CN117959419A - Genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells, and preparation method and application thereof - Google Patents

Genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells, and preparation method and application thereof Download PDF

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CN117959419A
CN117959419A CN202410386328.5A CN202410386328A CN117959419A CN 117959419 A CN117959419 A CN 117959419A CN 202410386328 A CN202410386328 A CN 202410386328A CN 117959419 A CN117959419 A CN 117959419A
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tumor
cell membrane
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饶浪
潘远伟
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Shenzhen Bay Laboratory
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Abstract

The invention discloses a genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells, a preparation method and application thereof. Wherein the genetically engineered cell membrane nanoparticle comprises: nanoparticles with the function of relieving tumor hypoxia; and a cell membrane vesicle coated on the nano-particle with the function of relieving tumor hypoxia, wherein the cell membrane vesicle is a cell membrane vesicle derived from a tumor stem cell of which the gene engineering overexpression SIRP alpha variant is performed. By applying the technical scheme of the invention, the genetically engineered cell membrane nano-particles for inducing the immunity of the anti-tumor stem cells have the functions of hypoxia alleviation and blocking of CD47-SIRP alpha immune check points, and can induce strong anti-tumor immunity so as to remove the CSCs and inhibit tumor growth.

Description

Genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells, and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to a genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells, a preparation method and application thereof.
Background
Tumor stem cells (CSCs) are a subset of tumor cells that have the ability to repair DNA damage and to resist apoptosis, are highly resistant to traditional chemotherapy and radiation therapy, leading to clinical recurrence and death, and are considered to be major obstacles to current cancer therapies.
Immune Checkpoint Blocking (ICB) therapies can produce a sustained immune response in a variety of malignancies and have attracted considerable attention in inhibiting CSCs. Immune checkpoint CD47 is upregulated in CSCs, thereby escaping macrophage phagocytosis through the CD 47-sirpa "don't eat me" signaling pathway. Therapeutic antibodies against CD47 have been shown to be active against CSCs, while immune evasion limits their clinical outcome.
Recent studies have shown that tumor hypoxia is associated with immune evasion, which also limits the effectiveness of immunotherapy against CSCs. CSCs exhibit a low differentiation state in hypoxia inducible factor 1 alpha (HIF-1 alpha) mediated processes to maintain self-renewal. More importantly, HIF-1 a can directly activate transcription of the CD47 gene in CSCs, thereby evading phagocytosis by macrophages. At present, therapeutic antibodies against CD47 show anti-CSCs activity, but the effect of the hypoxic microenvironment on immune escape is not considered. Although there are reports of improving the efficacy of anti-CSCs chemotherapy by relieving CSCs hypoxia through hyperbaric oxygen (Nano Today 2021, 40, 101248; adv. Sci. 2023, 2301278.), the use of the methods for relieving CSCs hypoxia through hyperbaric oxygen is limited.
Therefore, improving the therapeutic effect of anti-CSCs immunotherapy remains a currently urgent problem to be solved.
Disclosure of Invention
The invention aims to provide a genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells, a preparation method and application thereof, so as to improve the curative effect of anti-tumor immunotherapy.
In order to achieve the above object, according to one aspect of the present invention, there is provided a genetically engineered cell membrane nanoparticle for inducing immunity against tumor stem cells. The genetically engineered cell membrane nanoparticle comprises: nanoparticles with the function of relieving tumor hypoxia; and a cell membrane vesicle coated on the nano-particle with the function of relieving tumor hypoxia, wherein the cell membrane vesicle is a cell membrane vesicle derived from a tumor stem cell of which the gene engineering overexpression SIRP alpha variant is performed.
Further, the nano-particles with the function of relieving tumor hypoxia are one or more of hMnO 2 nano-particles, catalase nano-particles, MOF nano-particles, graphite alkyne nano-particles and ferromanganese nano-particles.
Further, the cell membrane vesicles from the tumor stem cells are one or more of cell membrane nanovesicles from melanoma stem cells, breast cancer stem cells, brain glioma stem cells and liver cancer stem cells.
Further, the mass ratio of the nano particles with the function of relieving tumor hypoxia to the cell membrane vesicles is 1:1-1:4.
According to another aspect of the invention, there is provided the use of genetically engineered cell membrane nanoparticles for inducing immunity against tumor stem cells in the preparation of an anti-tumor medicament.
According to a further aspect of the present invention there is provided a medicament for inducing immunity against tumour stem cells comprising an effective dose of any one of the genetically engineered cell membrane nanoparticles described above and a pharmaceutically acceptable carrier.
According to another aspect of the present invention, there is provided a method for preparing genetically engineered cell membrane nanoparticles of any of the above-mentioned methods for inducing immunity against tumor stem cells. The preparation method comprises the following steps: synthesizing nano particles with the function of relieving tumor hypoxia; synthesizing cell membrane vesicles derived from tumor stem cells genetically engineered to overexpress SIRP alpha variants; and wrapping the cell membrane vesicles derived from the tumor stem cells on the nano-particles with the function of relieving tumor hypoxia, thus obtaining the genetically engineered cell membrane nano-particles.
Further, the nano-particles with the function of relieving tumor hypoxia are one or more of hMnO 2 nano-particles, catalase nano-particles, MOF nano-particles, graphite alkyne nano-particles and ferromanganese nano-particles.
Further, the cell membrane vesicles from the tumor stem cells are one or more of cell membrane nanovesicles from melanoma stem cells, breast cancer stem cells, brain glioma stem cells and liver cancer stem cells.
Further, wrapping the tumor stem cell-derived cell membrane vesicles on the nanoparticle having the function of relieving tumor hypoxia comprises: the cell membrane vesicle from the tumor stem cells and the nano-particles with the function of relieving tumor hypoxia are mixed and then extruded by a micro extruder to form the genetically engineered cell membrane nano-particles.
Further, the membrane pore size of the micro-extruder was 400nm and 200nm.
By applying the technical scheme of the invention, the genetically engineered cell membrane nano-particles for inducing the immunity of the anti-tumor stem cells have the functions of hypoxia alleviation and blocking of CD47-SIRP alpha immune check points, and can induce strong anti-tumor immunity so as to remove the CSCs and inhibit tumor growth.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 shows a schematic representation of genetically engineered CSC membrane coated hollow manganese dioxide (hMnO 2 @ gCMs) nanoparticles for inducing anti-tumor immunity against CSCs;
FIG. 2 shows a schematic drawing of the synthesis process of hMnO 2 nanoparticles and TEM images of the nanoparticles corresponding to the steps;
FIG. 3 shows graphs of the results of the preparation and characterization of hMnO 2 @ gCM;
FIG. 4 shows graphs of the degradation behavior of hMnO 2, wherein A is a graph of the ultraviolet-visible spectrum measurement of the degradation behavior of hMnO 2, and B is a photograph of the changes of H 2O2 solution, hMnO 2 solution and hMnO 2 solution added with H 2O2;
FIG. 5 shows optical micrographs and statistical results of B16F 10-SIRPalpha-adherent cells and tumor globus cells, wherein A is an optical micrograph of B16F 10-SIRPalpha-adherent cells and tumor globus cells; b is a statistical result graph of CD133+ percentage in B16F10-SIRP alpha adherent cells and tumor spheroid cells;
FIG. 6 is a graph showing the results of the correlation of the biocompatibility of hMnO 2 @ gCM and the ability to target CSCs;
FIG. 7 shows a graph of the results of whole blood testing following intravenous injection in healthy C57BL/6 mice;
FIG. 8 shows H & E stained slice images of major organs (heart, liver, spleen, lung and kidney) after intravenous injection in healthy C57BL/6 mice;
FIG. 9 shows CLSM images after incubation of B16F10 tumor cells with hMnO 2 @ gCM;
FIG. 10 shows in vivo fluorescence imaging of intravenous B16F10-CSCs tumor-bearing mice;
FIG. 11 is a graph showing the results of an hMnO 2 @ gCMs hypoxia relief experiment;
FIG. 12 shows an optical micrograph of tumor sphere formation taken after treatment with PBS and hMnO 2;
FIGS. 13-1 and 13-2 show graphs of results relating to the activation of hMnO 2 @ gCM by anti-tumor immunity;
FIG. 14 shows TNF- α (A in FIG. 14) and IFN- γ (B in FIG. 14) levels in mouse tumor tissue after various treatments;
FIG. 15 is a graph showing experimental results of hMnO 2 @ gCM inhibiting tumor growth and eliminating CSCs;
FIG. 16 is a graph showing the results of H & E staining of groups of B16F10-CSCs tumor-bearing mice at the end of treatment;
FIG. 17 shows H & E staining images of major organs (heart, liver, spleen, lung, kidney) of each group of tumor-bearing mice at the end of treatment; and
Figure 18 shows a schematic representation of hydrated particle size results of nanoparticle and cell membrane vesicle coating at different ratios according to an embodiment of the application.
