A tumor microenvironment-responsive fusion protein-siRNA complex prodrug system and application thereof

Abstract

The present application relates to a fusion protein, in particular to a tumor microenvironment responsive fusion protein-siRNA complex prodrug system and application thereof. The carrier is a fusion protein coupled by PSTAG shielding sequence, tumor microenvironment enzyme responsive sequence, RNA binding protein sequence and human heavy chain ferritin. The carrier can bind and deliver siRNA drugs into cells to play a gene silencing effect.

Description

Technical Field

[0001] This invention relates to fusion proteins, and more specifically to a tumor microenvironment-responsive fusion protein-siRNA complex prodrug system and its application. Background Technology

[0002] Small interfering RNA (siRNA) is a double-stranded RNA molecule composed of approximately 21-23 nucleotides. It is a short molecule formed by the cleavage of double-stranded RNA and can specifically bind to specific mRNA sequences, leading to the cleavage and degradation of the target mRNA, thereby inhibiting gene translation and expression. siRNA mediates gene silencing or downregulation by targeting and binding to specific genes in tumor cells, thus inhibiting tumor cell growth and proliferation. Compared to traditional treatments such as chemotherapy and radiotherapy, siRNA therapy for tumors has many advantages, including high specificity, persistence, reversibility, no drug resistance, and compatibility with other treatment methods, making it a very promising therapeutic approach.

[0003] siRNA is approximately 7–8 nm in length, 2–3 nm in diameter, and has a molecular weight of approximately 13–16 kDa. Its molecules are too large to cross cell membranes, yet are rapidly cleared by glomerular filtration, making it difficult to distribute to target tissues. siRNA is readily degraded by widely distributed nucleases in the body and possesses high water solubility and polyanionic properties. After intravenous injection, it is rapidly excreted through renal filtration, with a plasma half-life of <10 min. Therefore, an efficient delivery system must be used for siRNA.

[0004] siRNA delivery vectors include liposomes, polymers, metal nanoparticles, viral vectors, and extracellular vesicles. These vectors have different advantages and disadvantages. For example, N-acetylgalactosamine (GalNAc) ligands are widely used for siRNA delivery. GalNAc-siRNA conjugates, exhibiting low adverse reactions and limited toxicity, have been shown to have a high binding affinity for the sialic acid glycoprotein receptor (ASGPR). ASGPR is mainly found on hepatocytes and rarely expressed on other cells. Drug formulations based on this conjugate can only treat liver diseases and are prone to degradation and other complications. Liposomes are widely used as gene delivery vectors, but liposome-based formulations still have some drawbacks in terms of transfection efficiency, in vivo stability, insufficient targeting ability, toxicity, and immune response. Cationic polymers have the problem of high cytotoxicity caused by high positive charge.

[0005] Therefore, finding novel siRNA delivery systems is a pressing technical problem that needs to be solved. Among them, protein-based siRNA delivery systems have significant advantages over the aforementioned delivery vectors: (1) Stability: Proteins can protect siRNA from enzymatic degradation by forming complexes or encapsulating it, thereby improving the stability and half-life of siRNA; (2) Targeting: Proteins have specific structures that enable targeted delivery to specific cells or tissues; (3) Low toxicity: As a natural biological macromolecule, the metabolic products of proteins generally do not produce toxicity to cells and tissues. Compared with chemically synthesized compounds or liposomes, protein-based siRNA delivery has lower toxicity, which can reduce adverse reactions and toxic side effects during treatment; (4) Large-scale preparation: Using gene recombination technology, protein vectors can be prepared on a large scale, which is scalable and feasible.

[0006] Therefore, the present invention provides a novel protein carrier system for delivering siRNA. Summary of the Invention

[0007] Purpose of the invention

[0008] The purpose of this invention is to provide a tumor microenvironment-responsive fusion protein-siRNA complex prodrug system, overcoming the shortcomings of existing technologies.

[0009] Technical solution

[0010] A human heavy chain ferritin derivative, characterized in that the sequence from the N-terminus to the C-terminus includes a PSTAG shielding sequence as shown in SEQ ID NO: 2, a tumor microenvironment enzyme response sequence as shown in SEQ ID NO: 3, an RNA-binding protein as shown in SEQ ID NO: 4, and human heavy chain ferritin as shown in SEQ ID NO: 5.

[0011] The human heavy chain ferritin derivative is characterized in that the amino acid sequence of the human heavy chain ferritin derivative is SEQ ID NO.1.

[0012] The application of the human heavy chain ferritin derivative in the preparation of a drug delivery carrier.

[0013] A tumor microenvironment-responsive fusion protein-siRNA complex prodrug system, characterized in that the system comprises the aforementioned human heavy chain ferritin derivative and a small interfering RNA drug with gene silencing effect.

[0014] The system is characterized in that the drug carrier is a nanoparticle formed by the polymerization of human heavy chain ferritin derivatives, and the small interfering RNA drug binds to the carrier.

[0015] The system is characterized in that the small interfering RNA drug is SEQ ID NO: 6 and SEQ ID NO: 7; or SEQ ID NO: 8 and SEQ ID NO: 9.

