Bone targeting exosome polypeptide delivery system based on biological orthogonal strategy as well as preparation method and application of bone targeting exosome polypeptide delivery system

Abstract

The invention discloses a bone targeting exosome polypeptide delivery system based on a biological orthogonal strategy and a preparation method and application thereof, and relates to the technical field of biological medicine and drug delivery. The bone targeting exosome polypeptide delivery system based on the biological orthogonal strategy comprises an engineered exosome as a carrier and an osteogenesis-promoting therapeutic polypeptide, wherein the osteogenesis-promoting therapeutic polypeptide is anchored on the membrane surface of the engineered exosome; a fusion protein is displayed on the membrane surface of the engineered exosome, and the fusion protein is formed by fusing a bone targeting ligand and an industrial exosome transmembrane scaffold protein; the osteogenesis-promoting therapeutic polypeptide is connected with the industrial exosome through the flexible spacer arm, and the membrane surface steric hindrance is reduced through the flexible spacer arm so as to improve the accessibility of the active fragment of the polypeptide; the delivery system provided by the invention can improve the targeted enrichment and treatment efficiency of osteogenesis promoting drugs in bone tissues, and is suitable for intervention of bone metabolism related diseases such as osteoporosis, delayed fracture healing, bone defects and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and drug delivery technology, and in particular to a bone-targeting exosome peptide delivery system based on a bioorthogonal strategy, its preparation method, and its application. Background Technology

[0002] Osteoporosis is a systemic metabolic disease characterized by bone loss and microstructural damage, with its core pathology lying in the imbalance between osteoclast and osteogenic activities. Reshaping the bone microenvironment homeostasis and reversing the negative balance of bone remodeling are the fundamental goals of clinical treatment. Current osteoporosis treatments mainly include anti-resorption and bone-forming agents. While some drugs possess "dual regulatory" effects, they still primarily regulate through a single pathway, making it difficult to maintain bone remodeling homeostasis in the long term. Therefore, combined or sequential regulatory strategies of "promoting bone formation + inhibiting bone resorption" have gradually become a focus of clinical attention. However, the efficacy of this strategy is severely limited by current administration methods. Under systemic administration, drugs are widely distributed in non-target organs, resulting in extremely low local bioavailability in bone tissue. Especially for combination therapy, insufficient local effective load in bone tissue and low efficiency of combined administration limit the clinical benefits of this strategy, urgently requiring the development of novel delivery systems capable of targeted drug enrichment in bone tissue.

[0003] To improve the effective local exposure of drugs to bone tissue, synthetic nanocarriers have been extensively studied. However, they are easily cleared by the reticuloendothelial system in vivo and have difficulty penetrating the dense bone matrix. In contrast, exosomes, as natural extracellular vesicles, are considered a promising delivery carrier due to their low immunogenicity and excellent tissue penetration. Addressing the delivery needs of bone tissue, patent CN107034188B uses gene editing to fuse the aspartic acid sequence (Asp)8 with the exosome membrane surface scaffold protein LAMP2B, which enhances the affinity of exosomes for hydroxyapatite and bone-related cells, and has initially achieved exosome homing to bone tissue.

[0004] However, simple targeted delivery is insufficient to meet the increasingly complex clinical treatment needs. As mentioned earlier, osteoporosis treatment has evolved towards a dual mechanism of "promoting bone formation and inhibiting bone resorption." Existing research indicates that therapeutic peptides such as teriparatide, as downstream effectors, can directly activate osteogenic pathways, while nucleic acid drugs such as microRNAs, as upstream regulators, can precisely intervene in the osteoclast differentiation process. The two exhibit significant complementarity in terms of target and regulatory level. To achieve synergistic therapeutic goals, the technical strategy of co-loading peptides and nucleic acids using exosome platforms has been explored in fields such as tumor and wound repair. Patent CN111920700B discloses the co-loading of melanoma-targeting peptides and miR-137 onto exosomes for tumor treatment; additionally, studies have shown the use of silk fibroin-binding peptides and miR-146a to promote diabetic wound healing. However, the peptides in these existing technologies primarily function as targeting ligands or functional scaffolds, with relatively low requirements for spatial conformation and biological activity. In contrast, teriparatide, as a therapeutic drug with well-defined pharmacological activity, relies strictly on the precise binding of its N-terminal three-dimensional conformation to the PTH1 receptor for its osteogenic effect. Current technologies lack stable loading strategies for such conformation-sensitive therapeutic peptides and have not resolved the process compatibility issues related to their co-delivery with nucleic acid drugs, making it difficult to meet the clinical needs for dual-pathway regulation of bone remodeling.

[0005] Existing bone-promoting peptides, such as teriparatide, have poor stability and insufficient targeting in vivo, requiring high-frequency administration to achieve therapeutic effects. Meanwhile, existing exosome delivery methods generally suffer from low loading efficiency, easy obscuring of peptide active regions, and difficulty in achieving bone tissue enrichment. They cannot simultaneously achieve both targeting and conformational accessibility, thus limiting the therapeutic effect of drugs.

[0006] Current technologies for exosome surface modification of therapeutic peptides, especially conformationally sensitive peptides, still have significant limitations. On the one hand, traditional gene-engineered fusion expression strategies are constrained by the complex folding mechanisms of membrane proteins, and exogenous peptides are prone to losing their original biological activity due to steric hindrance or conformational changes; while simple physical adsorption is difficult to maintain stability in vivo due to weak binding forces. On the other hand, traditional chemical coupling strategies used to compensate for the shortcomings of physical adsorption often involve harsh reaction conditions and lack selectivity. This not only easily leads to denaturation of the natural functional proteins on the exosome surface but also disrupts the integrity of the exosome lipid bilayer. This disruption of the membrane structure not only affects the stability of the exosome itself but also limits its potential as a universal carrier for encapsulating other hydrophilic drugs (such as nucleic acid drugs). Therefore, there is an urgent need in this field to develop a novel surface modification technology with mild reaction conditions, high site specificity, and the ability to balance the activity of exogenous drugs with the integrity of the exosome membrane structure to meet increasingly complex clinical delivery needs.

