Cage-like nanocarrier for targeted delivery of sirna, and preparation method therefor and use thereof

Abstract

A cage-like nanocarrier for targeted delivery of siRNA, and a preparation method therefor and the use thereof. The preparation method comprises: (A) mutating a negatively charged or uncharged amino acid on the inner surface of a ferritin into a positively charged amino acid; and any one or more of the following steps: (B) coupling the N-terminus of the ferritin to a functional peptide having nucleic acid affinity; (C) coupling the N-terminus of the ferritin to a functional peptide promoting lysosomal escape; (D) truncating the E-helix at the C-terminus of the ferritin; (E) coupling the C-terminus of the ferritin to a functional peptide having nucleic acid affinity; and (F) coupling the C-terminus of the ferritin to a functional peptide promoting lysosomal escape. A new nucleic-acid-loaded protein nanocage carrier is constructed by means of modifying a negatively charged inner cavity of ferritin to make same positively charged. By means of electrostatic adsorption, a negatively charged siRNA can be efficiently loaded into a ferritin nanocage, thereby significantly improving the in-vivo and in-vitro delivery stability of siRNA, lysosomal escape functions, and efficacy of targeted therapy.

Description

A cage-like nanocarrier for targeted delivery of siRNA, preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to ferritin biological nanomaterials, in particular, to a cage-like nanocarrier for targeted delivery of siRNA, preparation method and application thereof. BACKGROUND

[0002] Nucleic acid drugs can specifically target pathogenic genes and precisely regulate diseases at the gene level. In recent years, nucleic acid drugs have received extensive attention in the field of precision and personalized treatment due to their safety, high specificity, high efficiency, and low production cost. In 1998, the phenomenon of RNA interference was first discovered. RNAi forms an RNA-induced silencing complex in the cytoplasm by introducing double-stranded RNA (i.e., small interfering RNA, siRNA) with the same sequence as the endogenous target gene, and pairs with its homologous mRNA to degrade it, achieving post-transcriptional gene silencing. This method is relatively safe, as siRNA cannot be integrated into the genome and can be enzymatically degraded after exerting its effect, without affecting gene transcription and protein expression in other normal cells. However, there are still great challenges in the current field. Free siRNA has poor stability and can easily cause immune problems in the body. First, siRNA, as a relatively short nucleotide sequence, is easily degraded by nucleases after entering the blood circulation and is easily cleared by the kidneys, with a short half-life in the body. At the same time, it has immunogenicity and can easily cause an immune response in the human body. In addition, siRNA is a negatively charged hydrophilic molecule with low cellular uptake efficiency. Free siRNA also lacks targeting ability and cannot achieve precise delivery. Furthermore, siRNA cannot escape the lysosome after entering the cell and thus cannot function in the cytoplasm. siRNA cannot effectively target tumors, which also limits their application in vivo. To overcome these difficulties, researchers have designed various carriers, such as lipid nanoparticles (LNPs), GalNAc-based nanocarriers, cationic polymers, and micelles. These carriers have to some extent solved the problems of poor stability and low delivery efficiency of siRNA, but due to the lack of tumor targeting ability, the therapeutic effect is not ideal. Therefore, in order to achieve safe and efficient, precise targeting nucleic acid therapy, it is urgent to develop a safe and efficient siRNA drug delivery carrier in vivo.

[0003] Among the numerous nucleic acid delivery systems, cage-like proteins have attracted extensive attention in the field of drug carriers due to their unique water solubility, high dispersibility, symmetry and uniformity. Ferritin is a representative natural cage-like protein. Mammalian ferritin has 24 subunits and can form a hollow spherical nanoprotem cage with an inner diameter of 8 nm and an outer diameter of 12 nm through a reversible self-assembly process. Ferritin has many advantages as a drug carrier, including: 1. uniform nanosize and hollow cage structure; 2. high stability, low immunogenicity and high biocompatibility; 3. reversible self-assembly properties, which can mediate the disassembly and self-assembly of the protein cage by adjusting the pH of the buffer or adding a denaturing agent, thereby realizing the intracavity loading of drugs; 4. easy to modify its properties and functions through genetic engineering or chemical modification; 5. ferritin has transferrin receptor 1 (TfR1) mediated endocytosis and inherent tumor targeting. Ferritin has been used to deliver drugs in many studies, but further research is needed for siRNA delivery. SUMMARY

[0004] In order to solve the problems of siRNA lysosome escape, loading and lack of tumor targeting, the present application provides a cage-like nanocarrier for targeted delivery of siRNA, a preparation method and application thereof, which can efficiently and stably load siRNA into the intracavity of the cage-like protein and realize lysosome escape.

[0005] In order to achieve the above-mentioned purpose, the present application provides a preparation method of a cage-like nanocarrier for targeted delivery of siRNA, which comprises:

[0006] (A) mutating the negatively charged or uncharged amino acids distributed on the inner surface of ferritin in space to positively charged amino acids; and any one or several of the following steps:

[0007] (B) coupling a functional peptide with nucleic acid affinity to the N terminus of the ferritin;

[0008] (C) coupling a functional peptide that promotes lysosome escape to the N terminus of the ferritin;

[0009] (D) truncating the E helix of the C terminus of the ferritin;

[0010] (E) coupling a functional peptide with nucleic acid affinity to the C terminus of the ferritin;

[0011] (F) coupling a functional peptide that promotes lysosome escape to the C terminus of the ferritin.

