Virus-like structure nanometer adapter and application thereof

Abstract

The invention discloses a virus-like structure nano adapter and application thereof, on the basis of hepatitis B virus core protein Uniprot P03146 of which nucleic acid binding domains at 145-183 sites of a C tail end are removed, CBP as shown in SEQ ID NO.01 is respectively inserted into a spike part and an N tail end of the hepatitis B virus core protein Uniprot P03146. The invention is constructed based on collagen combined polypeptide CBP designed by bionic decorrin, aims to imitate the interaction between natural decorrin and collagenous fiber, can efficiently cross over corneal epithelium barrier and repair corneal stroma in a non-photo-crosslinking manner, and solves the limitation of existing clinical treatment.

Description

Technical Field

[0001] The present invention belongs to the technical fields of pharmaceutical carriers and ophthalmic therapeutic drugs, and particularly relates to a viroid-like structure nano adapter and its application. Background Art

[0002] Corneal ectatic diseases are characterized by corneal weakness and irregular remodeling of the corneal structure, specifically manifested as corneal thinning, protrusion, and irregular astigmatism. Keratoconus is a bilateral, progressive, and non-inflammatory corneal ectatic disease. This disease causes an increase in the steepness of the paracentral cornea and an expansion of the corneal thinning area, thereby leading to a continuous increase in myopia, irregular astigmatism, and vision loss, and forming corneal scars in the late stage. As the disease progresses, visual impairment usually appears starting from adolescence and continues to progress significantly until around 35 years old, which significantly reduces the quality of life of patients. In daily life, performing simple activities such as driving, recognizing the faces of people in the distance, or reading road signs may become difficult. Therefore, there is an urgent need to immediately provide effective treatment techniques for patients with keratoconus. Currently, clinical treatments for keratoconus include surgical and non-surgical methods, which focus on restoring vision and terminating the progression of the disease. In the early stage of the disease, non-surgical treatment options such as glasses or contact lenses can improve vision; however, in the late stage of the disease, auxiliary devices cannot maintain vision.

[0003] Corneal cross-linking (CXL) is the only clinically proven effective treatment for halting the progression of keratoconus. This procedure increases corneal biomechanical stiffness, potentially helping to preserve vision in the early stages. CXL achieves biomechanical strengthening of the cornea by enhancing crosslinks between collagen structures within the corneal stroma. Current clinical approaches typically use riboflavin as a photoinitiator and ultraviolet (UV) light to induce crosslinks between collagen fibrils within the corneal stroma. In healthy corneas, copper and lysine oxidase can induce collagen crosslinks, but significantly reduced corneal lysine oxidase levels lead to insufficient collagen crosslinking. In clinical CXL procedures involving riboflavin / UV light, crosslinking of free histidine groups on collagen promotes the formation of additional covalent crosslinks in keratoconus. Although riboflavin / UV light-based CXL is the most effective nonsurgical option for treating keratoconus, it still has significant limitations and complications. For example, riboflavin-based CXL regimens are not suitable for patients with corneal thicknesses of 400 μm or less. This is because the corneal endothelial cells beneath the corneal stroma are negatively affected by UV exposure, leading to irreversible endothelial toxicity and corneal edema. Furthermore, clinical CXL surgery can also present postoperative complications such as permanent corneal haze, persistent corneal edema, and corneal endothelial failure. Riboflavin-based CXL regimens are ineffective in approximately 8% of patients with keratoconus, leaving keratoplasty as the only possible treatment option. Despite various improvements to the standard CXL procedure, none have significantly improved the efficacy of CXL treatment. Therefore, the development of novel cross-linking technologies for keratoconus is a critical scientific issue that urgently needs to be addressed and represents an international research frontier in this field.

[0004] The corneal stroma is primarily composed of parallel bundles of collagen fibers, each composed of smaller collagen fibrils. The spacing between each collagen fiber bundle is very uniform, and they extend in nearly the same direction. This arrangement ensures that light passes through the cornea in a straight line without scattering, thus maintaining visual clarity. Several bioactive proteins play a key role in assisting collagen fiber arrangement. For example, members of the small leucine repeat proteoglycan (SLRP) family are widely distributed in the corneal stroma. These proteins interact with each other in a compensatory and synergistic manner, regulating the formation and assembly of collagen fibers in the stroma and maintaining a highly ordered collagen fiber arrangement. This regular arrangement is one of the key factors in maintaining corneal transparency. In summary, the orderly arrangement of collagen fibers in the corneal stroma is crucial for maintaining the functional properties of the cornea. Any disruption of this natural order can lead to a loss of corneal transparency. Therefore, a deeper understanding of the assembly and repair mechanisms of the corneal stroma is of great clinical significance.

[0005] In addition, during corneal repair treatment, how to deliver drugs across the corneal epithelial barrier into the corneal stroma is an important challenge. Corneal epithelial cells form an effective barrier that restricts the penetration of most molecules. According to relevant research, the corneal epithelium consists of 6 to 8 layers of cells, which form tight junctions between these cells, and the paracellular pore size is about 2 nanometers (nm), which means that only small molecules with a molecular weight below 500 Daltons (Dalton, Da) can pass through this tight structure. Therefore, the molecules that can penetrate the intercellular spaces of corneal epithelial cells must be very small. For example, small molecules such as water and oxygen can easily cross this barrier, while larger molecules such as polypeptides or proteins are difficult to penetrate naturally. To improve the ability of drugs to penetrate the cornea, researchers have explored various methods, such as using penetration enhancers to temporarily increase the permeability of the cornea. Borneol, as a small molecule lipophilic terpene, has been proven to have a selective penetration-enhancing effect on hydrophilic and macromolecular drugs. It can promote penetration by changing the stratum corneum of the skin and loosening the tight junction structure between endothelial cells. In addition, EDTA can open the tight junctions between epithelial cells, thereby promoting the penetration of drugs through the paracellular pathway; while Tween 80 can improve the cell membrane permeability by disturbing the phosphatidyl chains. However, when considering any means to enhance corneal permeability, caution must be exercised to ensure that the natural protective function of the cornea is not damaged and unnecessary side effects are avoided. Therefore, it is very important for researchers who hope to develop an ocular drug delivery system to understand corneal physiology and its barrier characteristics. At the same time, when designing new drug delivery technologies, a balance also needs to be achieved between safety and effectiveness. Summary of the Invention

[0006] The object of the present invention is to overcome the defects of the prior art and provide a viroid-like structure nanoadapter.

