Intracellular delivery system and application thereof

Abstract

The invention relates to natural ferritin, in particular to an intracellular delivery system and application thereof. The system can effectively deliver various proteins into cells, keeps the biological activity of the proteins, and provides important technical support for developing protein therapy with intracellular targets as cores. The method shows significant advantages in the fields of protein intracellular transport promotion, intracellular protein interaction research, protein level regulation and control and the like. The delivery system has wide applicability, is simple and convenient to operate, is safe and reliable, and becomes an important tool for future intracellular protein research and application.

Description

Technical Field

[0001] The present invention relates to natural ferritin, and in particular to an intracellular delivery system and application thereof. Background Art

[0002] Natural ferritin (Fn) is a widely existing iron storage protein, composed of 24 subunits, which aggregate to form a cage-like structure with an outer diameter of 12nm and an inner cavity of 8nm. Various ferritin variants can be obtained by modifying its subunits. Compared with natural ferritin, these subunits have new structures and functions. In addition, the 8nm cavity provides a location for the loading of drugs / biomacromolecules and the synthesis of nanozymes. However, Fn enters cells through a unique CD71-dependent pathway. Because CD71 is not expressed in all cells or the expression of CD71 in some cells is low, and the efficiency of entry into cells is low, Fn makes it difficult to become a universal protein carrier. Therefore, natural ferritin needs to be modified to obtain more flexible and diverse functional applications.

[0003] Therefore, it is expected in the art to transform hollow proteins that do not have the ability to enter cells into a universal intracellular protein delivery carrier that can mediate the formation of a complex between the protein and the carrier through multiple interactions, safely and efficiently deliver the cargo protein into the cell, while fully retaining the biological activity of the protein in the cell. Summary of the invention

[0004] Purpose of the Invention

[0005] The purpose of the present invention is to provide a safe, efficient, universal and easy-to-operate intracellular protein delivery system to overcome the defects of existing delivery vectors.

[0006] Technical Solution

[0007] A supercharged ferritin Fn+, characterized in that the supercharged ferritin Fn+ is obtained by performing 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 to 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 as shown in SEQ ID No: 1.

[0008] A PBA-containing supercharged ferritin pFn+, characterized in that pFn+ is prepared by introducing a PBA group into the N-terminus of the supercharged ferritin Fn+. Specifically, a plasmid of an aminoacylated tRNA synthetase pair of an unnatural amino acid PBA (4-boron-L-phenylalanine) and a plasmid of a supercharged ferritin (Fn+) are co-transformed into an expression system to obtain pFn+.

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

[0010] A host cell containing the expression vector;

[0011] The PBA-containing supercharged ferritin pFn+, the expression vector or the host cell according to claim 4 are used 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 performing site mutation on the amino acid sequence of the prototype ferritin HFn, the site mutation is screened according to the Rosetta molecular modeling program and the Alphafold2 program, wherein the surface mutation site is determined by the energy minimization of the HFn crystal structure in Rosetta and the smaller RMSD value after the Alphafold2 mutation, including but not limited to the following sequence:

[0016] The amino acid sequence of HFn is:

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

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

[0019]

[0020]

[0021] The system is characterized in that the universal protein carrier is capable of CD71-independent intracellular entry and the lumen can deliver small molecule drugs (doxorubicin), nucleic acid drugs (siRNA) or metal drugs (metal ion manganese);

[0022] The system is characterized in that a PBA group is introduced at the N-terminus of the supercharged ferritin amino acid sequence, and 24 PBA groups are added to the modified ferritin, thereby improving the protein drug loading capacity and stability;

[0023] The application is characterized in that the delivered protein is a protein with drug activity, labeling effect or targeting effect, such as antigenic peptides, enzyme proteins, toxin proteins, antibodies, Trim21 and Cas9; after simple mixing with active proteins, it can form particles of uniform size and about 100 nm, and has good stability and maintains efficient cell entry activity;

[0024] The application is characterized in that a system containing PBA supercharged ferritin is used in the preparation of a carrier for intracellular protein delivery;