Detailed Description
It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other. The application will be described in detail below with reference to the drawings in connection with embodiments.
Noun interpretation:
hMnO 2 nanoparticles: hollow manganese dioxide nanoparticles.
GCMs/gCM: CSC membrane nanovesicles genetically engineered to overexpress sirpa variants.
CSC/CSCs: tumor stem cells.
Aiming at the technical problems in the background art, the application provides a brand-new, efficient, low-cost and convenient genetically engineered cell membrane nanoparticle strategy.
According to an exemplary embodiment of the present application, a genetically engineered cell membrane nanoparticle is provided that induces immunity against tumor stem cells. The genetically engineered cell membrane nanoparticle comprises: nanoparticles with the function of relieving tumor hypoxia; and a cell membrane vesicle coated on the nano-particle with the function of relieving tumor hypoxia, wherein the cell membrane vesicle is a cell membrane vesicle derived from a tumor stem cell of which the gene engineering overexpression SIRP alpha variant is performed.
The nanoparticle with the function of relieving tumor hypoxia can be hMnO 2, and can also be other nanomaterials with peroxidase (CAT) enzyme property, such as catalase, MOF (Metal-organic framework material-organic framework), graphite alkyne, ferromanganese oxygen nanomaterials and the like, which can produce O 2 through catalyzing and decomposing endogenous H 2O2 of tumor. The cell membrane vesicles derived from the tumor stem cells of the genetically engineered over-expressed SIRP alpha variants can be melanoma stem cell membrane nanovesicles of the over-expressed SIRP alpha, and the person skilled in the art can understand that the cell membrane vesicles can also be cell membrane vesicles derived from other tumor types, such as breast cancer stem cells, brain glioma stem cells, liver cancer stem cells and the like.
In the present application, the SIRPalpha variant may be a variant having increased affinity for CD47 (relative to the wild type), such high affinity binding being effective to block the CD 47-SIRPalpha signaling pathway. In a preferred embodiment of the application, the sirpa variant has the amino acid sequence of SEQ ID NO:1, it will be appreciated by those skilled in the art that this is merely an example and that other variants having increased affinity for CD47 are equally suitable for use in the present application.
In a preferred embodiment of the application, the nanoparticle having tumor hypoxia relieving function is hMnO 2, and the cell membrane vesicle derived from tumor stem cells genetically engineered to overexpress sirpa variants is CSC cell membrane nanovesicle of overexpressed sirpa, taking this as an example to explain the mechanism of the application. The genetically engineered cell membrane nanoparticle consists of a genetically engineered CSC membrane coated hollow manganese dioxide nanoparticle (hMnO 2 @ gCMs) wherein the gCMs outer shell exhibits efficient CSCs specific targeting due to homologous targeting of the CSC membrane, while the hMnO 2 core effectively relieves tumor hypoxia by inducing the breakdown of endogenous H 2O2 of the tumor, thereby inhibiting CSCs and reducing expression of CD 47. The excessive expression SIRPalpha on gCMs effectively blocks the signal path of CD 47-SIRPalpha 'to eat me' in cooperation with the downregulation of the CD47 induced by hypoxia relief, and cooperatively induces strong anti-tumor immune response. In a murine model of melanoma carrying B16F10-CSCs, tumor hypoxia remission combined with synergistic inhibition of the CD47-SIRP alpha signaling pathway exhibits enhanced therapeutic effects in eradicating CSCs and inhibiting tumor growth.
Without any cell membrane coating, the charge shielding effect due to the presence of ions in the PBS buffer resulted in significant aggregation of the naked nanoparticles. As the proportion of cell membranes increases, the effect is smaller. At a ratio of nanoparticle to cell membrane vesicle of 1:1, the nanoparticle did not cause aggregation in PBS buffer, see FIG. 18. In an embodiment of the application, the mass ratio of the nanoparticle having the function of relieving tumor hypoxia to the cell membrane vesicle is 1:1-1:4, for example, 1:1, 1:2, 1:3, 1:4, preferably 1:1. In a subsequent embodiment of the application, the ratio is chosen to be 1:1.
Referring to fig. 1, a schematic representation of genetically engineered CSC film coated hollow manganese dioxide (hMnO 2 @ gCMs) nanoparticles for inducing anti-tumor immunity against CSCs is shown. Therein, figure 1 a shows CSCs over-expressing sirpa variants obtained by genetic engineering methods and isolated to CSC membranes (gCMs) which are then coated onto the surface of manganese dioxide (hMnO 2) nanoparticles. FIG. 1B shows a schematic representation of the generation of a powerful anti-tumor immune response by hMnO 2 @ gCM through CD 47-SIRPalpha blockade and hypoxia remission.
According to an exemplary embodiment of the present invention, there is provided an application of any one of the genetically engineered cell membrane nanoparticles for inducing immunity of anti-tumor stem cells in preparing an anti-tumor drug.
According to an exemplary embodiment of the present invention, a medicament for inducing immunity against tumor stem cells is provided. The medicament comprises an effective dose of genetically engineered cell membrane nanoparticles of any one of the above and a pharmaceutically acceptable carrier. Further, pharmaceutically acceptable carriers include PLGA nanoparticles, liposomes, and the like.
According to an exemplary embodiment of the present invention, a method for preparing the genetically engineered cell membrane nanoparticle for inducing immunity of anti-tumor stem cells is provided. The preparation method comprises the following steps: synthesizing nano particles with the function of relieving tumor hypoxia; synthesizing cell membrane vesicles derived from tumor stem cells genetically engineered to overexpress SIRP alpha variants; and wrapping the cell membrane vesicles derived from the tumor stem cells on the nano-particles with the function of relieving tumor hypoxia, thus obtaining the genetically engineered cell membrane nano-particles.
The nanoparticle with the function of relieving tumor hypoxia can be hMnO 2, and can also be other nanomaterials with peroxidase (CAT) enzyme property, such as catalase, MOF, graphite alkyne, ferromanganese oxygen nanomaterials and the like, which can produce O 2 by catalyzing and decomposing endogenous H 2O2 of tumors. The cell membrane vesicles of the tumor stem cell source of the genetically engineered overexpression SIRP alpha variant can be CSC cell membrane nanovesicles of the overexpression SIRP alpha, and can also be cell membrane vesicles of other tumor type sources, such as breast cancer stem cells, brain glioma stem cells, liver cancer stem cells and the like.
In an exemplary embodiment of the present application, encapsulating tumor stem cell-derived cell membrane vesicles on nanoparticles having tumor hypoxia relieving function comprises: the cell membrane vesicles from the tumor stem cells and the nano-particles with the function of relieving tumor hypoxia are mixed and then extruded by a micro extruder to form the genetically engineered cell membrane nano-particles, for example, the membrane pore diameter of the micro extruder is 400nm and 200nm.
