Recombinant fusion protein-polyester nano drug delivery system as well as preparation method and application thereof

Abstract

The invention relates to a nano drug delivery system for delivering at least one drug and a preparation method of the nano drug delivery system. The nano drug delivery system is formed by assembling a recombinant fusion protein and hydrophobic degradable polyester or a derivative thereof with an albumin hydrophobic structural domain in the recombinant fusion protein through weak acting force and loading at least one drug, the medicine is a small molecule medicine used clinically. The hydrophobic degradable polyester or the derivative thereof is selected, different drug molecules and specific recombinant fusion protein are prepared into the nano drug delivery system for targeted delivery of at least one drug, the stability is excellent, the efficiency of targeted delivery of the drug to a tumor site can be improved, the anti-tumor curative effect is enhanced, and toxic and side effects are reduced. The preparation method of the nano drug delivery system is mild and controllable, and has high clinical application potential. The nano drug delivery system delivers at least one drug to different types of tumors in a targeted manner, and can be applied to tumor treatment.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a recombinant fusion protein-polyester nano drug delivery system and a preparation method and application thereof. Background Art

[0002] Nano drug delivery system is a new type of drug preparation that uses nano-carrier technology to deliver small molecule or macromolecular drugs. It can solve the problems of poor water solubility of small molecule drugs, non-specific tissue distribution, poor stability of macromolecular drugs, and less accumulation in lesion sites to a certain extent. A variety of nano drugs based on liposomes, albumin and polymer micelles have been successfully developed for clinical cancer treatment. For example, the doxorubicin liposome (Doxil) approved in 1995 effectively overcomes the cardiotoxicity of monomeric drugs and prolongs the half-life to 2 days. The albumin-bound paclitaxel (Abraxane) approved in 2005 solves the problem of high hypersensitivity reaction of hydrophobic drugs during intravenous injection, and at the same time greatly reduces the use of excipients with large side effects, making it have a higher tolerance dose and faster administration speed. For another example, the paclitaxel polymer micelle (Genexol-PM) approved for marketing in South Korea in 2007 achieves slow and controlled release of drugs, releasing 65% within 24 hours and 95% within 48 hours.

[0003] Although nanomedicines have important application prospects in tumor treatment, they have not significantly improved the survival of patients. A large number of clinical and preclinical studies have shown that approved nanomedicines have significant advantages in improving the solubility of chemotherapy drugs, promoting the circulation cycle and reducing side effects, but the improvement in overall response rate, efficacy and patient survival has been minimal. The reason may be related to the limited drug loading of these nanomedicines, poor in vivo stability, insufficient targeting ability, and the lack of specific accumulation in primary and metastatic tumors, and / or targeted and efficient drug release in tumor tissues / cells. For example, the drug loading of doxorubicin liposomes is 11%, and the large amount of lipid materials used poses a safety hazard; albumin-bound paclitaxel has the characteristics of rapid dissolution and release after entering the blood, in which free paclitaxel is transported by free diffusion, while albumin-bound paclitaxel complexes are transported into the tumor interstitium by binding to albumin receptors on tumor vascular endothelial cells, so they face the clinical problem of high-frequency and high-dose injections that are prone to adverse reactions; although paclitaxel polymer micelles have improved in vivo stability, they lack tumor internalization mechanisms compared to albumin-bound paclitaxel, and have no significant improvement in the treatment of solid tumors. Therefore, combined with the development history of nanomedicines that have been marketed, polymers are an important means to improve the stability of nanomedicines, and increasing the enrichment and internalization of drugs in tumor tissues / cells is expected to become an important strategy to further improve the efficacy of anti-tumor nanomedicines and prolong patient survival.

[0004] Modification of antibodies or their functional fragments is an important strategy to improve the targeting of nanomedicines, effectively improve the efficiency of drug delivery to the target, and enhance the enrichment and internalization of tumor tissues / cells. Among them, the fixation method of chemically bonded antibodies or their functional fragments is widely used and achieves good targeting effects in a variety of drug delivery systems. For example, Professor Gu Zhen's team at Zhejiang University modified trastuzumab on the surface of drug-loaded nanoparticles through amide bonds, which improved the efficiency of drug internalization into human epidermal growth factor receptor 2 (HER2)-positive tumor cells, and confirmed in a gallbladder cancer mouse model that this antibody modification strategy can increase the enrichment of drugs in tumor tissues / cells and exert a more obvious tumor killing function.

[0005] However, we found in our research that due to the limitation of the number of active groups on the surface of nano drug carriers, chemically bonded antibodies or their functional fragments have great limitations in type and quantity; on the other hand, in order to ensure the activity of antibodies, the reaction process is complicated and the reaction conditions are more stringent, and quality control is difficult, which brings great challenges to the clinical transformation of antibody fixation methods such as chemical bonding. If we can use genetic engineering technology to express different types of targeted recombinant fusion proteins, that is, single-chain antibody-albumin recombinant fusion proteins that recognize and bind to different cell surface antigens, and further use nanocarrier technology to selectively assemble recombinant fusion proteins, degradable polyesters and drugs, then construct a stable, high-drug-load recombinant fusion protein-polyester nano drug delivery system to improve the efficiency of drug delivery to the target and the anti-tumor efficacy. Summary of the invention

[0006] Based on this, one of the purposes of the present invention is to provide a recombinant fusion protein-polyester nano drug delivery system for targeted delivery of at least one drug, which can be used to target more than one tumor type, has good stability, and has better therapeutic effects than existing drugs.

[0007] A recombinant fusion protein-polyester nano drug delivery system, which is composed of a recombinant fusion protein and a hydrophobic degradable polyester or its derivatives assembled with the albumin hydrophobic domain in the recombinant fusion protein through weak interaction, and loaded with at least one drug; wherein the nano drug delivery system comprises:

[0008] a) a recombinant fusion protein, comprising an albumin having a hydrophobic domain and a single-chain antibody, wherein the albumin having the hydrophobic domain and the single-chain antibody are directly connected or connected via a peptide linker; the single-chain antibody has the function of recognizing a cell surface antigen but not covalently binding; the cell surface antigen and the single-chain antibody have the same species origin;

[0009] b) a degradable polyester, wherein the degradable polyester is an aliphatic polyester, or an aliphatic polyester modified with polyethylene glycol;

[0010] c) at least one drug, each of which is a small molecule drug used clinically.

[0011] The second object of the present invention is to provide a method for preparing the above-mentioned nano drug delivery system, comprising the following steps:

[0012] (1) mixing the recombinant fusion protein with water or physiological saline at a concentration of 0.5 to 20 mg / mL, preferably 10 to 15 mg / mL, to obtain an aqueous phase;

[0013] (2) mixing the hydrophobic degradable polyester or its derivative with an organic solvent at a concentration of 0.5 to 200 mg / mL, preferably in the range of 10 to 100 mg / mL, to obtain an oil phase A;

[0014] The drug is mixed with an organic solvent at a concentration of 0.5 to 200 mg / mL, preferably 20 to 100 mg / mL, to obtain an oil phase B;

[0015] Mixing the oil phase A with the oil phase B to obtain oil phase C;

[0016] The volume ratio of the oil phase A to the oil phase B is 1:1 to 10:1, preferably 5:1 to 10:1;

[0017] (3) preparing an oil-in-water emulsion from the aqueous phase in step (1) and the oil phase C in step (2), wherein the volume ratio of the aqueous phase to the oil phase is preferably 1:1 to 20:1, preferably 5:1 to 10:1;

[0018] (4) separating and purifying the emulsion to obtain the nano drug delivery system.

[0019] The third object of the present invention is to provide the use of particles assembled by recombinant fusion protein and hydrophobic degradable polyester or its derivatives in the above-mentioned nano drug delivery system through weak interaction with the hydrophobic domain of albumin in the recombinant fusion protein as carriers of targeted nano drugs in preparations for delivering at least one drug, wherein the drug is a small molecule drug for treating tumors.

[0020] The fourth objective of the present invention is to provide the use of the above nano drug delivery system in preparing drugs for treating cancer or autoimmune diseases.

[0021] The present invention has found in the research that the particles assembled by the hydrophobic degradable polyester or its derivatives with the hydrophobic domain of albumin in the recombinant fusion protein through weak forces can be used as delivery carriers of small molecule compound drugs, and are used to prepare a nano drug delivery system for delivering at least one drug. Moreover, by controlling the appropriate dosage, it is possible to obtain particles with appropriate particle size, good stability, and therapeutic activity superior to albumin-carried small molecule drugs for treating cancer or autoimmune diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the plasmid map of pX17.4(+)-αHER2-HSA.

[0023] Figure 2 This is a computational simulation diagram of the recombinant fusion protein αHER2-HSA.

[0024] FIG3 shows SDS-PAGE and Western Blot analysis of purified αHER2-HSA.

[0025] Figure 4 Western Blot analysis of purified αEGFR-HSA.

[0026] FIG5 is an ELISA assay for detecting the affinity of αHER2-HSA and αEGFR-HSA for HER2 and EGFR antigens, respectively.

[0027] Fig. 6A Confocal microscopy images showing the specific binding of αHER2-HSA to breast cancer BT474 or ovarian cancer SKOV3 cells.

[0028] Figure 6B The binding of cells to proteins was determined by flow cytometry.

[0029] Figure 7 Schematic diagram of the preparation of recombinant fusion protein-polyester nanodrug delivery system.

[0030] Fig. 8A Different ratios of recombinant fusion protein and different ratios of paclitaxel and PLGA were used to 105K The diameter size of the nano drug delivery system obtained by preparation.

[0031] Figure 8B The stability of the nano drug delivery system prepared by using different ratios of polyester and paclitaxel.

[0032] Figure 8C This is a schematic diagram of the effects of different concentrations of polyester on the diameter and polydispersity index of nanoparticles in Example 7.

[0033] Figure 9 shows the diameter of the nano drug delivery system prepared using different types of polyester polymers: (A) PLGA 12K Preparation; (B) using PLGA 53K Preparation; (C) using PLLA 7.2K Preparation; (D) using PLLA 36K Preparation; (E) using PLLA 130K preparation.

[0034] Fig.10 The paclitaxel loading rate of the recombinant fusion protein-polyester nano drug delivery system obtained under different preparation formulas.

[0035] Figure 11 is a recombinant fusion protein-polyester nano drug delivery system (A) NP αHER2-HSA / PLGA@PTX and (B)NP αEGFR-HSA / PLGA@Iri+Pab Hydration particle size distribution diagram.

[0036] Fig.12 NPs assembled from PLGA of different molecular weights αHER2-HSA-PLGA@PTX Transmission electron microscopy image of .

[0037] Fig.13 NP HSA-PLGA@PTX Random optical microscopy image of .

[0038] Fig.14 NPs assembled from PLGA of different molecular weights αHER-HSA / PLGA@PTX Plot of hydrated particle size distribution over 72 hours.

[0039] Fig.15 Albumin-bound paclitaxel and NP αHER2-HSA / PLGA@PTX Transmission electron microscopy image of .

[0040] Figure 16 shows NP αHER2-HSA@PTX and NP αHER2-HSA / PLGA@PTX Serum stability analysis of NPs: (A) Signal area of ​​particles larger than 100 nm in particle size standard samples and the proportion of particles larger than 100 nm in 90% FBS without added particles; (B) NPs αHER2-HSA@PTX and NP αHER2-HSA / PLGA@PTX The proportion of particles larger than 100 nm after incubation with serum for different time periods; (C) NP αHER2-HSA@PTX and NP αHER2-HSA / PLGA@PTX The proportion of particles with a size less than 100 nm after incubation with serum for different time periods; (D) NP αHER2-HSA@PTX and NP αHER2-HSA / PLGA@PTX Flow cytometric analysis after incubation with serum for different time periods.

