A human recombinant single-chain antibody, nanoparticles, and nanomedicine composition and their applications

Abstract

This invention relates to the field of antibody drug technology, and more particularly to a human recombinant single-chain antibody, nanoparticles, and nanomedicine compositions and their applications. This invention provides a human recombinant single-chain antibody specifically targeting PHD2, the amino acid sequence of which is shown in SEQ ID NO:2. The human recombinant single-chain antibody obtained by this invention is more stable than INP and exhibits better organ and tissue targeting. Combining this human recombinant single-chain antibody with mesoporous silica nanoparticles and erythrocyte membranes to prepare a nanomedicine composition, this nanomedicine composition has better therapeutic efficacy.

Description

Technical Field

[0001] This invention relates to the field of antibody drug technology, and in particular to a human recombinant single-chain antibody, nanoparticles, and nanomedicine compositions and their applications. Background Technology

[0002] Hypoxia-inducible factor-1 (HIF-1) is an oxygen-sensitive transcriptional activator that specifically binds to the hypoxia response element. HIF-1 is a heterodimer composed of HIF-1α and HIF-1β, with HIF-1β constitutively expressed and distributed in the nucleus, while HIF-1α is mainly distributed in the cytoplasm. Under hypoxic conditions, HIF-1α can be transported to the nucleus and combine with HIF-1β to form the HIF-1 heterodimer; this step is a prerequisite for HIF-1 to function.

[0003] HIF-1 can activate over 100 genes under hypoxic conditions. These genes are involved in various cellular adaptation processes to hypoxia, including angiogenesis, erythropoiesis, energy metabolism, cell proliferation, and cell cycle control. HIF-1 mitigates cell death induced by hypoxia by regulating the expression of these genes. For example, HIF-1 regulation of glycolysis or angiogenesis genes constitutes an early adaptive response to oxidative stress. Secondly, hypoxia and HIF-1 can induce the expression of growth factors such as insulin-like growth factor-2 (IGF-2) and transforming growth factor-α (TGF-α). These growth factors bind to their corresponding receptors, activating intracellular signaling pathways related to cell proliferation and survival. Furthermore, numerous genes involved in angiogenesis have been shown to increase in expression under hypoxic conditions. Therefore, the presence of HIF-1 plays a crucial role in the recovery process from injuries and other diseases.

[0004] However, under normoxic conditions, HIF-1α is continuously degraded and is extremely unstable. This oxygen-dependent degradation is mediated by prolyl hydroxylases (PHDs). Hydroxylation of HIF-1α at prolyl residues 402 or 564 promotes binding to the von Hippel-Lindau protein (VHL), which recruits an E3 ubiquitin-protein ligase complex, thereby promoting the ubiquitination and degradation of HIF-1α. Prolyl hydroxylases include PHD1, PHD2, and PHD3. The biochemical characteristics of PHDs are similar to collagen-proline-4-hydroxylases; they belong to the 2-OG-dependent dioxygenases, utilizing molecular oxygen (O2) and 2-oxoglutarate as co-substrates to hydroxylate two proline residues of HIF-1α, thereby mediating the degradation and inactivation of HIF-1α. Among them, PHD2 is considered the rate-limiting enzyme regulating the prolyl hydroxylation and subsequent ubiquitination and degradation of HIF-1α.

[0005] Studies have shown that HIF-1 is associated with the development of a variety of diseases. PHD inhibitors are currently widely used, but they often lack subtype specificity and have significant toxic side effects. Therefore, there is an urgent need to develop new, highly effective and specific PHD inhibitors.

[0006] Antibodies are an important component of the human immune system. Due to their superior targeting specificity, pharmacokinetic and pharmacodynamic properties, safety and toxicity characteristics, and adaptability through multifunctional engineering, they have become one of the fastest-growing drugs for treating various human diseases, including cancer. Advances in genetic engineering, humanization, transgenic mice, and antibody screening technologies have promoted the development of antibody technology and provided opportunities to construct various small molecule antibodies, such as single-chain variable fragments (scFvs, also known as single-chain antibodies). In medical diagnostics and treatment, antibody fragments are increasingly being used as alternatives to monoclonal whole antibodies. Single-chain antibodies (scFvs) are recombinant molecules composed of the variable regions of the light chain (VL) and heavy chain (VH) of IgG, along with a flexible linker sequence connecting them. Due to their small molecular weight, strong tissue penetration, and high targeting specificity, they have been widely used in disease diagnosis and treatment.

[0007] Although single-chain antibodies are widely used, unmodified single-chain antibodies suffer from drawbacks such as poor organ or tissue targeting and easy clearance, leading to poor therapeutic effects and potential toxicity. Furthermore, single-chain antibodies cannot effectively penetrate cell membranes to act on intracellular target molecules. These issues limit their further clinical application. Therefore, extending their in vivo half-life, achieving tissue or organ-targeted enrichment, and improving the cellular entry efficiency of single-chain antibodies have become the main research directions in the field of single-chain antibodies targeting intracellular molecules.

[0008] Based on this, the present invention is proposed. Summary of the Invention

[0009] The purpose of this invention is to develop a human recombinant single-chain antibody, nanoparticles, and nanomedicine composition and their applications.

[0010] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0011] This invention provides a human recombinant single-chain antibody, the amino acid sequence of which is shown in SEQ ID NO:2.