In a preferred embodiment of the present invention, the genetically engineered cell membrane nanoparticle (hMnO 2 @ gCMs) for inducing immunity of anti-tumor stem cells is prepared by the following method:
The preparation of hMnO 2 @ gCMs genetically engineered cell membrane nanoparticles comprises three steps, wherein the first step is to synthesize hMnO 2 nanoparticles, the second step is to synthesize CSC membrane nanovesicles (gCMs) of genetically engineered overexpressed SIRP alpha variants, and the third step is to wrap gCMs on hMnO 2 nanoparticles. To synthesize hMnO 2 nanoparticles, first, solid silica nanoparticles (SiO 2) were synthesized as templates. The method comprises the following specific steps: triton X-100 (5.3 mL), cyclohexane (22.5 mL) and n-hexanol (5.4 mL) were added to the 100 mL flask and stirred for 5 minutes (600 rpm). Ammonia (0.75 mL) and H 2 O (1 mL) were then added and stirring continued for half an hour. Thereafter, TEOS (0.5 mL) and APTES (0.1 mL) were mixed and added dropwise to the above solution, and the reaction was continued at room temperature for 24 hours. After the reaction was completed, siO 2 nanoparticles were collected by centrifugation (12000 rpm,10 minutes) and washed twice with ethanol and once with deionized water. The obtained SiO 2 nanoparticles were dispersed in deionized water (20 mg, 1 mg/mL). To further obtain SiO 2@MnO2 nanoparticles, KMnO 4 solution (10 mg/mL,15 mL) was added dropwise to the SiO 2 nanoparticle solution under ultrasonic conditions and the ultrasonic reaction was continued in a water bath ultrasonic apparatus for 6 hours. The resulting SiO 2@MnO2 nanoparticles were collected by centrifugation (12000 rpm,30 minutes) 2-3 times. Finally, siO 2@MnO2 nanoparticles were etched with Na 2CO3 solution (2 m,20 mL) overnight at 60 ℃, and hollow mesoporous MnO 2 nanoparticles (hMnO 2) were obtained by centrifugation and washing with water several times.
The SIRP variant CV1 is transfected on melanoma cells (B16F 10) by using lentiviruses, the B16F10 is cultured by using a suspension culture mode, and the CSCs are obtained by flow sorting cells positive for CD133 +, so that the CSCs cells (CSCs-SIRP alpha) which over express the SIRP variant are obtained. Expression of sirpa was confirmed using immunofluorescent staining and flow cytometry. To obtain CSCs cell membranes over-expressing sirpa variants, CSCs-sirpa cells were disrupted with a homogenizer, treated with DNase and RNase (Invitrogen) and centrifuged (3200 g,5 min). After collecting the supernatant, the solution was further centrifuged (20000 g,30 min). The pellet was then discarded and the supernatant (80000 g,2 hours) was centrifuged again with an ultracentrifuge (Optima MAX-XP, beckman Coulter). The protease-inhibited tablet mix was washed with PBS and 400 nm and 200 nm nm hole polycarbonate films were extruded using an Avanti mini-extruder (Avanti Polar Lipids) to obtain gCM nm vesicles. hMnO 2 nanoparticles were mixed with gCM and then sequentially extruded through a 200 nm-nanopore polycarbonate membrane to obtain hMnO 2 @ gCM genetically engineered cell membrane nanoparticles, which were collected using centrifugation and stored at 4℃for subsequent experiments.
The beneficial effects of the invention will be further described below in connection with experimental data. The steps or reagents described in the examples below, if not described in detail, may be accomplished using methods or reagents conventional in the art.
Example 1
1. Experimental materials
1. Reagent(s)
Triton X-100, n-hexanol and cyclohexane were purchased from ALFA AESAR. Tetraethylorthosilicate (TEOS) and (3-aminopropyl) triethoxysilane (APTES) are available from Merck. Potassium permanganate (KMnO 4), sodium carbonate (Na 2CO3) and 30 wt% hydrogen peroxide (H 2O2) were purchased from the national pharmaceutical group chemical Co., ltd. Dulbecco's Modified Eagle Medium (DMEM) and DMEM/F12 are available from Gibco. B-27 ™ supplement, epidermal Growth Factor (EGF) and basal Fibroblast Growth Factor (FGF) were purchased from Thermo FISHER SCIENTIFIC. Fetal Bovine Serum (FBS), penicillin and streptomycin were purchased from Invitrogen. Bovine Serum Albumin (BSA) was purchased from merck. 4', 6-diamidino-2-phenylindole (DAPI) was obtained from Life Technologies and phalloidin-FITC was obtained from Abcam. All reagents were used as received. Milli-Q water (18.2M. Omega. Cm) was used for all experiments.
2. Mouse tumor model
HMnO2@gCMs gene engineering cell membrane nano particles are used for eradicating the CSCs, and the tumor model of the mice is B16F10-CSCs tumor-bearing mice.
2. Experimental method
1. Synthesis of hMnO 2 nanoparticles
First, solid silica nanoparticles (SiO 2) were synthesized as templates. Triton X-100 (5.3 mL), cyclohexane (22.5 mL) and n-hexanol (5.4 mL) were added to the 100 mL flask and stirred for 5 minutes. Then, ammonia (0.75 mL) and H 2 O (1 mL) were immediately added, and the mixed solution was stirred for 30 minutes. Thereafter, TEOS (0.5 mL) and APTES (0.1 mL) were mixed and added to the above solution, and the reaction was continued at room temperature for 24 hours. The SiO 2 nanoparticles were collected by centrifugation and washed 3 times. The obtained SiO 2 nanoparticles were dispersed in ultrapure water (20 mg,1 mg. ML -1). KMnO 4 aqueous solution (10 mg ·ml -1, 15 mL) was added dropwise to the above solution, followed by continuous sonication for 6 h. KMnO 4 was reduced by the organosilicon on the surface of SiO 2 to give a uniform mesoporous MnO 2 layer (MnO 2@SiO2). The resulting nanoparticles were collected by centrifugation 2-3 times and etched with Na 2CO3 solution (2 m,20 mL) at 60 ℃ overnight to give hollow mesoporous MnO 2 nanoparticles (hMnO 2).
2. Synthesis of hMnO 2 @ gCMs nanocomposite
The SIRP alpha variant (SEQ ID NO: 1) is transfected on B16F10 cells by using lentiviruses, so that the B16F10 cells (refer to adv. Mater 2020, 32, 2004853) which over express the SIRP alpha variant by genetic engineering are prepared. B16F10-SIRP alpha tumor cells rich in CSCs were obtained by suspension culture on a low-adsorption plate using serum-free medium (cf. ACS Nano 2021, 15, 15069-15084). Sorting and identification of CD133 + CSCs was performed using BD Aria III. To obtain gCM, the CSCs of B16F 10-sirpa were broken up by a Dounce homogenizer. The whole solution was treated with DNase and RNase (Invitrogen) and centrifuged (3200 g,5 min). The supernatant was collected and further centrifuged (20000 g,30 min). The pellet was discarded and the supernatant was centrifuged again (80000 g,2 h) using a ultra high speed centrifuge (Optima MAX-XP, beckman Coulter, USA) to obtain a pellet. The pellet was washed with PBS mixed with protease inhibitor tablets and extruded sequentially through 400 nm and 200 nm nm hole polycarbonate membranes using an Avanti mini-extruder (Avanti Polar Lipids, usa) to give gCM. Subsequently gCM and hMnO 2 were mixed and then extruded sequentially through a 200 nm orifice. The resultant hMnO 2 @ gCMs was collected by centrifugation and stored at 4 ℃ for future use.
3. Characterization of
Transmission Electron Microscope (TEM) images were characterized by Talos F200X. Zeta potential experiments were performed using a Zetasizer Nano-ZS (Malvern Instruments). The UV-visible absorption spectrum was measured using a UV-visible spectrophotometer (Lambda 950, perkin Elmer). Inductively coupled plasma emission spectroscopy (ICP-OES) was performed using an Optima 2100 instrument of PERKIN ELMER. X-ray photoelectron spectroscopy (XPS) was performed on an Axis Ultra DLD instrument. Dissolved oxygen was measured with an oxygen probe (ST 300D, OHAUS).
4. SDS-PAGE analysis
For sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gCM, hMnO 2 and hMnO 2 @ gCM were added to the protein extraction buffer and the protein content was measured using the bicinchoninic acid (BCA) kit (Sigma-Aldrich). The samples were heated at 95 ℃ for 5 minutes and 20 μg of each sample was loaded into a 10% SDS-polyacrylamide gel. Samples were run at 120V for 2 hours, the gel was stained with coomassie blue for 4 hours and then destained overnight before observation.
5. Cell culture
B16F10 cells were cultured in DMEM and supplemented with 10% fbs and 100 units mL -1 penicillin and 100 μg mL -1 streptomycin. Cells were incubated at 37 ℃ with 5% CO 2. For tumor spheroid cell culture, B16F10 cells (20,000 mL -1) were suspended in an ultra-low adhesion plate and cultured with serum-free DMEM/F12 supplemented with B27 (purchased from gibco, diluted 1x for use), 0.4% BSA, EGF of 20 ng mL -1 and FGF of 20 ng mL -1. After 10 days of incubation, tumor balls were collected for further experiments.