[0041] Fig.17 NP αHER2-HSA / PLGA@PTX Confocal microscopy images after co-incubation with breast cancer BT474 or SKOV3 cells for different time periods.

[0042] Fig.18 NP αHER2-HSA / PLGA@PTX Changes in intracellular paclitaxel concentration after co-incubation with BT474 cells for different time periods.

[0043] Fig.19 NPs assembled from PLGA of different molecular weights αHER2-HSA / PLGA@PTX Cytotoxicity against BT474 cells.

[0044] Fig. 20 NP αHER2-HSA / PLGA12K@PTX Study on the cellular uptake mechanism.

[0045] Fig.21 The in vivo distribution of αHER2-HSA at the animal level.

[0046] Fig. 22 NP αHER2-HSA / PLGA@PTX Figure 3 Body weight changes of treated BT474 tumor-bearing mice.

[0047] Fig.23 NP αHER2-HSA / PLGA@PTX Tumor growth curve, tumor weight and tumor images of treated BT474 tumor-bearing mice. DETAILED DESCRIPTION

[0048] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0049] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0050] To facilitate understanding of the present technology, certain terms and phrases are defined below.

[0051] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0052] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0053] Homology: In biological phylogeny, two or more structures are said to be homologous if they have a common ancestor.

[0054] Antibody affinity refers to the binding strength between the antibody's antigen binding cluster and the antigenic determinant cluster of the antigen, or the binding force between the antibody and the antigen epitope or antigenic determinant cluster. It is essentially a non-covalent force that includes the attraction between amino acids, hydrogen bonds, hydrophobic forces, etc.

[0055] Single-chain antibody (scFv) is an antibody composed of the heavy chain variable region and the light chain variable region of the antibody connected by a short peptide (linker) of 15 to 20 amino acids. It is an artificially synthesized antibody expressed in Escherichia coli using genetic engineering technology. It has only one chain of the complete antibody, which includes a single-chain fragment that recognizes and binds to cell surface antigens.

[0056] Cell surface antigen refers to a class of molecules located on the cell surface that can be recognized by specific antibodies and can be used to identify specific cell subpopulations. It can be a certain cell surface receptor that induces specific effects in cells after interacting with ligands; it can also be a molecule with a certain antigenic determinant cluster that only has a recognition function. Cell surface antigens can be specifically expressed in tumor cells or immune cells, or they can be expressed in tumor cells and other cells at the same time or in immune cells and other cells.

[0057] In one embodiment, the present invention relates to a nano drug delivery system for delivering at least one drug, wherein the recombinant fusion protein comprises an albumin having a hydrophobic region connected to a single-chain antibody via a peptide linker.

[0058] The single-chain antibody of the recombinant fusion protein is consistent with the functional site of the cell surface antigen that is recognized and bound, so the single-chain antibody of the recombinant fusion protein can recognize and bind to the cell surface antigen through non-covalent bonding, and they have the same species source. The nano drug delivery system assembled by the recombinant fusion protein can recognize and bind to the cell surface antigen because the single-chain antibody has affinity with the targeted cell surface antigen and binds in an antigen-antibody manner.

[0059] For example, the cell surface antigen can be a tumor-specific receptor, which recognizes and non-covalently binds to the single-chain antibody of the recombinant fusion protein through the affinity between the antigen and the antibody.

[0060] The single-chain antibody includes but is not limited to: targets CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD70, CD79b, CD276, CTLA-4, HER2, HER3, Netin-4, TROP2, BCMA, EGFR, TissueFactor, FRα, PDL1, PSMA, B7H3, B7H4, Claudin Single chain antibodies against 18.2, FOLR1, c-Met, CD279, ROR1, MSLN, CEACAM5, DDL3, GPRC5D, HLA-DR, VTCN1, CDH3, DR5, MUC1, NT5E, IL2R, ZIP6, FAPα, LIV-1, ITB6, STn, ALPP, ALPPL2, KAGG1, AXL, ADAM9, Napi2b, TROR-2, ARX788, ARX305 and other antibodies.

[0061] In some embodiments, the recombinant fusion protein comprises albumin, a peptide linker and a single-chain antibody, and the albumin fragment includes but is not limited to serum albumin from mouse, bovine, rabbit, chicken and human sources.

[0062] The connecting peptide can be a peptide sequence conventionally used to connect polypeptides, which can connect two polypeptides and fold them naturally into a desired structure. Usually, it is a short peptide with a hydrophobicity and a certain degree of extensibility. The purpose of the present invention is to separate the two fused proteins to alleviate the mutual interference between the two. The peptide linker can be flexible or rigid. In certain embodiments, a flexible peptide linker may be advantageous, which can connect two protein / polypeptide components and maintain their respective activity and function. For example, the peptide linker includes but is not limited to: (GlyGlyGlyGlySer)n and (GluAlaAlaAlaLys)n, where n is an integer, for example, an integer of 1-6.

[0063] Single-chain antibody: Each recombinant fusion protein includes a single-chain antibody that binds to a type of cell surface antigen. Different single-chain antibodies determine different types of recombinant fusion proteins. Single-chain antibodies have fragments that the corresponding antibody recognizes and binds to the corresponding cell surface antigen through affinity.

[0064] The recombinant fusion protein at least has a single-chain antibody and a serum albumin fragment, and can be assembled into a nanosystem with at least one of a hydrophobic degradable polyester and its derivatives and a small molecule drug through weak forces.

[0065] In some preferred embodiments, the recombinant fusion protein comprises the full-length or partial fragment of albumin, a peptide linker and a single-chain antibody, or the above-mentioned protein with substitution, deletion, mutation and / or addition of one or more naturally occurring, non-naturally occurring or modified amino acids.

[0066] In some preferred embodiments, the single-chain antibody fragment is derived from a functional fragment of an antibody, preferably including a light chain variable region (VL) and a heavy chain variable region (VH) of an antibody.

[0067] In some preferred embodiments, the recombinant fusion protein is any one of VL-VH-albumin C-terminus, VL-VH-albumin N-terminus, VH-VL-albumin C-terminus, VH-VL-albumin N-terminus, VL-VH-peptide linker-albumin C-terminus, VL-VH-peptide linker-albumin N-terminus, VH-VL-peptide linker-albumin C-terminus, and VH-VL-peptide linker-albumin N-terminus from N-terminus to C-terminus.

[0068] In some embodiments, the recombinant fusion protein is composed of human serum albumin HSA and human single-chain antibody, or composed of mouse serum albumin MSA and mouse single-chain antibody.

[0069] The sequence of the human serum albumin HSA may be as shown in SEQ ID No.1, the sequence of the human single-chain antibody αHER2 may be as shown in SEQ ID No.2 and SEQ ID No.3, and the sequence of the human single-chain antibody αEGFR may be as shown in SEQ ID No.4.

[0070] In some embodiments of the present invention, the preparation method of the fusion protein comprises the following steps: (a) constructing a recombinant Chinese hamster ovary cell line; (b) inducing the expression of the recombinant fusion protein in its growth medium for 10 days, with the expression level reaching 300-500 mg / L; (c) purifying the protein expressed in step (b).

[0071] Polynucleotides encoding various albumins with hydrophobic domains, such as serum albumin, and polynucleotides encoding single-chain antibodies can be obtained by methods well known in the art, such as PCR, RT-PCR, artificial synthesis, and methods for constructing and screening cDNA libraries. The mRNA or cDNA used as PCR templates and for constructing cDNA libraries can be derived from any tissue, cell, and library containing the corresponding mRNA or cDNA, such as obtained from human fetal liver cDNA libraries. It can also be obtained by artificial synthesis. Codons preferred by the host can be selected during artificial synthesis, which often improves the expression of the product. Polynucleotides encoding IL1RA can be obtained by artificial synthesis. The fusion of polynucleotides encoding serum albumin and polynucleotides encoding single-chain antibodies can be obtained by various methods well known in the art, such as introducing restriction endonuclease recognition sites on both sides of the coding sequence by PCR, generating sticky ends by enzyme cutting, and then connecting the sticky ends with DNA ligase to obtain genes encoding recombinant fusion proteins; recombinant fusion gene fragments can also be obtained by overlap PCR. If necessary, polynucleotides can be introduced on both sides of the gene encoding the recombinant fusion protein of the present invention, and the introduced polynucleotides can have restriction endonuclease recognition sites. The nucleic acid containing the sequence encoding the recombinant fusion protein can be cloned into various expression vectors by methods known in the art. The host expressing the recombinant fusion protein can be yeast, mammalian cells, bacteria, animals, plants, etc. The recombinant fusion protein or polypeptide can exist in the host cell, or it can be secreted from the host, preferably it is secreted from the host. The signal peptide used for secretion is preferably the yeast α-factor signal peptide or the natural serum albumin signal peptide, or analogs of these two signal peptides. More preferably, the signal peptide of natural serum albumin is used, and the expression level of the recombinant fusion protein is higher when the signal peptide is used. The recombinant fusion protein or polypeptide can also be expressed in an intracellular soluble form in mammalian cells without the use of a signal peptide. The nucleic acid encoding the recombinant fusion protein can be inserted into the host chromosome, or exist in the form of a free plasmid.

[0072] The desired nucleic acid can be transformed into host cells by conventional methods, such as electroporation, preparation of competent spheroplasts, etc. Successfully transformed cells, i.e. cells containing the DNA construct of the present invention, can be identified by well-known techniques, such as collecting and lysing the cells, extracting the genome, and then identifying it by PCR, or the protein in the cell culture supernatant or cell lysate can be detected by anti-serum albumin or anti-antibody.

[0073] The recombinant fusion protein of the present invention can be produced by culturing a host containing the DNA construct of the present invention, such as recombinant yeast, recombinant mammalian cells, recombinant bacteria, transgenic plants and animals, etc. The specific culture method can be a shake flask or a bioreactor, etc., preferably a bioreactor during production. The culture medium should be able to provide the substances required for cell (or thalline) growth and product expression, and should contain nitrogen source, carbon source, pH buffer components, etc. The culture medium formula should generally be obtained by experiment according to different culture objects. Cultivation can be divided into two stages, the first stage is mainly used for the growth of cells (or thalline), and the second stage is mainly used for the expression of products.

[0074] After collecting the cell culture medium by centrifugation and concentrating the culture medium volume by a tangential flow device, the recombinant fusion protein can be separated and purified from the cell culture containing the DNA construct of the present invention by various protein separation methods, such as ultrafiltration, liquid chromatography and combinations of these techniques. Liquid chromatography can use gel exclusion, affinity, ion exchange, hydrophobic, reverse phase and other chromatography techniques.

[0075] In some embodiments of the present invention, a recombinant fusion protein-polyester nano drug delivery system for delivering at least one drug is involved, wherein the nano drug delivery system is composed of the above-mentioned at least one drug, at least one recombinant fusion protein and at least one hydrophobic degradable polyester and its derivatives assembled through weak interactions. For each different recombinant fusion protein, the single-chain antibody must be different, and the albumin with hydrophobic regions therein can be different or the same. The construction principle can be found in Figure 7 .