[0025] The PBA introduction described above utilizes genetic code expansion (GCE) technology to enable the stop codon (usually the amber codon UAG) to perform the coding function and introduce a new amino acid. The plasmid expressing the aminoacyl tRNA synthetase and transfer RNA is transformed into the expression strain, so that the nonsense codon (usually the amber codon UAG) can perform the coding and introduce the unnatural amino acid at the corresponding site;

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

[0027] The present invention also relates to a method for preparing the intracellular protein delivery vector. Specifically, the method comprises 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 the screening of surface mutation sites. As the number of module runs and the energy assignments of positively charged amino acids increased, the number of HFn surface mutation sites increased. Finally, multiple potential surface mutation sites of HFn were screened out. Subsequently, the R / K values ​​of the surface mutation sites were selected. The structure prediction software Alphafold2 and Chimerax's Matchmaker selected positively charged amino acids by calculating the average distance of atomic deviation (RMSD) of the mutated structure.

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

[0031] The glycerol-preserved bacteria expressing supercharged ferritin 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 220rpm. The bacteria were collected and ultrasonically disrupted under ice bath conditions until the viscosity of the bacterial solution decreased significantly. The ultrasonic bacterial solution was centrifuged at 4°C and 12000rpm for 15min, and the ultrasonic precipitation sample was collected. The bacterial precipitate was resuspended with the inclusion body washing buffer at a mass volume ratio of 1:20, and the magnetic stirrer was fully stirred for 30min at room temperature until the ultrasonic precipitation sample was fully resuspended. After the sample was fully resuspended, it was centrifuged at 4°C and 12000rpm for 10min to collect the precipitation sample. The washed precipitation sample was fully stirred at room temperature with a dissolution buffer for 60min until the sample was fully dissolved. Then the dissolution supernatant was collected by centrifugation at 4°C and 12000rpm for 20min, and then purified using the AKTA-PURE system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using 12% (v / v) polyacrylamide gel. Then, the purest fraction was collected, ultrafiltered using a 50 kDa centrifugal ultrafiltration tube, and the buffer was replaced with a protein refolding solution to obtain a fusion protein concentrate.

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

[0033] Fluorescein labeling: FITC-labeled rabbit serum-derived IgG and Cas9 protein, incubated overnight at 4°C at a FITC: target protein molar ratio of 5:1, and unreacted FITC fluorescent 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 target protein was gently added to the appropriate concentration of pFn+ and incubated at room temperature for 30 minutes, during which the suspension was gently aspirated to ensure the formation of a uniform complex; Complex characterization: The particle size and potential of the complex were detected by the nanoparticle sizer Zetasizer Nano ZS90.

[0034] principle:

[0035] like Fig.11 The schematic diagram of the principle shown uses super-strong ferritin (Fn+) with a diameter of about 12 nanometers, and introduces phenylboronic acid (PBA) on the surface of Fn+ nanocages through genetically encoded expansion (GCE) technology to obtain the carrier pFn+. pFn+ allows mixing with target proteins or biomolecules to form stable complexes through NB coordination, ion interactions, and multi-protein binding sites. These complexes are then efficiently internalized by cells and released into the cytoplasm, ensuring the activity of the delivered molecules and performing their biological functions.

