CN111840324B - Au DENPs-macrophage complex applied to osteosarcoma cell imaging or treatment - Google Patents

Au DENPs-macrophage complex applied to osteosarcoma cell imaging or treatment Download PDF

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CN111840324B
CN111840324B CN202010547721.XA CN202010547721A CN111840324B CN 111840324 B CN111840324 B CN 111840324B CN 202010547721 A CN202010547721 A CN 202010547721A CN 111840324 B CN111840324 B CN 111840324B
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王悍
尹芳芳
史向阳
范钰
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Abstract

The invention discloses a preparation method of an Au DENPs-macrophage complex applied to cell imaging of osteosarcoma and cell therapy at the same time, which comprises the following steps: the fifth generation dendrimer is used as a template, and the polyethylene glycol monomethyl ether activated by the maleimide is connected with the G5.NH through the interaction between chemical bonds 2 Is provided. The above is obtainedBy physical adsorption of HAuCl 4 Integrated together and reused with NaBH 4 Through in-situ reduction, finally, the amino groups on the surface of the dendrimer are totally acetylated through acetic anhydride to obtain 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 complexes, and the Au DENPs-macrophage complexes can be used for cell therapy of mouse osteosarcoma or CT imaging in microenvironment, and have potential application value in the tumor diagnosis and treatment field.

Description

Au DENPs-macrophage complex applied to osteosarcoma cell imaging or treatment
Technical Field
The invention relates to the field of nano medical diagnosis and treatment reagents, in particular to an Au DENPs-macrophage complex applied to cell imaging or cell treatment of osteosarcoma and a preparation method thereof.
Background
Nano-medicine is gradually changing cancer treatment methods and brings new hopes for diagnosis and treatment of tumors. For example, some nano-drug delivery systems with passive targeting or active targeting on tumors can realize accurate delivery of drugs in organisms, so that toxic effects on normal tissues are avoided, metabolic processes of the drugs in the bodies are prolonged, and the curative effect of the drugs is further enhanced.
Realizing the reaction imaging of tumors to immune nano-drugs is the key to design, development and application of effective targeted therapies. To verify the accuracy of the target, the distribution of the nanomedicine in clinically relevant tissues must be monitored, preferably using non-invasive means, to determine the absorption by the target tissue and the minimum absorption by non-target tissues. The most straightforward way to achieve this is to label the nanomaterial so that the nanomaterial 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 tumor to treatment and other related reactions, such as the immune cell infiltration of the tumor. Interactions between the nanoparticle and the surrounding environment and the range of cell types that may be interacting 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 lead to a variety of effects such as altering signaling pathways, disrupting cell-infecting viruses and cell-endocrine molecules, proteins and chromatin complexes. In addition, the nanoparticles may stimulate innate and adaptive immune responses through direct or indirect effects such as induction of cellular necrosis.
In the past, the research of cancer nano medicine focuses on targeting tumor or stromal cells, and the nano particles are used as an effective immunostimulant with anti-tumor response effect, namely cancer immunotherapy, become a new direction of 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 physical and chemical properties and can be used as potential immune activators. Stimulation of the innate and adaptive immune system through rationally designed cancer immune nanostructures would be important for enhancing anti-tumor effects.
The immune environment of the host is remodelled during the occurrence and progression of tumors, wherein certain immune cells such as macrophages play a role in eliminating germs and playing a defensive role in a normal organism. Tumor-associated macrophages (tumor associated macrophage, TAM) are a key component of the tumor microenvironment, accounting for approximately 50% of tumor weight. In the course of tumorigenesis and development, circulating macrophages can be recruited into the tumor microenvironment, playing a prominent role in the immune surveillance of tumor evading organisms. Studies have shown that TAMs can be broadly divided into two categories: m1 type and M2 type macrophages, and are stimulated differently, transformation can be achieved between the two types. Wherein, the M1 type macrophage can highly express CD86, iNOS, TNF-alpha and the like, and can play a role in inhibiting tumor. While TAM in tumor tissues mostly shows M2 type, which can promote the growth, angiogenesis and invasion and metastasis of tumors. Numerous studies have shown that tumor-associated macrophages play an important role in the progression of various tumors, such as lung cancer, pancreatic cancer, breast cancer, prostate cancer, glioblastoma, osteosarcoma, and the like. These evidences indicate that TAM is a viable strategy for tumor treatment. These strategies can be divided into three categories: 1) Inhibition of tumor recruitment TAM; 2) Directly killing TAM; 3) TAMs were then educated to shift from the M2-tumor-promoting phenotype to the M1 anti-tumor phenotype. Therefore, the development of new nanomedicine against TAM, reversing the immunosuppressive tumor microenvironment, is a promising opportunity.
The engineering design of novel nanomedicines 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 tumor microenvironments rich in macrophages in favor of tumor cells into antitumor types. In addition, the development of imaging nanostructures targeting TAMs can be used to study macrophage content in solid tumors, respond to therapeutic efficacy of treatment, and prognostic information, thereby better diagnosing and prognosticating cancer.
Computer tomography (computed tomography, CT) has the advantages of wide applicability, relatively low cost, high resolution of bone and lung tissue, etc., and is the most widely used cancer screening imaging technique. Most of the CT contrast agents commonly used in clinic are iodine agent small molecules, but the imaging agents generally have the defects of short circulation half-life, poor targeting property and the like. While the iodine or gold (or other large atoms) form a nano structure, so that the stability of the nano structure can be improved, the circulation time of the nano structure can be prolonged, and the nano structure can be modified to realize specific targeting to tumor sites. The nanometer material applied to tumor CT imaging comprises gold nanometer rods, dendritic molecules, iodine-containing liposome, lanthanum oxide nanometer particles and the like. Gold has a higher X-ray absorption coefficient than iodine. In addition, polyethylene glycol (PEG) is loaded on the surface of the gold nanoparticles, so that the circulation time of the gold nanoparticles in blood can be prolonged.