[0076] The drug is a small molecule drug currently used in clinical practice, and also includes new small molecule drugs to be further developed and used in the future, which can be adsorbed and combined with the hydrophobic region of the albumin part and the polyester molecule in the above-mentioned recombinant fusion protein.

[0077] Small molecule drugs include chemically synthesized drugs, such as organic compounds with a molecular weight of less than 1000. The present invention finds that under appropriate conditions, small molecule drugs can target a certain protein molecule or gene fragment of a known cancer at the cellular level, allowing targeted drugs to enter the body for binding, thereby blocking cancer cell signal transduction, killing cancer cells and inhibiting tumor growth, thereby achieving the purpose of treatment, and can be used to treat cardiovascular and cerebrovascular diseases, immune system diseases and other diseases.

[0078] The recombinant fusion protein constructed in the previous study of the present invention has a complete albumin structure and a single-chain antibody fragment. The albumin has a hydrophobic binding site for small molecule drugs, which is assembled and bound through hydrophobic interaction or electrostatic interaction.

[0079] Therefore, after a large number of experimental investigations and verifications, the present invention uses particles assembled with hydrophobic degradable polyester or its derivatives by weak forces and the hydrophobic domain of albumin in the recombinant fusion protein as carriers in a nano drug delivery system for delivering at least one drug, wherein the drug is a small molecule drug for treating tumors. The weak force can be a hydrophobic binding site on albumin with a small molecule drug effect, which is assembled and bound by hydrophobic interaction or electrostatic interaction.

[0080] In some embodiments, the drug is at least one of irinotecan, paclitaxel, doxorubicin, SN38, rapamycin, docetaxel, and palbociclib. The at least one is two, and the weight of the two drugs is any ratio in the range of 1:10 to 10:1, and can be prepared according to actual needs.

[0081] The hydrophobic degradable polyester and its derivatives are currently known degradable biomaterials, and also include new degradable biomaterials to be further developed in the future, which can be intertwined and combined with the hydrophobic region of the albumin part of the above-mentioned recombinant fusion protein and small molecule drugs. The polyester is an aliphatic polyester or its derivatives, or an aliphatic polyester modified with polyethylene glycol or its derivatives.

[0082] In some of the embodiments, the aliphatic polyester is at least one of polylactide, polyglycolide, poly(glycolide-co-lactide) and polycaprolactone; or the polyethylene glycol-modified aliphatic polyester is at least one of polyethylene glycol-modified polylactide, polyethylene glycol-modified polyglycolide, polyethylene glycol-modified poly(glycolide-co-lactide) and polyethylene glycol-modified polycaprolactone.

[0083] In some of the embodiments, the aliphatic polyester is poly(glycolide-co-lactide); the glycolide / lactide ratio of the poly(glycolide-co-lactide) is selected from any ratio within the range of 95 / 5 to 50 / 50; the end group of the poly(glycolide-co-lactide) is at least one of a lipid group, a carboxyl group and a hydroxyl group; the molecular weight range of the poly(glycolide-co-lactide) is 3000 to 1100000 Daltons.

[0084] Preferably, the glycolide / lactide ratio of the poly(glycolide-co-lactide) is any ratio in the range of 75 / 25 to 50 / 50; the terminal group of the poly(glycolide-co-lactide) is a carboxyl group or a hydroxyl group; and the molecular weight range of the poly(glycolide-co-lactide) is 3000 to 105000 Daltons.

[0085] It is preferably 12K Dalton to 130K Dalton, and more preferably 12K Dalton to 105K Dalton.

[0086] In some embodiments, the nano drug delivery system is a nanoparticle with a particle size ranging from 60 to 200 nm, preferably ranging from 80 to 150 nm.

[0087] In some embodiments of the present invention, a method for preparing the above-mentioned nano drug delivery system is provided, comprising the following steps:

[0088] (1) mixing the recombinant fusion protein with water or physiological saline at a concentration of 0.5 to 20 mg / mL, preferably 10 to 15 mg / mL, to obtain an aqueous phase;

[0089] (2) mixing the hydrophobic degradable polyester or its derivative with an organic solvent at a concentration of 0.5 to 200 mg / mL, preferably in the range of 10 to 100 mg / mL, to obtain an oil phase A;

[0090] The drug is mixed with an organic solvent at a concentration of 0.5 to 200 mg / mL, preferably 20 to 100 mg / mL, to obtain an oil phase B;

[0091] Mixing the oil phase A with the oil phase B to obtain oil phase C;

[0092] The volume ratio of the oil phase A to the oil phase B when mixed is 100:1 to 1:100, preferably 1:10 to 10:1, more preferably 1 to 10:1;

[0093] (3) preparing an oil-in-water emulsion from the aqueous phase in step (1) and the oil phase C in step (2), wherein the volume ratio of the aqueous phase to the oil phase is preferably 1 to 20:1, preferably 5 to 10:1;

[0094] (4) separating and purifying the emulsion to obtain the nano drug delivery system.

[0095] In some embodiments, the nanoparticles are prepared without any additional stabilizer.

[0096] In some of these embodiments, the nanoparticles can be separated from free proteins and nanoparticles by at least one of centrifugation, tangential flow dialysis (dialysis under tangential shear force through a tangential flow device) and exclusion chromatography (based on the molecular weight of the nanoparticles and free proteins).

[0097] In some of the embodiments, the method of preparing the water phase and the oil phase into an oil-in-water emulsion comprises ultrasonic emulsification or high-pressure homogenization emulsification or microfluidics.

[0098] In some embodiments, the drug loading percentage of the nano drug delivery system is 1-20% by weight, preferably 5-10% by weight.

[0099] In some embodiments, the affinity of the nano drug delivery system to the corresponding cell surface antigen is 10 -13 ~10 -7 Mole per liter, preferably 10 -10 ~10 -8 Mole per liter.

[0100] In some embodiments, the mass ratio of the drug to the recombinant fusion protein is 0.1:30 to 1:5, preferably 0.1:10 to 1:10.

[0101] The mass ratio of the hydrophobic degradable polyester or its derivative to the recombinant fusion protein is in the range of 1:2.5 to 750; preferably 1:1 to 300; more preferably 1:2.5 to 30, 1:2 to 15, more preferably 1:5 to 10, more preferably 1:5 to 7.5.

[0102] In some embodiments, the mass ratio of the drug to the hydrophobic degradable polyester or its derivative polyester is (100-1):(1-100), preferably the ratio range is (3-1):(1-100), or 1:0.1-20, preferably 1:1-1:10, or the weight ratio of polyester to drug is 1:0.01-1, preferably 1:0.1-1.

[0103] In some embodiments, the volume ratio of the water phase to the oil phase is 1:1 to 20:1, preferably 5:1 to 10:1.

[0104] In some embodiments, the at least one drug delivered is irinotecan and palbociclib. Preferably, the mass ratio of irinotecan to palbociclib is any ratio in the range of 1:10 to 10:1, preferably any ratio in the range of 1:1-5:1.

[0105] In one embodiment of the present invention, the above-mentioned nano drug delivery system is applied to tumor treatment targeted drugs or diagnostic contrast agents.

[0106] In some embodiments, the tumor treatment targeted drug or diagnostic contrast agent is a breast cancer treatment targeted drug or diagnostic contrast agent.

[0107] In some of the embodiments, the nano drug delivery system of the present invention can be assembled from FDA-approved high molecular weight polyester, clinically used small molecule drugs and albumin recombinant fusion protein, and has excellent biocompatibility.

[0108] In some embodiments, the tumor includes but is not limited to acute leukemia, lymphoma, breast cancer, lung cancer, gastric cancer, colon cancer, liver cancer, pancreatic cancer, head and neck malignancies, ovarian cancer, nasopharyngeal cancer, cervical cancer, melanoma, multiple myeloma, sarcoma, bladder cancer, sarcoma, renal cell carcinoma, nervous system tumors, etc.

[0109] The single-chain antibody-albumin recombinant fusion protein of the recombinant fusion protein of the present invention binds to cell surface antigens by antibody-antigen specific recognition, improves the efficiency of targeted drug delivery to the target, enhances the enrichment of the lesion site, and provides a new idea and convenient solution for tumor targeted therapy.

[0110] By combining the unique advantages of antibody targeting function and serum albumin, single-chain antibody-albumin recombinant fusion protein is prepared by genetic engineering technology to improve the targeting of nano-drugs. The preparation method is simple and controllable, and does not require complex fixing methods such as chemical bonding. By selecting hydrophobic degradable polyester or its derivatives, different drug molecules and specific recombinant fusion proteins, a recombinant fusion protein-polyester nano-drug delivery system for targeted delivery of at least one drug is prepared. The hydrophobic degradable polyester or its derivatives are entangled and assembled with the albumin hydrophobic domain in the recombinant fusion protein through weak forces, and have excellent stability. The recombinant fusion protein-polyester nano-drug delivery system can reduce the leakage of free drugs during long circulation in the body, improve the efficiency of targeted drug delivery to the tumor site, enhance the anti-tumor efficacy and reduce toxic side effects. The types and proportions of polyester or its derivatives, drugs and recombinant fusion proteins in the nano-drug delivery system can be adjusted, and at least one drug can be targeted and delivered to different types of tumors to achieve the purpose of increasing efficacy and reducing toxicity. The components of the nano-drug delivery system are simple and safe, the preparation process is mild and controllable, and it has a high potential for clinical application. The present invention is the first to apply a recombinant fusion protein-polyester nano drug delivery system that can deliver at least one drug to different types of tumors in a targeted manner to the treatment or imaging of tumors and autoimmune diseases. The at least one drug may be 2 drugs or 3 drugs.

[0111] In addition, the nano drug delivery system of the present invention can also expose the single-chain antibody outward, thereby retaining the targeting recognition function of the single-chain antibody to the greatest extent.

[0112] Serum albumin is a helical protein containing three homologous α-helical domains, which are gathered together to form an asymmetric heart-shaped structure. Serum albumin has seven hydrophobic domains, which are pocket structures formed by hydrophobic and positively charged groups. Each domain contains two subdomains, and each subdomain is composed of 6 helical structures.

[0113] Human epidermal growth factor receptor 2 (HER2), also known as Neu, ErbB-2, CD340 (cluster of differentiation 340), is a protein encoded by the ERBB2 gene. About 25%-30% of breast cancer patients are affected by HER2, interfering with their prognosis.

[0114] Polylactic acid, also known as polylactide, polylactide,; polylactic acid, (C3H4O2) n It is a polyester polymer obtained by polymerization with lactic acid as the main raw material and is a new type of biodegradable material.

[0115] Poly(DL-lactide-co-glycolide), also known as poly(lactic acid-co-glycolic acid), poly(lactic acid-co-glycolic acid), poly(DL-lactide-co-glycolide, C6H8O4) m (C4H4O4) n It is a new type of biodegradable material made by random polymerization of two monomers, lactic acid and glycolic acid. It is widely used in the pharmaceutical field and is officially included in the pharmacopoeia as a pharmaceutical excipient.

[0116] The present invention is further described in detail below with reference to specific embodiments.

[0117] The related sequences used in the following examples.

