阅读说明：本技术 应用于骨肉瘤细胞成像或治疗的Au DENPs-巨噬细胞复合物 (Au DENPs-macrophage compound applied to imaging or treating osteosarcoma cells ) 是由 王悍 尹芳芳 史向阳 范钰 于 2020-06-16 设计创作，主要内容包括：本发明公开了一种应用于骨肉瘤的细胞成像以及同时细胞治疗的Au DENPs-巨噬细胞复合物的制备方法：以第五代树状大分子为模板,将经马来酰亚胺活化的聚乙二醇单甲醚通过化学键间相互作用连接在G5.NH<Sub>2</Sub>的一端。上述得到的化合物通过物理吸附作用将HAuCl<Sub>4</Sub>整合在一起,再用NaBH<Sub>4</Sub>经过原位还原,最后,通过乙酸酐将树状大分子表面的氨基全部乙酰化以得到最终产生[(Au<Sup>0</Sup>)<Sub>100</Sub>-G5.NHAc-mPEG]DENPs,简称为Au DENPs。将Au DENPs与小鼠单核巨噬细胞RAW264.7细胞共培养获得Au DENPs-巨噬细胞复合物,可以用于小鼠骨肉瘤的细胞治疗或微环境中的CT成像,在肿瘤诊疗领域具有潜在的应用价值。(The invention discloses a preparation method of an Au DENPs-macrophage compound applied to cell imaging and simultaneous cell therapy of osteosarcoma, which comprises the following steps: taking fifth generation dendrimer as template, and connecting polyethylene glycol monomethyl ether activated by maleimide to G5.NH through chemical bond interaction 2 To one end of (a). The obtained compound is used for adsorbing HAuCl by physical adsorption 4 Integrated together, re-using NaBH 4 After in situ reduction, the amino groups on the surface of the dendrimer are fully acetylated by acetic anhydride to give the final product [ (Au) 0 ) 100 ‑G5.NHAc‑mPEG]DENPs, abbreviated as Au DENPs. Au DENPs and mouse mononuclear macrophage RAW264.7 cells are co-cultured to obtain Au DENPs-macrophage compound, the Au DENPs-macrophage compound can be used for cell therapy of mouse osteosarcoma or CT imaging in microenvironment, and has potential application value in the field of tumor diagnosis and treatment.)

1. A preparation method of Au DENPs-macrophage compound applied to cell imaging and cell therapy of osteosarcoma is characterized by comprising the following steps:

(1) adding G5 polyamide-amine dendrimer into a solvent to obtain a solution containing G5 polyamide-amine dendrimer, wherein the concentration of the solution is 3-4 mmol/L, then adding 11.8-12.5 mg/mL mPEG-MAL, and stirring for reaction for 3d to obtain G5.NH ₂-mPEG solution;

(2) dilution of G5.NH with Water₂-mPEG solution and addition of HAuCl₄The solution is stirred vigorously to obtain a gold/dendrimer mixture, the HAuCl₄Solution with G5.NH₂-mPEG molar ratio of 100;

(3) after the solution is vigorously stirred for 30min, 24-30 mg/mLNaBH is added₄Stirring the solution for 2h to obtain [ (Au)⁰)₁₀₀-G5.NH₂-mPEG]DENP；

(4) Triethylamine was added to [ (Au) under magnetic stirring⁰)₁₀₀-G5.NH₂-mPEG]Continuously stirring for 30min in DENP, adding acetic anhydride, and reacting for 24 h;

(5) dialyzing the mixed solution for 3d to remove excessive reactants, and freeze-drying to obtain a final product, namely Au DENPs;

(6) culturing mouse mononuclear macrophage RAW264.7 cells by using a DMEM cell culture medium containing 10% fetal calf serum, adding Au DENPs into the culture medium, and culturing for 12-48h to obtain Au DENPs-macrophage compound.

2. The method of claim 1, further comprising:

step (7) using LPS as a positive control, further using the technologies including flow, PCR, immunofluorescence and ELISA to detect the marker of M1 type macrophage in Au DENPs-macrophage complex, and judging the polarization state;

step (8), the Au DENPs-macrophage compound and mouse osteosarcoma K7 cells are incubated together, and the apoptosis condition of the mouse osteosarcoma K7 cells is detected by using the technology comprising flow, immunofluorescence and WesternBlot.

