Protein-polyester-lipid-based nucleic acid delivery system, preparation method therefor, and use thereof

Abstract

Provided are a protein / polyester / lipid-nucleic acid delivery system for delivering a nucleic acid drug, a preparation method therefor, and use thereof. The system is prepared from an aqueous phase comprising a nucleic acid and a protein, and an organic phase comprising a hydrophobic degradable aliphatic polyester and a derivative thereof, and an ionizable lipid and / or a cationic lipid material. The preparation method comprises mixing an aqueous phase and an organic phase by using a single emulsification technology or a high-pressure homogenization technology to obtain a protein nanoassembly for targeted nucleic acid delivery. The protein is albumin or a single-chain antibody-albumin fusion protein. The stable protein nanoassembly is applied to a targeted nucleic acid delivery platform, and is applied to a nucleic acid treatment drug for tumors, autoimmune diseases, or inflammatory diseases.

Description

Protein-polyester-lipid-based nucleic acid delivery systems, their preparation methods and applications

[0001] This invention claims priority to Chinese Patent Application No. 2024114180906, filed on October 11, 2024, entitled “Nucleic Acid Delivery System of Protein-Polyester and its Preparation Method and Application”, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This invention relates to the field of pharmaceutical technology, specifically to a protein / polyester / lipid-nucleic acid delivery system for delivering nucleic acid molecules, its preparation method, and its applications. Background Technology

[0003] Nucleic acid drugs (short-chain nucleic acids such as small interfering RNA (siRNA), antisense oligonucleotides (ASO), and microRNA (miRNA), and long-chain nucleic acids such as messenger RNA (mRNA), circular RNA, and self-replicating RNA) have enormous application potential as therapeutic agents. They can achieve sustained or even curative effects by intervening in gene expression, overcoming the difficulties of small molecule drugs and antibody drugs for many disease targets. They are applicable to diseases such as cancer, genetic diseases, infectious diseases, and viral infections. Nucleic acid molecules are easily degraded by nucleases in serum and have difficulty penetrating cell membranes. Therefore, they are highly dependent on the protection and delivery capabilities of the delivery carrier. The delivery system is one of the key factors for the efficacy of nucleic acid drugs. Currently, the most widely used nucleic acid delivery carrier in preclinical and clinical research is lipid nanoparticles (LNPs). Ionizable lipids, cholesterol, auxiliary lipids, and PEGylated lipids together form a cell membrane-like structure, which can encapsulate nucleic acid drugs to prevent nuclease degradation and help nucleic acids escape from the endosomes into the cytoplasm to intervene in protein expression. Lipoproteins (LNPs), composed of various lipids, specifically adsorb onto lipoproteins, immunoglobulins, and coagulation factors in the blood, forming a protein corona. This protein corona alters the surface physicochemical properties of the particles, playing a crucial role in their biodistribution and phagocytosis. Apolipoprotein E (ApoE), a component of the protein corona, is a major factor influencing LNP delivery. After ApoE adsorption, LNPs are readily phagocytosed by hepatocytes that highly express low-density lipoprotein receptors (LDLR). Therefore, when LNPs are administered intravenously, the particles tend to accumulate in the liver. Long-term accumulation of LNPs in organs such as the liver and spleen can lead to chronic toxicity (such as lipid metabolism abnormalities). This limitation restricts the dosage and frequency of LNP administration, thereby limiting the indications and therapeutic window for nucleic acid drugs in disease treatment. Currently, only three drugs using nanocarriers as nucleic acid drug delivery carriers have been approved for marketing, all of which use LNPs (Alnylam's siRNA drug, Moderna's COVID-19 mRNA vaccine, and Pfizer / BioNTech's). However, these technologies have significant drawbacks in practical applications. The main challenges in using nanocarriers for nucleic acid drug delivery currently lie in delivery efficiency, safety, stability, and production and preservation processes.

[0004] First, the natural accumulation effect of LNP preparations and GalNAc-modified nucleic acids in the liver limits the application of nucleic acid drugs in non-hepatic diseases, and liver accumulation can easily lead to hepatotoxicity.

[0005] Secondly, the immunogenicity of LNP is also an important challenge in its clinical application. Many studies have shown that although PEG modification can prolong the circulation time of LNP, repeated injections can easily cause the body to produce anti-PEG antibodies, leading to accelerated blood clearance and activation of the complement system to trigger an innate immune response, resulting in systemic inflammation.

[0006] In addition, liposomes and polymer nanoparticles have been developed to improve the stability and pharmacokinetic properties of nucleic acid drugs, but they still face challenges such as low delivery efficiency, immunogenicity, and difficulty in large-scale manufacturing.

[0007] To meet the delivery needs of nucleic acid drugs and achieve efficient delivery of non-liver-targeted nucleic acid drugs and multiple dosing as needed for disease treatment, albumin-based delivery technology holds promise for further optimization and design to solve existing delivery challenges for nucleic acid drugs. This aims to achieve highly efficient, safe, stable, and specific therapeutic effects in various diseases, providing an efficient and safe delivery platform for the clinical application of nucleic acid drug formulations. Summary of the Invention

[0008] Based on this, the purpose of this invention is to provide a protein / polyester / lipid nanoassembly system (i.e., nucleic acid drug formulation) that can exert high efficiency, safety, stability and specificity, as well as its preparation method and application.

[0009] The first aspect of this invention is to provide a protein / polyester / lipid nanoassembly system for delivering nucleic acid molecules, comprising a carrier and nucleic acid molecules, wherein the carrier is a protein-polyester nanoassembly, and the nucleic acid molecules are single-stranded or double-stranded nucleic acid molecules such as small interfering RNA, antisense oligonucleotides, microRNA, and mRNA; the protein-polyester nanoassembly comprises three types of materials: protein, hydrophobic biodegradable polyester or its derivatives, and lipid; the biodegradable polyester is an aliphatic polyester.

[0010] The protein is preferably albumin or a fusion protein;

[0011] The fusion protein includes albumin and at least one single-chain antibody targeting a cell antigen, wherein the albumin and the single-chain antibody are directly linked or linked through a linker peptide.

[0012] The lipids include ionizable lipids and / or cationic lipids.

[0013] A second aspect of the present invention is a method for preparing the above-mentioned protein / polyester / lipid-nucleic acid delivery system for delivering nucleic acid molecules, comprising the following steps:

[0014] (1) The buffer solution is mixed with the protein and nucleic acid respectively to prepare the protein solution and the nucleic acid solution as the aqueous phase. The buffer solution is sodium acetate or sodium citrate buffer.

[0015] (2) Mix the hydrophobic biodegradable polyester or its derivative and lipid with an organic solvent respectively to prepare the hydrophobic biodegradable polyester or its derivative solution and lipid solution as the organic phase;

[0016] (3) Mix the aqueous phase described in step (1) and the organic phase described in step (2), and then perform ultrasonic emulsification on the resulting mixture, or perform high-pressure homogenization after mixing to obtain an emulsion;

[0017] (4) Remove the organic solvent from the emulsion, separate and purify to obtain the aqueous solution of the nano-assembly, freeze-dry the aqueous solution of the nano-assembly to obtain the nano-drug delivery system.

[0018] A third aspect of the present invention is the application of a protein / polyester / lipid-nucleic acid delivery system for delivering nucleic acid molecules in the treatment of tumors, inflammation, genetic diseases, and autoimmune diseases.

[0019] A fourth aspect of the present invention is to provide a treatment for tumors, inflammation, hereditary diseases or autoimmune diseases, comprising administering to a subject an appropriate dose of any of the above-described protein / polyester / lipid-nucleic acid delivery systems.

[0020] The protein / polyester / lipid-based nucleic acid delivery system constructed in this invention can effectively load and protect nucleic acid molecules, achieve efficient delivery and intracellular release of nucleic acid drugs, exert efficient function within cells, and ultimately improve therapeutic efficacy.

[0021] The protein / polyester / lipid-nucleic acid delivery system described in this invention can achieve efficient delivery of nucleic acid drugs without targeting the liver and multiple dosings according to disease needs. It realizes the delivery of nucleic acid drugs with high efficiency, safety, stability and specificity, and provides an efficient and safe delivery platform for the application of nucleic acid drug formulations.

[0022] This invention is the first to creatively apply this constructed protein / polyester / lipid-nucleic acid delivery system to therapeutic drugs for tumors, inflammation, genetic diseases and autoimmune diseases, which has broad application prospects. Attached Figure Description

[0023] Figure 1 is a schematic diagram of the preparation process of the protein / polyester / lipid-nucleic acid delivery system loaded with nucleic acid molecules, where A: preparation of the protein / polyester / lipid-nucleic acid delivery system by ultrasonic emulsification; B: preparation of the protein / polyester / lipid-nucleic acid delivery system by high pressure homogenization.

[0024] Figure 2 shows the particle size distribution of the protein / polyester / lipid-nucleic acid delivery system. In Figure 2, A represents the particle size distribution of the albumin / polyester / lipid-nucleic acid delivery system prepared by ultrasonic emulsification; B represents the particle size distribution of the αHER2 scFv-HSA fusion protein / polyester / lipid-nucleic acid delivery system prepared by ultrasonic emulsification; and C represents the particle size distribution of the albumin / polyester / lipid-nucleic acid delivery system prepared by high-pressure homogenization.

[0025] Figure 3 shows the characterization of the protein / polyester / lipid-nucleic acid delivery system prepared by removing different components. In the figure, A: agarose gel electrophoresis detection of the encapsulation of siRNA by nanoparticles; B: particle size and PDI of nanoparticles; C: potential of nanoparticles.

[0026] Figure 4 shows the characterization of the protein / polyester / lipid-nucleic acid delivery system by changing the ratio of two lipids in sodium acetate buffer. A: Agarose gel electrophoresis to detect the encapsulation of siRNA by nanoparticles; B: Particle size and PDI of nanoparticles.

[0027] Figure 5 shows the characterization of the protein / polyester / lipid-nucleic acid delivery system by changing the ratio of the two lipids in PBS buffer. A: Agarose gel electrophoresis to detect the encapsulation of siRNA by nanoparticles; B: Particle size and PDI of nanoparticles.

[0028] Figure 6 shows the effects of changing the pH of sodium acetate, the ionizable lipid solvent, and the mixing order on the encapsulation of siRNA in the protein / polyester / lipid-nucleic acid delivery system.

[0029] Figure 7 shows the effect of different buffer solutions on the protein / polyester / lipid-nucleic acid delivery system. In this figure, A represents the particle size of the nanoparticles, and B represents the encapsulation of siRNA by the nanoparticles as detected by agarose gel electrophoresis.

[0030] Figure 8 shows the knockdown efficiency of siRNA delivery in protein / polyester / lipid-nucleic acid delivery systems prepared with different buffers.

[0031] Figure 9 shows the effect of different N / P ratios on the nucleic acid encapsulation efficiency of the protein / polyester / lipid-nucleic acid delivery system.

[0032] Figure 10 shows the particle size and gene knockdown efficiency of protein / polyester / lipid-nucleic acid delivery systems prepared by PLLA with different molecular weights.

[0033] Figure 11 shows the characterization of the protein / polyester / lipid-nucleic acid delivery system prepared by replacing DOTAP with high-purity cholesterol (CHO-HP) and phosphocholine (DSPC). In the figure, A: particle size distribution of nanoparticles; B: agarose gel electrophoresis to detect the encapsulation of siRNA by nanoparticles.

[0034] Figure 12 shows the characterization of protein / polyester / lipid-nucleic acid delivery systems prepared using different ionizable lipids, where A: nanoparticle size and PDI; B: nanoparticle potential; C: agarose gel electrophoresis to detect the encapsulation of siRNA by nanoparticles.

[0035] Figure 13 shows the knockdown efficiency of siRNA delivery in protein / polyester / lipid-nucleic acid delivery systems prepared from different ionizable lipids.

[0036] Figure 14 shows the quantitative detection of the encapsulation efficiency of siRNA in the protein / polyester / lipid-nucleic acid delivery system. A: Schematic diagram of quantification of siRNA in the protein / polyester / lipid-nucleic acid delivery system using the Quant-iT RiboGreen RNA kit; B: Standard curve of quantification of siRNA using the Quant-iT RiboGreen RNA kit; C: Encapsulation efficiency of siRNA in the albumin / polyester / lipid-nucleic acid delivery system and the αHER2scFv-HSA fusion protein / polyester / lipid-nucleic acid delivery system; D: Encapsulation efficiency of siRNA in different protein-lipid nanoassemblies.

[0037] Figure 15 shows scanning electron microscope (SEM) images of the protein / polyester / lipid-nucleic acid delivery system. In Figure 15, A is an SEM image of the albumin / polyester / lipid-nucleic acid delivery system, and B is an SEM image of the αHER2 scFv-HSA fusion protein / polyester / lipid-nucleic acid delivery system. Scale bar: 100 nm.

[0038] Figure 16 is a transmission electron microscope image of the fusion protein / polyester / lipid-nucleic acid delivery system. Scale bar: 50 nm.

[0039] Figure 17 shows the HPLC quantitative detection of protein assembly efficiency in the protein / polyester / lipid-nucleic acid delivery system; where A: schematic diagram of quantifying siRNA in the protein / polyester / lipid-nucleic acid delivery system; B: standard curve of quantifying siRNA using the Quant-iT RiboGreen RNA kit; C: protein assembly efficiency in the αCD20 scFv-HSA fusion protein / polyester / lipid-nucleic acid delivery system.

[0040] Figure 18 shows the functional components of the fusion protein / polyester / lipid-nucleic acid delivery system detected by nanoflow cytometry, where A: schematic diagram of nanoflow cytometry detection; B: nanoflow cytometry diagram of the fusion protein / polyester / lipid-nucleic acid delivery system.

[0041] Figure 19 shows the stability of the protein / polyester / lipid-nucleic acid delivery system in different solutions.

[0042] Figure 20 shows the effect of different N / P albumin / polyester / lipid-nucleic acid delivery systems on cell viability.

[0043] Figure 21 shows the uptake of protein-polyester nanoparticles by cells; where A: uptake of albumin-lipid nanoparticles by MDA-MB-231 cells at different time points; B: uptake of different fusion protein-lipid nanoparticles by DB cells.

[0044] Figure 22 shows the imaging flow cytometry verification of cell uptake of the fusion protein / polyester / lipid-nucleic acid delivery system. The flow cytometry plot of the cells after uptake of the fusion protein / polyester / lipid-nucleic acid delivery system (left) and the microscopic image of some cells (right) are shown. Scale bar: 7 μm.

[0045] Figure 23 shows the confocal microscopy verification of endosome escape in the protein / polyester / lipid-nucleic acid delivery system. Scale bar: 10 μm.

[0046] Figure 24 shows the silencing effect of protein / polyester / lipid-nucleic acid delivery systems on luciferase gene in tumor cells; where A: the silencing effect of siLuc encapsulated in different protein / polyester / lipid-nucleic acid delivery systems on luciferase gene in BT474-Luc cells; B: the silencing effect of siLuc encapsulated in different protein / polyester / lipid-nucleic acid delivery systems on luciferase gene in MDA-MB-231-Luc cells.

[0047] Figure 25 shows the silencing effect of the fusion protein / polyester / lipid-nucleic acid delivery system on the luciferase gene in suspended cells.

[0048] Figure 26 shows the hemophagocytic curves of LNP and the protein / polyester / lipid-nucleic acid delivery system.

[0049] Figure 27 shows the production of APA in mice after multiple injections of LNP and the protein / polyester / lipid-nucleic acid delivery system.

