An in vitro photon domesticated macrophage system and method

Abstract

The application discloses an in-vitro photon domesticated macrophage system and method, which comprises an in-vitro photon regulation unit for emitting and regulating photons, an input unit for inputting a macrophage sample into the system, and a domesticated culture unit in communication with the input unit for domesticating the macrophage sample under the irradiation of the regulated photons. The in-vitro photon regulation unit comprises, in sequence according to the light path direction, a laser, a light path adjusting assembly, an energy regulator and a light beam expander. The input unit comprises a microinjector and a sample input pipeline in communication with the microinjector, and the domesticated culture unit has a culture bin, a conveying belt arranged on a carrier table in the culture bin and a culture dish arranged on the conveying belt. The culture dish is in communication with the sample input pipeline of the input unit in an initial state, and after the sample to be domesticated and cultured is obtained, the culture dish is transported by the conveying belt to a position irradiated by the light beam expander of the in-vitro photon regulation unit.

Description

Technical Field

[0001] This disclosure relates to the field of biomedical technology, and in particular to an in vitro photon-induced macrophage system and method. Background Technology

[0002] The in vitro photon-induced macrophage system is a cell modification and construction system combining micro- and nano-photonic materials and light field modulation technology. This system offers advantages such as speed, non-biological nature, high efficiency, and functional targeting. In most biomedical research, macrophage polarization is induced by biologically induced molecules, which faces problems such as low polarization rates and instability after polarization. The photon-induced macrophage system represents a novel research technique for macrophage modification engineering.

[0003] Unlike traditional macrophage modification biochemical techniques, photon-dominated macrophage systems offer superior optical performance and controllability. Most importantly, by combining micro / nano photonic materials and light field manipulation, photons can selectively regulate macrophage metabolism, thereby promoting or inhibiting gene expression and ultimately controlling macrophage phenotype. Since photon macrophage modulation technology does not involve any drug molecules, cell damage is virtually zero. Based on these advantages, photon-dominated macrophage systems offer controllability and plasticity in macrophage modification engineering, enhancing specific macrophage functions and thus holding a significant position in cell engineering and medical disease treatment technologies. Compared to the controllability and efficiency issues of existing cell modification techniques, photon-dominated macrophages open up a completely new avenue for cell modification engineering, showing bright prospects in disease treatment, cell engineering, and biochemical technologies.

[0004] In existing literature, macrophage polarization has been mediated through drug molecular regulation of metabolic pathways, and the intricate interactions between metabolic pathways and immune cell function have been revealed. Studies have shown how metabolic reprogramming affects the immune response to atherosclerosis, including changes in glucose metabolism, glycosaminoglycan metabolism, and retinoic acid signaling during the M1 and M2 phenotypic polarization induction phases. These studies have been provided by proteomics and phosphorylated proteomics and transcriptomics.

[0005] Therefore, altering and regulating macrophage metabolic pathways is essential for obtaining and establishing an effective immune response. For example, M1 macrophages rely on aerobic glycolysis to generate ATP, increasing glucose and glutamine consumption, but they inhibit oxidative metabolism. Conversely, M2 macrophages maintain an intact tricarboxylic acid (TCA) cycle and tend towards oxidative metabolism, particularly fatty acid oxidation (FAO), as a mode of ATP production. Within atherosclerotic plaques, monocyte-derived M2 macrophages, through the uptake of modified lipoproteins, particularly LDL, shift towards an anti-inflammatory, pro-fibrotic phenotype, potentially conferring protection by promoting tissue repair, alleviating inflammation, and limiting lesion progression. Therefore, understanding how cellular pathways and metabolites coordinate macrophage activation is crucial. For instance, α-KG, produced from glutamine hydrolysis, is an anti-inflammatory metabolite that maintains the M2 phenotype and controls the metabolic reprogramming of M2 macrophages through a jmjd3-dependent mechanism. Lysosomal acid lipase (LAL)-induced lipolysis is important for increased oxidative phosphorylation, enhanced standby respiratory capacity (SRC), prolonged survival, and the expression of genes that co-determine M2 activation. In mouse models, mitogen-activated protein kinase (MEK) and histone deacetylase (HDAC) also effectively promote M2-phenotypical macrophage polarization, angiogenesis, and wound healing.

