Targeted delivery of molecules, particles and methods of making and using the same
By synthesizing targeted delivery molecules under mild conditions using a mercapto-acrylamide click chemistry method, the problem of easily destroyed peptide activity has been solved, enabling targeted treatment of osteoarthritis and reducing drug consumption and cytotoxicity.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- YUMEI MINGDE (CHENGDU) BIOMEDICAL TECH CO LTD
- Filing Date
- 2021-06-10
- Publication Date
- 2026-05-15
AI Technical Summary
In existing technologies, the activity of peptides is easily destroyed during targeted delivery, resulting in poor targeting and modification effects and making it difficult to achieve precise treatment of osteoarthritis.
Targeted delivery molecules were synthesized under mild conditions using a mercapto-acrylamide click chemistry method. Targeted delivery molecules that do not impair peptide activity were prepared by reacting polyethylene glycol molecules with maleamide and hydrophobic groups with peptide targeting factors. These molecules were then inserted into lipid bilayer particles to form targeted delivery particles.
It achieves the maintenance of peptide activity under mild conditions, enhances targeting, reduces drug loss and toxic side effects on normal cells, and improves the targeted efficacy of osteoarthritis treatment.
Smart Images
Figure CN113332442B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biomedical materials technology, specifically to a targeted delivery molecule, particle, its preparation method, and its application. Background Technology
[0002] Osteoarthritis is a panarticular disease involving changes in the hyaline articular cartilage, subchondral bone, ligaments, synovium, and periarticular muscle structures. The pathogenesis of osteoarthritis is complex, involving mechanical, inflammatory, and metabolic factors that ultimately lead to structural damage and synovial joint failure. Traditional non-surgical treatments can improve symptoms to some extent but cannot restore articular cartilage regeneration or alter the degenerative process. Surgical joint replacement surgery can improve function and quality of life in the long term; however, instability and infection are the most common limitations, often requiring further revision surgery.
[0003] In recent years, research on the use of stem cells and exosomes in the treatment of osteoarthritis has increased significantly. Studies have shown that stem cells can inhibit the migration, activation, and production of inflammatory cytokines in immune cells and reduce inflammation in osteoarthritic joints. Exosome-mediated repair of osteochondral defects is characterized by increased cell proliferation and infiltration, enhanced matrix synthesis, and a regenerative immunophenotype. Liposomes, as drug delivery carriers, have also been reported in the treatment of osteoarthritis. However, to achieve precise and effective drug delivery, the carrier or drug is usually physicochemically modified. These techniques should enhance their targeting ability to specific tissues or cells while minimizing the impact on the original properties of the drug. However, because peptides are biologically active substances, targeted modification of peptides often results in the destruction of peptide activity due to reaction conditions, thus limiting the application of peptides. Summary of the Invention
[0004] This invention provides a targeted delivery molecule, particle, preparation method, and application that does not destroy the activity of the polypeptide and effectively achieves targeted action.
[0005] The technical solution adopted in this invention is:
[0006] A method for preparing a targeted delivery molecule, comprising the following steps:
[0007] Step 1: Add a solution of peptide targeting factor B containing thiol groups to a polyethylene glycol molecule A solution containing maleamide groups and hydrophobic groups, and stir until fully reacted; wherein the molar ratio of A to B is 1:1;
[0008] Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule.
[0009] Furthermore, the polypeptide targeting factor is a polypeptide associated with articular cartilage or osteoarthritis diseases.
[0010] Furthermore, the polypeptide targeting factor includes one or a mixture of two of chondrocyte homing peptides and type II collagen targeting peptides in any proportion.
[0011] Furthermore, the reaction time in step 1 is 2 to 6 hours.
[0012] Furthermore, the hydrophobic group is one or two of oleoyl and cholesterol in any proportion.
[0013] The targeted delivery molecule obtained by any one of the methods described in claims 1 to 5.
[0014] A targeted delivery particle, wherein the targeted delivery particle is composed of a particle in which a hydrophobic group in any one of the targeted delivery molecules of claims 1 to 5 is inserted into a lipid bilayer.