In the field of CT imaging, CT contrast is enhanced and natural killer cell attack on neuroblastoma and melanoma is stimulated by gold nanoparticles targeting GD2 antibodies (Jiao P F, et al J Mater Chem B.2016;4 (3): 513-520). In another model of melanoma using mice bearing human melanoma xenografts, whole body CT imaging involving T cells transduced to express melanoma specific T cell receptors effectively characterizes T cell distribution, migration and kinetics (Meir R, et al ACS Nano.2015;9 (6): 6363-6372.). A recent study showed that FDA-approved iron deficiency patients can successfully use ferulic acid (iron oxide nanoparticles) to induce the transition of TAM from immunosuppressive to pro-inflammatory phenotype, with anti-tumor and tumor metastasis reduction functions (Zanganeh, S, et al Nature Nanotech 2016;11, 986-994).
In previous work, dendrimer-encapsulated Au NPs (Au DENPs) have been designed and their surfaces functionalized for functional and structural imaging purposes, for CT/MR bimodal imaging, for CT imaging guided chemotherapy, and the like. The main advantages of Au DENPs are: 1) Au less than 5nm may be embedded in each dendrimer; 2) The dendrimer periphery may be further functionalized to achieve different imaging and therapeutic functions. However, at present, there is no report on the use of Au DENP for macrophage polarization and tracking for cell therapy applications.
In the current research, the mouse macrophage is utilized to phagocytose Au DENPs and further differentiate towards M1 type macrophages, so that CT imaging of the macrophages can be completed while cell immunotherapy on tumors can be realized.
Searching the literature and patent results about engineering macrophages by gold nanoparticles at home and abroad for tumor treatment, and finding: before the completion of the present invention, no report has been found on application of Au DENPs-based macrophage polarization to cell imaging of osteosarcoma and cell therapy research.
Accordingly, those skilled in the art have focused on developing 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 use Au DENPs-macrophage complexes for CT imaging of osteosarcoma and simultaneous cell therapy.
In order to achieve the above object, the present invention provides a preparation method of an Au DENPs-macrophage complex for CT imaging and cell therapy of osteosarcoma, comprising the steps of:
(1) Adding G5 polyamide-amine dendrimerAdding the mixture into a solvent to obtain a G5 polyamide-amine dendrimer solution with the concentration of 3-4 mmol/L, then adding 11.8-12.5 mg/mL of mPEG-MAL, and stirring for 3d to obtain G5.NH 2 -mPEG solution;
specifically, the concentration of the G5 polyamide-amine dendrimer-containing solution was 4mmol/L, and the concentration of mPEG-MAL was 12.3mg/mL.
(2) Dilution of G5.NH with Water 2 -mPEG solution and adding HAuCl 4 The solution was stirred vigorously to obtain a gold/dendrimer mixture, the HAuCl 4 Solution with G5.NH 2 -mPEG at a molar ratio of 100;
specifically, the diluted G5.NH2-mPEG solution has a volume of 40mL.
(3) Stirring the solution vigorously for 30min, and then adding 24-30 mg/mLNaBH 4 Stirring the solution for 2 hours to obtain [ (Au) 0 ) 100 -G5.NH 2 -mPEG]DENP；
Specifically, the concentration of NaBH4 was 29mg/mL.
(4) Triethylamine was added to [ (Au) with magnetic stirring 0 ) 100 -G5.NH 2 -mPEG]Adding acetic anhydride into DENP after continuously stirring for 30min, and reacting for 24h;
specifically, the triethylamine concentration was 730.38. Mu.g/. Mu.L, and the acetic anhydride concentration was 1078.125. Mu.g/. Mu.L.
(5) Dialyzing the mixed solution for 3d to remove excessive reactants, and freeze-drying to obtain a final product, namely Au DENPs;
specifically, the dialyzed solution was PBS buffer and water.
(6) The culture medium of DMEM cell containing 10% fetal bovine serum is used for culturing the mouse mononuclear macrophage RAW264.7 cells, and the Au DENPs are added into the culture medium for culturing for 12-48 hours to obtain the Au DENPs-macrophage complex.
Specifically, the incubation time was 24 hours, and the quantitative standard of Au DENPs was 200. Mu.M.
Further, the method further comprises the following steps:
step (7) using LPS as positive control, further using techniques including flow, PCR, immunofluorescence and ELISA to detect markers of M1 type macrophages in the Au DENPs-macrophage complex, and judging the polarization state of the markers;
specifically, the concentration of LPS was 10ng/mL, and markers of M1 type macrophages tested included CD86, TNF-. Alpha.and iNOS.
And (8) incubating the Au DENPs-macrophage complex with the mouse osteosarcoma K7 cells, and detecting apoptosis of the mouse osteosarcoma K7 cells by using technologies including flow, immunofluorescence and WesternBlot.
Specifically, the index for judging the apoptosis of the K7 cells 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 instead of adhering to the surfaces of the macrophages, the Au DENPs-macrophage complex expresses M1 type macrophage markers, the M1 type macrophage markers are CD86, TNF-alpha and iNOS, and the Au DENPs-macrophage complex is prepared by the preparation method.
Further, the gold nano particle size 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.
In one aspect, the invention provides the use of an Au DENPs-macrophage complex in the preparation of a CT contrast agent for targeting osteosarcoma.
