A cell-penetrating peptide-oligonucleotide delivery system prepared based on bio-orthogonal click chemistry reaction and a preparation method and application thereof

Abstract

The application discloses a cell-penetrating peptide-oligonucleotide delivery system prepared based on a biological orthogonal click chemical reaction and a preparation method and application thereof. The cell-penetrating peptide with a cell membrane penetration function and the oligonucleotide with a biological function are connected through the biological orthogonal click chemical reaction, so that the biological activity of the biomolecule can be better maintained, a high fidelity is provided for a wide range of functional groups, no obvious toxic side effects are caused, and the oligonucleotide delivery is efficiently realized, thereby providing a new approach for preventing and treating osteoclast activation diseases.

Description

Technical Field

[0001] This invention discloses a cell-penetrating peptide-oligonucleotide delivery system based on bioorthogonal click chemistry, its preparation method, and its application. Background Technology

[0002] Oligonucleotide therapy is considered an effective means of treating gene-related diseases. Due to the special properties of deoxyribonucleotides and ribonucleotides, most oligonucleotides can interact with their homologous target molecules through Watson-Crick specific base pairing, thus enabling intervention at the genetic level. Theoretically, any disease caused by the overexpression of pathogenic genes can be treated with specific oligonucleotides. Oligonucleotide therapy offers promise for treating a variety of genetic and acquired diseases; however, achieving efficient oligonucleotide delivery remains a major challenge in this field, and further therapeutic applications of oligonucleotides are still limited by delivery efficiency. For successful cytoplasmic oligonucleotide delivery, excellent cell permeability, lysosomal escape, and release of oligonucleotides into the cytoplasm are prerequisites.

[0003] Cell-penetrating peptides (CPPs) are short amphiphilic or cationic peptides (typically less than 30 amino acids) used to deliver chemical drugs, nucleic acids, peptides, and macromolecules, or in combination with other carriers (e.g., LNPs, polymeric vesicles, and exosomes) for the entry of exogenous substances into cells and even organelles. Currently, the main method for delivering oligonucleotides using CPPs is to combine them with effector nucleic acids to form non-covalent nanoparticles, while methods for covalently binding cell-penetrating peptides with effector nucleic acids have been less explored. The heterogeneity and potential for aggregation of nanoparticles pose challenges to delivery, and the high molar ratio of CPPs to oligonucleotides leads to higher cytotoxicity. In contrast, covalent coupling offers well-defined structure and yield, higher reproducibility, a lower CPP to oligonucleotide molar ratio, and less cytotoxicity. MicroRNAs (miRNAs) are fundamental regulators of various biological and pathological environments and have recently gained increasing clinical attention as promising therapeutic targets or biomarkers. Recent studies have demonstrated that inhibiting miRNA-182 has a significant protective effect against pathological bone loss.

[0004] Bioorthogonal click chemistry, a novel click chemistry approach, such as the dibenzylcyclooctyne-azide (N3-DBCO) cycloaddition chemistry, offers advantages in terms of reaction speed, thoroughness, and specificity. Its high fidelity, applicable to a wide range of functional groups, allows for better preservation of the bioactivity of biomolecules. Therefore, developing effective delivery strategies to improve the safety and therapeutic efficacy of oligonucleotides is crucial. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides an oligonucleotide with membrane-penetrating function, its preparation method, and its applications. A clickable cell-penetrating peptide oligonucleotide delivery system is obtained through bioorthogonal click chemistry. The Mal-DBCO-modified R9 cell-penetrating peptide can be rapidly, thoroughly, and specifically bioorthogonally clicked covalently coupled with Azido-modified oligonucleotide sequences. This provides a new method for oligonucleotide delivery and offers a novel approach for the prevention and treatment of osteoclast activation diseases.