[0036] Beneficial Effects

[0037] Fn enters cells through a unique CD71-dependent pathway. The CD71 pathway has the following disadvantages: it can only enter cells specifically, and it cannot enter cells without expressing CD71, and it cannot achieve universality and cannot escape lysosomes. For this reason, the inventors, based on the inspiration that positively charged proteins in CN112608366A can promote cell entry, mutated Fn into amino acids to make it a supercharged cavity ferritin Fn+. Among them, supercharged proteins are a class of proteins with more than one net charge per kilodalton molecular weight. Supercharged proteins in a folded state have important biological functions, including but not limited to DNA binding, transcriptional regulation, protein synthesis, antibacterial activity, and signal transduction. Biomacromolecule proteins are coordinated by electrostatic effects and other intermolecular forces to achieve their biological activity. Supercharging can achieve control of the primary structure of proteins, while providing the ability to regulate charge density, molecular weight, and change the charge position of supercharged proteins. In addition, various charge-induced interactions can be introduced into these types of supercharged proteins, and supercharged proteins can be easily expressed by fusion with other functional proteins. Finally, supercharged proteins can be encoded by genes, which allows them to be expressed in target cells. Supercharged proteins, especially those that are superpositively charged by rational mutation into positively charged amino acids (R\K), can significantly improve the efficiency of cellular internalization. The number of mutant charges, molecular weight and protein folding state are important structural features that determine the uptake pathway of mammalian cells. Experimental results show that if the total positive charge is higher than +168, its intracellular performance increases, and the intracellular efficiency increases with the increase of charge. After superpositive charge modification, the active intracellular ability becomes stronger, independent of the CD71 pathway, bypassing the late endosome / lysosome, and escaping from the early endosome before accumulating in the cytoplasm, expanding the scope of application.

[0038] However, the packaging capacity of Fn+ obtained by simply mutating amino acids is limited. In order 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 group with unique chemical properties. As an electron-deficient group, PBA usually exists in two forms of neutral and acidified hydroxyboronic acid anions in aqueous solution. In this state, the boron atom presents a planar structure and can form a stable complex with Lewis base or oxygen and nitrogen groups with electron-donating ability, thereby generating a coordination bond. In addition, the PBA group can also combine with positively charged substances through electrostatic interactions and benzene ring conjugation in the anionic state. The surface of proteins usually contains a variety of cationic groups, such as amine, imidazole, guanidine and anionic carboxylic acid groups. The PBA group can use its chemical properties to complex with these groups in proteins in a covalent or non-covalent manner, thereby playing a key role in the construction of nanoparticles and the development of protein delivery systems. The PBA group can bind to diols, electron-donating groups, positively charged ions, etc. through dynamic covalent bonds, nitrogen-boron coordination, cation-π interactions, electrostatic interactions, etc., plus the PBA group responds to pH, active oxygen, diols, etc.

[0039] Experiments show that the supercharged cavity 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, toxic proteins, etc., and the supercharged cavity ferritin pFn+ is highly safe and does not produce hemolytic reaction.

[0040] The present invention constructs an efficient, universal, safe and simple intracellular protein delivery system. The delivery system of the protein carrier can effectively deliver a variety of proteins into the cell and exert their biological activity, which helps to provide technical support for the development of protein therapy for intracellular targets. It provides new application means for subsequent protein cell pathway research, intracellular protein interaction research, intracellular protein level regulation and other ideas. The present invention has the following advantages:

[0041] 1. Efficient protein delivery: Using GFP as a model protein, studies have shown that the pFn+@GFP complex can achieve efficient cell entry and endosomal escape in a variety of cell lines;

[0042] 2. Broad 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1This is the result of secondary mass spectrometry analysis of peptides containing PBA groups.

[0045] Figure 2 The physicochemical properties of supercharged ferritin were identified, and its intracellular and small molecule delivery efficiency was studied. A is the SEC-HPLC elution curve of pFn+; B is the dynamic light scattering measurement of purified pFn+; C is the intracellular efficiency study of Fn+ with different charges; D is the delivery efficiency study of Fn+ encapsulated with different small molecule compounds.

[0046] Figure 3 Flow cytometry results of pFn+@GFP and PULSin@GFP cellular uptake in MDA-MB-231 cells.

[0047] Figure 4 The intracellular localization of fluorescent protein delivered by supercharged ferritin and the co-localization of fluorescent protein with endosomal markers in cells. A is the delivery and intracellular localization of pFn+@GFP; B is the co-localization curve of endosomal markers and GFP.

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

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

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

[0051] Figure 8 This is a study of intracellular delivery of Cas9 / sgRNA complexes. A is the detection of gene editing efficiency of pFn+@RNP complexes. B is in vitro tissue live imaging of pFn+@RNP delivery in vivo.