6. In vitro biocompatibility
To assess in vitro biocompatibility, CCK8 assays were performed on B16F10 tumor spheroid cells. Briefly, B16F10 tumor cells were seeded in ultra-low attachment 96-well plates for 24 hours and then further cultured with medium containing 20-100 μg mL -1 hMnO2、gCMs、hMnO2 @ gCMs for 24 hours. CCK8 assays were then performed to quantify cell viability.
7. In vivo biocompatibility
Female C57BL/6 mice (4-5 weeks old) were used in this example. All animal experiments were conducted according to guidelines approved by the ethical committee for animal experiments in Shenzhen Bay laboratory (license number: AERL 202101). To assess the in vivo toxicity of the nanocomposite, 12 healthy female C57BL/6 mice were randomly divided into two groups (n=6). Mice were intravenously injected with 100 μl of PBS or hMnO 2 @ gCM. The dose of nanocomposite was 20 mg kg -1. After 4 weeks, a mouse blood sample was drawn for whole blood detection and blood biochemical examination. Major organs of mice, including heart, liver, spleen, lung and kidney, were harvested and stained with hematoxylin and eosin (H & E) for histological analysis.
8. Cell uptake assay
Tumor spheroid cells (2×10 4 cells per well) were seeded in confocal plates and incubated with hMnO 2 @ gCMs for an additional 2,4 or 8 hours, hMnO 2 @ gCMs were previously stained with 1, 1-dioctadecyl-3, 3' tetramethyl indole dicarbacyanine, 4-chlorobenzenesulfonate (DiD, thermo FISHER SCIENTIFIC). Cells were then collected and washed with PBS to remove free nanocomposite. Finally, cells were stained with DAPI and phalloidin-FITC and measured by confocal laser scanning microscopy (CLSM, ZEISS LSM 980). To determine cellular uptake using flow cytometry, tumor spheroid cells were seeded and incubated with hMnO 2 @ gCM labeled with 1,1 '-dioctadecyl-3, 3' -tetramethylindole dicarbacyanine perchlorate (DiO, thermo FISHER SCIENTIFIC), and cells were then collected and stained with PE anti-mouse CD133 (Biolegend, 141203) to identify CSCs. Finally, the cells were transferred to a tube and examined by flow cytometry (CytoFLEX LX, beckman Coulter).
9. Determination of HIF-1 alpha expression at cellular levels
To assess the proportion of HIF-1 a after hypoxia relief, tumor cells were seeded under hypoxia in 12-well ultra-low adhesion plates (1×10 5 cells mL -1) for 48 hours. The cells were then incubated with hMnO 2 @ gCMs for 24 hours under hypoxic conditions. Immunofluorescent staining detects HIF-1 alpha expression levels in different treated cells.
10. Expression of in vitro post-hypoxia-remission dryness-associated genes
Tumor cells were seeded and incubated with hMnO 2 as described above. The total RNA of the cells was then isolated by TRI REAGENT according to the manufacturer's instructions and then quantified by spectrophotometry (Thermo Fisher; nano-Drop). Reverse transcription was performed with 1 μg total RNA using SuperScriptTM IV VILOTM Master Mix (Invitrogen). To assess the relative mRNA levels of OCT4, NANOG, and SOX2, quantitative real-time polymerase chain reaction (qRT-PCR) was performed on a QuantStudio 5 real-time PCR system (Applied Biosystems) using SYBRTM GREEN PCR MASTER Mix (Thermo Fisher). Primer sequences for qRT-PCR are shown in Table 1 and are normalized according to housekeeping gene GAPDH. Finally, mRNA levels of OCT4, NANOG, and SOX2 and cells treated with PBS alone were normalized.
TABLE 1 qRT-PCR target gene primer sequences.
11. In vitro tumor sphere formation assay
For in vitro tumor spheroid formation assays, tumor spheroid cells were seeded and incubated with hMnO 2 as described above. Cells were then harvested with trypsin, and then 2000 or 4000 cells per group were seeded into new 96-well ultra-low attachment plates and maintained for 10 days. Finally, the number of tumor spheres (> 50 μm) was counted and an optical micrograph taken under a microscope.
12. Determination of the proportion of CSCs after in vitro hypoxia remission
To assess the proportion of CSCs post-treatment, tumor spheroid cells were incubated with hMnO 2 or hMnO 2 @ gCM and treated as described above. After that, the cells were digested by trypsin, washed with PBS and stained with PE anti-mouse CD 133. The proportion of CD133 + cells was then determined as CSCs by flow cytometry and analyzed by FlowJo software.
13. In vivo CSCs tumor model
A model of murine melanoma with B16F10-CSCs was established and used. Briefly, B16F10 tumor spheroid cells (2.5X10 5 cells per mouse) were inoculated subcutaneously into the back of female C57BL/6 mice. After the tumor volume reached 50-100 mm 3, tumor-bearing mice were used for further experiments.
14. In vivo hypoxia relief assay
B16F10-CSCs tumor-bearing mice were injected intravenously with 100. Mu.L of PBS or hMnO 2, respectively. After 24 hours, mice were sacrificed and tumors were harvested. The expression levels of HIF-1. Alpha., CD47, CD133 in tumor tissues were detected by immunofluorescent staining.
15. In vivo biodistribution
The B16F10-CSCs tumor-bearing mice were intravenously injected with 100. Mu.L of hMnO 2 or hMnO 2 @ gCMs, respectively. After 24 hours, mice were sacrificed, major organs and tumors were harvested and weighed, and then the Mn amount was determined by ICP-OES analysis. For in vivo fluorescence imaging, hMnO 2 and hMnO 2 @ gCM were first labeled with IR 780. After IR 780-labeled hMnO 2 and hMnO 2 @ gCM were intravenously injected into B16F10-CSCs tumor-bearing mice, the mice were imaged by a small animal imaging system (IVIS Lumina III PERKINELMER). To assess targeting ability of CSCs in vivo, hMnO 2 @ gCMs was first labeled with DID and then hMnO 2 @ gCM labeled by intravenous injection of DID. After 24 hours, tumors were dissected and stained with CSCs markers CD133 and DAPI.
16. In vivo anti-tumor study
B16f10—cscs tumor-bearing mice were randomly divided into four groups (n=5) and received three doses of PBS, hMnO 2, gCMs, or hMnO 2@gCMs（hMnO2 doses=10 mg kg −1,gCMs=10 mg kg−1) intravenously, once every other day. Tumor volume and body weight were measured every two days from the start of treatment. Tumor volume was measured using a digital caliper, tumor volume= (tumor length) × (tumor width) 2/2. Two weeks after the first treatment, mice were sacrificed and the major organs, including heart, liver, spleen, lung and kidney were dissected and stained for H & E.
17. In vivo therapeutic effect and CSCs marker expression staining
The B16F10-CSCs tumor-bearing mice received three doses of intravenous injection. PBS, hMnO 2, gCMs, or hMnO 2@gCMs（hMnO2 dose = 10 mg kg −1,gCMs=10 mg kg−1) injected every other day. Two weeks later, the tumors were dissected and stained with H & E and Ki-67. Furthermore, immunohistochemical staining detects the expression levels of CSCs markers CD133, SOX2, NANOG in tumor tissues.
18. Flow cytometry
For flow cytometry analysis, tumor tissue is first harvested and sectioned after treatment. Single cell suspensions were prepared by homogenization in cold PBS supplemented with digestive enzymes and then filtered with a 70 μm cell filter. For macrophage polarization, cells were stained with anti-CD 45-Cy5.5, anti-CD 11b-FITC, anti-F4/80-AlexaFluor 647, anti-CD 80-Bv510 and anti-CD 206-Bv785 antibodies according to the manufacturer's protocol. CD11b +F4/80+CD80+ and CD11b +F4/80+CD206+ cells were defined as M1 and M2 phenotype macrophages, respectively. For T cell assessment, cells were stained with anti-CD 3-Bv510, anti-CD 4-Bv650 and anti-CD 8-AlexaFluor750 antibodies according to the manufacturer's protocol. Samples were measured using CytoFLEX flow cytometer (Beckman) and the results were analyzed using FlowJo.