[0007] Another object of the present invention is to provide the application of the above viroid-like structure nanoadapter.

[0008] The technical solution of the present invention is as follows:

[0009] A viroid-like structure nanoadapter is based on the hepatitis B virus core protein Uniprot P03146 with the nucleic acid binding domain at positions 145 - 183 of the C-terminus removed. The spike part and the N-terminus are respectively inserted with CBP as shown in SEQ ID NO.01.

[0010] In a preferred embodiment of the present invention, its amino acid sequence is as shown in SEQ ID NO.02.

[0011] More preferably, its nucleotide sequence is as shown in SEQ ID NO.03.

[0012] Use of the viroid - like structure nano - linker in the preparation of a composition for promoting corneal stroma repair.

[0013] A composition for promoting corneal stroma repair, the active ingredient of which comprises the above - mentioned viroid - like structure nano - linker.

[0014] The beneficial effects of the present invention are as follows:

[0015] 1. The present invention makes full use of the advantages of the viroid - like structure, not only enhancing the binding efficiency of the biomimetic decorin polypeptide CBP to collagen, but also realizing an efficient transcytosis process mediated by the caveolae pathway, and precisely transporting the present invention into the corneal stroma.

[0016] 2. Inside the corneal stroma, the present invention promotes the stable connection of collagen fibers through a non - covalent binding mode that mimics the formation of natural stroma, thereby achieving the effect of stabilizing the corneal stroma and repairing damaged corneas.

[0017] 3. The present invention can complete the repair process without light conditions, has higher safety and lower operation complexity, providing new possibilities for future clinical treatment. Description of the Drawings

[0018] Figure 1 ipTM (a) and pTM (b) scores showing the predicted VLS NEs dimer and tetramer complex structures in Example 1 of the present invention.

[0019] Figure 2 Showing the differences between the dimer (a) and tetramer (b) complex structures of VLS NEs and the wild - type HBc VLPs structures in Example 1 of the present invention, with indicators including Interface_PAE and RMSD, etc.

[0020] Figure 3 Multiple indicators for evaluating the quality of the dimer (a) and tetramer (b) complex structures of VLS NEs in Example 1 of the present invention, including dG / dSASA, BUNS, and packstat, etc.

[0021] Figure 4 Showing the molecular weights of VLS NEs and its backbone protein HBc149 detected by SDS - PAGE in Example 1 of the present invention.

[0022] Figure 5 Showing the TEM analysis (a) and DLS analysis (b) of VLS NEs in Example 1 of the present invention.

[0023] Figure 6 Showing the thermodynamic stability of VLS NEs analyzed by DSC (a) and DSF (b) in Example 1 of the present invention.

[0024] Figure 7 Show the interaction of CBP(a), CBP-dimer(b) and VLS NEs(c) with type I collagen in Example 2 of the present invention by SPR analysis.

[0025] Figure 8 Show the residence of VLS NEs in corneal tissue by IVIS analysis in Example 3 of the present invention, scale bar: 20 μm.

[0026] Figure 9 Show the fluorescence microscopy analysis of corneal tissue sections after drug treatment in Example 3 of the present invention, scale bar: 20 μm.

[0027] Figure 10 Show the process of VLS NEs penetrating corneal tissue from corneal epithelium inward in Example 3 of the present invention, scale bar: 20 μm.

[0028] Figure 11 Show the cell population histogram of the uptake of VLS NEs by HCECs pretreated with blocking agents at different time points in Example 4 of the present invention.

[0029] Figure 12 Show the proportion of positive cell populations and the mean fluorescence intensity of the uptake of VLS NEs by HCECs pretreated with different blocking agents (M-β-CD, EIPA and CHL) at different time points in Example 4 of the present invention.

[0030] Figure 13 Show the change trends of the proportion of positive cell populations (a) and the mean fluorescence intensity (b) of the uptake of VLS NEs by HCECs pretreated with different blocking agents at different time points from the time dimension in Example 5 of the present invention, and the uptake inhibition relative to the positive control group (c-d).

[0031] Figure 14 Show the efficiency study of the crossing of VLS NEs across the HCECs cell barrier at different time points and with different blocking agents (M-β-CD, EIPA, CHL, BRE.A, MON, BAF.A1) in Example 5 of the present invention by Transwell experiment.

[0032] Figure 15 Show the heat map analysis of the efficiency study of the crossing of VLS NEs across the HCECs cell barrier at different time points and with different blocking agents (M-β-CD, EIPA, CHL, BRE.A, MON, BAF.A1) in Example 5 of the present invention by Transwell experiment.

[0033] Figure 16Show the effects of different sealants (M-β-CD, EIPA, CHL, BRE.A, MON, BAF.A1) on VLS NEs of HCECs cells in the confocal experiment of Example 5 of the present invention.

[0034] Figure 17 Show the results of the rat corneal lysis experiment at different time points and after drug treatment in Example 6 of the present invention.