[0006] However, despite the use of specific kinase and inhibitor drugs to coordinate macrophage polarization, M2 macrophages remain susceptible to dysfunction due to complex biological crosstalk in the local microenvironment. Therefore, exploring a rapid and effective method to achieve loyal M2 macrophages is crucial. Summary of the Invention

[0007] To address the problems of existing technologies, this disclosure proposes an in vitro photon-induced macrophage training system, which includes,

[0008] An external photon modulation unit is used for the emission and modulation of photons;

[0009] An input unit is used to input macrophage samples into the system;

[0010] The domestication and culture unit, connected to the input unit, is used to domesticate the macrophage sample under regulated photon irradiation.

[0011] Furthermore, the external photon manipulation unit includes, in sequence according to the optical path direction: a laser, an optical path adjustment component, an energy regulator, and a beam expander.

[0012] Furthermore, the input unit includes a micro-injector and a sample input channel connected to the micro-injector.

[0013] Furthermore, the acclimatization and cultivation unit has a cultivation chamber with a conveyor belt on a stage within the chamber, and a culture dish placed on the conveyor belt. Initially, the culture dish is connected to the sample input pipe of the input unit. After obtaining the sample to be acclimatized and cultivated, the culture dish is transported by the conveyor belt to a position irradiated by the beam expander of the external photon modulation unit. The cultivation chamber is equipped with temperature and CO2 controllers.

[0014] Furthermore, the optical path adjustment component includes, in sequence according to the optical path, a broadband beam splitter, a polarizer, and a quarter-wave plate. The photon frequency modulation range is in the visible light band, and the quarter-wave plate modulates the photon phase.

[0015] This disclosure presents a method for in vitro photon-induced macrophage training, comprising the steps of: extracting and differentiating macrophages; and administering medication to stimulate macrophages. The macrophages are extracted from mouse bone marrow. The extraction and differentiation of mouse bone marrow macrophages includes...

[0016] (1) Separate the tibia and femur of the mouse;

[0017] (2) Flushing the bone marrow;

[0018] (3) Remove impurities, including red blood cells;

[0019] (4) Perform cell seeding plate operation.

[0020] The photon domestication, or optical domestication, in this disclosure refers to the domestication process using photons. The meaning of domestication derives from its original concept, which involves using a selective function that does not alter the original nature while simultaneously enhancing and shaping it to achieve a specific effect, such as the domestication of wolves by humans. In this disclosure, it can refer to the specific effects of photon energy on macrophages.

[0021] The in vitro photon-induced macrophage system disclosed herein is a precise, stable, and functionally enhanced non-biochemical optical polarization system for macrophages. It utilizes micro-nano photonics to precisely target macrophages, and light field modulation technology can rapidly initiate macrophage metabolism. The metabolic process of macrophages can be controlled by the coordinated use of photon frequency, polarization, energy, and frequency. It features high macrophage directional polarization efficiency, high purity, rapid self-proliferation of macrophages, and direct usability.

[0022] Therefore, the beneficial effects of this disclosure include:

[0023] 1. Laser treatment is used to induce and regulate immune cell receptor molecules, and to cause immune cell gene reprogramming to modify cell function;

[0024] 2. Precise screening of receptor molecules ensures that the function of macrophages under laser induction and regulation is defined and stable;

[0025] 3. Lasers can simultaneously induce and regulate multiple highly correlated receptor molecules, thereby enabling non-invasive regulation of cellular metabolic functions;

[0026] 4. Immune cells induced and regulated by laser can maintain stability and be reinfused into the donor body for specific treatment techniques. Attached Figure Description

[0027] The above and other objects, features, and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of the invention are illustrated in the drawings by way of example and not limitation, wherein:

[0028] Figure 1 A schematic diagram of an in vitro photon-induced macrophage system according to one embodiment of the present invention.

[0029] Figure 2 A schematic diagram of micro / nano photonic materials and light field modulation of RAW264.7 M0 macrophages to M2 type under in vitro stimulation according to one embodiment of the present invention.

[0030] Figure 3 A schematic diagram illustrating the clearing effect of RAW264.7 cells under different stimulation treatments according to one embodiment of the present invention on apoptotic erythrocytes, high-density lipoprotein and low-density lipoprotein and other substances that easily induce vascular plaque formation.

