CN115252782B - Oxygen-carrying bionic molecular probe, preparation method thereof and application thereof in HIFU and immune synergistic treatment of cancers - Google Patents
Oxygen-carrying bionic molecular probe, preparation method thereof and application thereof in HIFU and immune synergistic treatment of cancers Download PDFInfo
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- CN115252782B CN115252782B CN202210899284.7A CN202210899284A CN115252782B CN 115252782 B CN115252782 B CN 115252782B CN 202210899284 A CN202210899284 A CN 202210899284A CN 115252782 B CN115252782 B CN 115252782B
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Abstract
The invention relates to the technical field of nano immunoregulation and cancer treatment reagents, in particular to an oxygen-carrying biomimetic molecular probe, a preparation method thereof and application thereof in HIFU and immune cooperative treatment of cancers. The oxygen-carrying bionic molecular probe comprises a shell, wherein liquid fluorocarbon and oxygen are wrapped in the shell; the outer shell is wrapped with cancer cell membranes; superparamagnetic iron oxide is inlaid in the shell. The probe can realize homologous targeting, enhance the killing of HIFU to in-situ tumor tissues, induce and release more tumor-related antigens, and stimulate the anti-tumor immunity of organisms. Meanwhile, the probe can also release oxygen, improve the hypoxic environment of tumors and relieve immunosuppression. The oxygen-carrying biomimetic molecular probe has wide application prospect in image-guided tumor enhancement treatment, can cooperate with HIFU and PD-L1 monoclonal antibody immunotherapy, and can inhibit tumor growth and prevent tumor metastasis.
Description
Technical Field
The invention relates to the technical field of nano immunoregulation and cancer treatment reagents, in particular to an oxygen-carrying biomimetic molecular probe, a preparation method thereof and application thereof in HIFU and immune cooperative treatment of cancers.
Background
High-intensity focused ultrasound (HIFU) is generally considered to be one of the most representative non-invasive therapeutic strategies for thermal ablation and mechanical destruction of tumor tissue using focused Ultrasound (US). This technique has shown considerable potential in the treatment of solid tumors, including malignant tumors that grow in the breast, pancreas, liver, kidney and prostate. However, as the tissue penetration depth and blood perfusion rate increase, the ultrasonic energy decays exponentially, resulting in insufficient energy accumulation in the target area, affecting the therapeutic effect of HIFU. Classical strategies to improve HIFU cancer ablation efficacy are to increase acoustic power or extend ablation time, but these options may increase the risk of adverse biological effects such as transient pain, skin burns or nerve damage. Several studies have demonstrated that the introduction of potentiators (synergistic agents, SAs) is an effective alternative to enhance the efficacy of HIFU surgery.
Microbubbles (MBs) are one of the most commonly used synergists, which can effectively alter the acoustic environment and increase ultrasonic energy deposition by enhancing thermal and cavitation effects. However, their micron-sized size prevents them from penetrating tumor tissue. Meanwhile, the microbubbles have poor stability, short half-life and uncontrollable cavitation range, so that the clinical application of the microbubbles is limited. In recent years, various Perfluorocarbon (PFC) materials capable of liquid-gas phase transition are prepared into HIFU potentiators, and compared with microbubbles, the HIFU potentiators have remarkable advantages and potential such as long blood circulation time, strong stability and easy modification. Theoretically, perfluorocarbons based on nano-size can enter tumor tissue through vascular circulation and be converted into microbubbles in situ by HIFU excitation, thereby improving ablation efficiency. Applicant team has successfully developed temperature responsive phase change nanoparticles in previous work for accurate ultrasound examination and enhanced HIFU procedures.
On the other hand, however, HIFU ablation therapy alone may inevitably lead to the presence of residual tumors, which in turn lead to recurrence and metastasis of the tumor. How to avoid the occurrence of tumor recurrence and metastasis, how to fully utilize the advantages of the HIFU binding potentiator in tumor therapy and prevent the occurrence of side effects, and how to activate specific and non-specific anti-tumor immune responses while HIFU therapy is a technical problem to be solved by the skilled person.
Disclosure of Invention
The invention aims to provide an oxygen-carrying bionic molecular probe for solving the technical problem that the single HIFU ablation treatment mode in the prior art leads to recurrence and metastasis of tumors to a certain extent.
In order to achieve the above purpose, the invention adopts the following technical scheme:
an oxygen-carrying bionic molecular probe comprises a shell, wherein liquid fluorocarbon and oxygen are wrapped in the shell; the outer shell is wrapped with cancer cell membranes; superparamagnetic iron oxide is inlaid in the shell.
The scheme also provides a preparation method of the oxygen-carrying biomimetic molecular probe, which comprises the following steps in sequence:
s1, preparing P-SP nano particles: dissolving polylactic acid-glycolic acid copolymer in methylene dichloride; then adding perfluorohexane for the first time of sound vibration emulsification; adding PVA, and performing a second sound vibration emulsification; finally adding isopropanol and magnetically stirring; washing to obtain P-SP nanoparticles;
s2 extraction of cancer cell membranes: obtaining a cell membrane of a breast cancer 4T1 cell;
s3, preparing a M@P-SOP molecular probe: mixing cancer cell membranes and P-SP nanoparticles to obtain a mixed solution; pushing the mixed solution for multiple times by a lipid extruder, and washing to obtain M@P-SP nanoparticles; oxygenation was performed in M@P-SP nanoparticles to obtain M@P-SOP molecular probes.
The scheme also provides application of the oxygen-carrying biomimetic molecular probe in preparing an immune activation system.
The scheme also provides application of the oxygen-carrying biomimetic molecular probe in preparing an anti-tumor system, wherein the anti-tumor system comprises the oxygen-carrying biomimetic molecular probe, high-intensity focused ultrasound equipment and a PD-L1 antibody.