[0052] Fig. 9This is a study on the intracellular delivery of antibody / Trim21 complex. Laser confocal microscopy analysis of the intracellular targeted degradation of pFn+@Trim-away complex (scale bar: 10μm)

[0053] Fig.10 The biosafety study of supercharged ferritin. A is the MTT analysis of the cytotoxicity of pFn+ at different concentrations (n=5, mean±SD); B is the hemolytic activity analysis of pFn+ at different concentrations (n=5, mean±SD).

[0054] Fig.11 It is a schematic diagram of the principle of the present invention. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0056] The following embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Based on the specific embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work belong to the protection scope of the present invention.

[0057] In the examples 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 examples of the present invention, unless otherwise specified, the technical means used are conventional means well known to those skilled in the art.

[0058] HFn represents ferritin without mutation;

[0059] Fn+ represents the site where the mutation was introduced and the supercharged ferritin of the mutated amino acid;

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

[0061] Example 1 Screening and preparation of supercharged ferritin (Fn+) and supercharged ferritin (pFn+) mutation sites introduced by PBA groups:

[0062] (1) The PDB structure of ferritin HFn was uploaded to the Rosetta supercharge module for the screening of surface mutation sites. As the number of module runs and the energy assignments of positively charged amino acids increased, the number of HFn surface mutation sites increased, and eventually multiple potential surface mutation sites of HFn were screened out. 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, and the predicted structure was analyzed for prediction accuracy. Chimerax's Matchmaker selects positively charged amino acids by calculating the mean relative distance of atomic displacement (RMSD) of the mutated structure.

[0063] result:

[0064] The change in the average distance of atomic offset of the structure after mutation is shown in Table 1, which is the RMSD value when each mutation site mutates to R or K, and then the mutation site of the protein is determined; the smaller the RMSD of R or K, the smaller the impact on ferritin, and the properties are not easily changed, so the mutant amino acids and their sites with smaller RMSD are selected.

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

[0066]

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

[0068] The gene sequence of HFn was obtained from NCBI. After gene synthesis of the gene sequence expressing the HFn fusion protein with a His tag at the N-terminus, the target fragment was obtained by double digestion with XbaⅠ and HindⅢ, and ligated to the pET28a(+) plasmid with the same sticky end using T4 ligase. The plasmid expressing the HFn protein after circularization was transformed into BL21(DE3) competent cells, and coated on a complete nutrient solid medium containing kanamycin sulfate resistance using a coating rod, and cultured at 37°C for 12 hours. A single colony was picked from the solid plate, inoculated into a test tube containing 2 mL of LB liquid medium with Kan resistance, and cultured overnight at 37°C and 220 rpm in a constant temperature shaker for 8 hours. The cultured bacterial liquid was extracted from the plasmid. The point mutation kit FastMutagenesis Kit V2 was used for point mutation, and the point mutation product was purified and transformed into BL21(DE3) competent cells to obtain an expression strain expressing the supercharged ferritin Fn+.

[0069] The bacteria with supercharged ferritin Fn+ 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 220rpm. The bacteria were collected and ultrasonically disrupted under ice bath conditions until the viscosity of the bacterial solution decreased significantly. The ultrasonic bacterial solution was centrifuged at 4°C and 12000rpm for 15min, and the ultrasonic precipitation sample was collected. The bacterial precipitate was resuspended with the inclusion body washing buffer at a mass volume ratio of 1:20, and the magnetic stirrer was fully stirred for 30min at room temperature until the ultrasonic precipitation sample was fully resuspended. After the sample was fully resuspended, it was centrifuged at 4°C and 12000rpm for 10min to collect the precipitation sample. The washed precipitation sample was fully stirred at room temperature with a dissolution buffer for 60min until the sample was fully dissolved. Then the dissolution supernatant was collected by centrifugation at 4°C and 12000rpm for 20min, and then purified using the AKTA-PURE system. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using 12% (v / v) polyacrylamide gel. Then, the purest fraction was collected, ultrafiltered using a 50 kDa centrifugal ultrafiltration tube, and the buffer was replaced with a protein refolding solution to obtain a fusion protein concentrate.