[0007] In summary, existing single modification strategies are insufficient to simultaneously meet the multiple requirements of "precise targeting of bone tissue," "preservation of therapeutic peptide activity," and "integrity of exosome membrane structure." Therefore, there is an urgent need in this field to develop a bone-targeting exosome peptide delivery system based on a bioorthogonal strategy, along with its preparation method and applications. Summary of the Invention

[0008] In view of this, the present invention addresses the deficiencies of the existing technology, and its main objective is to provide a bone-targeting exosome peptide delivery system based on a bioorthogonal strategy, its preparation method, and its application. This system endows exosomes with bone-targeting capabilities through genetic engineering and uses a bioorthogonal chemical strategy to anchor bone-promoting therapeutic peptides to the membrane surface in a specific conformation. It is also compatible with the loading of bone metabolism regulatory nucleic acids, thus constructing a precise delivery platform that promotes bone growth and inhibits bone resorption, in order to meet the therapeutic drug needs of osteoporosis and other bone metabolic diseases.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: A bone-targeting exosome peptide delivery system based on a bioorthogonal strategy includes engineered exosomes as carriers and bone-promoting therapeutic peptides: the bone-promoting therapeutic peptides are anchored to the membrane surface of the engineered exosomes via covalent bonds formed through a bioorthogonal reaction. The membrane surface of the engineered exosomes displays a fusion protein, which is formed by fusing a bone-targeting ligand with an engineered exosome transmembrane scaffold protein, thereby endowing the engineered exosomes with the ability to target and enrich bone matrix (such as hydroxyapatite) or bone microenvironment cells (such as osteoblasts, osteoclasts, and osteoendothelial cells). The osteogenic therapeutic peptide is linked to the exosome via a flexible spacer arm. The flexible spacer arm is attached to the end or side chain of the osteogenic therapeutic peptide. The flexible spacer arm reduces steric hindrance on the membrane surface to improve the accessibility of the active peptide fragment.

[0010] As a preferred embodiment, the coupling structure of the osteogenic therapeutic polypeptide and engineered exosomes is characterized as follows: The amino groups on the surface of the engineered exosome membrane are connected to diphenylcyclooctyne (DBCO) groups via amide bonds; The bone-promoting therapeutic polypeptide has an N-terminus connected to an azide group (-N3) and the flexible spacer arm. The azide group and the diphenylcyclooctyn group are covalently linked to form a triazole ring via a strain-promoted azide-yne ​​cycloaddition reaction (SPAAC).

[0011] As a preferred embodiment, the modified structural formula of the osteogenic therapeutic polypeptide is: N3-PEG. n - Peptide sequence -X; where PEG nThe flexible linker is PEG2 to PEG12; the polypeptide sequence is teriparatide (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF); X is a C-terminal modification group, which is selected from lysine, lysine-fluorescein isothiocyanate (Lys-FITC), biotin, or hydroxyl. This modification strategy ensures that the polypeptide is displayed on the exosome surface in an N-terminal outward and C-terminal inward orientation or in a freely extended conformation.

[0012] As a preferred embodiment: the bone-targeting ligand is selected from the aspartic acid repeat sequence (Asp)n (n≥6) (where n is an integer of 6–14), SDSSD sequence, DSS6 sequence or CREDVW sequence; the exosome transmembrane scaffold protein is selected from LAMP2B, CD63, CD9 or CD81, preferably LAMP2B.

[0013] As a preferred embodiment: the osteogenic therapeutic polypeptide is selected from teriparatide (PTH 1-34), the active fragment of parathyroid hormone-related protein (PTHrP) or its osteogenic analogues; the flexible spacer arm is a polyethylene glycol (PEG) chain with a repeating unit number n of 2 to 12 (more preferably PEG2 to PEG4).

[0014] As a preferred embodiment, the delivery system further comprises a regulatory nucleic acid; the regulatory nucleic acid is a bone metabolism regulatory nucleic acid, which is loaded in the lumen of the engineered exosome or anchored to the membrane structure of the engineered exosome; the regulatory nucleic acid is a miRNA, siRNA, or antisense oligonucleotide capable of inhibiting osteoclast differentiation.

[0015] As a preferred embodiment, the miRNA comprises miR-200b-3p or a homologous sequence having at least 90% homology with the mature sequence of miR-200b-3p, and this regulatory nucleic acid can significantly inhibit osteoclast differentiation and bone resorption.

[0016] A pharmaceutical composition comprising the bone-targeting exosome peptide delivery system based on a bioorthogonal strategy as described in claim 6 and a pharmaceutically acceptable carrier; The pharmaceutical composition is selected from any of the following forms: (i) Integrated formulation: The delivery system is a single-particle dual-load exosome simultaneously loaded with the bone-promoting therapeutic peptide and bone metabolism-regulating nucleic acid; (ii) Combination formulation: comprising a first component and a second component, wherein the first component is a bone-targeted engineered exosome loaded with a bone-promoting therapeutic polypeptide, and the second component is a bone-targeted engineered exosome loaded with a bone metabolism regulatory nucleic acid; the first component and the second component are individually packaged or mixed formulations.

[0017] A method for preparing a bone-targeting exosome peptide delivery system based on a bioorthogonal strategy as described in any one of claims 1-5, comprising the following steps: S1. Construct donor cells expressing bone-targeting fusion proteins, and collect and purify engineered exosomes; S2. The first bioorthogonal reactive group is introduced by reacting the amino groups on the surface of the engineered exosomes obtained in step S1 with the heterobifunctional crosslinking agent. S3. Synthesize osteogenic therapeutic peptides with a second bioorthogonal reactive group and a flexible spacer arm at the N-terminus. S4. The osteogenic therapeutic peptide and the engineered exosomes are mixed in a liquid system to carry out a bioorthogonal coupling reaction.

[0018] As a preferred embodiment, after step S4 is completed, the following step is also included: loading bone metabolism regulatory nucleic acids into the engineered exosome cavity by electroporation or transfection.

[0019] The application of the bone-targeting exosome peptide delivery system based on a bioorthogonal strategy in the preparation of drugs for treating bone metabolic diseases, including osteoporosis, delayed fracture healing, or bone defects.