The principle and the advantages of the scheme are as follows:
the technical proposal prepares the O-carrying liquid fluorocarbon and ferroferric oxide coated by cancer cell membrane for the first time 2 Molecular probes (M@P-SOP). The introduction of the cancer cell membrane coating can lead the molecular probe to have the capability of targeting homologous tumor cells and actively enrich in tumor tissues. Low boiling point liquid fluorocarbon carryover O 2 After being irradiated by HIFU, the HIFU is converted into microbubbles, and O is released while improving the ablation effect of the HIFU 2 Relieving the anoxic microenvironment. Therefore, the phase-change PFH nanoparticle not only can be used as an ideal synergist for HIFU treatment, but also can be used as a good oxygen carrier, and can relieve the tumor hypoxia microenvironment.
Under the action of HIFU, the molecular probe of the scheme can induce the death of immunogenic cells (immunogenic cell death, ICD) and relieve hypoxia to improve the tumor immune environment in a combined way, thereby activating specific and non-specific anti-tumor immune responses. Therefore, the oxygen-carrying biomimetic molecular probe is used for preparing an immune activation system (immune suppression relieving system) and is specially used for improving the killing capacity of the immune system to cancer cells and inducing tumor-related giant phagocytosisThe cells are polarized towards the M1 type, so that the polarization of the M2 type is reduced, and the immunosuppression is relieved. Also simultaneously induces stronger immunogenic death of tumor cells, releases more tumor-associated antigens, stimulates DC cell maturation and activates CD8 + T cells stimulate the antitumor immunity of the body.
The scheme combines HIFU, hypoxia relieving (oxygen carrying biomimetic molecular probe) and anti-PD-L1 monoclonal antibody for tumor treatment for the first time, and the three form an effective anti-tumor system. By combining immune checkpoint blockade with HIFU-induced anti-tumor immune response and using perfluorocarbon nanoparticle-carried O 2 Relieving hypoxia, generating strong synergistic effect and providing a new strategy for cancer treatment. The particles coated by cancer cell membranes (cancer cell membrane, CCM) have affinity ligand inherited from parent cells, can actively target homologous tumors, and ensure that perfluorocarbon nanoparticles are effectively accumulated in tumor tissues before HIFU irradiation. By increasing T cell responses in combination with checkpoint therapy (PD-L1 mab), M@P-sop+hifu treatment can produce a significant distant effect, inhibiting the growth of distant metastases.
Furthermore, M@P-SOP can also enable complementary multi-modality imaging, including ultrasound imaging of PFH, photoacoustic imaging (PAI) of superparamagnetic iron oxide (SPIO), and T2-weighted Magnetic Resonance Imaging (MRI).
In conclusion, the study successfully prepares the oxygen-carrying biomimetic molecular probe M@P-SOP coated by cancer cell membranes and carrying SPIO and PFH, and the breast cancer is treated by synergistic HIFU ablation and synergistic PD-L1 antibody. The probe can realize homologous targeting, gather at tumor site, increase killing of HIFU to in-situ tumor tissue, induce stronger tumor cell immunogenic death, release more tumor related antigen, stimulate DC cell maturation, activate CD8 + T cells stimulate the antitumor immunity of the body. Meanwhile, the probe can also release oxygen, improve the hypoxic environment of tumors, induce tumor-related macrophages to polarization of M1 type, reduce polarization of M2 type and relieve immunosuppression. After the PD-L1 antibody is cooperated, tumor cells and CD8 can be cut off + The PD-1/PD-L1 immune inhibition pathway between T cells further amplifies the generated immune response and spreads to the whole body, effectively inhibiting the generation of metastasis. In addition, M @The P-SOP can also perform ultrasonic/photoacoustic/magnetic resonance multi-mode imaging and guide treatment in real time. Therefore, the combined treatment strategy can effectively kill the primary tumor and inhibit the metastasis, and has a certain clinical reference value.
Further, the shell is polylactic acid-glycolic acid copolymer; the cancer cell membrane is from a 4T1 breast cancer tumor cell; the liquid fluorocarbon is perfluorohexane. Polylactic acid-glycolic acid copolymers are the first choice for drug delivery because of their low toxicity, high biocompatibility, ease of chemical modification and food products, and approval by the U.S. Food and Drug Administration (FDA).
Further, the ratio of the polylactic acid-glycolic acid copolymer, the perfluorohexane and the superparamagnetic iron oxide was 50mg:200 μl: 50. Mu.L. The above proportion can realize good encapsulation of medicines such as perfluorohexane, superparamagnetism ferric oxide and the like.
Further, in S1, the first and second sonic emulsification are performed under ice bath conditions, and the parameter conditions are: 60W, 3min. The adoption of the above-mentioned sound vibration emulsification condition can promote the formation of nano particles with proper grain size.
Further, in S2, the method for obtaining the cell membrane of the breast cancer 4T1 cell is as follows: culturing breast cancer 4T1 cells to logarithmic phase, collecting cells, centrifuging and collecting cell precipitate; after cell precipitation is cracked and repeatedly frozen and thawed, centrifuging to obtain supernatant; and centrifuging to obtain cancer cell membrane precipitate, and freeze-drying the cancer cell membrane precipitate for later use. The conventional means in the prior art for obtaining the cell membrane of the breast cancer 4T1 cell by a cell lysis method is easy to operate, and the obtained cell membrane has good targeting capability.
Further, parameters of the high-intensity focused ultrasound equipment are set to 120W and 3s; the dosage of the oxygen carrying biomimetic molecular probe is 200 μl; the amount of PD-L1 antibody used was 1.5 mg/kg.BW. Under the action of HIFU, the oxygen-carrying biomimetic molecular probe realizes enhanced ablation of tumor tissues, releases more tumor-related antigens, stimulates DC cell maturation and activates CD8 + T cells stimulate the antitumor immunity of the body. The oxygen-carrying bionic molecular probe can also release oxygen, improve the anoxic environment of the tumor and induceTumor-associated macrophages polarize towards M1, reducing polarization of M2, alleviating immunosuppression. After the PD-L1 antibody is cooperated, tumor cells and CD8 can be cut off + The PD-1/PD-L1 immune inhibition pathway between T cells further amplifies the generated immune response and spreads to the whole body, effectively inhibiting the generation of metastasis.