[0016] The application of the system in the preparation of antitumor drugs.

[0017] Specifically:

[0018] The masking sequence of PSTAG 216 (P216), consisting of 216 amino acids, is as follows:

[0019] GPSATPGSTGPTSAPGSTGPATPSGTSGPSTAGPTSGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPAGSTPSTGPSAPTGSTGPAGSTPSTGPTSGPTASGPSATPGSTGPTSAPGSTGPSATPGTSGPSGTSAPTGPSGTPASTGPATPSGTSGPSATPGTSGPSGTSPTAGPSATPGSTGPTSAPGSTGPSTGSPATGPSTPAGST, see SEQ ID NO: 2

[0020] The tumor microenvironment MMP2 enzyme response sequence is PLGLAG, see SEQ ID NO: 3;

[0021] The RNA-binding protein sequence is as follows:

[0022] GDLSAGFFMEELNTYRQKQGVVLKYQELPNSGPPHDRRFTFQVIIDGREFPEGEGRSKKEAKNAAAKLAVEILNKEKKAVSPLLLTTTNSSEGLSMGNYIGLINRIAQKKRLTVNYEQVASGVHGPEGFHYKVKMGQKEYSIGTGSTKQEAKQLAAKLAYLQILSEETSV, see SEQ ID NO: 4;

[0023] The amino acid sequence of human heavy chain ferritin is:

[0024] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES, see SEQ ID NO:5.

[0025] Preferably, the PSTAG shielding sequence, tumor microenvironment enzyme response sequence, RNA-binding protein, and ferritin are coupled via GG or GGGGS linking sequences;

[0026] The system is characterized in that the amino acid sequence of the human heavy chain ferritin derivative is:

[0027] See SEQ ID NO: 1.

[0028] Table 1 (Sequence List): Description of Fusion Protein Sequences

[0029]

[0030]

[0031] The method for preparing the system is characterized by comprising the following steps: expression and purification of the fusion protein, and binding of the fusion protein to siRNA to form a complex.

[0032] The system described is used in the delivery of anti-tumor siRNA drugs.

[0033] The application is characterized in that the tumor is liver cancer, the target is the oncogene HBx, and the P216-MMP2-RBP-HFn vector delivers si-HBx, which has an in vitro anti-tumor effect after treatment. The application is further characterized in that the vector delivers siRNA gene therapy drugs, achieving specific killing of the tumor by efficiently targeting and silencing key targets related to tumor growth, metastasis, angiogenesis, and drug tolerance.

[0034] This invention also relates to a method for preparing the aforementioned fusion protein-siRNA complex prodrug system, specifically, the method comprising the following steps:

[0035] Step (1): Express and purify the recombinant ferritin derivative.

[0036] The full-length sequence of human H-ferritin was obtained by searching the NCBI database and references. PSTAG 216, the tumor microenvironment MMP2 enzyme response site, and the RNA-binding protein sequence were introduced at its N-terminus to design the protein sequence of the fusion protein. After codon optimization with E. coli as the host, the DNA of the fusion protein was constructed into the expression vector pET-28a(+)(GenScript). Then, using the E. coli prokaryotic expression system, the plasmid of the fusion protein was transformed into the competent cells (Vazyme) of the expression strain E. coli BL21(DE3). Expression was induced by IPTG, and the bacterial culture was collected after fermentation for the isolation and purification of the fusion protein.

[0037] The specific methods for expression and purification are as follows: Glycerol-containing bacteria were activated in test tubes, expanded in small conical flasks, and then inoculated into LB liquid medium containing 1‰ Kan resistance at a volume ratio of 1:100. The culture was carried out at 37℃ and 220 rpm on a shaker, with the addition of 1 mM IPTG to induce the expression of the target protein. After culturing for another 24 h, the culture was centrifuged at 4℃ and 8000 rpm for 15 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial pellet was resuspended in 20 mM Tris-HCl (pH 8.0) Q-column equilibration buffer, and bacterial lysis was obtained by sonication, followed by centrifugation (10000 rpm, 20 min, 4℃). The supernatant was collected and loaded onto a Q-column using a peristaltic pump. The flow-through was collected, and the column was washed with Q-column equilibration buffer, with continued collection of the eluent until the G250 index no longer showed a blue color. Q-flow impregnation was performed to prepare a solution of 20 mM Tris and 2 mM MgCl2, followed by the addition of nuclease (Sinobiological 500 Units / g bacteria). The solution was incubated at 37°C for 30 min to remove residual nucleic acid and prevent RBP from binding to other RNAs. The solution was then further impregnated with Q-flow impregnation to prepare a solution of 20 mM Tris and 500 mM NaCl (NiCl2). 2+ Column balancing fluid, Ni loaded using a peristaltic pump 2+ On the column, Ni 2+ The column was washed with column impurity removal buffer (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 8.0), followed by Ni... 2+ Column elution buffer (20 mM Tris, 500 mM NaCl, 200 mM imidazole, pH 8.0) eluted the bound Ni 2+ The target protein on the column was removed, and nucleases without His tags were eliminated.