[0020] Compared with existing technologies, this invention has significant advantages and beneficial effects. Specifically, as can be seen from the above technical solution, this invention provides a bone-targeting synergistic delivery system by constructing engineered exosomes that display bone-targeting ligands on the membrane surface and promote click chemical coupling to facilitate bone polypeptide delivery, while also being compatible with the loading of nucleic acids that regulate bone metabolism. It utilizes bone-affinity ligands on the exosome membrane surface to achieve specific targeting and enrichment of bone tissue, exhibiting strong specificity. Furthermore, it employs a flexible spacer-assisted bioorthogonal strategy to anchor therapeutic polypeptides, effectively avoiding steric hindrance and maintaining the bioactivity of conformation-sensitive polypeptides such as teriparatide. By combining bone-promoting peptides with bone-inhibiting nucleic acids for delivery (or combined administration), dual regulation of bone metabolism balance can be achieved. The preparation process is mild, protects the integrity of the carrier, and has good clinical translation potential. By displaying bone-targeting ligands on the exosome membrane surface, the enrichment efficiency of exosomes on bone tissue is improved. And by using a flexible spacer arm-mediated bioorthogonal coupling method, the bone-promoting therapeutic peptides are anchored on the exosome surface in a more stable and spatially accessible form, thereby improving their in vivo bioavailability. This provides a delivery platform with higher therapeutic efficacy and better adaptability for osteoporosis and other bone metabolism-related diseases.

[0021] To more clearly illustrate the structural features and effects of the present invention, a detailed description is provided below in conjunction with the accompanying drawings and specific embodiments. Attached Figure Description

[0022] Figure 1 This is a flow cytometry image showing the expression of engineered cell fusion protein in Example 1 of the present invention. Figure 2 This is a Western blotting image of the fusion protein and exosome marker protein in Example 1 of the present invention; Figure 3 This is a nanoparticle tracking analysis diagram of the bone-targeting exosome particle size in Example 1 of the present invention; Figure 4 This is a transmission electron microscope image of bone-targeted exosomes in Embodiment 1 of the present invention.

[0023] Figure 5 This is a fluorescence imaging image of the binding ability of bone-targeting exosomes to hydroxyapatite in Embodiment 2 of the present invention; Figure 6 In Embodiment 2 of the present invention Figure 5 The corresponding quantitative statistical chart of binding capacity.

[0024] Figure 7 This is a graph showing the loading efficiency of teriparatide under different reaction conditions, as detected by nanoflow cytometry in Example 3 of the present invention.

[0025] Figure 8 This is a μCT image of bone tissue after engineered exosome treatment in Embodiment 4 of the present invention; Figure 9 In Embodiment 4 of the present invention Figure 8 Corresponding statistical chart of bone microstructure parameters; Figure 10 HE and Masson staining images of bone tissue after engineered exosome treatment in Embodiment 4 of the present invention; Figure 11 This is an HE staining image of various tissues and organs after treatment with engineered exosomes in Example 4 of the present invention.

[0026] Figure 12 This is a TRAP staining image of osteoclasts after miRNA treatment in Example 5 of the present invention; Figure 13 In Embodiment 5 of the present invention Figure 12 Corresponding TRAP cell statistics; Figure 14 This is a graph showing the expression of osteoclast-related genes after miRNA action in Example 5 of the present invention; Figure 15This is a comparison chart showing the impact of different loading strategies on the loading efficiency of miR-200b-3p in Embodiment 6 of the present invention. Detailed Implementation

[0027] The present invention is as follows Figure 1 As shown in Figure 15, a bone-targeting exosome peptide delivery system based on a bioorthogonal strategy includes engineered exosomes as carriers and bone-promoting therapeutic peptides: the bone-promoting therapeutic peptides are anchored to the membrane surface of the engineered exosomes via covalent bonds formed by a bioorthogonal reaction. The membrane surface of the engineered exosomes displays a fusion protein, which is formed by fusing a bone-targeting ligand with an engineered exosome transmembrane scaffold protein, giving the engineered exosomes the ability to target and recognize bone matrix or bone microenvironment cells. The osteogenic therapeutic peptide is linked to the exosome via a flexible spacer arm. The flexible spacer arm is attached to the end or side chain of the osteogenic therapeutic peptide. The flexible spacer arm reduces steric hindrance on the membrane surface to improve the accessibility of the active peptide fragment.

[0028] The coupling structure of this bone-promoting therapeutic peptide with engineered exosomes is characterized as follows: The amino groups on the surface of the engineered exosome membrane are linked to diphenylcyclooctyne (DBCO) groups via amide bonds; The bone-promoting therapeutic polypeptide has an N-terminus connected to an azide group (-N3) and the flexible spacer arm; The azide group and the diphenylcyclooctyn group are covalently linked to form a triazole ring via a strain-promoted azide-yne ​​cycloaddition reaction (SPAAC).

[0029] The modified structural formula of the bone-promoting therapeutic peptide described above is: N3-PEG n - Peptide sequence -X; where PEG n For PEG2 to PEG12, there is a flexible linker arm; the polypeptide sequence is teriparatide (SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF); X is a C-terminal modification group, which is selected from lysine, lysine-fluorescein isothiocyanate (Lys-FITC), biotin, or hydroxyl.

[0030] The bone-targeting ligand is selected from the aspartic acid repeat sequence (Asp)n (n≥6), SDSSD sequence, DSS6 sequence, or CREDVW sequence; the exosome transmembrane scaffold protein is selected from LAMP2B, CD63, CD9, or CD81.

[0031] The osteogenic therapeutic peptide is selected from teriparatide (PTH 1-34), the active fragment of parathyroid hormone-related protein (PTHrP) or its osteogenic analogues; the flexible spacer arm is a polyethylene glycol (PEG) chain.

[0032] The delivery system also includes a regulatory nucleic acid; the regulatory nucleic acid is a bone metabolism regulatory nucleic acid, which is loaded in the lumen of the engineered exosome or anchored to the membrane structure of the engineered exosome; the regulatory nucleic acid is a miRNA, siRNA or antisense oligonucleotide capable of inhibiting osteoclast differentiation.

[0033] The miRNA contains miR-200b-3p or a homologous sequence that has at least 90% homology with the mature sequence of miR-200b-3p.