[0012] The present invention also provides nucleotides encoding the amino acid sequence of the human recombinant single-chain antibody, said nucleotides being shown in SEQ ID NO:1.

[0013] The present invention also provides a primer set for amplifying the nucleotides, the primer set comprising primer pair 1 and primer pair 2;

[0014] The primer pair 1 is shown in SEQ ID NO:3-4;

[0015] Primer pair 2 is shown in SEQ ID NO:5-6.

[0016] The present invention also provides the use of the aforementioned human recombinant single-chain antibody in the preparation of prolyl hydroxylase inhibitors, reagents for activating hypoxia-inducible factor-1, or drugs for protecting and treating liver injury.

[0017] The present invention also provides an expression vector comprising the aforementioned nucleotides.

[0018] The present invention also provides an expression strain comprising the aforementioned nucleotides.

[0019] The present invention also provides the use of the expression vector or the expression strain in the preparation of prolyl hydroxylase inhibitors, reagents for activating hypoxia-inducible factor-1, or drugs for protecting and treating liver injury.

[0020] The present invention also provides a nanoparticle comprising the aforementioned human recombinant single-chain antibody.

[0021] The present invention also provides a nanomedicine composition comprising the aforementioned nanoparticles and a drug carrier.

[0022] The present invention also provides the use of the nanoparticles or the nanomedicine composition described herein in the preparation of prolyl hydroxylase inhibitors, reagents for activating hypoxia-inducible factor-1, or drugs for protecting and treating liver injury.

[0023] This invention provides a human recombinant single-chain antibody, nanoparticles, and a nanomedicine composition, as well as their applications. The human single-chain antibody INP described in this invention is a human single-chain antibody with low immunogenicity, high tissue penetration efficiency, and strong specificity, making it suitable as a therapeutic drug. However, as a large molecule drug, it is difficult for it to effectively enter cells to exert its effect. This invention improves the cellular entry efficiency of INP by conjugating the single-chain antibody INP with a cell-penetrating peptide, successfully constructing a recombinant single-chain antibody IRB and verifying the feasibility of this method. Furthermore, unmodified single-chain antibodies have drawbacks such as poor organ or tissue targeting and easy clearance by in vivo proteases. To utilize this recombinant single-chain antibody for disease treatment, this invention also prepares it into a nanomedicine composition. The nanoparticles in this nanomedicine composition exhibit a size effect during in vivo transport; nanoparticles of different sizes have different retention capacities for different organs or tissues. Controlling the particle size of the nanoparticles can effectively control their organ or tissue targeting, thereby effectively treating diseases. The nanomedicine composition constructed in this invention has a particle size of about 70 nm, which effectively enhances the targeting of human recombinant single-chain antibodies to the liver (nanoparticles of 6-100 nm are more likely to remain in the liver). This nanomedicine composition has good biosafety and long retention time at lesion sites, and can effectively treat diseases. Attached Figure Description

[0024] Figure 1 Preparation of human recombinant single-chain antibody IRB (Figure A shows IRB amplification and expression plasmid identification; Figure B shows IRB expression and purification, where 1 represents protein marker, 2 represents bacterial lysate without IPTG induction, 3 represents bacterial lysate after induction with 1 mM IPTG at 30℃ for 20 h, 4 represents protein sample solution loaded onto column, 5 represents effluent from nickel column loading, 6 represents effluent from binding buffer passing through nickel column, 7 represents effluent from washing nickel column with washing buffer, and 8-13 represent effluent from elution buffer eluting protein).

[0025] Figure 2Characterization of the human recombinant single-chain antibody IRB (A: ELISA identification of INP and IRB (ns represents no significant difference, ** represents P<0.01, the ordinate represents absorbance value at 492nm); B: Western blot identification of IRB, where 1 represents pre-induction cell lysate, 2 represents post-induction cell lysate, 3 represents protein sample, and 4 represents purified target protein; C: ELISA analysis of the binding activity of INP and IRB with recombinant PHD2 (*** represents P<0.001), the ordinate represents absorbance value; D: Affinity curves of INP and IRB with PHD2, the abscissa represents... Concentration, with the ordinate representing absorbance values; E indicates Co-IP detection of the ability of human recombinant single-chain antibody IRB to bind to intracellular PHD2; F indicates MTT assay confirming that INP and IRB are not cytotoxic (# represents no significant difference between the control group (0 μM group) and other experimental groups, with the ordinate representing relative cell viability); G indicates Western blot assay confirming the cell entry efficiency of INP and IRB; H indicates immunofluorescence assay of IRB intracellular localization (red fluorescence represents PHD2, green fluorescence represents IRB, blue fluorescence represents the cell nucleus, and the scale bar is 20 μm); I indicates Western blot assay of IRB in vitro biological activity.

[0026] Figure 3 Construction and characterization of the drug-loaded system coated with erythrocyte membrane (A: particle size and potential of mesoporous silica nanoparticles (MSN), erythrocyte membrane (RM), and erythrocyte membrane-coated mesoporous silica nanoparticles (MSN@RM); B: transmission electron microscopy image of MSN@RM; C: SDS-PAGE image of erythrocyte membrane proteins in the nanoparticles; D: transmission electron microscopy image of MSN; E: EDS analysis of MSN and IRB@MSN and the mass percentage of C, N, O, and S in the two nanoparticles; F: encapsulation efficiency of IRB-loaded MSN (IRB@MSN) and erythrocyte-coated IRB-loaded MSN (IRB@MSN@RM) (vertical axis: encapsulation efficiency %); G: drug loading of IRB@MSN and IRB@MSN@RM (vertical axis: drug loading %)).