In another aspect, the invention provides the use of an Au DENPs-macrophage complex in the preparation of a medicament for targeting osteosarcoma CT contrast agents and simultaneously treating osteosarcoma.
The invention also provides application of the Au DENPs-macrophage complex combined antitumor drug in preparation of osteosarcoma drugs.
The results of characterizing the Au DENPs-macrophage complexes obtained by the present invention using NMR (nuclear magnetic resonance), UV-Vis (ultraviolet visible spectrum), TEM (transmission electron microscope), DLS, CCK-8, CT imaging device, ICP-AES, optical glasses, flow, immunofluorescence, westernBlot (western blot), ELISA (enzyme linked immunosorbent assay) are as follows, respectively:
(1) Results of NMR test
1 The H NMR spectrum indicates the type and number of dendrimer surface groups. Reference is made to figure 2 of the accompanying drawings. Indicating that mPEG has been successfully modified at G5.NH 2 The surface of the dendrimer has no influence on the acetylation of the terminal amino group.
(2) UV-Vis test results
The UV-Vis test results show that: the nano particles prepared by the method have obvious absorption peaks at about 520 nm. Reference is made to figure 3 of the accompanying drawings.
(3) TEM test results
TEM test results show the size and size distribution of gold nanoparticles. Reference is made to figure 4 of the accompanying drawings. The gold nanoparticles have a size of 1 to 6nm and exhibit good monodispersity.
TEM was also used to show the distribution of gold nanoparticles in subcellular compartments. Reference is made to figure 5 of the accompanying description. FIG. 5B clearly illustrates that more electronically stained particles can be found in the cytoplasm of cells after incubation with Au DENPs. In contrast, FIG. 5A shows that no electronically stained particles are found in the cytoplasm of RAW264.7 without Au DENPs culture, in sharp contrast to FIG. 5B. TEM confirmed that Au DENPs internalized within the cell rather than adhering to the cell surface.
(4) DLS test results
The DLS results showed a size distribution of hydrated particle sizes of Au DENPs, and with reference to Table 1, the average hydrated particle size was about 142.6nm, indicating that Au DENPs had good water solubility.
(5) Cytotoxicity test results
The cytotoxicity test results show that in the range of 0-400 mu M, the nano particles have no influence on RAW264.7 cell activity and do not show obvious cytotoxicity. Reference is made to figure 6 of the accompanying drawings.
(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 FIG. 7 of the specification, it can be seen that the amount of gold nanoparticles in RAW264.7 cells increases linearly with increasing [ Au ], and that when [ Au ] reaches 200. Mu.M, the cell uptake can reach 10pg/cell.
(6) Results of flow assay for CD86 expression by Au DENPs-macrophage complexes
Detection of Au DENPs-macrophage Complex expression CD11b Using flow + CD86 + Refer to figure 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., the Au DENPs-macrophage complex, was 41.2% (p < 0.05). The Au DENPs-macrophage complex was demonstrated to highly express the marker CD86 of M1 type macrophages.
(7) ELISA detection of results of expression of TNF-alpha by Au DENPs-macrophage complexes
The ELISA was used to detect TNF- α, and reference is made to FIG. 9 of the specification. As can be seen, the Au DENPs-macrophage complexes secreted TNF-. Alpha.in amounts comparable to LPS, much higher than those of the Blank group (p < 0.01). The Au DENPs-macrophage complex was demonstrated to highly express the M1-type macrophage marker TNF- α.
(8) Immunofluorescence detection of Au DENPs-macrophage Complex expression iNOS results
In the case of using immunofluorescence to detect TNF- α, reference is made to FIG. 10 of the specification. It can be seen that the Au DENPs-macrophage complex expressed iNOS in an amount comparable to LPS, whereas the Blank group hardly seen expression of iNOS protein. The Au DENPs-macrophage complex was demonstrated to highly express the M1 type macrophage marker iNOS.
(9) Results of flow detection of the influence of the Au DENPs-macrophage complex on apoptosis of mouse osteosarcoma K7 cells
In normal living cells, phosphatidylserine (PS) is located inside the cell membrane, whereas in the early stages of apoptosis, PS can evert from inside the cell membrane to the surface of the cell membrane, exposing to the extracellular environment. Annexin V is a calcium-dependent phospholipid-binding protein capable of binding specifically to PS with high affinity. Thus, the flow assay for cells may be used, see FIG. 11 of the specification. It can be seen that almost no apoptosis was detected in the control group; the apoptosis rate of the individual macrophage group is 9% and is lower than 10%; apoptosis rate in lps+macrophage group was 14.38%; whereas the apoptosis rate of K7 cells in Au DENPs group was significantly increased, as much as 29.5%. From this, it can be seen that the Au DENPs-macrophage complex can promote apoptosis of K7 cells.
(10) Flow detection of expression results of Au DENPs-macrophage complexes on Caspase 3 expression of mouse osteosarcoma K7 cells
Caspase 3 plays a key role in apoptosis, is an effector of apoptosis, and after being activated, can cause cells to perform some biochemical reactions, can also cause the morphology of the cells to change, and finally leads to apoptosis of the cells. Therefore, we selected to further examine the expression of activated Caspase 3 in K7 cells after incubation with various macrophages using immunofluorescence and immunoWestern blotting, see FIGS. 12 and 13 of the specification. It can be seen that the Control group and the individual macrophage group did not see significant Caspase 3 expression, whereas protein expression could be detected in the LPS+macrophage group and the Au DENPs-macrophage complex group, where the amount of Caspase 3 expression was higher in the Au DENPs-macrophage complex group than in the LPS+macrophage group. In combination with the above results, it can be seen that the Au DENPs-macrophage complexes can promote apoptosis of K7 cells.