[0006] To address the problems in the existing technology, the technical solution adopted by this invention is as follows:

[0007] A cell membrane penetration peptide-oligonucleotide delivery system based on bioorthogonal click chemistry is disclosed. The system comprises a Mal-DBCO-modified R9 cell membrane penetration peptide and an Azido-modified oligonucleotide. The Mal-DBCO-modified R9 cell membrane penetration peptide and the Azido-modified oligonucleotide are prepared via bioorthogonal click chemistry. The structural formula of the Mal-DBCO-modified R9 cell membrane penetration peptide is shown below.

[0008]

[0009] The above-mentioned method for constructing a cell-penetrating peptide-oligonucleotide delivery system based on bioorthogonal click chemistry includes the following steps:

[0010] Step 1: DBCO-modified R9 cell membrane-penetrating peptides were synthesized using the Fmoc solid-phase synthesis method.

[0011] Step 2: Synthesize Azido-modified oligonucleotides and modify them with the fluorescent group Cy3;

[0012] Step 3: The Mal-DBCO-modified R9 cell-penetrating peptide and the Azido-modified oligonucleotide are covalently linked through a bioorthogonal click chemistry reaction to obtain a cell-penetrating peptide-oligonucleotide delivery system.

[0013] The above-mentioned cell-penetrating peptide-oligonucleotide delivery system is used in the preparation of drugs for the prevention or treatment of osteoclast-related diseases.

[0014] As an improvement, the osteoclast-related disease is osteoporosis.

[0015] A pharmaceutical composition wherein the effective carrier of the pharmaceutical composition is a cell-penetrating peptide-oligonucleotide delivery system.

[0016] Beneficial effects:

[0017] Compared with existing technologies, this invention provides a cell-penetrating peptide-oligonucleotide delivery system based on bioorthogonal click chemistry, its preparation method, and its application. By linking cell-penetrating peptides with cell membrane penetration function to biologically functional oligonucleotides through bioorthogonal click chemistry, the biomolecules' biological activity can be better maintained. It has high fidelity to a wide range of functional groups, no obvious toxic side effects, and efficiently achieves oligonucleotide delivery. The oligonucleotide core sequence can inhibit miR-182, thereby inhibiting osteoclast activation and providing a new approach for the prevention and treatment of osteoclast activation diseases. Attached Figure Description

[0018] Figure 1 The results are from the HPLC detection of cell-penetrating peptides.

[0019] Figure 2 ESI-MS results for cell-penetrating peptides;

[0020] Figure 3 The main peaks detected by 1H-NMR analysis of cell membrane-penetrating peptides;

[0021] Figure 4 The results are HPLC detection results for two oligonucleotides: (a) is a bioclickable azide-modified oligonucleotide, and (b) is a bioclickable azide-modified oligonucleotide modified with Cy3 fluorescent group.

[0022] Figure 5 MS detection results for two oligonucleotides: (a) bioclickable azide-modified oligonucleotide, (b) bioclickable azide-modified oligonucleotide modified with Cy3 fluorescent group;

[0023] Figure 6 Urea-PAGE gel electrophoresis of cell-penetrating peptides and oligonucleotides after a bioorthogonal reaction: 1 is oligonucleotide, 2 is cell-penetrating peptide, 3 is cell-penetrating peptide-oligonucleotide, 4 is oligonucleotide-Cy3, 5 is cell-penetrating peptide, 6 is cell-penetrating peptide-oligonucleotide-Cy3, M is RNA Ladder, and nt is the number of bases.

[0024] Figure 7 The results of CCK-8 toxicity assay for cell membrane-penetrating peptide-oligonucleotides are as follows: ns, no statistical significance.

[0025] Figure 8 The results of cell live / dead staining for each experimental group;

[0026] Figure 9 The results of the erythrocyte hemolysis experiment for each experimental group;

[0027] Figure 10The results of flow cytometry analysis of the number of Cy3-positive cells in each group;

[0028] Figure 11 The staining results for cell membrane-penetrating peptide-oligonucleotide-Cy3 and lysosomes at 3h, 6h, 12h and 24h are shown. Cy3 is an oligonucleotide labeled red; Lyso is a lysosomal labeled green; and DAPI is a nuclear label blue.

