An intracellular delivery system and uses thereof

Abstract

The present application relates to natural ferritin, and particularly relates to an intracellular delivery system and application thereof. The system can effectively deliver various proteins into cells and maintain the biological activity thereof, and provides important technical support for the development of protein therapy with intracellular target as core. The method exhibits significant advantages in promoting intracellular protein transport, intracellular protein interaction research and protein level regulation. The delivery system has wide applicability, is simple to operate and safe and reliable, and becomes an important tool for future intracellular protein research and application.

Description

Technical Field

[0001] This invention relates to natural ferritin, and more specifically to an intracellular delivery system and its applications. Background Technology

[0002] Natural ferritin (Fn) is a widely distributed iron storage protein composed of 24 subunits that aggregate to form a cage-like structure with an outer diameter of 12 nm and an inner lumen of 8 nm. Various ferritin variants can be obtained by modifying its subunits, exhibiting novel structures and functions compared to natural ferritin. Furthermore, the 8 nm cavity provides a suitable location for loading drugs / biomolecules and synthesizing nanozymes. However, Fn enters cells via a specific CD71-dependent pathway. Because CD71 is not expressed in all cells or is expressed at low levels in some cells, and because its entry efficiency is low, Fn is difficult to use as a universal protein carrier. Therefore, natural ferritin needs to be modified to achieve more flexible and diverse functional applications.

[0003] Therefore, there is a desire in the art to modify hollow proteins that do not have the ability to enter cells into a universal intracellular delivery protein carrier that can safely and efficiently deliver cargo proteins into cells through a variety of interactions that mediate the formation of complexes between the protein and the carrier, while fully preserving the biological activity of the protein in the cell. Summary of the Invention

[0004] Purpose of the invention

[0005] The purpose of this invention is to provide a safe, efficient, universal, and easy-to-operate intracellular protein delivery system that overcomes the shortcomings of existing delivery vectors.

[0006] Technical solution

[0007] A supercharged ferritin Fn+, characterized in that the supercharged ferritin Fn+ is obtained by site mutation based on the prototype ferritin, wherein one or more of the following positions are mutated to arginine R or lysine K: aspartic acid D at position 15, alanine A at position 18, asparagine N at position 25, aspartic acid D at position 45, aspartic acid D at position 84, glutamate E at position 94, asparagine N at position 98, cysteine ​​C at position 102, histidine H at position 105, asparagine N at position 109, glutamate E at position 116, aspartic acid D at position 123, and glutamate E at position 162; wherein the total charge is greater than or equal to +168, and the amino acid sequence of the prototype ferritin is shown in SEQ ID No:1.

[0008] A PBA-containing supercharged ferritin pFn+ is characterized in that pFn+ is prepared by introducing a PBA group at the N-terminus of the supercharged ferritin Fn+. Specifically, pFn+ is obtained by co-transforming a plasmid of an aminoacylated tRNA synthetase of the non-natural amino acid PBA (4-boron-L-phenylalanine) with a plasmid of supercharged ferritin (Fn+) into an expression system.

[0009] An expression vector for the supercharged ferritin Fn+ or the PBA-containing supercharged ferritin pFn+;

[0010] A host cell containing the expression vector;

[0011] The application of the PBA-containing supercharged ferritin pFn+, the expression vector, or the host cell of claim 4 in the preparation of a drug delivery system.

[0012] The application is characterized in that the drug delivery system can achieve intracellular delivery.

[0013] The application is characterized in that the drug is a small molecule compound, nucleic acid, metal drug, or protein.

[0014] Specifically:

[0015] A supercharged ferritin (Fn+), characterized in that the supercharged ferritin is obtained by site mutation of the amino acid sequence of the prototype ferritin HFn. The site mutations are screened according to the Rosetta molecular modeling program and the Alphafold2 program. In Rosetta, the surface mutation sites are determined by minimizing the energy of the HFn crystal structure and the smaller RMSD value after Alphafold2 mutation. The mutation sites include, but are not limited to, the following sequences:

[0016] The amino acid sequence of HFn is as follows:

[0017] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES, such as SEQ ID Shown as No:1

[0018] Table 1. Mutation sites, corresponding amino acids, and charge changes.

[0019]

[0020]

[0021] The system is characterized in that the universal protein carrier is capable of non-CD71-dependent cell entry and can deliver small molecule drugs (doxorubicin), nucleic acid drugs (siRNA), or metal drugs (metal ions manganese) into the lumen.

[0022] The system is characterized in that a PBA group is introduced at the N-terminus of the supercharged ferritin amino acid sequence, thereby increasing the protein drug loading capacity and stability by 24 PBA groups on the modified ferritin.