19. Multispectral immunohistochemistry
Tumor histological sections were rehydrated with alcohol, washed 3 times with PBS, then recovered in an autoclave with sodium citrate, blocked with PBS buffer containing 2.5% BSA for 1 hour at 37 ℃, followed by incubation with primary and secondary antibodies. The following antibodies were used in immunofluorescence: anti-CD 8 rabbit monoclonal antibodies (CELL SIGNALING Technology) and anti-F4/80 rabbit monoclonal antibodies (CELL SIGNALING Technology), goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen), goat anti-rabbit IgG Alexa Fluor 647 (Invitrogen) and DAPI.
20. Cytokine detection
B16F10-CSCs tumor bearing mice were randomly divided into four groups (n=4): PBS, hMnO 2, gCMs, or hMnO 2 @ gCMs. Mice received three doses of different nanoparticles (hMnO 2 dose = 10 mg kg −1,gCMs=10 mg kg−1) by intravenous injection, once every other day. Two weeks later, different groups of tumor tissue were obtained. Levels of TNF- α and IFN- γ in tumors were measured using corresponding ELISA kits according to manufacturer's guidelines.
21. Statistical analysis
All results are expressed as mean ± standard error of mean (s.e.m.) of independent experiments. Statistical analysis was performed on experimental data using Origin 9.0 software using one-way analysis of variance (ANOVA) and Tukey significant difference post hoc test. A p value of 0.05 is considered a significant level, and data classification as (×) indicates p <0.05, (×) indicates p < 0.01, (×) indicates p < 0.001.
3. Experiment
1. Synthesis of hMnO 2
As shown in fig. 2, first, siO 2 nanoparticles were obtained by hydrolyzing TEOS, and freshly prepared SiO 2 was immediately used as a hard template for preparing SiO 2@MnO2 nanoparticles. And carrying out ultrasonic mixing on KMnO 4 and SiO 2 nano particles to obtain a layer of manganese dioxide (MnO 2) on the surfaces of the SiO 2 nano particles, and carrying out centrifugal cleaning to obtain the SiO 2@MnO2 nano particles. Finally, the Na 2CO3 solution is used for etching SiO 2 in the SiO 2@MnO2 nano particles to finally obtain the hollow MnO 2 nano particles (hMnO 2).
As shown in the transmission electron microscope diagram of FIG. 2, the prepared SiO 2、SiO2@MnO2 and hMnO 2 nano-particles are spherical and uniform in size (scale bar, 50 nm), and the TEM diagram clearly shows that the synthesized hMnO 2 nano-particles are hollow structures.
FIG. 3 shows graphs of the results of the preparation and characterization of hMnO 2 @ gCM; therein, a in fig. 3 shows a TEM image of hMnO 2 nanoparticles. Scale bar, 50 nm. The XPS full measurement spectrum and the Mn 2p high resolution spectrum of the hMnO 2 nanoparticles are shown in FIGS. 3B and C, respectively. TEM images of hMnO 2 nanoparticles after incubation in PBS (pH 7.4) and PBS (pH 5.5) containing H 2O2 (100. Mu.M) are shown in FIGS. 3D and E, respectively. Scale bar, 100 nm. The change in O 2 concentration after incubation of different concentrations of hMnO 2 and H 2O2 solutions (100 μm) is shown in fig. 3F. Immunofluorescence imaging and flow cytometry quantification of sirpa expression on primary and genetically engineered B16F10 cells are shown at G in fig. 3. Scale bar, 10 μm. A TEM image of hMnO 2 @ gCMs nanoparticles is shown in fig. 3H. Scale bar, 50 nm. SDS-PAGE analysis of hMnO 2, gCM and hMnO 2 @ gCM is shown at I in FIG. 3. The Zeta potentials of hMnO 2, gCMs and hMnO 2 @ gCMs nanoparticles are shown in fig. 3J.
As shown in the TEM image (a in fig. 3), hMnO 2 shows a spherical morphology and a hollow structure. The surface chemistry of hMnO 2 nanoparticles was analyzed by XPS to elucidate their elements and the corresponding valence states. XPS spectra of hMnO 2 nanoparticles showed signals of O and Mn elements (B in FIG. 3), and characteristic peaks at 642.4 and 654.2eV in Mn 2p spectra could be attributed to Mn 2p 3/2 and Mn 2p 1/2 (C in FIG. 3), respectively. These values indicate that the valence state of Mn in hMnO 2 nanoparticles is +4.Mn 2+ ions can be rapidly excreted through the kidney, and the long-term toxicity of hMnO 2 is reduced. As can be seen from TEM images, there was no significant change in hMnO 2 nanoparticle morphology in PBS at ph=7.4 (D in fig. 3). Whereas in the acidic environment containing H 2O2, the hMnO 2 nanoparticles undergo significant degradation (E in fig. 3). hMnO 2 can trigger H 2O2 existing in tumor microenvironment to be decomposed into water and oxygen, and generated oxygen can improve hypoxia of CSCs. To verify the ability of hMnO 2 nanoparticles to catalyze the production of oxygen by H 2O2, different concentrations of hMnO 2 nanoparticles were added to the solution of H 2O2 and the dissolved oxygen in the mixed solution was measured by an oxygen probe. The inventors found that hMnO 2 could trigger O 2 production rapidly in a concentration-dependent manner (F in fig. 3). While O 2 bubbles generated by hMnO 2 were visually observed (fig. 4B shows the photographic changes of H 2O2 solution, hMnO 2 solution, and hMnO 2 solution with H 2O2 added).
Meanwhile, fig. 4a shows the degradation behavior of hMnO 2 incubated in PBS (left) and H 2O2 (right) by uv-vis spectroscopy, the degradation rate being further determined by the change in absorbance band characteristic of hMnO 2, which is stable at pH 7.4, but rapidly decreases in the presence of acidic H 2O2 solution.
To construct a genetically engineered CSC cell membrane (gCM), sirpa variants were first transfected onto mouse melanoma B16F10 cells by lentiviruses. To obtain CSCs-enriched B16F 10-sirpa tumor cells CSCs were enriched by suspension culture (fig. 5a shows an optical micrograph of B16F 10-sirpa adherent cells and tumor cells; scale bar, 100 μm; fig. 5B shows statistics of cd133+ percentages in B16F 10-sirpa adherent cells and tumor cells). And CD133 + CSCs were separated out by a flow sorter. And further confirmed the expression of sirpa variants on CSCs cells by immunofluorescence imaging and flow cytometry (G in fig. 3). In order to construct the biomimetic nanomaterial with the hMnO 2 coated by the CSC cell membrane, the CSC cell membrane which over-expresses SIRP alpha is prepared by hypotonic lysis and differential centrifugation, and then the hMnO 2 @ gCM nanoparticle is obtained by coating the hMnO 2 surface with the gCM membrane by a physical extrusion method. TEM images showed that hMnO 2 @ gCM nanoparticles coated gCM film, compared to hMnO 2 nanoparticles, showed a coating (H in FIG. 3). The surface charge of hMnO 2 @ gCM was verified by zeta potential technology and the potential of hMnO 2 nanoparticles after coating gCM films increased to approximately the same level as gCM (J in fig. 3). To further verify the successful coating of gCM membranes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) assays were performed. As shown in fig. 3I, hMnO 2 @ gCM nanoparticles retained the integrity of the protein on the gCM membrane.
2. Biocompatibility and CSCs targeting evaluation of hMnO 2 @ gCMs genetically engineered cell membrane nanoparticles
Fig. 6 shows a graph of the results relating the biocompatibility of hMnO 2 @ gCM and the ability to target CSCs. Wherein, in fig. 6 a shows cell viability of B16F10 tumor cells after incubation with hMnO 2, gCM and hMnO 2 @ gCM. The biochemical blood test of hMnO 2 @ gCMs is shown in fig. 6B. ALT: alanine aminotransferase, ALP: alkaline phosphatase, AST: aspartate aminotransferase, BUN: urea nitrogen in blood, TP: total protein. CLSM images and corresponding line spectra of B16F10 tumor cells incubated with hMnO 2 @ gCM for 2 hours, 4 hours, and 8 hours are shown in fig. 6C; scale bar, 10 μm. FIG. 6D shows the degree of cellular uptake of hMnO 2 @ gCM in CSCs quantified by flow cytometry for incubation times of 2 hours, 4 hours, and 8 hours, respectively; in vivo fluorescence imaging and quantitative analysis of B16F10-CSCs tumor bearing mice by intravenous injection of hMnO 2 and hMnO 2 @ gCM, respectively, are shown in FIGS. 6E and F. FIG. 6G, H shows the biodistribution of hMnO 2 and hMnO 2 @ gCM nanocomposites in different organs of B16F10 tumor bearing mice. Results of confocal microscopy observation of hMnO 2 @ gCM and CSCs internalization and corresponding line spectra are shown in figure 6I, J, CSCs stained with CD 133. Scale bar, 50 μm.