[0118] SEQ ID No: 1

[0119] HSA

[0120] DAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVADESAE

[0121] NCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEV

[0122] DVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKAACLLP

[0123] KLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTK

[0124] VHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPAD

[0125] LPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCC

[0126] AAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPT

[0127] LVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLV

[0128] NRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQL

[0129] KAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLSEQ ID No:2

[0130] αHER2 ECD4 scFv-HSA (abbreviated as αHER2-HSA)

[0131] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGCAPKLLIYSASFLYSGVPSR

[0132] FSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGSGGSGGCPPCGGG

[0133] GSEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYT

[0134] RYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGCGTLV

[0135] TVSSGGGGSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKL

[0136] VNEVTEFAKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFL

[0137] QHKDDNPNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAA

[0138] FTECCQAADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFP

[0139] KAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLE

[0140] KSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVL

[0141] LLLRAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNA

[0142] LLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEK

[0143] TPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETTFHADICTLSEKERQIKKQTAL

[0144] VELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLEQKL

[0145] ISEEDLNSAVDHHHHHHH

[0146] SEQ ID No.: 3

[0147] αHER2 ECD2 scFv-HSA

[0148] DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPSRF

[0149] SGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKGGGGSGGGGSGGGGSEV

[0150] QLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGGSIYNQ

[0151] RFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLVTVSSGGG

[0152] GSGGGGSGGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF

[0153] AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDN

[0154] PNLPRLVRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQA

[0155] ADKAACLLPCLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEV

[0156] SKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEV

[0157] ENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTY

[0158] ETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYFQNALLVRYTKK

[0159] VPQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVT

[0160] KCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHK

[0161] PKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLEQKLISEEDLNS

[0162] AVDHHHHHH

[0163] SEQ ID No:4

[0164] αEGFR-HSA

[0165] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPS

[0166] RFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGGGSGGGGSGGGG

[0167] SQVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDY

[0168] NTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSA

[0169] GGGGSDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEFAKTCVA

[0170] DESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRL

[0171] VRPEVDVMCTAFHDNEETFLKKYLYEIARRHPYFYAPELLFFAKRYKAAFTECCQAADKA

[0172] ACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLV

[0173] TDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVEND

[0174] EMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYET

[0175] TLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKV

[0176] PQVSTPTLVEVSRNLGKVGSKCCKHPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTK

[0177] CCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGLHHHHHHHHHH.

[0178] Raw materials and sources used in the examples:

[0179] αHER2-HSA, αEGFR-HSA recombinant fusion protein: expressed by recombinant Chinese hamster ovary cell line (CHO-ZN) and purified by NGC protein purifier. The peptide linker in the recombinant fusion protein is GlyGlyGlyGlySer.

[0180] Polylactic acid PLA 7.2K 、PLA 36K 、PLA 137K , with molecular weights of 7200Da, 36000Da, and 137000Da, respectively, all terminated with ester groups, purchased from Jinan Daigang Biotechnology Co., Ltd.

[0181] Poly(glycolide-co-lactide) PLGA 12K , PLGA 53K , PLGA 105K , with molecular weights of 11700Da, 53000Da, and 105000Da, respectively. The ratio of glycolide:lactide was 75:25, and the end-capping was carboxyl. They were purchased from Nanjing Well Biotechnology Co., Ltd.

[0182] Chloroform: purchased from Guangzhou Chemical Reagent Factory.

[0183] Anhydrous ethanol: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0184] Transmission electron microscope copper grid: purchased from Hyde Venture (Beijing) Biotechnology Co., Ltd.

[0185] Anti-His-tag antibody (HRP, mouse antibody): purchased from Beijing Sino Biological Technology Co., Ltd.

[0186] Anti-HSA antibody (mouse antibody): purchased from Beijing Sino Biological Technology Co., Ltd.

[0187] ELISA colorimetric solution: purchased from Beijing Sino Biological Technology Co., Ltd.

[0188] HER2 antigen: purchased from ACRObiosystem, USA.

[0189] Goat anti-rat IgG HRP antibody: purchased from Beijing Sino Biological Technology Co., Ltd.

[0190] Polystyrene plates used in ELISA were purchased from Corning, USA.

[0191] Cy3-succinimidyl ester: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0192] Cy5-succinimidyl ester: purchased from Xi’an Dianhua Biotechnology Co., Ltd.

[0193] Silica-rhodamine: purchased from Xi'an Dianhua Biotechnology Co., Ltd.

[0194] Acetonitrile, chromatography grade: purchased from Anhui Zesheng Technology Co., Ltd.

[0195] The experimental instruments and models used in the examples are:

[0196] Ultrasonic cell disruptor: VCX130, Sonics, USA.

[0197] Rotary evaporator: RV 10digital V digital display model, IKA, Germany.

[0198] Microchannel reactor: 1300SERIES A2, Corning, USA.

[0199] Nanoparticle size and Zeta potential instrument: Nano ZSE, Malvern, UK.

[0200] Tabletop refrigerated microcentrifuge: Microfuge 20R, Beckman Coulter, USA.

[0201] Transmission electron microscope: Talos L120C, Thermo Fisher Scientific, USA.

[0202] ELISA reader: BioTek, USA.

[0203] Flow cytometer: BD FACSCelesta TM , BD Biosciences, USA

[0204] Confocal microscope: LSM 880, Zeiss, Germany

[0205] Example 1 Design of recombinant fusion protein gene

[0206] The antibody sequence was retrieved from the IMGT database to obtain the selected VH and VL Kappa or Lamda segments of the corresponding antibody, and the VH and VL fragments were connected with (GlyGlyGlyGlySer)3 to obtain the corresponding single-chain antibody peptide sequence; the single-chain antibody was connected to the HSA or MSA peptide sequence without a signal peptide through (GlyGlyGlyGlySer)3 to obtain the corresponding recombinant fusion protein peptide sequence; the peptide sequence was converted into the corresponding nucleotide sequence through the Yunzhou Biological website or other similar websites, and the codons were optimized, and the optimized species was selected as Chinese hamster. The designed sequence was handed over to Sangon for synthesis.

[0207] The sequence of αHER2 scFv-HSA constructed by the above method is shown in SEQ ID No. 2;

[0208] αHER2 constructed by the above method ECD2 The scFv-HSA sequence is shown in SEQ ID No. 3;

[0209] The sequence of αEGFR scFv-MSA constructed by the above method is shown in SEQ ID No. 4;

[0210] Example 2 pX17.4(+)-αHER2 scFv-HSA plasmid transfection into CHO-ZN cells

[0211] Take 30 μg of pX17.4(+)-αHER2-HSA linearized plasmid (such as Figure 1 ), and containing 10 7800μL CHO Fusion medium of CHO-ZN cells were mixed and transferred to the electroporation cup after mixing; the electroporation conditions were set as follows: voltage (V): 280; duration (ms): 20.0; number of pulses: 2.0; pulse interval (s): 0.1; electroporation volume (μL): 800.0. After electroporation, the cells were allowed to stand at room temperature for 5 minutes, and then transferred to a 10mm plate containing 12mLCD CHO FUSION medium (4mM glutamine for cell recovery), and placed in a constant temperature incubator at 37°C and 5% CO2 for static culture; after 48 hours, the medium was replaced with CHO BM03 medium containing 25μM L-methionine sulfoximide, and static culture was carried out for 5 to 7 days; after the cell viability recovered to more than 90%, the cells were transferred to a shake flask for continued pressurized culture, and the supernatant was collected after 7 days of culture.

[0212] Example 3 Purification of recombinant fusion protein

[0213] After connecting the nickel column to the NGC protein purification system, use ultrapure water to balance for 10 column volumes, and then use Binding Buffer to balance for 10 column volumes. The culture supernatant is filtered through a 0.45μm filter membrane and loaded. Wash Buffer (containing low concentration imidazole) is used for 10 column volumes, and then eluted with Elution Buffer (containing high concentration imidazole) for 10 column volumes to obtain the recombinant fusion protein. The eluate is collected and desalted to obtain the purified αHER2 scFv-HSA (abbreviated as αHER2-HSA; unless otherwise specified, other recombinant fusion proteins are abbreviated in the same way) recombinant fusion protein. See the structural simulation diagram of αHER2-HSA for details. Figure 2 , SDS-PAGE, Western-Blot characterization results, see Figure 3A and 3B ; Characterization results of αEGFR-MSA, see Figure 4 .

[0214] Example 4 Affinity Characterization of Recombinant Fusion Protein

[0215] The affinity of the recombinant fusion protein to the corresponding antigen was characterized by ELISA. The human HER2 or mouse EGFR antigen was diluted to 0.5 μg / mL using coating buffer and added to the ELISA plate at 100 μL / well. The plate was incubated overnight at 4°C and washed 3 times with PBST. Blocked with skim milk powder and washed 3 times with PBST. Different concentrations of αHER2-HSA or αEGFR-MSA recombinant fusion protein were added at 100 μL / well, incubated at 37°C for 2 hours, and washed 6 times with PBST. Anti-His Tag (HRP) was used as the secondary antibody, incubated at 37°C for 1 hour, and washed 6 times with PBST. ELISA colorimetric solution was added, 100 μL per well, reacted in the dark for 10 minutes, and the reaction was terminated with 2M sulfuric acid. The absorbance of OD450-OD630 was detected using an enzyme reader. The results are shown in Figure 5. Figure 5A and 5B The affinities of the recombinant fusion proteins are αHER2-HSA and αEGFR-HSA, respectively. The affinities of the recombinant fusion proteins are similar to those of the source antibodies.

[0216] Example 5 Characterization of the binding ability of the recombinant fusion protein to target cells

[0217] Human SKOV3 ovarian cancer cell line, human BT474 breast cancer cell line, and human MDA-MB-231 breast cancer cell line were all from the American Type Culture Collection (ATCC).

[0218] αHER2-HSA or human serum albumin was fluorescently labeled with sulfo-Cy5-NHS; 100,000 SKOV3 cells or BT474 cells expressing HER2 antigen were added to a glass-bottomed culture dish per well, placed in a 37°C incubator overnight to adhere to the wall, and then incubated with fluorescently labeled αHER2-HSA or human serum albumin for 15 minutes; mouse anti-HER2 IgG was used to label HER2 on the cell membrane surface, and anti-mouse IgG FITC was used as a secondary antibody; Hoechst 33342 was used to label the cell nucleus; and the binding of the recombinant fusion protein was observed using a confocal fluorescence microscope. The results are shown in FIG. Fig. 6A As shown, in SKOV3 cells and BT474 cells expressing HER2, αHER2-HSA was able to successfully bind to the cell membrane, while no significant binding of human serum albumin was observed.

[0219] 50,000 SKOV3 cells, BT474 cells or MDA-MB-231 cells were seeded in a 24-well plate and placed in a 37°C incubator overnight to adhere to the wall; 10 μg / mL trastuzumab was added or not added for 15 minutes, and then fluorescently labeled αHER2-HSA or human serum albumin was added for 15 minutes, washed with PBS, and digested with trypsin into single cells. The binding of cells to proteins was determined by flow cytometry. The results are as follows: Figure 6B As shown in the figure, αHER2-HSA has a stronger average fluorescence intensity in SKOV3 cells or BT474 cells expressing HER2, indicating that αHER2-HSA has the ability to bind to HER2 and can target this type of cells, and this binding function can be blocked by the αHER2-derived antibody trastuzumab; while in MDA-MB-231 cells that do not express HER2, αHER2-HSA has similar fluorescence intensity to human serum albumin, indicating that its binding effect is related to HER2 and is specific.