[0031] Figure 4 A schematic diagram of gene expression and signaling pathways in RAW264.7 cells under different stimulation treatments according to one embodiment of the present invention.

[0032] Figure 5 Phenotypic changes in RAW264.7 cells under different stimulation treatments and the action of signaling pathway inhibitors, according to one embodiment of the present invention.

[0033] 1—Laser, 2—Broadband beam splitter, 3—Polarizer, 4—Quarter-wave plate, 5—Energy controller, 6—Beam expander, 7—Temperature and CO2 controller, 8—System chamber stage (including petri dishes and conveyor belt), 9—Expanded beam, 10—Microsyringe, 11—Sample input tubing (made of plastic or stainless steel). Detailed Implementation

[0034] Existing methods for polarizing macrophages using biopharmaceutical molecules rely on molecular interactions, resulting in a single mode of action. This leads to M2 macrophages being easily repolarized into M1 macrophages after polarization. Furthermore, the polarization efficiency is low, the time is long, and the reprogramming of macrophage genes cannot be controlled. To address these shortcomings, the in vitro photon-based macrophage domestication system disclosed herein aims to:

[0035] 1. Lasers promote changes in cell function by modulating receptor molecules, ultimately achieving re-editing of cell genes. They can induce and regulate multiple receptor molecules at multiple points without damaging cells.

[0036] 2. Under laser induction and control, targeted and direct action can be achieved, with the action time being several orders of magnitude shorter than that of drugs;

[0037] 3. Lasers interact with specific frequencies, causing no damage to surrounding tissues or metabolic systems. They do not require drug metabolism processes and do not have differences in drug dosage.

[0038] 4. Laser-induced function of the body's own immune cells avoids the side effects caused by the entry of foreign drug molecules into the body.

[0039] According to one or more embodiments, an in vitro photon-induced macrophage system includes an in vitro photon modulation device, a nanomaterial input and cell culture device, specifically comprising:

[0040] A light source module for emitting photons, including a tunable laser and beam control components;

[0041] The optical path adjustment module is used to control the photons emitted by the light source and to process the beam, and to adjust the incident light illumination range.

[0042] Cell and sample input module, used to culture and acclimatize macrophages using the photon acclimatization system;

[0043] The cell domestication and culture module is used to domesticate macrophages under photon irradiation after regulation and to test the polarization of macrophages.

[0044] The photon modulation components of the aforementioned in vitro photon acclimation system include photon energy modulation, photon polarization modulation, photon frequency modulation, and photon phase modulation. The components are arranged and assembled in the aforementioned order. The light source module has a wavelength range of 300-1400 nm, a photon energy modulation range of 1%-99%, and a photon frequency modulation range in the visible light band. A quarter-wave plate modulates the photon phase. The cell and sample input module includes a rapid and precise micro-injector with an injection rate in the range of nl / s-ml / s. The syringe tip is connected to a microchannel and directly enters the system chamber, vertically injecting into the system culture dish. The injection rate is controllable, and the position is adjustable, ensuring that the number of injected samples and cells remains within a controllable range. The cell acclimation and culture module involves transferring the culture dish containing the sample into the sample injection system chamber via a conveyor belt to be irradiated under a pre-expanded light beam.

[0045] Figure 1This is a description of the in vitro photon-induced macrophage system described in this disclosure. Figure 1 In this system, the laser, as the core component of the light source module, provides the source for emitting photons. A broadband beam splitter is used to split the laser beam into two or more beams, facilitating subsequent beam processing. A polarizer is used to control the polarization state of the photons, allowing the beam to be adjusted to a specific polarization direction. A quarter-wave plate is primarily used to control the phase of the photons, enabling more complex photon phase manipulation. An energy regulator controls the energy of the photons, with a range of 1%-99%, allowing the system to adjust the photon energy intensity as needed. A beam expander expands the beam, increasing its diameter. This helps to uniformly irradiate macrophages in the culture dish, ensuring that the cells receive uniform photon irradiation. A temperature and CO2 controller controls the temperature and CO2 concentration within the system's culture chamber, crucial for maintaining the stability of the cell culture environment, as macrophage growth and metabolism require suitable temperature and CO2 concentrations. Within the system's culture chamber, the stage (including the culture dish and conveyor belt) holds the culture dish, while the conveyor belt moves it to a suitable position to receive the light beam. A microsyringe is used to inject macrophages or other samples into the system at injection rates ranging from nl / s to ml / s, enabling rapid and precise sample input. The syringe tip connects to the sample input microchannel, directly entering the system's culture chamber to ensure accurate sample injection into the culture dish. The sample input tubing (plastic or stainless steel) is used to transfer samples from the outside into the system. The choice of tubing material (plastic or stainless steel) considers biocompatibility and durability, ensuring that samples are not contaminated during transport.