[0027] Figure 4The in vitro safety and immune escape of the drug delivery system coated on the erythrocyte membrane were evaluated (A: MTT assay of MSN and MSN@RM cytotoxicity (# represents the control group (0 μM group) with no significant difference compared with other experimental groups (x-axis represents nanoparticle concentration, y-axis represents relative cell viability %)); B: Live and dead cell staining assay of MSN and MSN@RM cytotoxicity (red fluorescence represents dead cells, green cells represent live cells, scale bar is 200 μm); C: Hemolysis assay of MSN and MSN@RM (*** represents the positive control group (Water group) with P < 0.001 compared with other experimental groups (x-axis represents nanoparticle concentration, y-axis represents absorbance at 540 nm); D: Fluorescence assay of macrophage uptake of MSN@RM (red fluorescence represents Rhodamine-labeled nanoparticles, blue fluorescence represents DAPI-labeled cell nuclei, scale bar is 50 μm)).

[0028] Figure 5 The in vivo circulation and biosafety of the drug delivery system encapsulated in erythrocyte membranes are shown in Figure 1 (A represents the blood half-life of MSN and MSN@RM in mice (x-axis represents time, y-axis represents the relative fluorescence intensity of fluorescent nanoparticles in serum); B represents the distribution of MSN and MSN@RM in various tissues and organs of mice (heart, liver, spleen, lungs, and kidneys)); CF represents the levels of ALT (C), AST (D), UA (E), and CRE (F) in mice after drug injection (# represents no significant difference between the control group (Ctrl group) and other experimental groups); G represents the H&E staining images of paraffin sections of various organs and tissues of mice after drug injection (scale bar is 50 μm)).

[0029] Figure 6The therapeutic effect of a red blood cell membrane-encapsulated drug delivery system on APAP-induced acute liver injury was evaluated. (A represents the ALT level in mice injected with the drug at 8h, 24h, 48h, and 72h after administration of the drug to AILI (* indicates P < 0.05 compared to other experimental groups (except Crtl group) at the same time point); B represents the AST level in mice injected with the drug at 8h, 24h, 48h, and 72h after administration of the drug to AILI (* indicates P < 0.05 compared to other experimental groups (except Crtl group) at the same time point, ** indicates P < 0.05 compared to other experimental groups (except Crtl group) at the same time point). Compared with other experimental groups (except Crtl group) at the same time point, the RM group showed P<0.01; C represents H&E stained images of paraffin sections of mouse liver 8h, 24h, 48h and 72h after injection of AILI (scale bar is 50μm); DG represents the levels of MDA(D), GSH(E), CAT(F) and SOD(G) in mice 24h after injection of AILI (ns represents no significant difference between IRB@MSN@RM and Ctrl group, * represents P<0.05 between IRB@MSN@RM group and other experimental groups (except Ctrl group)). Detailed Implementation

[0030] In this invention, the amino acid sequence of the human recombinant single-chain antibody is shown in SEQ ID NO:2, SEQ ID NO:2: EIVMTQSPLSLPVTPGEPASISCRSSQSLLHSNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGLQTPTFGQGTKLTVLGSGGSTITSSDVYHTKLSSSGTQVQLVESGGGVVQPGRSLRLSCAASGFTFSDYGMHWVRQAPGKGLEWVAVTSYDGSDKYYGDSVKGRFTISRDNSKNTLYLQMNSLRAEDMAVYYCAKSGGYNGYGVDYWGQGTLVTVSSEAAAKEAAAKEAAAKKLPVM.

[0031] In the present invention, the nucleotide encoding SEQ ID NO:2 is as shown in SEQ ID NO:1, SEQ ID NO:1: GAAATTGTGATGACGCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAGAGCCGGCCTCCATCTCTTGCAGGTCTAGTCAGTCCCTCCTGCATTCTAATGGATATAACTATTTGGACTGGTACCTGCAGAAGCCAGGGCAGTCTCCACAGCTCCTGATTTATTTGGGTAGCAATCGGGCCTCTGGAGTCCCAGACAGGTTTAGTGGCAGTGGGTCAGGTACTGATTTCACACTGAAAATCAGCAGGGTGGAGGCTGAGGATGTTGGAGTTTATTACTGCATGCAAGGTCTACAAACTCCGACGTTCGGCCAAGGGACCAAGCTCACCGTCCTAGGTTCCGGAGGGTCGACCATAACTTCGTCTGATGTATACCATACGAAGTTATCCTCGAGCGGTACCCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGCTTCACCTTCAGTGACTACGGAATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAGTTACATCATATGATGGATCTGATAAGTACTACGGAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTACCTGCAAATGAACAGCCTGAGAGCCGAGGACATGGCTGTGTACTATTGTGCGAAGTCAGGCGGATATAATGGTTACGGAGTTGACTACTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCAGAGGCTGCAGCTAAGGAGGCTGCTGCTAAGGAGGCTGCTGCTAAGAAACTGCCGGTTATG。

[0032] The primer set for amplifying the nucleotide as described in SEQ ID NO:1 includes primer pair 1 and primer pair 2;

[0033] Primer pair 1 is shown in SEQ ID NO:3-4; primer pair 2 is shown in SEQ ID NO:5-6. Forward primer SEQ ID NO:3: TTGGCGCGCATGCCGAAATTGTGATGACGCAG; Reverse primer SEQ ID NO:4: GCCTCCTTAGCAGCAGCCTCCTTAGCTGCAGCCTCTGAGGAGACAGTG ACCAG; Forward primer SEQ ID NO:5: TTGGCGCGCATGCCGAAATTGTGATGACGCAG; Reverse primer SEQ ID NO:6: CTAGCTAGCCATAACCGGCAGTTTCTTAGCAGCAGCCTCCTTAGCAGCA GCC.