[0220] Example 6 Preparation of recombinant fusion protein-bound paclitaxel

[0221] Take 50 μL of 20 mg / mL paclitaxel chloroform solution, add 5 μL of ethanol solution, and mix well; add 500 μL of αHER2-HSA aqueous solution, and the purified αHER2-HSA is determined by Nanodrop One ultra-micro UV spectrophotometer to prepare an aqueous solution with a protein concentration of 15 mg / mL; ultrasonic emulsification is performed in an ice water bath by an ultrasonic cell disruptor. The specific ultrasonic conditions are as follows: ultrasonic power: 130 W, amplitude: 25%, ultrasonic time: 3 minutes, pause for 2 seconds every 5 seconds of ultrasonication, and the size of the ultrasonic probe is 3 mm. After the ultrasonication is completed, the chloroform is removed by decompression using a rotary evaporator. The rotary evaporation conditions are as follows: speed: 120 rpm; vacuum: 200 mbar, 2 minutes; 100 mbar, 2 minutes; 75 mbar, 1 minute; 50 mbar, 15 minutes. After the rotary evaporation is completed, centrifuge at 3000 g for 5 minutes to remove the unencapsulated paclitaxel. Collect the recombinant fusion protein-bound paclitaxel for later use, recorded as NP αHER2-HSA@PTX The preparation method of the recombinant fusion protein-polyester nano drug delivery system using other different types of fusion proteins to load paclitaxel is referred to the above.

[0222] Example 7

[0223] Preparation of recombinant fusion protein-polyester nano-drug delivery system with different ratios of paclitaxel

[0224] Take 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 105K) chloroform solution in a 5mL centrifuge tube, add 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μL of 20 mg / mL paclitaxel chloroform solution, then add 90, 80, 70, 60, 50, 40, 30, 20, 10, 0 μL of chloroform respectively, so that the total volume of the chloroform solution is 200 μL, and mix well; add 1mL of αHER2-HSA aqueous solution, the protein concentration of the purified αHER2-HSA is determined by Nanodrop One ultra-micro UV spectrophotometer, and the aqueous solution with a protein concentration of 5, 10, 15 mg / mL is prepared; ultrasonic emulsification is performed in an ice water bath by an ultrasonic cell disruptor, and the specific ultrasonic conditions are as follows: ultrasonic power: 130 W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, ultrasonic probe size is 3 mm. After the ultrasonication, the chloroform was removed by decompression using a rotary evaporator. The rotary evaporation conditions were as follows: rotation speed: 120 rpm; vacuum degree: 200 mbar, 2 minutes; 100 mbar, 2 minutes; 75 mbar, 1 minute; 50 mbar, 15 minutes. After the rotary evaporation, the free drug and polyester polymer that were not encapsulated were removed by centrifugation at 3000 g for 5 minutes. Figure 7 The schematic diagram of the preparation process is shown in Figure 2. The diameter of the recombinant fusion protein-polyester nano drug delivery system prepared with different proportions of paclitaxel was measured by a nanoparticle size analyzer. Fig. 8A As shown, within the selected dosage ratio of paclitaxel to recombinant fusion protein of 1:2.5 to 1:75, a nano-scale drug delivery system with a diameter of 100-250nm can be prepared; the preferred dosage ratio range is 1:10 to 1:75, and within this range a nano-drug delivery system with a diameter of 100-150nm can be prepared.

[0225] Screening of proteins at different concentrations

[0226] 1 mL of αHER2-HSA aqueous solution was added, and the protein concentration of the purified αHER2-HSA was determined by Nanodrop One ultra-micro UV spectrophotometer, and aqueous solutions with protein concentrations of 5, 10, and 15 mg / mL were prepared; 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 105K) chloroform solution in a 5mL centrifuge tube, add 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μL of 20mg / mL paclitaxel chloroform solution, then add 90, 80, 70, 60, 50, 40, 30, 20, 10, 0 μL of chloroform respectively, so that the total volume of the chloroform solution is 200 μL, mix well; ultrasonic emulsification is performed by ultrasonic cell disruptor in an ice water bath, and the specific ultrasonic conditions are as follows: ultrasonic power: 130W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, ultrasonic probe size is 3mm. After the ultrasonic is completed, the chloroform is removed by decompression by a rotary evaporator, and the rotary evaporation conditions are as follows: speed: 120rpm; vacuum degree: 200mbar, 2 minutes; 100mbar, 2 minutes; 75mbar, 1 minute; 50mbar, 15 minutes. After the rotary evaporation, the free drug and polyester polymer that were not encapsulated were removed by centrifugation at 3000g for 5 minutes. The diameter of the recombinant fusion protein-polyester nano drug delivery system prepared with different proportions of paclitaxel was measured by a nanoparticle size analyzer. Fig. 8A As shown, at the same paclitaxel to recombinant fusion protein dosage ratio, such as a dosage ratio of 1:12.5, the selection of different concentrations of recombinant fusion protein has a significant effect on the size of the prepared drug delivery system. For example, 5 mg / mL of recombinant fusion protein makes the prepared nano drug delivery system have a larger diameter, among which 10 mg / mL and 15 mg / mL of protein concentrations are preferred conditions.

[0227] Preparation of recombinant fusion protein-polyester nano-drug delivery system with different ratios of paclitaxel and poly (glycolide-co-lactide).

[0228] 100, 100, 100, 90, 80, 75, 50, 25, 20, 10, 5, 1 μL of 20 mg / mL paclitaxel chloroform solution was taken into a 5 mL centrifuge tube, and 1, 2, 5, 10, 20, 25, 50, 75, 80, 90, 100, 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 12K ) chloroform solution of paclitaxel, and then add 100 μL of chloroform to make paclitaxel and PLGA 12K的The mass ratio ranges from 100:1 to 1:100, and the mixture is mixed. 1 mL of αHER2-HSA aqueous solution is added, and the protein concentration of the purified αHER2-HSA is determined by a NanodropOne ultra-micro UV spectrophotometer, and the aqueous solution with a protein concentration of 15 mg / mL is prepared. Ultrasonic emulsification is performed in an ice-water bath by an ultrasonic cell disruptor. The specific ultrasonic conditions are as follows: ultrasonic power: 130 W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, and ultrasonic probe size is 3 mm. After the ultrasonic is completed, the chloroform is removed by decompression by a rotary evaporator. The rotary evaporation conditions are as follows: speed: 120 rpm; vacuum degree: 200 mbar, 2 minutes; 100 mbar, 2 minutes; 75 mbar, 1 minute; 50 mbar, 15 minutes. After the rotary evaporation, the free drug and polyester polymer that were not encapsulated were removed by centrifugation at 3000g for 5 minutes. The diameter of the recombinant fusion protein-polyester nano-drug delivery system prepared with different proportions of paclitaxel was measured by a nanoparticle sizer, and then diluted 20 times and measured again by a nanoparticle sizer. As shown in the figure, the paclitaxel and PLGA were adjusted at the same protein concentration. 12K The nano drug delivery system can be prepared by adding different proportions, and different stability nano drug delivery systems can be obtained. The unstable nano drug delivery system is characterized by a significant decrease in diameter with the dilution of the sample. Figure 8B As shown, the stability of the carrier increases with the decrease of the mass ratio of paclitaxel:PLGA. The preferred mass ratio of paclitaxel:PLGA is 3:1 to 1:100, and more preferably 1:3 to 1:100.

[0229] Screening of polyester with different concentrations

[0230] Take 20 μL of chloroform solution containing 100 mg / mL poly(glycolide-co-lactide) (PLGA12K) and 20 mg / mL paclitaxel in a 5 mL centrifuge tube, add 0, 20, 80, 180, 380, 780, 1980, 3980 μL of chloroform to make the final concentration of PLGA 100, 50, 20, 10, 5, 2.5, 1, 0.5 mg / mL, and the amount of paclitaxel is one fifth of the polymer; then 1 mL of αHER2-HSA aqueous solution, the purified αHER2-HSA was washed with a Nanodrop One ultra-trace UV spectrophotometer was used to determine the protein concentration, and an aqueous solution with a protein concentration of 15 mg / mL was prepared. Except for the experimental groups with polymer concentrations of 100, 50, and 20 mg / mL, the other experimental groups were supplemented with ultrapure water to a water phase: oil phase volume ratio of 5:1; ultrasonic emulsification was performed in an ice water bath using an ultrasonic cell disruptor, and the specific ultrasonic conditions were as follows: ultrasonic power: 130 W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, and ultrasonic probe size was 3 mm. After the ultrasonication was completed, the chloroform was removed by decompression using a rotary evaporator, and the rotary evaporation conditions were as follows: speed: 120 rpm; vacuum: 200 mbar, 2 minutes; 100 mbar, 2 minutes; 75 mbar, 1 minute; 50 mbar, 15 minutes. After the rotary evaporation was completed, 3000 g centrifugation was used for 5 minutes to remove the free drugs and polyester polymers that were not encapsulated. Figure 7 The schematic diagram of the preparation process is shown in Figure 2. The diameter of the recombinant fusion protein-polyester nano drug delivery system prepared with different proportions of paclitaxel was measured by a nanoparticle size analyzer. Figure 8C As shown, when the PLGA concentration is in the range of 0.5-100 mg / mL, the diameter of the prepared nano drug delivery system first decreases gradually and then increases gradually. The appropriate PLGA concentration can obtain a nano drug delivery system with a smaller diameter. When the PLGA concentration is greater than 5 mg / mL, the prepared nanometer polydispersity index is less than 0.3, indicating that the uniformity of its diameter is better. The preferred PLGA concentration range is 10-50 mg / mL. A nano drug delivery system with a smaller diameter and better uniformity can be prepared when the concentration is within this range.

[0231] Example 8 Preparation of recombinant fusion protein-polyester nano drug delivery system of different types of polyester polymers

[0232] Take 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 12K PLGA 53K ) chloroform solution or polylactic acid (PLLA 7.2K or PLLA 36K or PLLA 130K) chloroform solution in a 5mL centrifuge tube, add 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μL of 20 mg / mL paclitaxel chloroform solution, then add 90, 80, 70, 60, 50, 40, 30, 20, 10, 0 μL of chloroform, so that the total volume of the chloroform solution is 200 μL, and mix well; add 1mL of αHER2-HSA aqueous solution, determine the protein concentration of the purified αHER2-HSA by Nanodrop One ultra-micro UV spectrophotometer, and prepare an aqueous solution with a protein concentration of 15 mg / mL; perform ultrasonic emulsification in an ice water bath using an ultrasonic cell disruptor, and the specific ultrasonic conditions are as follows: ultrasonic power: 130 W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, and ultrasonic probe size 3 mm. After the ultrasound was completed, the chloroform was removed by decompression using a rotary evaporator. The rotary evaporation conditions were as follows: speed: 120 rpm; vacuum: 200 mbar, 2 minutes; 100 mbar, 2 minutes; 75 mbar, 1 minute; 50 mbar, 15 minutes. After the rotary evaporation, the free drug and polyester polymer that were not encapsulated were removed by centrifugation at 3000 g for 5 minutes. The diameter of the nano drug delivery system prepared with different formulations was measured by a nanoparticle size analyzer.