[0035] Figure 18 Show the effects of VLS NEs treatment on various tissue structures of the mouse eye in Example 7 of the present invention, including direct observation, fluorescein fundus angiography, OCT, and fundus imaging, etc.

[0036] Figure 19 Show the evaluation of the organizational structure integrity of corneal endothelial cells in Example 7 of the present invention. Scale bar: 100μm

[0037] Figure 20 Show the H&E staining images of the heart, liver, spleen, lung, and kidney tissue sections of mice in Example 7 of the present invention. Scale bar: 100μm Detailed implementation manners

[0038] The technical solutions of the present invention will be further described and illustrated below through specific implementation manners in conjunction with the accompanying drawings.

[0039] In the following examples, the female Balb / c mice and C57BL / 6 mice used were purchased from the Experimental Animal Center of Shandong First Medical University. The feeding of the mice and the animal experiments were all carried out in accordance with the requirements of the Ethics Committee of the Experimental Animal Center of Shandong First Medical University.

[0040] In the following examples, the preparation of common solutions is as follows:

[0041] (1) Phosphate buffer solution (PBS): Phosphate buffer solution (PBS): Accurately weigh 8g of NaCl, 1.44g of Na2HPO4, 0.2g of KCl, and 0.24g of KH2PO4, dissolve them in 900mL of deionized water, and stir well until completely dissolved. Use NaOH or HCl to adjust the pH to 7.4, and then make up the volume to 1L with deionized water. After preparation, autoclave the PBS solution at 121°C for 30 minutes, and store it for later use after cooling.

[0042] (2) 1M Tris-HCl (pH 8.0) stock solution: Accurately weigh 124.07g of Tris base, add it to 900mL of deionized water and stir well until completely dissolved. Adjust the pH value of the solution to 8.0 with concentrated hydrochloric acid, and finally make up the volume to 1L with deionized water, and store it for later use.

[0043] (3) 5×SDS-PAGE Electrophoresis Buffer Stock Solution: Weigh 94 g of glycine, 5 g of sodium dodecyl sulfate (SDS), and 15.1 g of Tris separately, mix and dissolve them in 900 mL of deionized water. After stirring evenly, make up the volume to 1 L with deionized water. This stock solution needs to be diluted five-fold to obtain the working concentration when in use.

[0044] (4) Ampicillin Stock Solution: After equilibrating the ampicillin dry powder at room temperature, accurately weigh 5 g, dissolve it in 40 mL of deionized water, and then make up the volume to 50 mL. This solution should be filtered and sterilized through a 0.22 μm filter membrane in a laminar flow hood and aliquoted and stored at -20 °C for subsequent use.

[0045] (5) High-Salt Lysis Buffer: Weigh 58.45 g of NaCl, add 20 mL of 1 M Tris-HCl (pH 8.0) stock solution, 100 mL of glycerol, and 1 mL of Triton X-100, then add 700 mL of deionized water and stir well until all components are completely dissolved. Finally, make up the volume to 1 L with deionized water, mix well, and it can be used for the cell lysis step.

[0046] (6) Coomassie Brilliant Blue Staining Solution: Weigh 1.0 g of Coomassie Brilliant Blue R-250, first dissolve it in 450 mL of methanol, then add 100 mL of glacial acetic acid, and finally make up the volume with 450 mL of deionized water. After mixing well, the staining solution is obtained and is suitable for protein gel staining.

[0047] (7) Decolorizing Solution: Mix according to the ratio of 450 mL of methanol, 100 mL of glacial acetic acid, and 450 mL of deionized water. After stirring well, it can be used as a decolorizing solution to remove the excess dye in the gel for easy observation of protein bands.

[0048] (8) Endotoxin Affinity Chromatography Medium Cleaning Buffer: Weigh 10 g of sodium deoxycholate, first dissolve it in 800 ml of deionized water, and stir well until all components are completely dissolved. Finally, make up the volume to 1 L with deionized water, mix well, and it can be used for cleaning the endotoxin affinity chromatography medium.

[0049] In the following examples, the methods of some experiments involved are as follows:

[0050] (1) Observation of the uptake of VLS NEs by HCECs cells using a laser confocal microscope:

[0051] Label VLS NEs with FITC fluorescence as follows: Accurately weigh 2 mg of sulfo-FITC, add 400 μL of DMSO, take 10 μL after ultrasonic dissolution, add it to 5 mL of VLS NEs (1 mg / ml), react at room temperature for 90 min, load it into a 3500 Da dialysis bag, and dialyze in PBS solution for 24 h to obtain FITC-VLS NEs. Pay attention to avoiding light during the process.

[0052] Seed the induced HCECs cells into a 30 mm cell culture dish dedicated for laser confocal microscopy, and seed 5×10 4 cells per well and culture overnight. Add FITC-VLS NEs to the serum-free cell culture medium to make the final concentration of VLS NEs 1 μg·mL -1 . After culturing in the cell incubator for 2 h, remove the supernatant, wash the culture dish once with sterile PBS, add Hoechst33342 staining solution, stain at room temperature for 15 min, and wash the culture dish once with sterile PBS (For the subcellular localization of BMDCs, the cells are also stained with a red lysosome fluorescent probe according to the instructions). Then add 500 μL of cold 4% paraformaldehyde solution to fix for 10 min, wash the culture dish 3 times with sterile PBS, drop anti-fluorescence quenching mounting medium on the cell surface, and observe under a laser confocal microscope. Operate under dark conditions throughout the process.