[0038] Samples were taken at each stage, and protein size and purity were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using 12% (v / v) polyacrylamide gel electrophoresis. High-purity fractions were collected, placed in 50 kDa dialysis bags, and dialyzed overnight to 1xPBS buffer. The target protein was then concentrated using a 50 kDa centrifugal ultrafiltration tube to obtain a concentrated fusion protein. Using bovine serum albumin as a standard, protein concentration was determined using the BCA protein assay kit (Beyotime). The calculated yield reached 50 mg / L of fermentation broth. The broth was aliquoted and stored at -20°C.

[0039] Step (2): Use fusion protein to bind siRNA

[0040] DNA templates were designed based on different target sequences, and siRNA was transcribed in vitro and purified by centrifugation column according to the steps of the in vitro transcription kit (Vazyme) (New England Biolabs).

[0041] By incubating P216-MMP2-RBP-HFn protein with the corresponding siRNA at 37°C for 1 hour at an N / P (monomer protein to nucleic acid molar ratio) ratio of 2, a fusion protein-siRNA complex can be obtained.

[0042] principle:

[0043] like Figure 7 As shown, the tumor-targeting ferritin derivative carrier of this invention comprises, from the N-terminus to the C-terminus, a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an RNA-binding protein sequence, and human heavy chain ferritin, forming a fusion protein. Human heavy chain ferritin self-assembles into a spherical cage-like structure with the N-terminus of the ferritin subunits facing outwards. Fusion expression at the N-terminus is necessary to expose other functional proteins on the outer surface of the spherical ferritin.

[0044] Each part performs the following functions:

[0045] ① PSTAG shielding sequence: Extends the half-life of the fusion protein, improves its stability, enhances its in vivo distribution, reduces off-target liver accumulation, and lowers off-target toxicity. ② Tumor microenvironment response site: Can be specifically recognized and cleaved by the highly expressed enzyme MMP2 in the tumor microenvironment, removing the PSTAG shielding sequence and releasing the fusion protein-siRNA complex, RBP-HFn / siRNA, which exerts anti-tumor effects. ③ RNA-binding protein: Can recognize and bind dsRNA. ④ Ferritin: Targets tumor cells and performs cellular entry. The above sequences are linked by flexible linkers GG, EF, or GGGGS to allow the proteins on both sides to fold correctly and achieve their respective independent functions.

[0046] The nanoparticles formed by the polymerization of the human heavy chain ferritin derivative have siRNA that binds to the RNA-binding protein sequence in the vector, forming a fusion protein-siRNA complex prodrug system. This system has a strong gene silencing effect. Its mechanism of action is as follows: after the complex enters the body, it is cleaved by MMP2 in the tumor microenvironment, which removes the PSTAG shield and exposes RBP-HFn / siRNA. Then, it is introduced into the cell by TfR1, some lysosomes escape, and the vector is degraded to release siRNA, which exerts the gene silencing effect.

[0047] Beneficial effects

[0048] RNA-binding proteins (RBPs) interact with siRNA duplexes in a charge-independent manner through structural recognition. Protein kinase R (PKR) has two double-stranded RNA-binding domains that bind to dsRNA in a structure-dependent, rather than sequence-dependent, manner. The dsRBD consists of two dsRNA-binding motifs and a highly flexible, unstructured linker that allows the two motifs to be positioned in a close-packed manner on opposite faces of the dsRNA duplex. The dsRBD protein was the first ribonucleoprotein vector explored for siRNA delivery.

[0049] Heavy ferritin (HFn) is a spherical cage-like structure composed of 24 subunits, with an outer diameter of approximately 12 nm and an inner diameter of approximately 8 nm. Due to its unique spherical cage structure, controllable self-assembly, excellent safety, thermal stability, biocompatibility, well-defined in vivo biological processes, and favorable physicochemical properties, HFn has been used for the loading and delivery of small molecule drugs, metal drugs, and nucleic acid drugs. As a biological endogenous carrier, ferritin cages can specifically recognize and bind to transferrin receptor 1 (TfR1) overexpressed on the surface of tumor cells, achieving active targeting and internalization by tumor cells. The ferritin subunits can be functionalized through genetic engineering or chemical conjugation. However, HFn carriers still have problems. First, TfR1 is significantly expressed in the liver, causing HFn to be preferentially intercepted by the liver rather than targeting the tumor. Second, HFn has a relatively short plasma half-life.

[0050] PSTAG is a structureless, randomly coiled polypeptide, similar to the chemical PEG polymer. PSTAG is engineered and screened as a low-immunogenic polypeptide, composed of five amino acids: Ala, Gly, Pro, Ser, and Thr. It is typically expressed in recombinant form with enzymatically cleavable linkers and therapeutic peptides or proteins to increase the drug's in vivo half-life and stability and mask its activity, forming a prodrug system. In tumors, protease expression is often dysregulated, disrupting physiological processes that are often based on complex interactions between the tumor and its microenvironment. Proteases are upregulated in many tumors and play a crucial role in cancer development and progression by promoting extracellular matrix (ECM) remodeling, tumor invasion, and tumor metastasis.