[0015] Furthermore, the lipid bilayer-containing particles are one of the following: exosomes, liposomes, or cells that have a therapeutic effect on osteoarthritis.
[0016] An application of targeted delivery particles, wherein the targeted delivery particles serve as carriers for drugs used to treat osteoarthritis.
[0017] Furthermore, the targeted particles serve as gene vectors for targeted therapy of osteoarthritis.
[0018] The beneficial effects of this invention are:
[0019] (1) The present invention does not cause the peptide to lose its activity under relatively mild conditions; and the targeted delivery molecule for osteoarthritis is synthesized by mercapto-acrylamide click chemistry without the use of metal catalysts. The reaction conditions are mild and can maintain the bioactivity of the peptide to the greatest extent.
[0020] (2) The targeted delivery molecule obtained by the present invention inserts hydrophobic groups into particles containing liposome membranes, which can achieve targeted modification and endow them with the corresponding particle targeting effect; the particles can carry drugs or genes for treating osteoarthritis, etc., to achieve targeted treatment of osteoarthritis;
[0021] (3) The targeted delivery particles obtained by the present invention can reduce drug loss and toxic side effects on normal cells, and have good application prospects in the treatment of osteoarthritis. Attached Figure Description
[0022] Figure 1 This refers to the polyethylene glycol molecule containing maleamide groups and hydrophobic groups used in Example 1 of this invention.
[0023] Figure 2 This is a synthetic route diagram of the targeted delivery molecule in Example 1 of the present invention.
[0024] Figure 3 This is a schematic diagram of the targeted delivery particle synthesis process in this invention.
[0025] Figure 4 The NMR spectra of the targeted delivery molecules obtained in Examples 2 and 3 of this invention are shown.
[0026] Figure 5 This describes the application process of the targeted delivery particles obtained in Embodiment 4 of the present invention.
[0027] Figure 6 This is a fluorescence micrograph of the targeted delivery particles obtained in Example 4 of the present invention after co-culturing with chondrocytes.
[0028] In the figure: 1-lipid bilayer, 2-targeted delivery molecule, 3-exosome modified with CAP homing peptide, 4-chondrocyte, 5-CAP peptide binding site on chondrocyte membrane. Detailed Implementation
[0029] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.
[0030] A method for preparing a targeted delivery molecule, comprising the following steps:
[0031] Step 1: Add a solution of peptide targeting factor B containing thiol groups to a polyethylene glycol molecule A solution containing maleamide groups and hydrophobic groups, and stir until fully reacted; wherein the molar ratio of A to B is 1:1;
[0032] The peptide targeting factor is a peptide associated with articular cartilage or osteoarthritis diseases; including one or a mixture of two of the following in any proportion: chondrocyte homing peptide (CAP-C, sequence: DWRVIIPRPSA-C) and type II collagen targeting peptide (sequence: WYRGRL-C). Other types of peptides may also be selected as the peptide targeting factor.
[0033] Polyethylene glycol containing maleamide groups and hydrophobic groups has a molecular weight of 500 to 5000. The hydrophobic groups can be oleoyl groups, cholesterol, etc.
[0034] Solution A contains polyethylene glycol (PEG) molecules with maleamide and hydrophobic groups in a ratio of 1–2 mg to 0.4–0.6 mL of solvent. The PEG molecules are added to the solvent and dissolved by shaking at room temperature to obtain a PEG solution containing maleamide and hydrophobic groups. Solution B contains a thiol-containing peptide targeting factor in a ratio of 1–2 mg to 0.1–0.2 mL of solvent. The targeting peptide factor is dissolved by adding solvent and gently shaking to obtain a thiol-containing peptide solution.
[0035] After the addition is complete, stir the reaction at room temperature for 2–6 hours. The solvent is one of dimethyl sulfoxide, chloroform, N,N-dimethylformamide, and phosphate buffer solution.
[0036] Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule.
[0037] Polyethylene glycol molecules containing maleamide and hydrophobic groups and peptide targeting factors containing thiol groups are obtained using existing preparation methods, or ready-made products can be purchased directly.