[0012] Ferritin "HFn" refers to any ferritin that can form a cage structure, which can be a naturally derived ferritin, or a recombinantly expressed ferritin, or a mutant thereof, which can be derived from a prokaryote, a protist, a fungus, a plant, or an animal, such as a bacterium, a fungus, an insect, a reptile, a bird, an amphibian, a fish, a mammal, such as a rodent, a ruminant, a non-human primate, or a human, such as a mouse, a rat, a guinea pig, a dog, a cat, a cow, a horse, a sheep, a monkey, a gorilla, a human. From bacteria to human, although the amino acid sequences of ferritins of different organisms have great differences, their structures are similar, and they can all form a protein shell structure.

[0013] In some embodiments, the ferritin is a human heavy chain ferritin HFn, and the amino acid sequence thereof is SEQ ID NO. 1.

[0014] The present application constructs a novel nucleic acid-loaded protein nanocage carrier by genetically engineering the negatively charged inner cavity of ferritin to be positively charged. Through electrostatic adsorption, negatively charged siRNA can be efficiently loaded into the interior of the ferritin nanocage, significantly improving the in vitro and in vivo delivery stability, cellular uptake efficiency, and targeted therapy efficacy of siRNA, while achieving lysosome escape function (as shown in FIG. 15).

[0015] Specifically, in step (A), the mutation sites of the ferritin are Glu61, Glu64, Glu140, Glu147, D177, D179, and E181.

[0016] Preferably, in step (A), the positively charged amino acid is selected from any one of arginine, lysine, and histidine.

[0017] Specifically, step (B) is any one of the following:

[0018] (B1) coupling a functional motif with a nucleic acid binding peptide to the N-terminus of the ferritin;

[0019] (B2) coupling a functional motif with a positively charged peptide to the N-terminus of the ferritin;

[0020] Step (C) is: coupling a functional motif with a cell penetrating peptide to the N-terminus of the ferritin;

[0021] Step (E) is any one of the following:

[0022] (E1) coupling a functional motif with a nucleic acid binding peptide to the C-terminus of the ferritin;

[0023] (E2) coupling a functional motif with a positively charged peptide to the C-terminus of the ferritin;

[0024] Step (F) is coupling a functional motif with a cell-penetrating peptide to the C-terminus of the ferritin.

[0025] Preferably, in steps (B1) and (E1), the functional motif sequence with a nucleic acid binding peptide is as shown in SEQ ID NO. 9.

[0026] In steps (B2) and (E2), the functional motif sequence with a positively charged peptide is as shown in SEQ ID NO. 10-15.

[0027] In steps (C) and (F), the functional motif sequence with a cell-penetrating peptide is as shown in SEQ ID NO. 16-31.

[0028] The second aspect of the present application provides a ferritin cage nanocarrier for targeted delivery of siRNA prepared by the preparation method of the first aspect of the present application, wherein the inner cavity of the ferritin cage nanocarrier is positively charged.

[0029] In some embodiments, the ferritin cage nanocarrier contains mutation sites of Glu61, Glu64, Glu140, Glu147, D177, D179 and E181.

[0030] In some embodiments, the ferritin cage nanocarrier is truncated at different degrees at the C-terminus, and the final sequence is SEQ ID NO. 5-8.

[0031] In some embodiments, the ferritin cage nanocarrier contains an amino acid sequence of SEQ ID NO. 9-31.

[0032] In some embodiments, the ferritin cage nanocarrier has an amino acid sequence of any one or more of SEQ ID NO. 1-8.

[0033] In some embodiments, the ferritin cage nanocarrier has an siRNA loading capacity of 2-3 nucleic acid molecules per protein cage.

[0034] The third aspect of the present application provides an siRNA delivery system, which comprises siRNA and the ferritin cage nanocarrier for targeted delivery of siRNA of the second aspect of the present application.

[0035] In some embodiments, the molar mass ratio of the ferritin cage nanocarrier to siRNA is 1: (1-5), preferably 1:2.

[0036] The fourth aspect of the present application provides a method for loading siRNA using the siRNA delivery system of the third aspect, which comprises the following steps:

[0037] S1, preparing and purifying the ferritin cage nanocarrier in the siRNA delivery system;

[0038] S2, dissolving the siRNA in DEPC water and diluting to a certain concentration;

[0039] S3, adding the ferritin nanocage carrier obtained in step S1 into an acidic buffer solution with pH 1-3, and incubating at 4°C for 20-30 minutes to obtain an acid depolymerization system;

[0040] S4, adding the siRNA solution prepared in step S2 into an alkaline buffer solution with pH 9-11, mixing uniformly, then adding the acid depolymerization system obtained in step S3, and incubating at 4°C for 2-3 hours to reassemble the ferritin nanocage with siRNA loaded in the lumen, thereby obtaining the ferritin nanocage.

[0041] In some embodiments, the acidic buffer solution in step S3 is an HCl solution, preferably an HCl solution with pH 1.5-1.6.

[0042] In some embodiments, the alkaline buffer solution in step S4 includes but is not limited to Na2CO3 / NaHCO3, Na2CO3, NaHCO3, Tris, NaOH solution, etc., preferably the alkaline buffer solution is a Na2CO3 / NaHCO3 solution with pH 9-10.

[0043] In some embodiments, the molar ratio of the protein cage nanocarrier to siRNA in step S4 is 1:1-1:5, preferably 1:2.

[0044] The fifth aspect of the present application provides the cage-shaped nanocarrier for targeted delivery of siRNA according to the second aspect of the present application, and the siRNA delivery system according to the third aspect of the present application for use in siRNA delivery.

[0045] The sixth aspect of the present application provides the cage-shaped nanocarrier for targeted delivery of siRNA according to the second aspect of the present application, and the siRNA delivery system according to the third aspect of the present application for use in the preparation of a drug for treating anti-tumor or anti-viral and related genetic diseases.