[0023] The application is characterized in that the delivered protein is a protein with pharmacological activity, labeling function, or targeting function, such as: antigenic peptide, enzyme protein, toxin protein, antibody, Trim21, and Cas9; after simple mixing with the active protein, it can form particles of uniform size of about 100 nm, and has good stability and maintains high cell entry activity.

[0024] The application is characterized by the use of a system containing PBA supercharged ferritin in the preparation of a carrier for intracellular protein delivery.

[0025] The aforementioned PBA introduction utilizes genetic code expansion (GCE) technology to enable stop codons (usually amber codons UAG) to perform coding functions, introducing novel amino acids. Plasmids expressing aminoacylated tRNA synthetase and transfer RNA are transformed into expression strains, enabling nonsense codons (usually amber codons UAG) to perform coding functions, allowing the introduction of non-natural amino acids at the corresponding sites.

[0026] The preparation method of the system is characterized by the following steps: selecting positively charged amino acids by calculating the mean atomic offset distance (RMSD) of the mutated structure according to Chimerax's Matchmaker, followed by inducing expression and purifying supercharged ferritin to obtain an efficient, universal, safe and simple intracellular protein delivery system.

[0027] This invention also relates to a method for preparing the aforementioned intracellular protein delivery vector, specifically, the method comprising the following steps:

[0028] Step (1) Screening of surface mutation sites of supercharged ferritin

[0029] The PDB structure of supercharged ferritin was uploaded to the Rosetta supercharge module for screening surface mutation sites. With increasing module runs and energy assignments of positively charged amino acids, the number of surface mutation sites in HFn increased. Several potential surface mutation sites in HFn were ultimately identified. Subsequently, the R / K values ​​of the surface mutation sites were selected. The structure prediction software Alphafold2 and Chimerax's Matchmaker were used to select positively charged amino acids by calculating the mean distance of atomic offsets (RMSD) of the mutated structures.

[0030] Step (2) Construction, isolation and purification of supercharged ferritin

[0031] Glyceryl cultured bacteria expressing supercharged ferritin were inoculated into LB medium at a ratio of 1:200 (v / v) and cultured overnight in a shaker at 37°C and 220 rpm. The bacterial cells were collected and sonicated on ice until the viscosity of the culture significantly decreased. The sonicated culture was centrifuged at 4°C and 12,000 rpm for 15 min, and the precipitate was collected. The bacterial precipitate was resuspended in inclusion body washing buffer at a mass-to-volume ratio of 1:20 and stirred thoroughly with a magnetic stirrer for 30 min at room temperature until fully resuspended. After resuspending, the precipitate was centrifuged at 4°C and 12,000 rpm for 10 min, and the precipitate was collected. The washed precipitate was dissolved in dissolving buffer and stirred thoroughly at room temperature for 60 min until fully dissolved. The supernatant was then collected by centrifugation at 4°C and 12,000 rpm for 20 min and subsequently purified using the AKTA-PURE system. Analysis was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (v / v) polyacrylamide gel. Then, the purest components were collected, ultrafiltered using a 50kDa centrifugal ultrafiltration tube, and the buffer was replaced with protein refolding solution to obtain a concentrated fusion protein.

[0032] Step (3) Preparation and characterization of supercharged ferritin and protein complex

[0033] Fluorescein labeling: Rabbit serum-derived IgG and Cas9 protein were labeled with FITC at a FITC:target protein molar ratio of 5:1 and incubated overnight at 4°C. Unreacted FITC was removed by dialysis and ultrafiltration. The labeled target protein was collected and concentrated by ultrafiltration, and the sample concentration was determined using a BCA kit. Complex preparation: 1 μM of target protein was gently added dropwise to an appropriate concentration of pFn+ and incubated at room temperature for 30 min, during which the suspension was gently aspirated to ensure the formation of a homogeneous complex. Complex characterization: The particle size and potential of the complex were detected using a Zetasizer Nano ZS90 nanoparticle size analyzer.

[0034] principle:

[0035] like Figure 11 The schematic diagram illustrates how superferritin (Fn+) with a diameter of approximately 12 nanometers is used to introduce phenylboronic acid (PBA) onto the surface of Fn+ nanocages via gene-encoded extension (GCE) technology to obtain the carrier pFn+. pFn+ allows for mixing with target proteins or biomolecules to form stable complexes through NB coordination, ion interactions, and multiple protein binding sites. These complexes are then efficiently internalized by the cell and released into the cytoplasm, ensuring the activity of the delivered molecules and enabling them to perform their biological functions.