Drawings
Fig. 1 is a schematic diagram of the preparation flow and structure of the oxygen-carrying biomimetic molecular probe of example 1.
FIG. 2 is an electron microscope image of M@P-SOP of example 1.
FIG. 3 is a map of the Malvern particle size potential of M@P-SOP of example 1.
FIG. 4 shows the results of SDS-PAGE of cancer cell membrane protein fraction of example 1
FIG. 5 is a heated phase change coating of the molecular probe of example 1.
FIG. 6 is a graph showing the results of oxygen release experiments using the oxygen dissolution apparatus of example 1.
Fig. 7 shows the results of the laser confocal scanning microscopy test of experimental example 1 (in vitro homology targeting test).
FIG. 8 shows the results of flow cytometry analysis (in vitro homology targeting experiments) of Experimental example 1.
FIG. 9 is an in vivo fluorescence imaging image (in vivo homology targeting) of tumor-bearing mice of Experimental example 1.
FIG. 10 shows the results of the in vitro safety CCK-8 test of Experimental example 2.
FIG. 11 is an in vivo safety H & E stained section image of Experimental example 2.
Fig. 12 is an in vitro ultrasonic imaging experimental result of experimental example 3.
Fig. 13 is an in vivo ultrasonic imaging experiment result of experimental example 3.
Fig. 14 is an in vitro photoacoustic imaging experimental result of experimental example 3.
Fig. 15 is an in vivo photoacoustic imaging experimental result of experimental example 3.
Fig. 16 shows the results of in vitro MRI imaging experiments of experimental example 3.
Fig. 17 shows the in vivo MRI imaging experiment result of experimental example 3.
Fig. 18 shows the results of the experimental results of the HIFU in vitro bovine liver ablation synergy of experimental example 4.
FIG. 19 shows the results of the HIFU in vivo targeted ablation synergy experiment of Experimental example 4.
FIG. 20 shows the results of experiments (DC cells) on the ability of HIFU+ M@P-SOP treatment of Experimental example 4 to improve tumor immunity microenvironment.
FIG. 21 shows the results of a test for the ability of HIFU+ M@P-SOP treatment of Experimental example 4 to improve tumor immunity microenvironment (CD 8 + T cells).
FIG. 22 shows the results of an in vitro experiment of the ability of HIFU+ M@P-SOP treatment of Experimental example 4 to improve tumor hypoxia microenvironment.
FIG. 23 shows the results of in vivo experiments in which HIFU+ M@P-SOP treatment of Experimental example 4 improves the ability of tumor hypoxia microenvironment.
FIG. 24 flow cytometer analyses of infiltration of (A) M1 type macrophages and (B) M2 type macrophages in mice tumors and corresponding quantitative analyses.
FIG. 25 shows the results of HIFU+ M@P-SOP in combination with PD-L1 antibody of Experimental example 5 for treating breast cancer.
FIG. 26 shows the results of the immune mechanism evaluation test of Experimental example 5.
Detailed Description
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available.
Example 1: preparation and characterization of molecular probes
The technical scheme is that 4T1 breast cancer tumor cell membrane (cancer cell membrane, CCM) is extracted by chemical cracking and repeated freeze thawing, then CCM coated carrier superparamagnetism ferric oxide (superparamagnetic iron oxide, SPIO, american Ocean Nano company, product number: F0718SOR, particle diameter 10 nm) and polylactic acid-glycolic acid copolymer (polylactic acid-glycolic acid copolymer, PLGA, jinan Dacron Co., product number: DG-50DLGH018, lactic acid and glycolic acid ratio 50/50) bionic molecular probe is prepared by a double-emulsion method combined membrane extrusion methodNeedle (M@P-SP) is oxygenated by oxygen saturation to obtain oxygen-carrying oxygen (oxygen, O) 2 ) Biomimetic molecular probes (M@P-SOP).
The preparation method is more specifically as follows:
(1) Preparation of oxygen-carrying biomimetic molecular probe M@P-SOP
(1.1) preparation of P-SP nanoparticles carrying SPIO and PFH
Accurately weighing 50mg of PLGA dissolved in 2ml of CH 2 Cl 2 While adding 50. Mu.L (25 mg/ml) of SPIO, the ultrasonic cleaner was shaken until completely dissolved.
(1.2) 200. Mu.l of PFH solution was added and the primary emulsification (60W, 3 min) was carried out by an sonicator under ice bath conditions to give a dark brown primary emulsion.
(1.3) 8ml of PVA solution (4 wt.%, formulated with double distilled water using powdered PVA) was added again and the second emulsification (60W, 3 min) was carried out by a sonicator under ice bath conditions to give a pale brown multiple emulsion.
(1.4) 10ml of isopropanol solution (2 vol.% obtained by diluting isopropanol with double distilled water) was added and placed in small magnetic beads under ice bath conditions and magnetically stirred in a fume hood for 4h.
(1.5) washing with double distilled water (10000 rpm,8 min) 3 times to obtain PLGA-SPIO/PFH nanoparticle, namely P-SP.
In the preparation, PFH is replaced by double distilled water to obtain the P-S nanoparticles; on CH 2 Cl 2 Adding 10 mu l of DiI dye or DiR dye, and obtaining the DiI or DiR marked P-SP nanoparticle with unchanged residue.
(2) Extraction of cancer cell membrane CCM
(2.1) conventional culture of breast cancer 4T1 cells, collection of cells in the logarithmic growth phase with a cell scraper, centrifugation (3000 r/min,5 min) and removal of supernatant.