(11) CT imaging results of in vitro Au DENPs, au DENPs-macrophage complexes
The results of CT imaging of in vitro Au DENPs are shown in FIG. 14. CT imaging was performed on different Au DENPs concentrations (7.5, 15, 30, 60 and 80 mM), respectively. The results showed that the brightness (fig. 14A) and signal intensity (CT signal intensity is CT value, fig. 14B) of the CT image increased linearly with increasing concentration of Au DENPs.
The results of CT imaging of in vitro Au DENPs-macrophage complexes are shown in FIG. 15 of the specification. After incubation of different concentrations of Au DENPs with macrophages, cell suspensions were prepared and CT imaged. It can be seen that the CT number of the Au DENPs-macrophage complexes increases linearly with increasing [ Au ]. The above results are combined to demonstrate that Au DENPs-macrophage complexes can be used for CT imaging.
(12) CT imaging results of in vivo Au DENPs-macrophage complexes
FIG. 16 shows CT imaging of bone and meat tumors of mice before and after intravenous injection of Au DENPs-macrophage complexes (FIGS. 16A and 16B). Compared with the injection before, the CT value of the tumor part is gradually increased with the lapse of time, and the tumor part still maintains a higher signal value until 4 hours after the injection. The results demonstrate that the Au DENPs-macrophage complexes can reach the tumor site with circulation and CT imaging of the tumor can be achieved.
(13) Antitumor effects of in vivo Au DENPs-macrophage complexes
FIGS. 17-22 show that the Au DENPs-macrophage complexes have a therapeutic effect on osteosarcoma, and that the combination of the Au DENPs-macrophage complexes with DOX has a therapeutic effect superior to that of the chemotherapeutic group alone, demonstrating that the Au DENPs-macrophage complexes can further enhance the therapeutic effect of DOX on osteosarcoma.
Technical effects
(1) The preparation process is mild, simple and easy to implement;
(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, specific structure, and technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, features, and effects of the present invention.
Drawings
FIG. 1 is a schematic diagram of an Au DENPs of a preferred embodiment of the invention;
FIG. 2 is a schematic diagram of an Au DENPs of a preferred embodiment of the invention 1 H NMR spectrum;
FIG. 3 is a UV-Vis spectrum of Au DENPs of a preferred embodiment of the invention;
FIG. 4 is a TEM image (a) of Au DENPs of a preferred embodiment of the invention, and a corresponding size distribution histogram (b);
FIG. 5 is an electron microscope image (A) of a normal mouse mononuclear macrophage and a bioelectrical microscope image (B) of an Au DENPs-macrophage complex prepared according to the present invention, and white arrows represent Au DNENPs phagocytosed by macrophages;
FIG. 6 is a graph showing the results of CCK-8 after co-culturing Au DENPs prepared in accordance with the present invention with RAW264.7 cells at various concentrations (0, 1,5, 10, 25, 50, 75, 100, 200 and 400. Mu.M);
FIG. 7 shows the uptake of gold nanoparticles by cells after co-culturing Au DENPs of a preferred embodiment of the invention with RAW264.7 cells at various concentrations (0, 25, 50, 75, 100 and 200. Mu.M);
FIG. 8 shows the results of a flow assay for Au DENPs-macrophage complexes and CD86 expression from various control groups in accordance with a preferred embodiment of the invention;
FIG. 9 shows ELISA results for the expression of TNF- α by Au DENPs-macrophage complexes and various control groups in accordance with a preferred embodiment of the present invention;
FIG. 10 shows immunofluorescence of Au DENPs-macrophage complexes and iNOS expressed by each control group according to a preferred embodiment of the present invention;
FIG. 11 is a flow chart of the Au DENPs-macrophage complexes and the results of the flow assay of K7 apoptosis for each control group in accordance with a preferred embodiment of the invention;
FIG. 12 shows immunofluorescence of Au DENPs-macrophage complexes and K7 apoptosis protein Caspase 3 expression from various control groups according to a preferred embodiment of the invention;
FIG. 13 shows the results of WB detection of K7 apoptosis protein Caspase 3 expression by Au DENPs-macrophage complexes and respective control groups according to a preferred embodiment of the present invention. A is a western blot detection strip, B is a statistical analysis result of the expression of each group of cell proteins;
FIG. 14 is a CT image (A) and CT values (B) of Au DENPs of different concentrations (7.5, 15, 30, 60, 80 mM) according to a preferred embodiment of the invention;
FIG. 15 shows CT images (A) and CT values (B) of Au DENPs-macrophage complexes obtained after incubation of Au DENPs with macrophages at different concentrations (25, 50, 75, 100, 200. Mu.M) according to a preferred embodiment of the invention;
FIG. 16 is a graph (A, B) and CT values (C) of CT images of Au DENPs and Au DENPs-macrophage complexes in an in situ model of mouse osteosarcoma according to a preferred embodiment of the invention;
FIG. 17 shows the results of survival of K7 cells using CCK-8 after incubation of K7 cells with different concentrations of DOX alone or with Au DENPs-macrophage complexes in accordance with a preferred embodiment of the present invention;
FIG. 18 shows the results of knee volume changes during treatment in mice of different treatment groups according to a preferred embodiment of the present invention;
FIG. 19 shows the results of weight change during treatment of mice of different treatment groups according to a preferred embodiment of the present invention;
FIG. 20 shows pathological H & E detection and TUNEL fluorescence detection results of tumor sites of mice of different treatment groups according to a preferred embodiment of the present invention;
FIG. 21 shows the results of enzyme-linked immunosorbent assay (ELISA) of TNF- α in mice of different treatment groups according to a preferred embodiment of the invention;
FIG. 22 shows visceral H & E staining results of mice of different treatment groups according to a preferred embodiment of the invention.