[0029] Figure 12 The results were used to quantify the fluorescence intensity of cell membrane-penetrating peptide-oligonucleotide-Cy3. **, p<0.01, indicating statistical significance.

[0030] Figure 13 TRAP staining results of osteoclasts in each experimental group;

[0031] Figure 14 Genetic results related to osteoclasts generated from high-throughput RNA sequencing data;

[0032] Figure 15 The top 20 results for pathway enrichment from the Kyoto Encyclopedia of Genes and Genomes (KEGG);

[0033] Figure 16 Gene set enrichment analysis (GSEA) showed that cell-penetrating peptide-oligonucleotides significantly downregulated the enrichment of osteoclast differentiation pathways. Detailed Implementation

[0034] The following embodiments are provided to enable those skilled in the art to more fully understand the present invention, but do not limit the invention in any way.

[0035] I. Materials and Methods

[0036] 1. Materials

[0037] 1.1 Reagents and Experimental Equipment

[0038] 1.1.1 Main Medicines and Reagents

[0039] The Mal-DBCO-modified R9 cell membrane-penetrating peptide and the Azido-modified oligonucleotide (sequence design derived from Zhao et al. (Bone protection by inhibition of microRNA-182. Nat Commun. 2018 Oct5; 9(1):4108.)) were synthesized with the assistance of Qiangyao Biotechnology Co., Ltd. (Suzhou), China; CCK-8 and live / dead staining kits were purchased from Yeasen, China; TRAP staining kits were purchased from Bizhong Biotechnology, China; Lyso-Tracker Green (lysosomal green fluorescent probe), TRIzol, paraformaldehyde, and Urea-PAGE gel preparation kits were purchased from Beyotime Biotechnology Co., Ltd., China. Lipofectamine 2000 was purchased from Thermo Fisher Scientific, USA. Recombinant mouse nuclear factor κB receptor activator ligand (RANKL) was purchased from R&D Systems, USA.

[0040] 1.1.2 Main Instruments

[0041] The equipment included an Axiovert 40C optical microscope (Zeiss, Germany), an ELISA reader (Biotec, USA), and a cell culture system.

[0042] 1.2 Experimental Cells

[0043] RAW264.7 macrophages were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences, and the cells were passaged and cultured according to the guidelines provided by the bank.

[0044] 2. Experimental Methods

[0045] 2.1 Dissolution of cell-penetrating peptides and oligonucleotides and bioorthogonal reactions

[0046] Cell-penetrating peptides and oligonucleotides were dissolved in DEPC water at a concentration of 2000 nM. Equal volumes and molar masses of the cell-penetrating peptides and oligonucleotides were mixed, and a bioorthogonal reaction was carried out at room temperature. The resulting solution was then temporarily stored in a refrigerator at 4°C for later use.

[0047] 2.2 Urea-PAGE gel electrophoresis

[0048] Prepare the Urea-PAGE gel according to the instructions: Completely dissolve 30% Acr-Bis (29:1), TBE (5X), and urea, or add an appropriate amount of DEPC water to completely dissolve them, then bring the volume to a final level with DEPC water. After the gel solidifies, load the sample and perform electrophoresis at a constant voltage of 120V. After electrophoresis, remove the gel and place it in a clean container to wash with DEPC water. Stain with nucleic acid staining solution on a shaker at room temperature. After washing with DEPC water, observe the staining results after electrophoresis using a gel imaging device.

[0049] 2.3 Cytotoxicity assay (CCK-8)

[0050] RAW264.7 macrophages were seeded in 96-well plates and cultured overnight. After discarding the original culture medium, it was replaced with a medium containing different concentrations of cell-penetrating peptides-oligonucleotides, specifically serially diluted from 1000 nM to 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM, and 7.8125 nM. After culturing in a cell culture incubator for 2 days, the medium was replaced with CCK-8 solution for incubation, and the OD value at 450 nm was measured using a microplate reader to determine cytotoxicity.