[0053] (2) Detect the endocytic pathway of HCECs cells' uptake of VLS NEs by flow cytometry

[0054] To clarify the intracellular transport mechanism of VLS NEs, analyze its dependent pathway through specific endocytic pathway inhibitor blocking experiments. Select corneal epithelial cells and seed them in a 24-well plate. After the cell confluence reaches 80%, pretreat them with M-β-CD (2 mM, caveolin-mediated endocytosis inhibitor), EIPA (100 μM, macropinocytosis inhibitor), and chloroquine (100 μM, clathrin-dependent pathway inhibitor) for 1 h (37 °C), and then add the fluorescently labeled VSEs-drug complex and co-incubate for 2 h. After the cells are washed with PBS and resuspended, quantitatively analyze the fluorescence intensity change by flow cytometry, calculate the uptake efficiency decay rate based on the inhibitor-free group, and quantify the contribution of each pathway to the endocytosis of VSEs through a dose-effect curve.

[0055] (3) Detect the ability of VLS NEs to cross the HCECs cell barrier by Transwell

[0056] A corneal epithelial barrier model was constructed based on the Transwell system, and the integrity of the cell monolayer was monitored by transendothelial electrical resistance (TEER). In the experimental groups, M-β-CD (Methyl-β-cyclodextrin, a caveolin-mediated endocytosis inhibitor, 2 mM), EIPA (Ethylisopropylamiloride, a macropinocytosis inhibitor, 100 μM), and chlorquine (a clathrin-dependent pathway inhibitor, 100 μM) were used to inhibit different endocytic pathways; Monensin (50 μM), brefeldin A (90 μM), and bafilomycin A1 (150 μM) were used to inhibit different transport pathways. After pretreatment for 1 h (37 °C), VLS NEs drug was added to the upper chamber. After 2 h, the medium in the lower chamber was collected, and the drug concentration was quantitatively analyzed by fluorescence spectrophotometry. The inhibition rate was calculated with the transport efficiency of the control group (without inhibitor) as 100%, and combined with the data of blocking endocytosis and trans-organelle transport, a transmembrane transport path network model of VLS NEs was established to clarify the cooperative mechanism of sub-organelles it depends on.

[0057] (4) Corneal anti-enzymatic lysis experiment

[0058] Healthy female Sprague-Dawley rats with intact corneal epithelium were selected. After evaluation by slit lamp microscopy, they were randomly divided into 5 groups (n = 5 / group), and the right eyes received the following interventions: ① The control group was instilled with 200 μL of PBS buffer (pH 7.4); ② The RF + UV group was topically given a 1% riboflavin solution (30 μg / mL) followed by ultraviolet irradiation (3 mW / cm 2 , 30 min); ③ The experimental groups were instilled with wild-type HBc VLPs and VLS NEs at the same concentration, and each group maintained a 30-min contact time on the ocular surface. At the end of the experiment, euthanasia was performed by cervical dislocation, and the corneal tissue was completely dissected along the limbus by microsurgical techniques. The excised corneas were placed in 6-well plates containing 0.2% type II collagenase and incubated at 37 °C with constant shaking (175 rpm) until complete dissolution. During the dynamic monitoring stage, the residual corneal morphology was recorded every 5 min through a standardized imaging system, and the tissue area decay rate was quantitatively analyzed using Image J software to establish an enzymatic lysis kinetic curve.

[0059] (5) Evaluation of the ocular surface residence ability of VLS NEs

[0060] C57BL / 6 mice (n = 10) were selected. After anesthesia induction, they were randomly divided into an experimental group and a control group (n = 5 / group). The right eyes of the experimental group received 10 μL of VLS NEs-FITC composite nano-suspension (30 μg / mL), and the control group was given an equal volume of free FITC solution (30 μg / mL). Both groups maintained corneal infiltration for 30 min. A PerkinElmer IVIS Lumina III in vivo imaging system equipped with a near-infrared filter set (excitation / emission wavelength 750 / 773 nm) was used to perform dynamic fluorescence tracing on the craniofacial region. Imaging data were collected at 0 and 6 h time points by continuously monitoring for 6 h starting from drug administration.

[0061] (6) Ex vivo corneal permeability test of VLS NEs

[0062] The mice were sacrificed by cervical dislocation. The eyeballs were completely removed using corneal scissors, rinsed with 0.9% saline, and placed in a mold containing OCT tissue embedding medium. The position of the eyeballs was adjusted to keep them horizontal and then immediately frozen in liquid nitrogen.

[0063] The completely frozen eyeball tissue embedding blocks were taken out, sliced (10 μm) along the coronal plane of the eyeballs using a cryostat, and the tissue sections were attached to glass slides. Fluorescence images were observed and taken using an inverted fluorescence microscope, and fluorescence intensity quantitative analysis was performed using ImageJ.

[0064] (7) Tissue sectioning and hematoxylin-eosin staining

[0065] After the observation period, the hearts, livers, spleens, lungs, kidneys, and tumor sections of mice in each group were collected, fixed with 4% paraformaldehyde solution, then paraffin-embedded, sectioned with a thickness of 7 μm, stained with H&E, and observed and photographed under a microscope.

[0066] Example 1

[0067] I. Computer-aided sequence design of VLS NEs

[0068] The construction process of VLS NEs in this example includes:

[0069] (1) Using the hepatitis B virus capsid protein (HBcAg) as the backbone domain to achieve self-assembly of virus-like structure nanoparticles. The C-terminal nucleic acid binding domain (amino acid residues 150 - 183) of HBcAg was deleted to obtain HBc 149, thereby reducing immunogenicity and the difficulty of recombinant expression;

[0070] (2) The collagen-binding polypeptide CBP (such as that shown in SEQ ID NO.01: LRELHLNNN) was fused to the spike region (between amino acid residues 78 - 80) of HBc 149, and finally VLS NEs with the amino acid sequence shown in SEQ ID NO.02 were obtained, which can bind to collagen and assist in the formation of a complete three-dimensional structure.