Detailed Description
The following description of the preferred embodiments of the present invention refers to the accompanying drawings, which make the technical contents thereof more clear and easy to understand. The present invention may be embodied in many different forms of embodiments and the scope of the present invention is not limited to only the embodiments described herein.
As used herein, "G5 polyamide-amine dendrimer" refers to a fifth generation polyamide-amine dendrimer.
Example 1
First, according to the previous study of the subject group, a fifth generation polyamide-amine dendrimer (G5. NH 2 ) 40mg, dissolved in 5mL of deuterated water to obtain a solution. Magnetic stirring will contain 61.5mg mPEG-The solution of MAL was added dropwise to the above-mentioned solution. Stirring with strong magnetic force for 3 days to complete the reaction to obtain G5.NH 2 -mPEG solution. Then, the solution was diluted to 40mL and added with G5.NH under strong magnetic stirring 2 HAuCl with a mPEG molar ratio of 100 4 A solution. After 30min of reaction, ice containing 29mgNaBH was added 4 Stirring was continued for 2 hours to complete the reaction to give [ (Au) 0 ) 100 -G5.NH 2 -mPEG]DENPs。
Then, the [ (Au) 0 ) 100 -G5.NH 2 -mPEG]The DENPs are further acetylated to neutralize amino groups at the end of the dendrimer. 117.2. Mu.L of solution containing 85.6mg of triethylamine was added to [ (Au) with magnetic stirring 0 ) 100 -G5.NH 2 -mPEG]DENPs solution. After 10min of reaction, acetic anhydride (69 mg,64 μl) was added to the Au DENPs/triethylamine mixture solution with stirring, and the mixture was reacted for 24h. The reaction mixture was then extensively dialyzed against PBS buffer (4L) and water (4L) for 3 days by cleavage of a membrane having a molecular weight of 5000 to remove excess reactants, and then lyophilized to obtain the final product [ (Au) 0 ) 100 -G5.NHAc-mPEG]DENPs (abbreviated as Au DENP).
1 H NMR spectrum (FIG. 2) demonstrates-COCH 3 Has been successfully modified in mPEG-G5.NH 2 On the surface of the dendrimer. The absorption peak at 520nm in the ultraviolet spectrum is the absorption peak of gold nanoparticles (figure 3).
TEM test results show that the particle size distribution of the prepared Au DENPs is 1-6 nm (figure 4), and the agglomeration phenomenon does not occur. The DLS results show that the hydration particle size of the Au DENPs is about 142.6nm, the polydispersity index is 0.698, and the surface charge is 6.44+ -0.2 mV (see Table 1), which indicates that the Au DENPs has good monodispersity and good water solubility.
TABLE 1 hydrodynamic size and surface charge of Au DENPs
Figure BDA0002541345070000091
Example 2
Mouse mononuclear macrophage RAW264.7 cells were cultured using DMEM containing 10% fetal bovine serum. According to 3X 10 5 Is inoculated in 6-well plates at 37℃with 5% CO 2 Is cultured in a cell culture incubator for 24 hours. Au DENPs containing gold at a concentration of 200. Mu.M were added to the RAW264.7 broth to be cultured for 24 hours. The medium was then discarded, the cells were rinsed with PBS buffer, the cells were digested with pancreatin, centrifuged, and then rinsed 3 more times with PBS buffer. Then, the mixture was fixed with 1% osmium acid 0.1M phosphate buffer PBS (pH 7.4) at room temperature (20 ℃) for 2 hours, and rinsed with 0.1M phosphate buffer PBS (pH 7.4) 3 times for 15 minutes each. Sequentially adding the to-be-detected substances into 50% alcohol-70% alcohol-80% alcohol-90% alcohol-95% alcohol-100% acetone for upstream dehydration for 15min each time. And then using acetone:812 embedding medium=1:1, 2-4h, penetrating acetone:812 embedding medium=1:2 overnight, pure 812 embedding medium for 5-8h, pouring pure 812 embedding medium into the embedding plate, and inserting the sample into the embedding plate and then using an oven at 37 ℃ overnight. Then, the mixture was polymerized in an oven at 60℃for 48 hours. The 60-80 nm ultra-thin slice is cut by an ultra-thin slicer. Uranium-lead double staining (2% uranium acetate saturated alcohol solution, lead citrate, 15min each) was used and the sections were dried overnight at room temperature. Finally, observation under a transmission electron microscope is performed.
The distribution of nanoparticles in subcellular organelles was shown by TEM (fig. 5). FIG. 5B clearly shows that stained particles of nanoparticles can be found in subcellular organelles of cells after co-culture with Au DENPs. No electronically stained particles were found in subcellular organelles of RAW264.7 cells not co-cultured with Au DENPs shown in fig. 5A. TEM confirmed that Au DENPs internalized within the cell rather than adhering to the cell surface.
Example 3
According to 1X 10 4 Cell/well density cultured RAW264.7 cells were seeded in 96-well plates and cultured in a cell incubator for 24 hours. Washed 3 times with PBS, and then added with different concentrations of Au DENPs ([ Au)]=0, 1,5, 10, 25, 50, 75, 100, 200, 400 μm), 6 duplicate wells per group, and incubation was continued for 24h. Subsequently, the medium was discarded, the cells were washed 3 times with PBS, and 90. Mu.L of fresh DMEM medium and 10. Mu.L of CCK-8 solution were added to each well, and the culture was continued in a cell incubator2h. Then, absorbance at 450nm was detected using a microplate reader.
