Fusion proteins of human heavy chain ferritin or its variants with polyhedral or granulosome proteins and their applications

Abstract

This invention relates to the field of pharmaceutical technology, and more particularly to fusion proteins of human heavy chain ferritin or its variants with polyhedral or granular proteins and their applications. This invention fuses a ferritin variant with a baculovirus polyhedral protein subunit to obtain particles with a diameter <20 nm, consisting of a polyhedral protein outer layer and a ferritin inner layer. The particles have an outer diameter of 17 nm, an inner lumen of 13 nm, and are positively charged. pH adjustment enables the depolymerization and recombination of the outer and inner layers, allowing the encapsulation of negatively charged insulin molecules with a diameter >8 nm. Proteins fused with polyhedral or granular protein subunits to wild-type or mutant ferritin can self-assemble into bilayer nanoparticles. The inner layer is a cage-like structure with a positive or negatively charged lumen and a size of 8 nm or 13 nm; the outer layer is a polyhedral or granular protein layer, exhibiting resilience, resistant to high temperatures and dehydration, and resistant to digestion by pepsin and trypsin. These nanoparticles can serve as oral carriers for various active molecules.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to bio-nanoparticles formed by fusing baculovirus polyhedrosome protein or granulosome protein with wild-type human heavy chain ferritin or mutants, and their application in the preparation of oral drug carriers. Background Technology

[0002] Currently, drugs with nucleic acids, peptides, or proteins as their main active ingredients are rapidly emerging in fields such as oncology, genetic diseases, and metabolic diseases due to their advantages in high specificity and strong targeting, and have become an important development trend in the biomedical field. For example, mRNA vaccines have achieved great success in many areas such as cancer, autoimmune diseases, and viral infections, and insulin plays a crucial role in the treatment of diabetes.

[0003] However, the vast majority of nucleic acid, peptide, and protein drugs cannot be administered orally. The core reason lies in the multiple physiological barriers present in the gastrointestinal tract: the acidic environment of the stomach damages the drug structure, and nucleases and proteases in the gastrointestinal tract rapidly degrade the drug, causing it to lose its activity. Simultaneously, the large molecular weight and strong hydrophilicity of these drugs make it difficult for them to penetrate the intestinal mucus layer and epithelial cell barrier. Even if a small amount is absorbed, its bioavailability is significantly reduced due to lysosomal retention in the small intestinal epithelial cells and the first-pass effect. Therefore, these drugs currently rely mainly on injection administration, which, while ensuring efficacy, suffers from problems such as poor patient compliance and high medical costs.

[0004] Oral delivery refers to the delivery of drugs or other active molecules into the digestive tract through the mouth, where they are absorbed into the bloodstream via the gastrointestinal mucosa. To achieve oral delivery of nucleic acid or protein drugs, the drugs need to be encapsulated in a carrier. Currently, commonly used carriers for oral administration of nucleic acid and protein drugs include lipid nanoparticles, cationic polymers such as chitosan and polyethyleneimine, and metal-organic frameworks. These carriers can protect the drugs from gastrointestinal degradation and promote intestinal absorption; however, they often have drawbacks such as poor biocompatibility, incompatibility with the human physiological environment, and high lysosomal retention rates leading to extremely low bioavailability.

[0005] Previous research has proposed using baculovirus polyhedrosis protein as a novel "bio-nano-brick," with it serving as the outer layer and the self-assembling mammalian ferritin natural heavy chain cage structure as the inner layer. This resulted in the first preparation of nanoparticles formed from these two fusion proteins, with wild-type polyhedrosis protein as the outer layer and wild-type ferritin as the inner layer. However, the particle's cavity was only 8 nm in size and negatively charged, limiting the molecules it could encapsulate due to space and charge constraints. Subsequent studies investigated the electrical properties and size of the ferritin-formed cavity, but further improvements are needed to enhance its encapsulation capabilities for nucleic acids, peptides, and protein drugs. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a fusion protein of human heavy chain ferritin or its variants with polyhedral or granular proteins and its use in the preparation of oral drug carriers.

[0007] The present invention provides a packaging carrier having a double core-shell structure, wherein the inner layer is a positively charged ferritin cage structure with 48 subunits, and the outer layer is a polyhedral protein layer or a particulate protein layer.

[0008] The ferritin cage structure is formed by wild-type human heavy chain ferritin or by a mutant of human heavy chain ferritin. Preferably, the ferritin cage structure is formed by a mutant of human heavy chain ferritin.

[0009] The packaging carrier prepared by the invention has a bilayer protein core-shell structure. The inner layer is ferritin, which can regulate the size and charge of the lumen through amino acid mutation. It can encapsulate active molecules with different charges and sizes no larger than 13 nm. The outer layer is a polyhedral protein layer. Polyhedral subunits fused with ferritin can aggregate to form the first layer. By aggregating with individual expressed or wild-type polyhedral protein subunits, a series of polyhedral protein outer layers of different thicknesses can be formed to obtain different degrees of stress resistance. It can not only stabilize active molecules, but also resist digestion and degradation in the gastrointestinal tract. This bilayer protein core-shell structure allows both the inner and outer layers to depolymerize and repolymerize by adjusting the pH, and the different pH response ranges of the inner and outer layers provide operability for packaging exogenous active molecules. The overall particle size of less than 20 nm also allows the carrier to reach the lymphatic circulation or lamina propria through the absorption of Pyell's knots, and it also has a greater chance of breaking through the lamina propria through the gaps between the small intestinal epithelial villi cells to reach the blood. Then it is degraded by enzymes present in the circulatory system, especially the liver, thereby releasing the drug. This can significantly improve the immune response and bioavailability of oral administration of active molecules such as protein drugs, peptide drugs, and mRNA.

