A gold-protein nanocomposite and its preparation method and application

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to a gold-protein nanocomplex and its preparation method and application. The present invention provides for the first time a method for constructing an in situ biosynthesized gold-protein / peptide nanocomplex based on a tumor microenvironment. Compared with the traditional method, the method does not require labeling of fluorescent fuel, is green and environmentally friendly, safe, has strong bioadaptability, and has high delivery efficiency. The obtained gold-protein nanocomplex can be used as a drug treatment, or as a nanoprobe for imaging to solve problems such as real-time monitoring and imaging of tumor treatment. The gold-p53 nanoprotein complex prepared by the present invention has high bioadaptability and low toxicity, can inhibit tumor cell proliferation, migration and invasion, promote T cell proliferation and activation, and inhibit liver cancer lung metastasis and tumor size in a mouse model. The combined use of the complex with PD-L1 and metformin has stronger application potential.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to a gold-protein nanocomposite and a preparation method and application thereof. Background Art

[0002] Cancer is the number one killer of humans and a worldwide problem in modern medicine. The three major mysteries of cancer are tumor metastasis, drug tolerance, and the role of non-tumor tissues, which are also the three major challenges faced by tumor research in clinical practice. Traditional therapies mainly act on most tumor cells, and the residual "seed" tumor stem cells can lead to tumor resistance, recurrence, and metastasis. There is an urgent need to discover new targets or new treatment strategies to enhance the sensitivity of chemotherapy / immunotherapy.

[0003] In 1982, the FDA approved insulin as a therapeutic drug, which greatly promoted the development of protein therapy. In 2023 alone, 16 antibody therapies were approved for the first time. Many other protein drugs are also being explored for the treatment of various diseases. However, the widespread application of protein therapy in cancer treatment has been hindered by various obstacles, such as the inability to deliver to the tumor site, low cell membrane permeability, and endosomal / lysosomal uptake. Considering the protection of carrier proteins and the diversity of in vivo applications, nano / microcarriers can be regarded as one of the most promising protein delivery methods, with the help of nanocarriers, proteins can be aggregated at the tumor site and absorbed by tumor cells.

[0004] As a star member of nanomaterials, metal nanoclusters have attracted much attention for their excellent performance and molecular-like properties. They exhibit unique optical, electrical and chemical properties, including excellent photostability, biocompatibility and low toxicity. The delivery of some proteins, such as cytokines and tumor suppressor genes, often relies on viruses or transfection reagents to enter cells, but the immunogenicity and side effects caused are also worrying. For example, gold nanoclusters (Au NCs) have the characteristics of adjustable size and easy surface modification. Proteins can be stably encapsulated in them through covalent or non-covalent interactions without affecting the original structure and function of the protein. In recent years, in situ self-assembly synthesis strategies of metal nanoprobes have been gradually reported. This method is not only green and environmentally friendly, does not require the addition of any fluorescent dyes, effectively delivers target genes to the disease area, but also can achieve real-time detection of target cells. Therefore, constructing metal cation in situ self-assembled precious metal-protein nanocomplexes, delivering exogenous active recombinant proteins (such as tumor suppressor protein p53) into tumor cells, and having visual functionalized nanoprobes can not only exclude the effects on non-tumor, but also effectively combine bioimaging technology with treatment methods. In addition, this new anti-cancer therapy helps chemotherapy / immunotherapy responsiveness, and adopting a multidisciplinary comprehensive treatment strategy is expected to achieve efficient individualized targeted precision treatment.

[0005] Immunotherapy shows unprecedented promise in the treatment of cancer. For example, immune checkpoint inhibitors such as PD-1 / PD-L1 are becoming promising treatment options for cancer patients. However, their therapeutic effects are far from satisfactory and are only effective in a small number of patients with immunologically "hot" tumor characteristics. How to increase the heat of "cold" tumors and transform them into "hot" tumors is the key to breaking through the existing treatment bottleneck. A large number of studies have shown that "cold-hot" tumors are directly related to the effect of PD-1 / L1 immunotherapy. Nanomedicines can enhance the immunogenicity of PD-1 / L1 by inducing immunogenic death to drive T cells into "cold" tumors and assist in the transformation of "cold-hot" tumors. Due to the complexity of the disease, drug optimization combined treatment strategies are also important. The combined therapeutic application value of precious metal-protein nanocomplexes and other drugs cannot be ignored.