[0070] Example 2: Detection of the physicochemical properties of Fn+ cavity encapsulation and delivery efficiency and supercharged ferritin (pFn+) introduced with PBA groups

[0071] A series of ferritins with different positive charges were purified for intracellular entry and delivery efficiency studies. The digested single cell suspension was inoculated in a 48-well plate. After overnight culture, GFP-modified Fn+ with different charges was added and cultured for 24 hours to promote intracellular delivery. Subsequently, flow cytometry was performed to evaluate the delivery efficiency. In addition, in order to further verify the delivery efficiency, different small molecules, including doxorubicin (DOX), small interfering RNA (siRNA) and manganese ions (Mn ions), were encapsulated in the cavity, and a series of tests were performed. Among them, the delivery efficiency of DOX and siRNA was quantitatively analyzed by flow cytometry, while the measurement of Mn ions was quantitatively measured by formaldehyde oxime photometry.

[0072] Referring to the literature (DOI: 10.1002 / anie.200803240), the plasmid of the aminoacylated tRNA synthetase pair of the unnatural amino acid PBA (4-boron-L-phenylalanine) and the plasmid of the supercharged ferritin (Fn+) were co-transformed into the expression system, and then coated on a double-resistance plate containing kanamycin sulfate and chloramphenicol resistance, and after overnight culture at 37°C, a single clone was picked and expanded for culture. Glycerol with a final concentration of 10% (v / v) was added to the freshly cultured bacterial solution, mixed well, and the glycerol bacteria were stored at -80°C for later use. The strain was induced to express, and the glycerol-preserved bacteria expressing the supercharged ferritin (pFn+) introduced with the PBA group were inoculated in 2 mL LB medium at a ratio of 1:200 (v / v), which contained kanamycin sulfate solution and chloramphenicol at a final concentration of 50 μg / mL, and cultured overnight in a constant temperature shaker at 37°C and 220 rpm until the OD600 value was 0.6-0.8, and then inoculated into the LB medium in a small conical flask (20 mL) at the same inoculation ratio, and fermented in a constant temperature incubator at 37°C and 220 rpm for 7 hours, and then the fermented seed stock solution was inoculated into a large LB medium (200 mL) with kanamycin sulfate and chloramphenicol resistance to continue fermentation. When the bacterial solution OD600 was 0.6-0.8, IPTG, 1 mM arabinose, and 1 mM non-natural amino acids at a final concentration of 1 mM were added to induce protein expression, and the protein expression was induced in a shaker at 37°C and 220 rpm for 7 hours. The strains were verified for inducible expression and introduction of unnatural amino acids.

[0073] According to the above purification method, the supercharged ferritin (pFn+) introduced by the PBA group was purified, and the purified protein sample was filtered with a 0.22μm filter head and used for SEC-HPLC analysis. The target protein was diluted to 0.5mg / mL, and the hydrodynamic volume and purity of the protein were analyzed using a molecular sieve chromatography column Zenix-C 300, and 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 to ensure that no bubbles were generated, and then the particle size of the complex was detected using a Zetasizer Nano ZS90 nanoparticle sizer.

[0074] result:

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

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

[0077] Example 3 Study on the cellular universality of intracellular delivery of fluorescent proteins

[0078] Referring to Example 2, pFn+216 was used to encapsulate GFP to form pFn+216@GFP (Note: +216 means the total charge of Fn+ is 216), which was added to MDA-MB-231 cells and incubated for 12 hours. PULSin was used as a positive control for fluorescent protein delivery, and the cells were washed three times with PBS to remove the unabsorbed proteins. The cell uptake efficiency of pFn+@GFP was detected by flow cytometry.