[0034] In this invention, the human recombinant single-chain antibody is constructed based on the human single-chain antibody INP. The human single-chain antibody INP is prepared according to the method disclosed in application number 201711449190.5, patent title "A Human Single-Chain Antibody and Its Application", and the amino acid sequence is shown as SEQ ID NO:1 in 201711449190.5.

[0035] In this invention, the specific anti-PHD2 human recombinant single-chain antibody (IRB) is prepared by adding a rigid linker (EAAAK) ×3 and a cell-penetrating peptide BIP gene to the 3' end of the human single-chain antibody INP gene to obtain the NP-RL-BIP (IRB) gene, which is then expressed and purified in prokaryotic cells in *E. coli* HB2151 to prepare the specific anti-PHD2 human recombinant single-chain antibody. The gene sequence of the cell-penetrating peptide BIP is shown in SEQ ID NO:7, and the amino acid sequence of the cell-penetrating peptide BIP is shown in SEQ ID NO:8.

[0036] SEQ ID NO:7: AAACTGCCGGTTATG;

[0037] SEQ ID NO:8: KLPVM.

[0038] In this invention, when preparing the expression vector, it is preferable to add nucleotide sequences to the pINP vector for construction.

[0039] In this invention, when preparing the expression strain, it is preferable to add nucleotides to Escherichia coli HB2151 for construction.

[0040] In this invention, the nanoparticles include not only human recombinant single-chain antibodies, but also nanoparticles, including metal nanoparticles, oxide nanoparticles, carbon-based nanomaterials, polymer nanomaterials, etc., preferably oxide nanoparticles, and more preferably mesoporous silica nanoparticles.

[0041] In this invention, the drug carrier in the nanomedicine composition includes liposomes, nanomicelles, cell membranes, etc., preferably red blood cell membranes.

[0042] The following detailed description of the solutions provided by the present invention, in conjunction with the embodiments, should not be construed as limiting the scope of protection of the present invention.

[0043] The human single-chain antibody INP described in the examples was prepared according to the method disclosed in the patent application No. 201711449190.5 entitled "A Human Single-Chain Antibody and Its Application".

[0044] Example 1

[0045] Preparation of human recombinant single-chain antibody specifically against PHD2

[0046] I. Experimental Materials

[0047] 1.1 Escherichia coli HB2151 and XL1-Blue were purchased from Beijing Dingguo Biotechnology Co., Ltd.; INP / XL1-Blue and INP / HB2151 strains were prepared and preserved in our laboratory; BSSH II and Nhe I restriction endonucleases were purchased from Promega; T4 DNA ligase and its corresponding buffer were purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0048] 1.2 PCR primers used in this step:

[0049] Primers for PCR amplification of human recombinant single-chain antibodies were synthesized by Shanghai Sangon Biotech Co., Ltd., and the primers included primer pair 1 and primer pair 2.

[0050] Primer pair 1 is shown in SEQ ID NO:3-4; primer pair 2 is shown in SEQ ID NO:5-6.

[0051] II. Specific Implementation Steps

[0052] 1. Construction and identification of prokaryotic expression vector for human recombinant single-chain antibody (IRB)

[0053] To prepare the human recombinant single-chain antibody IRB conjugated with the cell-penetrating peptide BIP and a rigid linker (EAAAK) ×3, a recombinant expression vector for IRB needs to be constructed. First, the recombinant plasmid (pINP) of INP is extracted using an alkaline lysis method. Using the pINP plasmid as a template, a BIP with a rigid linker (RL) at the N-terminus, i.e., RL-BIP, is ligated to the C-terminus of the INP using two rounds of overlapping extension PCR. The PCR reaction program is as follows: (1) 95℃ pre-denaturation for 2 min; (2) 95℃ for 20 s; (3) 60℃ for 20 s; (4) 72℃ for 10 s; (5) 72℃ extension for 5 min; wherein (2)-(4) are performed for 35 cycles. For the specific construction method of the recombinant plasmid (pINP), please refer to the method disclosed in patent application number 201711449190.5, entitled "A Human Single-Chain Antibody and Its Application".

[0054] After digesting the pINP plasmid vector and IRB fragment with BSSH II and Nhe I enzymes, the target fragment was then inserted forward between the BSSH II and Nhe I restriction sites on the pINP plasmid vector using T4 DNA ligase to construct a secretory recombinant IRB prokaryotic expression vector. The ligation product was transformed into E. coli XL1-Blue to construct cloning bacteria. Positive clones were selected, cultured, and the plasmid was extracted, then identified by double enzyme digestion and 1% agarose gel electrophoresis. Figure 1 As shown in Figure A, the secretory prokaryotic expression vector pIRB for IRB was successfully constructed.