[0046] Through the above technical solutions, the present application achieves the following beneficial effects:

[0047] The application constructs a novel nucleic acid protein nanocage carrier by engineering the negative inner cavity of ferritin to be positive. Through electrostatic adsorption, the negatively charged siRNA can be efficiently loaded into the ferritin nanocage, and the in vitro and in vivo delivery stability, cell uptake efficiency and targeted therapy effect of siRNA are significantly improved by measures such as E-helix truncation and addition of lysosome escape peptide, while realizing the lysosome escape function. The ferritin cage nanocarrier constructed in the application can be used as a universal siRNA loading platform to realize lysosome escape and is widely used in antitumor, antiviral and related gene disease treatment. BRIEF DESCRIPTION OF DRAWINGS

[0048] Figure 1 is a schematic diagram of the construction and characterization of the inner cavity positive mutation and C-terminal truncated protein cage nanocarrier in the embodiment 2 of the application, wherein (A) is the size exclusion chromatography analysis of wild type HFn and series of mutant tHFn(+) proteins, (B) is the 15% SDS-PAGE analysis of wild type HFn and series of mutant tHFn(+) proteins after purification, (C) is the Native-PAGE analysis of wild type HFn and series of mutant tHFn(+) proteins after purification, (D) is the Zeta potential analysis of wild type HFn and series of mutant tHFn(+) proteins, (E) is the particle size characterization of wild type HFn and series of mutant tHFn(+) proteins by DLS, and (F) is the TEM characterization of wild type HFn and series of mutant tHFn(+) proteins;

[0049] Figure 2 is the lysosome escape function evaluation of tHFn(+) in the embodiment 3 of the application, wherein (A-B) are the CLSM analysis of the endosome localization of HFn and tHFn(+) in U87MG cells, (C) is the quantitative analysis of the endosome localization of HFn and tHFn(+) in U87MG cells by Pearson correlation coefficient, (D-E) are the CLSM analysis of the lysosome localization of HFn and tHFn(+) in U87MG cells, and (F) is the quantitative analysis of the lysosome localization of HFn and tHFn(+) in U87MG cells by Pearson correlation coefficient;

[0050] Figure 3 is the characterization of siRNA@tHFn(+) in the embodiment 4 of the application, wherein (A) is the comparison of siRNA loading capacity of protein mutants and HFn, (B) is the protein recovery rate of protein mutants loaded with siRNA, (C) is the particle size measurement of siRNA@tHFn(+) and (D) is the transmission electron microscope characterization of siRNA@tHFn(+);

[0051] Figure 4 is the evaluation of the cellular uptake efficiency of siRNA@tHFn(+) in Example 5 of the present application, wherein (A) is the CLSM analysis of the siRNA uptake in U87MG cells, and (B-C) are the FCM analysis and quantification of the siRNA uptake in U87MG cells;

[0052] Figure 5 is the evaluation of the cellular level knockdown ability of siRNA@tHFn(+) in Example 6 of the present application, (A-B) are the knockdown of TERT in U87MG cells by siTERT@tHFn(+) and the gray value analysis, and (C-D) are the knockdown of EGFR in U87MG cells by siEGFR@tHFn(+) and the gray value analysis;

[0053] Figure 6 is the evaluation of the tumor targeting ability of siRNA@tHFn(+) in Example 7 of the present application, (A) is the binding ability of the protein to TfR1 receptor, and (B) is the tumor targeting of siRNA@tHFn(+) in vivo in mice;

[0054] Figure 7 is the anti-tumor efficacy of siTERT@tHFn(+) against glioma in mice in Example 8 of the present application, (A-B) are the bioluminescence intensity and quantification of brain tumors, and (C) is the body weight of mice;

[0055] Figure 8 is the anti-tumor efficacy of siEGFR@tHFn(+) against glioma in mice in Example 8 of the present application, (A-B) are the bioluminescence intensity and quantification of brain tumors, and (C) is the body weight of mice;

[0056] Figure 9 is the DLS (A) and TEM characterization (B) of the nucleic acid binding peptide modified ferritin after purification in Example 9 of the present application;

[0057] Figure 10 is the comparison of the siRNA loading capacity of the nucleic acid binding peptide modified ferritin and HFn in Example 10 of the present application;

[0058] Figure 11 is the Native-PAGE (A) analysis and TEM characterization (B) of the positively charged peptide modified ferritin after purification in Example 11 of the present application;

[0059] Figure 12 is the CLSM analysis of the lysosome localization of the positively charged peptide modified ferritin mutant in U87MG cells in Example 12 of the present application;

[0060] Figure 13 is the TEM characterization of the cell penetrating peptide modified ferritin after purification in Example 13 of the present application;

[0061] Figure 14 is the CLSM analysis of the lysosome localization of the cell penetrating peptide modified ferritin mutant in U87MG cells in Example 14 of the present application;

[0062] Figure 15 is a schematic diagram of the present application for modifying ferritin and loading siRNA. Embodiments of the present application

[0063] The embodiments of the technical solutions of the present application will be described in detail below. The following examples are only used to more clearly illustrate the technical solutions of the present application, and therefore are only examples and cannot limit the protection scope of the present application. It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by the skilled in the art to which the present application belongs.

[0064] Example 1 Construction of lumen positive mutation and C-terminal truncated ferritin cage nanocarrier recombinant plasmid

[0065] Design based on amino acid mutation on the inner surface of the protein cage and C-terminal truncation of ferritin: through genetic engineering means, the negative amino acids located on the inner surface of HFn (SEQ ID NO. 1) are point mutated, i.e. the glutamic acid at positions 61, 64, 140, 147, 181 and the aspartic acid at positions 177, 179 of the HFn subunit are specifically replaced by positively charged lysine and arginine (SEQ ID NO. 2-4, HFn(4+), HFn(5+), HFn(7+)), the specific mutations are E61K / E64R / E140K / E147K / E181K / D177K / D179K, and the C-terminal of ferritin is truncated to different degrees (C-terminal truncated to 179, 174, 162, 159). According to the amino acid sequence of tHFn(+) (SEQ ID NO. 5-8), the cDNA sequence thereof is designed, which is cloned into the E. coli expression vector pET30a plasmid with NdeI and BamHI restriction enzyme sites, and the sequence is identified by DNA sequencing.