[0046] In this embodiment, a laser emits photons of a specific wavelength, which are then split by a broadband beam splitter. Polarization and phase modulation are achieved using a polarizer and a quarter-wave plate, followed by energy adjustment via an energy control device. The modulated beam is then expanded by a beam expander to uniformly irradiate macrophages in a culture dish. Macrophages are injected into the culture dish within the system compartment via a microsyringe and sample input channel. The syringe's rate and position are adjustable to ensure controllable cell number and distribution. The culture dish containing macrophages is conveyed via a conveyor belt to the expanded beam for irradiation and acclimatization. During acclimatization, a stable cell culture environment is maintained using temperature and CO2 controllers. Finally, the polarization of the macrophages is tested using the cell acclimatization culture module.

[0047] Therefore, the in vitro photon-induced macrophage acclimatization system of this disclosure possesses comprehensive control over photon energy, frequency, polarization, and phase, enabling the simulation of various photon conditions and providing rich experimental methods for studying the photon response of macrophages. The design of the microsyringe and sample input tubing ensures rapid and precise sample input, improving experimental efficiency and accuracy. The conveyor belt and temperature / CO2 controller achieve automation and environmental stability for cell culture and photon acclimatization, reducing human error. This system provides an efficient, precise, and controllable experimental and operational platform for studying the behavior and polarization of macrophages under different photon conditions.

[0048] For ease of understanding, the English abbreviations used in the accompanying drawings and specifications are summarized below:

[0049] LPS—Lipopolysaccharide;

[0050] iNOS—inositol—inositol;

[0051] il-6—interleukins 6—interleukin-6;

[0052] TNFα—tumor necrosis factor α;

[0053] Fizz 1 – Foundin in flammatory zone 1;

[0054] il-10—interleukins 10—interleukin-10;

[0055] YM 1 — Chitinase 3 Like 3 — Chitinase 3;

[0056] CD163 -- Cluster of Differentiation 163 — Differentiation antigen 163;

[0057] MCH II — Mean Corpuscular Hemoglobin II;

[0058] Unstained;

[0059] il-4—interleukins 4—interleukin-4;

[0060] SSC-A – Side Scatter Area;

[0061] PPIX-L — protoporphyrin IX dots-Light — protoporphyrin quantum dots-light;

[0062] PPIX -- protoporphyrin IX dots;

[0063] OX-LDL—oxidized low-density lipoprotein;

[0064] LDL—low-density lipoprotein;

[0065] BRC—blood red cells—hemoglobin;

[0066] PPAR—peroxisome proliferator-activated receptor;

[0067] α-KG—α-ketoglutarate—α-ketoglutarate;

[0068] Arg 1—Tyrosine kinase 1;

[0069] PPARα—peroxisome proliferator-activated receptor α;

[0070] m-TOR—mammalian target of rapamycin;

[0071] PPARδ—peroxisome proliferator-activated receptor δ;

[0072] Prdm1 — PR Domain Containing 1 — Contains PR domain protein 1;

[0073] CXCL-CXC motif 1-CXC chemokine;

[0074] NF-κbia—Nuclear Factor kappa-light-chain enhancer of activated B cells;

[0075] PPAR gc1a — peroxisome proliferator-activated receptor gc1a;

[0076] Norathyriol—1,3,6,7-tetrahydroxyoxanthone;

[0077] PP242 — 2-(4-amino-1-isopropyl-1H-pyrazolo[3,4-D]pyrimidin-3-yl)-1H-indol-5-ol;

[0078] GKT831—Setanaxib—Setanaxib;