3. The method according to claim 1, wherein the concentration of triethylamine in the step (4) is 730.38 μ g/μ L, and the concentration of acetic anhydride is 1078.125 μ g/μ L.

4. The method of claim 1, wherein the quantitative standard for Au DENPs in step (6) is 200. mu.M [ Au ].

5. The method of claim 2, wherein the concentration of LPS in step (7) is 10ng/mL, and the markers of M1-type macrophages detected include CD86, TNF- α, and iNOS.

6. The method according to claim 2, wherein the indicator used in step (8) for determining apoptosis of K7 is Annexin V-FITC/PI or Caspase 3 protein.

7. An Au DENPs-macrophage complex for use in imaging or treating osteosarcoma, wherein Au DENPs in the Au DENPs-macrophage complex are internalized within macrophages but not adhered to the surfaces of the macrophages, wherein the Au DENPs-macrophage complex expresses M1 type macrophage markers, wherein the M1 type macrophage markers are CD86, TNF- α and iNOS, and wherein the Au DENPs-macrophage complex is configured to be prepared by the preparation method of claim 1.

Use of Au DENPs-macrophage complex for the preparation of CT contrast agents for targeted osteosarcoma.

The application of the Au DENPs-macrophage compound in preparing a targeted osteosarcoma CT contrast agent and a medicine for treating osteosarcoma at the same time.

The application of the Au DENPs-macrophage compound and an anti-tumor drug in preparing a drug for treating osteosarcoma.

Technical Field

The invention relates to the field of nano medical diagnosis and treatment reagents, in particular to an Au DENPs-macrophage compound applied to cell imaging or cell therapy of osteosarcoma and a preparation method thereof.

Background

Nanomedicine is gradually changing cancer treatment methods and bringing new hopes for the diagnosis and treatment of tumors. For example, some nano-drug delivery systems that passively target or actively target tumors can achieve accurate delivery of drugs in the body, thereby avoiding toxic effects on normal tissues, prolonging the metabolic processes of drugs in the body, and further enhancing the therapeutic effects of drugs.

Realizing the reaction imaging of tumors to immune nano-drugs is the key to designing, developing and applying effective targeted therapies. To verify the accuracy of the target, the distribution of the nanomedicine in the clinically relevant tissue must be monitored, preferably using non-invasive means, to determine the absorption in the target tissue and the minimum absorption in non-target tissues. The most straightforward way to achieve this is to label the nanomaterial so that it can be detected in vivo using an imaging system, such as MRI (magnetic resonance imaging), CT (computed tomography). This allows not only the biodistribution of the nanoparticles to be determined, but also the responsiveness of the tumour to treatment and other relevant responses, such as immune cell infiltration of the tumour, to be monitored. Interactions between nanoparticles and the surrounding environment and the range of cell types that may interact may occur. For example, the physical properties of nanoparticles can affect the immune system in terms of immunosuppression, hypersensitivity, immunogenicity, and autoimmunity. These interactions can result in a variety of effects, such as altering signaling pathways, destroying viruses that infect cells, and molecules, proteins, and chromatin complexes that are secreted from cells. Furthermore, nanoparticles can stimulate innate and adaptive immune responses through direct or indirect effects such as induction of cell necrosis.

The focus of previous cancer nanomedicine research is to target tumors or stromal cells, and cancer immunotherapy, which is a nanoparticle used as an effective immunostimulant with an anti-tumor response effect, is a new direction for cancer treatment. In contrast to traditional therapies, cancer immunotherapy specifically detects, recognizes and destroys malignant cells by enhancing the host's own immune system. The nano-particles have various physicochemical properties and can be used as potential immune activators. Stimulation of the innate and adaptive immune system by rationally designed cancer immune nanostructures would be important to enhance anti-tumor effects.