[0055] 2. Purification of IRB

[0056] The correctly sequenced secretory prokaryotic expression vector pIRB was transformed into *E. coli* HB2151 to obtain the prokaryotic expression strain IRB / HB2151. The prokaryotic expression strain was induced with IPTG, and soluble expression was achieved using a human recombinant IRB single-chain antibody. The supernatant rich in the target protein was collected. Ammonium sulfate was added, and the precipitate was collected. After dissolving the precipitate and dialysis, the protein solution was obtained. Because the C-terminus of IRB carries a His tag, the obtained solution was subjected to HisTrap HPNi... 2+ The target protein IRB (SEQ ID NO:2, the nucleotide encoding this amino acid is shown in SEQ ID NO:1) was obtained by affinity chromatography purification. The purified IRB was identified by SDS-PAGE gel electrophoresis. Figure 1 As shown in Figure B, the target protein was successfully induced to express, and there was a clear protein band at 31 kDa, consistent with the expected size of IRB, indicating that the human recombinant single-chain antibody IRB specifically against PHD2 was successfully purified.

[0057] Example 2

[0058] Identification and activity characterization of IRB, a human recombinant single-chain antibody specifically against PHD2

[0059] I. Experimental Materials

[0060] Human single-chain antibody INP and human recombinant single-chain antibody IRB prepared in Example 1.

[0061] II. Specific Implementation Steps

[0062] 2.1 ELISA identification of IRB

[0063] Because IRB has a V5 tag at its C-terminus, it was used as an example to identify IRB. The purified single-chain antibody was added to an ELISA plate, coated overnight at 4°C, and then blocked. Mouse anti-V5 monoclonal antibody and rabbit anti-mouse HRP-conjugated IgG were added sequentially for incubation. The success of IRB protein purification was confirmed using an ELISA assay. The results are as follows: Figure 2 As shown in A, Figure 2 Results A showed that the human recombinant single-chain antibody IRB was successfully purified.

[0064] 2.2 Western blot identification of IRB

[0065] The prokaryotic expression strain IRB / HB2151 before induction, the prokaryotic expression strain IRB / HB2151 after IPTG induction, the protein sample solution, and the purified IRB were mixed with loading buffer and boiled. The protein samples were then separated by SDS-PAGE gel electrophoresis. Western blot was used to confirm the successful purification of the IRB protein. The results are shown in Figure 2B, further demonstrating the successful acquisition of a 31 kDa human recombinant single-chain antibody, IRB.

[0066] 2.3 ELISA detection of PHD2 binding ability of IRB

[0067] To ensure the effective function of the recombinant human single-chain antibody, the recombinant human PHD2 protein was expressed and purified (using the purification method disclosed in Example 1 of patent application number 201711449190.5) to obtain purified recombinant human PHD2 protein rhPHD2. The binding ability of the IRB to PHD2 was detected using ELISA. 1–10 μg / mL of recombinant human PHD2 was added to an ELISA plate, coated overnight at 4°C, and then blocked. Subsequently, 100 μL of purified single-chain antibody (10 μg / mL), 100 μL of mouse anti-V5 monoclonal antibody (0.02 μg / mL), and 100 μL of rabbit anti-mouse HRP-conjugated IgG (0.01 μg / mL) were added sequentially. The binding ability of the IRB to PHD2 was then detected using ELISA. The results are as follows: Figure 2 As shown in Figure C, this demonstrates that IRB can effectively bind to PHD2.

[0068] 2.4 Determination of antigen affinity for IRB

[0069] The affinity of IRB for rhPHD2 was tested. 100 μL of serially diluted rhPHD2 was added to each well of an ELISA plate and incubated overnight at 4°C. The plates were then blocked with 3% skim milk powder-PBST. Next, 100 μL of serially diluted purified human recombinant single-chain antibody IRB was added. Then, 100 μL of mouse anti-V5 monoclonal antibody (0.02 μg / mL) and 100 μL of rabbit anti-mouse HRP-conjugated IgG (0.01 μg / mL) were added sequentially. After adding OPD chromogenic buffer and stopping with H2SO4, the readings were read at 492 nm. The affinity constant (K) of the single-chain antibody was determined. aff The formula for calculating K is aff = (n-1) / (n[Ab′]-[Ab]), n = [Ag] / [Ag′], where [Ab] and [Ab′] refer to the antibody concentrations used to achieve half of the maximum absorbance at a wavelength of 492 nm, calculated using a nonlinear regression method, for the pores coated with [Ag] and [Ag′] respectively. The results are as follows: Figure 2 As shown in Figure D, this indicates that IRB can effectively bind PHD2 and has a good affinity for PHD2, with an affinity of 1.69 × 10⁻⁶. 7 M -1 .

[0070] 2.5 Co-IP assay of IRB's ability to bind to intracellular PHD2

[0071] To further verify whether IRB can effectively bind to endogenously expressed PHD2, 100 μg of IRB was added to the extracted cellular proteins of HepG2 cells. Then, 20 μL of Protein A / G agarose beads (SANTACRUZ BIOTECHNOLOGY) pre-incubated with anti-V5 monoclonal antibody (5 μg) was added to the mixture. After incubation overnight, the agarose beads from each group were collected for Western blot analysis. The results are as follows: Figure 2 As shown in Figure E, PHD2 and V5 were detected in the IRB group of the IP, indicating that the IRB can effectively bind intracellular PHD2.