[0010] The packaging carrier of the present invention is prepared by self-assembly of a fusion protein, wherein the fusion protein comprises fragment A and fragment B;

[0011] Fragment A is a polyhedral protein or a granuloprotein;

[0012] Fragment B is a mutant of the human heavy chain ferritin.

[0013] In this invention, the amino acid sequence of wild-type human heavy chain ferritin is shown in SEQ ID NO:1.

[0014] In this invention, the polyhedral protein or particulate protein is derived from invertebrate baculoviruses.

[0015] In some embodiments, the amino acid sequence of the mutant human heavy chain ferritin is shown in SEQ ID NO:2.

[0016] In some embodiments, the polyhedral protein has an amino acid sequence as shown in SEQ ID NO: 3.

[0017] In some embodiments, the particulate protein has an amino acid sequence as shown in SEQ ID NO: 4.

[0018] In some specific embodiments, the fusion protein, from the N-terminus to the C-terminus, sequentially includes the polyhedral protein shown in SEQ ID NO: 3, the granuloprotein shown in SEQ ID NO: 4, and the human heavy chain ferritin mutant shown in SEQ ID NO: 2.

[0019] The present invention provides a mutant of human heavy chain ferritin, wherein at least one of the following sites is mutated: position 62, position 65, position 103, position 131, positions 139-144, position 148, position 151C, or position 182.

[0020] In this invention, the mutation includes the substitution, addition, and / or deletion of amino acids. The substitution refers to replacing the original amino acid residue with any other amino acid residue or peptide segment; the deletion refers to the removal of one or more amino acid residues; and the addition refers to the addition of one or more amino acid residues.

[0021] In this invention, the mutant includes at least one of the following mutations: E62K, E65R, C103A, C131A, E148K, D151C, E182K or ∆139NEQVKA.

[0022] In some embodiments, wild-type human heavy chain ferritin has the amino acid sequence shown in SEQ ID NO:1, or has an amino acid sequence with at least 80% identity to SEQ ID NO:1. The at least 80% identity means identity greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, or greater than 99.9%. In some specific embodiments, the amino acid sequence of wild-type human heavy chain ferritin is as shown in SEQ ID NO:1.

[0023] In some embodiments, the amino acid sequence of the mutant human heavy chain ferritin is as shown in SEQ ID NO:2, or has an amino acid sequence with at least 80% identity to SEQ ID NO:2. The at least 80% identity means identity greater than 80%, greater than 85%, greater than 90%, greater than 95%, greater than 97%, greater than 98%, greater than 99%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, or greater than 99.9%. In some specific embodiments, the amino acid sequence of the mutant human heavy chain ferritin is as shown in SEQ ID NO:2.

[0024] This invention prepares a positively charged ferritin with a larger lumen composed of 48 subunits through point mutation, insertion, and deletion mutations. Furthermore, this invention fuses ferritin with polyhedral or granular proteins. Ferritin acts as a scaffold, while the polyhedral or granular proteins aggregate to form inner and outer layers. Both layers can be depolymerized and repolymerized through pH adjustment. The fused polyhedral protein subunits can also bind to expressed or wild-type polyhedral protein subunits to form a thicker, more resilient outer layer. The inner layer overcomes the limitations of charge and space, allowing it to encapsulate more molecules. Through cross-combination, eight designed particles can achieve molecules with dimensions no greater than 13 nm, with no charge limitations.

[0025] The present invention also provides a fusion protein comprising fragment A and fragment B;

[0026] Fragment A is a polyhedral protein or a granuloprotein;

[0027] Fragment B is a mutant as described above.

[0028] In some embodiments, fragment A of the fusion protein is a polyhedral protein or particulate protein derived from an invertebrate virus. As a feasible example, it is a nucleopolyhedral protein subunit derived from the baculovirus *Agrostis spp.*. In some specific embodiments, the polyhedral protein has the amino acid sequence shown in SEQ ID NO: 3; the particulate protein has the amino acid sequence shown in SEQ ID NO: 4.

[0029] In this invention, the outer layer of the polyhedral or granular protein in the fusion protein is derived from invertebrate viruses that pose no biosafety risk, while the ferritin is derived from the human body itself. Therefore, the resulting fusion protein exhibits good safety and biocompatibility.

[0030] In this invention, the structure of the fusion protein is as follows:

[0031] [(fragment A)a-(fragment B)b]c or [(fragment B)b-(fragment A)a]c;

[0032] Where: a represents the number of repetitions of fragment A, b represents the number of repetitions of fragment B, and c represents the number of repetitions of the unit composed of fragments A and B in the fusion protein. a, b, and c are independently selected from non-zero integers. For example, 1 ≤ a ≤ 10, 1 ≤ b ≤ 10, and 1 ≤ c ≤ 10. a, b, and c can be equal or unequal, and this invention does not limit this. Taking a = 1, b = 1, and c = 1 as an example, the structure of the fusion protein of this invention is fragment A-fragment B or fragment B-fragment A. Preferably, fragment A is located at the N-terminus of fragment B.

[0033] In some embodiments, the fusion protein of the present invention further includes a linker fragment; in the present invention, the linker is located between two adjacent fragments, and optionally, the length of the linker is 2-20 amino acids, or 5-15 amino acids, or 10-15 amino acids, and the present invention does not limit this. The linker is (GGGGS)n, where n is a non-zero integer, preferably n=3.

[0034] In this invention, the fusion protein, from N-terminus to C-terminus, sequentially comprises the polyhedral protein shown in SEQ ID NO: 3, (GGGGS)3, and the human heavy chain ferritin mutant shown in SEQ ID NO: 2;

[0035] Or, in sequence, it includes the granuloprotein shown in SEQ ID NO:4, (GGGGS)3, and the human heavy chain ferritin mutant shown in SEQ ID NO:2.