During the development and progression of tumors, the immune environment of a host is remodeled, and certain immune cells such as macrophages play roles in removing germs and defending in normal organisms. Tumor Associated Macrophages (TAMs) are a key component of the tumor microenvironment, and they account for approximately 50% of the tumor weight. During tumorigenesis, circulating macrophages can be recruited into the tumor microenvironment, playing a prominent role in the tumor evasion of immune surveillance of the body. Studies have shown that TAMs can be roughly divided into two categories: macrophages of the M1 type and M2 type, and are stimulated differently, with a shift between the two types being achieved. Wherein, M1 type macrophage can highly express CD86, iNOS, TNF-alpha and the like, and can play a role in inhibiting tumors. Most TAMs in tumor tissues appear as M2 type, and promote tumor growth, angiogenesis, invasion and metastasis. Many studies have shown that tumor-associated macrophages play an important role in the progression of many tumors, such as lung cancer, pancreatic cancer, breast cancer, prostate cancer, glioblastoma, osteosarcoma, and the like. These evidence suggest that targeting TAM is a viable strategy for tumor therapy. These strategies can be divided into three categories: 1) inhibition of tumor recruitment TAM; 2) direct killing of TAM; 3) TAM was re-educated to switch from the M2-pro-tumor phenotype to the M1 anti-tumor phenotype. Therefore, developing new nano-drugs against TAM, reversing the immunosuppressive tumor microenvironment is a promising opportunity.

Engineering of novel nano-drugs with the ability to target and kill or re-educate tumor-associated macrophages is a strategy for cancer immunotherapy that can induce efficient conversion of the tumor microenvironment rich in macrophages, which is conducive to tumor cell proliferation, into an anti-tumor type. In addition, the development of imaging nanostructures targeting TAMs can be used to study macrophage content in solid tumors, response to therapeutic efficacy, and prognostic information, to better diagnose and predict cancer.

Computed Tomography (CT) has the advantages of wide applicability, relatively low cost, high resolution of bone and lung tissues, and the like, and is the most widely used cancer screening imaging technique. Most of clinical common CT contrast agents are iodine agent micromolecules, but the imaging agents generally have the defects of short circulation half-life, poor targeting property and the like. The nanostructure formed by iodine or gold (or other large atoms) can increase the stability and prolong the circulation time, and can realize the specific targeting of tumor sites by modification. The nano material applied to tumor CT imaging comprises gold nanorods, dendritic molecules, iodic liposomes, lanthanum oxide nanoparticles and the like. Gold has a higher X-ray absorption coefficient than iodine. In addition, the loading of polyethylene glycol (PEG) on the surface of the gold nanoparticles can prolong the circulation time of the gold nanoparticles in blood.

In the field of CT imaging, the contrast of CT can be enhanced and natural killer cells can be stimulated to attack neuroblastoma and melanoma by gold nanoparticles targeted to GD2 antibody (Jiano P F, et al. J Mater Chem B.2016; 4(3): 513-. In another melanoma model using mice bearing human melanoma xenografts, whole-body CT imaging involving transduced T cells expressing melanoma-specific T cell receptors effectively characterized T cell distribution, migration and kinetics (Meir R, et al. ACS Nano.2015; 9(6): 6363-. A recent study has shown that FDA-approved iron deficiency patients can successfully use ferulic acid (iron oxide nanoparticles) to induce the transition of TAM from the immunosuppressive to the pro-inflammatory phenotype with anti-tumor and tumor metastasis reducing functions (Zanganeh, S, et al. Nature Nanotech 2016; 11, 986-.

In previous work, dendrimer encapsulated aunps (au denps) have been designed and surface functionalized for functional and structural imaging purposes, for CT/MR bimodal imaging, and for CT imaging guided chemotherapy, among others. The main advantages of Au DENPs are: 1) less than 5nm of Au can be embedded in each dendrimer; 2) the dendrimer periphery can be further functionalized to achieve different imaging and therapeutic functions. However, there are currently no reports of the use of audenps for macrophage polarization and tracking for cell therapy applications.

In the current research, the mouse macrophage is utilized to phagocytize Au DENPs and further differentiate the Au DENPs to M1 type macrophages, so that the CT imaging of the macrophages can be completed while the tumor is subjected to cellular immunotherapy.