The CCK-8 test results are shown in FIG. 6, and Au DENPs do not show cytotoxicity to RAW264.7 cells in the experimental concentration range (0-400 mu M), and show good cell compatibility.
Example 4
According to 3X 10 5 Cell/well density cultured RAW264.7 cells were seeded in 6-well plates, cultured for 24 hours, and then Au DENPs with gold concentrations of 0, 25, 50, 75, 100, 200 μm were added, and co-cultured with the cells for 24 hours. After 3 times of washing with PBS, the cells were trypsinized, collected by centrifugation, and decomposed by aqua regia. Each experiment was performed in triplicate. Uptake of the nanoprobe by cells was quantified using ICP-AES (FIG. 7). The results show that as the gold concentration increases, the amount of gold nanoparticles taken up by the cells appears to increase linearly with it. When the gold concentration reaches 200. Mu.M, the cell uptake can reach 10pg/cell.
Example 5
RAW264.7 cells were plated at 1X 10 per well 5 Planting in six-hole plate at density of/mL, culturing for 24 hr, discarding culture solution, replacing fresh culture solution, and adding Au DENPs ([ Au)]=200 μm), LPS (10 ng/ml), blank group served as control, and incubation was continued for 24h. The cultured cells were discarded, the supernatant was washed 3 times with PBS, the cells were digested with pre-chilled PBS, and the cell suspension was collected in an EP tube and centrifuged at 1000rpm for 3min at room temperature; the supernatant was discarded, 90. Mu.L of streaming buffer (5% FBS+95% PBS) and 5. Mu.L of Anti-mouse CD86 and 5. Mu.L of Anti-mouse CD11b were added to each EP tube, the blank was not added with any antibody, the isotype control was added to the isotype control of Anti-mouse CD86, gently mixed with a 100. Mu.L pipette, and incubated at room temperature in the absence of light for 15-20min. The results are shown in FIG. 8, CD11b in Blank group + CD86 + The proportion of cells with double positive is 7.82%, and the proportion of double positive in LPS group and Au DENPs is 31.3% and 41.2%, respectively, so that the proportion of cells with double positive in LPS group and Au DENPs group is obviously increased, which proves that both the cells successfully induce M1 type differentiation of macrophages, and the effect of Au DENPs is slightly obvious compared with that of LPS induced M1 type macrophage differentiation.
Example 6
RAW264.7 cells were plated at 1X 10 per well 5 Planting in six-hole plate at density of/mL, culturing for 24 hr, discarding culture solution, replacing fresh culture solution, and adding Au DENPs ([ Au)]=200 μm), LP (10 ng/mL), blank as control, and incubation was continued for 24h. ELISA detected the TNF- α content in the supernatant of 24h after incubation of RAW264.7 cells with Au DENPs, LPS and PBS, and the results are shown in FIG. 9, in which the expression level of TNF- α in the Au DENPs group and LPS group was significantly increased (p < 0.001) compared to that in the Blank group. It was found that TNF-. Alpha.expression levels were significantly increased in the LPS and Au DENPs groups, indicating that both successfully induced M1 type differentiation of macrophages.
Example 7
RAW264.7 cells were plated at 1X 10 per well 5 Planting in six-hole plate at density of/mL, culturing for 24 hr, discarding culture solution, replacing fresh culture solution, and adding Au DENPs ([ Au)]=200 μm), LPS (10 ng/mL), blank group served as control, and incubation was continued for 24h. The amount of iNOS expressed by each cell was measured using immunofluorescence, and the results are shown in fig. 10. Compared with the Blank group, the expression of the iNOS protein in the LPS group and the Au DENPs group is obviously increased, and especially the expression of the Au DENPs group protein is more than that of the LPS group. It was found that the expression level of iNOS was significantly increased in the LPS and Au DENPs groups, indicating that both successfully induced M1 type differentiation of macrophages.
Example 8
RAW264.7 cells were plated at 1X 10 per well 5 Planting in six-hole plate at density of/mL, culturing for 24 hr, discarding culture solution, replacing fresh culture solution, and adding Au DENPs ([ Au)]=200 μm), LPS (10 ng/ml), blank group served as control, and incubation was continued for 24h. Co-culturing macrophages of different treatment groups with K7 cells for 24 hours, and collecting a supernatant of a K7 cell culture solution; digesting the cells with pancreatin without EDTA, and stopping digestion with the culture medium; centrifuging at 1000rpm for 3min at room temperature using a solution containing no P, mg 2+ Cells were washed with PBS (dPBS); diluting Binding buffer (10 x) in the apoptosis kit by 10 times by using double distilled water; the Dpfs were discarded and the cells were resuspended with 300. Mu.l binding buffer (1X) per sample; FITC and PI independent staining groups were set, one group without any fluorescent dye added (single staining group for adjustment of compensation, no staining group for adjustment of voltage), 100. Mu.l of each cell suspension was taken from each group to a new EP tubeIn (a) and (b); adding FITC and PI marked fuel into an independent dyeing group and each treatment group respectively, wherein no dye is added into the dyeing group, and incubating for 20min at room temperature in a dark place; to each EP tube, 400. Mu.L of 1 Xbinding buffer was added, and the cells were resuspended and checked on the machine. The results are shown in FIG. 11: it can be seen that almost no apoptosis was detected in the control group; the apoptosis rate of the individual macrophage group is 9% and is lower than 10%; apoptosis rate in LPS+Macs group was 14.38%; whereas the apoptosis rate of K7 cells in Au DENPs group was significantly increased, as much as 29.5%. From this, it can be seen that the Au DENPs-macrophage complex can promote apoptosis of K7 cells.