[0071] SEQ ID NO.02:

[0072]

[0073] To verify that the recombinant protein sequence can maintain the integrity of the viroid-like structure, in this example, the AF3 model was used to perform dimer and tetramer (basic structural unit) structure simulation and evaluation analysis on it. The structure simulation results are as Figure 1 shown. The pTM (Predicted Template Modeling) and ipTIM (Interface Predicted Template Modeling) values of the dimer and tetramer simulation results are about 0.6 and about 0.4 respectively. Generally speaking, the AF3 model believes that the simulation result of the dimer is better than that of the tetramer and is more credible. Since the tetramer is a key transition state structure in the self-assembly mechanism of HBcVLPs, and 0.4 is only slightly lower than the normal standard, therefore, in subsequent research and analysis, this example simultaneously performed dimer and tetramer analyses.

[0074] In this example, a variety of computational tools and models (such as Rosetta) were used to conduct a detailed analysis of the structures of these complexes and their interaction interfaces. The evaluation indicators include but are not limited to:

[0075] (1) RMSD (Root Mean Square Deviation): Evaluate the deviation between the predicted structure and the experimental structure;

[0076] (2) Average pairwise minimum interface PAE (Interface_PAE): Quantify the uncertainty of the predicted structure;

[0077] (3) Binding free energy per unit area (dG / dSASA): Characterize the energy density on the binding interface;

[0078] (4) Number of buried unsatisfied polar atoms (BUNS): Reveal potential interface-unstable residues;

[0079] (5) Packing density (packstat): Evaluate the filling degree of the protein interface.

[0080] As Figure 2 shown in a, the analysis of the Interface_PAE (Interface Predicted Aligned Error) indicates that the inter-chain residue position alignment error values of the VLS NEs dimer are all lower than indicating a high accuracy in the prediction of the complex structure, which is consistent with the analysis results of the interface prediction template modeling score (ipTM). It is worth noting that the inter-chain residue position alignment error of the tetramer conformation increases significantly to around, suggesting that the prediction accuracy of the current AF3 model for the conformation of multimeric proteins is relatively limited compared to oligomeric structures. In addition, the results of the structural similarity analysis ( Figure 2 b) show that the RMSD values obtained from the dimer conformation alignment of VLS NEs and wild-type HBc VLPs are all less than The RMSD values of the tetramer conformation alignment are also generally lower than without significant conformational deviation. These data together demonstrate that the engineering modification has not significantly changed the conformational characteristics of the key intermediates in the self-assembly process of VLS NEs, supporting its potential for virus-like particle self-assembly comparable to that of the wild-type from a structural biology perspective.

[0081] Based on the Rosetta energy analysis toolkit, this example systematically evaluated the differences in the inter-chain interactions of the multimers between VLS NEs and wild-type HBc VLPs (as Figure 3 shown). Quantitative analysis shows that the difference in the interfacial binding free energy per unit area of the dimer conformation is small, without significant energy perturbation; while the difference in the interaction energy of the tetramer conformation is statistically significant, but its average deviation is less than 1, suggesting that the impact of this energy fluctuation on the self-assembly process may be limited. In addition, through the double-index verification of BUNS and packstat, it is found that wild-type HBc VLPs show better numerical characteristics in both parameters. Specifically, the BUNS value of VLS NEs is about 10 higher than that of the wild-type, and the packstat score drops by about 0.15 units. This difference may be due to the redistribution of the surface electrostatic potential caused by the engineering modification. Based on the above results, it can be seen that the VLS NEs prepared in this example have the assembly kinetic basis for forming complete virus-like particles, but their conformational stability may be weaker than that of the wild-type system, which provides a clear direction for subsequent stability optimization research.

[0082] II. Expression, purification and characterization of VLS NEs

[0083] In this example, the pET43.1A plasmid carrying the nucleotide sequence encoding VLS NEs (shown as SEQ ID NO.03: Atggacatcgaccattataaagagttcggcgcgagcgtggaattgctgtcgttcctgccgagcgactttttcccgagcattcgtgatctgctggataccgcttctgcactctaccgcgaggccttggagagcccggaacattgcagcccgcatcacaccgcactgcgtcaggcgattctgtgttggggtgaattaatgaacctcgctacctgggtgggttccaacttggaggacggcacgtctggctcatctggttcaggctctggtggttcaggtagtggtggcttgcgtgaactgcacctgaacaacaacggctcaggctcaggtggcagcggtagtggtagcagcggcagtacgggtagccgtgaacttgtagttggttatgttaatgttaacatgggtctgaagatccgccaaatcttatggtttcatatcagctgcctgacgtttggccgtgagacagttttggaatacctggtctccttcggggtgtggattcgtaccccgccggcgtatcgccctccgaatgccccaattctgtccaccctgccgggtagtggtggcttgcgtgaactgcacctgaacaacaaccaccaccaccaccaccac) was transformed into LPS-deficient BL21 Escherichia coli by heat shock method. After expression and multiple steps of purification, the final product was verified by SDS-PAGE (as Figure 4 shown), and the results showed that highly pure VLS NEs were obtained, and the endotoxin content was all lower than 1 EU / ml. The molecular weight of the purified protein was about 25 kDa, which was significantly higher than that of HBc149, and this was consistent with the theoretical value predicted by the online tool ProtParam. These results indicate that the purification process of this example can efficiently obtain the target protein, ensuring the quality and reliability of subsequent experiments.