[0034] A pharmaceutical composition comprising the bone-targeting exosome peptide delivery system based on a bioorthogonal strategy of claim 6 and a pharmaceutically acceptable carrier; The pharmaceutical composition is selected from any of the following forms: (i) Integrated formulation: The delivery system is a single-particle dual-load exosome that simultaneously loads the osteogenic therapeutic peptide and the bone metabolism regulatory nucleic acid; (ii) Combination formulation: comprising a first component and a second component, wherein the first component is a bone-targeted engineered exosome loaded with a bone-promoting therapeutic peptide, and the second component is a bone-targeted engineered exosome loaded with a bone metabolism regulatory nucleic acid; the first component and the second component are individually packaged or mixed formulations.

[0035] A method for preparing the bone-targeting exosome peptide delivery system based on any one of claims 1-5, comprising the following steps: S1. Construct donor cells expressing bone-targeting fusion proteins, and collect and purify engineered exosomes; S2. The first bioorthogonal reactive group is introduced by reacting the amino groups on the surface of the engineered exosomes obtained in step S1 with the heterobifunctional crosslinking agent. S3. Synthesize osteogenic therapeutic peptides with a second bioorthogonal reactive group and a flexible spacer arm at the N-terminus. S4. The osteogenic therapeutic peptide and the engineered exosome are mixed in a liquid system to carry out a bioorthogonal coupling reaction.

[0036] After step S4 is completed, the following steps are also included: loading bone metabolism regulatory nucleic acids into the engineered exosome cavity by electroporation or transfection.

[0037] The application of this bone-targeting exosome peptide delivery system based on a bioorthogonal strategy in the preparation of drugs for treating bone metabolic diseases, including osteoporosis, delayed fracture healing, or bone defects.

[0038] Example 1: Construction and characterization of bone-targeted engineered exosomes This embodiment provides a method for constructing engineered exosomes with bone-targeting function. This embodiment uses umbilical cord mesenchymal stem cells (MSCs) as the exosome source cells, and obtains engineered exosomes with bone-targeting delivery capability by expressing a bone-targeting fusion protein.

[0039] (1) Construction of fusion expression vector: First, an expression vector containing a bone-targeting fusion sequence is constructed. The fusion protein sequence includes the Lamp2 signal peptide region, glycosylation modification motif, spacer peptide sequence, SDSSD bone-targeting polypeptide, immune detection tag (such as FLAG or HA tag) and LAMP2B transmembrane domain for anchoring to the exosome membrane surface. The above sequence is inserted into the pLenti-CMV-Puro lentiviral vector through EcoRI and BamHI restriction sites to construct the recombinant lentiviral plasmid pLenti-SDSSD-LAMP2B.

[0040] (2) Obtaining stably expressing cells: Human umbilical cord mesenchymal stem cells were infected with packaged lentiviral solution (MOI=10). 48 hours after infection, 2 μg / mL puromycin was added for resistance selection. This selection was repeated for 7 days to obtain engineered cell lines (BT-MSCs) stably expressing the fusion protein. To verify the expression of the fusion protein in host cells, flow cytometry was used to detect the expression of the fusion protein on the cell surface using anti-FLAG-FITC antibody. The results showed that compared with the uninfected control group, the FLAG positivity rate of BT-MSCs was significantly increased, such as... Figure 1 As shown.

[0041] (3) Isolation and purification of exosomes: BT-MSCs were purified at 8000 / cm³. 2 The cells were seeded into a cell factory and cultured for 48-72 hours in complete medium containing exosome-free serum. The cell culture supernatant was collected and centrifuged sequentially as follows: 500 g for 10 min to remove cells; 2,000 g for 20 min to remove dead cells; and 10,000 g for 30 min to remove cell debris. The supernatant was then concentrated by tangential flow, filtered through a 0.22 μm filter, and finally centrifuged at 120,000 g for 70 min to precipitate the exosomes. The precipitate was resuspended in PBS and washed again by ultracentrifugation to obtain purified bone-targeting exosomes (BT-Exo).

[0042] (4) Characterization and identification of exosomes: Western blotting was used to detect the expression of FLAG tags and exosome marker proteins in exosome samples. The results showed that FLAG tag proteins and exosome marker proteins CD63 and TSG101 could be detected in BT-Exo, as shown in the figure. Figure 2 As shown; subsequently, nanoparticle tracking analysis was used to detect the particle size distribution of the exosomes, as shown. Figure 3 As shown, the obtained exosomes have a particle size of approximately 50–200 nm and exhibit a typical saucer-like, double-membrane vesicle structure as observed by transmission electron microscopy. Figure 4 As shown.

[0043] Example 2: Verification of the binding ability of bone-targeted exosomes to hydroxyapatite To verify the affinity of the engineered exosomes for bone tissue-related materials in vitro, this embodiment uses hydroxyapatite (HAp) as the surface material of the simulated bone matrix, and examines the binding ability of bone-targeted exosomes and control exosomes to HAp.

[0044] (1) Fluorescent labeling of exosomes: BT-Exo and Ctrl-Exo (protein concentration adjusted to 1 mg / mL) prepared in Example 1 were respectively added with lipophilic fluorescent dye DiR (final concentration 1 μM) and incubated at 37°C in the dark for 15 minutes. After incubation, the mixture was purified by Merck Millipore (100 kDa MWCO) ultrafiltration tube to remove unbound free dye and obtain DiR-labeled exosomes.

[0045] (2) The above DiR-labeled exosomes were subjected to NTA quantification. 200 μL of 10 mg / mL HAp microspheres were placed in a 1.5 mL centrifuge tube, and 1×10⁻⁶ HAp microspheres were added to each tube. 9 DiR-labeled BT-Exo or Ctrl-Exo solutions in PBS (total volume adjusted to 500 μL). The mixture was incubated in a 37°C shaker at 100 rpm for 1 hour to ensure adequate contact between exosomes and HAp. After incubation, the sample was centrifuged at 2,000 g for 5 minutes, the supernatant was discarded, and the precipitate was washed three times with PBS buffer to remove unspecifically bound exosomes.

[0046] (3) The precipitate was resuspended in 100 μL PBS and transferred to a 96-well plate. The fluorescence signal was detected using the IVIS small animal in vivo imaging system (Ex=748 nm, Em=780 nm). The fluorescence intensity of the bound exosomes was quantitatively analyzed using image analysis software to evaluate the degree of binding between the exosomes and HAp.

[0047] Image analysis results show (see) Figure 5 and Figure 6The surface fluorescence intensity of HAp particles in the BT-Exo group was significantly higher than that in the Ctrl-Exo group, indicating that bone-targeting exosomes were endowed with excellent hydroxyapatite targeting binding ability by displaying SDSSD peptides on the surface.