(2.2) hypotonic lysis buffer (available from Biyundian Co., ltd., cat# 042819190617) containing membrane protein extractant and phenylmethanesulfonyl fluoride (PMSF) was added, and the mixture was ice-washed for 15min.
(2.3) repeated freeze thawing for 3 times, centrifuging (700 g,10 min) at low temperature of about 4deg.C, and collecting supernatant.
(2.4) centrifuging at low temperature (14000 g,30 min), collecting precipitate to obtain CCM, and lyophilizing.
(3) Preparation of CCM-coated oxygen-carrying biomimetic molecular probe M@P-SOP
(3.1) mixing the prepared P-SP nanoparticles with CCM in a mass ratio of 1:2 by vortex to obtain a mixed solution.
(3.2) A polycarbonate film (1 μm) was added to a lipid extruder, and the mixture was pushed back and forth 10 to 12 times using the extruder.
(3.3) double steaming and washing (10000 rpm,8 min), removing redundant free tumor membranes, and obtaining the bionic molecular probe CCM@PLGA-SPIO/PFH coated by CCM, namely M@P-SP.
(3.4) another syringe of 20ml was filled with oxygen (about 20ml in excess) and connected to a two-way switch valve to close the valve.
(3.5) the other end of the two-way switch valve is connected with a container filled with M@P-SP or P-SP, the valve is opened, oxygen in the injector is injected, the valve is closed by a Liima, and the mixture is left standing for 2 to 3 hours at the low temperature of about 4 ℃ to obtain the oxygen-carrying biomimetic molecular probe CCM@PLGA-SPIO/O 2 PFH, M@P-SOP; or oxygen-carrying molecular probe PLGA-SPIO/O 2 PFH, P-SOP. A schematic diagram of the molecular probe obtained in this scheme is shown in FIG. 1.
The prepared molecular probe is characterized as follows: observing the shape of the material by a transmission electron microscope and a laser confocal microscope; the Malvern particle size potential measuring instrument detects particle size and potential and stability thereof; measuring the SPIO content of the molecular probe by an inductively coupled plasma emission spectrometer (inductively coupled plasma optical emission spectrometer, ICP-OES), and calculating the encapsulation efficiency; measuring the PFH encapsulation efficiency of the molecular probe by a trichloromethane ball breaking centrifugation method; protein gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE) is used for detecting cancer cell membrane proteins, and the membrane coating condition is verified; observing the heated phase change condition of the molecular probe on a microscope heating table; the dissolved oxygen meter monitors the oxygen release condition of the molecular probe.
The characterization results of the molecular probe are as follows: the M@P-SOP molecular probe is in a spherical shell-core structure under a transmission electron microscope, and the molecular probe has good dispersibility and uniform size under a laser confocal microscope (figure 2); the Malvern particle diameter potential measuring instrument detects that the particle diameter is (233.37 +/-2.20) nm, the potential is (-19.43+/-0.12) mV, and the particle diameter size stability is good (figure 3); the calculated SPIO encapsulation efficiency is 70.08% by ICP measurement; the encapsulation rate of PFH is calculated to be 50.17% by a methane dissolution centrifugation method; SDS-PAGE showed M@P-SOP retained the oncocyte membrane protein component (FIG. 4); the molecular probe under the microscope heating table is subjected to thermal phase transformation, and the phase transformation is most obvious at 70 ℃ (figure 5); the results of the oxygen dissolution instrument show that the molecular probe successfully carries oxygen and can release oxygen in a hypoxic environment, so that the local environmental oxygen concentration is remarkably improved (figure 6).
Experimental example 1: in vivo and in vitro homology targeting
Co-culturing DiI marked non-targeted P-SOP and targeted M@P-SOP molecular probes with 4T1 breast cancer cells respectively, observing the condition of targeting the molecular probes to the 4T1 cells under a laser confocal microscope, and detecting the binding rate by a flow cytometry; and then co-culturing the targeting M@P-SOP molecular probe with melanoma cells F16B10, breast cancer cells MCF-7, MDA-MB-231 and 4T1 cells respectively, observing the combination condition of the molecular probe and the cell targeting under a laser confocal microscope, and detecting the combination rate by using a flow cytometry. Establishing a 4T1 breast cancer in-situ tumor-bearing mouse (Balb/c) model, injecting DiR-marked non-targeting P-SOP and targeting M@P-SOP molecular probes through tail vein, performing in-vivo fluorescence imaging on the mice at different time points, measuring fluorescence signals at the tumor positions of the mice, and evaluating the performance of the M@P-SOP molecular probes in targeting the tumor positions of the mice.
The confocal laser microscope shows (figure 7) that more DiI marked red M@P-SOP molecular probes are visible around 4T1 cell nuclei, and the fluorescence intensity of the M@P-SOP group is strongest when the time is prolonged, and the peak value is reached at 4 hours; similarly, only a small amount of red M@P-SOP molecular probe aggregation was seen around the nuclei of F16B10, MCF-7 and MDA-MB-231, while a large amount of M@P-SOP homology targeting aggregation was seen around the nuclei of 4T 1; flow cytometer detection and confocal microscopy observations remained consistent (fig. 8). In vivo fluorescence imaging of tumor-bearing mice shows that the M@P-SOP molecular probe can be distributed in the tumor area of the mice in a targeted manner, the fluorescence signal is obviously higher than that of a non-target group, the fluorescence signal is gradually enhanced along with the extension of the injection time, and the fluorescence signal is reduced after reaching a peak value for 24 hours (figure 9).