[0072] Example 3

[0073] In vitro biosafety and membrane penetration efficiency of the human recombinant single-chain antibody IRB specifically against PHD2.

[0074] I. Experimental Materials

[0075] Human single-chain antibody INP and human recombinant single-chain antibody IRB prepared in Example 1.

[0076] II. Specific Implementation Steps

[0077] 3.1 MTT assay for IRB cytotoxicity

[0078] The recombinant single-chain antibody INP or IRB was added to RAW264.7 or LO-2 cells at concentrations of 10 μM, 5 μM, 2 μM, 1 μM, and 0.1 μM, respectively. The cytotoxicity of this single-chain antibody was detected using the MTT assay. The cytotoxicity results of this human recombinant single-chain antibody are as follows: Figure 2 As shown in F, INP and IRB at a concentration of 10 μM did not show any cytotoxicity to LO-2 and Raw 264.7 cells. These results indicate that IRB and INP are not cytotoxic to either RAW264.7 or LO2 cells.

[0079] 3.2 Detection of IRB permeation efficiency

[0080] To determine whether the addition of the transmembrane peptide effectively improved the cell entry efficiency of the single-chain antibody INP, INP and IRB were co-incubated with HepG2 cells for 9 hours. Total cellular protein was then extracted and analyzed by Western blot. The results are as follows: Figure 2 As shown in Figure G, the level of IRB in HepG2 cells was significantly higher than that of INP, demonstrating that the addition of the membrane-penetrating peptide BIP effectively improved the membrane-penetrating efficiency of the single-chain antibody INP. To further confirm this result, IRB was co-incubated with HepG2 cells for 12 hours, and the intracellular localization of IRB was detected by immunofluorescence assay. The results are shown in Figure G. Figure 2 As shown in Figure H, IRB effectively entered HepG2 cells and had good co-localization with PHD2.

[0081] 3.3 Intracellular bioactivity analysis of IRB

[0082] To demonstrate that intracellular IRB can exert its biological activity by inhibiting PHD2 function and increasing intracellular HIF-1α levels, HepG2 cells were co-incubated with IRB for 12 h, and total protein was extracted from the HepG2 cells for Western blot analysis. The results are as follows: Figure 2 As shown in Figure I, the PHD2 level in HepG2 cells remained unchanged, but the HIF-1α level in HepG2 cells with added IRB increased significantly, demonstrating that IRB effectively inhibits PHD2 enzyme activity upon entering the cells and has good biological activity.

[0083] Example 4

[0084] Construction and characterization of membrane-camouflaged nanosystems (nanomedicine compositions)

[0085] I. Experimental Materials

[0086] Human single-chain antibody INP and human recombinant single-chain antibody IRB prepared in Example 1.

[0087] II. Specific Implementation Steps

[0088] 4.1 Synthesis of mesoporous silica nanoparticles loaded with single-chain antibodies

[0089] First, mesoporous silica nanoparticles (MSN) were synthesized using a template method: Deionized water was added to a round-bottom flask, followed by the addition of hexadecyltrimethylammonium chloride (CTAC) and triethanolamine (TEA) (in a ratio of deionized water: 60 mL, CTAC: 6 g, TEA: 0.18 g), and the mixture was slowly stirred at 30 °C for 1 h. Then, 20 mL of a cyclohexane-tetraethyl orthosilicate (TEOS) solution (cyclohexane to tetraethyl orthosilicate volume ratio of 2:5) was added dropwise to the above liquid at 30 °C, and the mixture was stirred slowly at 30 °C for 12 h. After stirring, an equal volume of ethanol was added to precipitate the mesoporous silica. The mixture was centrifuged at 12000 rpm for 10 min, and the precipitate was collected. The collected product was refluxed with 100 mL of 1% hydrochloric acid-ethanol solution at 80 °C for 6 h to remove the template agent; this process was repeated three times. The final product was washed three times with ethanol and water and then lyophilized to obtain mesoporous silica nanoparticles (MSN), which were then stored.

[0090] After synthesizing MSN, MSN and IRB were added to PBS to a final concentration of 1 mg / mL. The mixture was then incubated at 4 °C for 12 h by rotation, centrifuged at 12000 rpm for 10 min, and the precipitate was collected to obtain mesoporous silica nanoparticles loaded with IRB, i.e., nanoparticles (IRB@MSN). The precipitate was washed three times with PBS for later use. The preparation method of mesoporous silica nanoparticles loaded with INP (INP@MSN) was the same as that of IRB@MSN.

[0091] 4.2 Extraction of Red Blood Cell Membrane

[0092] Mice were anesthetized and blood was collected via the heart. After centrifugation, the supernatant serum was discarded, and the mice were washed. The precipitate was added to 40 volumes of 0.25×PBS to induce swelling. The precipitate collected after centrifugation was red blood cell membrane, which was ready for use after washing.

[0093] 4.3 Construction of membrane-camouflaged nanosystems (nanomedicine compositions)

[0094] MSN was prepared by mixing MSN and erythrocyte membrane at a mass ratio of 3:1 and then sonicating for 3 minutes.

