1 and RAW 264.7 cell lines was studied. After incubation of cells with PLNP-Asp and PLNP-Asp / ALN at all concentrations (10, 20, 50, and 100 μg / mL) for 24 hours, the cell viability remained above 90%, indicating that both PLNP-Asp and PLNP-Asp / ALN have low cytotoxicity.

[0097] 5.5 Cellular Uptake of PLNP-Asp / ALN

[0098] As attached Figure 13 Shown are cell imaging images of PLNP-Asp / ALN. MC3T3-E1 and RAW 264.7 cells were co-cultured with DAPI, Lysotracker Green, and PLNP-Asp / ALN, respectively. A clear, long-lasting fluorescence signal from the nanomaterial was observed in the cytoplasm. The colocalized yellow color (Pearson correlation coefficient of 0.84 for MC3T3-E1 cells and 0.82 for RAW 264.7 cells) indicates that PLNP-Asp / ALN primarily enters lysosomes. Scale bar, 10 μm. Further investigation was conducted to determine whether PLNP-Asp / ALN enters MC3T3-E1 and RAW 264.7 cells differently. A clear, persistent fluorescence signal was observed within 5 seconds of incubation in RAW 264.7 cells, while a significant signal was not observed until after 15 seconds in MC3T3-E1 cells. The rapid uptake and increased intracellular uptake of PLNP-Asp / ALN by RAW 264.7 cells suggest that surface modification and drug loading promote the internalization of PLNP-Asp / ALN, enabling more specific osteoporosis treatment. Scale bar: 5 μm.

[0099] 5.6 Effect of PLNP-Asp / ALN on ALN ​​Release at the Cellular Level

[0100] As attached Figure 14 Figure 2 shows the results of TRAP staining after cells were co-incubated with ALN, PLNP-Asp, PLNP-PAMAM / ALN, and PLNP-Asp / ALN. ALN increased the tartrate-resistant acid phosphatase activity of the cells. ALN, PLNP-PAMAM / ALN, and PLNP-Asp / ALN all inhibited osteoclast differentiation of primary bone marrow mononuclear macrophages, with PLNP-Asp / ALN showing the greatest inhibitory effect. Scale bar: 400 μm.

[0101] 5.7 Biosafety of PLNP-Asp / ALN

[0102] As attached Figure 15 Figure 2 shows the hemolysis analysis of PLNP-Asp / ALN at different concentrations. Even at the highest dose (100 μg / mL), the hemolysis rate was less than 2.0%, indicating that PLNP-Asp / ALN has good biocompatibility.

[0103] As attached Figure 16 and attached Figure 17 Shown are serum biochemical assays and histological studies of ovariectomized mice following tail vein injection of the long-lasting nanocomplex. Staining of major organs, including the heart, liver, spleen, lung, and kidney, was performed on ovariectomized mice pre-injected with ALN, PLNP-Asp, PLNP-PAMAM / ALN, and PLNP-Asp / ALN. Serum biochemical assays and hematoxylin-eosin (HE)-stained sections demonstrated no toxicity in the liver, kidney, and other organs in all experimental groups. Furthermore, three months after injection, there were no significant changes in activity, body weight, or survival rate of the ovariectomized mice. These data demonstrate the biosafety of PLNP-Asp / ALN and its potential for further in vivo biological applications. Scale bar represents 200 μm.

[0104] 5.8 Bone Targeting and Biodistribution of PLNP-Asp / ALN

[0105] As attached Figure 18 Figure 2 shows ovariectomized mice injected with PLNP-PAMAM, PLNP-Asp, and PLNP-Asp / ALN via the tail vein. In vivo imaging was performed at 30 minutes and 4 hours, respectively. Six hours later, afterglow imaging of major organs and bones was performed. PLNP-Asp / ALN exhibited a higher bone retention signal than PLNP-PAMAM and PLNP-Asp. The afterglow signal of PLNP-Asp / ALN was primarily distributed in the liver, spleen, kidney, and bone, whereas the afterglow signal of ovariectomized mice injected with PLNP-PAMAM was primarily concentrated in the liver and spleen, rather than in the bone. This suggests that PLNP-Asp / ALN possesses the best bone targeting ability, which is corroborated by the afterglow imaging patterns of various tissues. These results demonstrate the bone targeting ability of PLNP-Asp / ALN and its great potential for osteoporosis imaging applications.

[0106] As attached Figure 19 To further validate the bone-targeting ability of PLNP-Asp / ALN, immunofluorescence staining was performed on femoral sections of ovariectomized mice treated with PLNP-Asp / ALN via the tail vein. Merging the fluorescence images of the osteoclast marker CTSK and the osteoblast marker OCN with those of PLNP-Asp / ALN revealed that PLNP-Asp / ALN primarily targeted osteoclasts, rather than osteoblasts. The scale bar is 10 μm.

[0107] As attached Figure 20 The figure shows two-photon microscopic imaging of the femur of mice in vivo after PLNP-Asp / ALN injection. The imaging depth exceeds 500 μm, which can achieve three-dimensional imaging of osteoclasts. The scale is 100 μm.