[0233] Fig. 9A -E respectively displays PLGA 12K , PLGA 53K ,PLLA 7.2K ,PLLA 36K ,PLLA 130K The diameter of the recombinant fusion protein-polyester nano-drug delivery system prepared by adding different proportions of paclitaxel and different proportions of αHER2-HSA recombinant fusion protein is as follows: the recombinant fusion protein-polyester nano-drug delivery system can be constructed by selecting the above different types of polyester polymers, and the preferred polyester polymer type is PLGA 12K , PLGA 53K , PLGA 105K , preferably the weight ratio of polyester polymer to recombinant fusion protein is 1:5-7.5.

[0234] Example 9 Determination of drug loading efficiency of recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel in different formulations

[0235] Take 1 mL of the purified nano drug delivery system solution in Example 7-8, transfer 10 μL and add 200 μL of acetonitrile solution, vortex for 1 minute, and destroy the particles by ultrasound for 15 minutes to dissolve the encapsulated paclitaxel, transfer to a high-performance liquid chromatography sample bottle, and use a high-performance liquid chromatography-ultraviolet detector to determine the paclitaxel content therein, and the remaining untreated 990 μL particles are freeze-dried and weighed. High-performance liquid chromatography conditions: chromatographic column: XBridge@C185μm 4.6×150mm; mobile phase: acetonitrile / water 70 / 30v / v; flow rate: 0.8mL / min; column temperature: 30°C; detection wavelength: 227nm; sample volume: 10μL. The concentration standard solution is an acetonitrile solution containing 500, 250, 125, 62.5, 31.3, 15.6, 7.8, 3.9, 1.9, and 1.0 μg / mL of paclitaxel. The linear region of the standard concentration sample-peak area is linearly fitted, and the paclitaxel content in the particles is calculated accordingly. The drug loading rate of paclitaxel is then calculated by the following formula. The actual drug loading rate obtained by measurement and calculation is as follows: Fig.10 shown.

[0236] Paclitaxel loading rate = total mass of paclitaxel / total mass of freeze-dried particles × 100%

[0237] Example 10 Preparation of Paclitaxel-Loaded Recombinant Fusion Protein-Polyester Nano-Drug Delivery System

[0238] Take 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 12K PLGA 53K PLGA 105K ) chloroform solution in a 15mL centrifuge tube, add 20μL 20mg / mL paclitaxel chloroform solution, add 80μL chloroform solution, mix well; add 1mL αHER2-HSA aqueous solution, the purified αHER2-HSA is determined by Nanodrop One ultra-micro UV spectrophotometer to prepare an aqueous solution with a protein concentration of 15mg / mL; ultrasonic emulsification is performed by an ultrasonic cell disruptor in an ice water bath, and the specific ultrasonic conditions are as follows: ultrasonic power: 130W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, and ultrasonic probe size is 3mm. After the ultrasonic is completed, the chloroform is removed by decompression by a rotary evaporator, and the rotary evaporation conditions are as follows: speed: 120rpm; vacuum: 200mbar, 2 minutes; 100mbar, 2 minutes; 75mbar, 1 minute; 50mbar, 15 minutes. After the rotary evaporation is completed, 3000g centrifugation is used for 5 minutes to remove the free drugs and polyester polymers that are not encapsulated. Collect the recombinant fusion protein-polyester nano drug delivery system for later use, denoted as NP αHER2-HSA / PLGA12K@PTX NP αHER2-HSA / PLGA53K@PTX NP αHER2-HSA / PLGA105K@PTXThe preparation method of the recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel using other different types of fusion proteins and different types of polyesters is referred to the above.

[0239] Example 11 Preparation of SN38-loaded recombinant fusion protein-polyester nanoparticle drug delivery system

[0240] Take 100 μL of 20 mg / mL poly-L-lactic acid (PLLA 36K ) chloroform solution in a 15mL centrifuge tube, add 10μL 5mg / mL SN38 chloroform solution, add 90μL chloroform solution, mix; add 1mL αHER2-HSA aqueous solution, the purified αHER2-HSA is determined by Nanodrop One ultra-micro UV spectrophotometer to prepare an aqueous solution with a protein concentration of 10mg / mL; ultrasonic emulsification is performed by an ultrasonic cell disruptor in an ice water bath, and the specific ultrasonic conditions are as follows: ultrasonic power: 130W, amplitude: 35%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, and ultrasonic probe size is 3mm. After the ultrasonic is completed, the chloroform is removed by decompression by a rotary evaporator, and the rotary evaporation conditions are as follows: speed: 120rpm; vacuum: 200mbar, 2 minutes; 100mbar, 2 minutes; 75mbar, 1 minute; 50mbar, 15 minutes. After the rotary evaporation is completed, 3000g centrifugation is used for 5 minutes to remove the free drugs and polyester polymers that are not encapsulated. Collect the recombinant fusion protein-polyester nano drug delivery system for use, which is NP αHER2-HSA / PLLA36K@SN38 The preparation method of the recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel using other different types of fusion proteins and different types of polyesters is referred to the above.

[0241] Example 12 Preparation of recombinant fusion protein-polyester nano drug delivery system loaded with doxorubicin

[0242] Doxorubicin hydrochloride was treated with triethylamine to remove the hydrochloride, dried and weighed, and then prepared into a 10 mg / mL solution in chloroform. Take 100 μL of 10 mg / mL poly (L-lactic acid) (PLLA 7.2K) chloroform solution in a 15mL centrifuge tube, add 20μL 10mg / mL SN38 chloroform solution, add 80μL chloroform solution, mix; add 1mL αHER2-HSA aqueous solution, the purified αHER2-HSA is determined by Nanodrop One ultra-micro UV spectrophotometer to prepare an aqueous solution with a protein concentration of 10mg / mL; ultrasonic emulsification is performed by an ultrasonic cell disruptor in an ice water bath, and the specific ultrasonic conditions are as follows: ultrasonic power: 130W, amplitude: 35%, ultrasonic time: 2.5 minutes, ultrasonic 5 seconds pause 2 seconds, and ultrasonic probe size is 2mm. After the ultrasonic is completed, the chloroform is removed by decompression by a rotary evaporator, and the rotary evaporation conditions are as follows: speed: 120rpm; vacuum: 200mbar, 2 minutes; 100mbar, 2 minutes; 75mbar, 1 minute; 50mbar, 15 minutes. After the rotary evaporation is completed, 3000g centrifugation is used for 5 minutes to remove the free drugs and polyester polymers that are not encapsulated. Collect the recombinant fusion protein-polyester nano drug delivery system for use, which is NP αHER2-HSA / PLLA7.2K@DOX The preparation method of the recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel using other different types of fusion proteins and different types of polyesters is referred to the above.

[0243] Example 13 Preparation of multi-targeted recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel

[0244] Take 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 12K、 ) chloroform solution in a 15 mL centrifuge tube, add 20 μL of 20 mg / mL paclitaxel chloroform solution, add 80 μL of chloroform solution, mix well; add 500 μL of αHER2-HSA aqueous solution and 500 μL of αHER2 ECD2 -HSA, which is purified αHER2-HSA and αHER2 ECD2 -HSA protein concentration was determined by Nanodrop One ultra-micro UV spectrophotometer, and an aqueous solution with a protein concentration of 15 mg / mL was prepared; ultrasonic emulsification was performed by an ultrasonic cell disruptor in an ice-water bath, and the specific ultrasonic conditions were as follows: ultrasonic power: 130 W, amplitude: 30%, ultrasonic time: 1.5 minutes, ultrasonic 5 seconds pause 2 seconds, ultrasonic probe size 3 mm. After the ultrasonication was completed, the chloroform was removed by decompression using a rotary evaporator, and the rotary evaporation conditions were as follows: speed: 120 rpm; vacuum: 200 mbar, 2 minutes; 100 mbar, 2 minutes; 75 mbar, 1 minute; 50 mbar, 15 minutes. After the rotary evaporation was completed, centrifugation at 3000 g for 5 minutes was used to remove the unencapsulated free drugs and polyester polymers. The recombinant fusion protein-polyester nano drug delivery system was collected for later use and recorded as NP bi-αHER2-HSA / PLGA12K@PTXThe preparation method of the recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel using other different types of fusion proteins and different types of polyesters is referred to the above.

[0245] Example 14 Preparation of recombinant fusion protein-polyester nano drug delivery system loaded with irinotecan and palbociclib

[0246] Take 100 μL of 20 mg / mL poly(glycolide-co-lactide) (PLGA 6.2K PLGA 12K PLGA 105K ) chloroform solution in a 15mL centrifuge tube, add 10μL 10mg / mL irinotecan chloroform solution and 10μL 10mg / mL palbociclib dimethyl sulfoxide solution, add 80μL chloroform solution, mix well; add 1mL αEGFR-HSA aqueous solution, which is the purified αEGFR-HSA protein concentration determined by Nanodrop One ultra-micro UV spectrophotometer, and prepare a protein concentration of 15mg / mL aqueous solution; ultrasonic emulsification is performed by ultrasonic cell disruptor in an ice water bath, and the specific ultrasonic conditions are as follows: ultrasonic power: 130W, amplitude: 30%, ultrasonic time: 2.5 minutes, ultrasonic 5 seconds pause 2 seconds, ultrasonic probe size is 3mm. After the ultrasonic is completed, the chloroform is removed by decompression by a rotary evaporator, and the rotary evaporation conditions are as follows: speed: 120rpm; vacuum: 200mbar, 2 minutes; 100mbar, 2 minutes; 75mbar, 1 minute; 50mbar, 15 minutes. After the rotary evaporation, the free drug and polyester polymer that were not encapsulated were removed by centrifugation at 3000g for 5 minutes. The recombinant fusion protein-polyester nano drug delivery system was collected for use, which was NP αEGFR-HSA / PLGA12K@Iri+Pab The preparation method of the recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel using other different types of fusion proteins and different types of polyesters is referred to the above.

[0247] Example 15 Characterization of particle size and surface potential of recombinant fusion protein-polyester nano drug delivery system

[0248] Take 200 μL of the recombinant fusion protein-polyester nano drug delivery system prepared in Examples 10-14, and use a nanoparticle size and Zeta potential instrument to measure the hydrated particle size distribution or surface potential of the nano drug delivery system. It can be seen that the size distribution of the prepared nano drug delivery system is in the range of 100-200 nm. Fig.11A The hydrated particle size distribution of the recombinant fusion protein-polyester nano drug delivery system loaded with paclitaxel can be obtained by the method. The average particle size and surface potential of the recombinant fusion protein-polyester nano drug delivery system loaded with irinotecan and palbociclib are as follows Fig. 11BAs shown in the table, the dual-drug loaded nano drug delivery system liquid can be successfully prepared with good size uniformity, which does not change significantly due to the adjustment of the type of loaded drug, indicating that the recombinant fusion protein-polyester nano drug delivery system has a wide range of applicability and can be used to load various types of hydrophobic small molecule drugs.

[0249] Example 16 Morphology and structural characterization of recombinant fusion protein-polyester nano drug delivery system

[0250] Take the purified and resuspended particle solution of Example 10 and dilute it appropriately, take 10 μL and drop it onto a 200-mesh carbon support film copper grid, let it stand for 5 minutes, then suck away the solution, drop 10 μL of 1% phosphotungstic acid solution (pH=5.5) and dye for 30 seconds, then suck away the solution, dry it naturally overnight and observe it under a transmission electron microscope. Fig.12 As shown, NP αHER2-HSA / PLGA12K@PTX and NP αHER2-HSA / PLGA53K@PTX It is nearly spherical.