Example 9
Caspase 3 plays a key role in apoptosis, is an effector of apoptosis, and after being activated, can cause cells to perform some biochemical reactions, can also cause the morphology of the cells to change, and finally leads to apoptosis of the cells. Therefore, we chose to further examine the expression of activated Caspase 3 in K7 cells after incubation with various macrophages using immunofluorescence and immunoWestern blotting. RAW264.7 cells were plated at 1X 10 per well 5 Planting in six-hole plate at density of/mL, culturing for 24 hr, discarding culture solution, replacing fresh culture solution, and adding Au DENPs ([ Au)]=200 μm), LPS (10 ng/mL), blank group served as control, and incubation was continued for 24h. After the macrophages of different treatment groups are co-cultured with K7 cells for 24 hours, immunofluorescence and western blotting are used for detecting the influence of each group of macrophages on osteosarcoma cell apoptosis, and as shown in the results of fig. 12 and 13, it can be seen that the Control group and the macrophage group have no obvious Caspase 3 expression, while the expression of protein can be detected in the LPS+macrophage group and the Au DENPs-macrophage complex group, wherein the expression level of Caspase 3 in the Au DENPs-macrophage complex group is higher than that in the LPS+macrophage group. In combination with the above results, it can be seen that the Au DENPs-macrophage complexes can promote apoptosis of K7 cells.
Example 10
Au DENPs of different concentrations ([ Au ] = 7.5, 15, 30, 60, 80 mM) were mounted in EP tubes and CT imaged using 120KV with a layer thickness of 0.625 mM. The results are shown in FIG. 14. It can be seen that as the gold nanoparticle concentration increases, the CT image brightness of the Au DENPs also increases, and the CT value exhibits a linear increase. The above results demonstrate that Au DENPs have good CT imaging effects.
Example 11
RAW264.7 cells were co-cultured with Au DENPs at different concentrations ([ Au ] =25, 50, 75, 100, 200 μm) for 24h. Cells were then trypsinized, centrifuged, and resuspended in 1.5mL EP tubes by washing 3 times with PBS. CT imaging was performed using the same scanning conditions as in example 10. The results are shown in FIG. 15. It can be seen that the brightness of the CT image of the Au DENPs-macrophage complex increases as [ Au ] increases, and that the CT value of the Au DENPs-macrophage complex can reach 85HU when [ Au ] reaches 200. Mu.M, indicating that the Au DENPs-macrophage complex can be used for CT imaging.
Example 12
A Balb/C mouse was used to establish a mouse osteosarcoma in situ animal model for CT imaging of the Au DENPs-macrophage complex. Mouse osteosarcoma K7 cells were cultured at 37℃in 5% CO 2 Selecting cells in logarithmic phase, discarding culture solution, washing with PBS for 3 times, centrifuging to obtain cell precipitate, adding appropriate amount of PBS to adjust cell density to 1×10 8 /mL. The alcohol cotton ball wipes the knee joint of the mice and keeps the knee joint in a bent state, a micro-injector is used for sucking the cell suspension, the cell suspension is penetrated into the bone marrow cavity of the tibia from the tibia plateau, and 10 mu L of the cell suspension is injected into each mouse, so that the injection is gently and slowly completed. After 2 weeks, tumor formation at the knee site of the mice was seen. Two groups were separated, one group was injected with Au DENPs ([ Au]=200 μm), another group was injected with Au DENPs-macrophage complexes. Obtaining and Au DENPs ([ Au)]After injection of Au DENPs-macrophage complexes (200 μl of PBS diluted cell suspension) via the tail vein =200 μΜ), images were taken at different time points and CT values of tumor sites were measured, as shown in fig. 16. FIG. 16A shows an Au DENPs-macrophage complex injection group, FIG. 16B shows an Au DENPs injection group, and the dotted line encircled part in the drawing shows a tumor part, and it can be seen that the CT value of the Au DENPs injection group reaches a peak value 90min after injection, and then the CT value has a descending trend; while the CT value of the tumor part of the Au DENPs-macrophage complex group is always increased until 4 hours, no consideration is given toDecrease in CT value. The above results demonstrate that the time of aggregation of the Au DENPs-macrophage complexes at the tumor site is longer than that of Au DENPs alone under the condition of equal amount of gold nanoparticles, and FIG. 16C is a graph showing the time-dependent trend of CT values of animal imaging of the Au DENPs group and the Au DENPs-macrophage complexes group.
Example 13
Doxorubicin (DOX) is a conventional chemotherapeutic agent for osteosarcoma treatment, and to further enhance the therapeutic effect, we used DOX in combination with Au DENPs-macrophage complexes for treatment. On the cell level, after incubation of mouse mononuclear macrophage RAW264.7 cells with Au DENPs for 24 hours, cell culture supernatant was obtained. The viability of each group of K7 cells was tested using CCK-8 cell proliferation assay kit after incubation with K7 cells for 24h, alone and mixed with Au DENPs-macrophage complex supernatant (50% V/50% V) at 1:1, at different concentrations of DOX (0, 1,5, 10, 25, 50. Mu.g/mL), as shown in FIG. 17. It can be seen that the viability of both groups of K7 cells decreased with increasing concentration of DOX. Overall, K7 cell viability was lower in the DOX-in combination with the Au DENPs-macrophage complex group than in the DOX group alone, indicating that the Au DENPs-macrophage complex can enhance the inhibitory effect of DOX on osteosarcoma cell proliferation.