The results of searching domestic and foreign literatures and patents related to the utilization of gold nanoparticle engineering for macrophage transformation for tumor treatment find that: before the completion of the present invention, no report on the application of macrophage polarization based on Au DENPs in cell imaging of osteosarcoma and cell therapy research has been found.

Therefore, those skilled in the art are working to develop a method for macrophage polarization based on Au DENPs and applied to cell imaging and cell therapy of osteosarcoma.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the technical problem to be solved by the present invention is how to CT-image osteosarcoma with Au DENPs-macrophage complex and perform cell therapy at the same time.

In order to achieve the above object, the present invention provides a method for preparing AuDENPs-macrophage complex for use in CT imaging and cell therapy of osteosarcoma, comprising the steps of:

(1) adding G5 polyamide-amine dendrimer into a solvent to obtain a solution containing G5 polyamide-amine dendrimer, wherein the concentration of the solution is 3-4 mmol/L, then adding 11.8-12.5 mg/mL mPEG-MAL, and stirring for reaction for 3d to obtain G5.NH ₂-mPEG solution;

specifically, the concentration of the G5 PAMAM dendrimer-containing solution was 4mmol/L, and the concentration of mPEG-MAL was 12.3 mg/mL.

(2) Dilution of G5.NH with Water₂-mPEG solution and addition of HAuCl₄The solution is stirred vigorously to obtain a gold/dendrimer mixture, the HAuCl₄Solution with G5.NH₂-mPEG molar ratio of 100;

specifically, the volume of the diluted G5.NH2-mPEG solution is 40 mL.

(3) After the solution is vigorously stirred for 30min, 24-30 mg/mLNaBH is added₄Stirring the solution for 2h to obtain [ (Au)⁰)₁₀₀-G5.NH₂-mPEG]DENP；

Specifically, the concentration of NaBH4 was 29 mg/mL.

(4) Triethylamine was added to [ (Au) under magnetic stirring⁰)₁₀₀-G5.NH₂-mPEG]Continuously stirring for 30min in DENP, adding acetic anhydride, and reacting for 24 h;

specifically, the concentration of triethylamine is 730.38 mug/muL, and the concentration of acetic anhydride is 1078.125 mug/muL.

(5) Dialyzing the mixed solution for 3d to remove excessive reactants, and freeze-drying to obtain a final product, namely AuDENPs;

specifically, the dialyzed solution was PBS buffer and water.

(6) Culturing mouse mononuclear macrophage RAW264.7 cells by using a DMEM cell culture medium containing 10% fetal calf serum, adding Au DENPs into the culture medium, and culturing for 12-48h to obtain Au DENPs-macrophage compound.

Specifically, the incubation time was 24 hours, and the quantitative standard of Au DENPs was 200. mu.M [ Au ].

Further, the method also comprises the following steps:

step (7) using LPS as a positive control, further using the technologies including flow, PCR, immunofluorescence and ELISA to detect the marker of M1 type macrophage in Au DENPs-macrophage complex, and judging the polarization state;

specifically, the concentration of LPS is 10ng/mL, and the markers of M1 type macrophages to be detected comprise CD86, TNF-alpha and iNOS.

Step (8), the Au DENPs-macrophage compound and mouse osteosarcoma K7 cells are incubated together, and the apoptosis condition of the mouse osteosarcoma K7 cells is detected by using the technology comprising flow, immunofluorescence and WesternBlot.

Specifically, the index for judging K7 cell apoptosis is Annexin V-FITC/PI or Caspase 3 protein.

The invention also provides an Au DENPs-macrophage complex applied to cell imaging and cell therapy of osteosarcoma, which is characterized in that Au DENPs in the Au DENPs-macrophage complex are internalized in macrophages but not adhered to the surfaces of the macrophages, the Au DENPs-macrophage complex expresses an M1 type macrophage marker, the M1 type macrophage marker is CD86, TNF-alpha and iNOS, and the Au DENPs-macrophage complex is prepared by the preparation method.

Furthermore, the size of gold nanoparticles in the Au DENPs is 1-6 nm, and the average value of the hydration particle size of the Au DENPs is 142.3-142.6 nm.