Specifically, in this example, the biocompatibility of hMnO 2, gCM and hMnO 2 @ gCM was first examined at the cellular level (a in fig. 6). Analysis by CCK-8 experiments (CCK-8 is a short term for Cell Counting Kit-8 and is a cell proliferation and cytotoxicity method) shows that when the concentrations of hMnO 2, gCM and hMnO 2 @ gCM are 100 mug/mL, the cell activity is above 80%, and obvious toxicity is not caused to B16-F10 mouse skin melanoma cells. The result shows that the prepared nano material has good biocompatibility. After demonstrating good biocompatibility of hMnO 2 @ gCM at the cellular level, the biocompatibility at the hMnO 2 @ gCM animal level was further examined. First, mice did not show significant weight loss and death after 1 month of observation by intravenous injection of hMnO 2 @ gCM. Meanwhile, a blood sample of the mouse is collected, and subjected to blood routine detection and blood biochemical index detection, and the obtained results are shown in a graph B and a graph 7in FIG. 6 (FIG. 7 shows whole blood detection after intravenous injection of healthy C57BL/6 mice: saline injection or hMnO 2 @ gCMs.WBC: white blood cells, RBC: red blood cells, HGB: hemoglobin, HCT: hematocrit, MCV: mean red blood cell volume, MCH: mean red blood cell hemoglobin, MCHC: mean red blood cell hemoglobin concentration, PLT: platelets, MPV: mean platelet volume, lym: lymphocytes, RDW: red blood cell distribution width). Experimental results show that white blood cells, red blood cells, hemoglobin, hematocrit, average red blood cell volume, average hemoglobin amount, average hemoglobin concentration, platelets, average platelet volume, lymphocyte and red blood cell distribution width are not significantly different from those of the control group after intravenous injection of hMnO 2 @ gCM. In the biochemical indexes of the mouse blood, compared with a control group, the total protein, albumin, serum globulin, alkaline phosphatase, urea nitrogen, aspartic acid aminotransferase and alanine aminotransferase have no obvious difference, and the result shows that hMnO 2 @ gCM does not cause damage to the liver and kidney of the mouse. H & E staining of major organs was evaluated for hMnO 2 @ gCM biotoxicity in vivo. As shown in FIG. 8 (H & E stained slice images of major organs (heart, liver, spleen, lung and kidney) after intravenous injection of healthy C57BL/6 mice, saline or hMnO 2 @ gCMs were injected), no tissue damage and no apparent morphological changes were observed in the major organs of the mice after material injection compared to the control group. The prepared hMnO 2 @ gCM has good in vivo biocompatibility.
To evaluate the targeting ability of hMnO 2 @ gCM nanoparticles to CSCs, B16-F10 tumor spheroid cells were cultured by a pellet 3D suspension culture technique and incubated with DID-stained hMnO 2 @ gCM nanoparticles and the phagocytosis of different time materials was observed by fluorescent confocal microscopy. By staining the nuclei with DAPI, DAPI appears blue under excitation by the 405 nm laser. Cell actin was stained by phalloidin (FITC), which appears green under excitation by 488nm laser. The DID is excited to red with 639 nm excitation light. As a result, as shown in FIG. 6C, the B16-F10 tumor cells showed a red fluorescence that became stronger gradually over time. And red fluorescence gradually penetrates into the interior of the tumor sphere. Since CSCs are generally located inside the tumor, it is derived that hMnO 2 @ gCM possesses the ability to target CSCs located inside the tumor sphere. Furthermore, different levels of B16-F10 tumor cells were scanned (FIG. 9A, B, C shows CLSM images of B16F10 tumor cells incubated with hMnO 2 @ gCM at 2, 4, 8 hours, respectively). After 2 hours of incubation, the nanomaterial was only present at the outermost periphery of the cells at the different cell layers. Over time, hMnO 2 @ gCM can be observed inside the different cell layers when the incubation time reaches 8 hours. The hMnO 2 @ gCM has good tumor sphere penetrating capacity. To further quantify the amount of CSCs phagocytosing hMnO 2 @ gCM, B16-F10 tumor cells and DID-stained hMnO 2 @ gCM were incubated for different times. Wherein CSCs are fluorescently labeled by CD133-PE and the phagocytosis of different time materials in CSCs is analyzed by flow cytometry. The results obtained are shown in FIG. 6D, with the amount of CSCs phagocytosing hMnO 2 @ gCM gradually increasing over time, and the amount of phagocytosis increased by about twice over 8 hours of incubation compared to 2 hours.
To observe and analyze the in vivo distribution of hMnO 2 @ gCM at various times, IR 780-stained hMnO 2 and hMnO 2 @ gCM were intravenously injected into B16-F10 tumor-bearing mice, and fluorescence from IR780 was collected to obtain in vivo fluorescence images (fig. 6E, F and 10, fig. 10 shows in vivo fluorescence imaging of intravenous B16F10-CSCs tumor-bearing mice, and hMnO 2 and hMnO 2 @ gCM were injected). The signal of the nano material in the tumor gradually increases and reaches the maximum value after 24 hours along with the time extension, which indicates that the material is slowly enriched in the tumor part. The fluorescence intensity of hMnO 2 @ gCM is obviously stronger than that of hMnO 2, which shows that the coating of the CSC cell membrane can improve the enrichment of the nano material at the tumor part. In order to quantitatively analyze the distribution of hMnO 2 @ gCM in organisms, hMnO 2 and hMnO 2 @ gCM are intravenously injected into mice bearing tumors of B16-F10, and the main organs of the mice, including liver, spleen and tumors, are tested for the content of Mn by ICP technology, so that the enrichment of nano materials in each organ is calculated. As shown by G and H in FIG. 6, the enrichment in both liver and spleen was reduced compared to hMnO 2,hMnO2 @ gCM. Enrichment at the liver and spleen sites is due to phagocytosis of material by macrophages, and the CSCs cell membrane coated nanomaterial has low immunogenicity, which can reduce clearance of hMnO 2 @ gCM by immune cells. Most importantly, the enrichment of hMnO 2 @ gCM at the tumor site is higher than that of hMnO 2, which proves that the coating of the CSCs cell membrane improves the enrichment of the nanomaterial at the tumor site. The CSC cell membrane has a homologous targeting function, and can enhance the targeting effect of the bionic nano-material on the CSCs. To verify the effect of hMnO 2 @ gCM on targeting CSCs. The results of the immunofluorescent staining of the mice tumor sections collected 24 hours after intravenous injection of hMnO 2 @ gCM and the immunofluorescent staining of CSCs showed a clear co-localization of hMnO 2 @ gCM and CSCs as shown in fig. 6I and J, demonstrating that hMnO 2 @ gCM can well target CSCs in tumors.
3. Evaluation of hMnO 2 @ gCMs genetically engineered cell membrane nanoparticles for tumor hypoxia relief
The results are shown in FIG. 11 (hMnO 2 @ gCMs-buffered hypoxia-related results), where FIG. 11A shows an immunofluorescence image of HIF-1α in B16F10 tumor cells after treatment with PBS and hMnO 2 @ gCMs. Scale bar, 10 μm. The statistics of the percentage of CD133 + in B16F10 tumor cells after hypoxia was relieved by various nanocomposites are shown in fig. 11B. The expression of the stem related genes (OCT 4, NANOG and SOX 2) in B16F10 tumor cells after various treatments is shown in FIGS. 11C-E. 2000 or 4000 re-seeded cells (F in FIG. 11) and number (G in FIG. 11, >50 μm). Scale bar, 100 μm. Immunohistochemical staining and relative immunohistochemical staining intensity of HIF-1α (H, K in fig. 11), CD133 (I, L in fig. 11), CD47 (J, M in fig. 11) in tumor bearing mice with B16F10-CSCs after intravenous injection of saline or hMnO 2. Scale bar, 50 μm.