[0084] Subsequently, in this example, TEM (transmission electron microscopy) and DLS technology (dynamic light scattering) were used to characterize the structural features of VLS NEs in detail ( Figure 5)。TEM images show (5a) that VLSNEs can assemble into complete viroid-like structures, and statistical analysis indicates that these particles have a uniform size, with an average diameter of approximately 31 nm. In addition, the results of DLS measurements ( Figure 5 b) further confirm this finding, showing that the particle size of VLS NEs in aqueous solution is close to 32 nm, and at the same time, its particle size distribution is narrow. By combining these two complementary technical means, this example not only verifies the morphological integrity of VLS NEs but also precisely quantifies their actual size in the dry state and their hydrated size under physiological conditions.

[0085] In addition, this example uses DSC (Differential Scanning Calorimetry) and DSF (Differential Scanning Fluorimetry) to systematically evaluate the thermal stability of VLS NEs. According to the DSC analysis ( Figure 6 a), VLS NEs exhibit a significant Tm, approximately 68 °C; while the Tm value obtained from the DSF test is close to 64 °C ( Figure 6 b). The results of the above two methods consistently show that the VLS NEs prepared in this example have good thermal stability, which not only reflects the firmness of their structure but also provides favorable conditions for long-term preservation.

[0086] Example 2

[0087] To explore the effect of viroid structure surface display on receptor-ligand affinity, this example uses SPR technology (Surface Plasmon Resonance) to measure the binding constants K of CBP, CBP-dimer (dimer), and the VLS NEs prepared in Example 1 with type I collagen D .

[0088] The experimental results are as Figure 7 shown in and Table 1. The K D values of CBP, CBP-dimer, and VLS NEs with type I collagen are 1.912E-6 M, 6.482E-6 M, and 1.991E-10 M, respectively. Among them, compared with the monomer and dimer forms of CBP, VLS NEs exhibit a binding strength approximately four orders of magnitude higher; at the same time, there is no obvious affinity difference between CBP and CBP-dimer.

[0089] Table 1

[0090]

[0091] Further analysis of ka and k d It was found that the reasons for this significant difference include:

[0092] (1) Soluble CBP exhibits a faster dissociation rate after binding to type I collagen. This phenomenon may be attributed to the fact that CBP contains only binding sites without additional stable domains, resulting in a relatively large k d relatively large;

[0093] (2) CBP contains a relatively small number of binding domains, resulting in a relatively small k a relatively small. In contrast, VLS NEs not only provide more binding sites but also enhance the overall structural stability through their unique viroid-like backbone, thus significantly improving the binding efficiency of CBP to type I collagen.

[0094] Notably, although CBP dimerization increases the number of potential binding sites, it does not significantly improve its affinity for type I collagen, suggesting that simple addition of binding sites is not sufficient to enhance affinity. Instead, the multivalent display mechanism - the presence of multiple CBP copies on each VLS NE - greatly facilitates efficient binding to type I collagen. This effect is similar to the interaction pattern between viruses and the surface receptors of their host cells in nature.

[0095] In summary, the research in this example shows that using the viroid structure as a vector can significantly enhance the binding efficacy between soluble polypeptides or proteins and target molecules.

[0096] Example 3

[0097] The corneal epithelial barrier is the outermost protective structure of the eye, composed of multiple layers of non-keratinized stratified squamous epithelial cells. These cells are rich in lipids and are tightly connected, forming an effective barrier against most hydrophilic and some lipophilic drugs, restricting the drug delivery efficiency into the eye. In addition, the normal corneal epithelial barrier is not only crucial for maintaining the health of the ocular surface but also plays an important role in maintaining corneal transparency and visual quality.

[0098] Such as Figure 8As shown, in this example, the VLS NEs prepared in Example 1 labeled with FITC fluorescence were applied to the surface of the mouse eye by the eye drop method. At 0 and 6 h respectively, the fluorescence distribution on the cornea was analyzed using an In Vivo Imaging System (IVIS). The experimental results showed that approximately 30% of the VLS NEs were able to achieve long-term retention in the corneal tissue instead of being rapidly cleared by the tear fluid, which was in sharp contrast to the result that the small molecule FITC was rapidly and completely cleared. These findings revealed that VLS NEs have the ability to cross the epithelial barrier and rapidly penetrate into the corneal stromal layer, providing favorable conditions for subsequent treatment.

[0099] To further explore the penetration and distribution of VLS NEs in the corneal tissue, in this example, the excised corneal tissue was cross-sectioned and observed under a fluorescence microscope technique. The results are as Figure 9 shown. After 6 h of eye drop application of VLS NEs on the ocular surface, they could be evenly infiltrated and distributed in the corneal stroma, while FITC could not be observed. This result was consistent with the previous observation, indicating that VLS NEs have significantly superior trans-barrier delivery performance compared to small molecules. This property of VLS NEs is of great significance for the development of new treatment methods for corneal diseases. The treatment of corneal diseases often faces challenges in drug delivery because many drugs are difficult to penetrate the corneal epithelium, thus limiting their therapeutic effects. As a carrier that can penetrate the corneal epithelium, VLS NEs can improve the bioavailability of drugs, enabling the drugs to reach the lesion site more effectively, thereby improving the therapeutic effect.

[0100] In addition, based on the above experimental results, in this example, the time point approximately 2 h after eye drop application on the ocular surface was selected for observation, aiming to analyze in detail the behavior pattern of VLS NEs crossing the barrier and diffusing in the corneal stroma. The research results are as Figure 10 shown. A large amount of VLS NEs were endocytosed by corneal epithelial cells and gradually transcytosed into the corneal stroma. Subsequently, VLS NEs gradually diffused in the stromal layer until they were taken up by corneal endothelial cells. From the trend of fluorescence intensity change, it can be seen that the whole penetration process is time-dependent.