[0051] This invention provides a stable, safe, efficient, and tumor-targeting protein carrier for in vivo siRNA delivery, namely the fusion protein PSTAG-MMP2-RBP-HFn. The PSTAG-MMP2-RBP-HFn carrier delivers si-HBx knockdown to the HBx target in Hep3B cells, achieving in vitro antitumor activity. This provides a new research approach for the targeted in vivo delivery of siRNA to tumor cells. It also offers new technical means for the development of intracellular siRNA delivery.

[0052] This invention constructs a novel prodrug system based on a fusion protein-siRNA complex. This system comprises a tumor-targeting ferritin derivative carrier and an siRNA gene-silencing drug. The tumor-targeting ferritin derivative carrier, P216-MMP2-RBP-HFn, is a fusion protein formed by conjugating a PSTAG shielding sequence, a tumor microenvironment enzyme response sequence, an RNA-binding protein sequence, and human heavy chain ferritin. This invention has the following advantages:

[0053] 1. For example Figure 7 As shown, this fusion protein carrier is designed as a prodrug structure that can be activated by enzymes in the tumor microenvironment. It uses a PSTAG shielding sequence to reduce liver accumulation, overcome the defect in existing technologies where ferritin is preferentially intercepted by the liver rather than targeting the tumor, and improves safety; it also prolongs the half-life; the tumor microenvironment enzyme response sequence enhances tumor targeting; ferritin is in 24-mer form, and one fusion protein expresses 24 RBPs, which can theoretically bind 24 siRNAs, resulting in high drug loading capacity.

[0054] 2. For example Figure 1 As shown, the fusion protein obtained by prokaryotic expression, isolation and purification has high yield (50 mg / L fermentation broth), high purity (>80%), and simple and easy preparation process.

[0055] 3. For example Figure 2 As shown, the P216-MMP2-RBP-HFn vector can bind well to siRNA to form a complex. Figure 5 As shown, the P216-MMP2-RBP-HFn vector binds to siRNA to form a complex, protecting the siRNA and facilitating its delivery, and significantly improving the serum stability and RNase A stability of the siRNA.

[0056] 4. For example Figure 3 As shown, the P216-MMP2-RBP-HFn vector has a significant cell entry effect and can escape from lysosomes; the vector effectively delivers siRNA into cells, and after the RBP-HFn / siRNA escapes from lysosomes, RBP-HFn is degraded and releases siRNA.

[0057] 5. For example Figure 4 , 5 As shown, the P216-MMP2-RBP-HFn vector delivered siRNA into cells and exerted gene silencing activity, verifying the feasibility of the vector.

[0058] 6. For example Figure 5 , 6 As shown, the P216-MMP2-RBP-HFn vector delivered si-HBx, knocking down the oncogene HBx in Hep3B cells and inhibiting the proliferation and migration of liver cancer cells. The P216-MMP2-RBP-HFn vector is an endogenous biological vector that is degradable and has no significant cytotoxicity.

[0059] 7. For example Figure 7 As shown, the fusion protein vector of the present invention can deliver siRNAs with different targets, thereby increasing the anti-tumor effect and providing a safe, targeted, and easy-to-prepare novel protein vector for anti-tumor siRNA drugs. Attached Figure Description

[0060] Figure 1 Preparation and characterization of the fusion protein. a) P216-MMP2-RBP-HFn protein purification and analysis by 12% SDS-PAGE (lanes: 1: pre-induction; 2: post-induction (25℃); 3: superposition (20mM Tris); 4: superposition; 5: Q flow-through; 6: Q elution; 7: Q flow-through (20mM Tris + 2mM MgCl2 + 500U / g nuclease, 37℃ for 30min); 8: Ni flow-through; 9: Ni 50mM imidazole for impurity removal; 10: Ni 200mM imidazole elution); b) Western blotting of P216-MMP2-RBP-HFn protein. Blot identification; c is the purity of P216-MMP2-RBP-HFn as determined by RP-HPLC; d is the transmission electron microscopy characterization of P216-MMP2-RBP-HFn; e is the temperature stability verification of P216-MMP2-RBP-HFn by SDS-PAGE; f is the in vitro cleavage verification of MMP2 in P216-MMP2-RBP-HFn.

[0061] Figure 2Preparation and characterization of the fusion protein-siRNA complex. a) 2% agarose gel electrophoresis of transcribed siRNA (lanes: 1: primer annealing (template 1); 2: primer annealing (template 2) 47bp; 3, 4: transcription 21bp; 5, 6: DNase I, RNase T1 double digestion; 7, 8: purification); b) gel retardation assay of P216-MMP2-RBP-HFn / siRNA complexes with different N / P ratios (lanes: 1: siRNA; 2: P216-MMP2-RBP-HFn; 3: N / P (monomer protein / nucleic acid) = 1; 4: N / P = 2).

[0062] Figure 3 This study investigated the cell entry and lysosomal escape efficiency of the fusion protein-siRNA complex. a) Flow cytometry was used to detect the cell uptake efficiency of FAM-P216-MMP2-RBP-HFn; b) Flow cytometry was used to detect the intracellular fluorescence intensity of P216-MMP2-RBP-HFn / Alexa546-siRNA; c) Laser confocal microscopy was used to detect the cell entry and lysosomal escape of P216-MMP2-RBP-HFn / Alexa 546-siRNA.