[0006] Protein therapy has high specificity, selectivity, strong therapeutic effects, and low toxicity and side effects, and can effectively treat serious human diseases such as diabetes, cardiovascular disease, and malignant tumors. However, due to the degradation and metabolism of various enzymes in the body, protein drugs have shortcomings such as short half-life, instability, and variability in the body, which greatly limits their clinical application.

[0007] At present, the use of nanocarriers for cytoplasmic protein delivery is the most widely used delivery method. Most methods for cytoplasmic protein delivery require covalent modification of natural proteins, which usually involves complex synthetic processes and may lead to reduced protein biological activity and other biosafety issues. Therefore, the development of efficient and safe protein intracellular delivery systems is of great significance and has opened up new avenues for the development of anti-cancer therapy based on intracellular proteins. Summary of the invention

[0008] The purpose of the first aspect of the present invention is to provide a method for preparing a gold-protein nanoparticle complex.

[0009] The second aspect of the present invention aims to provide a gold-p53 protein nanocomplex.

[0010] The third aspect of the present invention aims to provide a composition comprising a gold-p53 protein nanocomplex and a PD-L1 inhibitor.

[0011] The fourth aspect of the present invention is to provide a composition comprising a gold-p53 protein nanocomplex and metformin.

[0012] The fifth aspect of the present invention aims to provide an application.

[0013] The sixth aspect of the present invention aims to provide a cell delivery method.

[0014] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0015] The first aspect of the present invention provides a method for preparing a gold-protein nanocomposite, comprising the following steps: incubating a gold salt precursor, a protein and tumor cells to obtain.

[0016] Preferably, the gold salt precursor comprises chloroauric acid.

[0017] Preferably, the tumor cells include at least one of HepG2 cells, HCCLM3 cells and Hepa1-6 cells.

[0018] Preferably, the protein comprises at least one of an enzyme, a growth factor, a hormone, a receptor, a transcription factor, an HLA-antigen polypeptide, a drug peptide, and a vaccine.

[0019] Preferably, the protein comprises p53 protein.

[0020] Preferably, the incubation temperature is 35-40°C;

[0021] Preferably, the incubation gas environment is 3-7 (v / v)% CO 2 ;

[0022] Preferably, the incubation time is 12 to 96 hours.

[0023] Preferably, the preparation method further comprises the steps of collecting cells, lysing, separating and purifying.

[0024] The second aspect of the present invention provides a gold-p53 protein nanocomplex prepared by the preparation method of the first aspect of the present invention.

[0025] Preferably, the particle size of the gold-p53 protein nanocomplex is 1 to 7 nm.

[0026] Preferably, the particle size of the gold-p53 protein nanocomplex is 4 to 5 nm.

[0027] The third aspect of the present invention provides a composition comprising the gold-p53 protein nanocomplex of the second aspect of the present invention and a PD-L1 inhibitor; or comprising the gold salt precursor, protein and PD-L1 inhibitor of the first aspect of the present invention.

[0028] Preferably, the gold salt precursor comprises chloroauric acid.

[0029] Preferably, the protein comprises p53 protein.

[0030] The fourth aspect of the present invention provides a composition comprising the gold-p53 protein nanocomplex of the second aspect of the present invention and metformin; or comprising the gold salt precursor, protein and metformin of the first aspect of the present invention.

[0031] Preferably, the gold salt precursor comprises chloroauric acid.

[0032] Preferably, the protein comprises p53 protein.

[0033] The fifth aspect of the present invention provides the use of the gold-p53 protein nanocomplex of the second aspect of the present invention, and / or the composition of the third aspect of the present invention, and / or the composition of the fourth aspect of the present invention in the preparation of a product for treating tumors.