[0050] Figure 28 shows the enrichment of albumin or fusion protein / polyester / lipid-nucleic acid delivery system in mouse tumors.

[0051] Figure 29 shows the antitumor effect of the albumin / polyester / lipid-nucleic acid delivery system in animals; where A: changes in tumor volume in different treatment groups; B: changes in body weight of mice in different treatment groups.

[0052] Figure 30 illustrates the effects of the albumin / polyester / lipid-nucleic acid delivery system on the tumor microenvironment and gene silencing in tumor cells; where A represents the CD8+ expression in tumors of different treatment groups. + The proportion of T cells; B: PDL1 in tumor cells. + Cell proportion; C: CD47 in tumor cells + The proportion of cells.

[0053] Figure 31 shows the antitumor effect of the fusion protein / polyester / lipid-nucleic acid delivery system in animals; where A: changes in tumor volume in different treatment groups; B: changes in body weight of mice in different treatment groups.

[0054] Figure 32 shows the inhibitory effect of the fusion protein / polyester / lipid-nucleic acid delivery system on tumors; where A: tumor images of different treatment groups; B: tumor quality of different treatment groups.

[0055] Figure 33 is a schematic diagram of the preparation process of the albumin / polyester / lipid-mRNA delivery system.

[0056] Figure 34 shows the nucleic acid loading efficiency of the albumin / polyester / lipid-mRNA delivery system. A: Nucleic acid loading efficiency of particles before purification; B: Nucleic acid loading efficiency of particles under different centrifugation conditions.

[0057] Figure 35 shows the particle size and transfection efficiency of the albumin / polyester / lipid-mRNA delivery system recovered under different high-speed centrifugation conditions.

[0058] Figure 36 shows the effect of different protein concentrations on the albumin / polyester / lipid-mRNA delivery system, where A: nanoparticle size and PDI; B: agarose gel electrophoresis to detect the encapsulation of mRNA by nanoparticles; C: gene expression efficiency in B16-F10 cells; and D: gene expression efficiency in SK-OV-3 cells.

[0059] Figure 37 shows the effect of different polylactic acid concentrations on the albumin / polyester / lipid-mRNA delivery system. In the figure, A: nanoparticle size and PDI; B: agarose gel electrophoresis to detect the encapsulation of mRNA by nanoparticles; C: gene expression efficiency in B16-F10 cells; D: gene expression efficiency in SK-OV-3 cells.

[0060] Figure 38 shows the effect of different polylactic acid molecular weights on the albumin / polyester / lipid-mRNA delivery system. In the figure, A: nanoparticle size and PDI; B: agarose gel electrophoresis to detect the encapsulation of mRNA by nanoparticles; C: gene expression efficiency in RAW264.7 cells; D: gene expression efficiency in B16-F10 cells.

[0061] Figure 39 shows the effects of different buffers on the albumin / polyester / lipid-mRNA delivery system. In the figure, A represents the particle size and PDI of the nanoparticles; B represents the encapsulation of mRNA by the nanoparticles as detected by agarose gel electrophoresis; C represents the gene expression efficiency in RAW264.7 cells; and D represents the gene expression efficiency in B16-F10 cells.

[0062] Figure 40 shows the effect of different concentrations of buffer solution on the albumin / polyester / lipid-mRNA delivery system. In the figure, A: nanoparticle size and PDI; B: agarose gel electrophoresis to detect the encapsulation of mRNA by nanoparticles; C: gene expression efficiency in MDA-MB-231 cells; D: gene expression efficiency in Jurkat cells.

[0063] Figure 41 shows the effects of different ionizable lipids on the albumin / polyester / lipid-mRNA delivery system. A: Nanoparticle size and PDI; B: Agarose gel electrophoresis of nanoparticle encapsulation of mRNA; C: Effect of the albumin / polyester / lipid-mRNA delivery system on cell viability; D: Gene expression efficiency of the albumin / polyester / lipid-mRNA delivery system. Figure 42 shows the effects of different lipid contents on the albumin / polyester / lipid-mRNA delivery system. A: Nanoparticle size and PDI; B: Agarose gel electrophoresis of nanoparticle encapsulation of mRNA; C: Gene expression efficiency of the albumin / polyester / lipid-mRNA delivery system in different cell types.

[0064] Figure 43 shows the in vitro expression efficiency of different types of mRNA delivered by the protein / polyester / lipid-mRNA delivery system.

[0065] Figure 44 shows the in vitro expression efficiency of mEGFP delivered by the protein / polyester / lipid-mRNA delivery system in different cells.

[0066] Figure 45 shows the gene expression in mice using albumin / polyester / lipid-mRNA delivery systems prepared with different ionizable lipids.

[0067] Figure 46 shows the gene expression and Luc luminescence intensity statistics of albumin / polyester / lipid-mRNA delivery systems prepared with different ionizable lipids in mice in major organs.

[0068] Figure 47 shows the expression percentage and average fluorescence intensity of the protein / polyester / lipid-mRNA delivery system in cells of various organs in vivo.

[0069] Figure 48 shows the percentage of immune cell expression and average fluorescence intensity in major organs in vivo using the protein / polyester / lipid-mRNA delivery system.

[0070] Figure 49 shows the editing efficiency of the protein / polyester / lipid-mRNA delivery system on lung cells at different time points. Detailed Implementation

[0071] Unless otherwise specified, the experimental methods described in the following embodiments of the present invention are generally performed under conventional conditions or as recommended by the manufacturer. All commonly used chemical reagents used in the embodiments are commercially available products.

[0072] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention.

[0073] To facilitate understanding of this technology, some terms and phrases are defined below.

[0074] The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, apparatus, product, or device that includes a series of steps is not limited to the steps or modules listed, but may optionally include steps not listed, or may optionally include other steps inherent to such process, method, product, or device.

[0075] In this invention, "multiple" refers to two or more. "And / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A alone, A and B simultaneously, or B alone. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0076] Homologous: In the phylogenetic theory of biology, if two or more structures have the same ancestor, they are said to be homologous.

[0077] Antibody affinity refers to the binding strength between the antigen-binding cluster of an antibody and the antigenic determinant cluster, or the binding force between an antibody and an antigenic epitope or antigenic determinant cluster. It is essentially a non-covalent interaction force, including attraction between amino acids, hydrogen bonds, hydrophobic interactions, etc.

[0078] In this article, nucleic acid molecules refer to natural or artificially constructed single-stranded or double-stranded nucleic acids, which can be DNA or RNA, such as small interfering RNA (siRNA), antisense oligonucleotides, microRNA, messenger RNA (mRNA), circular RNA, and self-replicating RNA. They can be double-stranded or single-stranded, and can be linear or circular.

[0079] As used in this article, the term "prevention" refers to stopping or delaying the onset of disease.

[0080] As used in this article, the term “treatment” means curing or at least partially halting the progression of a disease, or alleviating the symptoms of a disease.

[0081] In this invention, a "therapeutic effective amount" includes a protein / polyester / lipid-nucleic acid delivery system of the present invention that, when administered to a patient for a disease (e.g., tumor, inflammation, hereditary disease, or autoimmune disease), is sufficient to cause treatment of the disease (e.g., by weakening, alleviating, or maintaining an existing disease, or one or more symptoms of the disease). This "therapeutic effective amount" may vary depending on the nucleic acid formulation, how it is administered, the disease and its severity, and the patient's medical history, age, weight, family history, genetic makeup, type of prior or combined treatment (if any), and other independent characteristics.

[0082] In this invention, a "preventive effective dose" refers to an amount of the protein / polyester / lipid-nucleic acid delivery system sufficient to prevent or alleviate the disease or one or more symptoms of the disease when administered to a subject who has not yet experienced symptoms of the disease but may be susceptible to it. Alleviating the disease includes slowing its progression or reducing the severity of subsequent disease development. This "preventive effective dose" may vary depending on how the protein / polyester / lipid-nucleic acid delivery system described in this invention is administered, the degree of disease risk, and the patient's medical history, age, weight, family history, stage of the genetic pathological process, type of prior or combined treatment (if any), and other independent characteristics.

[0083] "Therapeutic effective dose" or "preventive effective dose" also includes the amount of one of the aforementioned protein / polyester / lipid-nucleic acid delivery systems, with a reasonable benefit-risk ratio acceptable to any treatment.

[0084] As those skilled in the art will know, the described protein / polyester / lipid-nucleic acid delivery system can be used in combination with known drugs corresponding to the disease, depending on the needs of the test subject's condition.

[0085] The protein / polyester / lipid-nucleic acid delivery system for delivering nucleic acids described in this invention treats diseases in which the nucleic acid and the entire delivery system are the active ingredients of the drug. These diseases are treated by administering appropriate amounts to the subject for prevention or treatment. These include, but are not limited to, diseases such as tumors, inflammation, genetic diseases, or autoimmune diseases.

[0086] The tumors mentioned in this article include masses formed by the abnormal proliferation of body cells, which can be divided into benign tumors and malignant tumors (cancer).

[0087] In this article, inflammation also refers to inflammatory diseases, including those caused by bacteria, viruses, fungi, parasites, etc., or physical damage, chemical stimulation, or immune response, which are the body's defensive responses to stimuli, such as sepsis.

[0088] In this article, autoimmune diseases include those caused by the immune system abnormally attacking its own tissues, such as rheumatoid arthritis.

[0089] In this article, hereditary diseases include those caused by abnormalities in genetic material (DNA), such as single-gene or polygenic hereditary diseases.

[0090] This invention provides a nucleic acid delivery system based on albumin or albumin fusion proteins, hydrophobic degradable aliphatic polyesters or their derivatives, ionizable lipids and / or cationic lipids, prepared using ultrasonic emulsification or high-pressure homogenization techniques. Unlike nucleic acid drugs delivered by lipid nanocarriers, which primarily exert their effects in the liver, the nanosystem prepared in this invention can target nucleic acid drugs to different cell types, such as tumor cells, dendritic cells, and T cells, by controlling the types of proteins involved.

[0091] In some embodiments of the present invention, a protein / polyester / lipid-nucleic acid delivery system for delivering nucleic acids is disclosed. This system is a nucleic acid drug formulation prepared by using a solution containing nucleic acids and proteins as the aqueous phase and a hydrophobic degradable aliphatic polyester or its derivatives, ionizable lipids, and / or cationic lipids as the organic phase. The total amino group ratio of the ionizable lipids and cationic lipids to the N / P ratio of the nucleic acid is 4 to 12:1, preferably 6 to 10:1.

[0092] The protein / polyester / lipid-nucleic acid delivery system, as a nucleic acid drug formulation, can be in the form of an oral dosage form, injection, inhalation dosage form, implant, or cavity delivery. It can be used for local or systemic drug administration.

[0093] Injectable drugs can be administered via various methods, including intravenous, intramuscular, subcutaneous, and intradermal (ID) injections.

[0094] In this document, "subject" or "patient" refers to a human or a non-human animal such as a mammal. "Subject" can include any animal, including horses, dogs, cats, pigs, goats, rabbits, hamsters, monkeys, guinea pigs, rats, mice, lizards, snakes, sheep, cattle, fish, and birds. Human subjects may be referred to as patients.

[0095] Some embodiments of the present invention relate to a protein having at least a serum albumin fragment that can bind to a hydrophobic degradable polymer through hydrophobic interactions. In the present invention, the serum albumin may be at least one of human serum albumin, bovine serum albumin, mouse serum albumin, rat serum albumin, rabbit serum albumin, or chicken ovalbumin.

[0096] In some preferred embodiments, the protein is a human serum albumin protein having at least three or four hydrophobic domains, the protein having hydrophobic domains is a human serum albumin protein having at least five hydrophobic domains, or the above-mentioned protein having its hydrophobic domains intact through substitution, deletion and / or addition of one or more amino acids, more preferably, a human serum albumin protein having six or seven hydrophobic domains.

[0097] Some embodiments of the present invention relate to a fusion protein containing albumin, wherein the fusion protein comprises albumin and a single-chain antibody targeting a cell antigen, the albumin and the single-chain antibody being directly linked or linked via a linker peptide. The antitumor effect of the fusion protein is superior to that of albumin.

[0098] In some embodiments, the fusion protein has at least a single-chain antibody (scFv) targeting a specific cellular antigen, a linker peptide, and serum albumin.

[0099] The single-chain antibody is derived from a functional fragment of an antibody and includes at least one light chain variable region (VL) and / or heavy chain variable region (VH) of the antibody.

[0100] The structure of the recombinant fusion protein is any one of the following: N-terminus-VL-VH-albumin C-terminus, C-terminus-VL-VH-albumin N-terminus, N-terminus-VH-VL-albumin C-terminus, C-terminus-VH-VL-albumin N-terminus, N-terminus-VL-VH-peptide linker-albumin C-terminus, C-terminus-VL-VH-peptide linker-albumin N-terminus, N-terminus-VH-VL-peptide linker-albumin C-terminus, and C-terminus-VH-VL-peptide linker-albumin N-terminus.

[0101] In some embodiments, cellular antigens include intracellular antigens as well as cell surface antigens. For example, the single-chain antibody targets at least one of the following antigens: HER2, HER3, EGFR, GPC3, CEA, PSMA, FAP, EpCAM, BCMA, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD30, CD38, CD56, CD123, CD138, CD11c, and F4 / 80, derived from humans, mice, or other species.

[0102] The linker peptide can be a conventional peptide sequence used to link polypeptides, capable of linking two polypeptides and naturally folding them into the desired structure. It is typically a short peptide with hydrophobicity and a certain degree of extensibility. The purpose in this invention is to separate the two fused proteins to mitigate their mutual interference. The linker peptide can be flexible or rigid. In some embodiments, a flexible linker peptide may be advantageous, as it can link two protein / peptide components while maintaining their respective activities and functions. In some embodiments, the hydrophobic regions of the fused protein and the functional fragment of the immunomodulatory antibody are linked by a linker peptide, which in some embodiments, for example, uses [GlyGlyGlyGlySer]n, where n is an integer from 0 to 4, more preferably 1, 2, or 3. When n is 0, it means that the hydrophobic regions of the protein and the functional fragment of the immunomodulatory antibody are directly linked.

[0103] In some embodiments of the present invention, hydrophobic biodegradable polyesters and their derivatives are involved, which are currently known biodegradable biomaterials, and also include new biodegradable biomaterials to be further developed in the future, which can bind to the hydrophobic regions of the serum albumin moiety described above. The polyester is an aliphatic polyester or a derivative thereof.

[0104] In some embodiments, the aliphatic polyester is polylactide or a poly(lactide-co-glycolic acid) copolymer; the polylactide is levorotatory polylactide (PLLA), dextrorotatory polylactide (PDLA), or racemic polylactide (PDLLA); the end groups of the polylactide are at least one selected from ester, carboxyl, and hydroxyl groups. Preferably, the end groups of the polylactide are ester groups, which have stronger hydrophobicity. The block molecular weight of the polylactide is 1 kDa-1,100 kDa, and the lactic acid / glycolic acid ratio in the poly(lactide-co-glycolic acid) copolymer ranges from 95 / 5 to 50 / 50.

[0105] In some embodiments, the polylactic acid is L-polylactic acid, and the end groups of the L-polylactic acid are ester groups.

[0106] In some embodiments, the molecular weight of the L-polylactide ranges from 1 K Dalton to 1,100 K Dalton, preferably from 17 K Dalton to 220 K Dalton, and even more preferably from 36 K Dalton to 130 K Dalton.