[0036] Beneficial effects

[0037] Fn enters cells via a specific CD71-dependent pathway. However, the CD71 pathway has several drawbacks: it only allows for specific cell entry; without CD71 expression, entry is impossible, lacking versatility, and it prevents lysosomal escape. To address this, inspired by the positively charged protein pathway promoted by CN112608366A, the inventors mutated Fn to create supercharged cavitary ferritin Fn+. Supercharged proteins are a class of proteins with a net charge exceeding one net charge per kilodalton. Supercharged proteins in their folded state possess important biological functions, including but not limited to DNA binding, transcriptional regulation, protein synthesis, antibacterial activity, and signal transduction. Biological macromolecules, such as proteins, achieve their biological activity through electrostatic interactions and coordination with other intermolecular forces. Supercharging allows for control over the primary structure of proteins, providing the ability to regulate charge density, molecular weight, and alter the charge position of supercharged proteins. Furthermore, various charge-induced interactions can be introduced into these types of supercharged proteins, and they can be easily fused with other functional proteins for expression. Finally, supercharged proteins can be genetically encoded, allowing for their expression in target cells. Supercharged proteins, especially those rationally mutated to positively charged amino acids (R / K) to achieve superpositive charge, significantly improve intracellular entry efficiency. The amount, molecular weight, and folding state of the mutated charge are important structural features determining the uptake pathway in mammalian cells. Experimental results showed that when the total positive charge exceeds +168, its entry performance increases, and the entry efficiency increases with increasing charge. Superpositive modification enhances active entry capability, independent of the CD71 pathway, bypassing late endosomes / lysosomes, and escaping from early endosomes before accumulating in the cytoplasm, thus expanding its application range.

[0038] However, simply mutating amino acids to obtain Fn+ packaging capacity has limited effectiveness. To package more compounds or proteins, the inventors reacted it with phenylboronic acid to form a complex of Fn+ and phenylboronic acid. The phenylboronic acid (4-boron-L-phenylalanine, PBA) is a chemically unique group. As an electron-deficient group, PBA typically exists in aqueous solution as a neutral and acidified hydroxyboronic acid anion. In this state, the boron atom exhibits a planar structure, enabling it to form stable complexes with Lewis bases or oxygen and nitrogen groups containing electron-donating capabilities, thereby generating coordinate bonds. Furthermore, in its anionic state, the PBA group can also bind to positively charged substances through electrostatic interactions and benzene ring conjugation. Protein surfaces typically contain various cationic groups, such as amino, imidazole, guanidinium, and anionic carboxylic acid groups. The PBA group can utilize its chemical properties to complex with these groups in proteins covalently or non-covalently, thus playing a crucial role in the construction of nanoparticles and the development of protein delivery systems. PBA groups can bind to diols, electron-donating groups, and positively charged ions through various mechanisms such as dynamic covalent bonds, nitrogen-boron coordination, cation-π interactions, and electrostatic interactions. In addition, PBA groups respond to pH, reactive oxygen species, and diols.

[0039] Experiments show that the supercharged cavitary ferritin pFn+ introduced by the PBA group can form a complex with the delivered protein, and the activity of the delivered protein remains unchanged after entering the cell, such as enzymes, antibodies, and toxic proteins. Moreover, the supercharged cavitary ferritin pFn+ has high safety and does not produce a hemolytic reaction.

[0040] This invention constructs a highly efficient, universal, safe, and simple intracellular protein delivery system. This protein carrier delivery system can effectively deliver various proteins into cells and allow them to exert their biological activities, providing technical support for the development of protein therapies targeting intracellular targets. It also provides new application methods for subsequent research on protein-cell pathways, intracellular protein interactions, and the regulation of intracellular protein levels. This invention has the following advantages:

[0041] 1. Highly efficient protein delivery: Using GFP as a model protein, studies have shown that the pFn+@GFP complex can achieve highly efficient cell entry in various cell lines and can also achieve endosome escape.

[0042] 2. Wide adaptability: pFn+ can bind to a variety of proteins to form nanoparticles with uniform particle size, while maintaining the biological activity of the delivered protein;

[0043] 3. Safety: Cytotoxicity and hemolytic activity tests showed that pFn+ has good preliminary biosafety. Attached Figure Description

[0044] Figure 1The results are from a secondary mass spectrometry analysis of a peptide containing a PBA group.

[0045] Figure 2 This study aims to identify the physicochemical properties of supercharged ferritin, investigate its cell entry efficiency, and evaluate its small molecule delivery efficiency. A shows the SEC-HPLC elution curve of pFn+; B shows the dynamic light scattering measurement of purified pFn+; C shows the cell entry efficiency of Fn+ with different charges; and D shows the delivery efficiency of Fn+ encapsulated with different small molecule compounds.