[0066] Example 2 Expression, purification and characterization of lumen positive mutation and C-terminal truncated protein cage nanocarrier

[0067] tHFn(+) protein expression: the plasmids obtained in Example 1 were transformed into expression strain BL21(DE3) respectively, and were grown and expanded in LB medium containing 100 mg / L kanamycin or ampicillin, and protein induction expression was carried out by adding IPTG with a final concentration of 0.5 mM and culturing at 25°C, 200 rpm for 12h.

[0068] tHFn(+) protein purification: The bacterial solution was collected and centrifuged at 4000 g to collect the bacterial cells, which were resuspended in 20 mM Tris-HCl (pH 8.0) buffer. After breaking the bacteria by high-pressure homogenization, the E. coli residues were removed by centrifugation at 12000 g, and the supernatant was collected and heated at 60°C for 10 min to denature and precipitate most of the impurities. The supernatant was collected by centrifugation at 12000 g. The mutant supernatant was first subjected to preliminary purification by hydrophobic chromatography column Phenyl sepharose, and then further purified by Superdex 200 molecular sieve.

[0069] The protein concentrations of wild-type HFn and mutant tHFn(+) were determined by BCA method, and the purity of HFn and tHFn(+) was identified by 15% SDS-PAGE electrophoresis. The 24-mer assembly of mutant tHFn(+) was identified by Native-PAGE electrophoresis, and the morphology and particle size of tHFn(+) were further characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS).

[0070] The experimental results were analyzed, as shown in Figure 1, size exclusion chromatography analysis showed that the mutant tHFn(+) 1-179 , tHFn(+) 1-174 , tHFn(+) 1-162 , tHFn(+) 1-159 The peak position was basically the same as that of WT. SDS-PAGE results showed that the protein bands of tHFn(+) series mutants with different degrees of C-terminal truncation appeared at 17-21 kDa molecular weight, which was consistent with the theoretical value. On the Native-PAGE gel, the protein bands of the series mutant HFn(+) and wild-type HFn were at similar positions, indicating that the lumen modification and C-terminal truncation did not affect the assembly ability of ferritin, and the tHFn(+) subunit could successfully self-assemble to form a 24-mer. TEM and DLS analysis showed that the series mutant tHFn(+) with lumen positive amino acid mutation and C-terminal truncation had a nanocage structure with a diameter of about 13 nm. Zeta potential analysis showed that the surface properties of tHFn(+) series were basically the same as those of WT.

[0071] Example 3 Verification of the lysosome escape function of tHFn(+) nanocarrier

[0072] The protein mutant tHFn(+) 1-159 with the largest degree of truncation in Example 2 was selected for subsequent exploration (SEQ ID NO. 8), which was referred to as tHFn(+) hereinafter.

[0073] Endosome co-localization: U87MG was plated in confocal dishes, and Cy5.5-HFn and Cy5.5-tHFn(+) were added respectively, with the concentration of Cy5.5 being 1 μM. After co-incubation for 15, 30 min, the samples were taken out, stained with EEA1 for endosome, stained with DAPI for nucleus, and observed by CLSM to characterize the localization of drugs and endosome.

[0074] Lysosome co-localization: U87MG was plated in confocal dishes, and Cy5.5-HFn and Cy5.5-tHFn(+) were added respectively, with the concentration of Cy5.5 being 1 μM. After co-incubation for 15, 30, 60, 120 min, the samples were taken out, stained with LAMP-1 for lysosome, stained with DAPI for nucleus, and observed by CLSM to characterize the localization of drugs and lysosome.

[0075] The experimental results were analyzed, and the results are shown in Figure 2. From the CLSM results and the Pearson correlation coefficient analysis, it can be seen that HFn first enters the endosome and then accumulates in the lysosome, and tHFn(+) first enters the early endosome and then escapes without entering the lysosome, indicating that the tHFn(+) nanocarrier can realize the escape function in tumor cells.

[0076] Example 4 Method for loading siRNA by protein cage nanocarrier

[0077] siRNA is an oligonucleic acid with a double-stranded RNA structure. The following specifically describes a method for loading siRNA by a protein cage nanocarrier: equal volume of 10 mg / mL protein cage nanocarrier tHFn(+) solution was added to an HCl solution (pH 1-3), mixed thoroughly, and then co-incubated at 4°C for 20-30 min to mediate the depolymerization of the protein cage in a strong acid environment. The siRNA solution was pre-mixed with a Na2CO3 / NaHCO3 solution (pH 9-11), and then added to the acid depolymerization system of the protein cage and mixed thoroughly, and the system was neutralized to neutral (pH 6.5-7.5), and co-incubated at 4°C for 2-3 hours. After taking out, the sample solution was ultrafiltrated to remove free siRNA. Finally, the concentration of siRNA was quantified by the microRNA kit of Qubit 4 Fluorometer, the concentration of protein was quantified by the BCA kit, and the siRNA loading rate, protein recovery rate and siRNA utilization rate of tHFn(+) were calculated.

[0078] The experimental results were analyzed, and the results are shown in Figure 3. With the gradual increase of the C-terminal truncation degree, the siRNA rate and the siRNA utilization rate also increased in turn. Compared with HFn, the nucleic acid loading capacity of tHFn(+) was greatly improved. At the same time, the lumen modification did not affect the stability of the ferritin cage itself, and the protein recovery rate of tHFn(+) was basically consistent with that of wild-type HFn. TEM and DLS analysis showed that siRNA@tHFn(+) could be assembled into a protein cage structure with a diameter of about 13.7 nm, which was consistent with the structure of tHFn(+) without loading siRNA.