Experimental example 2: in vivo and in vitro safety
In vitro safety: after P-SOP and M@P-SOP molecular probes with different concentrations are respectively co-cultured with 4T1 cells for 24 hours, cytotoxicity of each group is evaluated by a CCK-8 method. In vivo safety: the Kunming mice were randomly divided into control group and experimental group (n=3), after the control group was injected with physiological saline for 28d, and after the experimental group was injected with M@P-SOP molecular probes (1 d, 3d, 5d, 7d, 14d, 28 d), blood samples of the mice were collected from the orbit for blood routine and blood biochemical analysis, while important viscera (heart, liver, spleen, lung and kidney) were collected for H & E staining section observation.
The CCK-8 results showed that the different concentrations of P-SOP and M@P-SOP molecular probes were not significantly cytotoxic to 4T1 cells (FIG. 10). The results of H & E staining sections of the vital organs with conventional/biochemical indicators of mouse blood showed no significant abnormalities in each experimental group compared to the control group (fig. 11).
Experimental example 3: oxygen-carrying biomimetic molecular probe ultrasound/photoacoustic/magnetic resonance multi-mode imaging
(1) In vivo and in vitro ultrasound imaging (USI)
After the diluted M@P-SOP, M@P-SP and M@P-S are irradiated by HIFU, respectively adding the diluted M@P-SOP, M@P-SP and M@P-S into a gel model, and observing the development conditions of an ultrasonic Mode (B-Mode) and a contrast Mode (CEUS) of each group by using a PBS group as a control group. Establishing a 4T1 breast cancer in-situ tumor-bearing mouse model, and randomly dividing into: non-target group (P-SOP) and target group (M@P-SOP), after 24h of tail vein injection, the development change conditions of tumor region B-Mode and CEUS before and after HIFU irradiation are collected by an ultrasonic diagnostic apparatus, and the ultrasonic signals of each group of ultrasonic images are measured by DFY quantitative analysis software.
After HIFU irradiation, in the B-Mode and CEUS modes, no ultrasound enhancement signal was found in the PBS control group, only a few signal enhancement was found in the M@P-S group without PFH phase change material, whereas the ultrasound signals of the M@P-SOP and M@P-SP groups loaded with PFH phase change material were significantly enhanced, and the analysis results of the DFY quantitative analysis software were consistent with the ultrasound image (fig. 12); before HIFU irradiation, the ultrasound signals of the tumor area of the mice in the non-targeted group (P-SOP) and the targeted group (M@P-SOP) are not obviously different, while after HIFU irradiation, the ultrasound signals of the tumor area of the mice in the targeted group (M@P-SOP) are obviously enhanced, and the ultrasound signals of the tumor area of the mice in the non-targeted group (P-SOP) are not obviously changed, which is consistent with the analysis result of the DFY quantitative analysis software (fig. 13).
(2) In vivo and in vitro photoacoustic imaging (PAI)
Different concentrations of M@P-SOP were placed in a gel model, photoacoustic signals were acquired via a photoacoustic imager, and their relationship to M@P-SOP concentration was evaluated. Establishing a 4T1 breast cancer in-situ tumor-bearing mouse model, and randomly dividing the model into two groups: after molecular probes are injected into tail vein, photoacoustic signals of tumor areas are respectively collected at different time points (pre, 1h, 3h, 6h, 24h and 48 h) and quantitatively analyzed.
In vitro photoacoustic results show that M@P-SOP can enhance photoacoustic imaging, and photoacoustic signal values thereof are in linear relation with concentration, and enhance with increasing concentration (fig. 14); after the tumor-bearing mice are injected with molecular probes through tail veins, the photoacoustic signals of the tumor areas of the target group (M@P-SOP) are gradually enhanced, are obviously higher than those of the non-target group, and gradually decline after reaching a peak value for 24 hours. The quantitative analysis result is consistent with the photoacoustic image (fig. 15).
(3) In vivo and in vitro Magnetic Resonance Imaging (MRI)
Different concentrations of M@P-SOP were placed in a 2ml EP tube, signals were acquired via T2-weighted magnetic resonance imaging and evaluated for their relationship to M@P-SOP concentration. Establishing a 4T1 breast cancer in-situ tumor-bearing mouse model, and randomly dividing into: and (3) respectively collecting magnetic resonance imaging T2 weighted signals of the tumor area at different time points (pre, 1h, 3h, 6h, 24h and 48 h) after the molecular probes are injected into the tail vein of the non-targeted group (P-SOP) and the targeted group (M@P-SOP), and carrying out quantitative analysis.
In vitro MRI imaging results showed that T2-weighted signal intensity decreased with increasing M@P-SOP concentration, increased negatively, and that relaxation rate increased with increasing concentration in linear relationship with M@P-SOP concentration (FIG. 16); after the tumor-bearing mice are injected with molecular probes through tail veins, T2 weighted magnetic resonance signals of tumor areas of a targeting group (M@P-SOP) are gradually reduced, the signals are obviously lower than those of non-targeting groups, and the signals are gradually increased to be negative enhancement after reaching the lowest value for 24 hours. The quantitative analysis results were consistent with the T2 weighted image (fig. 17).
In conclusion, the prepared M@P-SOP molecular probe carrier has good US/PA/MR imaging enhancement effect, can effectively target and enrich the M@P-SOP in a tumor-bearing mouse model, and can be used as an ideal contrast agent for enhancing US/PA/MRI multi-mode imaging.
Experimental example 4: oxygen-carrying bionic molecular probe synergistic HIFU ablation and tumor microenvironment improvement
(1) HIFU in vitro bovine liver ablation synergy
Fresh beef liver is purchased, the relatively small parts of blood vessels and bile ducts are selected, and the beef liver is cut into a plurality of square blocks of 15cm multiplied by 10cm, and is subjected to degassing treatment. The experimental components are M@P-SOP, M@P-S and M@P, the dosage of the substance to be detected is 200 μl/melting point, wherein the concentration of each nanoparticle is 5mg/ml. HIFU power is divided into 90W, 120W, 150W, all for 3 seconds. After ablation of the ablation points is completed, cutting the ablation points layer by layer along the long axis direction of the acoustic beam, finding the maximum section of the coagulated necrosis tissue, observing the coagulated necrosis condition of the liver tissue, and evaluating the optimal HIFU treatment power. The high intensity focused ultrasound device (HIFU device) model JC200, period/frequency=1 ms/1000Hz, duty cycle 100%.