[0048] Example 3: Exosome loading and detection of teriparatide (PTH1-34) via click chemistry This embodiment uses teriparatide (PTH1-34) as a model drug and employs a copper-free click chemistry (SPAAC) strategy. By introducing DBCO groups onto the surface of exosomes and reacting them with azidated teriparatide, engineered exosomes (BT-Teri) with dual bone-targeting and osteopromoting activities are constructed. The specific operational steps are as follows: (1) Azide modification of teriparatide: Teriparatide modified with bioorthogonal groups was prepared by solid-phase peptide synthesis (SPPS). An azide-modified flexible linker arm (N3-PEG2) was introduced at the N-terminus of the peptide, and a fluorescein-labeled lysine (Lys-FITC) was linked at the C-terminus to obtain a conformation-dependent therapeutic peptide of N3-PEG2-SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF-Lys(FITC). The N-terminal N3-PEG modification of this invention is located outside the receptor-binding region. By providing sufficient conformational freedom, it avoids adverse effects on the spatial exposure of the N-terminal active core fragment of teriparatide and the specific binding of the PTH1 receptor, thereby maintaining its native three-dimensional conformation and pharmacological activity.

[0049] (2) Construction of DBCO-functionalized exosomes: The engineered exosomes prepared in Example 1 were counted, and the total amount was 1×10⁻⁶. 12 The particles were resuspended in 1 mL of pH 8.0 PBS buffer and transferred to a 1.5 mL enzyme-free centrifuge tube. Considering the large molecular weight and high steric hindrance of PTH peptides, this embodiment drives the coupling reaction by increasing the reactant concentration. DBCO-sulfo-NH (Sigma-Aldrich, 762040) was added to achieve a final concentration of 90 μM (i.e., a molar ratio of exosome particles to DBCO-sulfo-NHS of 10:1). 12 (90 nmol). Centrifuge tubes were fixed on a rotary mixer and reacted at room temperature in the dark for 4 hours. The reaction utilizes the reaction of NHS esters with amino groups of proteins on the surface of exosomes to introduce DBCO groups. After the reaction, the product was transferred to an ultrafiltration tube (100 kDa, Merck Millipore), centrifuged at 4,000 × g for 10 min, the filtrate was discarded and PBS was added. The ultrafiltration washing was repeated 3 times to obtain DBCO-modified exosomes (DBCO-Exo).

[0050] (3) Click chemical conjugation of teriparatide: Combine DBCO-Exo obtained in step (2) with azido-Teriparatide synthesized in step (1) at a ratio of 1×10 12 Each exosome particle was added with 45 nmol of peptide. The centrifuge tube was fixed on a rotary mixer and incubated at 4°C for 12 hours. This optimized high-concentration reaction system significantly increased the probability of molecular collisions in the reaction system, effectively overcoming the steric hindrance effect of the peptide. After the reaction was completed, unbound free peptides were removed by centrifugation at 4,000 × g for 10 min using an ultrafiltration tube, and the mixture was washed three times with PBS. Finally, the precipitate was resuspended in an appropriate amount of PBS to obtain exosomes loaded with teriparatide (PTH1-34) (BT-Teri).

[0051] (4) Detection of teriparatide loading efficiency: Dilute BT-Teri samples to 1×10⁻⁶. 8 The particles / mL were measured at the single-particle level using a Beckman Nano flow cytometer (CytoFLEX Nano).

[0052] Test results as follows Figure 7 As shown, under conventional low-concentration (3 μM) reaction conditions, the loading efficiency on the exosome surface is extremely low (positive rate <5%); however, under the optimized reaction conditions in this embodiment (90 μM), the exosome population exhibits a significant shift in fluorescence signal, with the proportion of FITC channel positive particles reaching 55.6%. These results demonstrate that by optimizing reaction kinetics and combining it with a flexible spacer arm strategy, membrane surface steric hindrance was successfully overcome, achieving efficient loading of the macromolecule teriparatide onto the exosome surface and constructing an ideal drug presentation conformation with the N-terminus facing outwards and the activity exposed.

[0053] Example 4: In vivo efficacy and safety evaluation of engineered exosomes in an osteoporosis model This embodiment uses an ovariectomy (OVX)-induced mouse osteoporosis model to evaluate the in vivo bone regeneration capacity of PTH1-34 loaded with engineered exosomes (i.e., BT-Teri) prepared in Example 3. The synergistic effect of this delivery system compared to free drugs and exosomes alone was investigated under low-dose, low-frequency administration conditions.

[0054] (1) Animal model construction and grouping: Eight-week-old female C57BL / 6J mice were randomly divided into five groups: Sham group (sham operation group), OVX group (model group), BT-Exo group (carrier-only group), Teri group (free drug group), and BT-Teri group (experimental group). Modeling method: In the Sham group, only the dorsal skin and peritoneum of the mice were cut, without removing the ovaries, and the wound was sutured; the other four groups of mice underwent bilateral ovariectomy. After surgery, the mice were fed normally for 8 weeks, and drug treatment was started after the osteoporosis model was successfully established.

[0055] (2) Dosing regimen: All treatment groups received tail vein injection three times a week for eight consecutive weeks. To verify the ability of the engineered exosomes described in this invention to improve drug bioavailability, a treatment regimen with a significantly lower dose than conventionally reported was established. The effective dose of PTH was set at 10 μg / kg (Note: the treatment doses reported in conventional literature are usually in the range of 40-80 μg / kg). The specific dosages are as follows: 1) Sham group: Injected with an equal volume of physiological saline.

[0056] 2) Model group (OVX group): Injected with an equal volume of physiological saline. 3) Free drug group (Teri group): Free PTH1-34 was injected at a dose of 10 μg / kg.

[0057] 4) Empty exosome group (BT-Exo group): Bone-targeted engineered exosomes without drug loading (prepared in Example 1) were injected at a dose of 2 × 10⁻⁶. 9 particles / only / time.

[0058] 5) Drug-loaded exosome group (BT-Teri group): Drug-loaded engineered exosomes (prepared in Example 3) were injected, with an effective PTH dose of 10 μg / kg (equal to the Teri group), and the exosome carrier amount was approximately 2 × 10⁻⁶. 9 particles / each / time (equal to the BT-Exo group).