[0107] In some embodiments, the cationic lipid is characterized by one or more cationic heads, linkers, and hydrophobic tails, possessing at least a permanently charged property, and may be at least one of DOTAP (2,3-dioleoylpropyl)-trimethylammonium chloride), DODMA (1,2-dioleoyl-3-dimethylamino-propane), DOTMA (1,2-bisoctadecenoxy-3-methylammonium propane), DC-Chol (3β-[N-(N',N'-dimethylaminoethyl)aminoformylcholesterol), etc. Simultaneously, the ionizable lipid exhibits neutrality at physiological pH but carries a positive charge in the acidic environment of the endostomy. Due to the significant improvement in efficacy and toxicity characteristics, the pH-dependent ionization capability makes ionizable lipids suitable materials for nucleic acid delivery. In this invention, the ionizable cationic lipid can be derived from a lipid consisting of one or more cationic heads, connecting bonds, and hydrophobic tails, wherein the cationic heads are neutral at physiological pH and protonated at acidic pH, such as SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate) and ALC-0315 ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0108] In some preferred embodiments, the ionizable lipid is DLin-MC3-DMA, chemically known as methyl 4-(N,N-dimethylamino)butyrate (dilinyl) ester.

[0109] In some preferred embodiments, the lipids include ionizable lipids and cationic lipids.

[0110] In some embodiments, the ratio of the cationic lipid to the ionizable lipid is (0-12):(12-0), preferably (1-12):(12-1).

[0111] The preferred ratio of cationic lipids to ionizable lipids is (1-3):(3:1), at which point the nucleic acid in the protein / polyester / lipid-nucleic acid delivery system is less likely to leak, resulting in good encapsulation effect.

[0112] When the nucleic acid is mRNA, the ratio of cationic lipid to ionizable lipid (1-2):(2:1) is the best for encapsulation.

[0113] In some embodiments, the nucleic acid in the protein / polyester / lipid-nucleic acid delivery system can bind to cationic lipids through electrostatic interactions. The nucleic acid is an oligonucleotide and messenger RNA (mRNA), including antisense oligonucleotides (ASO), small interfering RNA (siRNA), nucleic acid aptamers, microRNA (miRNA), self-replicating RNA (saRNA), circular RNA (circRNA), etc., which can be single-stranded or double-stranded.

[0114] In some embodiments, the nucleic acid is a small interfering RNA (siRNA), including modified and unmodified siRNA. In some embodiments, the siRNA is one of siNC, siLuc, siNC-Cy5, siPDL1, siPLK1, and siCD47. In some embodiments, the siRNA is used to inhibit tumor cell growth.

[0115] In the fusion protein of the present invention, the albumin fragment can be linked to one, two or three single-chain antibodies that target different antigens.

[0116] The nucleic acid targeted for delivery in this invention can be a nucleic acid modified by a chemical, enzyme, or peptide.

[0117] In some embodiments of the present invention, nucleic acids may be used alone or in appropriate combinations of two or more.

[0118] In some embodiments of the present invention, the preparation method of the protein / polyester / lipid-nucleic acid delivery system includes the following steps:

[0119] (1) The buffer solution is mixed with the protein and nucleic acid respectively to prepare protein solution and nucleic acid solution as aqueous phase. The buffer solution is sodium citrate buffer or sodium acetate buffer.

[0120] (2) Mix the hydrophobic biodegradable polyester or its derivative and the lipid with an organic solvent respectively to prepare a hydrophobic biodegradable polyester or its derivative solution and a lipid solution as the organic phase;

[0121] (3) Mix the aqueous phase described in step (1) and the organic phase described in step (2), and perform ultrasonic emulsification on the resulting mixture, or perform high-pressure homogenization after uniform mixing to obtain an emulsion;

[0122] (4) The organic solvent is removed by rotary evaporation of the emulsion, and the nanoparticle solution is obtained after separation and purification. The nanoparticle aqueous solution is freeze-dried to obtain the nano-drug delivery system.

[0123] In some embodiments, the separation and purification method is centrifugal precipitation.

[0124] In some embodiments, the final concentration of protein in the protein solution is 0.1 mg / mL to 20 mg / mL, preferably 0.1 mg / mL to 15 mg / mL, and more preferably 1 mg / mL to 10 mg / mL.

[0125] In some embodiments, the concentration of the hydrophobic biodegradable polyester or its derivative solution is 0.1 mg / mL to 20 mg / mL, preferably 0.1 mg / mL to 15 mg / mL, more preferably 1 mg / mL to 10 mg / mL, more preferably 2 mg / mL to 10 mg / mL; and even more preferably 5 mg / mL to 10 mg / mL.

[0126] In some embodiments, the concentration of cationic lipids in the lipid solution is 0.1 to 15 mg / mL, and the concentration of ionizable lipids is 5.0 mg / mL to 30 mg / mL, more preferably 10 mg / mL to 25 mg / mL.

[0127] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 2 to 10:1, preferably 4 to 8:1.

[0128] In some embodiments, when the nucleic acid is siRNA, the sodium citrate buffer or sodium acetate buffer is 25 μM to 1 mM.

[0129] In some embodiments, when the nucleic acid is mRNA, the sodium citrate buffer or sodium acetate buffer is 1mM to 25mM, preferably 10mM to 25mM, or preferably 5mM to 15mM sodium citrate buffer.

[0130] In some embodiments, the purified nucleic acid nanoparticle solution is further provided with the addition of a lyophilization protectant at a final concentration of 10% to 20% w / v, wherein the lyophilization protectant is at least one of the following: sucrose, glucose, trehalose, or mannitol.

[0131] In some embodiments, the mass ratio of nucleic acid to protein in the protein-polyester nucleic acid delivery system ranges from 1:5 to 1000, preferably from 1:50 to 500, more preferably from 1:50 to 250, more preferably from 1:50 to 150, and most preferably from 1:50 to 100.

[0132] In some embodiments, the mass ratio of nucleic acid to hydrophobic biodegradable polyester ranges from 1:1 to 200, preferably from 1:10 to 100, more preferably from 1:20 to 80, and even more preferably from 1:30 to 60.

[0133] In some embodiments, the weight ratio of polyester to protein is 1:0.1 to 1:30, preferably 1:1 to 1:25, more preferably 1:1 to 1:15, even more preferably 1:1 to 1:15, and even more preferably 1:1 to 10.

[0134] The organic solvent is at least one of chloroform, dichloromethane, ethyl acetate, methanol, and acetonitrile, preferably dichloromethane, ethyl acetate, and methanol, and more preferably dichloromethane and ethyl acetate.

[0135] In some embodiments, the nucleic acid delivery system is a nanoparticle with a particle size range of 50–550 nm, preferably 70 nm–250 nm, more preferably 80–180 nm, and even more preferably 90–150 nm.

[0136] Some embodiments of the present invention relate to the use of any of the above-described protein / polyester / lipid-nucleic acid delivery systems in the preparation of gene therapy drugs in which nucleic acids are one of the active ingredients. For example, the use of the protein / polyester / lipid-nucleic acid delivery system to deliver the nucleic acid molecules in tumors, inflammation, genetic diseases, and autoimmune diseases.

[0137] The present invention will be further described in detail below with reference to specific embodiments.

[0138] Raw materials and their sources used in the examples:

[0139] αHER2 scFv-HSA, αCD19 scFv-HSA, αCD20 scFv-HSA, and αCD22 scFv-HSA were expressed in recombinant CHO-ZN cells and processed... The protein was purified using a protein purification instrument.

[0140] SEQ ID No: 1

[0141] HSA

[0142] SEQ ID No: 2

[0143] αCD19 scFv-HSA

[0144] SEQ ID No: 3

[0145] αCD20 scFv-HSA

[0146] SEQ ID No: 4

[0147] αCD22 scFv-HSA

[0148] Human serum albumin (HSA) was purchased from Merck, Germany.

[0149] Polylactic acid, with molecular weights ranging from 1.5 to 1100 kDa, was purchased from Jinan Daigang Biotechnology Co., Ltd.

[0150] chloroform: purchased from Guangzhou Chemical Reagent Factory.

[0151] Anhydrous ethanol: purchased from Sinopharm Chemical Reagent Co., Ltd.

[0152] Sodium acetate was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0153] Acetic acid was purchased from Sinopharm Chemical Reagent Co., Ltd.

[0154] Diethyl pyrocarbonate (DEPC) was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0155] Transmission electron microscope copper mesh: purchased from Haide Venture (Beijing) Biotechnology Co., Ltd.

[0156] Protein-free blocking solution: purchased from Shanghai Sangon Biotech Co., Ltd.

[0157] DLin-MC3-DMA was purchased from Aivit Shanghai Pharmaceutical Technology Co., Ltd.

[0158] DOTAP was purchased from Aivito Shanghai Pharmaceutical Technology Co., Ltd.

[0159] siRNA and Cy5-siRNA were purchased from Suzhou Beixin Biotechnology Co., Ltd.

[0160] EGFP mRNA (mEGFP), Fluc mRNA (mLuc), and Cre mRNA (mCre) were purchased from Hefei Afana Biotechnology Co., Ltd., China.

[0161] Agarose was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0162] TAE was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0163] GelRed dye was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0164] Triton X-100, purchased from Merck, Germany.

[0165] Quant-iT TM RiboGreen TM RNA reagent, purchased from Invitrogen, USA.

[0166] CCK-8, purchased from Lanjieke Technology Co., Ltd.

[0167] D-fluorescein potassium salt was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0168] PE anti-human CD22 (clone number S-HCL-1) was purchased from BioLegend, Inc., USA.

[0169] Alexa Fluor 488NHS Ester, Thermo Fisher Scientific, USA.

[0170] The siRNAs used include the following:

[0171] siLuc:CUUACGCUGAGUACUUCGAdTdT)SEQ ID No: 5

[0172] siPDL1: AGACGUAAGCAGUGUUGAAdT(s)dT SEQ ID No: 6

[0173] siPLK1:UGAAGAAGAUCACCCUCCUUAdTdT SEQ ID No: 7

[0174] siCD47: GGACUUGGCCUCAUUGUAATTTdTdT SEQ ID No: 8.

[0175] Example 1

[0176] 1.1 Preparation method of protein / polyester / lipid-nucleic acid delivery system

[0177] (1) Ultrasonic emulsification method

[0178] Human serum albumin solution with a final concentration of 1 mg / mL and siRNA solution with a final concentration of 2 mg / mL were prepared using sodium acetate (50 μM, pH 4.0) buffer. Polylactic acid (PLA) solution with a final concentration of 5 mg / mL was prepared using chloroform. 130k ), 22.06 mg / mL of DOTAP and 20.27 mg / mL of DLin-MC3-DMA solution were used as the aqueous phase.

[0179] Take 1 mL of the above human serum albumin solution and 20 μL of siRNA solution into a 15 mL centrifuge tube, add the prepared organic phase: 0.2 mL of polylactic acid, 30 μL of DOTAP and 10 μL of DLin-MC3-DMA chloroform solution (i.e., the molar ratio of DOTAP to DLin-MC3-DMA is 3:1, and the N / P ratio of lipid to nucleic acid is 10:1), and mix.

[0180] 15 mL centrifuge tubes were immersed in an ice-water bath and ultrasonically emulsified using an ultrasonic cell disruptor. The ultrasonic power was 60 W, the amplitude was 35%, the ultrasonic time was 1.5 min, and the ultrasonic treatment lasted 10 s with a 2 s pause.

[0181] After sonication, the emulsion was transferred to a 100 mL round-bottom flask and connected to a rotary evaporator. Evaporation was performed sequentially at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar for a total time of 30 min to thoroughly remove chloroform. After rotary evaporation, the albumin / polyester / lipid-nucleic acid delivery system (NP) was collected. HSA The preparation methods for polylactic acid nanoparticles of different molecular weights and different types of proteins are the same as those described above. The preparation process flow of the ultrasonic emulsification method is shown in Figure 1A.

[0182] (2) High-pressure homogenization method

[0183] A 5 mg / mL human serum albumin solution and a 2 mg / mL siRNA solution were prepared using sodium acetate (50 μM, pH 4.0) buffer solution as the aqueous phase; a 5 mg / mL polylactic acid solution, a 20.27 mg / mL DLin-MC3-DMA solution, and a 22.06 mg / mL DOTAP solution were prepared using chloroform as the organic phase.

[0184] The 10 mL human serum albumin solution and 200 μL siRNA solution were sequentially added to a mixed solution consisting of 2 mL PLA, 300 μL LOTAP, and 100 μL LDLin-MC3-DMA. The mixture was then immersed in an ice-water bath and dispersed using an IKAT18 digital batch disperser (10,000 rpm, 1 min) to obtain a homogeneous initial emulsion. This emulsion was immediately transferred to a NanoGenizer 30K microfluidic high-pressure homogenizer for high-pressure homogenization at 10,000 psi for 4 cycles. After homogenization, the emulsion was immediately transferred to a rotary evaporator and maintained at room temperature under vacuum conditions of 400, 300, 200, 100, 50, and 25 mbar for 5 minutes sequentially to thoroughly remove chloroform, yielding the albumin / polyester / lipid-nucleic acid delivery system solution. The preparation process flow of the high-pressure homogenization method is shown in Figure 1B.

[0185] 1.2 Purification of the protein / polyester / lipid-nucleic acid delivery system

[0186] The nanoparticles prepared in Example 1 were centrifuged at low speed (400g, 5min, 4℃) using a benchtop micro-refrigerated centrifuge to remove unassembled polylactic acid and lipids. The supernatant of the particles was transferred to a new EP tube and centrifuged at high speed (20,000g, 1.5h, 4℃) to centrifuge the nanoparticles into a precipitate to remove free proteins from the supernatant. The lower precipitate of particles was resuspended in ultrapure water for later use.

[0187] Large-volume nanoparticles prepared by high-pressure homogenization are purified by tangential flow or hollow fiber column purification, with 10 times the volume of the particle solution replaced by ultrapure water. For storage, the nanoparticle aqueous solution can be lyophilized.

[0188] 1.3 Particle size characterization of protein / polyester / lipid-nucleic acid delivery system

[0189] An appropriate volume of nanoparticles prepared by the above ultrasonic emulsification method was added to the instrument's measuring cell. The average hydration particle size and polydispersity index (PDI) of the particles were measured using a nanoparticle size analyzer and a Zeta potential analyzer. The corresponding nanoparticle size distribution is shown in Figure 2. The NPHSA@siRNA particle size is about 105 nm and the PDI is about 0.109 (Figure 2A); the NPαHER2scFv-HSA@siRNA particle size is about 116 nm and the PDI is about 0.140 (Figure 2B), showing that the protein / polyester / lipid-nucleic acid delivery system has excellent dispersibility.

[0190] The nanoparticles prepared by the high-pressure homogenization method described in this embodiment have a particle size range of 100-180 nm. The particle size distribution of the corresponding nano-assemblies (protein / polyester / lipid-nucleic acid delivery system) is shown in Figure 2C. The particle size is about 134.6 nm, the PDI dispersion is 0.115, and the uniformity is good.

[0191] Example 2: Optimization of the preparation process of the protein / polyester / lipid-nucleic acid delivery system

[0192] 2.1 Investigation of particle preparation by removing different components in sodium acetate buffer solution

[0193] A 1 mg / mL human serum albumin solution was prepared using 50 μM sodium acetate buffer (pH 4.0), and the pH was adjusted to 3.5 with acetic acid. 2 mg / mL siRNA was also prepared. Separately, a 5 mg / mL polylactic acid (PLA) solution was prepared using chloroform. 130k ), 20.27mg / mL DLin-MC3-DMA, 22.06mg / mL DOTAP.