The results of the HIFU in-vitro bovine liver ablation synergy experiment show that the maximum section ablation area of the M@P-SOP group is obviously larger than that of other groups under the same HIFU power treatment condition. In the M@P-SOP treatment group, the maximum section ablation area shows a trend of 150W to 120W to 90W, but the long axial diameter of the 150W treatment group is too long, which is unfavorable for targeting ablation in vivo, and is easy to penetrate through a tumor area to damage the abdominal organs of mice (figure 18).
(2) HIFU living body targeted ablation synergy
A 4T1 breast cancer in situ tumor bearing mouse model was established, randomly divided into 4 groups (n=3): control groups (physiological saline), M@P-SOP, M@P-S, M@P, injection amount of 200. Mu.l/mouse, concentration of nanoparticles of 5mg/ml. Tumors of the above 4 groups of mice were subjected to HIFU (120W, 3 s) live ablation while the change of ultrasonic gray values of tumor areas was recorded. After the end of the HIFU ablation, each group of mice is sacrificed, after the tumor of the mice is cut through TTC dye staining, the mice are cut layer by layer along the long axis direction of the sound beam, the maximum section of the coagulated necrosis tissue is found, and the coagulated necrosis volume and the target area ultrasonic energy factor (EEF) are measured and calculated.
The results of the HIFU living body targeted ablation synergy experiment show (figure 19) that after the tumor of the mice injected with M@P-SOP through the tail vein is ablated through the HIFU, the ultrasonic gray value and the coagulation necrosis volume of the tumor target area are obviously larger than those of other groups, and the ultrasonic energy factor of the target area is obviously lower than those of other groups.
(3) HIFU+ M@P-SOP treatment improves the ability of tumor immune microenvironment
A 4T1 breast cancer in situ tumor bearing mouse model was established, randomly divided into 6 groups (n=3): control groups (normal saline), M@P-SP, M@P-SOP, HIFU, HIFU + M@P-SP, HIFU+ M@P-SOP, injection amount of 200 μl/mouse, nanoparticle concentration of 5mg/ml. After 24h of tail vein injection, the three groups of mice are all treated by HIFU (120W/3 s). On day 3 after treatment, the serum cytokine IL-12 and TNF-alpha content of each group of mice was detected, the tumors, tumor draining lymph nodes and spleens of the mice were dissected, single cell suspensions prepared from the draining lymph nodes and spleens were analyzed for Dendritic Cell (DC) maturity by flow cytometry, paraffin sections prepared from the tumors were examined by fluorescent staining for each group of induced tumor-immune cell death (Immunogenic cell death, ICD) and CD8 + Infiltration of cytotoxic T cells (Cytotoxic T lymphocytes, CTLs).
HIFU ablates tumor tissue, releases a large amount of tumor-associated antigens, greatly promotes DC cell maturation, and increases intratumoral CD8 + Infiltration of T cells and IL-12, TNF- α content in serum, while addition of M@P-SOP amplified ICD induction by HIFU and subsequent forward changes in immune mechanisms, greatly improved tumor immune microenvironment (FIGS. 20 and 21). In FIG. 21, green fluorescence indicates CD8 + T cells, blue fluorescence, represent nuclei. Maturation of DC cells under HIFU stimulation + The T cells are activated, and the addition of the phase change material PFH amplifies the immune forward effect, and the oxygen can further amplify the effect to a certain extent. In addition, oxygen can be filled simultaneously to relieve tumor hypoxia environment, thereby reducing polarization of macrophages to M2 type, promoting polarization of M1 type, and relieving hypoxia to CD8 + The inhibition of T cells promotes the mechanisms mutually, and enhances the anti-tumor effect.
(3) Capability of HIFU+ M@P-SOP treatment to improve tumor hypoxia microenvironment
Establishing an anoxic micro-environment 4T1 cell culture model, adding PBS into a negative control group, culturing under normal oxygen conditions, and culturing in a hypoxia model for 12h in a positive control group (PBS), a 2.5mg/ml M@P-SOP group and a 5mg/ml M@P-SOP group. 10mg/ml (Ru (dpp) 3 )Cl 2 Each of the above groups was added thereto, and the culture was continued for 12 hours. Observing the red fluorescence intensity of each group of cells, namely the hypoxia degree of the cells, by using an inverted fluorescence microscope; a 4T1 breast cancer in situ tumor bearing mouse model was established, randomly divided into 6 groups (n=6): control groups (normal saline), M@P-SP, M@P-SOP, HIFU, HIFU + M@P-SP, HIFU+ M@P-SOP, injection amount of 200 μl/mouse, nanoparticle concentration of 5mg/ml. After 24h of tail vein injection, the latter three groups were given HIFU (120 w,3 s) treatment. On day 3 after treatment, 3 mice were randomly taken from each group, the mice were sacrificed to dissect out tumors, paraffin sections were prepared, HIF-1α and pimonidazole expression were detected after fluorescent staining, and the improvement of the hypoxic microenvironment in the tumor area of the mice was evaluated. And simultaneously detecting the content of the residual mouse serum cytokine IL-10, dissecting out the tumor to prepare single cell suspension, and analyzing the polarization conditions of M1 type and M2 type of tumor-associated macrophages (tumor-associated macrophage, TAM) by flow cytometry.