[0038] The degree of substitution of the thiol-containing peptide targeting factor group is 50%–99%, and the degree of substitution of the hydrophobic group in the polyethylene glycol molecule containing maleamide group and hydrophobic group is 99%.
[0039] The preparation method of the thiol-containing polypeptide targeting factor group can be carried out according to the following method, but is not limited to the following method. Other existing methods can be used to prepare it.
[0040] 1) Select the resin based on the polypeptide sequence and determine the amount of resin to use (using 5g of resin as an example); weigh 5g of resin and place it in the reaction column, first soak it in about 50mL of dichloromethane for 1 minute, then dry it under vacuum. Add about 50mL of 20% hexahydropyridine DMF solution, and agitate under nitrogen at room temperature for 15 minutes. Then dry it under vacuum, wash the resin with DMF 9 times, and dry it under vacuum again.
[0041] 2) Add Fmoc-Ala-OH and the condensing agent TBTU according to the polypeptide sequence. Calculate the K value based on the weight of the resin and the degree of substitution.
[0042] K = Resin degree of substitution × Resin weight × 2 = 0.6 × 5 × 2 = 6
[0043] Fmoc-Ala-OH dosage = molecular weight × K / 1000 = 311.3 × 6 / 1000 = 1.86g
[0044] Condensing agent TBTU: 0.321 × K = 0.321 × 6 = 1.92 g
[0045] 0.5 mL of the alkaline reagent N-methylmorphine.
[0046] Add Fmoc-Ala-OH and TBTU to the reaction column according to the calculated amounts, then add about 50 mL of DMF and 0.5 mL of N-methylmorphine. Purge with nitrogen at room temperature for 25 minutes, then dry under vacuum. Wash the resin with DMF 6 times and dry under vacuum again.
[0047] Take about 10 to 30 resin particles and place them in a test tube. Add about 0.5 mL of Kaiser's reagent and heat in a dry incubator at 120°C for 1 to 2 minutes. The colorless resin indicates that the reaction is complete.
[0048] 3) Add about 50 mL of 20% hexahydropyridine DMF solution and agitate under nitrogen for 15 minutes at room temperature to remove Fmoc. Then dry the resin and wash it with DMF 9 times before drying.
[0049] 4) Synthesize the next amino acid sequentially according to the sequence, and repeat steps 2) and 3).
[0050] 5) After synthesizing the polypeptide according to the polypeptide sequence, wash the resin three times with dichloromethane and three times with diethyl ether, then dry it under vacuum and send it for cutting.
[0051] 6) Use 100 mL of TFA cutting fluid to perform a cutting reaction with the resin condensed with amino acids. Add diethyl ether to the filtrate to precipitate the peptide and obtain the crude product. After purification, the product is obtained.
[0052] Polyethylene glycol containing maleamide groups and hydrophobic groups, with cholesterol as an example of the hydrophobic group:
[0053] Cholesterol (0.25 mmol, 97 mg) was dissolved in 30 mL of tetrahydrofuran (THF), and K-Naphthalide solution was added under arsenic atmosphere until the solution turned slightly green and the stability time exceeded 10 minutes. Then, 200 mmol (10 mL) of ethylene oxide (dried on CaH2) was added.
[0054] The mixture was concentrated at -70°C to produce a choline solution. The reaction mixture was then allowed to be heated to room temperature. Polymerization of ethylene oxide proceeded for two days, producing a light brown, highly viscous solution. The polymer was recovered as diethyl ether via precipitation. The polymer was redissolved in chloroform, and precipitation was repeated four times to remove naphtha and unreacted monomers.
[0055] The resulting white powder was dissolved in benzene and lyophilized, yielding a polymer yield of approximately 90%. Cholesterol noted that the molecular weight of PEO always remained consistent with the initial monomer-to-initiator ratio. The method can be found in "Reversible Cell Aggregation Induced by Specific Ligand-Receptor Coupling".