[0079] result:

[0080] like Figure 3 The results showed that after pFn+216@GFP was incubated with MDA-MB-231 cells, flow cytometry showed that pFn+216@GFP had a higher cell uptake efficiency than the commercial protein transfection reagent PULSin, which means that it could better deliver GFP protein into cells. And after pFn+@GFP was co-incubated with a variety of cells, it was able to achieve intracellular delivery of GFP, indicating the cellular versatility of this protein intracellular delivery system.

[0081] Example 4 Study on the delivery mechanism of supercharged ferritin for fluorescent protein delivery

[0082] In order to study the delivery mechanism of supercharged ferritin for delivering fluorescent protein, the present 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: The intracellular localization and distribution of GFP protein delivered by pFn+216@GFP were observed using a laser confocal microscope. Figure 4B is the use of endosomal markers to explore the changes in intracellular transport pathways during the intracellular delivery of pFn+216@GFP. Immunofluorescence staining was used to identify the co-localization of intracellular GFP with the early endosomal marker Rab5 and the late endosomal marker LAMP1, and the lysosomal tracer Lysotracker Red was used to analyze the co-localization of GFP and lysosomes;

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

[0086] Example 5 Preparation and characterization of supercharged ferritin and protein complexes

[0087] In order to verify the versatility of pFn+ protein delivery, supercharged ferritin and protein complexes were prepared with reference 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 complex were detected using the nanoparticle sizer Zetasizer Nano ZS90.

[0088] result:

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

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

[0091] In addition to being effectively taken up by cells, an efficient protein delivery system also needs to retain the biological activity of the protein after entering the cell. Gently add 1μM β-galactosidase and horseradish peroxidase to the appropriate concentration of pFn+ and incubate at room temperature for 30 minutes. During this period, gently blow and aspirate the suspension to form a uniform complex. β-galactosidase can react with the substrate X-Gal to form a dark blue product, so that it is easy to observe the blue cells under an optical microscope. Horseradish peroxidase can also color and react with the substrate. The digested single cell suspension was inoculated in a 24-well plate. After 24 hours, the cells were treated with pFn+@β-Gal and pFn+@HRP complexes, and the substrate was colorized after 12 hours. Figure 6A shows that after the cells are treated with the pFn+@β-Gal complex, the substrate is used to incubate the cells for color development, and the reaction generates a dark blue product. Figure 6 B is a schematic diagram of optical microscope observation of DAB color development delivered by pFn+@HRP (scale: 50μm)

[0092] result:

[0093] like Figure 6 It shows that pFn+ can successfully internalize enzyme-active proteins into cells, and its enzyme catalytic activity is well retained. Its delivery and activity effects are better than PULSin protein transfection reagent.

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

[0095] In order to show that the biological activity of the protein is retained after entering the cell, the toxic proteins Saporin and RNase A were used to verify again because they exert cytotoxic effects in different ways. The digested single cell suspension was inoculated in a 96-well plate and placed in a cell culture incubator for overnight culture. The next day, pSC-HFn@Saporin or pSC-HFn@RNase A was added to the 96-well plate, and only Saporin or RNase A alone and the commercial protein transfection reagent PULSin were used as controls. After 12 hours, PBS was used to wash away the unabsorbed proteins. The culture medium was replaced with a complete culture medium containing 10% (v / v) FBS and cultured in a cell culture incubator for another 12 hours. MTT detection was then performed. Figure 7 A and B are the toxic 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+, Saporin and RNase can be effectively internalized into cells and inhibit cell proliferation. pFn+ can efficiently deliver biologically active proteins into cells while maintaining their biological activity.