[0079] DAPI — 4',6-Diamidinyl-2-phenylindole;

[0080] Phalloidin—phalloidin;

[0081] PpIX@CDs — a composite material composed of protoporphyrin IX (PpIX) and carbon dots (CDs);

[0082] ATP—adenosine triphosphate;

[0083] CO2 — carbon dioxide;

[0084] H2O——water;

[0085] H2O2—hydrogen peroxide;

[0086] PPARγ—Peroxisome proliferator-activated receptor γ;

[0087] α-KG—α-ketoglutaric acid;

[0088] g-KG—γ-ketoglutaric acid;

[0089] NADH—Nicotinamide adenine dinucleotide;

[0090] NAD ＋ —Nicotinamide adenine dinucleotide;

[0091] FADH2—flavin adenine dinucleotide;

[0092] FAD – Flavin adenine dinucleotide;

[0093] 1 O2—singlet oxygen;

[0094] ADP—adenosine diphosphate;

[0095] GTP—Guanine triphosphate;

[0096] GDP – Guanosine diphosphate;

[0097] GS1—Glutathione S-transferase 1;

[0098] CXCL1—Chemokine (CXC motif) ligand 1;

[0099] CXCL10—Chemokine (CXC motif) ligand 10;

[0100] NF-κBib — nuclear factor κBib;

[0101] NF-κBid — nuclear factor κBid;

[0102] PRDM14—PR domain protein 14;

[0103] PRDM13—PR domain protein 13;

[0104] PRDM1—PR domain protein 1;

[0105] HIF-1α—Hypoxia-inducible factor 1α.

[0106] This disclosure relates to macrophage RAW264.7, where RAW is an identifier used to distinguish different cell lines, and RAW264.7 is the name of a mouse macrophage line. This cell line was isolated from a peritonitis model and exhibits macrophage characteristics when cultured in vitro. The number "264.7" is commonly used to identify cell lines. Macrophages can be classified into different subtypes based on their activation status and functional characteristics, with M0, M1, and M2 being common classifications. M0 represents resting macrophages. These cells are in a quiescent state when not stimulated and do not exhibit significant pro-inflammatory or anti-inflammatory properties. M2 represents alternately activated macrophages, also known as anti-inflammatory macrophages. M2 macrophages play a role in immune regulation, tissue repair, and antiparasitic immunity. They are typically activated upon encountering specific cytokines (such as IL-4 and IL-13), exhibiting anti-inflammatory properties that contribute to tissue repair and suppress inflammatory responses. When RAW264.7 M0 cells are biased towards the M2 type, it means that these cells exhibit characteristics more similar to M2 macrophages under specific stimuli, such as participating in anti-inflammatory responses and tissue repair. Figure 2 This example illustrates how micro-nano photonic materials and light field modulation in vitro stimulated RAW264.7 M0 macrophages to lean towards the M2 morphology. (ad) Under the same culture conditions, (a) qPCR analysis of M1 marker gene mRNA expression levels after 12 h of stimulation with micro-nano photonic materials (PPIX), micro-nano photonic materials and light field modulation (PPIX-L), il-4, LPS, and controls. (b) qPCR analysis of M1 / M2 marker gene mRNA expression after stimulation with 100 ng / ml LPS following the PPIX-L step. (c) Morphological images of macrophages after 0 h, 6 h, and 12 h of stimulation with PPIX-L, LPS, PPIX-L+LPS, and controls, respectively, under the same culture conditions. (d) qPCR analysis of M1 / M2 marker gene mRNA expression levels with PPIX-L, LPS, PPIX-L+LPS, and controls. * p<0.05, ** p<0.01, *** p<0.001. Data represent 3 independent experiments, 5 samples per group (ad; mean ± sd). (h) Flow cytometry results. M0 macrophages were immunofluorescently stained with untreated, il-4, LPS, and PPIX-L antibodies, respectively, using APC anti-mouse IA / IE antibody and PE anti-mouse CD163 antibody.