[0059] (3) Micro-CT Imaging Analysis: After the treatment cycle, the mice were euthanized, the femurs were separated, and scanned and reconstructed using a Micro-CT system. For example... Figure 8 As shown, the model group (OVX group) exhibited typical osteoporosis characteristics, with reduced trabecular bone number, sparse structure, and impaired continuity. The free drug treatment group (Teri group) showed limited bone recovery under low-dose administration, suggesting that free drugs are unlikely to achieve effective exposure levels in bone tissue. In contrast, the BT-Teri treatment group showed a more intact and denser trabecular structure and significantly improved bone mass. Further quantitative results of bone microstructure are as follows... Figure 9As shown, compared with the model group, the BT-Teri group exhibited increased bone mineral density (BMD) and bone volume fraction (BV / TV), and decreased trabecular bone spacing (Tb.Sp). Specifically, the BT-Teri group showed significantly better BV / TV performance than the free PTH treatment group. These bone repair effects indicate that, under the same drug dosage conditions, the drug-loaded engineered exosomes have a superior tendency to promote bone healing. This result aligns with the expected therapeutic advantages of exosome-mediated tissue-directed distribution and membrane-fixed display of peptides, suggesting that the exosome delivery method described in this invention is beneficial for improving the effective utilization of peptides in target tissues, thereby achieving a more significant improvement in bone structure even at low dosages.

[0060] (4) Histological analysis: The femoral bone sample was stained with HE and Masson staining, such as... Figure 10 As shown, the model group (OVX group) exhibited typical osteoporosis pathological features, with fractured and sparsely arranged trabeculae accompanied by significant enlargement of the medullary cavity. The free drug group (Teri group) showed some improvement in trabeculae, but the overall structure remained relatively porous. In contrast, the BT-Teri group showed a more continuous and denser trabecular structure, a more compact arrangement, a significantly reduced medullary cavity area, and a degree of tissue structure recovery approaching that of the normal control group (Sham group). The empty exosome group also showed some improvement in bone tissue, but the effect was weaker than that of the BT-Teri group. This phenomenon is consistent with the potential therapeutic advantages of exosome-mediated tissue-directed delivery and membrane fixation of therapeutic peptides, suggesting that the drug-loaded exosome system helps achieve a more significant improvement in tissue structure within the bone microenvironment.

[0061] (5) In vivo biocompatibility assessment: Mouse weight and behavioral activity were monitored during treatment, and no significant differences were observed among the groups. After treatment, major organs (heart, liver, spleen, lung, and kidney) were collected for HE staining analysis. The results showed that the tissue structure of each organ in the BT-Teri group was intact, and no obvious pathological changes such as inflammatory infiltration, congestion, or necrosis were observed (see...). Figure 11 This demonstrates that the engineered exosome system has good biocompatibility.

[0062] Example 5: In vitro functional validation of regulatory nucleic acids and construction strategy of exosome-assisted delivery system To screen nucleic acid payloads suitable for the delivery system of this invention, this embodiment uses miR-200b-3p, a candidate molecule obtained through bioinformatics screening, to verify its regulatory effect on osteoclast differentiation. Candidate miRNAs were obtained through differential analysis and functional annotation screening of sequencing data from osteoporosis-related clinical samples.

[0063] Mouse mononuclear macrophage leukemia cells (RAW264.7) were used as precursor cells and seeded in 6-well culture plates at a density of approximately 2 × 10⁻⁶ cells / well. 4 Up to 4×10 4 Cells / well were cultured overnight in DMEM complete medium containing 10% FBS. Transfection was performed when the cells adhered to the well and reached a confluence of approximately 60%–70%.

[0064] Transfection Treatment: To ensure sufficient miRNA levels in cells before induction of differentiation, this embodiment employs a transfection-before-induction strategy. 24 hours before the addition of the inducing factor, miR-200b-3p mimics, inhibitors, or negative controls (NC) are transfected into cells using a liposome transfection reagent (miRNA Transfection Reagent). The working concentration for transfection is set in the range of 30–100 nM (50 nM is preferred in this embodiment). The transfection process is performed according to the reagent instructions. 6–8 hours after transfection, complete culture medium is added or replaced, and the cells are cultured for another 24 hours post-transfection.

[0065] Induction of Differentiation: After transfection (i.e., 24 hours later), discard the old culture medium and replace it with complete culture medium containing inducing factors. Add nuclear factor κB receptor activator ligand (RANKL) to the culture medium at a final concentration of 20–100 ng / mL (50 ng / mL in this example) to initiate the osteoclast differentiation program. Induction culture lasts for 7–10 days (7 days in this example). During this period, replace half or all of the culture medium containing the same concentration of RANKL every 2 days (i.e., Day 3) to maintain the induction environment and remove metabolic waste.

[0066] TRAP staining assay: After induction, cells were fixed and stained with TRAP. Cells that were TRAP-positive (purple-red) and had ≥3 nuclei were defined as mature osteoclasts. Figure 12 As shown, the negative control group exhibited a large number of well-fused TRAP⁺ multinucleated cells; however, after transfection with miR-200b-3p mimics, the number of TRAP-positive cells was significantly reduced, and both cell volume and degree of fusion decreased. Quantitative results are as follows. Figure 13 As shown, the mimics group TRAP + The proportion of cells was significantly lower than that of the control group, indicating that miR-200b-3p can inhibit the formation and maturation of osteoclasts.

[0067] Osteoclastic marker gene detection: Cells were collected on day 7 of induced differentiation. Total RNA was extracted using the TRIzol method and reverse transcribed into cDNA. The regulatory role of miR-200b-3p in osteoclastic differentiation was verified at the molecular level using qPCR. In this example, typical markers of different stages of osteoclastic differentiation were selected for detection, including the upstream signaling molecule Rank1, the key transcription factor Nfatc1, and the maturation marker Trap (Acp5). Figure 14 As shown, compared with the negative control group, the expression levels of the above genes were significantly reduced after transfection with miR-200b-3p mimics, suggesting that miR-200b-3p can intervene in osteoclast differentiation and functional maturation at multiple stages. This result is consistent with the aforementioned TRAP staining results.