[0251] HSA was labeled with AF488-succinimidyl ester and PLGA 12K The Cy3-labeled paclitaxel was labeled with silicon-rhodamine, and the nanoparticles with three fluorescent labels were prepared and purified by the method of Example 10. The structure of the recombinant fusion protein-polyester nano drug delivery system was characterized by random optical reconstruction microscopy. Fig.13 As shown, it can be seen that the recombinant fusion protein, polyester polymer and paclitaxel are assembled on the same nanoparticle, which is a main structure of the nanoparticle in the recombinant fusion protein-polyester nano drug delivery system.

[0252] Example 17 Stability of Paclitaxel-Loaded Recombinant Fusion Protein-Polyester Nano-Drug Delivery System in PBS

[0253] Take 8 portions of 10 μL of the purified and resuspended particle solution of Example 10, add 200 μL of 0.01M PBS (pH=7.4) buffered saline solution, and place in a shaker at 37°C. Take out at 0, 1, 2, 4, 8, 24, 48, and 72 hours, respectively, and measure the particle size by nanoparticle size analyzer and Zeta potential analyzer. Fig.14 As shown, within 72 hours, the particle sizes of the three paclitaxel-loaded recombinant fusion protein-polyester nano-drug delivery systems did not change significantly, indicating that the recombinant fusion protein-polyester nano-drug delivery system proposed in the present invention has good stability in PBS buffered saline solution.

[0254] Example 18 Stability of Paclitaxel-Loaded Recombinant Fusion Protein-Polyester Nano-Drug Delivery System in Serum

[0255] To evaluate the superiority of the present invention, we investigated the stability of the recombinant fusion protein-polyester nano drug delivery system in serum. First, we prepared the recombinant fusion protein-bound paclitaxel in Example 6. Fig.15 As shown, it has similar morphological features under transmission electron microscopy to the commercially available albumin-bound nanomedicine (Shijiazhuang Pharmaceutical Group), both of which only contain albumin or recombinant protein and paclitaxel.

[0256] Take 10 μL of the nanoparticles (labeled as NP αHER2-HSA@PTX ) and the nanoparticles in Example 10 (labeled as NP αHER2-HSA / PLGA12K@PTX ) Add 90 μL FBS and place in a shaker at 37°C for 5 minutes or 1 hour. Take 10 μL ultrapure water and add 90 μL FBS as a blank control group. Dilute the incubated nanoparticles 2000 times and measure them using a nanoflow cytometer. Fig.16A As shown in FIG. 1 , the particle size standard was used to obtain the range of nanoparticles with a particle size greater than 100 nm. By comparing the change in the proportion of particles with a particle size greater than 100 nm in the sample after being placed in a 37°C shaker for 5 minutes and 1 hour, as shown in FIG. Fig. 16B As shown, NP αHER2 scFv-HSA@PTX The proportion of particles larger than 100 nm decreased significantly, while NP αHER2-HSA / PLGA12K@PTX The proportion of particles larger than 100 nm did not change significantly, which indicates that the recombinant fusion protein-polyester nano drug delivery system proposed in the present invention has better serum stability. Fig. 16C and 16D As shown, NP αHER2-HSA@PTX The proportion of particles with a diameter less than 100 nm increased, and the average fluorescence intensity also increased, indicating that the particles disintegrated; while NP αHER2-HSA / PLGA12K@PTX The proportion of particles with a diameter less than 100 nm did not change much, and the average fluorescence intensity did not increase significantly, indicating that the nano drug delivery system provided by the present invention is more stable.

[0257] Example 19 Binding and uptake of recombinant fusion protein-polyester nano drug delivery system by tumor cells

[0258] In order to evaluate the ability of the present invention to specifically deliver drugs to target cells, we compared and evaluated the binding and uptake of the nanoparticles in Example 10 with commercially available albumin-bound paclitaxel and the nanoparticles in Example 6 with tumor cells. The albumin-bound paclitaxel or αHER2 scFv-HSA fusion protein was labeled with Cy3-succinimide to observe whether the protein was bound to tumor cells and taken up by tumor cells.

[0259] NP was obtained by using the preparation methods of Examples 6 and 10 respectively for Cy3-labeled αHER2 scFv-HSA recombinant fusion protein.αHER2-HSA@PTX and NP αHER2-HSA / PLGA12K@PTX The albumin-bound paclitaxel and NP were determined by ELISA. αHER2-HSA@PTX and NP αHER2-HSA / PLGA12K@PTX The fluorescence intensity of Cy3 in these three particles was calculated. The amount of NPs corresponding to the same fluorescence intensity was added to SKOV3 cells or BT474 cells, and incubated with them in a 37°C constant temperature incubator for 15 minutes, 1 hour, and 4 hours, and then washed with PBS to remove the NPs that were not bound or taken up. We used the cell membrane dye Cell MaskDeep Red, the lysosomal dye Lysotacker Green DND-26, and the cell nuclear dye Hoechst33342 to mark different parts of the cells. Then we observed them under a confocal fluorescence microscope. Fig.17 As shown in Figure 2, nab-paclitaxel hardly binds to the surface of SKOV3 cells or BT474 cells, and no protein uptake by cells was observed; while NP αHER2-HSA@PTX It can bind to the surface of SKOV3 cells or BT474 cells, and only a small amount is taken up by cells after 4 hours of incubation. Most of them have co-localization signals with the cell membrane, indicating that it binds to the cell surface; NP αHER2-HSA / PLGA12K@PTX After 15 minutes of incubation, the nanoparticles can bind to the surface of SKOV3 cells or BT474 cells, and the lysosomal co-localization signal appears after 1 hour of incubation, indicating that a large number of nanoparticles have been taken up by BT474 cells. Similarly, after 4 hours of incubation, SKOV3 cells also take up a large number of particles. The above results show that the recombinant fusion protein-polyester nano drug delivery system proposed in the present invention can not only effectively bind to the target cells, but also be taken up by the target cells faster and more into the cells, and can achieve the purpose of targeted drug delivery to the target cells.

[0260] Example 20 Determination of drug content taken up by tumor cells in the recombinant fusion protein-polyester nano drug delivery system

[0261] In order to evaluate whether the recombinant fusion protein-polyester nano drug delivery system proposed in the present invention can successfully deliver drugs into tumor cells, we used liquid chromatography-mass spectrometry to quantitatively determine the intracellular paclitaxel content over time.

[0262] 100,000 SKOV3 cells were seeded in each well of a 24-well plate. After 24 hours, albumin-bound paclitaxel, NP-containing paclitaxel, and 1 μg / mL paclitaxel were added. αHER2-HSA@PTX (prepared by the method of Example 6), NP HSA / PLGA12K@PTX (prepared by the method of Example 10) and NP αHER2-HSA / PLGA12K@PTX(Prepared by the method of Example 10) were treated for 15 minutes, 30 minutes, 4 hours and 8 hours, a total of 4 time points, and each NPs contained 3 replicates at each time point; after treatment, the medium containing paclitaxel was aspirated and PBS was added for washing 3 times; 100 μL of RIPA lysis solution was added to each well and lysed on ice for 30 minutes; the lysate was transferred to a 1.5 mL centrifuge tube, and then centrifuged at 4°C and 12000 rpm for 30 minutes; the supernatant was taken for BCA quantitative determination of protein concentration, and another 40 μL of supernatant was added with 10 μL of paclitaxel-D5 internal standard solution (200 ng / mL acetonitrile solution containing paclitaxel-D5) and 150 μL of acetonitrile, vortexed for 3 minutes, sonicated for 15 minutes, and then centrifuged at 12000 rpm for 10 minutes, 100 μL of supernatant was transferred to a sample bottle, and the paclitaxel content was determined by liquid chromatography-mass spectrometry.

[0263] Take 1 mg / mL acetonitrile solution of paclitaxel standard, add acetonitrile to dilute and prepare paclitaxel working solutions containing 20, 16, 8, 4, 2, 1, 0.6, 0.2, and 0.1 μg / mL of paclitaxel standard, respectively; take 40 μL blank matrix (supernatant of SKOV3 cell lysate without drug treatment), add 10 μL paclitaxel working solution of different concentrations, add 10 μL paclitaxel-D5 internal standard solution and 150 μL acetonitrile, vortex for 3 minutes, ultrasonicate for 15 minutes, then centrifuge at 12000 rpm for 10 minutes, take 100 μL supernatant and transfer it to a sample bottle, use liquid chromatography-mass spectrometry to determine the peak area, fit a linear curve within the linear range as the ratio of the peak area to the internal standard peak area, and calculate the paclitaxel concentration through the curve.

[0264] The amount of paclitaxel in cells treated with different NPs at different time points was divided by the protein content to calculate the amount of paclitaxel contained in each μg of protein, and the amount of paclitaxel taken up by tumor cells at different time points was further compared. Fig.18 As shown, compared with other nanoparticles, the NP αHER2-HSA / PLGA12K@PTX The highest paclitaxel uptake was detected after 8 hours of incubation with SKOV3, and the intracellular paclitaxel concentration exceeded that of NPs without HER2 targeting function after 30 minutes of incubation. HSA / PLGA12K@PTX ; Albumin-bound paclitaxel and NP αHER2-HSA@PTX The reason why more paclitaxel was taken up in the first 4 hours was that the two nanoparticles were easy to disintegrate, and the paclitaxel loaded in them was released in a free form and diffused into the cells passively; albumin-bound paclitaxel and NP αHER2-HSA@PTX There was no significant difference in the uptake of paclitaxel, indicating that this part of the drug was not delivered to the target cells through targeting. Therefore, the drug in the recombinant fusion protein-bound nanomedicine that does not contain polyester components may not be able to achieve targeted drug delivery through recombinant fusion proteins.

[0265] Example 21 Killing effect of recombinant fusion protein-polyester nano drug delivery system on target cells

[0266] BT474 cells were seeded in 96-well plates at 15,000 cells / well. After 24 hours, albumin-bound paclitaxel, NP- αHER2-HSA / PLGA12K@PTX (prepared by the method of Example 10), NP αHER2-HSA / PLGA53K@PTX (prepared by the method of Example 10) and NP αHER2-HSA / PLGA105K@PTX (prepared by the method of Example 10), after incubation for 1 hour, the drug was washed off and replaced with a medium without paclitaxel, followed by continued incubation for 72 hours in an incubator at 37°C containing 5% CO2; 10 μL CCK-8 was added, and after continued incubation for 2 hours, the absorbance at 450nm and 630nm was measured by an ELISA instrument, and the relative cell survival rate was calculated after deducting the absorbance of the blank control group, so as to evaluate the killing ability of the nanoparticles on the target cells. Fig.19 As shown, NP αHER2-HSA / PLGA12K@PTX NP αHER2-HSA / PLGA53K@PTX and NP αHER2-HSA / PLGA105K@PTX Both showed stronger target cell killing effects than albumin-bound paclitaxel.

[0267] Example 22 Mechanism of endocytosis of the recombinant fusion protein-polyester nano drug delivery system by target cells

[0268] BT474 cells were seeded in 24-well plates, with 100,000 cells per well. After 24 hours of seeding, amiloride, β-cyclodextrin, chlorpromazine, deoxyglucose, and wortmannin were added for 1 hour, and then NPs containing the same molar amount of protein were added. HSA / PLGA12K@PTX (prepared by the method of Example 10) and NP αHER2-HSA / PLGA12K@PTX (prepared by the method of Example 10) incubated in a 37°C constant temperature incubator for 4 hours; then washed with PBS 3 times to remove the NPs that were not internalized; BT474 cells were digested, fixed and perforated, and mouse anti-HSA-FITC IgG antibody was added, incubated at 4°C for 30 minutes, and then detected by flow cytometry. Fig. 20 As shown, NP HSA / PLGA12K@PTX and NP αHER2-HSA / PLGA12K@PTX The two NPs were internalized by BT474 cells through different mechanisms. αHER2-HSA / PLGA12K@PTX It is mainly internalized by cells through the clathrin-mediated uptake pathway.