[0036] The fusion protein described in this invention can assemble into a bilayer protein core-shell structure. The inner layer is ferritin, whose lumen size and charge can be adjusted through amino acid mutations, and can encapsulate active molecules with different charges and sizes no larger than 13 nm. The outer layer is a polyhedral protein layer. Polyhedral subunits fused with ferritin can aggregate to form the first layer. By aggregating with individual expressed or wild-type polyhedral protein subunits, a series of polyhedral protein outer layers of varying thicknesses can be formed to obtain different degrees of stress resistance. This not only stabilizes the active molecules but also resists digestion and degradation in the gastrointestinal tract. Both the inner and outer layers of this bilayer protein core-shell structure can depolymerize and repolymerize by adjusting the pH, and the different pH response ranges of the inner and outer layers provide operability for the packaging of exogenous active molecules. The overall particle size of less than 20 nm also allows the carrier to reach the lymphatic circulation or lamina propria through the Pyell's knots, and it also has a greater chance of penetrating the lamina propria through the intercellular spaces of the small intestinal epithelial villi to reach the bloodstream. It is then degraded by enzymes present in the circulatory system, especially the liver, thereby releasing the drug. This can significantly improve the immune response and bioavailability of oral administration of active molecules such as insulin and mRNA.

[0037] Furthermore, the present invention also provides nucleic acids that encode the mutants as described above, or encode the fusion proteins as described above.

[0038] The present invention also provides an expression unit comprising a promoter, a nucleic acid as described above, and a terminator.

[0039] The promoters are either prokaryotic or eukaryotic promoters. For example, the promoters are CMV, CAG, EF1a, PGK, U6, H1, EFS, CBh, SFFV, MSCV, SV40, UBC, or TRE promoters. The terminators are T7 phage terminators, T0 phage terminators, λ phage terminators, SV40 terminators, CMV terminators, rrnB terminators, bGH terminators, hGH terminators, or rbGlob terminators.

[0040] The expression unit described in this invention also includes enhancers, such as SV40 enhancer, CMV enhancer, SV-1 enhancer, ROSA26 enhancer, EF1α enhancer, HARE5 enhancer, UBC enhancer, EF1A enhancer, PGK enhancer, CAGG enhancer, COPIA enhancer or ACT5C enhancer.

[0041] Furthermore, the present invention also provides a plasmid vector comprising the nucleic acid as described above or comprising the expression unit as described above.

[0042] The plasmid vector described in this invention is a recombinant vector capable of stably replicating and expressing the inserted nucleic acid sequence or expression unit in a host cell. It is used for the preservation or amplification of nucleic acids as described above. In specific embodiments, the plasmid vector may be a cloning vector or expression vector backbone commonly used in the art. As a feasible example, the backbone of the plasmid vector is selected from, but is not limited to, pUC series plasmids (such as pUC18, pUC19), pBluescript series plasmids, pET series plasmids (such as pET-32a), pCDNA series plasmids (such as pCDNA3.1), pCMV series plasmids, pEGFP series plasmids, pSV2 series plasmids, pBI series plasmids, pRS series plasmids, pACYC184, pBR322, etc.

[0043] Furthermore, the present invention also provides a host cell that is transformed or transfected with the plasmid vector as described above; or whose genome includes the nucleic acid as described above or the expression unit as described above.

[0044] In this invention, the host cell is used for the preservation, amplification, or expression of the aforementioned plasmid vector or fusion protein. In this invention, the host is a prokaryotic or eukaryotic host. The eukaryotic host includes, but is not limited to, yeast, insect cells, and renal epithelial cells, while the prokaryotic host includes, but is not limited to, *Escherichia coli*. As feasible examples, the host is *Escherichia coli* BL21(DE3), BL21(DE3) pLysS, DH5α, JM109, JM110, TOP10, or HB101.

[0045] Furthermore, the present invention also provides a method for preparing the mutant or fusion protein as described above, comprising: culturing the host cells and harvesting a culture product containing the mutant or fusion protein.

[0046] Furthermore, the present invention also provides a method for preparing the packaging carrier as described above, which includes culturing the host cells, harvesting the culture product containing the fusion protein, and self-assembling the fusion protein to obtain the packaging carrier.

[0047] In the fusion protein described in this invention, both the outer layer of baculovirus polyhedromic or granular protein and the inner layer of ferritin can be depolymerized and reassembled through pH adjustment. The outer layer of baculovirus polyhedromic or granular protein can resist digestion by trypsin and pepsin. If the resistance time of a single polyhedromic or granular protein layer is insufficient, the outer layer can be further enhanced by depolymerizing it and then adding monomeric polyhedromic or granular protein subunits in a specific molar ratio. The inner layer of ferritin, with different sizes and charges, can encapsulate polypeptide and protein drug molecules of different charges and sizes. Figure 1 ).

[0048] Based on this, the present invention also provides the use of the mutants or fusion proteins described above in the preparation of oral drug carriers.

[0049] Furthermore, the present invention also provides an oral pharmaceutical formulation prepared from a target molecule and a mutant as described above, or a fusion protein as described above, or a packaging carrier as described above.

[0050] In this invention, the target molecule is a protein drug, a peptide drug, or a nucleic acid drug.

[0051] Among them, protein drugs can be selected from biologically active protein molecules such as insulin, growth hormone, interferon, and interleukin; peptide drugs can be selected from small molecule peptide compounds composed of amino acids linked by peptide bonds, such as leuprorelin, octreotide, and somatostatin; nucleic acid drugs can be selected from nucleic acid molecules that can regulate gene expression or have therapeutic effects, such as messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), microRNA (miRNA), antisense oligonucleotides (ASO), and plasmid DNA. In a specific embodiment, the protein drug is insulin.