The invention provides an application of Au DENPs-macrophage complex in preparing a CT contrast agent for targeting osteosarcoma.

The invention also provides application of the Au DENPs-macrophage compound in preparing a targeted osteosarcoma CT contrast agent and a medicine for treating osteosarcoma at the same time.

The invention also provides application of the Au DENPs-macrophage compound combined antitumor drug in preparation of osteosarcoma drugs.

The results of characterizing the Au DENPs-macrophage complex obtained by the invention by using NMR (nuclear magnetic resonance), UV-Vis (ultraviolet visible spectrum), TEM (transmission electron microscope), DLS, CCK-8, CT imaging equipment, ICP-AES, optical lens, flow, immunofluorescence, WesternBlot (western blot), ELISA (enzyme-linked immunosorbent assay) are respectively as follows:

(1) results of NMR measurement

¹H NMR spectra indicated the type and number of dendrimer surface groups. Reference is made to the description accompanying figure 2. Shows that mPEG has been successfully modified at G5.NH₂On the surface of the dendrimer, acetylation of terminal amino groups was not affected.

(2) UV-Vis test results

The UV-Vis test result shows that: the nano-particles prepared by the method have obvious absorption peaks around 520 nm. Reference is made to the description accompanying figure 3.

(3) TEM test results

TEM test results show the size and size distribution of the gold nanoparticles. Reference is made to the description accompanying figure 4. The size of the gold nanoparticles is 1-6 nm, and good monodispersity is shown.

TEM was also used to show the distribution of gold nanoparticles in the subcellular compartments. Reference is made to the description accompanying fig. 5. Figure 5B clearly illustrates that more electron stained particles can be found in the cytoplasm of the cells after incubation with Au DENPs. While in fig. 5A it is shown that no electron stained particles were found in the cytoplasm of RAW264.7 cultured without Au DENPs, in strong contrast to fig. 5B. TEM confirmed that Au DENPs were internalized inside the cell rather than adhering to the cell surface.

(4) DLS test results

The DLS results show the size distribution of the hydrated particle size of the Au DENPs, and referring to Table 1, the average value of the hydrated particle size is about 142.6nm, which shows that the Au DENPs have better water solubility.

(5) Results of cytotoxicity test

The cytotoxicity test result shows that the nano-particles do not influence the viability of RAW264.7 cells in the range of 0-400 mu M, and do not show obvious cytotoxicity. Reference is made to the description accompanying figure 6.

(5) ICP measurement of cellular uptake results

The amount of Au DENPs taken up by mouse mononuclear macrophage RAW264.7 cells was calculated by ICP-AES. Referring to the attached FIG. 7 of the specification, it can be seen that the amount of gold nanoparticles in RAW264.7 cells is measured to be linearly increased along with the increase of [ Au ], and when [ Au ] reaches 200 μ M, the cell uptake can reach 10 pg/cell.

(6) Results of flow-type detection of Au DENPs-macrophage complex expression CD86

Expression of CD11b Using flow assay Au DENPs-macrophage Complex⁺CD86⁺Refer to fig. 8 of the specification. It can be seen that the proportion of double positive cells in the negative control Blank group was 7.82%, the proportion of double positive cells in the positive control LPS (lipopolysaccharide) group was 31.3%, and the proportion of double positive cells in the Au DENPs group, i.e., Au DENPs-macrophage complex was 41.2% (p < 0.05) compared with the negative control Blank group. Indicating that the Au DENPs-macrophage complex highly expresses the marker CD86 of M1 type macrophages.

(7) Results of ELISA detection of TNF-alpha expression of Au DENPs-macrophage complex

The TNF-alpha was detected by ELISA, according to the description of FIG. 9. It can be seen that AuDENPs-macrophage complex secreted TNF- α in comparable amounts to LPS, much higher than the Blank group (p < 0.01). Indicating that AuDENPs-macrophage complex highly expresses TNF-alpha, a macrophage marker of type M1.