[0101] Example 4

[0102] This example explored the process of VLS NEs cell transendocytosis, which can be subdivided into two main stages: first, endocytosis, and second, exocytosis. For different types of endocytic pathways, specific inhibitory strategies were adopted in this example to evaluate their effects on the entry of VLS NPs into human corneal epithelial cells (HCECs).

[0103] Specifically, this example employed three different blockers - Methyl-β-Cyclodextrin (M-β-CD), 5-(N-Ethyl-N-Isopropyl)amiloride (EIPA), and Chloroquine (CHL). These reagents were used to block caveolae-mediated endocytosis, macropinocytosis, and clathrin-dependent endocytic mechanisms, respectively. In the experimental design, this example co-incubated Cy5.5 fluorescently labeled VLS NPs with HCECs pretreated with the above blockers, and set multiple time points (45 min, 90 min, and 120 min) to monitor the kinetic characteristics of VLS NPs uptake by cells.

[0104] Analysis by flow cytometry revealed that, within all the tested time periods, the M-β-CD pretreatment group showed the most significant inhibitory effect. As Figure 11 shown, starting from 45 min, the M-β-CD pretreatment samples exhibited obvious population separation, while no similar signs of uptake inhibition were observed in other control groups. Further, this example quantitatively analyzed the proportion of positive cell populations and their mean fluorescence intensity (see Figure 12 ). The results showed that, before 90 min, although there were certain differences in the uptake ratios among the treatment groups, these differences were not obvious; however, there were significant differences in the mean fluorescence intensity in terms of statistics, indicating that M-β-CD treatment significantly slowed down the rate of VLS NPs uptake by HCECs.

[0105] Example 5

[0106] To gain a deeper understanding of the time-dependent changes in the inhibitory effects of different blockers on cell pathways, this example further analyzed the data obtained by flow cytometry from the time dimension ( Figure 13 ). This time-series analysis helps to reveal the specific effects of each blocker on VLS NEs uptake over time. As Figure 13 shown in a and b, after 120 minutes of co-incubation, M-β-CD showed the most significant uptake inhibitory effect compared to the positive control group and other inhibitor groups. Specifically, Figure 13c and d show that M-β-CD is particularly prominent in inhibiting the uptake of VLS NEs by HCECs, with an inhibition ratio of up to about 20%, while the inhibitory effects of EIPA and CHL are relatively mild, and with the prolongation of time, the effects of these two blockers gradually weaken to almost zero. In terms of the change in average fluorescence intensity, only the M-β-CD treatment group showed obvious inhibitory characteristics. Generally speaking, as a specific caveolae inhibitor, M-β-CD can effectively hinder this process, while the action mechanisms of the other two blockers seem to have nothing to do with or have a weak association with this pathway. These results suggest that VLS NEs are likely to be mainly taken up by HCECs through the caveolae-mediated endocytosis pathway.

[0107] As a bioactive protein nanodrug, the intracellular metabolic pathway of VLS NEs mainly involves the lysosomal degradation system. This example finds that the endocytic uptake of VLS NEs by corneal epithelial cells significantly depends on the caveolin-mediated clathrin-independent endocytosis pathway ( Figure 14 ). To systematically clarify its trans-barrier transport mechanism, this example uses the Transwell co-culture model, combined with specific pathway inhibitors (M-β-CD, EIPA, CHL, BRE.A, MON, BAF.A1), to quantitatively analyze the functional contributions of the endocytosis and transcytosis pathways.

[0108] The results of the Transwell experiment ( Figure 14 ) show that different pathway inhibitors such as M-β-CD, EIPA, CHL, BRE.A, MON, and BAF.A1 have significantly different effects on the efficiency of VLS NEs penetrating the corneal epithelial barrier. Specifically, the endocytosis pathway inhibitor M-β-CD significantly reduces the trans-barrier ratio of VLS NEs, and this ratio increases with time, reaching more than 30% at 120 minutes. This finding is consistent with the previous endocytosis experiment results, further confirming that endocytosis is a key step for VLS NEs to cross the corneal epithelial barrier, and the caveolae endocytosis pathway is particularly important. In addition, the study also shows that when inhibiting the endoplasmic reticulum-Golgi delivery pathway and the Golgi-plasma membrane delivery pathway, the trans-barrier efficiency of VLS NEs will decrease by about 5%, and this effect weakens with the prolongation of time. Interestingly, after using BRF.A1 to inhibit lysosomal maturation, the transcytosis efficiency of VLS NEs is significantly improved, especially reaching a peak at about 90 minutes, with an increase close to double. This indicates that although a large number of endocytosed VLS NEs are taken up and degraded by lysosomes, over time, more and more VLS NEs are released by direct exocytosis, thus reducing the influence of the lysosomal pathway on it. Generally speaking ( Figure 15) VLSNEs mainly rely on caveolae-mediated endocytosis to efficiently cross the corneal epithelial barrier and then enter the corneal stromal layer through multiple transcytosis mechanisms. Notably, in the initial stage, some VLS NEs may undergo a degradation process caused by the lysosomal pathway. However, as the amount of endocytosis increases, this degradation phenomenon gradually decreases, showing the unique advantages of VLS NEs in crossing biological barriers and their potential broad application prospects.

[0109] This example also observed this process through a confocal microscope. The results are as Figure 16 shown. After treatment with the M-β-CD inhibitor, the uptake of VLS NEs by HCECs was significantly reduced, and the particles were mainly concentrated on the cell membrane surface and difficult to enter the cells, which was significantly different from the control group and the groups treated with other pathway inhibitors. This is consistent with the conclusions obtained in the endocytosis experiment and the transwell experiment in this example.