[0051] 2.4 Live and Dead Staining

[0052] RAW264.7 macrophages were cultured for 1 day in media containing cell-penetrating peptides, oligonucleotides, cell-penetrating peptide-oligonucleotides, Lipo2000, or Lipo2000 / oligonucleotides. The original media were discarded. 5 μL of Calcein-AM solution (2 mM) and 15 μL of PI solution (1.5 mM) were added to 5 mL of 1× reaction buffer, mixed thoroughly, and added to each well. The cells were incubated at 37°C for 15 min. Live cells (yellow-green fluorescence) and dead cells (red fluorescence) were simultaneously detected under a fluorescence microscope using a 490±10 nm excitation filter.

[0053] 2.5 Lysosomal escape staining

[0054] Add 1 μl of Lyso-Tracker Green to 13.33 ml of cell culture medium and incubate at 37°C for 30 minutes. Remove the cell culture medium, add the Lyso-Tracker Green staining working solution prepared in step 1 and incubated at 37°C, and incubate with the cells at 37°C for 60 minutes. Remove the Lyso-Tracker Green staining working solution, add fresh cell culture medium, and then observe using a fluorescence microscope.

[0055] 2.6 RANKL-induced osteoclast differentiation

[0056] RAW264.7 macrophages were seeded in 48-well plates and cultured overnight. After discarding the original culture medium, cell-penetrating peptides, oligonucleotides, and cell-penetrating peptide-oligonucleotides were added. After transfection overnight, the culture medium was removed, and fresh cell culture medium containing RANKL was added to induce osteoclast differentiation. The medium was changed every 2-3 days until osteoclast formation was achieved.

[0057] 2.7 Tartrate-resistant acid phosphatase staining

[0058] 2.7.1 Components of the reagent kit

[0059]

[0060] 2.7.2 Staining Procedure

[0061] (1) Discard the culture medium and wash the well plate with PBS buffer;

[0062] (2) Preheat two tubes of 8mL Solution I (labeled as tube A and tube B respectively) to 37°C;

[0063] (3) Add 200 μL of paraformaldehyde fixative to each well and fix at room temperature for 10 min;

[0064] (4) Discard the fixative and wash with PBS buffer;

[0065] (5) Preparation of colorimetric substrate: Take the preheated tube A above, add 80uL Solution II, and mix thoroughly;

[0066] (6) Add the chromogenic substrate to each well plate and incubate at 37°C for 30 min in a humidified chamber under dark conditions;

[0067] (7) 5 minutes before the end of the above steps, take 160uL of solution III and 160uL of solution IV, mix them thoroughly and let them stand at room temperature for 2 minutes.

[0068] (8) Preparation of display solution: Add the mixture from step 7 to the preheated tube B (containing 8 mL Solution I) and mix well;

[0069] (9) After removing the display substrate, add 200 μL of the chromogenic solution to each well plate and incubate at 37°C in the dark for 20-30 min. Adjust the incubation time until the TRAP staining results can be clearly displayed.

[0070] (10) Rinse each well with deionized water to terminate the reaction;

[0071] (11) Microscopic observation and analysis: osteoclasts were positive for TRAP staining and had three or more nuclei.

[0072] 2.8 RNA Sequencing Analysis

[0073] RAW264.7 macrophages were stimulated with RANKL with or without cell-penetrating peptide-oligonucleotide treatment. Total RNA was then extracted from the cells using TRIzol Reagent, and subsequent RNA sequencing analysis was performed by Genewiz (China). Readings were compared with reference genes using Hisat2 software, with differentially expressed genes maintaining a fold change >1.5 and p <0.05. KEGG enrichment analysis was performed using differentially expressed genes.

[0074] 2.9 Statistical Analysis

[0075] Statistical analysis and graphing were performed using Graphpad Prism version 8.0. All normally distributed data are expressed as mean ± standard deviation (Mean ± SD). Student's t-test was used to analyze statistical differences between two groups, and one-way ANOVA was used to analyze differences between more than two groups. A p-value < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01).