[0056] A targeted delivery particle is composed of particles in which hydrophobic groups of a targeted delivery molecule are inserted into a lipid bilayer. The lipid bilayer-containing particle can be the surface of vesicles composed of a lipid bilayer, such as cells, exosomes, and liposomes, which have therapeutic effects on osteoarthritis, and can target and deliver these cells and vesicles, along with their contents, to cartilage.
[0057] Targeted delivery particles serve as carriers for drugs used to treat osteoarthritis or as gene vectors for targeted treatment of osteoarthritis.
[0058] Example 1
[0059] A method for preparing a targeted delivery molecule, comprising the following steps:
[0060] Step 1: Add 0.4 mL of dimethyl sulfoxide to 1 mg of polyethylene glycol (CLS-PEG1000-MAL) containing maleamide and hydrophobic groups, and dissolve by shaking at room temperature to obtain a polyethylene glycol solution containing maleamide and hydrophobic groups. The hydrophobic group is cholesterol, and its molecular structure is as follows. Figure 1 As shown.
[0061] Add 0.1 mL of dimethyl sulfoxide to 1 mg of a thiol-containing peptide targeting factor, gently shake to dissolve, and obtain a thiol-containing peptide reagent solution.
[0062] A solution of polypeptide targeting factor B containing thiol groups was added dropwise to a polyethylene glycol molecule A solution containing maleamide groups and hydrophobic groups, and the reaction was stirred for 2 hours; wherein the molar ratio of A to B was 1:1.
[0063] Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule. The reaction process is as follows: Figure 2 As shown.
[0064] Example 2
[0065] A method for preparing a targeted delivery molecule, comprising the following steps:
[0066] Step 1: Add 0.5 mL of dimethyl sulfoxide to 1.5 mg of polyethylene glycol molecules (CLS-PEG1000-MAL) containing maleamide and hydrophobic groups, and shake to dissolve at room temperature to obtain a polyethylene glycol molecule solution containing maleamide and hydrophobic groups.
[0067] 0.15 mL of dimethyl sulfoxide was added to 1.5 mg of a thiol-containing peptide targeting factor, and the solution was gently shaken to dissolve, yielding a thiol-containing peptide reagent solution. The thiol-containing peptide targeting factor was a thiol-containing chondrogenic peptide (CAP-C, sequence DWRVIIPPRPSA-C).
[0068] A solution of polypeptide targeting factor B containing thiol groups was added dropwise to a polyethylene glycol molecule A solution containing maleamide groups and hydrophobic groups, and the reaction was stirred for 2 hours; wherein the molar ratio of A to B was 1:1.
[0069] Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule (CLS-PEG-DWRVIIPPRPSA).
[0070] Example 3
[0071] A method for preparing a targeted delivery molecule, comprising the following steps:
[0072] Step 1: Add 0.5 mL of dimethyl sulfoxide to 1.5 mg of polyethylene glycol molecules (CLS-PEG1000-MAL) containing maleamide and hydrophobic groups, and shake to dissolve at room temperature to obtain a polyethylene glycol molecule solution containing maleamide and hydrophobic groups.
[0073] 0.15 mL of dimethyl sulfoxide was added to 1.5 mg of a thiol-containing peptide targeting factor, and the solution was gently shaken to dissolve, yielding a thiol-containing peptide reagent solution. The thiol-containing peptide targeting factor was a thiol-containing type II collagen targeting peptide (sequence: WYRGRL-C).
[0074] A solution of polypeptide targeting factor B containing thiol groups was added dropwise to a polyethylene glycol molecule A solution containing maleamide groups and hydrophobic groups, and the reaction was stirred for 2 hours; wherein the molar ratio of A to B was 1:1.
[0075] Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule (CLS-PEG-WYRGRL).
[0076] NMR spectrum as follows Figure 4 As shown in the figure, the MAL peak of the targeted delivery molecule at 6.75 ppm is smaller, and a new peak appears at the characteristic peak of the peptide (around 1.5 ppm). This indicates that the targeted delivery molecule was successfully synthesized.