[0095] After the prepared IRB@MSN was mixed with the erythrocyte membrane at a mass ratio of 3:1, it was sonicated for 3 minutes to obtain IRB@MSN@RM.

[0096] 4.4 Characterization of membrane camouflage nanosystems

[0097] After coating the surface of MSN with erythrocyte membranes using ultrasound, the particle size was characterized using dynamic light scattering analysis, and the results are as follows: Figure 3 As shown in Figure A, the particle size of the nanoparticles increased from 63.57 nm to 77.77 nm after being coated with the erythrocyte membrane. Furthermore, the zeta potential analysis results for the nanoparticle surface potential are as follows... Figure 3 As shown in Figure A, the potential of MSN@RM was found to be similar to that of RM. Simultaneous TEM observation was performed, as shown... Figure 3 As shown in Figure B, the MSNs in the MSN@RM group exhibited a clear membrane coating, indicating that the erythrocyte membrane successfully coated the MSNs. Further analysis of erythrocyte membrane proteins before and after coating was performed using SDS-PAGE, as shown in... Figure 3 As shown in Figure C, it was found that the erythrocyte membrane proteins were well preserved after coating.

[0098] The structure of the mesoporous silicon nanoparticles was observed using transmission electron microscopy (TEM), and the results are as follows: Figure 3 As shown in Figure D, a distinct mesoporous structure can be observed. To verify that MSN can carry IRB, EDS energy dispersive spectroscopy analysis was performed on MSN and IRB@MSN, and the results are shown in Figure D. Figure 3 As shown in Figure E, after co-incubation with IRB, the proportions of C, N, and S elements in the nanoparticles significantly increased, while the proportion of O, an important component of MSN (monolithic nanoparticles), decreased. These results indicate that the MSN was successfully loaded with the protein sample IRB. The coating efficiency and drug loading of the two nanoparticles against single-chain antibodies were measured. Figure 3 F and 3G, where IRB@MSN had a coverage of 37.06% and a drug loading of 27.02%, and IRB@MSN@RM had a coverage of 46.70% and a drug loading of 25.86%.

[0099] Example 5

[0100] Biosafety and Immune Escape of Membrane-Clad Nanosystems (Nanomedicine Compositions) in Vitro I. Experimental Materials

[0101] MSN, MSN@RM, IRB@MSN@RM, INP@MSN@RM.

[0102] II. Specific Implementation Steps

[0103] 5.1 Cytotoxicity Detection of Membrane-Clad Nanosystems

[0104] Nanoparticles at concentrations of 1, 10, 50, and 100 μg / mL were added to LO-2, HepG2, or RAW264.7 cells, respectively. The cytotoxicity of the camouflage system was assessed using the MTT assay. Results are as follows: Figure 4 As shown in Figure A, the camouflage system did not exhibit significant cytotoxicity. This result was further verified using a live-dead cell staining assay, as shown in Figure A. Figure 4 As shown in Figure B, green fluorescence represents live cells, and no red fluorescence representing dead cells is observed in the field of view.

[0105] 5.2 Blood compatibility testing of membrane camouflage nanosystems

[0106] After blood collection, nanoparticles at concentrations of 1, 10, 50, and 100 μg / mL were added to the collected red blood cells. The blood compatibility of the camouflage system was assessed using a hemolysis assay. The results are as follows: Figure 4 As shown in C, the camouflage system has good blood compatibility.

[0107] 5.3 Detection of the Immune Escape Effect of Membrane Camouflage Nanosystems

[0108] CD47 on the erythrocyte membrane can evade macrophage recognition, thereby achieving immune escape and long-term circulation in vivo. Therefore, Rhodamine B was first used to label MSN (the synthesis method of Rhodamine-labeled MSN was based on the MSN synthesis method in Example 4, requiring the addition of 100 μL of Rhodamine B-modified 3-aminopropyltriethoxysilane APTES after the addition of TEOS). RAW264.7 cells were then treated with Rhodamine B-labeled MSN coated on the erythrocyte membrane. The uptake of nanoparticles by RAW264.7 macrophages was observed under a fluorescence microscope. The results are as follows: Figure 4 As shown in Figure D, the uptake of MSN by macrophages was significantly reduced after the erythrocyte membrane was coated, indicating that the camouflage system of the erythrocyte membrane is conducive to immune escape and effectively reduces the clearance of nanoparticles by the immune system.

[0109] Example 6

[0110] Membrane-camouflaged nanosystems (nanomedicine compositions) in vivo targeting and in vivo safety

[0111] I. Experimental Materials

[0112] Single-chain antibody INP, recombinant single-chain antibody IRB, MSN, MSN@RM, INP@MSN, IRB@MSN, INP@MSN@RM, IRB@MSN@RM.

[0113] II. Specific Implementation Steps

[0114] 6.1 In vivo circulation time of membrane-camouflaged nanosystems

[0115] Rhodamine B-labeled MSN and MSN@RM were injected intravenously into mice at a dose of 1 mg / kg. Blood samples were collected from the orbital sinus at 0.5, 1, 2, 3, 4, 7, and 24 hours, and the relative concentration of nanoparticles in the blood was detected using a fluorescence spectrophotometer. The results are shown in Figure 5A. The coating of the erythrocyte membrane prolonged the blood half-life of the nanoparticles.