[0063] Figure 4 The effect of knocking down EGFP by the fusion protein-siRNA complex. a) Flow cytometry detection of changes in EGFP levels after P216-MMP2-RBP-HFn / si-EGFP treatment; b) Quantitative intracellular mean fluorescence intensity (MFI) by flow cytometry.

[0064] Figure 5 This study investigated the in vitro knockdown effect and stability of the fusion protein-siRNA complex. a) RT-qPCR was used to detect changes in HBx mRNA levels after complex treatment; b) Western blotting was used to detect changes in intracellular HBx protein levels; c) 2% agarose gel electrophoresis was used to verify the serum stability of siRNA; d) 2% agarose gel electrophoresis was used to verify the RNase A stability of siRNA.

[0065] Figure 6 To assess the in vitro antitumor effect of the fusion protein-siRNA complex. a) Changes in cell proliferation activity detected by CCK8 assay; b) Cell migration assessed by scratch healing assay; c) Quantitative analysis by measuring scratch width.

[0066] Figure 7 This is a schematic diagram of the mechanism of action of the present invention. a is a schematic diagram of the monomer structure of the fusion protein; b is a schematic diagram of P216-MMP2-RBP-HFn binding to siRNA and delivering it into the cell to exert a gene silencing effect. Detailed Implementation

[0067] 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.

[0068] 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.

[0069] 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 specified, the technical means used are conventional means well known to those skilled in the art.

[0070] Example 1: Preparation and Characterization of Fusion Protein

[0071] (1) The full-length sequence of human H-ferritin (HFn) was obtained by querying the NCBI database and references. PSTAG 216 (P216), the tumor microenvironment MMP2 enzyme response site (MMP2), and the RNA-binding protein sequence (RBP) were sequentially introduced at its N-terminus to design the protein sequence of the fusion protein. After codon optimization with E. coli as the host, the DNA of the fusion protein was constructed into the expression vector pET-28a(+)(GenScript). Then, using the E. coli prokaryotic expression system, the plasmid of the fusion protein was transformed into the competent cells (Vazyme) of the expression strain E. coli BL21(DE3). Expression was induced by IPTG, and the bacterial culture was collected after fermentation for the isolation and purification of the fusion protein.

[0072] The specific methods for protein expression and purification are as follows: Glycerol-containing bacteria were activated in test tubes, expanded in small conical flasks, and then inoculated into LB liquid medium containing 1‰ Kan resistance at a volume ratio of 1:100. The culture was carried out at 37℃ and 220 rpm on a shaker, with the addition of 1 mM IPTG to induce the expression of the target protein. After culturing for another 24 h, the culture was centrifuged at 4℃ and 8000 rpm for 15 min, the supernatant was discarded, and the bacterial pellet was collected. The bacterial pellet was resuspended in 20 mM Tris-HCl (pH 8.0) Q-column equilibration buffer, and bacterial lysis was obtained by sonication, followed by centrifugation (10000 rpm, 20 min, 4℃). The supernatant was collected and loaded onto a Q-column using a peristaltic pump. The flow-through was collected, and the column was washed with Q-column equilibration buffer, with continued collection of the eluent until the G250 index no longer showed a blue color. Q-flow impregnation was performed to prepare a solution of 20 mM Tris and 2 mM MgCl2, followed by the addition of nuclease (Sinobiological 500 Units / g bacteria). The solution was incubated at 37°C for 30 min to remove residual nucleic acid and prevent RBP from binding to other RNAs. The solution was then further impregnated with Q-flow impregnation to prepare a solution of 20 mM Tris and 500 mM NaCl (NiCl2). 2+ Column balancing fluid, Ni loaded using a peristaltic pump 2+ On the column, Ni 2+ The column was washed with column impurity removal buffer (20 mM Tris, 500 mM NaCl, 50 mM imidazole, pH 8.0), followed by Ni... 2+ Column elution buffer (20 mM Tris, 500 mM NaCl, 200 mM imidazole, pH 8.0) eluted the bound Ni 2+ The target protein on the column was removed, and nucleases without His tags were eliminated.

[0073] Samples were taken from each purification stage, and protein size and purity were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 12% (v / v) polyacrylamide gel. High-purity fractions were collected, placed in 50 kDa dialysis bags, and dialyzed overnight to 1xPBS buffer. The target protein was then concentrated using a 50 kDa centrifugal ultrafiltration tube to obtain a concentrated fusion protein. Using bovine serum albumin as a standard, protein concentration was determined using the BCA protein assay kit (Beyotime). The calculated yield was 50 mg / L of fermentation broth. The broth was aliquoted and stored at -20°C.