[0077] Example 4
[0078] A method for preparing a targeted delivery molecule, comprising the following steps:
[0079] Step 1: Add 0.6 mL of dimethyl sulfoxide to 2 mg of polyethylene glycol molecules (CLS-PEG1000-MAL) containing maleamide and hydrophobic groups, and shake to dissolve at room temperature to obtain a polyethylene glycol molecule solution containing maleamide and hydrophobic groups.
[0080] 0.2 mL of dimethyl sulfoxide was added to 2 mg of a thiol-containing peptide targeting factor, and the solution was gently shaken to dissolve, yielding a thiol-containing peptide reagent solution. The thiol-containing peptide targeting factor was a thiol-containing chondrogenic peptide (CAP-C, sequence DWRVIIPPRPSA-C).
[0081] A solution of polypeptide targeting factor B containing thiol groups was added dropwise to a polyethylene glycol molecule A solution containing maleamide groups and hydrophobic groups, and the reaction was stirred for 2 hours; wherein the molar ratio of A to B was 1:1.
[0082] Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule.
[0083] By modifying exosomes with the aforementioned targeted delivery molecules, targeted exosomes can be obtained. For example... Figure 5 As shown.
[0084] In this embodiment, exosomes modified with chondrocyte homing peptide (CAP-EXO) and unmodified exosomes (EXO) were divided into two groups. The same amount of each group was co-cultured with chondrocytes for 24 hours. After discarding the culture medium, the cells were washed twice with PBS, and then fluorescent micrographs were taken of the cell surface. Figure 6 As shown in the figure, the exosomes modified with the targeted peptide exhibit a greater negative polarity onto chondrocytes (significantly more significant impact). Furthermore, they remain immobilized on the cells after PBS washing. In contrast, the unmodified exosomes show significantly weaker accumulation and retention on chondrocytes.
[0085] The targeted delivery molecule prepared in this invention has a chondrocyte-specific polypeptide targeting factor at one end, which can target cartilage tissue. The hydrophobic group at the other end can be physically inserted into a hydrophobic lipid bilayer. This allows the polypeptide targeting factor to be assembled onto the surface of vesicles composed of a lipid bilayer, such as cells, exosomes, and liposomes, which have therapeutic effects on osteoarthritis. These cells and vesicles, along with their contents, can be targeted and delivered to cartilage, showing promising application prospects in the treatment of osteoarthritis. Targeting cells and vesicles, along with their contents, to cartilage reduces drug loss and toxic side effects on normal cells. Furthermore, the reaction of this invention is carried out under relatively mild conditions, preserving the bioactivity of the polypeptide. The synthesis process does not use metal catalysts, achieving targeted modification and endowing the drug or its delivery carrier with targeted effects, thus improving the targeting efficacy against lesions.
Claims
1. A method for preparing targeted delivery particles, characterized in that, The targeted delivery particles are composed of particles in which a hydrophobic group is inserted into a lipid bilayer; one end of the targeted delivery molecule is connected to a polypeptide targeting factor, and the other end is a hydrophobic group. The targeted delivery molecule is prepared using the following method: Step 1: Add a solution of a thiol-containing polypeptide targeting factor B dropwise to a polyethylene glycol molecule A solution containing maleamide and hydrophobic groups, and stir until fully reacted; wherein the molar ratio of A to B is 1:1; the polypeptide targeting factor is a polypeptide related to articular cartilage or osteoarthritis; the polypeptide targeting factor includes one or a mixture of two of chondrocyte homing peptides and type II collagen targeting peptides in any proportion; the hydrophobic group is one or a mixture of two of oleoyl groups and cholesterol in any proportion; the reaction time in Step 1 is 2-6 hours, and the solvent for solutions A and B is one of dimethyl sulfoxide and phosphate buffer solution; Step 2: Dialyze and freeze-dry the solution obtained in Step 1 to obtain the desired targeted delivery molecule; The lipid bilayer-containing particles are exosomes.
2. The use of a targeted delivery particle prepared by the method described in claim 1 in the preparation of a carrier for a drug for treating osteoarthritis.
3. The application of a targeted delivery particle prepared by the method described in claim 1 in the preparation of a gene vector for targeted treatment of osteoarthritis.