[0052] Furthermore, the present invention also provides a method for preparing an oral pharmaceutical formulation as described above, comprising:

[0053] The fusion protein, as described above, is cleaved, mixed with a solution of the target molecule, and assembled to encapsulate the target molecule into the internal cavity of the fusion protein, thereby obtaining the formulation.

[0054] The polyhedral protein layer in the fusion protein of the present invention begins to dissociate at pH > 8, and the ferritin remains stable in dilute acids and alkalis at pH 2-12. The preparation method of the present invention utilizes the overlapping and non-overlapping pH regions of the two protein layers to perform synchronous and stepwise dissociation and recombination of the two layers, thereby achieving the encapsulation of target molecules.

[0055] As a feasible example, pH adjustment can be used to open only the polyhedral or granular protein layer without opening the ferritin inner layer. By binding expressed or wild-type polyhedral or granular proteins with fused polyhedral or granular proteins, the thickness of the outer polyhedral or granular protein layer of nanoparticles can be controlled to improve stress resistance.

[0056] Furthermore, the present invention also provides a medicament comprising a pharmaceutically acceptable excipient and at least one of the following:

[0057] 1) Mutants or fusion proteins as described above;

[0058] 2) The packaging carrier as described above;

[0059] 2) Oral drug preparations as described above.

[0060] The drug described in this invention is an oral preparation, and its dosage form includes, but is not limited to, tablets, capsules, granules, powders, oral liquids, suspensions, emulsions, and pills. Among them, tablets are ordinary tablets, enteric-coated tablets, sustained-release tablets, or controlled-release tablets; capsules are hard capsules, soft capsules, or enteric-coated capsules.

[0061] Furthermore, the present invention also provides a drug delivery method comprising: encapsulating a drug active ingredient in a packaging carrier as described above, and then delivering the drug-loaded packaging carrier to an organism via an oral route.

[0062] In this invention, the packaging carrier carrying the drug or active substance has an outer polyhedral protein layer that resists digestion by gastric acid, pepsin, and trypsin in the gastrointestinal tract. Since the overall particle size of the carrier is less than 20 nm, it can pass through the Pye's knots, and a larger proportion of it will pass through the gaps between the villi cells of the small intestinal epithelium. After breaking through the lamina propria, it reaches the blood. Then, enzymes in the circulatory system, especially in the liver, will degrade the carrier and release insulin.

[0063] Furthermore, the present invention also provides a method for preventing and treating diseases, comprising administering the drug as described above. The administration method includes oral administration.

[0064] In this invention, ferritin mutants were optimized and screened, and then polyhedral protein subunits and mutant ferritin subunits were fused and expressed to successfully prepare bio-nanoparticles with a particle size of less than 20 nm, consisting of a wild-type polyhedral protein outer layer and a mutant ferritin inner layer. The particle has an inner cavity size of 13 nm and carries a positive charge. The outer and inner layers can be depolymerized and recombined by pH adjustment, and can encapsulate a variety of nucleic acids, peptides, proteins and other active molecules. The outer polyhedral protein layer has stress resistance and can form outer layers of different thicknesses by interacting with polyhedral protein subunits, thereby increasing stress resistance and stabilizing the encapsulated active molecules. The overall particle size of no more than 20 nm has the potential to allow the encapsulated molecules to reach the lymphatic circulation system or lamina propria through the absorption of Pyell's knots, and can also cross the intestinal barrier through the paracellular pathway to reach the blood circulation system, providing new possibilities for the oral delivery of active molecules. Attached Figure Description

[0065] Figure 1 This demonstrates the design of oral carriers for nucleic acid / peptide / protein drugs based on polyhedral / granular proteins and ferritin;

[0066] Figure 2 This demonstrates an oral delivery pathway for nucleic acid / peptide / protein drugs based on polyhedral / granular proteins and ferritin;

[0067] Figure 3 The SDS-PAGE results of wild-type polyhedral protein are shown in the following order from left to right: lane 1 and lane 2. The target bands in lanes 1 and 2 are derived from the same gel. Lane 1 is the protein marker and lane 2 is the Pohl protein with a molecular weight of 46 kDa.

[0068] Figure 4The results of transmission electron microscopy (TEM) and histograms of particle size distribution of polyhedral protein (Polh) at different time points are shown. Figure A shows the particle size distribution of polyhedral protein (samples prepared immediately after the experiment), Figure B shows the histogram and fitted curve of the corresponding frequency distribution, Figure C shows the particle size distribution of polyhedral protein (stored at 4°C for about 18 days), and Figure D shows the histogram and fitted curve of the corresponding frequency distribution.

[0069] Figure 5 The results of SDS-PAGE, transmission electron microscopy, and frequency distribution histogram of wild-type ferritin (Fer-wt) are shown. In Figure A, from left to right: lane 1 and lane 2. The target bands in lanes 1 and 2 are from the same gel. Lane 1 is the protein marker; lane 2 is Fer-wt protein with a molecular weight of 38 kDa. Figure B is the particle size distribution map of wild-type ferritin. Figure C is the frequency distribution histogram and fitted curve of the corresponding statistics.

[0070] Figure 6 The results of SDS-PAGE, transmission electron microscopy, and histogram of particle size frequency distribution of mutant ferritin (Fer-mut) are shown. In Figure A, from left to right: lane 1 and lane 2. The target bands in lanes 1 and 2 are from the same gel. Lane 1 is the protein marker. Lane 2 is the Fer-mut protein with a molecular weight of 37 kDa. Figure B is the particle size distribution of mutant ferritin.

[0071] Figure 7 The results of SDS-PAGE, transmission electron microscopy, and histogram of particle size distribution for fusion protein 1 (Polh-(G4S)3-Fer-wt) are shown. In Figure A, from left to right: lanes 1 and 2; the target bands in lanes 1 and 2 originate from the same gel, lane 1 is the protein marker, and lane 2 is the Pohl-(G4S)3-Fer-wt protein with a molecular weight of 68 kDa. Figure B shows the particle size distribution of fusion protein 1, and Figure C shows the corresponding frequency distribution histogram and fitted curve.