Analysis: the H 2O2,hMnO2 @ gCM nano particles can be generated by cancer cells in the tumor, so that H 2O2 in the cancer cells can be decomposed into oxygen and water, and the generated oxygen can be used for relieving the hypoxia of the tumor in situ. To verify the ability of hMnO 2 @ gCM nanoparticles to relieve hypoxia, B16F10 tumor cells were co-incubated with hMnO 2 @ gCMs nanoparticles and culture continued in a hypoxic environment. B16F10 tumor cells, cultured under hypoxic conditions, express large amounts of HIF-1. Alpha. After incubation of hMnO 2 @ gCMs nanoparticles, HIF-1α expression of the cells was greatly reduced, indicating that oxygen produced by hMnO 2 @ gCMs nanoparticles significantly reduced hypoxia in tumor cells (FIG. 11A). Studies have shown that as hypoxia increases, the percentage of CSCs in tumors also increases. After successful verification that hMnO 2 nanoparticles can significantly improve hypoxia of B16F10 tumor cells, the proportion of CSCs in B16F10 tumor cells was further analyzed. As shown in fig. 11B, the proportion of CD133 + CSCs in B16F10 tumor cells was significantly reduced after incubation with hMnO 2 nanoparticles, indicating that hypoxia improvement can reduce the proportion of CSCs. OCT4, NANOG, and SOX2 are the three most common overexpressed dryness-related genes in CSCs. After B16F10 tumor cells were incubated with hMnO 2 nanoparticles, OCT4, NANOG, and SOX2 expression was analyzed by qRT-PCR. As shown by C-E in FIG. 11, expression of all three genes decreased after incubation of hMnO 2 nanoparticles. Indicating that the alleviation of hypoxia has better inhibition effect on the expression of the three dry genes. The ability of tumor balls to form is considered a hallmark feature of CSCs. In order to demonstrate the decrease in CSCs proportion after treatment, in vitro tumor spheroid formation experiments were performed. After B16F10 tumor spheroid cells are incubated with hMnO 2 nano-particles, the same number of cells are collected and inoculated into an ultra-low adhesion cell culture 96-well plate to form tumor spheroids. After 10 days of tumor ball growth, the number of tumor balls greater than 50 μm was counted. As shown in figure 11F, G and figure 12 (after treatment with PBS and hMnO 2, 2000 cells were seeded and maintained for 10 days, and then optical micrographs of tumor sphere formation were taken), PBS groups had no effect on tumor sphere formation, resulting in a large number of tumor spheres. However, hMnO 2 nanoparticles significantly reduced tumor sphere formation, small and fragmented tumor spheres were observed. Statistics of the number of tumor spheres also indicate that relief of hypoxia has an inhibitory effect on tumor sphere formation.
To verify the alleviation of tumor hypoxia in vivo of hMnO 2 nanoparticles, tumor-slice HIF-1α expression was analyzed in tumor-bearing mice by intravenous injection of hMnO 2 to B16F 10. Compared with the control group, the expression of HIF-1 alpha in the mouse tumor section of the experimental group injected with hMnO 2 is obviously reduced, which indicates that the hypoxia of the tumor tissue is obviously relieved (K in figure 11). The main reason is that H 2O2 in tumor tissue and hMnO 2 nano particles enriched in tumor tissue can react to generate oxygen so as to effectively reduce the hypoxia of the tumor tissue part. By semi-quantitative analysis of confocal images of tissue sections, it was further demonstrated that HIF-1α expression was significantly reduced after intravenous injection of hMnO 2, and tumor hypoxia was significantly improved (H in fig. 11) compared to the control group. Research shows that the hypoxia microenvironment drives the enrichment of CSCs, which are concentrated in the hypoxia zone of mouse tumors, and the process is mainly mediated by HIF-1 a. After intravenous injection of hMnO 2 nanoparticles, the change in expression of CSCs marker CD133 was compared by immunofluorescence histochemical staining. As shown in fig. 11I and L, after the hMnO 2 nanoparticles significantly improved tumor hypoxia, the proportion of CSCs was also significantly reduced, indicating that hypoxia alleviation can significantly reduce the proportion of CSCs in vivo. Increased expression of CD47 can allow cancer cells to evade phagocytosis of macrophages and promote the phenotype of CSCs through the CD 47-sirpa signaling pathway, and studies indicate that HIF-1 a regulates CD47 expression by directly activating transcription of the CD47 gene. In the clear that hMnO 2 nanoparticles significantly slowed hypoxia, we validated CD47 expression in tumors (M in fig. 11). After intravenous injection of hMnO 2 nanoparticles, CD47 expression was significantly reduced in tumors, indicating that hypoxia alleviation can reduce CD47 expression (J in fig. 11).
4. Evaluation of anti-tumor immune activation effect of hMnO 2 @ gCMs genetically engineered cell membrane nanoparticles
The experimental results of the evaluation of the anti-tumor immune activation effect of hMnO2@gCMs genetically engineered cell membrane nano particles are shown in figures 13-1 and 13-2, wherein A in figure 13-1 shows a schematic diagram of experimental design; FIGS. 13-1 and 13-2 show flow cytometry analysis of macrophages in F4/80 +CD11b+CD45+ cell gating (B in FIG. 13-1), M1-type macrophages (CD 80 +) (C in FIG. 13-1, D in FIG. 13-2) and M2-type macrophages (CD 206 +) (E in FIG. 13-1, F in FIG. 13-2). The ratio between the different sets of M1 and M2 macrophages is shown in FIG. 13-1 at G. The flow cytometric analysis of CD86 + dendritic cells in tumor tissue, gated on CD45 +CD11c+ cells, is shown at I in FIG. 13-1 and at H in FIG. 13-2. Flow cytometry analysis of CD3 +、CD8+ and CD4 + T cells in CD45 + cell tumor gating is shown in FIG. 13-1 at K, L, M and J in 13-2. N in fig. 13-1 shows immunohistochemical images of tumors showing infiltration of T cells and macrophages in different treatment groups. Scale bar, 20 μm.
Analysis: the research finds that the hypoxia microenvironment plays an important role in cancer immunotherapy. First, HIF-1 a regulates CSCs over-expressing CD47, thereby allowing cancer cells to evade phagocytosis of macrophages through the CD 47-sirpa "do not eat me" signaling pathway to achieve immune escape. On the other hand, the hypoxic microenvironment regulates the transition of tumor-associated macrophages (TAMs) to M2 phenotype cells, M2 TAMs being immunosuppressive cells, through the anti-tumor immune effect to promote tumor growth. hMnO 2 @ gCM nano particles can improve the immune suppression microenvironment by relieving the hypoxia of tumors, and can enhance the immune response by blocking the CD47-SIRP alpha 'eating me' signal path. After the end of treatment, the intratumoral TAM was analyzed for changes by flow cytometry (B in FIG. 13-1). Mice injected intravenously with hMnO 2 had increased TAM type M1 (C in fig. 13-1, D in fig. 13-2) and significantly decreased TAM type M2 (E in fig. 13-1, F in fig. 13-2) compared to PBS group. Improvement in hypoxia proved to improve TAM and thereby increase phagocytosis of macrophages. The intravenous hMnO 2 nanoparticle group had significant polarization of M2 toward M1 in TAM within the tumor compared to the other groups (G in fig. 13-1). The improvement of the hypoxia is shown to be capable of better enhancing the immunity, thereby achieving better treatment effect.
GCM increase infiltration of DC and T cells at the tumor site, thereby inhibiting tumor growth. Following treatment, the number of DC and T cells from different populations in the mouse tumor was examined by flow cytometry. The results are shown in FIG. 13-2H, J and FIG. 13-1 at I, K, L, M, with more DC and infiltration of CD4 + and CD8 + T cells in the mouse tumor after gCM nanoparticle-induced treatment. Meanwhile, we examined the changes in the content of CD8 + T cells and macrophages in the tumors of mice in different treatment groups by immunofluorescence histochemical staining. In the control group, T cell and macrophage infiltration was limited, and T cell and macrophage infiltration in tumor tissue was significantly improved by combination therapy of hMnO 2 @ CSCs-gCM nanoparticle-induced hypoxia improvement and CD47-SIRP alpha signaling pathway blocking (N in FIG. 13-1).