(8) Result of immunofluorescence detection of Au DENPs-macrophage complex expression iNOS

The use of immunofluorescence for the detection of TNF- α is described in the specification accompanying FIG. 10. As can be seen, the Au DENPs-macrophage complex expressed iNOS in an amount comparable to that of LPS, while the Blank group hardly observed the expression of iNOS protein. Indicating that the Au DENPs-macrophage compound highly expresses M1 type macrophage marker iNOS.

(9) Results of flow-type detection of influence of Au DENPs-macrophage complex on apoptosis of mouse osteosarcoma K7

In normal living cells, Phosphatidylserine (PS) is located inside the cell membrane, and in the early stages of apoptosis, PS can evert from the inside of the cell membrane to the surface of the cell membrane, exposing to the extracellular environment. Annexin v (annexin v) is a calcium-dependent phospholipid-type binding protein that specifically binds to PS with high affinity. Therefore, the case of flow-detecting cells can be used, with reference to FIG. 11 of the specification. As can be seen, apoptosis was hardly detected in the control group; the apoptosis rate of the independent macrophage group is 9 percent and is lower than 10 percent; the apoptosis rate in the LPS + macrophage group was 14.38%; and the apoptosis rate of K7 cells in the Au DENPs group is obviously increased and reaches as high as 29.5%. Therefore, the Au DENPs-macrophage complex can promote K7 cell apoptosis.

(10) Flow detection of expression result of Au DENPs-macrophage compound on Caspase 3 expressed by mouse osteosarcoma K7 cells

Caspase 3 plays a key role in apoptosis, belongs to an effector of apoptosis, can enable cells to generate certain biochemical reactions after being activated, and can also enable the morphology of the cells to be changed, and finally leads to the apoptosis of the cells. Therefore, we chose to use immunofluorescence and immunoblotting to further detect the expression of activated Caspase 3 in K7 cells after incubation with various macrophages, see FIG. 12 and FIG. 13 of the specification. It can be seen that no obvious Caspase 3 expression was observed in the Control group and the macrophage alone group, while protein expression could be detected in the LPS + macrophage group and Au DENPs-macrophage complex group, wherein Caspase 3 expression level in the Au DENPs-macrophage complex group was higher than that in the LPS + macrophage group. In combination with the above results, it can be seen that Au DENPs-macrophage complex can promote apoptosis of K7 cells.

(11) CT imaging results of Au DENPs and Au DENPs-macrophage complex in vitro

The CT imaging results of in vitro Au DENPs are shown in figure 14. CT imaging was performed for different Au DENPs concentrations (7.5, 15, 30, 60 and 80mM), respectively. The results show that the CT image brightness (fig. 14A) and the signal intensity (CT signal intensity is CT value, fig. 14B) increase linearly with the increase of the Au DENPs concentration.

The CT imaging results of in vitro Au DENPs-macrophage complex refer to the description attached figure 15. After Au DENPs with different concentrations and macrophages are incubated together, cell suspension is prepared and is subjected to CT imaging. It can be seen that the CT value of Au DENPs-macrophage complex increases linearly with the increase of [ Au ]. In combination with the above results, the Au DENPs-macrophage complex can be used for CT imaging.

(12) CT imaging results of Au DENPs-macrophage complex in vivo

Figure 16 shows CT imaging of mouse osteosarcoma before and after intravenous Au DENPs-macrophage complex injection (figure 16A and figure 16B). The CT values at the tumor sites increased gradually over time compared to before injection, and the tumor sites maintained higher signal values until 4h after injection. The results indicate that the Au DENPs-macrophage complex can reach the tumor site with circulation and CT imaging of the tumor can be achieved.

(13) Antitumor effect of in vivo Au DENPs-macrophage complex

FIGS. 17-22 show that Au DENPs-macrophage complex has a certain therapeutic effect on osteosarcoma, and the combined use of AuDENPs-macrophage complex and DOX has a better therapeutic effect than that of single chemotherapy group, which indicates that Au DENPs-macrophage complex can further enhance the therapeutic effect of DOX on osteosarcoma.