[0072] Figure 8 The results of SDS-PAGE, transmission electron microscopy, and histogram of particle size distribution for fusion protein 2 (Polh-(G4S)3-Fer-mut) are shown. Figure A, from left to right: lane 1 and lane 2; the target bands in lanes 1 and 2 are from the same gel, lane 1 is the protein marker; lane 2 is the Pohl-(G4S)3-Fer-mut protein with a molecular weight of 67 kDa; Figure B is the particle size distribution map of fusion protein 2; Figure C is the histogram of the corresponding frequency distribution and the fitted curve.

[0073] Figure 9The morphology and particle size distribution of iron core structure nanoparticles are shown in the figure. Figure A is a transmission electron microscope image, and Figure B is a particle size distribution statistical diagram. During the expression of ferritin in prokaryotes, some of it will assemble in the cytoplasm and synthesize iron-containing particles in the lumen. In negative staining electron microscopy, the electron beam does not pass through the iron core and appears as small black particles.

[0074] Figure 10 The morphology and particle size distribution of nanoparticles that reassembled from fusion proteins to restore the binuclear-shell structure are statistically shown. Figure A is a transmission electron microscope image, and Figure B is a particle size distribution statistical graph. The depolymerized particles reassembled after pH adjustment.

[0075] Figure 11 The standard curve and mass spectrum of insulin are shown. Figure A is the standard curve of insulin measured by LC-MS; Figure B is the mass spectrum of insulin standard sample (64 ng / mL); Figure C is the mass spectrum of the protein before dissociation (sample 1); Figure D is the mass spectrum of the protein dissociation releasing insulin (sample 2). The sample before dissociation was eluted by multiple rounds of ultrafiltration to remove adsorbed or residual insulin. Figures B, C, and D are re-graphed. Detailed Implementation

[0076] This invention provides human heavy chain ferritin mutants and their application in the preparation of oral drug carriers. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0077] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0078] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0079] The term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can mean: A exists alone, A and B exist simultaneously, or B exists alone. A and B can be singular or plural.

[0080] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0081] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0082] The test materials used in this invention are all commercially available products. The amino acid sequences involved include:

[0083] The amino acid sequence of wild-type human heavy chain ferritin SEQ ID NO:1

[0084] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSDNES*

[0085] The amino acid sequence of the mutant human heavy chain ferritin SEQ ID NO:2

[0086] MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDDDVALKNFAKYFLHQSHKERRHAEKLMKLQNQRGGRIFLQDIKKPDADDWESGLNAMEAALHLEKNVNQSLLELHKLATDKNDPHLADFIETHYLIKKLGCHVTNLRKMGAPESGLAEYLFDKHTLGDSDNKS*

[0087] Wild-type polyhedral protein sequence SEQ ID NO: 3:

[0088] MPDYSYRPTIGRTYVYDNKYYKNLGAVIKNAKRKKHFAEHEIEEATLDPLDNYLVAEDPFLGPGKNQKLTLFKEIRNVKPDTMKLVVGWKGKEFYRETWTRFMEDSFPIVNDQEVMDVFLVVN MRPTRPNRCYKFLAQHALRCDPDYVPHDVIRIVEPSWVGSNNEYRISLAKKGGGCPIMNLHSEYTNSFEQFIDRVIWENFYKPIVYIGTDSAEEEEILLEVSLVFKVKEFAPDAPLFTGPAY*

[0089] Wild-type granulosome protein sequence SEQ ID NO: 4:

[0090] MGYNRALRYSKHEGTTCVIDNQHYKSLGAVLKDVKHKKDRLREAEIEPVLDIADQYMVTEDPFRGPGKNVRITLFKECRRVEPDTLKLVCNWSGKEFLREMWTRFISEEFPITTDQQIMNMWFE IQVRPMQPNRCYKFTMQYALDAHPDYVPHDVIRAQDPYYIGPNNIERINLKKGFAFPLMCLQSVYNDNFETFFEDVLWPYFHRPLVYIGTTSSETEEILLEVSFLFKIKEFAPDVPLYTGPAY*

[0091] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0092] In this invention, a polyhedrin (Polh) protein is fused to the N-terminus of human heavy chain ferritin (Fer), which undergoes multiple insertions, deletions, and site mutations (Fer-mutations). The two are linked by a flexible linker (G4S)3 to ensure proper connection and folding. We successfully expressed the Polh-(G4S)3-Fer-mut fusion protein and then prepared nanoparticles assembled from these protein subunits. By adjusting the pH, we successfully encapsulated insulin molecules into these nanoparticles. This invention elucidates the design, preparation, and method of encapsulating insulin molecules using this carrier. Based on this invention, we will follow... Figure 2Further research was conducted. In artificial gastric and intestinal fluid resistance experiments, if the target resistance time was not reached, due to the nature of polyhedral proteins, wild-type or expressed polyhedral proteins were prepared, and then combined with fusion-type polyhedral proteins by pH adjustment to prepare Polh-layered bilayer nanoparticles until the target resistance time against acid and enzymatic degradation was achieved. Then, the effect of the oral carrier encapsulating insulin molecules was evaluated using a diabetic rat model. The invention is further illustrated below with reference to examples:

[0093] Example 1: Construction, expression, preparation and characterization of wild-type polyhedral protein (Polh) in Escherichia coli expression strains

[0094] The nucleopolyhedrome protein gene sequence of wild-type alfalfa silver-striped moth baculovirus (hereinafter referred to as: wild-type polyhedrome protein, amino acid sequence SEQ ID NO:3) was synthesized by gene synthesis method. The recombinant expression vector pET32a-Polh carrying the target sequence was transformed into Escherichia coli BL21(DE3) strain to obtain recombinant Escherichia coli 1.