[0034] Preferably, the tumor comprises liver cancer.

[0035] Preferably, the product includes at least one of a medicine and a probe.

[0036] Preferably, the drug further comprises other pharmaceutically acceptable excipients.

[0037] Preferably, the pharmaceutically acceptable excipients include but are not limited to at least one of a stabilizer, an antioxidant, a preservative, and a pigment.

[0038] Preferably, the medicine may also include other active ingredients.

[0039] Preferably, the p53 protein in the probe may also be modified with at least one of a fluorescent probe and a fluorescent antibody.

[0040] A sixth aspect of the present invention provides a cell delivery method:

[0041] The gold-p53 nanoprotein complex of the second aspect of the present invention is incubated with cells; or the gold salt precursor and protein of the first aspect of the present invention are incubated with cells.

[0042] Preferably, the protein comprises p53 protein.

[0043] Preferably, the cells comprise tumor cells.

[0044] Preferably, the tumor cells include at least one of HepG2 cells, HCCLM3 cells and Hepa1-6 cells.

[0045] Preferably, the gold salt precursor comprises chloroauric acid.

[0046] Preferably, the method is used for in vitro non-diagnostic, therapeutic purposes.

[0047] Preferably, the method is used for scientific research purposes.

[0048] The beneficial effects of the present invention are:

[0049] The present invention provides for the first time a method for constructing in situ biosynthesized precious metal gold-protein / peptide nanocomplexes based on the tumor microenvironment. Compared with the traditional method, this method does not require fluorescent dye labeling, is green and environmentally friendly, safe, has strong bioadaptability, and has high delivery efficiency. The gold-protein nanocomplex obtained based on this method can be used as a drug therapy or as a nanoprobe for imaging to solve the problems of real-time monitoring and imaging of tumor treatment.

[0050] The present invention prepares a gold-p53 nanoprotein complex, which has high biocompatibility and low toxicity, can inhibit tumor cell proliferation, migration and invasion, promote T cell proliferation and activation, and inhibit liver cancer lung metastasis and tumor size in a mouse model. The combined use of the complex with PD-L1 or metformin has stronger application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The uptake and intracellular localization of the in situ self-assembled Au NCs@p53 complex in HepG2 cells of the present invention.

[0052] Figure 2This is the particle size distribution result of the Au NCs@p53 complex prepared in HepG2 cells of the present invention.

[0053] Figure 3 It is the UV-visible absorption spectrum peak of the Au NCs@p53 complex prepared in HepG2 cells of the present invention.

[0054] Figure 4 The surface enhanced Raman spectrum band of the Au NCs@p53 complex prepared in HepG2 cells of the present invention.

[0055] Figure 5 Comparison of the inhibition of HCCLM3 cell proliferation by Au NCs@p53 complexes formed at different concentrations.

[0056] Figure 6 The in vivo distribution of Au NCs@p53 complex in HepG2 tumor-bearing mice 2 hours after treatment (using small animal in vivo imaging).

[0057] Figure 7 The statistical results of the weight of tumor tissue in HepG2 tumor-bearing mice after the treatment cycle of Au NCs@p53 complex was completed.

[0058] Figure 8 Statistical analysis results of the effect of Au NCs@p53 complex combined with PD-L1 inhibitor on the number of tumor cell metastatic nodules in Hepa1-6 tumor-bearing mice.

[0059] Fig. 9 Au NCs@p53 complex combined with metformin induces apoptosis of HepG2 tumor cells. DETAILED DESCRIPTION

[0060] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0061] Example 1 Preparation and Characterization of Gold-p53 Nanocomplex

[0062] 1) Preparation of gold-p53 nanocomplex

[0063] In this example, 1 g of chloroauric acid powder was dissolved in sterile distilled water to obtain 200 mM HAuCl 4Solution (gold ion precursor, CAS: 16903-35-8, pH = 6-8); the p53 recombinant protein from Escherichia coli was purified by affinity chromatography and the purity (≥90%) was detected by SDS-PAGE. In order to increase the stability and half-life of the p53 recombinant protein, a GST tag was modified at its N-terminus, with a total size of 82 kDa.