[0079] Example 5 Promotion of siRNA cellular uptake function by tHFn(+) nanocarrier (in vitro cell verification)

[0080] (1) Fluorescence confocal microscope (CLSM) method evaluation: U87MG was plated in a confocal dish, and the control group and the experimental group were added with Free Cy5-siRNA and Cy5-siRNA@tHFn(+), respectively, wherein the concentration of Cy5-siRNA in both was 1 μM. After 2h of co-incubation, it was taken out, the cell nucleus was stained with DAPI, and the Cy5-siRNA fluorescence inside the U87MG cell was observed with CLSM to characterize the cell uptake. (2) Flow cytometry evaluation: U87MG was plated in a 6-well plate, and Free Cy5-siRNA and siRNA@tHFn(+) were placed in 37°C for 2h and 4h of co-incubation, respectively. The cells in each group were collected, and the Cy5-siRNA fluorescence was detected and quantitatively analyzed by flow cytometry.

[0081] The experimental results were analyzed, and the results are shown in Figure 4. From the CLSM and flow analysis results, it can be seen that free siRNA is difficult to be taken up by cells due to its hydrophilicity and the difficulty in penetrating the cytoplasmic membrane. Flow quantitative analysis shows that the siRNA uptake amount of siRNA@tHFn(+) in U87MG cells is much higher than that of free siRNA. Almost no Cy5-siRNA fluorescence was observed in U87MG cells in the free siRNA group, while in the siRNA@tHFn(+) experimental group, the intracellularization efficiency of siRNA was significantly improved with the help of tHFn(+) mediated endocytosis.

[0082] Example 6 Verification of siRNA@tHFn(+) nanocarrier knocking down target protein function

[0083] U87MG was plated into 6-well plates, and tHFn(+), free siRNA, siNC@tHFn(+), siRNA@LNP, siRNA@tHFn(+) were added respectively. After 48 h of incubation, cells were collected, proteins were separated in 10% SDS-PAGE, then transferred to PVDF membrane, blocked with blocking solution for 1 h, TERT / EGFR primary antibody and Tubulin primary antibody were incubated at 4℃ overnight, secondary antibody was incubated for 1 h, and exposure was performed. The protein bands were analyzed by gray value to characterize the expression of TERT / EGFR protein.

[0084] The experimental results were analyzed, and the results are shown in Figure 5. From the WB results and gray value analysis, it can be seen that free siEGFR does not have a knockdown effect, while siRNA@LNP and siRNA@tHFn(+) can achieve knockdown of the target protein at the cellular level, indicating that tHFn(+) is an effective siRNA delivery carrier.

[0085] Example 7 Tumor targeting ability of tHFn(+) protein cage nanocarrier

[0086] Affinity of protein to TfR1 receptor (ELISA method): HFn and tHFn(+) protein samples were gradient diluted with carbonate buffer (pH 9.4) and added to the ELISA plate, incubated at 37℃ for 2 hours. Then 0.5% BSA solution was used for incubation at 37℃ for 1 hour. Subsequently, the plate was sequentially incubated with TfR1, TfR1 antibody and anti-mouse IgG (H+L) for 1 hour. Finally, TMB substrate was added to the plate, and the OD 652nm was read using an enzyme labeler.

[0087] Evaluation of tumor targeting ability of mice: 1×10 6 U87MG-luc-mCherry cells were intracranially injected into the brain of Balb / c-nu male mice by microsyringe to construct a brain tumor model. Cy5-labeled siRNA and siRNA@tHFn(+) were injected intravenously, and the final concentration of Cy5 was 15 μM. After 6, 12 and 24 hours of administration, Living Image was used for in vivo imaging.

[0088] The experimental results were analyzed, and the results are shown in Figure 6. According to the ELISA results and K D values were calculated, the K D values of HFn and tHFn(+) were 26.59 nM and 1.44 nM, respectively, indicating that tHFn(+) improves the affinity of the protein to TfR1, which is beneficial to the targeting of the carrier to tumor cells. According to the in vivo imaging of mice, free siRNA mainly enters the kidney and cannot be enriched in the brain tumor site, while siRNA delivered by tHFn(+) can cross the blood-brain barrier and target the brain tumor.

[0089] Anti-tumor function of tHFn(+) protein cage nanocarrier delivering siRNA (in vivo efficacy verification)

[0090] Anti-tumor efficacy evaluation (intracranial glioma model): 1 x 10 6 U87MG-luc-mCherry cells were injected intracranially into the brain of Balb / c-nu male mice to construct a brain tumor model. For TERT target-based treatment, tumor-bearing mice were randomly divided into groups and administered PBS, tHFn(+), siTERT, siNC@tHFn(+), siTERT@LNP, and siEGFR@tHFn(+) intravenously, respectively, with a siTERT dosage of 1 mg / kg. The mice were injected with drugs every other day, and their body weights were recorded. The mice were imaged every other day by IVIS Lumina3, and the bioluminescence intensity was analyzed to record the tumor size. For EGFR target-based treatment, the mice were divided into four groups: PBS, siEGFR, tHFn(+), and siEGFR@tHFn(+) for administration. The body weight and tumor volume were recorded according to the experimental design.

[0091] The experimental results were analyzed, as shown in FIGS. 7 and 8. The in vivo anti-tumor efficacy results showed that free siRNA and siRNA@LNP could not inhibit tumor growth. The tHFn(+) carrier itself did not have an anti-tumor effect, but by delivering siTERT or siEGFR, it showed an anti-tumor effect on intracranial glioma. In addition, the ferritin cage nanocarrier tHFn(+) and siRNA@tHFn(+) had good in vivo safety and did not cause side effects such as body weight loss.