[0095] Recombinant *E. coli* 1 was inoculated into LB medium containing ampicillin and cultured. The expression of soluble proteins was then induced at low temperature with an inducer. After induction, the bacterial cell pellet was washed with magnetic bead washing buffer and resuspended in the same buffer to obtain the induced bacterial culture. The culture was then sonicated and centrifuged in a high-speed refrigerated centrifuge to collect the supernatant. The supernatant was purified using NTA-Ni magnetic beads and eluted with elution buffer containing 500 mM imidazole. The flow-through and eluent were collected. The purified protein was analyzed by SDS-PAGE electrophoresis. The results are shown below. Figure 3 As shown, the purified wild-type polyhedral protein achieved a purity of over 95%. The protein was then concentrated to 1 ml via ultrafiltration and finally dialyzed into a ferritin assembly buffer to complete the assembly of the wild-type polyhedral protein. Protein concentration was quantified using a protein quantification kit. After determining the sample concentration, negative-stained transmission electron microscopy (TEM) samples were prepared.

[0096] The specific steps for preparing negatively stained transmission electron microscopy samples are as follows: Take out the copper screen, place one side of the carbon sheet in contact with the sample, absorb the excess sample with filter paper, then place the other side of the carbon sheet in contact with the phosphotungstic acid staining solution for staining, then absorb the excess staining solution with filter paper, and after drying, perform the analysis on the instrument.

[0097] Negative staining transmission electron microscopy results: Figure 4 Figure A shows the transmission electron microscopy (TEM) results of the Pohl sample prepared immediately according to the experimental procedure, while... Figure 4The C-type Pohl sample was stored at 4°C for approximately 18 days before the experiment, resulting in the final transmission electron microscopy (TEM) results. Particle size frequency statistics (count > 300 particles) were performed on the TEM results of both samples, and frequency distribution maps were constructed (corresponding to...). Figure 4 B and Figure 4 (D), perform a normality test on the frequencies. Figure 4 In Figure B, p=0.139 > 0.05. Figure 4 In the middle D, p=0.300>0.05, and both groups of frequencies conform to the characteristics of a normal distribution. Statistical analysis shows that... Figure 4 The mean outer diameter of A in the protein is 16.9 ± 3.0 nm, indicating that the particle size of the vast majority of particles in the protein is concentrated around 16.9 nm, and the standard deviation is 3.0 nm, indicating that the particle uniformity is good. Figure 4 The particle size of C is distributed between 25nm and 75nm, with an average particle size of 49.4±8.3nm, indicating that the particle size in the protein is not uniform, and the vast majority of particles are larger than 16.9nm. It is inferred that over time, the size of Polh has increased, and more polyhedral subunits have been bound to it through the surface salt bridge structure, forming a larger polymer.

[0098] Example 2: Construction, expression, preparation and characterization of wild-type ferritin (Fer-wt) cage structure in Escherichia coli expression strains

[0099] The natural mammalian ferritin heavy chain gene sequence (hereinafter referred to as wild-type ferritin, Fer-wt, amino acid sequence is SEQ ID NO:1) was used to synthesize the sequence through gene synthesis methods. The fusion gene recombinant expression vector pET32a-Fer with the target sequence was transformed into Escherichia coli BL21(DE3) strain to obtain recombinant Escherichia coli 2.

[0100] Following the method in Example 1, wild-type ferritin purified from NTA-Ni magnetic beads was obtained and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 5 As shown in Figure A, the purified wild-type ferritin achieved a purity of over 95%. After filtration through a 0.22 μm filter membrane, it was dialyzed into the ferritin assembly buffer. Finally, impurities were removed by centrifugation at 10,000 rpm, and the supernatant was collected to complete the assembly of wild-type ferritin. The protein concentration was quantified using a protein quantification kit. After determining the sample concentration, negative staining transmission electron microscopy (TEM) samples were prepared.

[0101] Negative staining transmission electron microscopy results: Figure 5 B and Figure 5 C represents the morphology and structure diagram of Fer-wt nanoparticles and the histogram of particle size frequency statistics, respectively. Particle size frequency statistics were performed on the transmission electron microscopy results (statistic count > 200 particles), a frequency distribution map was constructed, and the frequencies were tested for normality. Figure 5For B, p=0.361>0.05, the frequency of this group conforms to the characteristics of a normal distribution. Statistical analysis shows... Figure 5 The mean outer diameter of the C-cell electron microscope is 10.2 ± 1.1 nm, indicating that the particle size of most particles in the protein is concentrated around 10.2 nm, with a standard deviation of 1.1 nm, indicating good particle uniformity. This electron microscopy result is consistent with the structure of the natural mammalian ferritin heavy chain (24 ferritin subunits), which presents a hollow cage-like structure, and its outer diameter parameters also form a precise match.

[0102] Example 3: Construction, expression, preparation, and characterization of the mutant ferritin cage structure in Escherichia coli expression strains; wild-type polyhedral protein.

[0103] Point mutations, insertion mutations, and deletion mutations were performed on the natural mammalian ferritin heavy chain protein sequence according to the expected target (hereinafter referred to as: mutant ferritin, Fer-mut, amino acid sequence SEQ ID NO:2). The sequence was synthesized using gene synthesis methods, and the recombinant expression vector pET32a-Fer-mut carrying the target sequence was transformed into Escherichia coli BL21(DE3) strain to obtain recombinant Escherichia coli 3.