[0064] Principle of in situ self-assembled gold-p53 nanocomplex formation in tumor cells: Gold ion precursor solution consumes NADPH and H in the acidic tumor microenvironment 2 O 2 The gold nanoclusters are synthesized in situ by reducing substances such as Au, Au, O-Au, Au-S, etc., and then combined with p53 non-glycosylated polypeptide chains through physical adsorption, N-Au / O-Au / Au-S bonds and other non-covalent bonds to form gold nanoprotein complexes.

[0065] HAuCl was added to tumor cells (HepG2 cells, HCCLM3 cells, and Hepa1-6 cells) 4 Solution (10 μM for HepG2 cells, 30 μM for HCCLM3 cells, 20 μM for Hepa1-6 cells) and p53 protein (0.1 mg / mL, 50 μL) were incubated at 37°C and 5% CO 2 Incubate for 48 h under the same conditions.

[0066] This example tested the corresponding results in HepG2 cells, HCCLM3 cells and Hepa1-6 cells. Figure 1 The results of the formation and localization of gold-protein nanocomplexes in HepG2 cells are shown. The presence and cellular localization of gold nanoparticles-p53 (Au NCs@p53) complexes in HepG2 cells were observed after staining with p53 monoclonal antibody (ThermoScientific, MA515244). p53 protein was detected using p53 monoclonal antibody, and gold nanoclusters could be directly detected at 488nm due to their own fluorescence properties.

[0067] The above-mentioned HepG2 tumor cells were collected and repeatedly frozen and thawed 3-5 times until all cells were completely lysed, and sterile centrifuged at 2500 rpm for 20 min and filtered with a 0.22 μm filter to obtain a cytoplasmic extract containing a large amount of gold-p53 nanocomplexes.

[0068] 2) Characterization of physical and chemical properties

[0069] HepG2 cytoplasmic extracts containing gold-p53 nanocomplexes were visualized by transmission electron microscopy to observe the particle size of the nanocomplexes ( Figure 2). The optical, physical, chemical and biological properties were studied. Compared with the control group (only gold salt Au NCs group), the results showed that Au NCs@p53 had a significant UV-visible absorption spectrum peak at 277nm and a strong Raman spectrum fingerprint peak of the protein α-helix structure and CS bond ( Figure 3 and Figure 4 ).

[0070] Example 2 Gold-p53 Nanocomplex Inhibits Tumor Cell Proliferation

[0071] 1. Experimental methods

[0072] CCK8 proliferation assay was performed in HCCLM3 or HepG2 cells in vitro, indicating that HAuCl 4 After incubation of the solution (30 μM for HCCLM3 cells and 10 μM for HepG2 cells) and p53 protein (0.1 mg / mL 50 μL) for 120 h, Au NCs@p53 were self-assembled in situ and effectively inhibited the proliferation of HCCLM3 or HepG2 cells.

[0073] 2. Experimental results

[0074] Here we show the results of gold-p53 nanocomplexes in HCCLM3 cells. Figure 4 As shown, Au NCs@p53 effectively inhibited the proliferation of HCCLM3 cells ( Figure 4 ).

[0075] The Au NCs@p53 complex induces redox imbalance in tumor cells, triggering a large accumulation of ROS, leading to cell damage and even death.

[0076] ROS produced by oxidative stress can induce autophagy under various conditions. With the help of transmission electron microscopy, mitochondrial membrane potential and mitochondrial mass detection, it was found that Au NCs@p53 complex activated HepG2 cell mitochondrial autophagy through the PINK1 / Parkin signaling pathway, mediated endoplasmic reticulum stress, triggered immunogenic death and reshaped the immune microenvironment.