[0076] II. Results

[0077] 1. Characterization analysis of cell-penetrating peptides

[0078] By high performance liquid chromatography (HPLC, purity >95%), Figure 1 After purification, the synthesized molecules were detected by electrospray ionization mass spectrometry (ESI-MS). The monoisotopic mass number [M+5H]5+ of CPP-DBCO was determined to be 581.2, corresponding to its theoretical molecular weight of 2,899.2. Figure 2 ).

[0079] 1H-NMR analysis also revealed all diagnostic peaks for the main synthetic molecule, further confirming the success of the molecule. Figure 3 The above results confirm the successful chemical synthesis of bioclickable DBCO-modified cell-penetrating peptides.

[0080] 2. Oligonucleotide Characterization

[0081] To clearly identify the delivery effectiveness of the bio-clickable delivery strategy, a Cy3 fluorescent group was added to the end of the oligonucleotide. The oligonucleotide was purified by HPLC (purity >95%). Figure 4 ESI-MS results showed that the monoisotopic masses of the azide-modified oligonucleotide and the azide-modified oligonucleotide-Cy3 were 7,941.8 and 8,546.9 Da, respectively, corresponding to their theoretical molecular weights of 7,944.6 and 8,550.7 Da. Figure 5 The successful chemical synthesis of bio-clickable azide-modified oligonucleotides and azide-modified oligonucleotide-Cy3 was verified.

[0082] 3. Biological orthogonal reaction

[0083] The synthesized product was analyzed by Urea-PAGE gel electrophoresis after mixing cell-penetrating peptides and oligonucleotides. Figure 6As shown, after the reaction, both increased in mass, and their gel electrophoresis rates decreased compared to oligonucleotides alone, while the transmembrane peptide group did not show up. By comparing all the bands, it can be seen that the electrophoretic rate of oligonucleotide-Cy3 was slower than that of oligonucleotides alone, and the electrophoretic rate of oligonucleotide-Cy3, which underwent a bioorthogonal reaction connecting cell transmembrane peptides, was the slowest.

[0084] 4. Cytotoxicity analysis

[0085] Before investigating delivery efficiency, we first analyzed the cytotoxicity of our delivery system.

[0086] Firstly, the CCK-8 assay revealed that the cell-penetrating peptide-oligonucleotide showed no significant cytotoxicity and had no significant effect on cell proliferation. The absorbance at each concentration gradient was not statistically significant compared to the control group. Figure 7 Subsequently, live / dead staining was performed. It was observed that no significant cell death was seen when using cell-penetrating peptides and oligonucleotides alone, or when using cell-penetrating peptide-oligonucleotide combination. Conversely, using the classic transfection reagent Lipofectamine 2000 resulted in a relatively increased number of dead cells, indicating that the delivery system constructed in this invention has lower toxicity. Figure 8 The blood compatibility of different samples was assessed using a erythrocyte hemolysis assay. In stark contrast to the positive control results, no significant erythrocyte hemolysis was observed in the cell-penetrating peptide group, oligonucleotide group, and cell-penetrating peptide-oligonucleotide group. Figure 9 ).

[0087] 5. Membrane penetration efficiency analysis

[0088] After ruling out cytotoxicity, delivery efficiency was then assessed. Our synthesized oligonucleotides contain a Cy3 fluorescent group, allowing for detection by flow cytometry or fluorescence microscopy.

[0089] First, flow cytometry was used to count transfected macrophages at 6h, 12h, and 24h to determine delivery efficiency, with the classic transfection reagent Lipofectamine 2000 used as a control. It was observed that the efficiency of oligonucleotides entering cells autonomously was low, while the transmembrane peptide-oligonucleotide delivery system significantly increased delivery efficiency. The percentages of positive cells at 6h, 12h, and 24h were 53.7%, 54.1%, and 81.4%, respectively, while the percentages of positive cells in the Lipofectamine 2000 group were 27.1%, 38.8%, and 69.2%, respectively. These results indicate that the transmembrane peptide-oligonucleotide delivery system has superior intracellular delivery capability. Figure 10Simultaneously, lysosomal staining was performed to further examine the lysosomal escape capability of the transmembrane peptide-oligonucleotide delivery system. Immunofluorescence staining images showed that at 3h, 6h, 12h, and 24h, the fluorescence intensity of Cy3-labeled oligonucleotides increased with time, indicating that the oligonucleotides entered the cell. At the same time, green fluorescently labeled lysosomes were visible, and the oligonucleotides were not degraded by the lysosomes. Figure 11 and Figure 12 ).