[0092] Example 9: Construction, expression, purification, and characterization of recombinant plasmids of ferritin cage nanocarrier modified with nucleic acid binding peptide functional motifs

[0093] Design of ferritin N-terminal or C-terminal modification of nucleic acid binding peptide functional motifs: The C-terminus of wild-type ferritin (SEQ ID NO. 1) and the inner cavity positive point mutation ferritin (SEQ ID NO. 2) obtained in Example 1 was truncated and coupled with a nucleic acid binding peptide functional motif by genetic engineering means. According to the amino acid sequence of Dps16 (SEQ ID NO. 9), the cDNA sequence of the ferritin mutant was designed, cloned into the E. coli expression vector pET22b plasmid with NdeI and BamHI restriction enzyme sites, and the sequence was identified by DNA sequencing.

[0094] Protein expression: The plasmids obtained above were transformed into expression strain BL21 (DE3) respectively, and grown and expanded in LB medium containing 100 mg / L kanamycin or ampicillin, and protein induction expression was performed at 25°C, 200 rpm for 12 h after adding IPTG with a final concentration of 0.5 mM.

[0095] Protein purification: The bacterial solution was collected and centrifuged at 4000 g to collect the bacterial cells, which were resuspended in 20 mM Tris-HCl (pH 8.0) buffer. After breaking the bacteria by high-pressure homogenization, the E. coli residues were removed by centrifugation at 12000 g, and the supernatant was collected and heated in a 60°C water bath for 10 min to denature and precipitate most of the impurities. The supernatant was collected by centrifugation at 12000 g. The mutant supernatant was first subjected to preliminary purification by anion exchange column Q-Sepharose Fast Flow, and then further purified by Superdex 200 molecular sieve.

[0096] Protein characterization: DLS and TEM were used to characterize the particle size of the nucleic acid binding peptide functional motif modified ferritin mutant and the assembly of the nanocage.

[0097] The experimental results were analyzed, as shown in FIG. 9, and the DLS and TEM results showed that the ferritin could be successfully self-assembled to form nanocages with a nanodiameter of about 13 nm after being modified with the nucleic acid binding peptide.

[0098] Example 10 Verification of loading level of nucleic acid binding peptide functional motif modified ferritin cage nanocarrier

[0099] In an HCl solution (pH 1-3), an equal volume of 10 mg / mL Dps16 modified protein cage nanocarrier solution was added, thoroughly mixed, and then incubated at 4°C for 20-30 min to mediate the depolymerization of the protein cage in a strong acid environment. The siRNA solution was pre-mixed with a Na2CO3 / NaHCO3 solution (pH 9-11), and then added to the acid depolymerization system of the protein cage and thoroughly mixed, and the system was neutralized to neutral (pH 6.5-7.5) and incubated at 4°C for 2-3 h. After removal, the sample solution was ultrafiltrated to remove free siRNA. The concentration of siRNA was quantified using the microRNA kit of Qubit 4 Fluorometer, and the concentration of protein was quantified using the BCA kit, and the siRNA loading rate of the protein was calculated. The experimental results were analyzed, and the results are shown in FIG. 10. Compared with HFn, the nucleic acid loading capacity of HFn(+)-Dps16 was improved.

[0100] Example 11 Construction, expression and purification of positively charged peptide functional motif modified ferritin cage nanocarrier recombinant plasmid and characterization

[0101] Design based on N-terminal or C-terminal modification of the positively charged peptide functional motif of ferritin: The C-terminal of wild-type ferritin and the lumen positively charged mutant ferritin HFn(4+) constructed in Example 1 were truncated by genetic engineering and coupled with a positively charged peptide functional motif (SEQ ID NO. 10-15). According to the ferritin mutant with the amino acid sequence (SEQ ID NO. 10-15), the cDNA sequence was designed and cloned into the E. coli expression vector pET22b plasmid with NdeI and BamHI restriction enzyme sites, and the sequence was identified by DNA sequencing.

[0102] Protein expression: The above obtained plasmids were transformed into the expression strain BL21(DE3) respectively, and grown and expanded in LB medium containing 100 mg / L kanamycin or ampicillin, and the final concentration of IPTG was added to 0.5 mM, and the protein induction expression was carried out at 25°C, 200 rpm for 12h.

[0103] Protein purification: The bacterial solution was collected and centrifuged at 4000 g to collect the bacterial cells, which were resuspended with 20 mM Tris-HCl (pH 8.0) buffer. After breaking the bacteria by high pressure homogenization, the E. coli residues were removed by centrifugation at 12000 g, and the supernatant was collected and heated in a 60°C water bath for 10 minutes to denature and precipitate most of the impurities. The supernatant was collected by centrifugation at 12000 g. The mutant supernatant was first subjected to preliminary purification by anion exchange column Q-Sepharose Fast Flow, and then further purified by Superdex 200 molecular sieve.

[0104] Protein characterization: Native-PAGE electrophoresis and TEM were used to characterize the 24-mer assembly of the positively charged peptide functional motif modified ferritin mutant and the protein cage morphology.

[0105] The experimental results were analyzed, as shown in Figure 11, on the Native-PAGE gel, the protein bands of the positively charged peptide functional motif modified ferritin mutant and the wild-type HFn were at similar positions, indicating that the ferritin could successfully self-assemble into 24-mer after modification of the positively charged peptide. TEM analysis showed that the lumen positively charged peptide modified ferritin mutant had good nanocage structure.