[0046] Figure 3 The results are flow cytometry findings of pFn+@GFP and PULSin@GFP uptake in MDA-MB-231 cells.

[0047] Figure 4 This study investigates the intracellular localization of fluorescent protein delivered by supercharged ferritin and the co-localization of intracellular endosome markers of the fluorescent protein. A shows the delivery and intracellular localization of pFn+@GFP; B shows the co-localization curves of endosome markers and GFP.

[0048] Figure 5 The particle size and potential of the complex were detected using a Zetasizer Nano ZS90 nanoparticle size analyzer. A represents pFn+@β-Gal; B represents pFn+@HRP; C represents pFn+@Saporin; D represents pFn+@RNase; E represents pFn+@RNP; and F represents pFn+@IgG.

[0049] Figure 6 This study investigated the intracellular delivery and activity of β-galactosidase and horseradish peroxidase. A shows optical microscopy observation after intracellular delivery of pFn+@β-Gal (scale bar: 50 μm); B shows DAB colorimetric assay after delivery of pFn+@HRP (scale bar: 50 μm).

[0050] Figure 7 This study investigated the intracellular delivery and activity of the toxic proteins Saporin and RNase A. A represents the cytotoxicity assay of the pFn+@Saporin complex against HeLa cells (n=5, mean±SD); B represents the cytotoxicity assay of the pFn+@RNase complex (n=5, mean±SD).

[0051] Figure 8 This study investigates the intracellular delivery of the Cas9 / sgRNA complex. A shows the gene editing efficiency of the pFn+@RNP complex. B shows in vivo imaging of ex vivo tissues after pFn+@RNP delivery.

[0052] Figure 9For the study of intracellular delivery of the antibody / Trim21 complex. Intracellular targeted degradation of the pFn+@Trim-away complex was analyzed using laser confocal microscopy (scale bar: 10 μm).

[0053] Figure 10 This study investigates the biocompatibility of supercharged ferritin. A represents the cytotoxicity of different concentrations of pFn+ as determined by MTT assay (n = 5, mean ± SD); B represents the hemolytic activity assay of different concentrations of pFn+ (n = 5, mean ± SD).

[0054] Figure 11 This is a schematic diagram illustrating the principle of the present invention. Detailed Implementation

[0055] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0056] The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. All other embodiments obtained by those skilled in the art based on the specific embodiments of the invention without inventive effort are within the protection scope of the invention.

[0057] In the embodiments of the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art; in the embodiments of the present invention, unless specifically indicated, the technical means used are conventional means well known to those skilled in the art. Terminology:

[0058] HFn represents ferritin without mutations;

[0059] Fn+ represents supercharged ferritin with introduced mutation sites and mutated amino acids;

[0060] pFn+ represents supercharged ferritin introduced by the PBA group;

[0061] Example 1: Screening and preparation of supercharged ferritin (Fn+) mutant sites introduced by the PBA group:

[0062] (1) The PDB structure of ferritin HFn was uploaded to the Rosetta supercharge module for screening surface mutation sites. With increasing module runs and energy assignments of positively charged amino acids, the number of HFn surface mutation sites increased, ultimately identifying several potential surface mutation sites. The corresponding amino acids were mutated to arginine (R) or lysine (K), and the mutated ferritin sequence was uploaded to Alphafold2 for homology modeling to obtain the mutated PDB structure. The prediction accuracy of the predicted structure was then analyzed. Chimerax's Matchmaker selected positively charged amino acids by calculating the mean distance of atomic offsets (RMSD) of the mutated structure.

[0063] result:

[0064] The changes in the average atomic offset distance of the structure after mutation are shown in Table 1. These are the RMSD values ​​when each mutation site is mutated to R or K, thus determining the mutation sites of the protein. Among them, the smaller the RMSD of R or K, the smaller the impact on ferritin, and the less likely the properties will change. Therefore, the mutant amino acids and their sites with smaller RMSDs were selected.

[0065] Table 1: Changes in the mean distance of atomic offset (RMSD) of the structure after mutation

[0066]

[0067] (2) Preparation of supercharged ferritin Fn+.