[0194] 1) Take 1 mL of 1 mg / mL human serum albumin solution into a centrifuge tube, add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA).130k Chloroform solution, 30 μL 22.06 mg / mL DOTAP chloroform solution, and 10 μL 20.27 mg / mL DLin-MC3-DMA chloroform solution (i.e., albumin and PLA) 130k The mass ratio is 1:1, the molar ratio of DOTAP to DLin-MC3-DMA is 3:1, and the NP ratio of lipids to nucleic acids is 10.

[0195] 2) Take 1 mL of 1 mg / mL human serum albumin solution into a centrifuge tube, add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA). 130k ) Chloroform solution, 30 μL chloroform solution and 10 μL 20.27 mg / mL DLin-MC3-DMA chloroform solution (i.e., the DOTAP component was removed from the first centrifuge tube);

[0196] 3) Take 1 mL of 1 mg / mL human serum albumin solution into a centrifuge tube, add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA). 130k ) Chloroform solution, 0 μL 22.06 mg / mL DOTAP chloroform solution and 10 μL chloroform solution (i.e., the DLin-MC3-DMA component was removed from the first centrifuge tube);

[0197] 4) Take 1 mL of 1 mg / mL human serum albumin solution into a centrifuge tube, add 20 μL DEPC·H2O, 200 μL of 5 mg / mL polylactic acid (PLA130k) chloroform solution, 30 μL of 22.06 mg / mL DOTAP chloroform solution and 10 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution (i.e., remove the siRNA component from the first centrifuge tube).

[0198] The mixtures in four centrifuge tubes were ultrasonically emulsified in an ice-water bath using an ultrasonic cell disruptor. The ultrasonic power was 60W, the amplitude was 35%, and the ultrasonic time was 1.5 min, with a 10-second sonication followed by a 2-second pause (the pause time was not included in the sonication time). After sonication, the emulsion was transferred to a 100 mL round-bottom flask and rotary evaporated sequentially at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation. HSA@siRNA The nanoparticles were characterized for particle size, electrical potential, and nucleic acid encapsulation efficiency.

[0199] Weigh 0.6 g of agarose, pour it into 60 mL of 1×TAE solution, heat it in a microwave oven to dissolve, and after slightly cooling, add 6 μL of LGelRed. TMNucleic acid dye was mixed and poured into a mold, and the agarose gel was allowed to solidify. 20 μL of the particles prepared in Example 1 was mixed with 4 μL of loading buffer (6×). 0.5 μg of nucleic acid was quantitatively loaded into each well, with free siRNA as a control. Electrophoresis was performed at 110 V for 20 min. After electrophoresis, the gel was imaged using an ultrasensitive multi-functional imager (Amersham Imager 600, GE, USA). The gel imaging results are shown in Figure 3A. HSA After gel electrophoresis, the particles were blocked near the loading wells, and no obvious free siRNA band appeared. Particles without DOTAP could not load siRNA, resulting in free bands. As shown in Figure 3B, particles without DOTAP became larger, and therefore could not encapsulate nucleic acids. After adding DOTAP, NP... HSA With positive charge, NP without DOTAP HSA Negatively charged (Figure 3C).

[0200] 2.2 Preparation of particles by changing the ratio of two lipids in sodium acetate solution

[0201] Prepare a 1 mg / mL human serum albumin solution using 50 μM sodium acetate buffer (pH 4.0) and adjust the pH to 4 with acetic acid. Prepare a 2 mg / mL siRNA solution using 2 mg / mL polylactic acid (PLA) using chloroform solution. 130k ), 20.27 mg / mL DLin-MC3-DMA, 22.06 mg / mL DOTAP. Take 1 mL of 1 mg / mL human serum albumin solution into four centrifuge tubes, add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA). 130kChloroform solution. Add 20 μL of 22.06 mg / mL DOTAP chloroform solution and 20 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution to four centrifuge tubes, respectively; 10 μL of 22.06 mg / mL DOTAP chloroform solution and 30 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution; 5 μL of 22.06 mg / mL DOTAP chloroform solution and 35 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution; and 40 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution. Sonicate the mixtures in each of the four centrifuge tubes in an ice-water bath using an ultrasonic cell disruptor. The ultrasonic power was 60 W, the amplitude was 35%, and the ultrasonic time was 1.5 min, with a 10 s sonication followed by a 2 s pause (interruption time was not included in the sonication time). After sonication, the emulsion was transferred to a 100 mL round-bottom flask and subjected to rotary evaporation at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation. HSA@siRNA The nanoparticles were characterized for particle size and nucleic acid encapsulation efficiency. As shown in Figure 4A, in a sodium acetate buffer solution at pH 4.0, the amount of nucleic acid leakage increased with decreasing DOTAP content. As shown in Figure 4B, the particle size was larger when the DOTAP to DLin-MC3-DMA feed ratio was 2:6 and 1:7.

[0202] 2.3 Preparation of particles by changing the ratio of two lipids in 1×PBS solution

[0203] Prepare a 1 mg / mL human serum albumin solution using 1×PBS solution and adjust the pH to 7.4 and 4.0 respectively. Also prepare a 2 mg / mL siRNA solution. Separately, prepare a 5 mg / mL polylactic acid (PLA) solution using chloroform solution. 130k ), 20.27 mg / mL DLin-MC3-DMA, and 22.06 mg / mL LDOTAP. The preparation methods are as follows.

[0204] 1) Take 1 mL of 1 mg / mL human serum albumin solution (pH 7.4) into a centrifuge tube, add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA). 130k Chloroform solution, 20 μL DLin-MC3-DMA (20.27 mg / mL), and 20 μL LDOTAP (22.06 mg / mL).

[0205] 2) Take 1 mL of 1 mg / mL human serum albumin solution (pH 4.0) and add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA) to each of the four centrifuge tubes. 130k Chloroform solution. Add 20 μL of 22.06 mg / mL DOTAP chloroform solution and 20 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution to four centrifuge tubes, respectively; 10 μL of 22.06 mg / mL DOTAP chloroform solution and 30 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution; 5 μL of 22.06 mg / mL DOTAP chloroform solution and 35 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution; and 40 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution. Sonicate the mixtures in each of the five centrifuge tubes in an ice-water bath using an ultrasonic cell disruptor. The sonication power was 60 W, the amplitude was 35%, and the sonication time was 1.5 min, with a 10 s sonication interval followed by a 2 s pause (interruption time was not included in the sonication time). After sonication, the emulsion was transferred to a 100 mL round-bottom flask and subjected to rotary evaporation at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation. HSA@siRNA The particle size and nucleic acid encapsulation efficiency of the nanoparticles were characterized, and the results are shown in Figure 5. As the ratio of DOTOP and DLin-MC3-DMA decreased, the amount of nucleic acid leakage gradually increased.

[0206] 2.4 Preparation of granules by changing the pH, lipid solvent, and mixing order of sodium acetate.

[0207] A 1 mg / mL human serum albumin solution was prepared using 50 μM sodium acetate solution, and the pH was adjusted to 7.4 and 4.0 respectively with acetic acid. A 2 mg / mL siRNA solution was also prepared, and a 5 mg / mL polylactic acid (PLA) solution was prepared using chloroform solution. 130k 20.27 mg / mL DLin-MC3-DMA and 22.06 mg / mL LDOTAP were administered, and 20.27 mg / mL DLin-MC3-DMA was prepared separately with ethanol. 1 mL of 1 mg / mL human serum albumin solution (pH 7.4) was added to a 15 mL centrifuge tube, along with 20 μL of 2 mg / mL siRNA (pH 7.0) and 200 μL of 5 mg / mL polylactic acid (PLA). 130kChloroform solution, 20 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution, and 20 μL of 22.06 mg / mL LDOTAP chloroform solution were ultrasonically emulsified for 1.5 min in an ice-water bath using an ultrasonic cell disruptor. 20 μL of 2 mg / mL siRNA and 20 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution were mixed by pipetting in a centrifuge tube, and then 1 mL of 1 mg / mL human serum albumin aqueous solution (pH 7.4) was added. The mixture was then ultrasonically emulsified for 1.5 min in an ice-water bath using an ultrasonic cell disruptor. (This process was repeated three times in the original text.) Mix 20 μL of 2 mg / mL siRNA and 20 μL of 20.27 mg / mL DLin-MC3-DMA ethanol solution in a centrifuge tube by pipetting. Then add 1 mL of 1 mg / mL human serum albumin aqueous solution (pH 7.4) and sonicate for 1.5 min in an ice-water bath using an ultrasonic cell disruptor. Alternatively, mix 20 μL of 2 mg / mL siRNA and 20 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution in a centrifuge tube and sonicate for 1 min in an ice-water bath using an ultrasonic cell disruptor. Then add 1 mL of 1 mg / mL human serum albumin aqueous solution (pH 7.4) and sonicate for 30 s in an ice-water bath using an ultrasonic cell disruptor. The ultrasonic power was 60 W, the amplitude was 35%, and the sonication time was 10 s followed by a 2 s pause (the interruption time was not included in the sonication time). After sonication, the emulsion was transferred to a 100 mL round-bottom flask and subjected to rotary evaporation at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation. HSA@siRNA The encapsulation efficiency of the nanoparticles was characterized, and the results are shown in Figure 6. Changing the pH of sodium acetate, altering the lipid solvent, and adjusting the mixing order of several components all failed to achieve effective nucleic acid encapsulation. Therefore, the preferred preparation conditions are an aqueous phase at pH 4.0, an organic phase of chloroform, and a mixture of albumin solution, siRNA, polylactic acid, and the two lipids in that order, followed by sonication.

[0208] 2.5 Preparation of Particles Using Different Aqueous Phases

[0209] Human serum albumin was prepared into 1 mg / mL solutions using 25 μM, 50 μM, 250 μM, 500 μM, 1 mM, and 50 mM sodium acetate buffer and sodium citrate buffer, respectively. The pH was adjusted to 4.0 with acetic acid. 2 mg / mL siRNA was prepared accordingly. Separately, 5 mg / mL polylactic acid (PLA) was prepared using chloroform solution. 130k ), 20.27 mg / mL DLin-MC3-DMA, 22.06 mg / mL DOTAP. Take 1 mL of 1 mg / mL human serum albumin aqueous solution into a 15 mL centrifuge tube, add 20 μL of 2 mg / mL siRNA, 200 μL of 5 mg / mL polylactic acid (PLA) 130k A mixture of chloroform solution, 30 μL of 22.06 mg / mL DOTAP chloroform solution, and 10 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution (i.e., albumin to PLA130k mass ratio 1:1, DOTAP to DLin-MC3-DMA molar ratio 3:1, lipid to nucleic acid NP ratio 10) was ultrasonically emulsified in an ice-water bath using an ultrasonic cell disruptor. The ultrasonic power was 60 W, the amplitude was 35%, and the ultrasonic time was 1.5 min, with a 10 s ultrasonic interval followed by a 2 s pause (interruption time not included in the ultrasonic time). After ultrasonication, the emulsion was transferred to a 100 mL round-bottom flask and rotary evaporated at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation. HSA@siRNA The particle size and nucleic acid encapsulation efficiency of the nanoparticles were characterized. As shown in Figure 7, particles could not be prepared in a 50 mM sodium acetate or sodium citrate buffer solution. When the concentration of sodium acetate or sodium citrate was between 25 μM and 1 mM, the particle size of the prepared particles was all below 150 nm. The particles prepared when the concentration of sodium acetate or sodium citrate was between 25 μM and 1 mM could effectively encapsulate nucleic acids.

[0210] 2.6 Investigating the Luciferase gene knockdown effect after transfection of particles prepared with different aqueous phases into 4T1-Luc.

[0211] 4T1-Luc cells were seeded at 5000 cells per well in 96-well white plates and cultured for 12 h before drug administration. The experimental groups were: 1) PBS; 2) Free siLuc; 3) Lipo / siLuc; 4) GoldenTran-R; 5) iCLAN; 6) NPHSA@siLuc (sodium acetate 1 mM–25 μM); 7) NPHSA@siLuc (sodium citrate 25 μM–1 mM). Each experimental group had two replicates. After co-culturing at 37°C for 48 hours, the culture medium was discarded, and 100 μL of 150 μg / mL luciferase substrate was added to each well. The luminescence intensity was detected using a multi-mode microplate reader. The results are shown in Figure 8. The 25 μM–1 mM sodium acetate solution showed better knockdown than the 25 μM–1 mM sodium citrate solution.

[0212] 2.7 Preparation of particles with different N / P ratios

[0213] Prepare a 1 mg / mL human serum albumin solution using 50 μM sodium acetate buffer and adjust the pH to 4 with acetic acid. Prepare a 150 nmol / mL siRNA solution. Separately prepare a 5 mg / mL polylactic acid (PLA) solution using chloroform. 130k Different concentrations of DLin-MC3-DMA and DOTAP were tested. 1 mL of 1 mg / mL human serum albumin solution was taken, and 21 μL of 150 nmol / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA) were added. 130k Chloroform solution. Add 20 μL of DOTAP chloroform solution and 20 μL of DLin-MC3-DMA chloroform solution, with lipid concentrations of: 5.06 mg / mL DOTAP and 4.65 mg / mL DLin-MC3-DMA (N / P = 2), 10.12 mg / mL DOTAP and 9.30 mg / mL DLin-MC3-DMA (N / P = 4), 15.18 mg / mL DOTAP and 13.96 mg / mL DLin-MC3-DMA (N / P = 6), 20.27 mg / mL DOTAP and 22.06 mg / mL DLin-MC3-DMA (N / P = 8), and 25.31 mg / mL DOTAP and 23.24 mg / mL DLin-MC3-DMA (N / P = 10). Sonicate the mixtures in an ice-water bath using an ultrasonic cell disruptor. The ultrasonic power was 60W, the amplitude was 35%, and the ultrasonic time was 1.5 min, with a 10-second ultrasonic interval followed by a 2-second pause (the interruption time was not included in the ultrasonic time). After ultrasonication, the emulsion was transferred to a 100 mL round-bottom flask and rotary evaporated at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation.HSA@siRNA The nucleic acid encapsulation efficiency of nanoparticles was characterized. As shown in Figure 9, the amount of nucleic acid leakage decreased with the increase of N / P. When N / P was greater than or equal to 4, the nucleic acid encapsulation efficiency of nanoparticles reached over 98%.

[0214] 2.8 Changing the molecular weight and type of polyester

[0215] A 1 mg / mL human serum albumin solution was prepared using 50 μM sodium acetate buffer, and the pH was adjusted to 4.0 with acetic acid. 150 nmol / mL siRNA was also prepared. 20.27 mg / mL DLin-MC3-DMA and 22.06 mg / mL LDOTAP were prepared using chloroform solution. Additionally, 5 mg / mL of different types and molecular weights of polymers, including polylactic acid (PLA 19k, 36k, 130k, 250k) and polylactic acid-glycolic acid copolymer (PLGA 50 / 5053k, 75 / 2553k), were prepared using chloroform solution, with the same feed ratios and preparation methods as above. Nanoparticles were prepared using PLLA of different molecular weights under the same formulation conditions, as shown in Figure 10. The particle size of the nanoparticles tended to increase with increasing PLLA molecular weight.

[0216] 2.9 Changing the type of nucleic acid

[0217] A 1 mg / mL human serum albumin solution was prepared using 50 μM sodium acetate buffer and the pH was adjusted to 4.0 with acetic acid. 150 nmol / mL siRNA, ASO, mRNA, and pDNA were also prepared. 5 mg / mL PLA130k, 20.27 mg / mL DLin-MC3-DMA, and 22.06 mg / mL DOTAP were prepared using chloroform solution. The dosage ratios and preparation methods were the same as above (Table 1).