Secretion of related cytokines such as tumor necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) is detected by enzyme-linked immunosorbent assay (ELISA). Compared with the control group, the expression of TNF-alpha and IFN-gamma in the tumor after the hMnO 2 @ gCM nano particle treatment is obviously up-regulated. It was demonstrated that hMnO 2 @ gCM nanoparticles can alter the immune-suppressing microenvironment within tumors and induce cytotoxic T cell-mediated antitumor immunity after TME (fig. 14 shows TNF- α (a in fig. 14) and IFN- γ (B in fig. 14) levels in mouse tumor tissues after different treatments.
5. Evaluation of the effect of hMnO 2 @ gCMs Gene engineering cell Membrane nanoparticles on tumor growth inhibition and eradication of CSCs
Results fig. 15: hMnO 2 @ gCM inhibits tumor growth and eliminates CSCs. A treatment regimen diagram for a mouse model carrying B16F10-CSCs subcutaneously is shown in FIG. 15A. Individuals of each group of B16F10-CSCs tumor-bearing mice receiving different treatments (B in fig. 15), average tumor growth kinetics (C in fig. 15), and body weight changes (D in fig. 15) were recorded every 2 days. The relative immunohistochemical staining intensities of CD133 (E in FIG. 15), NANOG (F in FIG. 15), SOX2 (G in FIG. 15) in each group B16F10-CSCs tumor-bearing mice at the end of treatment are shown in FIGS. 15E-G. Immunohistochemical staining of tumors Ki 67, CD133, NANOG and SOX2 in each group of B16F10-CSCs tumor-bearing mice at the end of treatment is shown at H in FIG. 15. Scale bar, 50 μm.
Analysis: to investigate the effect of hMnO 2 @ gCM on CSCs tumor models on CSCs eradication and on tumor growth inhibition. First, constructing a CSCS tumor model by using B16F10 tumor spherical cells. In vivo treatment experiments, mice were divided into 4 groups: PBS, hMnO 2、gCM、hMnO2 @ gCM. After the tumor had grown to 50-100 mm 3, mice were dosed by intravenous injection, 3 times a day. The body weight of the mice and the tumor size were weighed every other day during the course of treatment. The tumor size detection results of the mice are shown as B and C in fig. 15, compared with the control group, the tumor growth of the mice in the experimental group is obviously delayed after 14 days of treatment, and the tumor growth inhibition effect of hMnO 2 @ gCM is optimal. The results of the weight change of the mice are shown as D in FIG. 15, and the weight change of the mice is not large after 14 days of treatment. Ki-67 staining of tumor sections showed that the positive proportion of tumor cells Ki-67 was reduced after treatment with hMnO 2 @ gCM, and proliferation of tumor cells was significantly inhibited (H in FIG. 15). At the same time, H & E staining of tumor sections also demonstrated that hMnO 2 @ gCM treatment group caused extensive damage to tumor cells (fig. 16).
CSCs are the primary cause of tumor recurrence and metastasis, and to verify the effect of hMnO 2 @ gCM nanoparticle treatment on CSCs eradication, we analyzed the expression of tumor-slice CSCs-related markers (CD 133, NANOG, and SOX 2) after the end of treatment (H in fig. 15). Compared with the control group, the expression of the related markers of the mice tumor sections CSCs in the experimental group injected with hMnO 2 @ gCM is obviously reduced, which indicates that the CSCs in tumor tissues are obviously inhibited. Further by semi-quantitative analysis of immunohistochemical images of tissue sections, the proportion of CSCs in tumor tissue after treatment was significantly reduced compared to control, with the most significant reduction in hMnO 2 @ gCM treatment group (E-G in fig. 15). After the end of the treatment, the major organs of the B16F10 tumor-bearing mice were tissue-sectioned for heart, liver, spleen, lung and kidney, and the results are shown in fig. 17, wherein the major organs of the treatment group were not distinguished from the control group, and no significant necrosis, fibrosis and inflammation occurred. The result of the tissue section shows that the hMnO 2 @ gCM nano particles have better biocompatibility in vivo and are safe in mice.
From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the above examples provide a genetically engineered CSC film coated hMnO 2 @ gCMs biomimetic nano platform that exhibits excellent hypoxia mitigation and immune checkpoint blocking capability. By decoration of gCM on the surface of hMnO 2, the biomimetic nano platform shows effective CSCs specific targeting ability through the homologous targeting ability of CSC membrane. The obtained hMnO 2 can induce the decomposition of endogenous H 2O2 of tumor, relieve tumor hypoxia, inhibit CSCs, reduce the expression of CD47, and enhance immune response. In addition, sirpa over-expressed on gCM enhances cancer immunotherapy by disrupting the CD 47-sirpa signaling pathway. Down-regulation of CD47 and immune checkpoint blocking therapy synergistically inhibit the CD47-SIRP alpha signaling pathway, eliciting a significant immune response. In the murine melanoma model carrying B16F10-CSCs, the alleviation of tumor hypoxia and the synergistic inhibition of the CD47-SIRP alpha signaling pathway have shown a powerful effect in eradicating CSCs and inhibiting tumor growth. Therefore, the bionic nano-platform shows great prospect of eliminating the CSCs.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (11)

1. A genetically engineered cell membrane nanoparticle for inducing immunity to tumor stem cells, comprising:
Nanoparticles with the function of relieving tumor hypoxia; and
And the cell membrane vesicle coated on the nanoparticle with the function of relieving tumor hypoxia is a cell membrane vesicle derived from a tumor stem cell of which the SIRP alpha variant is genetically engineered to be overexpressed.
2. The genetically engineered cell membrane nanoparticle of claim 1, wherein the nanoparticle having tumor hypoxia relieving function is one or more of hMnO 2 nanoparticle, catalase nanoparticle, MOF nanoparticle, graphite alkyne nanoparticle, and ferromanganese nanoparticle.
3. The genetically engineered cell membrane nanoparticle of claim 1, wherein the tumor stem cell-derived cell membrane vesicle is one or more of a cell membrane nanovesicle derived from a melanoma stem cell, a breast cancer stem cell, a brain glioma stem cell, and a liver cancer stem cell.
4. The genetically engineered cell membrane nanoparticle of claim 1, wherein the mass ratio of the nanoparticle having tumor hypoxia relieving function to the cell membrane vesicle is 1:1-1:4.
5. Use of genetically engineered cell membrane nanoparticles inducing immunity to anti-tumor stem cells according to any one of claims 1 to 4 for the preparation of anti-tumor drugs.
6. A medicament for inducing immunity to tumor stem cells, comprising an effective amount of the genetically engineered cell membrane nanoparticle of any one of claims 1 to 4 and a pharmaceutically acceptable carrier.
7. A method for preparing genetically engineered cell membrane nanoparticles for inducing immunity to anti-tumor stem cells according to any one of claims 1 to 4, comprising the steps of:
synthesizing nano particles with the function of relieving tumor hypoxia;
synthesizing cell membrane vesicles derived from tumor stem cells genetically engineered to overexpress SIRP alpha variants;
Wrapping the cell membrane vesicles derived from the tumor stem cells on the nano-particles with the function of relieving tumor hypoxia, so as to obtain the genetically engineered cell membrane nano-particles.
8. The method of claim 7, wherein the nanoparticle having a function of alleviating tumor hypoxia is one or more of hMnO 2 nanoparticle, catalase nanoparticle, MOF nanoparticle, graphite alkyne nanoparticle, and ferromanganese nanoparticle.
9. The method of claim 7, wherein the tumor stem cell-derived cell membrane vesicles are one or more cell membrane nanovesicles derived from melanoma stem cells, breast cancer stem cells, brain glioma stem cells and liver cancer stem cells.
10. The method of claim 7, wherein encapsulating the tumor stem cell-derived cell membrane vesicles on the nanoparticle having tumor hypoxia relieving function comprises:
and mixing the cell membrane vesicles derived from the tumor stem cells with the nano-particles with the function of relieving tumor hypoxia, and extruding the mixture through a micro extruder to form the genetically engineered cell membrane nano-particles.
11. The method of claim 10, wherein the micro-extruder has a membrane pore size of 400nm and 200nm.
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