[0068] The HFn gene sequence was obtained from NCBI. After synthesizing the gene sequence expressing the HFn fusion protein with an N-terminal His tag, the target fragment was obtained by double digestion with XbaI and HindIII. This fragment was then ligated into a pET28a(+) plasmid with the same sticky ends using T4 ligase. The circularized HFn protein expression plasmid was transformed into BL21(DE3) competent cells and spread on a complete nutrient solid medium containing kanamycin sulfate resistance. The cells were incubated statically at 37°C for 12 hours. Single colonies were picked from the solid plates and inoculated into 2 mL test tubes containing LB liquid medium with Kansin resistance. The cells were incubated overnight at 37°C and 220 rpm for 8 hours using a shaker. The plasmid was extracted from the cultured bacterial solution. Point mutation was performed using the FastMutagenesis Kit V2. The purified point mutation product was transformed into BL21(DE3) competent cells to obtain an expression strain expressing supercharged ferritin Fn+.

[0069] The supercharged ferritin Fn+ bacteria obtained above were inoculated into LB medium at a ratio of 1:200 (v / v) and cultured overnight in a constant temperature shaker at 37°C and 220 rpm. The bacterial cells were collected and sonicated under ice bath conditions until the viscosity of the bacterial solution decreased significantly. The sonicated bacterial solution was centrifuged at 4°C and 12,000 rpm for 15 min, and the precipitate was collected. The bacterial precipitate was resuspended in inclusion body washing buffer at a mass-to-volume ratio of 1:20 and stirred thoroughly with a magnetic stirrer at room temperature for 30 min until the sonicated precipitate was fully resuspended. After full resuspending, the sample was centrifuged at 4°C and 12,000 rpm for 10 min, and the precipitate was collected. The washed precipitate was stirred thoroughly with dissolution buffer at room temperature for 60 min until the sample was fully dissolved. Then, it was centrifuged at 4°C and 12,000 rpm for 20 min, and the supernatant was collected. Purification was then performed using the AKTA-PURE system. Analysis was performed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on a 12% (v / v) polyacrylamide gel. Then, the purest components were collected, ultrafiltered using a 50kDa centrifugal ultrafiltration tube, and the buffer was replaced with protein refolding solution to obtain a concentrated fusion protein.

[0070] Example 2: Detection of Fn+ cavity-loaded delivery efficiency and physicochemical properties of PBA-introduced supercharged ferritin (pFn+).

[0071] A series of ferritins with different positive charges were purified for cell entry and delivery efficiency studies. Digested single-cell suspensions were seeded in 48-well plates and cultured overnight. Then, GFP-modified Fn+ with different charges were added, and the plates were cultured for another 24 hours to promote intracellular delivery. Flow cytometry was then used to assess delivery efficiency. Furthermore, to further validate delivery efficiency, different small molecules, including doxorubicin (DOX), small interfering RNA (siRNA), and manganese ions (Mn ions), were loaded into the cavities, and a series of assays were performed. The delivery efficiency of DOX and siRNA was quantitatively analyzed by flow cytometry, while the Mn ion was quantified using formaldehyde oxime spectrophotometry.

[0072] Referring to the literature (DOI:10.1002 / anie.200803240), the plasmid of non-natural amino acid PBA (4-boron-L-phenylalanine) aminoacylated tRNA synthetase and the plasmid of supercharged ferritin (Fn+) were co-transformed into the expression system. The resulting culture was then plated on double-antibody plates containing kanamycin sulfate and chloramphenicol resistance, incubated overnight at 37°C, and single colonies were picked for expansion. A final concentration of 10% (v / v) glycerol was added to the freshly cultured bacterial suspension, mixed thoroughly, and the glycerol-containing bacteria were stored at -80°C for later use. The strain was induced to express supercharged ferritin (pFn+) introduced by the PBA group. Glycerol-preserved bacteria were inoculated at a ratio of 1:200 (v / v) into 2 mL of LB medium containing kanamycin sulfate solution and chloramphenicol at a final concentration of 50 μg / mL. The culture was incubated overnight at 37°C and 220 rpm in a shaker until the OD600 value reached 0.6–0.8. Then, the culture was inoculated into 20 mL of LB medium in a small conical flask at the same ratio and fermented at 37°C and 220 rpm for 7 h. The fermented seed culture was then inoculated into 200 mL of large LB medium containing kanamycin sulfate and chloramphenicol resistance for further fermentation. When the OD600 of the bacterial culture reached 0.6–0.8, 1 mM IPTG, 1 mM arabinose, and 1 mM non-natural amino acids were added to induce protein expression. The culture was then incubated at 37°C and 220 rpm for 7 h in a shaker to induce protein expression. The strain underwent validation for induced expression and validation for the introduction of non-natural amino acids.