[0074] (2) Western blotting of the purified fusion protein was performed using recombinant anti-ferritin heavy chain antibody (Abcam). The purified fusion protein was diluted to 0.5 mg / mL with 1×PBS and filtered through a 0.22 μm aqueous filter. The purity of the fusion protein was detected by RP-HPLC using a Sepax GP-C18 column. The column was first washed with 5% (v / v) acetonitrile. After the system stabilized, the flow rate was set to 1 mL / min and the detection wavelength was 214 nm. Within 30 min, the proportion of mobile phase B was increased from 5% (v / v) to 60% (v / v). The data were recorded and chromatograms were plotted using GraphPad.

[0075] (3) The morphology of the fusion protein was identified by transmission electron microscopy (TEM). The protein concentration was diluted to 0.5 mg / ml with PBS, filtered through a 0.22 μm filter membrane, and 10 μl was used for copper mesh sample preparation and imaged by TEM.

[0076] (4) To study the stability of the fusion protein, 200 μl of the fusion protein (1 mg / mL) was incubated at stable temperatures of 37 °C and 60 °C. At the required time points (0, 1, 2, 4, 8, 24, 48 h), 20 μl of the sample was taken and quantified by 12% SDS-PAGE.

[0077] (5) To verify that the fusion protein can be cleaved by MMP-2 enzyme, P216 was removed to release RBP-HFn for in vitro protein cleavage verification. 25 μg of protein was cleaved using 1 μl of 50 ng / μl MMP-2 pre-activated human (Sigma-Aldrich), incubated at 37°C for 2 h, and samples were taken for 12% SDS-PAGE gel analysis.

[0078] result:

[0079] Figure 1 a is the SDS-PAGE identification image of the purified fusion protein, which was obtained after separation and purification as P216-MMP2-RBP-HFn;

[0080] Figure 1 b is the Western Blot identification image of the fusion protein (DAB method), and the bands are of the correct size and are uniform;

[0081] Figure 1c represents the determination of the purity of the P216-MMP2-RBP-HFn protein purified by RP-HPLC. The results show that the peak shape of the P216-MMP2-RBP-HFn protein is symmetrical and the purity is greater than 80%.

[0082] Figure 1 d shows the fusion protein characterized using a HT7700 microscope (Hitachi, Japan), which exhibits good dispersibility and uniform particle size.

[0083] Figure 1 e shows the SDS-PAGE results of the fusion protein after storage for different times. It was found that the fusion protein was very stable at 37°C with minimal degradation; it also remained stable for 8 hours at 60°C.

[0084] Figure 1 f shows the SDS-PAGE characterization of the in vitro cleavage of the fusion protein MMP2. The results show that P216-MMP2-RBP-HFn was successfully cleaved by the MMP2 enzyme, releasing RBP-HFn.

[0085] Example 2: Preparation and characterization of the fusion protein-siRNA complex

[0086] (1) Design DNA templates according to different target sequences, perform in vitro transcription and centrifugation column purification of siRNA according to the steps of the in vitro transcription kit (Vazyme) (New England Biolabs), measure the concentration after purification with Nanodrop, freeze at -20℃, and take samples for identification by 2% agarose gel electrophoresis.

[0087] (2) The binding ability of the fusion protein to siRNA was verified by gel retardation assay. A 2% agarose gel was prepared, and the thickness of the gel after solidification was 6-8 mm. The P216-MMP2-RBP-HFn protein and the corresponding siRNA were incubated at 37°C for 1 h according to the N / P ratio of 0, 1, and 2, with 1 nmol of siRNA used. After electrophoresis, the gel was imaged using a UV transilluminator.

[0088] (3) Select different protein concentrations according to the experiment, mix P216-MMP2-RBP-HFn protein with the corresponding siRNA at a ratio of N / P (molar ratio of monomeric protein to nucleic acid) of 2, and incubate at 37℃ for 1 h to obtain the complex of fusion protein and siRNA.

[0089] result:

[0090] Figure 2 a) is the agarose gel electrophoresis verification of in vitro transcribed siRNA, showing that the band size is correct and the purity is high;

[0091] Figure 2 b shows the gel retardation assay of PMRH / siRNA complexes with different N / P ratios. The results show that the fusion protein can bind free siRNA well under the condition of N / P of 2, achieving a high drug loading capacity.

[0092] Example 3: Study on the cell entry and lysosomal escape effects of the fusion protein-siRNA complex.

[0093] (1) The P216-MMP2-RBP-HFn fusion protein was labeled with FAM fluorescent dye. Hep3B cells were divided into groups of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 cells per well in 24-well plates. Cells were incubated with 1 μM, 3 μM, and 5 μM MAM-P216-MMP2-RBP-HFn fusion protein at 37 °C for 24 h. Cells were digested and collected, washed three times with 1×PBS buffer to remove protein adsorbed on the cell surface, and the protein entry efficiency was detected by flow cytometry.

[0094] (2) After treating cells with free Alexa 546-siRNA, 3 μM P216-MMP2-RBP-HFn and transfection reagent Lipo2000 (Vazyme) (both protein and Lipo2000 were loaded with an equal amount of Alexa 546-siRNA) for 24 h, flow cytometry was used to detect whether P216-MMP2-RBP-HFn could successfully deliver Alexa 546-siRNA into cells.