[0116] 6.2 In vivo targeting of membrane camouflage nanosystems

[0117] Rhodamine B-labeled MSN and MSN@RM were injected intravenously into mice at a dose of 2.5 mg / kg. Heart, liver, spleen, lung, and kidney of the mice were harvested at 1.5, 6, 12, 24, and 48 hours, and the relative number of nanoparticles in each organ was observed using a small animal in vitro imaging system. Results are as follows: Figure 5 As shown in B, the coating of the erythrocyte membrane effectively enhances the accumulation and retention time of the camouflage system in the liver.

[0118] 6.3 In vivo safety of membrane-camouflaged nanosystems

[0119] IRB and each group of nanoparticles were injected intravenously into mice at a dose of 0.8 mg / kg of single-chain antibody (the dose for the MSN@RM group was based on the amount of MSN). PBS injection alone served as a negative control. Blood was collected from the orbital sinus at 24 hours to measure the levels of AST (aspartate aminotransferase), ALT (alanine aminotransferase), UA (uric acid), and CRE (creatinine). The results are as follows: Figure 5 As shown in C-5F, the levels of AST, ALT, UA, and CRE in mice in the IRB and nanoparticle administration groups were not significantly different from those in the control mice. Hearts, livers, spleens, lungs, and kidneys of the mice were harvested at 24 hours for H&E staining, and the results are as follows... Figure 5 As shown in G, no structural changes were observed in any organ of the mice in the drug-treated groups, further demonstrating that the nanoparticles are non-toxic and have good in vivo safety.

[0120] Example 7

[0121] The therapeutic effect of IRB membrane-camouflaged nanosystems (nanomedicine compositions) on acetaminophen (APAP)-induced acute liver injury

[0122] I. Experimental Materials

[0123] Single-chain antibody INP, recombinant single-chain antibody IRB, MSN, MSN@RM, INP@MSN, IRB@MSN, INP@MSN@RM, IRB@MSN@RM.

[0124] II. Specific Implementation Steps

[0125] Eight-week-old male Balb / c mice were selected. The mice were fasted the night before injection, but allowed free access to water to deplete glutathione in their livers. The following day, they were intraperitoneally injected with 250 mg / kg APAP solution. After injection, they were allowed to resume a normal diet, thus establishing an APAP-induced acute liver injury mouse model. IRB and nanoparticles of each group were injected intravenously via the tail vein at a dose of 0.8 mg / kg single-chain antibody (the dose for the MSN@RM group was based on the MSN amount). PBS injection alone served as a negative control. Blood samples were collected from the orbital sinus at 8, 24, 48, and 72 hours to measure ALT and AST levels. Results are as follows: Figure 6 As shown in A and 6B, the IRB@MSN@RM group exhibited significantly lower ALT and AST levels, demonstrating the best therapeutic effect. H&E staining and pathological analysis were performed on the livers of mice treated for 24, 48, and 72 hours, with results as follows: Figure 6 As shown in Figure C, the IRB@MSN@RM group significantly inhibited liver damage, and liver damage was repaired within 48 hours, demonstrating the best therapeutic effect. Furthermore, the 24-hour liver levels of GSH (glutathione), MDA (malondialdehyde), CAT (catalase), and SOD (superoxide dismutase) in each group are as follows: Figure 6 As shown in D-6G, this further demonstrates that the IRB@MSN@RM group significantly improved the oxidative stress state of the liver. These results all indicate that IRB@MSN@MSN has excellent therapeutic effects on APAP-induced acute liver injury.

[0126] As can be seen from the above embodiments, the present invention provides a human recombinant single-chain antibody, nanoparticles, and nanomedicine compositions and their applications. The present invention utilizes cell-penetrating peptides and cell membrane-coated drug delivery systems to improve the organ targeting and cell entry efficiency of the anti-PHD2 human single-chain antibody INP, thereby effectively inhibiting the hydroxylation of HIF-1α by PHD2 and increasing intracellular HIF-1 levels, achieving the treatment of diseases targeting PHD2, such as liver injury. The present invention constructs a nanomedicine composition with a particle size of approximately 70 nm, effectively enhancing the targeting of the single-chain antibody to the liver (nanoparticles of 6–100 nm are easily retained in the liver). This drug composition exhibits good biocompatibility, long liver retention time, and effectively promotes the recovery of liver injury.

[0127] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A human recombinant single-chain antibody, characterized in that, The amino acid sequence of the human recombinant single-chain antibody is shown in SEQ ID NO:

2.

2. The nucleotide encoding the human recombinant single-chain antibody of claim 1, characterized in that, The nucleotide is shown in SEQ ID NO:

1.

3. The use of the human recombinant single-chain antibody according to claim 1 in the preparation of a medicament for treating acute liver injury.

4. An expression carrier, characterized in that, Includes the nucleotides described in claim 2.

5. An expression strain, characterized in that, The expression strain comprises the nucleotides described in claim 2.

6. The use of the expression vector of claim 4 or the expression strain of claim 5 in the preparation of a medicament for treating acute liver injury.

7. A nanoparticle, characterized in that, The nanoparticles include the human recombinant single-chain antibody as described in claim 1.

8. A nanomedicine composition, characterized in that, This includes the nanoparticles described in claim 7 and the drug carrier.

9. The use of the nanoparticles of claim 7 or the nanomedicine composition of claim 8 in the preparation of a medicament for treating acute liver injury.

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