[0104] Following the method in Example 1, mutant ferritin purified from NTA-Ni magnetic beads was obtained and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 6 As shown in Figure A, trace amounts of impurities were present after purification, with staining depth significantly weaker than the target band. The protein purification buffer was then dialyzed into the ferritin assembly buffer. After dialysis, impurities were removed by centrifugation at 10,000 rpm, and the supernatant was collected to allow the mutant ferritin to complete its assembly. The protein concentration was confirmed using a protein quantification kit, and after dilution to an appropriate concentration, negatively stained transmission electron microscopy (TEM) samples were prepared according to the method in Example 1.

[0105] Negative staining transmission electron microscopy results: Figure 6 Figure B shows the morphological structure of the Fer-mut nanostructure at 100 nm. Due to the mutation of the natural mammalian ferritin heavy chain protein sequence, the mutant ferritin is composed of 48 subunits, which is less stable than the wild-type ferritin composed of 24 subunits, as shown by the red arrow in the figure. The resulting ferritin structure is less and less typical.

[0106] Example 4: Construction, expression, preparation, and characterization of wild-type polyhedrosis protein and wild-type ferritin in Escherichia coli expression strains.

[0107] Through genetic engineering, (G4S)3 was inserted as a linker between the N-terminus of the mammalian ferritin heavy chain subunit (amino acid sequence SEQ ID NO:1) and the C-terminus of the wild-type alfalfa silver-striped moth baculovirus nucleopolyhedrovirus subunit (amino acid sequence SEQ ID NO:3), forming a fusion protein 1 sequence (hereinafter referred to as: fusion protein 1, Polh-(G4S)3-Fer-wt). The recombinant expression vector pET32a-Polh-(G4S)3-Fer-wt carrying the fusion gene fusion gene was transformed into Escherichia coli BL21(DE3) strain to obtain recombinant Escherichia coli 4.

[0108] Following the method in Example 1, the purified fusion protein 1 from NTA-Ni magnetic beads was obtained and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 7 As shown in A, the purified fusion protein 1 has a purity of over 95%. Then, the protein volume is concentrated to 1 ml by ultrafiltration, and finally dialyzed into the ferritin assembly buffer to complete the assembly of fusion protein 1.

[0109] The assembled fusion protein 1 was centrifuged at 10,000 rpm and filtered through a 0.22 μm filter membrane to quantify the protein concentration and determine the protein concentration for sample preparation. The negative staining transmission electron microscopy (TEM) sample was then prepared according to the method in Example 1.

[0110] Negative staining transmission electron microscopy results: Figure 7 B and Figure 7 C represents the morphology and particle size frequency histogram of the Pohl-(G4S)3-Fer-wt nanostructure, respectively. Particle size frequency statistics (statistical count > 100 particles) were performed on the transmission electron microscopy results, and a frequency distribution map was constructed. Normality was then tested for the frequencies. Figure 7 For B, p=0.312>0.05, the frequency of this group conforms to the characteristics of a normal distribution. Statistical analysis shows... Figure 7 The mean outer diameter of B particles was 16.1 ± 2.5 nm, indicating that the vast majority of particles in the protein were concentrated around 16.1 nm, with a standard deviation of 2.5 nm. (Observation) Figure 7 According to electron microscopy results, the fusion protein 1 has the same structure as the natural mammalian ferritin heavy chain (24 ferritin subunits), exhibiting a hollow cage-like structure.

[0111] Example 5: Construction of the recombinant expression vector pET32a-Polh-(G4S)3-Fer-mut. Construction, expression, preparation, and characterization of the *E. coli* expression strain.

[0112] Following Example 3, the natural mammalian ferritin heavy chain was mutated. Then, following Example 4, the mutated ferritin (amino acid sequence SEQ ID NO: 2) was linked with the wild-type alfalfa silver-striped moth baculovirus nucleopolyhedrovirus protein baculovirus polyhedrovirus protein using a linker to form a fusion protein 2 sequence (hereinafter referred to as: fusion protein 2, Polh-(G4S)3-Fer-mut). The recombinant expression vector pET32a-Polh-(G4S)3-Fer-mut carrying the target sequence was transformed into Escherichia coli strain BL21(DE3) to obtain recombinant Escherichia coli 5.

[0113] Following the method in Example 1, the purified fusion protein 2 from NTA-Ni magnetic beads was obtained and analyzed by SDS-PAGE electrophoresis. The results are as follows: Figure 8 As shown in A, the purified fusion protein 2 has a purity of over 95%. Then, the protein volume is concentrated to 1 ml by ultrafiltration, and finally dialyzed into the ferritin assembly buffer to complete the assembly of fusion protein 2.

[0114] The assembled fusion protein 2 was centrifuged at 10,000 rpm and filtered through a 0.22 μm filter membrane to quantify the protein concentration and determine the protein concentration for sample preparation. The negative staining transmission electron microscopy (TEM) sample was then prepared according to the method in Example 1.

[0115] Negative staining transmission electron microscopy results: Figure 8 B and Figure 8 C represents the morphology and particle size frequency histogram of the Pohl-(G4S)3-Fer-mut nanostructure, respectively. Particle size frequency statistics (statistical count > 100 particles) were performed on the transmission electron microscopy results, and a frequency distribution map was constructed. Normality was then tested for the frequencies. Figure 8 For B, p=0.132>0.05, the frequency of this group conforms to the characteristics of a normal distribution. Statistical analysis shows... Figure 8 The mean outer diameter of B particles was 16.1 ± 1.8 nm, indicating that the vast majority of particles in the protein were concentrated around 16.1 nm, with a standard deviation of 1.8 nm. This was observed... Figure 8 B, a fusion protein 2 composed of baculovirus polyhedromes and mutant ferritin, forms a hollow cage-like structure consistent with the structure of the natural mammalian ferritin heavy chain (24 ferritin subunits) under the stabilizing effect of the polyhedromes.