[0073] The PBA-introduced supercharged ferritin (pFn+) was purified according to the above purification method. The purified protein sample was filtered through a 0.22 μm filter and used for SEC-HPLC analysis. The target protein was diluted to 0.5 mg / mL, and the hydrodynamic volume and purity of the protein were analyzed using a Zenix-C 300 molecular sieve chromatography column. The hydration kinetic diameter and surface potential were determined using DLS. When determining the hydration kinetic diameter, the protein sample was slowly added to a clean quartz cuvette, ensuring no air bubbles were generated. The particle size of the complex was then detected using a Zetasizer Nano ZS90 nanoparticle size analyzer.

[0074] result:

[0075] Figure 1 Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) analysis was performed on target peptides that may contain PBA groups after peptide separation. The results showed that the introduction efficiency of PBA groups was 69.4%, and they were successfully introduced into the succinate codon target site of the corresponding protein.

[0076] Figure 2 A is the SEC-HPLC elution curve of pFn+; Figure 2B represents the dynamic light scattering measurement of purified pFn+; Figure 2 C is a study on the cell entry efficiency of Fn+ with different charges detected and analyzed by flow cytometry. It was found that Fn+ with a charge greater than 168 has receptor-independent cell entry, and the cell entry efficiency increases with the increase of charge; cell entry does not occur or the cell entry efficiency is low when the charge is less than 168. Figure 2 Further research was conducted on the delivery efficiency of Fn+ carrying different small molecule compounds (DOX, siRNA, and Mn). The supercharged ferritin with amino acid mutations can enter cells through non-CD71 cells and the delivery efficiency of small molecule compounds is greatly improved.

[0077] Example 3: Cellular universality study of intracellular delivery of fluorescent proteins

[0078] Referring to Example 2, GFP was coated with pFn+216 to form pFn+216@GFP (Note: +216 means the total charge of Fn+ is 216). This was added to MDA-MB-231 cells and incubated for 12 hours. PULSin was used as a positive control for fluorescent protein delivery. Cells were washed three times with PBS to remove untaken proteins. Flow cytometry was used to detect the cellular uptake efficiency of pFn+@GFP.

[0079] result:

[0080] like Figure 3 The results showed that after incubation with MDA-MB-231 cells, flow cytometry analysis revealed that pFn+216@GFP exhibited higher cellular uptake efficiency compared to the commercially available protein transfection reagent PULSin, indicating a better delivery of GFP protein into cells. Furthermore, pFn+@GFP successfully delivered GFP intracellularly to various cell types after co-incubation, demonstrating the cellular versatility of this protein intracellular delivery system.

[0081] Example 4: Study on the delivery mechanism of fluorescent protein delivered by supercharged ferritin

[0082] To investigate the delivery mechanism of fluorescent protein delivered by supercharged ferritin, this invention uses laser confocal microscopy to observe the intracellular localization and distribution of GFP protein delivered by pFn+216@GFP.

[0083] result:

[0084] Figure 4 A shows the intracellular localization and distribution of GFP protein delivered by pFn+216@GFP using laser confocal microscopy. Figure 4B uses endosome markers to investigate changes in intracellular transport pathways during pFn+216@GFP intracellular delivery. Immunofluorescence staining was used to identify the colocalization of intracellular GFP with the early endosome marker Rab5 and the late endosome marker LAMP1, and the colocalization of GFP with lysosomes was analyzed using the lysosomal tracer Lysotracker Red.

[0085] Conclusion: This invention uses HeLa cells with low CD71 expression. The GFP delivered by pFn+216@GFP can be effectively taken up by the cells and evenly distributed within them. pFn+216@GFP bypasses late endosomes / lysosomes and escapes from early endosomes before accumulating in the cytoplasm, rather than using the non-CD71-dependent entry pathway specific to Fn.

[0086] Example 5: Preparation and Characterization of Supercharged Ferritin and Protein Complex

[0087] To verify the universality of pFn+ delivery proteins, supercharged ferritin and protein complexes were prepared according to Example 3, specifically pFn+@β-Gal, pFn+@HRP, pFn+@Saporin, pFn+@RNase, pFn+@RNP, and pFn+@IgG. The particle size and potential of the complexes were detected using a Zetasizer Nano ZS90 nanoparticle size analyzer.

[0088] result:

[0089] like Figure 5 The results show that pFn+ can form complexes with various proteins of suitable particle size, and the supercharged ferritin introduced by the PBA group can effectively bind to the target protein to form a complex with uniform and suitable nanoparticle size that is conducive to cellular uptake.