[0090] 6. Inhibits osteoclast formation

[0091] The biological effects of the transmembrane peptide-oligonucleotide delivery system were further analyzed. TRAP staining revealed that the control group had a high number and large area of ​​osteoclasts, while treatment with the transmembrane peptide-oligonucleotide system significantly reduced the number of osteoclasts and made the cells significantly smaller. Figure 13 Based on the above results, RANKL-induced macrophages, with or without cell-penetrating peptide-oligonucleotide treatment, were further collected for high-throughput RNA sequencing.

[0092] The results showed that the expression of osteoclastogenesis-related genes such as ACP5, MMP9, and ATP6V0D2 was relatively low after treatment with cell-penetrating peptide-oligonucleotides. Figure 14 Further analysis of transcriptome sequencing data using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that genes associated with osteoclast differentiation were among the top 20 most enriched pathways in the KEGG dataset. Figure 15 Furthermore, studies using gene set enrichment analysis (GSEA) on sequencing data further confirmed that the enrichment of osteoclast differentiation pathways in the cell membrane-penetrating peptide-oligonucleotide genome was significantly downregulated. Figure 16 ).

[0093] This invention utilizes bioorthogonal click chemistry to link cell-penetrating peptides with cell-penetrating capabilities to biologically functional oligonucleotides. The Mal-DBCO-modified R9 cell-penetrating peptide can be rapidly, thoroughly, and specifically bioorthogonally clicked covalently coupled with Azido-modified oligonucleotide sequences, thus better preserving the bioactivity of the biomolecules and achieving low-toxicity and high-efficiency oligonucleotide delivery. This provides a new strategy for the delivery of oligonucleotide drugs and the treatment of osteoclast activation diseases.

Claims

1. A cell-penetrating peptide-oligonucleotide delivery system prepared based on bioorthogonal click chemistry, characterized in that, The cell membrane penetration peptide-oligonucleotide delivery system comprises Mal-DBCO-modified R9 cell membrane penetration peptide and Azido-modified oligonucleotides; wherein, the Mal-DBCO-modified R9 cell membrane penetration peptide and Azido-modified oligonucleotides are prepared by bioorthogonal click chemistry reaction; the structural formula of the Mal-DBCO-modified R9 cell membrane penetration peptide is shown below: ， The oligonucleotide core sequence is capable of inhibiting miR-182, and the oligonucleotide sequence is 5′-TTCTACCATTGCCAA-3′.

2. A method for constructing a cell-penetrating peptide-oligonucleotide delivery system based on bioorthogonal click chemistry as described in claim 1, characterized in that, Includes the following steps: Step 1: DBCO-modified R9 cell membrane-penetrating peptides were synthesized using the Fmoc solid-phase synthesis method. Step 2: Synthesize Azido-modified oligonucleotides and modify them with the fluorescent group Cy3; Step 3: The Mal-DBCO-modified R9 cell-penetrating peptide and the Azido-modified oligonucleotide are covalently linked through a bioorthogonal click chemistry reaction to obtain a cell-penetrating peptide-oligonucleotide delivery system.

3. The application of the cell-penetrating peptide-oligonucleotide delivery system according to claim 1 in the preparation of drugs for the prevention or treatment of osteoclast-related diseases, characterized in that, The osteoclast-related disease is osteoporosis.

4. A pharmaceutical composition, characterized in that, The effective carrier of the pharmaceutical composition is the cell-penetrating peptide-oligonucleotide delivery system as described in claim 1.

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