[0106] Example 12 Verification of the lysosome escape function of the positively charged peptide functional motif modified ferritin nanocarrier

[0107] U87MG was plated into confocal dishes, and Cy5-labeled HFn+(LR)3 (SEQ ID NO. 13), HFn+(LR)4 (SEQ ID NO. 14), HFn+(LR)5 (SEQ ID NO. 15) were added respectively, with the concentration of Cy5 being 1 μM. After co-incubation for 15, 30, 120 min, the confocal dishes were taken out, stained with LAMP-1 for lysosomes, and stained with DAPI for cell nuclei, and the different fluorescence inside U87MG cells was observed by CLSM to characterize the localization of drugs and lysosomes.

[0108] The experimental results were analyzed, and the results are shown in Figure 12. From the CLSM results, it can be seen that HFn+(LR)3, HFn+(LR)4, and HFn+(LR)5 modified with cell penetrating peptide functional motifs are not co-localized with lysosomes. It is shown that after disassembly of the series of ferritin mutants in the lysosome, the lysosome escape function can be realized through the membrane disruption of the positively charged peptide.

[0109] Example 13 Construction, expression, purification, and characterization of recombinant plasmids of cell penetrating peptide functional motif modified ferritin cage nanocarriers

[0110] Design of cell penetrating peptide functional motifs based on N- or C-terminal modification of ferritin: through genetic engineering means, the N-terminus of some of the inner cavity positive point electric mutant ferritins HFn(4+) of Example 1 is coupled with a cell penetrating peptide functional motif, the C-terminus of some HFn(4+) is truncated and coupled with a cell penetrating peptide functional motif, and the C-terminus of some HFn(4+) is truncated and coupled with a cell penetrating peptide functional motif at the N-terminus (SEQ ID NO. 16-31). The cDNA sequence of the ferritin mutant according to the linking amino acid sequence (SEQ ID NO. 16-31) is designed, cloned into the E. coli expression vector pET22b plasmid with NdeI and BamHI restriction enzyme sites, and the sequence is identified by DNA sequencing.

[0111] Protein expression: the above obtained plasmids were transformed into expression strain BL21(DE3) respectively, and grown and expanded in LB medium containing 100 mg / L kanamycin or ampicillin, and the final concentration of added IPTG was 0.5 mM, and the culture was incubated at 25°C, 200 rpm for 12 h for protein induction expression.

[0112] Protein purification: the bacterial solution was collected, centrifuged at 4000 g to collect the bacterial cells, and the bacterial cells were resuspended with 20 mM Tris-HCl (pH 8.0) buffer. After breaking the bacteria by high pressure homogenization, the E. coli residues were removed by centrifugation at 12000 g, and the supernatant was collected and heated in a 60°C water bath for 10 minutes to denature and precipitate most of the impure proteins. The supernatant was collected by centrifugation at 12000 g.

[0113] Wherein the supernatant of part of the mutants is first subjected to preliminary purification by anion exchange column Q-Sepharose Fast Flow, and then subjected to further purification by Superdex 200 molecular sieve.

[0114] Wherein the supernatant of part of the mutants is first subjected to preliminary purification by hydrophobic chromatography column Phenyl sepharose, and then subjected to further purification by Superdex 200 molecular sieve.

[0115] Wherein the supernatant of part of the mutants is first subjected to preliminary purification by nickel column, and then subjected to further purification by Superdex 200 molecular sieve.

[0116] Protein characterization: TEM is used to characterize the 24-mer assembly and morphology of the positively charged peptide functional motif modified ferritin mutants.

[0117] After analyzing the experimental results, TEM shows that the series of mutants modified by cell penetrating peptide (SEQ ID NO. 16-31) all have good nanocage structures (part of the cell penetrating peptide functional motif results are shown in Figure 13, wherein GALA6 is at the C-terminal and H5WY is at the N-terminal).

[0118] Example 14 Verification of lysosome escape function of cell penetrating peptide functional motif modified ferritin nanocarrier

[0119] U87MG is plated into a confocal dish, and Cy5-labeled HFn(+)-GALA6 (SEQ ID NO. 19) and H5WY-HFn(+) (SEQ ID NO. 22) are added, wherein the concentration of Cy5 is 1 μM. After co-incubation for 15, 30 and 120 min, it is taken out, stained with LAMP-1 for lysosomes, stained with DAPI for cell nuclei, and observed by CLSM to characterize the positioning of drugs and lysosomes inside U87MG cells.

[0120] After analyzing the experimental results, the results are shown in Figure 14, and from the CLSM results, it can be seen that HFn(+)-GALA6 and H5WYG-HFn(+) modified with cell penetrating peptide functional motifs are not co-localized with lysosomes. It is shown that after the series of ferritin mutants are disassembled in the lysosome, the cell penetrating peptide can penetrate the lysosome membrane to realize the lysosome escape function.

[0121] The sequences described in the present application are as follows:

[0122] SEQ ID NO. 1 Human heavy chain recombinant ferritin HFn

[0123] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0124] SEQ ID NO. 2-4 Internal surface amino acid residue mutations

[0125] HFn (4+)

[0126] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES

[0127] HFn (5+)

[0128] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNKS

[0129] HFn (7+)

[0130] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMGAPESGLAEYLFDKHTLGKSKNKS

[0131] SEQ ID NO. 5-8 Internal surface amino acid residue mutations and C-terminal truncation to position 179, 174, 162, 159

[0132] tHFn(+) 1-179

[0133] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMGAPESGLAEYLFDKHTLGDSD

[0134] tHFn(+) 1-174

[0135] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMGAPESGLAEYLFDKHT

[0136] tHFn(+) 1-162

[0137] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMGAPE

[0138] tHFn(+) 1-159

[0139] TTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNKQVKAIKKLGDHVTNLRKMG