[0095] (3) Add 2×10 to the laser confocal glass substrate (35mm). 5Hep3B cells were cultured for 24 h. P216-MMP2-RBP-HFn was labeled with FAM, and free FAM was removed by ultrafiltration. 3 μM P216-MMP2-RBP-HFn was mixed with Alexa 546-siRNA at a 2:1 molar ratio and incubated at 37°C for 1 h. The culture medium was then replaced with OPTI-MEM, and FAM-P216-MMP2-RBP-HFn and P216-MMP2-RBP-HFn / Alexa 546-siRNA were added to separate dishes. After 24 h of incubation, the cells were washed three times with 1×PBS for 5 minutes each time. Lysosomes were stained with Lyso-Tracker Red / Green lysosomal tracer (Beyotime), followed by cell fixation with formaldehyde, and finally, the nuclei were stained with DAPI. Laser Scanning Confocal Microscopy (LSCM) was used to study the cell entry and lysosomal escape of the FAM-fusion protein, as well as the intracellular localization of the fusion protein / Alexa 546-siRNA, namely, the cell entry of the fusion protein into the cellular environment and the lysosomal escape of the Alexa 546-siRNA.

[0096] result:

[0097] Figure 3 a) Flow cytometry was used to detect the cellular uptake efficiency of FAM-P216-MMP2-RBP-HFn. The results showed that, compared with the Blank group, the FITC peaks of the fusion protein-treated groups were significantly shifted, indicating that P216-MMP2-RBP-HFn could successfully enter the cells in a concentration-dependent manner.

[0098] Figure 3 b shows the cellular uptake efficiency of the P216-MMP2-RBP-HFn / Alexa 546-siRNA complex as detected by flow cytometry. The results showed a significant shift in the PE peak of the complex group, indicating that P216-MMP2-RBP-HFn can deliver Alexa 546-siRNA into cells;

[0099] Figure 3 c shows the cell entry detection using laser confocal microscopy. The results show that FAM-P216-MMP2-RBP-HFn can enter the cell well, and the protein is not completely co-localized with the lysosome, indicating that the protein can escape from the lysosome; the fusion protein binds to Alexa546-siRNA and successfully delivers it into the cell. P216-MMP2-RBP-HFn / siRNA undergoes lysosomal escape, and the fusion protein is degraded by intracellular enzymes, releasing the siRNA to exert its function.

[0100] Example 4: Effect of fusion protein-siRNA complex on EGFP knockdown

[0101] A549 cells stably transfected with EGFP were selected as model cells to detect the knockdown efficiency of intracellularly expressed EGFP protein by the delivery of the fusion protein si-EGFP. In the experiment, 3 μM P216-MMP2-RBP-HFn was combined with its corresponding si-EGFP at an N / P ratio of 2:1 to form a complex, which was then co-incubated with 24-well A549-EGFP cells. After 48 h of drug administration, the silencing efficiency of intracellular EGFP in each drug-treated group was detected by flow cytometry to verify the activity of the vector-delivered siRNA.

[0102] result:

[0103] Figure 4 a) The change in EGFP protein level after treatment with the P216-MMP2-RBP-HFn / si-EGFP complex was detected by flow cytometry.

[0104] Figure 4 b represents the quantification of intracellular mean fluorescence intensity (MFI). The results showed that P216-MMP2-RBP-HFn / si-EGFP had a silencing effect on intracellular EGFP, knocking down the EGFP protein level by 25% compared with the Blank group.

[0105] Example 5: In vitro knockdown effect and stability study of the fusion protein-siRNA complex

[0106] (1) Hepatitis B virus (HBV) is a major pathogenic factor in the development and progression of liver cancer. The HBV genome encodes the HBx protein. The HBx protein is a multifunctional regulatory factor and one of the important factors involved in the HBV-induced hepatocellular carcinogenesis process, making it a potential target for the treatment of HBV-related liver cancer.

[0107] The si-HBx sequence from the references was transcribed in vitro to produce si-HBx. Hep3B liver cancer cells were selected as model cells to detect the knockdown efficiency of HBx protein in Hep3B cells by P216-MMP2-RBP-HFn delivery of si-HBx. In the experiment, 3 μM of P216-MMP2-RBP-HFn and si-HBx were incubated at an N / P ratio of 2:1 to form a complex, which was then co-incubated with 12-well Hep3B cells. After 48 h of incubation, RNA was extracted, and the knockdown level of HBx by the complex was detected using real-time quantitative PCR.

[0108] (2) To further identify the silencing efficiency of the P216-MMP2-RBP-HFn / si-HBx complex for intracellular HBx protein expression after delivery to Hep3B cells, the cells were incubated with the drug for 72 h. The difference in intracellular HBx expression was detected by Western blotting (Abcam Anti-Hepatitis B Virus X antigen antibody).

[0109] (3) Study on the serum stability of the P216-MMP2-RBP-HFn / si-HBx complex. 4 nmol of siRNA was incubated with the corresponding amount of P216-MMP2-RBP-HFn (N / P = 2) to form a complex. The complex and control siRNA were divided into four equal portions (1 nmol), and 10 μl of 100% FBS was added to each portion. The mixture was incubated at 37°C, and samples were taken at 0 h, 10 h, 18 h, and 24 h for 2% agarose gel electrophoresis to compare the degree of siRNA degradation in the presence of serum.