[0077] Example 3 Evaluation of the therapeutic effect and biosafety monitoring of gold-p53 nanocomplexes in vivo

[0078] 1. Experimental methods

[0079] 1×10 6 HepG2 or HCCLM3 cells were used to construct HepG2 and HCCLM3 tumor-bearing mouse models, respectively. When the tumor volume reached 50-100 mm 3At the same time, the tumor-bearing mice were randomly divided into three groups (control group, Au NCs group, Au NCs@p53 group, n=5 in each group). Before the experiment, the hemolysis test of the nanocomplex was performed to observe the tissue compatibility. The operation was as follows: fresh mouse blood (100-150 μL / mouse) was collected from the tail vein and placed in an EDTA anticoagulant tube and centrifuged at 3000 rpm for 10 minutes to obtain red blood cells. Subsequently, the red blood cells were cultured with different concentrations of nanomaterials (10 μM for HepG2 cells, 30 μM for HCCLM3 cells, 50 μL of p53 protein 0.1 mg / mL) at 37°C for 4 hours and centrifuged at 3000 rpm for 15 minutes. ddH 2 O and PBS were used as positive and negative controls, respectively. The supernatant of the samples was absorbed and the absorbance of the samples was detected at 542 nm using an enzyme labeling instrument.

[0080] Then PBS (100 μL), HAuCl 4 Solution (2mM 100μL) or HAuCl 4 The suspension of solution (2mM 100μL) and p53 (1-2mg / kg) was intravenously injected into tumor-bearing mice. The injection was intravenously injected once every three days for a total of 7 times. Live imaging of small animals was performed at 0h, 2h, 6h, 12h, and 24h after the first treatment. The tumor volume was weighed and monitored during the treatment (the calculation formula was length × width 2 / 2). All mouse experiments were approved by the hospital ethics review committee and performed in accordance with the ethics committee guidelines.

[0081] 2. Experimental results

[0082] The results suggest that in HAuCl 4 The solution has good blood compatibility at a concentration of 2 mM. 4 Solution (2mM 100μL) and p53 protein (1-2mg / kg) were administered every 3 days. Fluorescence ( Figure 6 ). At the end of the 21-day treatment cycle, Au NCs@p53 significantly inhibited the growth of HepG2 tumors compared with the control group ( Figure 7 ). Staining of major tissues and organs indicated that Au NCs@p53 did not cause liver and kidney damage and had low toxicity.

[0083] Example 4 Combination therapy of gold-p53 nanoprotein complex and PD-L1 inhibitor

[0084] 1. Experimental methods

[0085] C57BL / 6J mice were injected subcutaneously on one side of the abdomen with 1×10 65 × 10 Hepa1-6 cells were injected into the tail vein 5 days later. 5 Hepa1-6 cells were used to construct a mouse model of liver cancer lung metastasis / recurrence. When the primary tumor volume reached 50-100 mm 3 The mice were randomly divided into four groups (control group, PD-L1 inhibitor group, Au NCs@p53 group, and PD-L1 inhibitor + Au NCs@p53 group) and injected with HAuCl 4 Solution (2mM 100μL) and p53 protein (1-2mg / kg), intervention once every 3 days (injected on days 0, 3, 6, 9, 12, 15, and 18, for a total of 7 times); intraperitoneal injection of PD-L1 inhibitor (PD-1 / PD-L1-IN-8, GC62688, GlpBio, 50μg / mouse), intervention once every 4 days for a total of 5 times (injected on days 4, 8, 12, 16, and 20, for a total of 5 times). After the treatment (a total of 21 days), the mice were euthanized, and the main organs were harvested in vitro for staining and analysis, showing that the combined treatment strategy of the two inhibited lung metastasis / reduced recurrence.

[0086] 2. Experimental results

[0087] The results are as follows Fig. 9 As shown, the combination treatment of gold-p53 nanoprotein complexes with PD-L1 inhibitors significantly inhibited lung metastasis / reduced recurrence.

[0088] Example 5 Uptake and release of gold-p53 nanoprotein complexes

[0089] Referring to Example 1, HAuCl was added to HepG2 cells in sequence. 4 After the solution (10 μM) and p53 protein (0.1 mg / mL 50 μL) were incubated for 12 h, four recognized endocytosis inhibitors, chlorpromazine, Dynamin GTPase inhibitor (Dynasore), Rottlerin, and Genistein, were added, and the endocytosis mechanism of tumor cell uptake was further studied by flow cytometry.