[0218] 2.10 Preparation of Nanoassemblies Using Albumin Fusion Proteins

[0219] A fusion protein solution of 1 mg / mL albumin was prepared using 50 μM sodium acetate buffer and the pH was adjusted to 4.0 with acetic acid. 150 nmol / mL siRNA was also prepared. 5 mg / mL PLA130k, 20.27 mg / mL DLin-MC3-DMA, and 22.06 mg / mL LDOTAP were prepared using chloroform solution, with the same feed ratios and preparation methods as above.

[0220] NP preparations using different types and molecular weights of polyesters, different types of nucleic acids, and different proteins HSA Particle sizes are shown in Table 1.

[0221] Table 1. Particle size of nanocarriers prepared from different materials

[0222] 2.11 Modifying cationic lipids to prepare particles

[0223] A 1 mg / mL human serum albumin solution was prepared using 50 μM sodium acetate buffer, and the pH was adjusted to 4.0 with acetic acid. Separately, a 2 mg / mL siRNA solution was prepared using chloroform solution. 130k ), 20.27 mg / mL DLin-MC3-DMA, 22.06 mg / mL DOTAP, 10 mg / mL CHO-HP, and 10 mg / mL DSPC. Prepare three 15 mL centrifuge tubes. Take 1 mL of 1 mg / mL human serum albumin solution into each of the three 15 mL centrifuge tubes, add 20 μL of 2 mg / mL siRNA, and 200 μL of 5 mg / mL polylactic acid (PLA). 130k 20 μL of chloroform solution and 20.27 mg / mL DLin-MC3-DMA chloroform solution were added. 20 μL of 22.06 mg / mL LDOTAP chloroform solution was added to the first centrifuge tube; 18.8 μL of 10 mg / mL CHO-HP chloroform solution and 10 μL of 10 mg / mL DSPC chloroform solution were added to the second centrifuge tube; and 10 μL of 10 mg / mL DSPC chloroform solution was added to the third centrifuge tube. The mixtures in the three centrifuge tubes were then sonicated in an ice-water bath using an ultrasonic cell disruptor. The sonication power was 60 W, the amplitude was 35%, and the sonication time was 1.5 min, with a 10 s sonication interval followed by a 2 s pause (interruption time was not included in the sonication time). After sonication, the emulsion was transferred to a 100 mL round-bottom flask and subjected to rotary evaporation at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NP was collected after rotary evaporation. HSA@siRNA The nanoparticles were characterized for particle size and nucleic acid encapsulation efficiency, as shown in Figure 11A. Removing DOTAP from the formulation and adding CHO-HP and DSPC produced particles with a diameter of approximately 140 nm. As shown in Figure 11B, particles prepared by replacing DOTAP with CHO-HP and DSPC could not load nucleic acids. Replacing DOTAP with the same molar concentrations of DODMA, DOTMA, and DC-Chol effectively loaded nucleic acids.

[0224] 2.12 Modifying ionizable lipids to prepare particles

[0225] A 1 mg / mL human serum albumin solution was prepared using 50 μM sodium acetate buffer and the pH was adjusted to 3.5 with acetic acid. A 2 mg / mL siRNA solution was prepared using DEPC·H2O. Separately, a 5 mg / mL polylactic acid (PLA) solution was prepared using chloroform. 130k), 20.27 mg / mL DLin-MC3-DMA, 22.06 mg / mL DOTAP, 22.42 mg / mL SM-102 and 24.19 mg / mL LALC-0315. Prepare three 15 mL centrifuge tubes, take 1 mL of 1 mg / mL human serum albumin solution into each of the three 15 mL centrifuge tubes, add 20 μL of 2 mg / mL siRNA and 200 μL of 5 mg / mL polylactic acid (PLA). 130k 10 μL of 20.27 mg / mL DLin-MC3-DMA chloroform solution and 30 μL of 22.06 mg / mL DOTAP chloroform solution were added to the first centrifuge tube; 10 μL of 22.42 mg / mL SM-102 chloroform solution was added to the second centrifuge tube; and 10 μL of 24.19 mg / mL LALC-0315 chloroform solution (i.e., albumin and PLA) was added to the third centrifuge tube. 130k The mass ratio of DOTAP to ionizable lipids was 1:1, the molar ratio of DOTAP to ionizable lipids was 3:1, and the NP ratio of lipids to nucleic acids was 10. The emulsion was ultrasonically emulsified in an ice-water bath using an ultrasonic cell disruptor. The ultrasonic power was 60W, the amplitude was 35%, and the ultrasonic time was 1.5 min, with a 10-second ultrasonic interval followed by a 2-second pause (interruption time was not included in the ultrasonic time). After ultrasonication, the emulsion was transferred to a 100 mL round-bottom flask and rotary evaporated at vacuum levels of 400 / 200 / 100 / 50 / 25 mbar sequentially for a total evaporation time of 30 min to thoroughly remove chloroform. NPs were collected after rotary evaporation. HSA@siRNA The nanoparticles were characterized for particle size, potential, and nucleic acid encapsulation efficiency, as shown in Figure 12A. The nanoparticles prepared from three ionizable lipids—SM-102, ALC-0315, and DLin-MC3-DMA—(NP) were compared. HSA@siLuc (DLin-MC3-DMA), NP HSA@siLuc (SM-102), NP HSA@siLuc The particle size of (ALC-0315) is around 100 nm, the potential is around 20 mV (Figure 12B), and they can all effectively load nucleic acids (Figure 12C).

[0226] 2.13 Investigate the Luciferase gene knockdown effect after transfecting MDA-MB-231-Luc and TC-1-Luc cells with particles prepared from different ionizable lipids.

[0227] MDA-MB-231-Luc cells and TC-1-Luc cells were seeded at 5000 cells per well in 96-well blank plates and cultured for 12 hours before drug administration. The experimental groups were: 1) PBS; 2) Free siLuc; 3) Lipo / siLuc; 4) GoldenTran-R; 5) iCLAN; 6) NPHSA@siLuc (DLin-MC3-DMA); 7)NP HSA@siLuc (SM-102); 8)NP HSA@siLuc (ALC-0315). Each experimental group was set up with 2 replicates. After co-culturing at 37℃ for 48 hours, the culture medium in the well plate was discarded, and 100 μL of 150 μg / mL luciferase substrate was added to each well. The luminescence intensity was detected by a multifunctional microplate detector. The results are shown in Figure 13. The particles prepared by the three ionizable lipids SM-102, ALC-0315, and DLin-MC3-DMA showed good Luciferase gene knockdown after transfection into MDA-MB-231-Luc cells and TC-1-Luc cells.

[0228] Example 3: Nucleic acid encapsulation efficiency of the protein / polyester / lipid-nucleic acid delivery system

[0229] The nucleic acid encapsulation efficiency of nanoparticles was assessed using the Quant-iT RiboGreen RNA assay kit. 2% (v / v) Triton X-100 (demulsifier) ​​and Quant-iT were prepared using 1×TE Buffer. TM RiboGreen TM For the RNA nucleic acid dye working solution, dilute the RNA standard in the kit to 2 μg / mL, and then sequentially halve it to create seven concentration gradients (i.e., 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0 μg / mL). For the determination of total RNA: Add 100 μL of 2% Triton X-100 to an EP tube, add 100 μL of the diluted particulate sample, mix well, and let stand for 5 minutes. Add 100 μL of the mixture to a 96-well plate, and then add 100 μL of Quant-iT RiboGreen Reagent. For the determination of free RNA content: Add 100 μL of the diluted particulate sample to a 96-well plate, add 100 μL of Quant-iT RiboGreen Reagent, and incubate at room temperature in the dark for 5 minutes. Measure the fluorescence value using a multi-functional microplate reader with excitation at 480 nm and emission at 520 nm. See Figure 14A for a schematic diagram of the detection process. The standard curve of siRNA versus fluorescence intensity is shown in Figure 14B. The encapsulation efficiency of siRNA by nanoparticles was calculated using the following formula: Encapsulation efficiency (%) = (Total RNA - Free RNA before demulsification) / Total RNA. The encapsulation efficiencies of different nanoparticles for siRNA are shown in Figures 14C and D. The efficiency of siRNA loading by nanoparticles prepared from albumin and fusion protein both reached over 98%.

[0230] Example 4: Morphological characterization of the protein / polyester / lipid-nucleic acid delivery system

[0231] (1) Scanning electron microscope

[0232] The protein / polyester / lipid-nucleic acid delivery system was prepared according to the preparation method in Example 1. Unassembled, water-insoluble polyester and lipids were removed by centrifugation at low speed (400g, 4℃) for 5 min using a benchtop microcentrifuge. The supernatant was transferred to a new EP tube and centrifuged at high speed (20,000g, 4℃) for 90 min to separate free protein and nanoassemblies. Free albumin in the supernatant was removed, and the lower nanoassembly precipitate was resuspended in ultrapure water. After appropriate dilution, 2 μL was added dropwise onto a silicon wafer. After the water evaporated, gold was sputtered onto the wafer for 45 s, and the wafer was observed under a scanning electron microscope. As shown in Figure 15, the nanoassemblies exhibited a uniform spherical structure.

[0233] (2) Transmission electron microscopy

[0234] Take the purified and resuspended particle solution from Example 1, dilute it appropriately (by measuring nanoparticle size and Zeta potential, the particle solution was diluted to an attenuator of 8 and a count rate of approximately 200 kcps), take 2 μL and drop it onto a 200-mesh copper grid, allow it to air dry, then stain it with 1% (w / v) phosphotungstic acid for 30 s and observe it under TEM. As shown in Figure 16, NP HSA It has a regular spherical shape.

[0235] Example 5: Determination of protein assembly rate in a protein / polyester / lipid-nucleic acid delivery system

[0236] 100 μL of reaction buffer (1M sodium bicarbonate solution) was mixed with 900 μL of αCD20 scFv-HSA protein solution (protein concentration 6 mg / mL) to obtain 1 mL of protein labeling stock solution. 3.9 μL of AF 488 dye (molecular weight 643.40, concentration 10 mg / mL) was added to the protein solution at a molar ratio of solution B (dye) to solution A (protein) of 2:1. After reacting overnight at 4°C, the free dye was removed by ultrafiltration. NPAF488-αCD20scFv-HSA@siRNA nanoparticles were prepared using the fluorescently labeled fusion protein according to the method in Example 1. The supernatant after high-speed centrifugation was collected using a particle purification method. Standard curves with different concentrations and corresponding fluorescence intensities were prepared using high-performance liquid chromatography. The fluorescence intensity of the free protein in the fusion protein stock solution and the supernatant after high-speed centrifugation were measured, and the protein assembly efficiency of the particles was calculated to be 93% (Figure 17).

[0237] Example 6: Nanoflow cytometry characterization of functional components of protein / polyester / lipid-nucleic acid delivery system

[0238] Following the method in Example 1, fluorescently unlabeled NP was prepared. αCD20scFv-HSA@siRNANPAF488-αCD20scFv-HSA@siRNA nanoparticles were prepared by combining nanoparticles and AF488-labeled αCD20scFv-HSA fusion protein. These nanoparticles encapsulated Cy5-labeled siRNA. αCD20 scFv-HSA@Cy5-siRNA nanoparticles and NPAF488-αCD20scFv-HSA@Cy5-siRNA nanoparticles prepared by loading Cy5 fluorescently labeled siRNA and AF488-labeled αCD20scFv-HSA fusion protein were used. The fluorescence signals of the four types of particles were measured by nanoflow cytometry. Nanoparticles with double positive signals from AD488 and Cy5 were detected, demonstrating that the fusion protein and nucleic acid coexist within the nanoparticles (Figure 18).

[0239] Example 7: In vitro stability of the protein / polyester / lipid-nucleic acid delivery system

[0240] The purified particle solution from Example 1 was divided into 7 equal portions, resuspended in water, 5% (w / v) glucose, and 10% (w / v) sucrose respectively, and placed in a shaker at 37°C. One portion was taken at each time point (0, 1, 2, 3, 4, 5, 6, and 7 days) for particle size analysis using a nanoparticle size analyzer and a Zeta potential analyzer. As shown in Figures 19A and 19B, the particle size of the albumin / polyester / lipid-nucleic acid delivery system and the αHER2 scFv-HSA fusion protein nanoassembly did not change significantly over 7 days, indicating that the protein / polyester / lipid-nucleic acid delivery system of the present invention has good in vitro stability.

[0241] Example 8: CCK-8 assay for cytotoxicity of protein / polyester / lipid-nucleic acid delivery system

[0242] Nanoparticles with different N / P ratios were prepared according to the method in Example 2.7. Except for the N / P ratio, other parameters remained constant. B16 cells were seeded at 5000 cells per well in 96-well blank plates and cultured for 12 hours. Then, different doses of albumin / polyester / lipid-nucleic acid delivery systems with N / P ratios of 2, 3, 4, 6, and 8 were added, with three replicates for each experimental group. After co-culturing at 37°C for 48 hours, 10 μL of CCK-8 solution was added to each well, and after co-incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated as [OD(drug-added) - OD(blank)] / [OD(drug-added) - OD(blank)] × 100%. The results are shown in Figure 20. The IC50 (half-maximum inhibitory concentration) for cell viability gradually decreased with N / P ratios ranging from 2 to 8, indicating that a higher N / P ratio tended to result in higher toxicity.

[0243] Example 9: Cellular uptake of the protein / polyester / lipid-nucleic acid delivery system

[0244] Nanoparticles (NPs) were prepared using Cy5-labeled siRNA (Cy5-siRNA) according to the method described in Example 1.1. HSA@Cy5-siRNA MDA-MB-231 cells were introduced at a rate of 1 × 10⁻⁶ cells per well. 5 One seed was inoculated into a 24-well plate and cultured for 12 hours, then NP was added. HSA@Cy5-siRNA (Cy5-siRNA final concentration was 20 nM) Cells were incubated for 1 h, 3 h, 6 h, and 12 h, respectively. After incubation, the culture medium was discarded, and the cells were washed with 1 mL of 1×PBS. Then, 0.25% trypsin was added to digest the adherent cells, and digestion was terminated with 1 mL of complete culture medium. The cells were centrifuged and collected, resuspended in pre-cooled 1×PBS, and finally, the fluorescence signal of intracellular Cy5-siRNA in tumor cells was detected by flow cytometry (BDAccuri C6 plus). The average fluorescence intensity of intracellular Cy5-siRNA was analyzed using Flowjo software. The results are shown in Figure 21A. With increasing incubation time, the average fluorescence intensity of the cells gradually increased, demonstrating the synergistic effect of MDA-MB-231 cells on NP... HSA@Cy5-siRNA Their intake is gradually increasing.

[0245] DB cells were collected, centrifuged, and counted. A PBS control group and an NPαCD19scFv-HSA@cy5-siRNA particle group were set up. αCD20 The following groups of scFv-HSA@cy5-siRNA particles were prepared: NPαCD22scFv-HSA@cy5-siRNA particles, NPαCD19 / 20 / 22scFv-HSA@cy5-siRNA particles, and NPαCD19 / 20 / 22scFv-HSA@cy5-siRNA particles. Particles were added at transfection concentrations of 50 nmol / L and 100 nmol / L, respectively. After incubation at 37°C for 4 h, free particles were washed away with 1×PBS and resuspended. Fluorescence signals in the APC channel were detected by flow cytometry, and the average fluorescence intensity of intracellular Cy5-siRNA was analyzed using Flowjo software. The results are shown in Figure 21B. DB cells prepared NP5-siRNA particles from the three fusion proteins... αCD19 / 20 / 22 scFv-HSA@cy5-siRNA particles take up more uptake than particles prepared from a fusion protein.