Example 14
A Balb/C mouse was used to establish a mouse osteosarcoma in situ animal model for in vivo treatment of Au DENPs-macrophage complexes. The animal model was constructed in the same manner as in example 12. After model establishment, the tumor-bearing mice were grouped into 4 groups, a Control group, a chemotherapy (DOX) group, an Au DENPs-macrophage therapy group, and a combination therapy (Au DENPs-macrophage complex+DOX) group, respectively. Wherein Control group (100. Mu.L of physiological saline administration), DOX group (DOX administered to tail vein at concentration of 4.0mg/kg body weight), au DENPs-macrophage complex treatment group (Au DENPs-macrophage complex injected to tail vein of 1X 10) 6 Individual cells/100 μl), combination treatment group (equal amount of Au DENPs-macrophage complex was given first, 1 hour later, and the same dose of DOX) was injected every other day for one week. Observation ofDuring this period, knee joint volumes (fig. 18) and body weights (fig. 19) of mice of each treatment group were measured for 2 weeks to evaluate the treatment effect. It can be seen that the Control group had the greatest knee volume, and the Au DENPs-macrophage complex+DOX treated group had significantly less knee volume than the other groups. Due to the high malignancy of osteosarcoma, the body weight of each group of mice was generally reduced compared to the initial treatment. The Control group showed the greatest decrease in body weight, while the Au DENPs-macrophage complex treatment group showed the least decrease. After the treatment period is finished, serum of each group of mice is obtained by adopting an eyeball blood taking mode for quantitative detection of serum TNF-alpha, and then tumors and important internal organs (heart, liver, spleen, lung and kidney) of each group of mice are obtained for pathological detection. It can be seen that H of the tumor tissue of each group&In E and fluorescence TUNEL detection (figure 20), the tumor tissue structure of the Au DENPs-macrophage complex+DOX group is the most sparse, the apoptosis fluorescence intensity is the strongest, and the DOX group, the Au DENPs-macrophage complex group and the Control group are sequentially weakened. ELISA detection results of serum TNF-alpha (figure 21) show that the levels of TNF-alpha in the Control group and the DOX group are lower, the levels of TNF-alpha in the Au DENPs-macrophage complex treatment group are increased compared with the first two groups, and the content of TNF-alpha in the Au DENPs-macrophage complex plus DOX group is highest and is obviously higher than that in the other three groups. In addition, the major viscera H of the mice of the four treatment groups&The E staining results showed (FIG. 22) that the other three treatment groups did not damage the viscera of the mice compared to the Control group, indicating that the Au DENPs-macrophage complex did not damage the normal tissues of the body. Overall, the Au DENPs-macrophage complexes have a therapeutic effect on osteosarcoma, and the combination of the Au DENPs-macrophage complexes with DOX has a therapeutic effect superior to that of the chemotherapy group alone, indicating that the Au DENPs-macrophage complexes can further enhance the therapeutic effect of DOX on osteosarcoma.
The foregoing describes in detail preferred embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention without requiring creative effort by one of ordinary skill in the art. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Claims (4)

1. A method for preparing an Au DENPs-macrophage complex for use in cellular imaging and cellular therapy of osteosarcoma, comprising the steps of:
(1) Adding G5 polyamide-amine dendrimer into solvent to obtain G5 polyamide-amine dendrimer solution with concentration of 3-4 mmol/L, adding mPEG-MAL with concentration of 11.8-12.5 mg/mL, stirring for 3d reaction to obtain G5.NH 2 -mPEG solution;
(2) Dilution of G5.NH with Water 2 -mPEG solution and adding HAuCl 4 The solution was stirred vigorously to obtain a gold/dendrimer mixture, the HAuCl 4 Solution with G5.NH 2 -mPEG at a molar ratio of 100;
(3) Stirring the solution vigorously for 30min, and then adding 24-30 mg/mLNaBH 4 Stirring the solution for 2 hours to obtain [ (Au) 0 ) 100 -G5.NH 2 -mPEG]DENP；
(4) Triethylamine was added to [ (Au) with magnetic stirring 0 ) 100 -G5.NH 2 -mPEG]In DENP, stirring for 30min, adding acetic anhydride, and reacting for 24h, wherein the concentration of triethylamine is 730.38 mug/mu L and the concentration of acetyl anhydride is 1078.125 mug/mu L;
(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 bovine serum, adding Au DENPs into the culture medium, and culturing for 12-48 hours to obtain an Au DENPs-macrophage complex, wherein the quantitative standard of the Au DENPs is [ Au ] 200 mu M;
(7) Detecting markers of M1 type macrophages in the Au DENPs-macrophage complex by using LPS as a positive control and further using technologies including flow, PCR, immunofluorescence and ELISA to judge the polarization state of the markers, wherein the concentration of the LPS is 10ng/mL, and the detected markers of the M1 type macrophages comprise CD86, TNF-alpha and iNOS;
(8) The Au DENPs-macrophage complex is incubated with the mouse osteosarcoma K7 cells, and apoptosis of the mouse osteosarcoma K7 cells is detected by using a technology comprising flow, immunofluorescence and WesternBlot, wherein the index for judging apoptosis of the K7 cells is Annexin V-FITC/PI or Caspase 3 protein.
2. An Au DENPs-macrophage complex for osteosarcoma imaging or therapy prepared by the preparation method of claim 1, wherein Au DENPs in the Au DENPs-macrophage complex internalize within macrophages but not adhere to the surface of macrophages, the Au DENPs-macrophage complex expressing M1 type macrophage markers, the M1 type macrophage markers being CD86, TNF- α and iNOS.
3. Use of an Au DENPs-macrophage complex prepared by the preparation method of claim 1 in the preparation of a CT contrast agent for targeting osteosarcoma.
4. The use of the Au DENPs-macrophage complex combined antitumor drug prepared by the preparation method of claim 1 in the preparation of a medicament for treating osteosarcoma.
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