[0068] The above results demonstrate that miR-200b-3p can significantly inhibit osteoclast maturation and bone resorption-related functional phenotypes, and has the potential to be used as a therapeutic nucleic acid drug loaded into the exosome system of this invention.

[0069] Example 6: Optimization and Validation of Engineered Exosome Dual Loading Process and Construction of Delivery System To achieve efficient co-delivery of a therapeutic peptide (teriparatide) and a regulatory nucleic acid (miR-200b-3p) on the same exosome vector, this example investigated the impact of different combinations of two modification methods, "chemical transfection" and "bioorthogonal click reaction," on drug loading efficiency, aiming to screen for the optimal engineering construction strategy. The specific steps are as follows: This embodiment sets up a total of 5 experimental groups for comparative evaluation: (1) First spot transfection followed by transfection (BT-Teri-200m): The process route is to first perform bioorthogonal reaction to couple teriparatide, and then perform miR-200b-3p chemical transfection. (2) Transfection followed by spotting (BT-200m-Teri): The process route of first performing miR-200b-3p chemical transfection and then performing bioorthogonal reaction to couple teriparatide was adopted. (3) Simple transfection group (Exo + miRNA): used as a positive reference for internal loading efficiency; (4) Simple click group (Exo + Teri): used as a reference for biological orthogonal reaction; (5) Blank control group (Exo): BT-Exo was used as a negative control.

[0070] The core parameters of the reaction system were set as follows: exosome protein amount of approximately 50–200 μg, final concentration of DBCO-sulfo-NHS of 15 μM, final concentration of N3-PEG2-Teri of 6 μM, and feed amount of miR-200b-3p mimics of 1 μg.

[0071] Preparation process flow: (1) Preparation method of the first-point-then-transfer group (BT-Teri-200m): 1) The exosomes were mixed with DBCO-sulfo-NHS and incubated at 80 r / min for 1 hour under room temperature and dark conditions. After incubation, unreacted free DBCO was removed by centrifugation using a 30 kDa ultrafiltration tube to obtain the intermediate product. 2) Add N3-PEG2-Teri to the intermediate product, incubate at room temperature for 2 hours to carry out a click chemical reaction, and then wash by ultrafiltration again; 3) Finally, using the Exo-Fect transfection kit, the surface-modified exosomes were incubated with miR-200b-3pmimics at 37°C for 30–45 minutes (reaction volume 100–150 μL), and the product was recovered by the matching purification column.

[0072] (2) Preparation method of the first-transfer-then-point group (BT-200m-Teri): 1) Mix the original exosomes with miR-200b-3p mimics and Exo-Fect transfection reagent, and incubate at 37°C for 30–45 minutes; after incubation, recover the drug-loaded exosomes through a matching purification column to completely remove free nucleic acids; 2) Subsequently, the transfection product was incubated with DBCO-sulfo-NHS at room temperature for 1 hour, and free DBCO was removed by ultrafiltration; 3) Finally, N3-PEG2-Teri was added and incubated at room temperature for 2 hours. After ultrafiltration and washing, the final dual-carrier exosomes were obtained.

[0073] Loading efficiency detection: Total RNA was extracted from exosomes of each group and reverse transcribed into cDNA. U6 was used as an internal reference gene, and the relative enrichment of miR-200b-3p was detected by qPCR (calculated based on the Ct value of the 20x diluted sample).

[0074] Results Analysis: The test results are as follows: Figure 15As shown in Table 1, the miR-200b-3p expression level in the simple transfection group was in a high range, significantly higher than the baseline level of the blank control group. The miR-200b-3p expression level in the BT-200m-Teri group prepared by the "transfection-then-dip" process was similar to that in the simple transfection group, with no statistically significant difference between the two. The miR-200b-3p expression level in the BT-Teri-200m group prepared by the "dip-then-transfection" process was lower than that in the aforementioned two groups, with a relative expression level approximately 1 / 70th that of the "transfection-then-dip" group. These results indicate that pre-coupling a high-density teriparatide layer on the exosome surface can create a significant steric hindrance effect or alter the physicochemical properties of the membrane surface, thereby severely hindering the fusion of the transfection reagent with the exosome membrane.

[0075] Meanwhile, the final product prepared by the optimized process (transfection followed by spotting) was detected by ELISA, and the results confirmed the successful loading of teriparatide, indicating that internal nucleic acid transfection and external peptide modification can coexist and are compatible.

[0076] Conclusions and Application Extensions: In summary, this embodiment establishes "first performing internal nucleic acid chemical transfection, then performing surface peptide bioorthogonal modification" as the optimal preparation process of this invention, and successfully constructs bifunctional engineered exosomes that combine high-efficiency internal nucleic acid loading and surface peptide modification.

[0077] Table 1: Impact of different build strategies on miR-200b-3p loading efficiency

[0078] Based on the optimized dual-loading process and modular construction strategy described above, this invention proposes two types of exosome formulations adapted to different clinical needs: (i) Single-particle dual-load formulation: namely, BT-200m-Teri prepared in this embodiment, which co-loads therapeutic peptides and regulatory nucleic acids into the same exosome particle to achieve spatiotemporal synchronous co-delivery of the two drugs at the microscale; (ii) Combination Formulation: Bone-targeting exosomes loaded with teriparatide are prepared using the bioorthogonal strategy described in this invention, and bone-targeting exosomes loaded with miR-200b-3p are prepared using the nucleic acid loading strategy described in this invention (including but not limited to chemical transfection, electroporation, co-incubation, etc.). The two are mixed or used in combination according to the therapeutic dose ratio to achieve synergistic drug delivery. This combination form establishes a modular combination drug delivery strategy: targeting multiple pathological mechanisms of complex diseases, it removes the physical limitations of the loading ratio of the two drugs, allowing for flexible adjustment of the mixing ratio of the two functional exosomes according to different stages of disease development or individual patient differences, achieving truly personalized precision treatment. The successful verification of this embodiment shows that this delivery system is not only suitable for osteoporosis, but also has broad applicability and application prospects in the treatment of other complex diseases involving multi-target synergistic regulation.