[0108] 5.9 Application of PLNP-Asp / ALN in the Treatment of Osteoporosis

[0109] As attached Figure 21 and attached Figure 22 Figure 2 shows Micro-CT images of osteoclasts and statistical analysis of corresponding bone tissue parameters in ovariectomized mice following tail vein injection of ALN, PLNP-Asp, PLNP-PAMAM / ALN, and PLNP-Asp / ALN. The results show that the osteoporosis symptoms of ovariectomized mice treated with ALN showed some improvement, with PLNP-Asp / ALN demonstrating a significantly better therapeutic effect on osteoclasts than PLNP-PAMAM / ALN. Compared to ovariectomized mice treated with ALN alone, PLNP-Asp / ALN achieved the same therapeutic effect despite a lower ALN loading, suggesting that PLNP-Asp / ALN protects the ALN from the in vivo environment and enhances drug delivery to the target site.

[0110] As demonstrated in the above examples, the present invention utilizes a long-lasting nanomaterial surface coated with PAMAM, followed by targeted modification and drug loading, resulting in a core-shell structure. The long-lasting nanomaterial serves as the core, while the shell is modified with drug-loaded PAMAM and peptides. The long-lasting nanocomposite prepared by this method exhibits outstanding optical properties and excellent drug release, enabling targeting of osteoclasts for in vivo afterglow imaging and two-photon microscopy. It can also be loaded with ALN to inhibit osteoclast activity, potentially treating osteoporosis. It holds great promise for imaging and treating bone-related diseases.

[0111] Near-infrared long-lasting glow nanomaterials enable in situ excitation-free bioimaging, boasting a remarkable high signal-to-noise ratio. They are expected to enable highly sensitive osteoclast imaging and tracking of cell migration and distribution. Furthermore, long-lasting glow nanomaterials can be excited by femtosecond two-photon light to emit a long afterglow, potentially enabling multi-scale osteoclast imaging at both the in vivo and microscopic levels, reducing phototoxicity and enabling long-term tracking. Long-lasting glow nanomaterials can also be used to construct targeted drug delivery systems, extending the half-life of osteoporosis drugs and reducing their side effects, providing new options for the targeted release of anti-osteoporosis drugs.

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A polypeptide-modified long-lasting nanocomposite, characterized in that: It is a core-shell structure, which consists of an internal core and a shell surrounding the core. The core is a long-lasting nanomaterial, and the shell is a dendrimer modified with a polypeptide and a modifying molecule and loaded with a drug. wherein the dendrimer is an ethylenediamine core, generation 3.0; The polypeptide is D-Asp8-SH, i.e., a polypeptide formed by connecting 8 D-aspartic acids, and the C-terminus is connected to a sulfhydryl group; The modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS; The long afterglow nano material is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 .

2. The polypeptide-modified long-lasting nanocomposite according to claim 1, characterized in that: The drug is alendronate or a bisphosphonate drug for treating osteoporosis.

3. The polypeptide-modified long-lasting nanocomposite according to claim 1, characterized in that: The particle size of the core body is 12±5.7 nm, and the thickness of the shell is 2.4 nm.

4. A method for preparing a polypeptide-modified long-lasting nanocomposite, characterized in that: It consists of the following steps: Step 1: Weigh the long afterglow nanomaterial and disperse it in NaOH solution, stir, centrifuge, and freeze-dry to obtain PLNP-OH; Step 2: dissolve PLNP-OH in N,N-dimethylformamide, add 3-iodopropyltrimethoxysilane, stir and centrifuge to obtain iodinated long-lasting nanomaterial PLNP-I; Step 3: adding the dendrimer and triethylamine to the PLNP-I methanol dispersion, ultrasonicating, stirring, centrifuging, and washing to obtain the dendrimer PLNP-PAMAM whose core is a long-lasting nanomaterial; Step 4: reacting PLNP-PAMAM with the modified molecule in an N,N-dimethylformamide solution containing triethylamine; Step 5: Add the polypeptide dissolved in PBS, stir, wash, and freeze-dry in a dark environment at room temperature to obtain a dendrimer PLNP-Asp whose core is a long-lasting nanomaterial and is modified with the polypeptide and the modifying molecule; Step 6: Add PLNP-Asp to the drug saline solution, incubate at room temperature, centrifuge, and wash to obtain a dendrimer PLNP-Asp / ALN whose core is a long-lasting nanomaterial, modified with a polypeptide and a modifying molecule, and loaded with a drug; wherein the dendrimer is an ethylenediamine core, generation 3.0; The polypeptide is D-Asp8-SH, i.e., a polypeptide formed by connecting 8 D-aspartic acids, and the C-terminus is connected to a sulfhydryl group; The modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS; The long afterglow nano material is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 .

5. The method for preparing a polypeptide-modified long-lasting nanocomposite according to claim 4, characterized in that: The dendrimer is an ethylenediamine core, 3.0 generations, The polypeptide is D-Asp8-SH, i.e., a polypeptide formed by connecting 8 D-aspartic acids, and the C-terminus is connected to a sulfhydryl group; The modified molecule is maleimide polyethylene glycol active ester Mal-PEG-NHS, The drug is alendronate or a bisphosphonate drug for treating osteoporosis; The long afterglow nano material is Zn 1.2 Ga 1.6 Ge 0.2 O4:Cr 3+ 0.0075 .

6. A polypeptide-modified long-afterglow nanocomposite according to any one of claims 1 to 3 or a long-afterglow nanocomposite prepared by the preparation method of claims 4 to 5, for targeting osteoclasts to achieve in vivo afterglow imaging and microscopic two-photon imaging.

7. A polypeptide-modified long-lasting nanocomposite according to any one of claims 1 to 3 or a long-lasting nanocomposite prepared by the preparation method according to claims 4 to 5, for use in preparing a drug for treating osteoporosis.

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