Technical effects

(1) The preparation process is mild, simple and feasible;

(2) the gold nanoparticles prepared by the method have good stability and biocompatibility;

(3) the Au DENPs-macrophage complex prepared by the invention has good anti-tumor and CT imaging effects, and provides a new idea for tumor diagnosis and treatment and cancer immunotherapy.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 is a schematic diagram of Au DENPs according to a preferred embodiment of the present invention;

FIG. 2 is a drawing of the present inventionOf Au DENPs of a preferred embodiment¹H NMR spectrum;

FIG. 3 is a UV-Vis spectrum of Au DENPs of a preferred embodiment of the present invention;

FIG. 4 is a TEM picture of Au DENPs of a preferred embodiment of the present invention (a), and the corresponding size distribution histogram (b);

FIG. 5 is an electron micrograph of normal mouse mononuclear macrophages (A) and a bioelectronic micrograph of Au DENPs-macrophage complexes prepared according to the present invention (B), white arrows representing AuDNENPs phagocytosed by macrophages, according to a preferred embodiment of the present invention;

FIG. 6 shows the results of CCK-8 after co-culture of Au DENPs prepared by the present invention with RAW264.7 cells at various concentrations (0, 1, 5, 10, 25, 50, 75, 100, 200 and 400. mu.M) according to a preferred embodiment of the present invention;

FIG. 7 shows the gold nanoparticle uptake of Au DENPs in different concentrations (0, 25, 50, 75, 100 and 200 μ M) in the cells co-cultured with RAW264.7 according to a preferred embodiment of the present invention;

FIG. 8 shows the flow-based detection of the Au DENPs-macrophage complex and CD86 expression in each control group according to a preferred embodiment of the present invention;

FIG. 9 shows the results of ELISA assays for the expression of TNF- α in Au DENPs-macrophage complex and each control group according to a preferred embodiment of the present invention;

FIG. 10 shows the result of immunofluorescence assay of Au DENPs-macrophage complex and iNOS expression in each control group according to a preferred embodiment of the present invention;

FIG. 11 shows the Au DENPs-macrophage complex and the flow detection of K7 apoptosis in each control group according to a preferred embodiment of the present invention;

FIG. 12 shows the Au DENPs-macrophage complex and the immunofluorescence assay for K7 Caspase 3 expression for each control set in accordance with a preferred embodiment of the present invention;

FIG. 13 shows the Au DENPs-macrophage complex and WB assay of K7 Caspase 3 expression for each control set in accordance with a preferred embodiment of the present invention. A is a western blot detection strip, and B is a statistical analysis result of the protein expression of each group of cells;

FIG. 14 is a CT image (A) and CT values (B) of Au DENPs with different concentrations (7.5, 15, 30, 60, 80mM) according to a preferred embodiment of the present invention;

FIG. 15 is a CT image (A) and CT value (B) of Au DENPs-macrophage complex obtained by incubating Au DENPs and macrophages at different concentrations (25, 50, 75, 100, 200 μ M) according to a preferred embodiment of the present invention;

FIG. 16 is a CT image (A, B) and CT value (C) of Au DENPs and Au DENPs-macrophage complex in mouse osteosarcoma in situ model according to a preferred embodiment of the present invention;

FIG. 17 is a graph showing the survival rate of K7 cells measured by CCK-8 after different concentrations of DOX alone or in combination with Au DENPs-macrophage complex and K7 cells in accordance with a preferred embodiment of the present invention;

FIG. 18 shows the results of knee volume changes during treatment of mice of different treatment groups according to a preferred embodiment of the present invention;

FIG. 19 shows the results of weight changes during treatment of mice from different treatment groups according to a preferred embodiment of the present invention;

FIG. 20 shows the results of H & E pathology at the tumor site and TUNEL fluorescence in mice from different treatment groups according to a preferred embodiment of the present invention;

FIG. 21 shows the results of an enzyme-linked immunosorbent assay (ELISA) for detecting TNF- α in mouse serum from different treatment groups according to a preferred embodiment of the present invention;

FIG. 22 is a graph showing visceral H & E staining of mice from different treatment groups in accordance with a preferred embodiment of the present invention.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Reference herein to a "G5 polyamidoamine dendrimer" is a fifth generation polyamidoamine dendrimer.