[0090] Example 6: Intracellular delivery and activity assay of β-galactosidase and horseradish peroxidase

[0091] An efficient protein delivery system requires not only effective uptake by cells but also good preservation of the protein's biological activity after entry. 1 μM β-galactosidase and horseradish peroxidase were gently added dropwise to an appropriate concentration of pFn+ and incubated at room temperature for 30 min, during which the suspension was gently aspirated to form a homogeneous complex. β-galactosidase reacts with the substrate X-Gal to form a deep blue product, making the blue-colored cells easily observable under a light microscope. Horseradish peroxidase also reacts with the substrate to produce color. The digested single-cell suspension was seeded in 24-well plates. After 24 h, cells were treated with the pFn+@β-Gal and pFn+@HRP complex, and substrate color development was performed 12 h later. Figure 6A represents cells treated with the pFn+@β-Gal complex, followed by incubation with the substrate to develop a dark blue product. Figure 6 B is a schematic diagram of DAB colorimetric optical microscopy observation of pFn+@HRP delivery (scale bar: 50μm).

[0092] result:

[0093] like Figure 6 The results showed that pFn+ could successfully internalize the enzyme-active protein into the cell, and its enzyme catalytic activity was well preserved. Its delivery and activity performance were better than that of PULSin protein transfection reagent.

[0094] Example 7 Intracellular delivery and activity assay of toxic proteins Saporin and RNase A

[0095] To demonstrate that the biological activity of the protein was preserved after entering the cell, the different mechanisms by which the cytotoxic proteins Saborin and RNase A exerted their cytotoxic effects were used for further verification. Digested single-cell suspensions were seeded in 96-well plates and cultured overnight in a cell culture incubator. The next day, pSC-HFn@Saporin or pSC-HFn@RNase A was added to the 96-well plates, with Saborin or RNase A alone and the commercial protein transfection reagent PULSin used as controls. After 12 hours, untaken proteins were washed away with PBS. The culture medium was replaced with complete medium containing 10% (v / v) FBS, and the cells were cultured for another 12 hours in a cell culture incubator. MTT assays were then performed. Figure 7 A and B represent the cytotoxic effects of pFn+@Saporin and pFn+@RNase A on cells, respectively.

[0096] result:

[0097] like Figure 7 The results showed that the complex formed by pFn+ with saporin and RNase can be effectively internalized into cells and inhibit cell proliferation. pFn+ can efficiently deliver bioactive proteins into cells while maintaining their biological activity.

[0098] Example 8: Intracellular delivery and in vivo quantitative detection of the Cas9 / sgRNA complex

[0099] To determine the intracellular delivery and gene editing efficiency of the Cas9 / sgRNA complex, digested and stably transfected HEK-293T single-cell suspensions were seeded in 6-well plates and cultured overnight. The next day, 1 μM of pFn+@Cas9 / sgRNA complex was added, and after 12 hours of incubation, the cells were washed with PBS to remove untaken proteins, and then replaced with fresh complete culture medium for another 48 hours. After culture, cells were digested with trypsin and enriched by centrifugation. Genomic DNA was then extracted using a genomic DNA extraction kit, and the GFP gene was amplified from it. After the reaction, the GFP gene fragment was recovered using a DNA purification kit, and the gene editing efficiency was assessed using a T7 Endonuclease I (T7E1) assay. Figure 8 A showed that the pFn+@Cas9 / sgRNA complex could achieve efficient genome editing in stably transformed cells. Subsequently, in vivo delivery efficiency studies were conducted. In in vivo experiments, mice were injected via tail vein using a Cy7-labeled protein complex, and ex vivo tissue samples were collected for Cy7 signal detection and quantification. Figure 8 B shows the fluorescence quantitative results of pFn+@Cas9 / sgRNA after in vivo delivery.

[0100] result:

[0101] like Figure 8 The results show that the pFn+@Cas9 / sgRNA complex can not only internalize Cas9 / sgRNA into cells, but also has effective gene editing capabilities within cells.

[0102] Example 9 Intracellular delivery and detection of the antibody / Trim21 complex

[0103] Equimolar amounts of Trim21 protein and GFP antibody were placed in EP tubes and incubated at room temperature for 30 minutes, with gentle vortexing during incubation to prepare the Trim-away complex. Next, the digested HEK-293T(GFP) single-cell suspension was seeded into 35 mm laser confocal microscope culture dishes and cultured overnight. Subsequently, the pFn+@antibody / Trim21 complex was added to the culture dishes, with the commercial protein transfection reagent PULSin used as a control. After 12 hours of incubation, untaken protein was washed away with PBS. Following staining, changes in green fluorescence intensity in HEK-293T(GFP) cells were observed using laser confocal microscopy to verify the effectiveness of pFn+@antibody / Trim21 in knocking down GFP. Figure 9 A shows the laser confocal detection results of pFn+@antibody / Trim21.