[0116] Example 6: Insulin-loaded Polh-(G4S)3-Fer-mut fusion protein

[0117] 6.1 Open the cavity of the Pohl-(G4S)3-Fer-mut fusion protein

[0118] The fusion protein assembled in Example 5 was adjusted to a suitable concentration, such as 1 mg / mL. The pH was adjusted to 12.0 with dilute NaOH solution, and the protein was lysed in an ice bath for 1 hour. At this point, both the outer and inner polyhedral layers were open. Following the method in Example 1, samples were taken and characterized using negative staining electron microscopy (TEM). The particle size was then statistically analyzed using frequency distribution (count > 300 particles). Figure 9 Results: All particles in the field of view were black, and no formed protein structure was observed. Frequency statistics of the particles in the image were performed, and a frequency distribution map was constructed. The mean outer diameter was 2.3 ± 0.6 nm, which is the iron core of ferritin.

[0119] 6.2 Insulin Encapsulation

[0120] Insulin solution was prepared using hydrochloric acid. The insulin solution was rapidly added to the pretreated fusion protein 2 solution described in section 6.1 at a specific molar ratio (e.g., insulin to fusion protein subunit molar ratio of 1:10). Simultaneously, the pH was adjusted to 8.5 using dilute HCl, with gentle stirring to ensure uniform distribution of insulin in the fusion protein 2 solution. The mixture was incubated at room temperature on an ice bath for 4 hours, followed by overnight incubation at 4°C in the dark to allow for complete encapsulation of insulin into the internal cavity of fusion protein 2. Following the method described in Example 1, the fully encapsulated sample was characterized using negative staining electron microscopy (TEM), and the particle size frequency distribution was statistically analyzed (count > 300 particles). Figure 10 .

[0121] result: Figure 10 Image A shows the morphological structure of the fusion protein reassembled to restore the binuclear-shell structure. Frequency distribution plots were generated by counting the particles in the image. Figure 10 For sample B, based on the normal distribution statistics, p = 0.063 > 0.05, indicating that this set of data conforms to the characteristics of a normal distribution. The statistically significant mean outer diameter is 17.3 ± 2.7 nm, indicating that the particle size of the vast majority of particles in the protein is concentrated around 17.3 nm, with a standard deviation of 2.7 nm, suggesting good particle uniformity. The mean outer diameter of the particles and... Figure 8 The fusion proteins that were not subjected to alkaline hydrolysis had uniform particle size, confirming that alkaline hydrolysis can open the inner and outer layers of the fusion protein. Furthermore, when the pH was adjusted back to 8.5, the fusion protein restored its structure and retained its physiological activity.

[0122] 6.3 Removal of insulin-free capsules

[0123] After incubation, the insulin-encapsulated fusion protein solution was transferred to an ultracentrifuge tube for centrifugation. The supernatant was collected and filtered through a 0.22 μm filter membrane. Finally, the filtrate was ultrafiltered, washed, and concentrated. The process was repeated three times to remove unencapsulated insulin, resulting in a high-concentration fusion protein complex containing insulin. 500 μL of this complex was taken as sample 1 for testing.

[0124] 6.4 Characterizing whether insulin packaging was successful

[0125] Insulin concentration in solution was detected using LC-MS (liquid chromatography-mass spectrometry). First, a standard insulin stock solution was prepared. Preliminary mass spectrometry conditions were established, and full scan acquisition was performed to identify the precursor ion, obtaining the mass spectrometry acquisition parameters: daughter ion, fragmentation voltage, and collision energy. Next, isocratic elution was performed using a formic acid / acetonitrile mobile phase to determine the retention characteristics of insulin on a C18 column (300 Å). Gradient separation conditions were determined using isocratic separation characteristics. Finally, an LC-MS method for insulin detection was established. The standard insulin stock solution was diluted (Table 1) to establish a standard curve, as shown in Table 1. Figure 11 A. Take the standard insulin stock solution and the fusion protein complex sample from 6.3 after removing the unencapsulated insulin. Adjust the pH to 12.0, stir in an ice bath for 1 hour, and open the polyhedral protein layer and ferritin layer to release insulin from the complex. Take the supernatant as test sample 2. Use a mass spectrometer to detect the signal values ​​of the insulin stock solution, test sample 1, and test sample 2 (e.g., ...). Figure 11 (B, C, and D in the standard curve) to calculate the actual insulin content.

[0126] Results: After dilution, a standard curve was established using LC-MS for the insulin standard: y = 0.426370x - 0.771168, R0 2 =0.999. The signal values ​​of samples 1 and 2 were detected by mass spectrometry within 0.1~0.2 min. Finally, the actual concentration of insulin in samples 1 and 2 was obtained by standard curve. No insulin concentration was detected in sample 1, while the insulin concentration in sample 2 was 9.38 ng / mL, proving that insulin was encapsulated by the fusion protein. After adjusting the pH to open the fusion protein, insulin was released and detected by mass spectrometry.

[0127] Table 1. Dilution Table of Standard Insulin Stock Solution

[0128]

[0129] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an oral pharmaceutical preparation, characterized in that, The oral drug formulation is prepared from a target molecule and a fusion protein. The fusion protein, from the N-terminus to the C-terminus, includes, in sequence, the polyhedral protein shown in SEQ ID NO: 3, (GGGGS)3, and the human heavy chain ferritin mutant shown in SEQ ID NO:

2. The target molecule is a protein drug, a peptide drug, or a nucleic acid drug. The method for preparing the oral drug formulation includes: cleaving the fusion protein at pH 12.0, mixing it with a target molecule solution, adjusting the pH to 8.5, and assembling the target molecule into the internal cavity of the fusion protein to obtain the oral drug formulation.

2. The method for preparing an oral pharmaceutical formulation according to claim 1, characterized in that, The protein drug is insulin.

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