[0246] Following the method in Example 1, NP was prepared using Cy5-siRNA. αCD20scFv-HSA@Cy5-siRNA nanoparticles were incubated with SU-DHL-4 tumor cells at 37°C for 4 h, followed by centrifugation to remove free particles. After cell resuspending, PE anti-human CD22 was added and incubated on ice for 30 min to label the SU-DHL-4 cell membrane. Imaging flow cytometry was used to detect the fluorescence intensity of PE and APC channels, and the uptake of the αCD20 scFv-HSA fusion protein / polyester / lipid-nucleic acid delivery system by cells was simultaneously captured (Figure 22). This demonstrated that SU-DHL-4 tumor cells expressing CD22 uptake NPαCD20scFv-HSA@Cy5-siRNA into the cells.

[0247] Example 10: Intracellular endosome escape of protein / polyester / lipid-nucleic acid delivery system

[0248] SKOV3 cells were loaded at 3×10 4 Inoculate 1 / mL of the culture medium into laser confocal culture dishes and incubate at 37°C for 12 hours. Then replace with serum-free medium and add NP. HSA Incubate with Cy5-siRNA for 4 hours (Cy5-siRNA concentration: 100 nM). After incubation, discard the original culture medium and add LysoTracker Green DND-26 (1:1000 dilution), incubate in the dark for 45 minutes. Then wash adherent cells three times with PBS, add 200 μL Hoechst 33342 solution (2 μg / mL), incubate at room temperature in the dark for 10 minutes, wash twice more with PBS, and image NPs using a Zeiss LSM880 laser confocal microscope. HSA The endosome escape of @Cy5-siRNA was observed. Hoechst 33342 excitation and emission wavelengths were 346 nm and 460 nm, respectively; Cy5 excitation and emission wavelengths were 640 nm and 670 nm, respectively; and LysoTracker Green DND-26 excitation and emission wavelengths were 504 nm and 511 nm, respectively. Results were processed and exported using ZEN (Zeiss) software. As shown in Figure 23, after 2 hours of incubation, the red fluorescently labeled Cy5-siRNA was mainly distributed in the green fluorescently labeled acidic organelles, indicating that the nanoparticles were still within the endosomes or early lysosomes. However, with prolonged culture time, the green and red fluorescence within the cells gradually separated, indicating that the siRNA gradually escaped from the endosomes into the cytoplasm.

[0249] Example 11: The effect of protein / polyester / lipid-nucleic acid delivery system on gene silencing in tumor cells

[0250] HER2-positive BT474-Luc cells (Fig. 24A) and HER2-negative MDA-MB-231-Luc cells (Fig. 24B) were seeded at 5000 cells per well in 96-well white plates and cultured for 12 h. The experimental groups were set up as follows: 1) Untreated group with an equal volume of PBS; 2) Free siLuc; 3) Lipo / siLuc; 4) NP HSA@siLuc (1-100nM); 5)NP αHER2 scFv-HSA@siLuc (1-100 nM). Each experimental group was set up with 3 replicates. After co-culturing at 37℃ for 48 hours, the culture medium in the wells was discarded, and luciferase substrate at a concentration of 150 μg / mL was added. The luminescence intensity of the cells was detected using a multi-functional microplate reader. As shown in Figure 24, the albumin / polyester / lipid-nucleic acid delivery system (NP...) HSA@siLuc The knockdown effects were similar in both cell types, showing good knockdown efficiency. The αHER2 scFv-HSA fusion protein / polyester / lipid-nucleic acid delivery system (NP) demonstrated similar knockdown effects. αHER2scFv-HSA@siLuc Knockdown is more effective in HER2-positive cells.

[0251] After collecting and centrifuging suspended WSU-DLCL2-Luc cells, they were counted at a ratio of 5 × 10⁶ cells per well. 4 Cell / well density was consistent with that of seeding plates in 96-well white plates. The following groups were included: PBS control group, Lipo / siLuc group, GoldenTrans-R transfection reagent group, and NP group. HSA@siLuc Group, NP HSA@siN.C. Group, NP αCD19scFv-HSA@siLuc Group, NP αCD20scFv-HSA@siLuc Group, NP αCD22 The scFv-HSA@siLuc group and the NPαCD19 / 20 / 22scFv-HSA@siLuc group were prepared in triplicate. The transfection concentration was 150 nmol / L, and the mixtures were co-cultured at 37°C for 48 hours. Afterward, luciferase substrate was added, and the luminescence intensity was detected using a multi-functional microplate reader. The results are shown in Figure 25. The gene silencing efficiency of the nanoassemblies prepared by mixing the three fusion proteins was higher.

[0252] Example 12: Pharmacokinetics of a protein / polyester / lipid-nucleic acid delivery system

[0253] Healthy Balb / c mice were injected via tail vein with NP containing siRNA-cy5 HSA@siRNA-cy5 or LNP @siRNA-cy5Three mice were placed in each group, and the dosage of siRNA was 1 mg / kg. 0.15, 0.5, 1, 2, 4, and 6 hours after administration, 50 μL of blood was collected from each group of mice via the orbital venous plexus, and the fluorescence signal in the blood was detected using a multi-functional microplate reader. As shown in Figure 26, NP... HSA@siRNA-cy5 The half-life in blood is LNP. @siRNA-cy5 The effect was 2.93 times greater. Furthermore, after multiple administrations, the LNP experimental group produced a large amount of anti-PEG antibodies, leading to a further shortening of the blood circulation half-life of the siRNA drug (Figure 27).

[0254] Example 13: Enrichment of the protein / polyester / lipid-nucleic acid delivery system at tumor sites.

[0255] SK-OV-3 cells in good growth condition were digested with 0.25% trypsin, centrifuged at 4°C and 800 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 1×PBS and the concentration was adjusted to 1×10⁻⁶. 7 Cells / mL, use a disposable syringe to draw 100 μL of cell suspension (approximately 1 × 10⁶ cells / mL). 6 (Number of cells) were seeded subcutaneously in the right side of mice. The tumor volume was increased to approximately 200 mm². 3 At that time, nanoparticles loaded with siRNA-cy5 were injected via tail vein. The experimental setup was as follows: 1) PBS; 2) NP HSA@siRNA-cy5 ;3)NP αHER2 scFv-HSA@siRNA-cy5 was administered to three mice in each group, with a siRNA dosage of 1 mg / kg. Twenty-four hours after administration, tumor tissues were collected from each group of mice, and fluorescence signals in the tumor tissues were detected using small animal imaging. As shown in Figure 28, NP... HSA@siRNA-cy5 and NP αHER2 Both scFv-HSA and siRNA-cy5 were enriched at the tumor site, and NP αHER2scFv-HSA@siPLK1 In HER2 + It is more concentrated in tumor tissue.

[0256] Example 14: Antitumor effect of protein / polyester / lipid-nucleic acid delivery system at the animal level

[0257] Healthy MC38 cells were digested with 0.25% trypsin, centrifuged at 4°C and 800 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 1×PBS and the concentration was adjusted to 1×10⁻⁶. 7 Cells / mL, use a disposable syringe to draw 100 μL of cell suspension (approximately 1 × 10⁶ cells / mL). 6 (Number of cells) were inoculated subcutaneously on the right side of mice. The tumor size was measured using calipers, and the tumor volume was calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width2 When the tumor volume reaches 50-100 mm 3 Treatment was initiated at that time, and the experimental groups were set up as follows: 1) PBS; 2) NP. HSA@siN.C. ;3)NP HSA@siPD-L1 ;4)NP HSA@siCD47 Each group consisted of 5-7 mice. The siRNA dosage was 1 mg / kg, administered via tail vein injection, twice daily for a total of four doses. Mice were sacrificed on day 14. As shown in Figure 29A, compared with the PBS and NP groups... HSA@siN.C. Compared to the group, NP HSA@siPD-L1 Groups and NP HSA@siCD47 Tumor growth was significantly inhibited in all groups. As shown in Figure 29B, there was no significant change in body weight in any group of mice throughout the treatment process, indicating that none of the components had serious toxicity to mouse survival.

[0258] After each group of treatments, mice were sacrificed, tumors were removed, and approximately 0.5g of tumor tissue was excised into a 5mL centrifuge tube. The tissue was thoroughly minced with scissors, and 4mL of a digestion solution prepared with 1640 medium (1mg / mL collagenase IV, 100μg / mL DNase) was added. The mixture was incubated at 37℃ on a shaker at 180rpm for 15min. The digested tissue suspension was then filtered through a 200-mesh steel mesh into a 50mL centrifuge tube. Digestion was terminated with PBS (containing 0.2% BSA), and the suspension was transferred to a 15mL centrifuge tube. The tube was centrifuged at 500g for 5min, the supernatant was discarded, and the cell pellet was resuspended in PBS (containing 0.2% BSA). The pellet was then transferred through a 200-mesh nylon mesh to a new centrifuge tube for dilution and counting. Each sample was divided into tubes A and B, with 1.5×10⁶ cells collected from each tube. 6 Cells were placed in tubes A and B and centrifuged at 500g for 5 min at 4°C. The cell pellet was collected, and the cells were resuspended in 30 μL of CD16 / 32 antibody dilution buffer. The cells were blocked on ice for 15 min. After incubation, 30 μL of a mixture of anti-CD45, anti-CD4, and anti-CD8 antibodies labeled with different fluorescent dyes was added to tube A, mixed, and incubated on ice in the dark for 45 min. A mixture of anti-CD45, anti-CD47, and anti-PD-L1 antibodies labeled with different fluorescent dyes was added to tube B, mixed, and incubated on ice in the dark for 45 min. After incubation, the cells were washed once with 1 mL of PBS, resuspended in an appropriate amount of PBS, and transferred to flow cytometry tubes through a 200-mesh nylon mesh. The proportions of each cell type were detected by flow cytometry, and the data were analyzed and processed using FlowJo software. As shown in Figure 30A, compared with the PBS group and NP group... HSA@siN.C. Group, NP HSA@siPD-L1 and NP HSA@siCD47 Both can significantly increase intratumoral CD8 + T cell infiltration. As shown in Figures 30B and C, NPHSA@siPD-L1 It can downregulate intratumoral PD-L1 expression without affecting CD47 expression. NP HSA@siCD47 It only downregulated CD47 expression and did not affect PD-L1 expression, indicating that siRNA gene silencing has strong target specificity.

[0259] Example 15: The fusion protein / polyester / lipid-nucleic acid delivery system for HER + Anti-tumor effects on tumors

[0260] SK-OV-3 cells in good growth condition were digested with 0.25% trypsin, centrifuged at 4°C and 800 rpm for 3 min, the supernatant was discarded, and the cells were resuspended in 1×PBS and the concentration was adjusted to 1×10⁻⁶. 7 Cells / mL, use a disposable syringe to draw 100 μL of cell suspension (approximately 1 × 10⁶ cells / mL). 6 (Number of cells) were inoculated subcutaneously in the right side of the mouse. Once the tumor was first palpable, its size was monitored daily using calipers. The tumor volume was calculated using the following formula: Volume (mm²) 3 = 0.5 × length × width 2 When the tumor volume reaches 50-100 mm 3 Treatment was initiated at that time, and the experimental groups were set up as follows: 1) PBS; 2) NP. HSA@siPLK1 ;3)NP αHER2scFv-HSA@siPLK1 Five mice were placed in each group. The dosage of siRNA was 1 mg / kg, administered via tail vein injection, once every two days for a total of six administrations. Mice were sacrificed on day 16. As shown in Figure 31A, NP... HSA@siPLK1 and NP αHER2scFv-HSA@siPLK1 Both can inhibit tumor growth, and NP αHER2scFv-HSA@siPLK1 The inhibitory effect on tumors was superior. As shown in Figure 31B, the body weight of mice in each group did not change significantly throughout the treatment process, indicating that the components in each group had no serious toxicity to mouse survival.

[0261] After treatment, tumors from each group of mice were removed, photographed on clean plates, and the weight of each tumor was recorded. As shown in Figure 32A, NP... HSA@siPLK1 and NP αHER2scFv-HSA@siPLK1 The tumors in all mice were significantly reduced, demonstrating a tumor-suppressive effect. In contrast, NP... αHER2scFv-HSA@siPLK1 The tumor size in this group is the smallest. In addition, as shown in Figure 32B, NP... αHER2 The scFv-HSA@siPLK1 group also had the lightest tumor weight, further confirming the NP (negative tumor marker). αHER2scFv-HSA@siPLK1 It has the best anti-tumor effect.

[0262] Example 16, NP HSA @mRNA preparation and characterization

[0263] 16.1 NP HSA Preparation of mRNA nanoparticles

[0264] As shown in Figure 33, taking mLuc (mRNA encoding luciferase) as an example, HSA and mLuc were dissolved in 25mM sodium acetate solution (pH=4, prepared with DEPC water) to obtain concentrations of 5mg / mL and 1mg / mL, respectively, as the aqueous phase. Then, PLLA-COOH (PLLA) with a molecular weight of 36K was added... 36K HSA, mLuc, PLLA, MC3, and DOTAP were dissolved in chloroform to concentrations of 5 mg / mL, 20.4 mg / mL, and 22.2 mg / mL, respectively, forming the organic phase. The components were mixed in a volume ratio of 500:21:100:10:10, and then ultrasonically emulsified using a 45W ultrasonic cell disruptor for 90 seconds. Immediately afterwards, the chloroform was removed using an IKA rotary evaporator, yielding HSA / mLuc / PLLA / MC3 / DOTAP nanoparticles (abbreviated as NP). HSA @mLuc).

[0265] 16.2 NP HSA Characterization of mRNA nanoparticles

[0266] (1) RiboGreen detects particle recovery rate under different centrifugation conditions.

[0267] The collected particles were purified using high-speed centrifugation to remove free proteins, lipids, polylactic acid, nucleic acids, and other components from the solution. The content of recovered particles under different centrifugation conditions was determined using the RiboGreen method for detecting nucleic acid encapsulation efficiency.

[0268] The experimental method is as follows: Particles were aliquoted into multiple 1.5 mL EP tubes and centrifuged under different conditions (400g for 5 min, 12000g for 15 min, 15000g for 15 min, 15000g for 30 min, and 15000g for 60 min). The supernatant was collected and the total nucleic acid content (A1) and free nucleic acid content (B1) were measured using the RiboGreen assay described above. Untreated particles were also collected and the total nucleic acid content (A1) and free nucleic acid content (B1) in the solution were measured using the same RiboGreen assay. The relative particle content in the supernatant under different treatment conditions was calculated using formula (1-2) to determine whether centrifugation could recover most of the particles. Relative particle content (%) = (A1 - B1) / (AB) Formula (1-2)

[0269] As shown in Figure 34, NP HSAThe encapsulation efficiency of mRNA was close to 100%, and the particle content in the supernatant was below 6% after centrifugation for different times, indicating that high-speed centrifugation can recover more than 94% of the particles.