Only a small amount of red fluorescence is seen in the negative control group, the positive control group shows high-intensity red fluorescence, the red fluorescence intensity is weakened after M@P-SOP is added, and the fluorescence intensity of the 5mg/ml M@P-SOP group is obviously reduced compared with that of the 2.5mg/ml M@P-SOP group, so that the alleviation degree of hypoxia is positively correlated with M@P-SOP concentration (figure 22); the mouse tumor hypoxia areas are shown by anti-HIF-1 alpha and anti-pimonidazole fluorescent staining, the M@P-SOP group fluorescence is weaker than that of the control group, the HIFU+ M@P-SOP group fluorescence is weakest, meanwhile, M2 type macrophages in tumor-bearing mice are reduced, M1 type macrophages are increased, IL-10 in serum is reduced, and the tumor immunosuppression microenvironment is suggested to be relieved (figure 23). Fig. 24 shows the results of flow cytometry in different treatment groups, and it can be seen that the oxygen-carrying nanoparticles of the present embodiment can significantly increase the number of M1 macrophages and significantly decrease the number of M2 macrophages (n=3). The oxygen-carrying biomimetic molecular probe is used for preparing an immune activation system (immune suppression relieving system), is specially used for improving the killing capacity of the immune system on cancer cells, inducing tumor-related macrophages to M1 type polarization, reducing M2 type polarization and relieving immune suppression.
In conclusion, M@P-SOP can remarkably enhance the ablation effect of HIFU on tumors of isolated bovine livers and tumor-bearing mice. M@P-SOP targeting synergistic HIFU induces ICD effect, increases CD8 + Tumor infiltration of T cells improves the tumor immunosuppression microenvironment. M@P-SOP also can reduce tumor hypoxia microenvironment by releasing oxygen, polarize TAM into antitumor M1 type macrophages, and reduce polarization of tumor promoting M2 type macrophages. Simultaneously, IL-12 and TNF-alpha in serum are increased and IL-10 is reduced, so that the tumor immunosuppression microenvironment is further improved.
Example 5: synergistic HIFU and synergistic PD-L1 antitumor treatment of oxygen-carrying biomimetic molecular probe
(1) HIFU+ M@P-SOP synergistic PD-L1 antibody for treating breast cancer
Establishing a 4T1 breast cancer metastasis tumor-bearing mouse model (namely a bilateral in-situ tumor model, wherein the right side is in-situ tumor, the left side is artificial distant metastasis), and randomly dividing the mice into 6 groups (n=5) when the in-situ tumor grows to be 0.6-0.8cm in diameter: (1) control group, (2) PD-L1 group (PD-L1 antibody), (3) HIFU group, (4) HIFU+NPs group, (5) HIFU+PD-L1 group, (6) HIFU+NPs+PD-L1 group. Wherein NPs is M@P-SOP. Day0, 200 μl of physiological saline was administered to the tail of mice in groups (1) (2) (3) (5), and 200 μl of M@P-SOP (5 mg/ml) was administered to the tail of mice in groups (4) (6). Day1, HIFU treatment (120 w,3 s) was given to mice of groups (3), (4), (5), (6). Day2, 5, 8, mice of groups (2), (5), (6) were given a PD-L1 antibody (1.5 mg/kg, bioxcell Co., U.S.A., under the designation anti-mouse PD-L1 (B7-H1), cat# 804721J 3) by tail intravenous injection. Day15 was observed by recording the body weight of each group of tumor-bearing mice every 2 days. After the observation is finished, all tumor-bearing mice are sacrificed and tumors are taken out, the weight of the tumors is recorded, the primary tumors are stained with H & E, TUNEL paraffin sections, the metastases are stained with PCNA paraffin sections, and the apoptosis condition of the primary tumor cells and the proliferation condition of the metastases cells are observed.
The PD-L1 group has no statistical significance in comparison with the control group in terms of tumor weight, the pure HIFU group has slight tumor inhibition effect, the HIFU+NPs group and the HIFU+PD-L1 group have stronger tumor inhibition effect on primary tumors and metastases, and the HIFU+NPs+PD-L1 group has obvious tumor inhibition effect on primary tumors and metastases, and meanwhile, no obvious change in the body weight of tumor-bearing mice is observed in the treatment process. The primary tumor H & E staining showed that the tumor cells were visibly evident with nuclear shrinkage, fragmentation, disruption of cell morphology, maximum tumor cell apoptosis as shown by TUNEL staining, and minimal proliferation as shown by distant tumor PCNA staining (fig. 25). The graph in fig. 25 shows, from left to right, the weight of primary tumor in different treatments, the weight of metastasis in different treatments, and the change over time in the weight of mice in different treatments, respectively (1 st indicates primary tumor, and 2nd indicates metastasis). The 1st raw data of fig. 25 is (g): control:0.95, 0.62, 0.7, 0.53, 0.41; PD-L1:0.89, 0.53, 0.44, 0.4, 0.43; HIFU:0.39, 0.36, 0.33, 0.28; hifu+nps:0.37, 0.2, 0.27, 0.23, 0.24; hifu+pd-L1:0.23, 0.19, 0.21, 0.16, 0.18; hifu+nps+pd-L1:0.19, 0.15, 0.13, 0.1. The 2st raw data of fig. 25 is (g): control:0.29, 0.24, 0.21, 0.19, 0.14; PD-L1:0.19, 0.2, 0.18, 0.15, 0.13; HIFU:0.11, 0.1, 0.12, 0.09, 0.1; hifu+nps:0.1, 0.09, 0.1, 0.08; hifu+pd-L1:0.07, 0.06, 0.05; hifu+nps+pd-L1:0.07, 0.06, 0.03, 0.02, 0.01.
(2) Immune mechanism assessment
A 4T1 breast cancer metastasis tumor-bearing mouse model (i.e., a bilateral in situ tumor model, right: in situ tumor, left: artificial distant metastasis) was established. Tumor-bearing mice were randomly grouped (n=3) and treated as before, day7 dissected out bilateral tumor draining lymph nodes after treatment, and analyzed Dendritic Cell (DC) maturity by flow cytometry, while bilateral tumors were removed to make paraffin sections, and CD8 was detected for each group after fluorescent staining + Infiltration of cytotoxic T Cells (CTLs).