[0110] Example 6

[0111] In this example, a multi-species corneal injury model (such as rats and New Zealand rabbits) was established to systematically evaluate the corneal repair efficacy of VLSNEs and its mechanism of action. Photodynamic therapy (RF) and wild-type HBc (wtVLPs) were introduced as parallel controls in the experimental design to clarify the differential advantages of VLS NEs.

[0112] As Figure 17 shown, in the protease-mediated corneal stromal lysis model, the VLS NEs treatment group showed a significant stromal stabilization effect: the corneal tissue integrity maintenance rate was increased by about 40% compared with the Control group, and the dissolution inhibition rate was increased by about 20% compared with the RF light treatment group. Notably, limited by the physical barrier effect of the corneal epithelium, the RF photosensitizer caused complete corneal dissolution due to insufficient penetration 40 minutes after treatment (the dissolution time was 10 minutes earlier than that of the VLS NEs group). Although wild-type HBc VLPs could delay collagen degradation through a protease competitive inhibition mechanism (the dissolution rate was reduced by 12.5%), its collagen three-dimensional cross-linking ability was insufficient, resulting in a limited efficacy maintenance time. The specific reasons need to be further studied and analyzed.

[0113] These results indicate that the excellent performance of VLS NEs stems from its dual mode of action:

[0114] (1) Efficient barrier penetration: The viroid structure can mediate trans-epithelial transport, has the ability to efficiently penetrate the corneal epithelial barrier, and the bioavailability is significantly improved compared with traditional small molecule therapies;

[0115] (2) Based on the non-covalent interaction mechanism of natural proteins, VLS NEs can induce cross-linking of collagen fibers in the corneal stroma and achieve the reconstruction of a three-dimensional collagen network while avoiding phototoxic damage.

[0116] These characteristics enable VLS NEs to maintain corneal transparency while demonstrating superior efficacy and safety compared to first-line clinical therapies, providing a novel non-invasive treatment strategy for corneal regenerative medicine.

[0117] Example 7

[0118] In corneal repair treatment, damaged structures may lead to a significant decline in patients' vision. Therefore, it is particularly important to evaluate the biosafety of VLS NEs. In this example, through a series of rigorous experimental designs, the aim is to comprehensively evaluate the impact of VLS NEs on ocular tissues.

[0119] (1) To preliminarily understand the impact of VLS NEs on the ocular surface, in this example, an ocular surface drop treatment was performed on a mouse model, and the integrity and smoothness of the corneal surface were directly observed. The results are as Figure 18 shown. VLS NEs did not cause obvious physical damage or rough changes on the corneal surface, indicating its good biocompatibility.

[0120] (2) This example focused on the limbus cornea, which is the transitional area between the cornea and the sclera and is crucial for maintaining the corneal stem cell microenvironment. Through careful microscopic examination, it was found that the limbus cornea structure remained intact without any abnormal phenomena, further confirming the safety of VLS NEs.

[0121] To further verify whether VLS NEs would have an impact on the retinal vascular system, in this example, a fluorescein sodium injection method was used for fundus angiography (FFA). This method can visually display the morphology and permeability of retinal blood vessels. The experimental results are as Figure 18 shown. Even in high-resolution images, no signs of early or subtle lesions were seen, indicating that VLS NEs do not cause adverse reactions in the retinal vascular system.

[0122] (3) To more deeply explore the impact of VLS NEs on the internal structure of the eyeball, in this example, non-invasive detailed examinations were performed using optical coherence tomography (OCT) and fundus imaging techniques. These two techniques can provide high-resolution information on the corneal structure of the eye and images of the posterior pole including the retina, choroid, and optic disc. From the obtained data ( Figure 18 ), no abnormal changes were seen in either the cornea or the various layers of the posterior segment of the eyeball, proving that VLS NEs are not only safe for the external cornea but also harmless to the deep ocular structures.

[0123] In summary, through comprehensive analysis at multiple levels, this embodiment shows that VLS NEs have excellent biosafety and exhibit great potential in corneal repair treatment applications.

[0124] In this embodiment, to evaluate the effects of VLS NEs and RF on ocular tissues, after ocular surface drug administration was completed, corneal samples were further collected in this embodiment, and the integrity and health status of corneal endothelial cells were examined by microscopy. The observation results are as Figure 19 shown. Whether or not there is a complete corneal epithelium as a barrier, neither VLS NEs nor RF showed any obvious damaging effects on corneal endothelial cells.

[0125] In the final stage of this embodiment, the safety of the major organs of mice receiving ocular administration of VLS NEs and PBS was evaluated in this embodiment. Specifically, at the end of the experiment, the major organs of these mice were removed for tissue sectioning, and the integrity and health status of their tissue structures were observed by H&E staining. The pathological examination results are as Figure 20 shown. VLS NEs exhibited good biosafety, and no damage or abnormal changes in the structure of any major organs were observed.

[0126] As described above, it is only a preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A viroid-like structure nanoconnector, characterized in that: Based on the hepatitis B virus core protein Uniprot P03146 with the nucleic acid binding domain at positions 145 - 183 of the C-terminus removed, the CBP shown in SEQ ID NO.01 was inserted into its spike part and N-terminus respectively.

2. The viroid-like structure nano adaptor according to claim 1, characterized in that: Its amino acid sequence is shown in SEQ ID NO.

02.

3. The viroid-like structure nano adapter according to claim 2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

03.

4. Use of a viroid-like structure nano adapter according to any one of claims 1 to 3 in the preparation of a composition for promoting corneal stroma repair.

5. A composition for promoting corneal stroma repair, characterized in that: Its active ingredient includes a viroid-like structure nano adapter according to any one of claims 1 to 3.

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