[0140] SEQ ID NO. 9 nucleic acid binding functional peptide Dps16

[0141] QSTEKGAADKARRKSA

[0142] SEQ ID NO. 10-15 positive electric peptide

[0143] (LR)1: LRLR

[0144] (LR)2: LRLRGGLRLR

[0145] (LR)3: LRLRGGLRLRGGLRLR

[0146] (LR)4: LRLRGGLRLRGGLRLRGGLRLR

[0147] (LR)5: LRLRGGLRLRGGLRLRGGLRLRGGLRLR

[0148] (LR)6: LRLRGGLRLRGGLRLRGGLRLRGGLRLRGGLRLR

[0149] SEQ ID NO. 16-25 cell penetrating peptide

[0150] GALA: WEAALAEALAEALAEHLAEALAEALEALAA

[0151] GALA4: LAEALAEHLAEALAE

[0152] GALA5: LAEALAEHLAEALAEALAE

[0153] GALA6: LAEALAEHLAEALAEALAEALAE

[0154] GALA7: LAEALAEHLAEALAEALAEALAEALAE

[0155] H5WYG: GLFHAIAHFIHGGWHGLIHGWY

[0156] H5WY: HHHHHWY

[0157] YWH5: YWHHHHH

[0158] H5: HHHHH

[0159] H(GH3)4: HHHHHGHHHGHHHGHHHG

[0160] SEQ ID NO.26-31 positively charged cell penetrating peptides

[0161] TAT: GRKKRRQRRR

[0162] Penetratin: RQIKIWFQNRRMKWKK

[0163] CADY: GLWRALWRLLRSLWRLLWRA

[0164] (KH3)4: KHHHKHHHKHHHKHHH

[0165] H(KH3)3: HHHHHKHHHKHHHK

[0166] Mu: MRRAHHRRRRASHRRMRGG

[0167] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not limited thereto; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that the technical solutions recorded in the foregoing embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. A method for preparing a caged nanocarrier for targeted delivery of siRNA, characterized by, Comprising: (A) mutating the negatively charged or uncharged amino acids on the inner surface of the ferritin into positively charged amino acids; and any one or several of the following steps: (B) coupling a functional peptide with nucleic acid affinity to the N-terminus of the ferritin; (C) coupling a functional peptide promoting lysosomal escape to the N-terminus of the ferritin; (D) truncating the E-helix of the C-terminus of the ferritin; (E) coupling a functional peptide with nucleic acid affinity to the C-terminus of the ferritin; (F) coupling a functional peptide promoting lysosomal escape to the C-terminus of the ferritin.

2. The production method according to claim 1, characterized by, In step (A), the mutation sites of the ferritin are Glu61, Glu64, Glu140, Glu147, D177, D179 and E181.

3. The preparation method according to claim 1, characterized in that, In step (A), the positively charged amino acid is selected from any one of arginine, lysine and histidine.

4. The preparation method of claim 1, wherein Step (B) is any one of the following: (B1) coupling a functional motif with a nucleic acid binding peptide to the N-terminus of the ferritin; (B2) coupling a functional motif with a positively charged peptide to the N-terminus of the ferritin; Step (C) is: coupling a functional motif with a cell penetrating peptide to the N-terminus of the ferritin; Step (E) is any one of the following: (E1) coupling a functional motif with a nucleic acid binding peptide to the C-terminus of the ferritin; (E2) coupling a functional motif with a positively charged peptide to the C-terminus of the ferritin; Step (F) is: coupling a functional motif with a cell penetrating peptide to the C-terminus of the ferritin.

5. The preparation method according to claim 4, characterized in that, In steps (B1) and (E1), the sequence of the functional motif with a nucleic acid binding peptide is shown in SEQ ID NO. 9; In steps (B2) and (E2), the sequence of the functional motif with a positively charged peptide is shown in SEQ ID NO. 10-15; In steps (C) and (F), the sequence of the functional motif with a cell penetrating peptide is shown in SEQ ID NO. 16-31.

6. The ferritin cage nanocarrier for targeted delivery of siRNA prepared by the preparation method of any one of claims 1 to 5.

7. The ferritin cage nanocarrier of claim 6, wherein, The siRNA loading capacity of the ferritin cage nanocarrier is 2-3 nucleic acid molecules per protein cage.

8. A siRNA delivery system, characterized by, The ferritin cage nanocarrier for targeted delivery of siRNA of claim 6 or 7.

9. The siRNA delivery system of claim 8, wherein, The molar mass ratio of the ferritin cage nanocarrier to siRNA is 1: (1-5).

10. A method of encapsulating siRNA using the siRNA delivery system of claim 8 or 9, characterized in that, Comprising the following steps: S1, preparing and purifying the ferritin cage nanocarrier in the siRNA delivery system; S2, dissolving siRNA in DEPC water; S3, adding the ferritin nanocage carrier obtained in step S1 to an acidic buffer with pH 1-3, and incubating at 4°C for 20-30 minutes to obtain an acid depolymerization system; S4, adding the siRNA solution prepared in step S2 to an alkaline buffer with pH 9-11, mixing uniformly, then adding the acid depolymerization system obtained in step S3, and incubating at 4°C for 2-3 hours to reassemble the ferritin nanocage with siRNA loaded in the inner cavity, and the product is obtained.

11. The cage-like nanocarrier for targeted delivery of siRNA according to claim 6 or 7, the use of the siRNA delivery system according to claim 8 or 9 in the delivery of siRNA.

12. The ferritin cage nanocarrier for targeted delivery of siRNA according to claim 6 or 7, the use of the siRNA delivery system according to claim 8 or 9 in the preparation of a drug for treating tumors, viral infections, central nervous system diseases, cardiovascular diseases.

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