[0090] The results showed that the uptake of Au NCs@p53 complexes mainly involved the large protein cytoplasmic pathway, clathrin-mediated endocytosis and caveolae-mediated endocytosis pathways.

[0091] When the Au NCs@p53 complex enters tumor cells, co-localization with lysosomes suggests that most Au NCs@p53 can successfully complete endosomal escape. In addition, GST catalyzes GSH to form a GS-GST complex, which is beneficial for the release of p53 to form an active tetramer.

[0092] Example 6 Gold-p53 Nanoprotein Complex Activates Anti-tumor Immunity in Vitro

[0093] Referring to Example 1, HAuCl was added to the tumor cells Hepa1-6 in sequence. 4 After incubation with the solution (20 μM) and p53 protein (0.1 mg / mL 50 μL) for 48 hours, Hepa1-6 tumor cells in different treatment groups (Control, Au NCs and Au NCs@p53 group) were co-cultured with mouse bone marrow-derived dendritic cells (BMDCs) through a co-culture system. The results showed that the Au NCs@p53 complex treatment group significantly promoted the BMDCs maturation rate by 41.8% (the Au NCs group was only 7.75%).

[0094] Tumor cells from different treatment groups were co-cultured with THP-1 cells through a co-culture system to observe the phenotypic changes of tumor-associated macrophages (TAMs). Flow cytometry showed that the Au NCs@p53 complex-treated group could reduce the tumor-promoting M2 TAMs (16.5% in the Au NCs group and only 3.17% in the Au NCs@p53 group) and increase the anti-tumor M1 TAMs (10.4% in the Au NCs group and increased to 23.4% in the Au NCs@p53 group), thus inducing the polarization of macrophages M2 to M1.

[0095] The T cell immune response and effector T-mediated killing ability were further evaluated. It was confirmed that the tumor cells in the Au NCs@p53 complex-treated group could promote T cell proliferation and activation and stimulate the potential of the body's immune function.

[0096] Example 7 Combination therapy of gold-p53 nanoprotein complex and metformin

[0097] Referring to Example 1, HAuCl was added to the tumor cells HepG2. 4 The solution (10 μM) and p53 protein (0.1 mg / mL 50 μL), as well as low-dose metformin reagent (Metformin, 10 μM) were co-incubated for 48 h, and the apoptosis of tumor cells was detected by one-step TUNEL kit ( Fig. 9 ). Statistical results showed that combined therapy induced more tumor cell apoptosis.

Claims

1. A method for in situ biosynthesis of gold-protein nanocomplexes in tumor cells, characterized in that: incubating the gold salt precursor, protein and tumor cells; The gold salt precursor is chloroauric acid solution; The tumor cells are HepG2 cells, HCCLM3 cells or Hepa1-6 cells; The protein is a P53 protein with a GST tag modified at the N-terminus; The incubation temperature is 35-40°C; The incubation gas environment is 3-7 v / v% CO2; The incubation time is 12 to 96 hours; Among them, chloroauric acid solution and 50 μL of 0.1 mg / mL p53 protein solution were added to the tumor cells respectively; When the tumor cells are HepG2 cells, the chloroauric acid solution is 10 μM; When the tumor cells are HCCLM3 cells, the chloroauric acid solution is 30 μM; When the tumor cells are Hepa1-6 cells, the chloroauric acid solution is 20 μM; The method is an in vitro method for non-diagnostic and non-therapeutic purposes.

2. A method for inducing apoptosis of tumor cells in vitro for non-disease treatment purposes, the steps of which are as follows: Chloroauric acid solution, 50 μL of 0.1 mg / mL N-terminally modified GST-tagged P53 protein solution, and metformin were added to HepG2 cells successively and incubated for 48 h; The chloroauric acid solution is 10 μM; The metformin is 10 μM; The incubation temperature is 37°C; The incubation gas environment is 5 v / v% CO2.

Images