Compared with other groups, the HIFU+ M@P-SOP is used for cooperating with PD-L1 antibody treatment, thereby promoting the maturation of DC cells in bilateral tumor drainage lymph nodes and greatly increasing CD8 in bilateral tumors + Infiltration of T cells is statistically significant (P < 0)05) (fig. 26). In the figure, green fluorescence represents cd8+ T cells, and blue fluorescence represents nuclei.
In conclusion, M@P-SOP can target synergistic HIFU to ablate primary tumor, and combined with PD-L1 antibody to activate systemic immune reaction, so that the growth of metastatic tumor can be further effectively inhibited.
The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.
Claims (10)
1. The application of the oxygen-carrying biomimetic molecular probe in preparing an immune activation system is characterized in that: comprises an oxygen-carrying bionic molecular probe and high-intensity focusing ultrasonic equipment; the oxygen-carrying bionic molecular probe comprises a shell, wherein liquid fluorocarbon and oxygen are wrapped in the shell; the outer shell is wrapped with cancer cell membranes; superparamagnetic iron oxide is embedded in the shell; the shell is polylactic acid-glycolic acid copolymer.
2. The use of an oxygen-carrying biomimetic molecular probe according to claim 1 for the preparation of an immune activation system, characterized in that: the cancer cell membrane is from a 4T1 breast cancer tumor cell; the liquid fluorocarbon is perfluorohexane.
3. The use of an oxygen-carrying biomimetic molecular probe according to claim 2 for the preparation of an immune activation system, characterized in that: the dosage ratio of polylactic acid-glycolic acid copolymer, perfluorohexane and superparamagnetic iron oxide is 50mg:200 μl: 50. Mu.L.
4. The use of an oxygen-carrying biomimetic molecular probe according to claim 1 for the preparation of an immune activation system, characterized in that: the oxygen carrying biomimetic molecular probe is prepared by the following method:
s1, preparing P-SP nano particles: dissolving polylactic acid-glycolic acid copolymer and superparamagnetic iron oxide in methylene dichloride; then adding liquid fluorocarbon to carry out first sound vibration emulsification; adding PVA, and performing a second sound vibration emulsification; finally adding isopropanol and magnetically stirring; washing to obtain P-SP nanoparticles;
s2 extraction of cancer cell membranes: obtaining a cell membrane of a breast cancer 4T1 cell;
s3, preparing a M@P-SOP molecular probe: mixing cancer cell membranes and P-SP nanoparticles to obtain a mixed solution; pushing the mixed solution for multiple times by a lipid extruder, and washing to obtain M@P-SP nanoparticles; oxygenation was performed in M@P-SP nanoparticles to obtain M@P-SOP molecular probes.
5. The use of an oxygen-carrying biomimetic molecular probe according to claim 4 for the preparation of an immune activation system, wherein: in S1, the first sound vibration emulsification and the second sound vibration emulsification are carried out under the ice bath condition, and the parameter conditions are as follows: 60W, 3min.
6. The use of an oxygen-carrying biomimetic molecular probe according to claim 5 for the preparation of an immune activation system, wherein: in S2, the method for obtaining the cell membrane of the breast cancer 4T1 cell comprises the following steps: culturing breast cancer 4T1 cells to logarithmic phase, collecting cells, centrifuging and collecting cell precipitate; after cell precipitation is cracked and repeatedly frozen and thawed, centrifuging to obtain supernatant; and centrifuging to obtain cancer cell membrane precipitate, and freeze-drying the cancer cell membrane precipitate for later use.
7. The application of the oxygen-carrying biomimetic molecular probe in preparing an anti-tumor system is characterized in that: the anti-tumor system comprises an oxygen-carrying biomimetic molecular probe, high-intensity focused ultrasound equipment and a PD-L1 antibody; the oxygen-carrying bionic molecular probe comprises a shell, wherein liquid fluorocarbon and oxygen are wrapped in the shell; the outer shell is wrapped with cancer cell membranes; superparamagnetic iron oxide is embedded in the shell; the shell is polylactic acid-glycolic acid copolymer.
8. The use of an oxygen-carrying biomimetic molecular probe according to claim 7 for preparing an anti-tumor system, wherein: the oxygen carrying biomimetic molecular probe is prepared by the following method:
s1, preparing P-SP nano particles: dissolving polylactic acid-glycolic acid copolymer and superparamagnetic iron oxide in methylene dichloride; then adding liquid fluorocarbon to carry out first sound vibration emulsification; adding PVA, and performing a second sound vibration emulsification; finally adding isopropanol and magnetically stirring; washing to obtain P-SP nanoparticles;
s2 extraction of cancer cell membranes: obtaining a cell membrane of a breast cancer 4T1 cell;
s3, preparing a M@P-SOP molecular probe: mixing cancer cell membranes and P-SP nanoparticles to obtain a mixed solution; pushing the mixed solution for multiple times by a lipid extruder, and washing to obtain M@P-SP nanoparticles; oxygenation was performed in M@P-SP nanoparticles to obtain M@P-SOP molecular probes.
9. The application of the oxygen-carrying biomimetic molecular probe in preparing an anti-tumor system according to claim 8, which is characterized in that: the cancer cell membrane is from a 4T1 breast cancer tumor cell; the liquid fluorocarbon is perfluorohexane; the dosage ratio of polylactic acid-glycolic acid copolymer, perfluorohexane and superparamagnetic iron oxide is 50mg:200 μl: 50. Mu.L.
10. The use of an oxygen-carrying biomimetic molecular probe according to claim 9 for preparing an anti-tumor system, wherein: parameters of the high-intensity focused ultrasound equipment are set to 120W and 3s; the dosage of the oxygen carrying biomimetic molecular probe is 200 μl; the amount of PD-L1 antibody used was 1.5 mg/kg.BW.
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