[0110] (4) Study on the RNase A stability of the P216-MMP2-RBP-HFn / si-HBx complex. 3 nmol of siRNA was incubated with the corresponding amount of P216-MMP2-RBP-HFn (N / P = 2) to form a complex. The complex and control siRNA were divided into three equal portions (1 nmol), and 1 μl of 0.5 mg / ml RNase A was added to each portion. The mixture was incubated at 37°C, and samples were taken at 0 h, 1 h, and 2 h for 2% agarose gel electrophoresis to compare the degree of siRNA degradation in the presence of RNase A.

[0111] result:

[0112] Figure 5 a) RT-qPCR was used to detect changes in HBx mRNA levels after complex treatment. The results showed that, compared with the Blank group, treatment of Hep3B cells with P216-MMP2-RBP-HFn / si-HBx inhibited HBx mRNA levels by 60%.

[0113] Figure 5 b shows the changes in intracellular HBx protein levels detected by Western Blot. Western Blot results indicate that P216-MMP2-RBP-HFn / si-HBx has a silencing effect on intracellular HBx expression;

[0114] Figure 5 c represents the serum stability of siRNA verified by 2% agarose gel electrophoresis. The P216-MMP2-RBP-HFn vector, upon binding to siRNA, exhibits a protective effect, enhancing serum stability.

[0115] Figure 5 d represents the RNase A stability of siRNA verified by 2% agarose gel electrophoresis. The P216-MMP2-RBP-HFn vector, after binding to siRNA, has a protective effect on it and enhances RNase A stability.

[0116] Example 6: In vitro antitumor effect of the fusion protein-siRNA complex

[0117] (1) The effect of the complex on the proliferation ability of Hep3B cells was detected by the CCK8 assay. 2×10 4 Hep3B cells were plated in 96-well plates. The protein concentration was 3 μM, and the N / P ratio of protein to siRNA was 2:1. After 24 h of administration, 10 μl of CCK8 assay reagent was added to each well and the cells were incubated at 37 °C for 2 h. The absorbance (OD) at 450 nm was measured using a Spectra Max I3X microplate reader to calculate the change in cell proliferation capacity.

[0118] (2) To investigate whether the P216-MMP2-RBP-HFn / si-HBx complex could inhibit the migration of Hep3B cells after drug administration through a scratch healing assay. First, 5×10⁻⁶ cells were... 5 Cells were evenly seeded in 6-well plates and cultured overnight. The complex was then added for incubation, and the medium was changed to OPTI-MEM. After 24 hours of incubation, when the cells reached approximately 80% confluence, the cell monolayer was vertically scratched using a 10 μl pipette tip. Cell debris was washed away with PBS, and the medium was replaced with 1% FBS to minimize the impact on cell proliferation. Cells migrating to the scratch area were monitored using an inverted microscope at 0, 24, and 48 hours. To more visually represent the experimental results, ImageJ was used for quantitative analysis of the data.

[0119] result:

[0120] Figure 6 a represents the changes in cell proliferation activity detected by the CCK8 assay. The results showed that, compared to the Blank group, the P216-MMP2-RBP-HFn / si-HBx group inhibited cell proliferation, and the P216-MMP2-RBP-HFn fusion protein showed no significant cytotoxicity.

[0121] Figure 6 b is the scratch healing test to assess cell migration. Figure 6c represents the quantitative analysis of the scratch healing assay. The results showed that, compared to the Blank group, cells treated with P216-MMP2-RBP-HFn / si-HBx exhibited a wider wound area 48 hours after wound initiation, indicating a loss of migration. This further confirms that P216-MMP2-RBP-HFn / si-HBx has a silencing effect on HBx in Hep3B cells.

Claims

1. A human heavy chain ferritin derivative, characterized in that, The sequence from the N-terminus to the C-terminus consists of the PSTAG shielding sequence as shown in SEQ ID NO:2, the tumor microenvironment enzyme response sequence as shown in SEQ ID NO:3, the RNA-binding protein as shown in SEQ ID NO:4, and the human heavy chain ferritin as shown in SEQ ID NO:

5. Alternatively, the amino acid sequence of the human heavy chain ferritin derivative is SEQ ID NO.

1.

2. The application of the human heavy chain ferritin derivative according to claim 1 in the preparation of small interfering RNA drug delivery vectors.

3. A tumor microenvironment-responsive fusion protein-siRNA complex prodrug system, characterized in that, The system comprises the human heavy chain ferritin derivative of claim 1 and a small interfering RNA drug with gene silencing effect.

4. The system according to claim 3, characterized in that, The human heavy chain ferritin derivative is polymerized to form nanoparticles, which serve as carriers for the small interfering RNA drug to bind to the carriers.

5. The system according to claim 3 or 4, characterized in that, The small interfering RNA drugs mentioned are SEQ ID NO: 6 and SEQ ID NO: 7; or SEQ ID NO: 8 and SEQ ID NO:

9.

6. The application of the system according to claim 4 in the preparation of antitumor drugs.

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