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

[0099] In order to determine the intracellular delivery and gene editing efficiency of Cas9 / sgRNA complex. The digested HEK-293T single cell suspension stably transfected with GFP was inoculated in a 6-well plate and cultured overnight. On the second day, 1 μM pFn+@Cas9 / sgRNA complex was added. After incubation for 12 hours, it was washed with PBS to remove unabsorbed proteins, and then replaced with fresh complete medium for further culture for 48 hours. After the culture was completed, the cells were digested with trypsin and enriched by centrifugation. The genomic DNA was then extracted using a cell genomic DNA extraction kit, and the GFP gene was amplified from it. After the reaction was completed, the GFP gene fragment was recovered using a DNA purification kit, and the gene editing efficiency was detected by the T7 Endonuclease I (T7E1) test. Figure 8 A shows that the pFn+@Cas9 / sgRNA complex can achieve efficient genome editing in stably transfected cells. Subsequently, in vivo delivery efficiency studies were performed. In the in vivo experiment, the Cy7-labeled protein complex was injected into the tail vein of mice, and the ex vivo tissues of the mice were taken for Cy7 signal detection and quantitative analysis. Figure 8 B shows the fluorescence quantification results of pFn+@Cas9 / sgRNA after in vivo delivery.

[0100] result:

[0101] like Figure 8 It shows that the pFn+@Cas9 / sgRNA complex can not only internalize Cas9 / sgRNA into cells but also enable effective gene editing in cells.

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

[0103] An equimolar ratio of Trim21 protein and GFP antibody was placed in an EP tube and incubated at room temperature for 30 minutes, vortexing gently during the period to prepare the Trim-away complex. Next, the digested HEK-293T (GFP) single cell suspension was inoculated in a 35mm laser confocal glass-bottom culture dish and cultured overnight. Subsequently, the pFn + @ antibody / Trim21 complex was added to the culture dish, and the commercial protein transfection reagent PULSin was used as a control. After incubation for 12 hours, PBS was used to wash away the unabsorbed protein. After staining, the changes in green fluorescence intensity in HEK-293T (GFP) cells were observed using a laser confocal microscope to verify the effectiveness of pFn + @ antibody / Trim21 in knocking down GFP. Fig. 9 A shows the laser confocal detection results of pFn+@antibody / Trim21.

[0104] result:

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

[0106] Example 10 Biosafety Study of Supercharged Ferritin

[0107] To study the biosafety of pFn+, single cell suspensions of RAW264.7 cells and HEK-293T cells were inoculated in 96-well plates. After overnight culture, different concentrations of pFn+ were added and cultured for 24 hours. Subsequently, 10 μL of 5 mg / mL MTT solution was added to each well and incubated in a cell culture incubator for 4 hours, followed by the addition of 100 μL of DMSO and continued incubation. Finally, the absorbance was measured at 570 nm to evaluate the toxicity of pFn+ to cells. Fig.10 B is to study the hemolytic activity of pFn+. Different concentrations of pFn+, PBS and 0.1% (v / v) Triton X-100 were added to the red blood cell working solution as the control group, mixed and incubated in a 37°C water bath, and the hemolysis was observed and recorded regularly. The absorbance was read at 540nm using an ELISA reader to determine the amount of red blood cell destruction and calculate the hemolytic activity of pFn+.

[0108] result:

[0109] like Fig.10 The results showed that pFn+ had no obvious cytotoxicity in a variety of cell lines under a series of concentration gradients. Hemolysis experiments showed that the hemolytic activity of pFn+ was not significant over a long period of time and at different concentrations, which was no different from the PBS treatment group, indicating the biosafety of pFn+ as a protein delivery system.

Claims

1. A supercharged ferritin Fn+, characterized in that The supercharged ferritin Fn+ is obtained by performing site mutations on 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 to 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.

2. A PBA-containing supercharged ferritin pFn+, characterized in that pFn+ is prepared by introducing a PBA group into the N-terminus of the supercharged ferritin Fn+ described in claim 1.

3. An expression vector containing the supercharged ferritin Fn+ described in claim 1 or the PBA-containing supercharged ferritin pFn+ described in claim 2.

4. A host cell containing the expression vector according to claim 3.

5. Use of the PBA supercharged ferritin pFn+ containing according to claim 1, the expression vector according to claim 3 or the host cell according to claim 4 in the preparation of a drug delivery system.

6. The use according to claim 5, characterized in that: The drug delivery system can achieve intracellular delivery.

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

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