[0104] result:

[0105] The highly efficient intracellular delivery protein carrier pFn+ was used to deliver antibodies, validating its ability to promote antibody internalization and its potential application in antibody-mediated ubiquitin-proteasome degradation technology.

[0106] Example 10: Biosafety Study of Supercharged Ferritin

[0107] To investigate the biosafety of pFn+, single-cell suspensions of RAW264.7 and HEK-293T cells were seeded in 96-well plates and cultured overnight. Different concentrations of pFn+ were then added, and the cells were cultured for another 24 hours. Subsequently, 10 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated for 4 hours. Then, 100 μL of DMSO was added, and incubation continued. Finally, absorbance was measured at 570 nm to assess the cytotoxicity of pFn+. Figure 10 B. The hemolytic activity of pFn+ was studied. Different concentrations of pFn+, PBS, and 0.1% (v / v) Triton X-100 were added to the red blood cell working solution as a control group. After mixing, the solution was incubated in a 37°C water bath, and the hemolysis was observed and recorded at regular intervals. The absorbance was read at 540 nm using an ELISA reader to determine the amount of red blood cell destruction and to calculate the hemolytic activity of pFn+.

[0108] result:

[0109] like Figure 10 The results showed that pFn+ exhibited no significant cytotoxicity in various cell lines at a range of concentration gradients. Hemolysis assays demonstrated that pFn+ showed no significant hemolytic activity over extended periods and at different concentrations, consistent with the PBS-treated group, indicating the biocompatibility of pFn+ as a protein delivery system.

Claims

1. An overcharged ferritin Fn+, characterized in that, The supercharged ferritin Fn+ is obtained by site mutation on the basis of the prototype ferritin, wherein one or more of aspartic acid D at position 15, alanine A at position 18, asparagine N at position 25, aspartic acid D at position 45, aspartic acid D at position 84, glutamic acid E at position 94, asparagine N at position 98, cysteine C at position 102, histidine H at position 105, asparagine N at position 109, glutamic acid E at position 116, aspartic acid D at position 123 and glutamic acid E at position 162 are mutated into arginine R or lysine K; wherein the total charge is greater than or equal to +168, and the amino acid sequence of the prototype ferritin is shown in SEQ ID No: 1; Specifically as follows: Fn+168 D15K / D45R / D84K / E94R / E116R / D123K / E162R Fn+192 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R Fn+192 D15K / D45R / D84K / E94R / E116R / D123K / E162R / N25R Fn+192 D15K / D45R / D84K / E94R / E116R / D123K / E162R / N98R Fn+192 D15K / D45R / D84K / E94R / E116R / D123K / E162R / C102K Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N98R Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / C102K Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / H105K Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N109K Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / N25R / N98R Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / N25R / C102K Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / N25R / H105K Fn+216 D15K / D45R / D84K / E94R / E116R / D123K / E162R / N25R / N109K Fn+240 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / C102K Fn+240 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / H105K Fn+240 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N109K Fn+240 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N98R / C102K Fn+240 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N98R / H105K Fn+240 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N98R / N109K Fn+264 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N98R / C102K Fn+264 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N98R / H105K Fn+264 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N98R / N109K Fn+264 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N98R / C102K / N109K Fn+264 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N98R / C102K / H105K Fn+288 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N98R / C102K / H105K Fn+288 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N98R / C102K / N109K Fn+312 D15K / D45R / D84K / E94R / E116R / D123K / E162R / A18R / N25R / N98R / C102K / H105K / N109K. ​ 2. A PBA-containing supercharged ferritin pFn+ characterized in that pFn+ is prepared by introducing PBA group at the N-terminal of the supercharged ferritin Fn+ of claim 1, and is achieved by the following steps, PBA is a non-natural amino acid 4-boron-L-phenylalanine, the plasmid of 4-boron-L-phenylalanine aminoacyl tRNA synthetase and the plasmid of supercharged ferritin Fn+ are co-transformed into an expression vector to obtain pFn+.

3. An expression vector, characterized by, It can express the supercharged ferritin Fn+ of claim 1 or the PBA-containing supercharged ferritin pFn+ of claim 2.

4. A host cell containing the expression vector of claim 3.

5. Use of the PBA-containing supercharged ferritin pFn+ of claim 2, the expression vector of claim 3 or the host cell of claim 4 in the preparation of a drug delivery system.

6. Use according to claim 5, characterized in that, The drug is a small molecule compound, a nucleic acid, a metal drug or a protein.

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