[0269] Example 23 In vivo targeting ability of recombinant fusion protein

[0270] SPF female NOD-Scid mice, 4 weeks old, were purchased from Guangdong Yaokang Biotechnology Co., Ltd. The mice were raised in the Experimental Animal Center of South China University of Technology, and the animal experiment procedures followed the relevant provisions of the Regulations on the Management of Experimental Animals of South China University of Technology. BT474 cells were inoculated orthotopically in the second mammary gland of mice to construct a BT474 orthotopic breast cancer tumor model. When the tumor volume grew to 200-300mm 3 The drug was administered via tail vein.

[0271] The αHER2-HSA purified in Example 3 was labeled with Cy5, and human serum albumin was labeled in the same way. The fluorescence intensity was measured by an ELISA instrument. The protein with the same fluorescence intensity was injected into mice through the tail vein. The distribution of the protein in the tumor site was observed by a small animal in vivo imaging device at different time points. Fig.21 As shown, αHER2-HSA has more tumor enrichment than human serum albumin 4 hours after administration, and its residence time in the tumor site is as long as 72 hours, while human serum albumin is gradually metabolized and cleared 8 hours after administration. This result shows that the recombinant fusion protein constructed by the present invention can effectively target the tumor site and has the ability to achieve targeted drug delivery.

[0272] Example 24 Evaluation of the in vivo effectiveness of the recombinant fusion protein-polyester nano drug delivery system

[0273] SPF female NOD-Scid mice, 4 weeks old, were purchased from Guangdong Yaokang Biotechnology Co., Ltd. The mice were raised in the Experimental Animal Center of South China University of Technology, and the animal experiment procedures followed the relevant provisions of the Regulations on the Management of Experimental Animals of South China University of Technology. BT474 cells were inoculated orthotopically in the second mammary gland of mice to construct a BT474 orthotopic breast cancer tumor model. When the tumor volume grew to 80-100 mm 3 Then the drug was administered through the tail vein.

[0274] Twenty-four mice inoculated with BT474 tumors were randomly divided into four groups and injected with PBS or 5 mg / kg of albumin-bound paclitaxel, NP- αHER2-HSA / PLGA12K@PTX and NP αHER2-HSA / PLGA105K@PTX ; Among them, albumin-bound paclitaxel comes from Shijiazhuang Pharmaceutical Group, NP αHER2-HSA / PLGA12K@PTX and NP αHER2-HSA / PLGA105K@PTX Prepared by the method of Example 10. The drug was administered once every 3 days, for a total of 6 times; during the treatment, the weight change of the mice was weighed by an analytical balance and the size of the mouse tumor was measured by a vernier caliper, and the tumor volume was calculated by the following formula; after the treatment experiment, the weight of the mouse tumor was weighed and photographed.

[0275] Tumor volume (mm 3) = 0.5 × tumor length (mm) × tumor width 2 (mm 2 )

[0276] Fig. 22 Shows the changes in mouse weight during treatment, NP αHER2-HSA / PLGA12K@PTX and NP αHER2-HSA / PLGA105K@PTX No significant weight loss occurred after treatment, indicating that the components of the recombinant fusion protein-polyester nano-drug delivery system had no obvious toxicity; Fig.23 The growth curves and tumor weights of the tumors in the different treatment groups were shown. The results showed that NP αHER2-HSA / PLGA12K@PTX It has a better therapeutic effect than albumin-bound paclitaxel because NP αHER2-HSA / PLGA12K@PTX It can deliver more paclitaxel to the tumor site, achieving the targeted delivery of chemotherapy drugs.

[0277] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0278] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nano drug delivery system for delivering at least one drug, characterized in that: It is prepared by assembling the albumin hydrophobic domain in the recombinant fusion protein with the hydrophobic degradable polyester or its derivatives through weak interaction and loading at least one drug; wherein the nano drug delivery system comprises: a) a recombinant fusion protein, which is formed by directly linking albumin and a single-chain antibody or linking them through a peptide linker; the single-chain antibody has the function of recognizing and non-covalently binding to a cell surface antigen targeted by the antibody; the cell surface antigen and the single-chain antibody have the same species origin; b) a degradable polyester or a derivative thereof, wherein the degradable polyester is an aliphatic polyester or an aliphatic polyester modified with polyethylene glycol; c) at least one drug, each of which is a small molecule chemical drug.

2. The nano drug delivery system according to claim 1, characterized in that: The albumin is any one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, and chicken ovalbumin.

3. The nano drug delivery system according to claim 1, characterized in that: The single-chain antibody is selected from any one of the following: targeting CD3, CD19, CD20, CD22, CD25, CD30, CD33, CD37, CD38, CD70, CD79b, CD276, CTLA-4, HER2, HER3, Netin-4, TROP2, BCMA, EGFR, Tissue Single chain antibodies against Factor, FRα, PDL1, PSMA, B7H3, B7H4, Claudin18.2, FOLR1, c-Met, CD279, ROR1, MSLN, CEACAM5, DDL3, GPRC5D, HLA-DR, VTCN1, CDH3, DR5, MUC1, NT5E, IL2R, ZIP6, FAPα, LIV-1, ITB6, STn, ALPP, ALPPL2, KAGG1, AXL, ADAM9, Napi2b, TROR-2, ARX788, and ARX305.

4. The nano drug delivery system according to claim 1, characterized in that: The drug is selected from at least one of the following: irinotecan, paclitaxel, doxorubicin, SN38, rapamycin, docetaxel, and palbociclib.

5. The nano drug delivery system according to claim 1, characterized in that: The components of the recombinant fusion protein are connected via a peptide linker, the residue of the peptide linker is at least one of (GlyGlyGlyGlySer)n and (GluAlaAlaAlaLys)n, where n is any integer, preferably n is an integer of 1-6.

6. The nano drug delivery system according to claim 1, characterized in that: The single-chain antibody is derived from a functional fragment of an antibody, and preferably comprises at least one light chain variable region (VL) and / or heavy chain variable region (VH) of an antibody.

7. The nano drug delivery system according to claim 6, characterized in that: The recombinant fusion protein is any one of VL-VH-albumin C-terminus, VL-VH-albumin N-terminus, VH-VL-albumin C-terminus, VH-VL-albumin N-terminus, VL-VH-peptide linker-albumin C-terminus, VL-VH-peptide linker-albumin N-terminus, VH-VL-peptide linker-albumin C-terminus, and VH-VL-peptide linker-albumin N-terminus from N-terminus to C-terminus.

8. The nano drug delivery system according to claim 7, characterized in that: The recombinant fusion protein comprises serum albumin, a peptide linker and a single-chain antibody from the N-terminus to the C-terminus.

9. The nano drug delivery system according to claim 1, characterized in that: The degradable polyester is selected from any one of the following: polylactide, polyglycolide, poly(glycolide-co-lactide) and polycaprolactone.

10. The nano drug delivery system according to claim 9, characterized in that: The degradable polyester is poly(glycolide-co-lactide); preferably, the glycolide / lactide ratio of the poly(glycolide-co-lactide) is 95 / 5 to 50 / 50; the end group of the poly(glycolide-co-lactide) is at least one of an ester group, a carboxyl group and a hydroxyl group; the molecular weight range of the poly(glycolide-co-lactide) is 3000 Daltons to 1100000 Daltons.

11. The nano drug delivery system according to claim 10, characterized in that: The glycolide / lactide ratio of the poly(glycolide-co-lactide) is 75 / 25 to 50 / 50, the terminal group of the poly(glycolide-co-lactide) is a carboxyl group or a hydroxyl group, and the molecular weight ranges from 3000 Dalton to 105000 Dalton, more preferably from 12K Dalton to 130K Dalton, and more preferably from 12K Dalton to 105K Dalton.

12. The nano drug delivery system according to any one of claims 1 to 11, characterized in that: The mass ratio of the drug to the hydrophobic degradable polyester or its derivative during preparation is in the range of (100-1):(1-100), preferably in the range of (3-1):(1-100), preferably 1:3-100, more preferably 1:(1-10).

13. The nano drug delivery system according to any one of claims 1 to 11, characterized in that: The mass ratio of the drug to the recombinant fusion protein during preparation is 1:2.5-750, preferably 1:5-75, preferably 1:10-75, and more preferably 1:7.5-37.

5.

14. The nano drug delivery system according to claim 1, characterized in that: The nano drug delivery system contains nanoparticles composed of recombinant fusion protein, hydrophobic degradable polyester or its derivative and the drug, and the particle size ranges from 60 to 200 nm, preferably from 80 to 150 nm.

15. The nano drug delivery system according to claim 1, characterized in that: The mass percentage of the drug loading of the nano drug delivery system is 1-20%, preferably in the range of 5-10%.

16. Use of particles in which the hydrophobic degradable polyester or its derivatives as claimed in claim 1 are assembled with the hydrophobic domain of the albumin in the recombinant fusion protein as claimed in claim 1 through weak forces as carriers in the preparation of a nano drug delivery system for delivering at least one drug, wherein the drug is a small molecule compound drug for treating tumors.

17. A method for preparing the nano drug delivery system according to claim 1, characterized in that: The following steps are involved: (1) mixing the recombinant fusion protein with water or physiological saline at a concentration of 0.5 to 20 mg / mL, preferably 10 to 15 mg / mL, to obtain an aqueous phase; (2) mixing the hydrophobic degradable polyester or its derivative with an organic solvent at a concentration of 0.5 to 200 mg / mL, preferably in the range of 10 to 100 mg / mL, to obtain an oil phase A; The drug is mixed with an organic solvent at a concentration of 0.5 to 200 mg / mL, preferably 20 to 100 mg / mL, to obtain an oil phase B; Mixing the oil phase A with the oil phase B to obtain oil phase C; The volume ratio of the oil phase A to the oil phase B when mixed is 100:1 to 1:100, preferably 1:10 to 10:1, more preferably 1 to 10:1; (3) preparing an oil-in-water emulsion from the aqueous phase in step (1) and the oil phase C in step (2), wherein the volume ratio of the aqueous phase to the oil phase is preferably 1 to 20:1, preferably 5 to 10:1; (4) separating and purifying the emulsion to obtain the nano drug delivery system.

18. The method for preparing the nano drug delivery system according to claim 17, characterized in that: The mass ratio of the hydrophobic degradable polyester or its derivative to the recombinant fusion protein is in the range of 1:2.5 to 750; preferably, the weight ratio of the hydrophobic degradable polyester or its derivative to the recombinant fusion protein is 1:2.5 to 30, more preferably 1:5 to 7.

5.

19. The method for preparing the nano drug delivery system according to claim 17, characterized in that: The organic solvent is at least one of chloroform, dichloromethane, ethyl acetate, methanol and acetonitrile.

20. Use of the nano drug delivery system according to any one of claims 1 to 15 in the preparation of drugs for treating cancer or autoimmune diseases.

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