[0270] (2) The centrifuged particles were resuspended in DEPC water, and the particle size was detected by DLS under different centrifugation conditions, as shown in Figure 35A. The particle size after centrifugation was approximately 200 nm. The transfection efficiency of the particles before and after centrifugation was determined. The particles centrifuged at 12000g for 15 min were resuspended in 1×PBS and simultaneously transfected into SK-OV-3 and B16F10 cells along with unpurified particles. The experimental method was the same as above, and the difference in transfection efficiency before and after centrifugation was detected. As shown in Figure 35B, there was no significant difference in transfection efficiency before and after centrifugation, indicating that the centrifugation method for purifying the particles is feasible.

[0271] Example 17 NP HSA @Optimization of mRNA preparation conditions

[0272] 17.1 Change the HSA concentration (1, 5, 10 mg / mL)

[0273] (1) HSA was dissolved in 25 mM sodium acetate solution (pH = 4, prepared with DEPC water) to make concentrations of 1, 5, and 10 mg / mL; mRNA was dissolved in 25 mM sodium acetate solution (pH = 4, prepared with DEPC water) to make concentrations of 1 mg / mL; PLLA was dissolved in... 36K MC3 and DOTAP were dissolved in chloroform to concentrations of 5 mg / mL, 20.4 mg / mL, and 22.2 mg / mL, respectively. The mixtures were then combined according to a volume ratio of HSA:mLuc:PLLA:MC3:DOTAP of 500:21:100:10:10. The mixture was ultrasonically emulsified using a 45W ultrasonic cell disruptor for 90 seconds, and immediately the chloroform was removed using a rotary evaporator to obtain NPs prepared at different protein concentrations. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0274] (2) B16F10 and SK-OV-3 were seeded in white 96-well plates at a density of 5,000 cells / well and cultured for 12 hours until the cells adhered. Then, NP / mLuc particles were added to the final concentration of mLuc to 1 μg / mL. Lipo / mLuc was used as a positive control. After transfection for 24 hours, D-luciferin potassium salt was added to the final concentration of 150 μg / mL. After incubation at room temperature for 10 min, the fluorescence intensity was detected by microplate reader.

[0275] As shown in Figure 36A, HSA at concentrations of 1, 5, and 10 mg / mL could all be used to prepare particles, and changing the protein concentration alone had no significant effect on particle size and transfection efficiency. All three albumin concentrations could completely load mRNA (Figure 36B). In B16F10 cells, particles prepared with 1 mg / mL HSA showed better transfection efficiency than the other two concentrations (Figure 36C). In SK-OV-3 cells, particles prepared with the three albumin concentrations showed similar transfection efficiency (Figure 36D).

[0276] 17.2 Change the PLLA concentration (1, 5, 10 mg / mL)

[0277] (1) Dissolve HSA and mLuc in 25mM sodium acetate solution (pH=4, prepared with DEPC water) to a concentration of 1mg / mL; dissolve PLLA... 36K Dissolved in chloroform to concentrations of 1, 5, and 10 mg / mL; MC3 and DOTAP dissolved in chloroform to concentrations of 20.4 mg / mL and 22.2 mg / mL, respectively. The components were mixed at a volume ratio of 500:21:100:10:10, and then ultrasonically emulsified using a 45W ultrasonic cell disruptor for 90 seconds. The chloroform was immediately removed using a rotary evaporator to obtain NPs prepared with different PLLA concentrations. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0278] (2) B16F10 and SK-OV-3 were seeded in white 96-well plates at a density of 5,000 cells / well and cultured for 12 hours until the cells adhered. Then, NP / mLuc particles were added to the final concentration of mLuc to 1 μg / mL. Lipo / mLuc was used as a positive control. After transfection for 24 hours, D-luciferin potassium salt was added to the final concentration of 150 μg / mL. After incubation at room temperature for 10 min, the fluorescence intensity was detected by microplate reader.

[0279] As shown in Figure 37A, PLLA at concentrations of 1, 5, and 10 mg / mL can all be used to form particles. Increasing the PLLA concentration can effectively reduce the particle size and PDI. All three polylactic acid concentrations can fully load mRNA (Figure 37B). Increasing the PLLA concentration in both cell types can slightly enhance the transfection effect (Figures 37C and D).

[0280] 17.3 Changing the molecular weight of PLLA (17K, 66K, 130K, 220K)

[0281] (1) HSA and mLuc were dissolved in 10mM sodium citrate buffer (pH=4, prepared with DEPC water) to a concentration of 1 mg / mL; PLLA-COOH with molecular weights of 17K, 66K, 130K, and 220K were dissolved in chloroform to a concentration of 5 mg / mL; MC3 and DOTAP were dissolved in chloroform to concentrations of 20.4 mg / mL and 22.2 mg / mL, respectively. The components were mixed according to the volume ratio of HSA:mLuc:PLLA:MC3:DOTAP of 500:21:100:10:10. After ultrasonic emulsification using a 45W ultrasonic cell disruptor for 90 seconds, the chloroform was immediately removed using a rotary evaporator to obtain NPs prepared with different PLLA molecular weights. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0282] (2) RAW264.7 (mouse mononuclear macrophage leukemia cells) and B16F10 were seeded at a density of 5,000 cells / well in white 96-well plates and cultured for 12 hours until cell adhesion. NP / mLuc particles were then added to a final mLuc concentration of 1 μg / mL, with Lipo / mLuc used as a positive control. 24 hours after transfection, D-luciferin potassium salt was added to a final concentration of 150 μg / mL. After incubation at room temperature for 10 min, fluorescence intensity was detected using a microplate reader. As shown in Figure 38, PLLA at 17K, 66K, 130K, and 220K could all be formulated into particles, and the particle size increased with increasing PLLA molecular weight. All particles could fully load mRNA. Considering overall efficacy, the more suitable range was 66K-130K.

[0283] 17.4 Changing the aqueous solution and pH

[0284] (1) Dissolve HSA and mLuc in 25 mM sodium acetate solution (NP-1: pH=3, NP-2: pH=4, prepared with DEPC water) or 10 mM sodium citrate buffer (NP-3: pH=3, NP-4: pH=4, prepared with DEPC water) respectively, so that the concentration of each is 1 mg / mL; dissolve PLLA with a molecular weight of 36K Dissolve HSA in chloroform to a concentration of 5 mg / mL; dissolve MC3 and DOTAP in chloroform to concentrations of 20.4 mg / mL and 22.2 mg / mL, respectively. Mix the components according to a volume ratio of 500:21:100:10:10, and sonicate using a 45W ultrasonic cell disruptor for 90 seconds. Immediately afterward, remove the chloroform using a rotary evaporator to obtain NP prepared from different aqueous solutions. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0285] (2) RAW264.7 and B16F10 cells were seeded at a density of 5,000 cells / well in white 96-well plates and cultured for 12 hours until cell adhesion. NP / mLuc particles were then added to a final mLuc concentration of 1 μg / mL, with Lipo / mLuc used as a positive control. After 24 hours of transfection, D-luciferin potassium salt was added to a final concentration of 150 μg / mL, and the cells were incubated at room temperature for 10 minutes before fluorescence intensity was detected using a microplate reader. As shown in Figure 39, particles could be prepared from all four aqueous phases and were effectively loaded with mRNA. Particles prepared using 10 mM sodium citrate buffer (pH=4) as the aqueous phase showed the best transfection effect in both cell types.

[0286] 17.5 Change the concentration of sodium citrate buffer

[0287] (1) Dissolve HSA and mLuc in 5mM, 2mM, 1mM, and 500μM sodium citrate buffer (pH=4, prepared with DEPC water) respectively, to a concentration of 1mg / mL; dissolve PLLA... 36K Dissolve HSA in chloroform to a concentration of 5 mg / mL; dissolve MC3 and DOTAP in chloroform to concentrations of 20.4 mg / mL and 22.2 mg / mL, respectively. Mix the components at a volume ratio of 500:21:100:10:10 using a 45W ultrasonic cell disruptor and immediately remove the chloroform using a rotary evaporator to obtain NP buffers prepared with different concentrations of sodium citrate buffer. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0288] (2) MDA-MB-231 (human breast cancer cells) and Jurkat (human T lymphocyte leukemia cells) were seeded in white 96-well plates at a density of 5,000 cells / well and cultured for 12 hours until the cells adhered. NP / mLuc particles were then added to a final concentration of 1 μg / mL of mLuc. Lipo / mLuc was used as a positive control. 24 hours after transfection, D-fluorescein potassium salt was added to a final concentration of 150 μg / mL. After incubation at room temperature for 10 min, the fluorescence intensity was detected using an ELISA reader.

[0289] As shown in Figure 40, particles could be formed using all four concentrations of sodium citrate buffer. The particle size increased with increasing salt ion concentration, while the transfection effect decreased accordingly. Particles prepared using 5 mM sodium citrate buffer (pH=4, prepared with DEPC water) as the aqueous phase had a particle size of less than 200 nm.

[0290] 17.6 Changing the type of ionizable lipids

[0291] (1) HSA and mLuc were dissolved in 5mM sodium citrate buffer (pH=4, prepared with DEPC water) to a concentration of 1 mg / mL; PLLA-COOH with a molecular weight of 130K was dissolved in chloroform to a concentration of 5 mg / mL; MC3, SM102, ALC-0315, and DOTAP were dissolved in chloroform to concentrations of 20.4, 22.5, 24.3, and 22.2 mg / mL, respectively. The components were mixed according to a volume ratio of HSA:mLuc:PLLA:ionizable lipids:DOTAP of 500:21:100:10:10. The mixture was ultrasonically emulsified using a 45W ultrasonic cell disruptor for 90 seconds, and then the chloroform was immediately removed using a rotary evaporator to obtain NPs prepared from different ionizable lipids. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0292] (2) B16F10 cells were seeded at a density of 5,000 cells / well in white 96-well plates and 96-well clear plates and cultured for 12 hours until cell adhesion was achieved. NP / mLuc particles were then added to a final mLuc concentration of 0.5 μg / mL, with Lipo / mLuc used as a positive control. 24 hours after transfection, D-fluorescein potassium salt was added to the white 96-well plates to a final concentration of 150 μg / mL. After incubation at room temperature for 10 min, fluorescence intensity was detected using a microplate reader. 24 hours after transfection, the supernatant was aspirated from the clear 96-well plates, and 100 μL of CCK8 (Biosharp) diluted 10-fold with culture medium was added. After incubation at 37°C for 2 hours, the absorbance at 450 nm was measured using a microplate reader, and the relative cell viability was calculated.

[0293] As shown in Figure 41, all three ionizable lipids can be made into particles with a diameter of less than 200 nm, which can effectively load mRNA. Furthermore, SM102 or ALC-0315 can achieve good transfection results in vitro with minimal impact on cell viability.

[0294] 17.7 Reduce cationic lipid DOTAP

[0295] (1) HSA and mLuc were dissolved in 5mM sodium citrate buffer (pH=4, prepared with DEPC water) to a concentration of 1 mg / mL; PLLA-COOH with a molecular weight of 250K was dissolved in chloroform to a concentration of 5 mg / mL; SM102, ALC-0315, and DOTAP were dissolved in chloroform to concentrations of 22.5, 24.3, and 22.2 mg / mL, respectively. The components were mixed according to a volume ratio of HSA:mLuc:PLLA:lipids of 500:21:100:20. The mixture was ultrasonically emulsified using a 45W ultrasonic cell disruptor for 90 seconds, and then the chloroform was immediately removed using a rotary evaporator to obtain NP prepared with or without the addition of DOTAP. HSA / mLuc particles. The particle size was determined using a dynamic light scattering instrument, and the encapsulation of nucleic acids by the particles was assessed using an agarose gel retardation assay.

[0296] (2) B16F10 cells were seeded at a density of 5,000 cells / well in white 96-well plates and cultured for 12 hours until the cells adhered. Then, NP / mLuc particles were added to the final concentration of mLuc to 0.5 μg / mL or 1 μg / mL. Lipo / mLuc was used as a positive control. 24 hours after transfection, D-fluorescein potassium salt was added to the white 96-well plate to the final concentration of 150 μg / mL. After incubation at room temperature for 10 min, the fluorescence intensity was detected using an ELISA reader.

[0297] As shown in Figure 42, particles can be prepared with or without the addition of DOTAP. However, the group without the addition of DOTAP cannot effectively encapsulate mRNA and has no transfection effect. Therefore, DOTAP is an essential component.

[0298] Example 18, NP HSA In vitro expression efficiency of different mRNAs

[0299] NP HSA In vitro expression of @mEGFP.

[0300] HEK 293T (human embryonic kidney cells) were seeded at a density of 50,000 cells / well in clear 24-well plates and cultured for 12 hours until the cells adhered. NP was then added. HSA The amount of mEGFP particles added was 1 μg / mL. The intensity of the EGFP fluorescence signal was detected by 12-color flow cytometry (BD FACS) 24 or 36 hours after transfection.

[0301] NP HSA In vitro expression of @mLuc.

[0302] SK-OV-3 (human ovarian adenocarcinoma cells) and B16F10 (mouse skin melanoma cells) were seeded at a density of 5,000 cells / well in non-transparent 96-well plates and cultured for 12 hours until cell adhesion. NP / mLuc particles were then added to a final mLuc concentration of 1 μg / mL. Lipofectamine Messenger Max (Invitrogen) / mEGFP (Lipo / mLuc) was used as a positive control. 24 hours after transfection, D-fluorescein potassium salt (Beyotime) was added to a final concentration of 150 μg / mL. After incubation at room temperature for 10 min, fluorescence intensity was detected using a ELISA reader (Tecan Infinite).

[0303] As shown in Figure 43, NP HSA @mRNA can be effectively delivered to both types of tumor cells at the in vitro level.

[0304] Example 19 NP HSA @mRNA expression efficiency in different cells

[0305] (1) Collect and count B16F10, Jurkat, and SK-OV-3 cells, and seed 5 × 10⁶ cells per well. 4 Cells were placed in 24-well clear plates with 500 μL of culture medium per well and incubated at 37°C for 12 hours in a constant temperature incubator containing 5% CO2.

[0306] (2) Transfection was performed using NPHSA@mEGFP, LNP, and Lipo, with a dosage of 0.5 μg mRNA per milliliter of culture medium;

[0307] (3) 24 hours after transfection, remove the culture medium, digest each well with 300 μL of 0.25% trypsin digestion solution for 3 minutes, and then add 1 mL of PBA to stop the digestion. Jurkat cells are suspension cells and do not need to be digested. Collect all cells in a 1.5 mL centrifuge tube, centrifuge at 3000 rpm for 3 minutes at 4°C, discard the supernatant, and add 500 μL of PBA pre-chilled on ice to resuspend the cells.

[0308] (4) Cell clumps were filtered using a double-layered 200-mesh nylon mesh, and cells were transferred to flow cytometry tubes. Before detection, 100×DAPI-labeled dead cells were added to each tube. EGFP expression in the three cell types was detected using the FITC channel of a 12-color flow cytometer. Data were processed and analyzed using FlowJo V10 and GraphPad Prism 9.5. As shown in Figure 44, compared with the control group, NP... HSA It showed good transfection efficiency in all three cell types. And NP HSAThe transfection effect in B16F10 and SK-OV-3 adherent cells was better than that in Jurkat cells.

[0309] Example 20 NP HSA @mRNA expression distribution in vivo

[0310] Use the optimized NP HSA / mRNA delivery in vivo

[0311] (1) Particle preparation: HSA and mLuc were dissolved in 5mM sodium citrate buffer (pH=4, prepared with DEPC water) to a concentration of 1 mg / mL; PLLA-COOH with a molecular weight of 250K was dissolved in chloroform to a concentration of 5 mg / mL; SM102, ALC-0315, and DOTAP were dissolved in chloroform to concentrations of 22.5, 24.3, and 22.2 mg / mL, respectively. The components were mixed according to a volume ratio of HSA:mLuc:PLLA:ionizable lipids:DOTAP of 500:21:100:10:10. The mixture was ultrasonically emulsified using a 45W ultrasonic cell disruptor for 90 seconds, and then the chloroform was immediately removed using a rotary evaporator to obtain NP. HSA / mLuc particles.