[0079] The key design feature of this invention is that it constructs engineered exosomes that display bone-targeting ligands on their membrane surfaces and utilize click chemical coupling to promote bone-promoting peptides, while also being compatible with the loading of bone metabolism-regulating nucleic acids, thus providing a bone-targeting synergistic delivery system. The system utilizes bone-affinity ligands on the exosome membrane surface to achieve specific targeting and enrichment of bone tissue, exhibiting strong specificity. A flexible spacer-assisted bioorthogonal strategy is employed to anchor therapeutic peptides, effectively avoiding steric hindrance and maintaining the bioactivity of conformation-sensitive peptides such as teriparatide. The system also utilizes bone-promoting peptides and bone-inhibiting nucleic acids to achieve specific targeting and enrichment of bone tissue. The combined delivery (or combination drug administration) achieves dual regulation of bone metabolism balance; the preparation process is mild and protects the integrity of the carrier, showing good potential for clinical translation; by displaying bone-targeting ligands on the exosome membrane surface to improve the enrichment efficiency of exosomes on bone tissue, and by using a flexible spacer arm-mediated bioorthogonal coupling method, the bone-promoting therapeutic peptides are anchored on the exosome surface in a more stable and spatially accessible form, thereby improving their in vivo bioavailability, and providing a delivery platform with higher therapeutic efficacy and better adaptability for osteoporosis and other bone metabolism-related diseases.

[0080] The above description is merely a preferred embodiment of the present invention and does not constitute any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A bone-targeting exosome polypeptide delivery system based on a bio-orthogonal strategy, characterized in that: The delivery system comprises engineered exosomes as carriers and bone anabolic polypeptides: the bone anabolic polypeptides are anchored to the membrane surface of the engineered exosomes via covalent bonds formed by bio-orthogonal reactions; The membrane surface of the engineered exosomes displays fusion proteins, which are formed by fusing bone-targeting ligands with engineered exosome transmembrane scaffold proteins, and endow the engineered exosomes with the ability of directional recognition of bone matrix or bone microenvironment cells; The bone anabolic polypeptides are connected to the engineered exosomes via flexible spacers, which are connected to the terminal or side chain of the bone anabolic polypeptides, and reduce steric hindrance on the membrane surface to improve the accessibility of active fragments of the polypeptides.

2. The bone-targeting exosome polypeptide delivery system based on bio-orthogonal strategy of claim 1, wherein: The coupling structure of the bone anabolic polypeptides and the engineered exosomes is characterized in that: The amino groups on the membrane surface of the engineered exosomes are connected to diphenyl cyclooctyne groups via amide bonds; The N-terminus of the bone anabolic polypeptides is connected to azido groups and the flexible spacers; The azido groups are covalently connected to the diphenyl cyclooctyne groups via strain-promoted azido-alkyne cycloaddition.

3. The bone-targeting exosome polypeptide delivery system based on bio-orthogonal strategy of claim 1, wherein: The modified structure of the bone-promoting therapeutic polypeptide is: N3-PEG n - a polypeptide sequence - X; wherein, PEG n is a flexible linker arm of PEG2 to PEG12; the polypeptide sequence is teriparatide; X is a C-terminal modification group, X is selected from lysine, lysine-fluorescein isothiocyanate, biotin or hydroxyl.

4. The bone-targeting exosome polypeptide delivery system based on a bio-orthogonal strategy of claim 1, wherein: The bone-targeting ligands are selected from aspartic acid repeat sequences, SDSSD sequences, DSS6 sequences or CREDVW sequences; the exosome transmembrane scaffold proteins are selected from LAMP2B, CD63, CD9 or CD81.

5. The bone-targeting exosome polypeptide delivery system based on bioorthogonal strategy of claim 1, wherein: The bone anabolic polypeptides are selected from teriparatide, active fragments of parathyroid hormone-related proteins or analogs thereof having osteogenic activity; and the flexible spacers are polyethylene glycol chains.

6. The bone-targeting exosome polypeptide delivery system based on a bioorthogonal strategy of claim 1, wherein: The delivery system further comprises a regulatory nucleic acid; the regulatory nucleic acid is a bone metabolism regulatory nucleic acid, which is loaded in the lumen of the engineered exosomes or anchored to the membrane structure of the engineered exosomes; and the regulatory nucleic acid is an miRNA, siRNA or antisense oligonucleotide capable of inhibiting the differentiation of osteoclasts.

7. The bone-targeting exosome polypeptide delivery system based on bioorthogonal strategy of claim 6, wherein: The miRNA comprises miR-200b-3p or a homologous sequence having at least 90% homology with the mature sequence of miR-200b-3p.

8. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the bone-targeting exosome polypeptide delivery system based on bio-orthogonal strategies according to claim 6 and a pharmaceutically acceptable carrier; The pharmaceutical composition is in any one of the following forms: (i) an integrated preparation: the delivery system is a single-particle double-loaded exosome simultaneously loaded with the bone anabolic polypeptides and the bone metabolism regulatory nucleic acid; (ii) a combined preparation: comprising a first component and a second component, the first component is bone-targeting engineered exosomes loaded with bone anabolic polypeptides, and the second component is bone-targeting engineered exosomes loaded with bone metabolism regulatory nucleic acids; the first component and the second component are independently packaged or mixed preparations.

9. A method of preparing a bone-targeting exosome polypeptide delivery system based on a bio-orthogonal strategy according to any one of claims 1-5, characterized by: The method comprises the following steps: S1, constructing donor cells expressing bone-targeting fusion proteins, collecting and purifying engineered exosomes; S2, reacting with the amino groups on the surface of the engineered exosomes obtained in step S1 using a heterobifunctional crosslinking agent to introduce first bio-orthogonal reaction groups; S3, synthesizing bone anabolic polypeptides with second bio-orthogonal reaction groups and flexible spacers at the N-terminus; S4, mixing the osteogenic therapeutic polypeptide and the engineered exosome in a liquid phase system to perform a bio-orthogonal coupling reaction.

10. The method for preparing the bone-targeting exosome polypeptide delivery system based on a bioorthogonal strategy according to claim 9, characterized in that: After the step S4 is completed, the following step is further included: loading the bone metabolism regulating nucleic acid into the lumen of the engineered exosome by electroporation or transfection.

11. Use of the bone-targeting exosome polypeptide delivery system based on the bio-orthogonal strategy according to any one of claims 1-7 in the preparation of a drug for treating bone metabolic diseases. The bone metabolism disease includes osteoporosis, delayed fracture healing or bone defect.

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