[0312] (2)NP HSA / mLuc particles were centrifuged at 12000g for 15 min and resuspended to remove free lipids and proteins. BALB / c mice were injected via tail vein with particles diluted in 1×PBS at a dose of 0.1 mg / kg. 24 hours after tail vein injection, 200 μL of 15 mg / mL sterile D-fluorescein potassium solution was injected. Bioluminescence signals in mice were detected 3 minutes later using the IVIS Lumina III (Perkin Elmer) small animal imaging system. Figure 45 shows the results. NP HSA / mRNA can be efficiently delivered in vivo. To further investigate NP in detail HSA In mice, the expression sites were determined by sacrificing the mice and harvesting the heart, liver, spleen, lungs, kidneys, and inguinal lymph nodes for analysis. The results, shown in Figure 46, reveal in vivo imaging of the ex vivo organs, indicating that NP... HSA It is expressed at high levels in both the lungs and spleen.

[0313] Example 21 NP HSA @mRNA expression distribution in various organs and cells in vivo

[0314] (1) C56BL / 6J mice were injected via tail vein with NP at a dose of 1 mg / kg mRNA. HSA@mEGFP, 24 hours after administration, mice were sacrificed and their blood, spleen, liver, and lungs were collected for testing;

[0315] (2) Blood sample processing: Blood was collected from the eyeballs of mice and dropped into centrifuge tubes pretreated with 30 μL of 10 mg / mL heparin sodium solution. Blood cells were collected by centrifugation at 3000 rpm for 5 min at 4°C. 1 mL of erythrocyte lysis buffer was added, and the cells were lysed at room temperature for 5 min. The cells were collected by centrifugation under the same conditions, and the lysis was repeated 2-3 times with erythrocyte lysis buffer until the blood sample cells turned white. The cells were resuspended in PBA until the density was 5 × 10⁻⁶. 6 / mL;

[0316] (3) Spleen processing: Mouse spleens were harvested and ground on a 200-mesh nylon mesh using a 1 mL sterile syringe plunger until single-celled cells were formed. Spleen cells were collected, centrifuged at 3000 rpm for 5 min at 4°C, the supernatant was discarded, and 2 mL of erythrocyte lysis buffer was added. Lysis was performed on ice for 3 min. 10 mL of PBA solution was added to stop erythrocyte lysis, and the cells were centrifuged at 3000 rpm for 5 min at 4°C. After discarding the supernatant, the cells were resuspended in 1 mL of PBA solution, passed through a double layer of 200-mesh nylon mesh, and counted. Cells were diluted with PBA to a density of 5 × 10⁶ cells / mL. 6 / mL;

[0317] (4) Lung and Liver Processing: Mice were sacrificed, and the lungs and livers were removed from the thoracic and abdominal cavities, respectively. They were placed in 5 mL EP tubes, and 3 mL of digestion solution was added. The tissues were then minced with scissors. The tissue suspension was placed in a 37°C shaking incubator at 200 rpm for 1 hour for digestion. The tissue suspension was passed through a 200-mesh steel mesh, and any incompletely digested tissue was gently ground using a syringe plunger. The dissociated tissue suspension was collected in a 15 mL centrifuge tube and centrifuged at 3000 rpm for 5 min at 4°C to collect lung and liver cells. Red blood cell lysis was performed at room temperature using 2 mL of erythrocyte lysis buffer for 3 min. The lysis was then terminated with 10 mL of PBA solution. After centrifugation, the cells were resuspended in 3 mL of PBA solution, passed through a double-layer 200-mesh nylon mesh, and counted. The cells were diluted with PBA to a density of 5 × 10⁶ cells / mL. 6 / mL;

[0318] (5) Flow cytometry detection: Take 1×10⁶ samples from each of the above samples. 6 Cells were collected in 1.5 mL centrifuge tubes, 0.3 μL of anti-mouse CD16 / 32 antibody was added, and the cells were blocked on ice for 20 min. Blocking was terminated with 1 mL of LPA. Cells were collected by centrifugation at 3000 rpm for 5 min at 4 °C. Cells were transferred to flow cytometry tubes, and DAPI-labeled cells were added to each tube. The percentage of EGFP-positive cells was detected using the FITC channel of a 12-color flow cytometer. As shown in Figure 47, compared to untreated mice, NP... HSAWhile @mEGFP expression efficiency increased in blood, EGFP MFI did not significantly improve. NP... HSA The mice in the @mEGFP administration group showed the highest EGFP expression efficiency in their lungs, reaching 1.5%, and a significant increase in MFI, indicating that NP HSA @mEGFP can be spontaneously expressed in the lungs through passive targeting in vivo. Meanwhile, NP... HSA The expression efficiency of EGFP in the spleen and liver of mice in the @mEGFP administration group increased, but the overall EGFP expression was still not significant due to the large number of cells in the organs.

[0319] Example 22 NP HSA @mRNA transfection efficiency on immune cells in vivo

[0320] (1)NP HSA The preparation, in vivo transfection, and processing of various organs of @mEGFP are the same as in Example 23;

[0321] (2) Count the cells in each group and take 2×10⁻⁶ cells. 6 One cell was placed in a 1.5 mL EP tube, centrifuged at 3000 rpm for 5 min at 4 °C, the supernatant was discarded, and the cells were resuspended in 100 μL PBA.

[0322] (3) Add 0.3 μL of anti-mouse CD16 / 32 antibody to each tube, block on ice for 20 min, add 1 mL PBA to stop the blocking, centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, and resuspend the cells in 50 μL PBA.

[0323] (4) Stain with BV510 anti-mouse CD45, APC anti-mouse CD3, APC / Cy7 anti-mouse B220, and PE / Cy7 anti-mouse F4 / 80 flow cytometry antibodies, and incubate at room temperature in the dark for 30 min. Add 1 mL of PBA to stop the fluorescent labeling, centrifuge at 3000 rpm for 5 min at 4℃, discard the supernatant, resuspend the cells in 500 μL of PBA, transfer the cells to flow cytometry tubes, add DAPI to label cell viability, and detect the fluorescence signal of immune cells in each organ using a 12-color flow cytometer. As shown in Figure 48, NP HSA @mEGFP is mainly expressed in macrophages of the spleen, liver and lungs, and the EGFP positivity rate and MFI in B cells have also increased.

[0324] Example 23 NP HSA @mRNA gene editing in cells of various organs in vivo

[0325] Investigating NP using B6-G / R transgenic miceHSA The effect of in vivo delivery of mCre. (The text abruptly ends here, likely due to an incomplete translation or a HSA @mEGFP was administered via tail vein injection to B6-G / R mice at a dose of 1 mg mRNA / kg. Lungs, spleen, and liver were collected at 0, 24, 48, and 72 hours after administration for frozen section fluorescence observation. NP... HSA @mCre in vivo gene-edited mouse organ effects visualization. The results are shown in Figure 49. With increasing administration time, red fluorescent signals gradually appeared in the lungs, spleen, and liver of the mice, indicating that NP... HSA @mCre successfully delivered mCre and expressed Cre protein. The strongest red fluorescence signal was observed in the lungs, indicating NP... HSA @mCre is primarily expressed and performs gene editing in the lungs, which is consistent with NP. HSA The results of in vivo delivery of mLuc and mEGFP were consistent. The spleen region was located in NP... HSA @mCre showed a red fluorescence signal 48 hours after administration, which weakened at 72 hours; the red fluorescence signal was weakest in the liver region. Experimental results indicate that NP HSA It is primarily expressed in the lungs, followed by the spleen, with the liver showing the weakest expression.

[0326] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A protein / polyester / lipid-nucleic acid delivery system, characterized in that, The nucleic acid delivery system includes a carrier and nucleic acid molecules. The carrier is a nano-assembly composed of protein, polyester, or lipid, and the nucleic acid is a single-stranded or double-stranded nucleotide molecule, satisfying one or more of the following characteristics: (i) The carrier comprises proteins, hydrophobic biodegradable polyesters or derivatives thereof, and lipids; (ii) The protein contained in the vector is a natural protein or a fusion protein expressed through genetic engineering; (iii) The lipids in the carrier are ionizable lipids and / or cationic lipids; (iv) The nucleic acid molecule is at least one of small interfering RNA, antisense oligonucleotide, microRNA, messenger RNA, circular RNA, self-replicating RNA, and plasmid DNA.

2. The protein / polyester / lipid-nucleic acid delivery system according to claim 1, characterized in that, The vector contains a protein that is either serum albumin or a fusion protein of a single-chain antibody and albumin.

3. The protein / polyester / lipid-nucleic acid delivery system according to claim 2, characterized in that, The natural proteins and fusion proteins are derived from mammals; and / or The single-chain antibody-albumin fusion protein contains a single-chain antibody targeting a cell surface antigen, a linker peptide, and a full-length or partial fragment of serum albumin; the single-chain antibody targets at least one of the following antigens, including but not limited to: HER2, HER3, EGFR, GPC3, CEA, PSMA, FAP, EpCAM, BCMA, CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD30, CD38, CD56, CD123, CD138, CD11c, F4 / 80 derived from mammals, including but not limited to mice, rats, rabbits, chickens, cattle, or humans.

4. The protein / polyester / lipid-nucleic acid delivery system according to claim 3, characterized in that, The single-chain antibody targets one, two, or three antigens.

5. The protein / polyester / lipid-nucleic acid delivery system according to claim 1, characterized in that, The hydrophobic biodegradable polyester is an aliphatic polyester, including at least one of polylactide, polyglycolic acid, poly(glycolic acid-co-lactide), and polycaprolactone; preferably, the aliphatic polyester is polylactide or poly(glycolic acid-co-lactide); the block molecular weight of the polylactide is 1-1,100 kDa, the block molecular weight of the poly(glycolic acid-co-lactide) is 1-1,100 kDa, and the ratio of glycolide to lactide is in the range of 95 / 5 to 50 / 50.

6. The protein / polyester / lipid-nucleic acid delivery system according to claim 1, characterized in that, The ionizable lipid is a positively charged lipid with protonated groups under acidic conditions (pH<7) and uncharged under neutral conditions (pH=7), preferably selected from SM-102 (1-octylnonyl 8-[(2-hydroxyethyl)[6-O-6-(undecyloxy)hexyl]amino]octanoate), ALC-0315 ((4-hydroxybutyl)azadiyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), DLin-MC3-DMA (4-(N,N-dimethylamino)butyrate (dilinyl)methyl ester).

7. The protein / polyester / lipid-nucleic acid delivery system according to claim 1, characterized in that, The cationic lipid is a permanently positively charged lipid molecule containing an amine group, preferably at least one of the following: DOTAP (2,3-dioleoyl-propyl)-trimethylammonium chloride), DODMA (1,2-dioleoyl-3-dimethylamino-propane), DOTMA (1,2-bisoctadecenoxy-3-methylammonium propane), DC-Chol (3β-[N-(N',N'-dimethylaminoethyl)aminoformylcholesterol).

8. The protein / polyester / lipid-nucleic acid delivery system according to claim 1, characterized in that, The ratio of cationic lipid to ionizable lipid is (0-12):(12-0), preferably (1-12):(12-1), and more preferably (1-3):(3-1).

9. The protein / polyester / lipid-nucleic acid delivery system according to claim 8, characterized in that, The total amino group ratio of the ionizable lipids and cationic lipids to the N / P ratio of nucleic acids is 4 to 12:1, preferably 6 to 10:

1.

10. The protein / polyester / lipid-nucleic acid delivery system according to claim 1, characterized in that, The nucleic acid delivery system is composed of nanoparticles with a particle size range of 50nm to 550nm, preferably 70nm to 250nm, and more preferably 80nm to 180nm.

11. A method for preparing the protein / polyester / lipid-nucleic acid delivery system according to any one of claims 1-10, characterized in that, Includes the following steps: (1) The buffer solution is mixed with the protein and nucleic acid respectively to prepare protein solution and nucleic acid solution as aqueous phase. The buffer solution is sodium citrate buffer or sodium acetate buffer. (2) Mix the hydrophobic biodegradable polyester or its derivative and the lipid with an organic solvent respectively to prepare a hydrophobic biodegradable polyester or its derivative solution and a lipid solution as the organic phase; (3) Mix the aqueous phase described in step (1) and the organic phase described in step (2), and perform ultrasonic emulsification on the resulting mixture, or perform high-pressure homogenization after uniform mixing to obtain an emulsion; (4) Remove the organic solvent from the emulsion, separate and purify it, and resuspend it in ultrapure water to obtain an aqueous solution of nanoparticles.

12. The preparation method according to claim 11, characterized in that, The final concentration of protein in the protein solution is 0.1 mg / mL to 20 mg / mL, preferably 0.1 mg / mL to 15 mg / mL, and more preferably 1 mg / mL to 10 mg / mL; And / or the concentration of the hydrophobic biodegradable polyester or its derivative solution is 0.1 mg / mL to 20 mg / mL, preferably 0.1 mg / mL to 15 mg / mL, more preferably 1 mg / mL to 10 mg / mL, and even more preferably 5 mg / mL to 10 mg / mL.

13. The preparation method according to claim 11 or 12, characterized in that, The concentration of the cationic lipid is 0.1–15 mg / mL; and / or the concentration of the ionizable lipid is 5 mg / mL–30 mg / mL, more preferably 10 mg / mL–25 mg / mL.

14. The preparation method according to claim 11 or 12, characterized in that, In the nucleic acid solution, the final concentration of the nucleic acid is 0.1 mg / mL to 2 mg / mL; preferably 0.5 mg / mL to 2 mg / mL. The mass ratio of nucleic acid to protein is 1:5 to 1000, preferably 1:50 to 500; The mass ratio of nucleic acid to the polyester is 1:1 to 200, preferably 1:10 to 100.

15. The preparation method according to claim 11 or 12, characterized in that, The volume ratio of the aqueous phase to the organic phase is 2 to 10:1, preferably 4 to 8:

1.

16. The preparation method according to claim 11, characterized in that, When the nucleic acid is siRNA, the sodium citrate buffer or sodium acetate buffer is 25 μM to 1 mM.

17. The preparation method according to claim 11, characterized in that, When the nucleic acid is mRNA, the sodium citrate buffer or sodium acetate buffer is 1mM to 25mM, preferably 10mM to 25mM, or preferably 5mM to 15mM sodium citrate buffer.

18. The preparation method according to claim 11, characterized in that, The method also includes adding a lyophilization protectant with a final concentration of 10% to 20% w / v to the purified nucleic acid nanoparticle solution and then freeze-drying it. The lyophilization protectant is at least one of the following: sucrose, glucose, trehalose, or mannitol.

19. The use of the protein / polyester / lipid-nucleic acid delivery system according to any one of claims 1-10 in the preparation of gene therapy drugs in which nucleic acid is one of the active ingredients.

20. The application according to claim 19, characterized in that, The gene therapy drugs mentioned are used to prevent or treat tumors, inflammation, hereditary diseases, or autoimmune diseases.

21. A treatment method for tumors, inflammation, hereditary diseases, or autoimmune diseases, characterized in that, The treatment method includes administering an appropriate dose of the protein / polyester / lipid-nucleic acid delivery system according to any one of claims 1-10 to the subject.

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