[0107] Figure 2The process by which micro / nano photonic materials and light field modulation induce in vitro stimulation of RAW264.7 M0 macrophages to shift towards the M2 morphology is described, specifically including:

[0108] (1) such as Figure 2 Images (a) a1-a3 show the effects of micro / nano photonic materials (MD) on cells at different time points (20 minutes, 8 hours, 12 hours), as well as fluorescence images of cells after the final addition of LPS;

[0109] (2) For example Figure 2 The (bd) figure shows the relative changes in mRNA expression levels of M1 (e.g., iNOS, TNFα) and M2 (e.g., Fizz1, il-10, YM1, il-4) macrophage marker genes under different treatment conditions. These data indicate that micro / nano photonic materials and light field modulation can promote macrophage polarization towards the M2 type;

[0110] (3) such as Figure 2 (e) Flow cytometry results show the immunofluorescence staining results of macrophages under different treatment conditions. The figure shows the expression of CD163 and MHC II, which are markers of M2 macrophages. Flow cytometry results indicate that PPIX-L treatment can significantly increase the proportion of M2 macrophages;

[0111] (4) such as Figure 2 The (h) flow cytometry results were further analyzed using flow cytometry to examine the polarization state of macrophages under different treatment conditions. The results showed that PPIX-L treatment significantly increased the proportion of M2 macrophages, which is consistent with... Figure 2 The result of (e) is consistent.

[0112] Figure 3 This study demonstrates the clearance effect of RAW264.7 cells under different stimulation treatments on apoptotic erythrocytes, high-density lipoprotein (HDL), and low-density lipoprotein (LDL), substances that readily induce vascular plaque formation. 20 μg / ml OX-LDL: green for macrophages; 20 μg / ml LDL: pink; 10⁴ / ml RBC: red. After 5 h of incubation, observation was performed using a laser confocal scanning microscope. ns: no significant difference; *P<0.05, **P<0.01, ***P<0.001. No paired two-tailed Student's t-test was performed. Data represent 3 or 2 independent experiments, with 3 samples per group.

[0113] Figure 3This study demonstrates the clearance effect of RAW264.7 cells on specific substances under different stimulation treatments. These specific substances include apoptotic erythrocytes (BRCs), high-density lipoprotein (HDL), low-density lipoprotein (LDL), and oxidized low-density lipoprotein (OX-LDL), which readily induces vascular plaque formation. Figure 3 (a) shows cell fluorescence images under different treatment conditions. This includes a control group, and il-4 and PPIX-L are two different stimulation treatments. In the images, DAPI (blue) labels the cell nucleus, and OX-LDL (green) and FBR (red) label specific substances, respectively. By comparing the fluorescence intensity of different treatment groups, the uptake and clearance capacity of RAW264.7 cells for these substances can be assessed. Columns I, II, and III represent cell images under different treatment conditions, including the control group, the il-4 treatment group, and the PPIX-L treatment group. OX-LDL, LDL, and BRC represent different fluorescently labeled substances used to assess cellular uptake of these substances.

[0114] Figure 3 (b) The bar chart shows the relative fluorescence area of ​​OX-LDL, LDL, and BRC under different treatment conditions, reflecting the uptake of these substances by cells. In the bar chart, the control group, il-4, and PPIX-L represent different treatment conditions. The green OX-LDL bar chart shows the uptake of oxidized low-density lipoprotein by cells. The PPIX-L treatment group had the highest uptake, indicating that PPIX-L treatment can enhance the uptake and clearance of OX-LDL by RAW264.7 cells. The pink LDL bar chart shows the uptake of low-density lipoprotein by cells. The il-4 treatment group had the highest uptake, indicating that il-4 treatment can enhance the uptake and clearance of LDL by RAW264.7 cells. The red BRC bar chart shows the uptake of a certain substance (possibly apoptotic erythrocytes) by cells. The PPIX-L treatment group had the highest uptake, indicating that PPIX-L treatment can enhance the uptake and clearance of this substance by RAW264.7 cells.

[0115] Figure 3 The results indicate that PPIX-L treatment significantly enhanced the uptake and clearance of OX-LDL and apoptotic erythrocytes by RAW264.7 cells, suggesting that PPIX-L treatment may help reduce vascular plaque formation and promote the clearance of cell debris. il-4 treatment significantly enhanced the uptake and clearance of LDL by RAW264.7 cells, suggesting that il-4 treatment may help reduce the deposition of low-density lipoprotein in the vascular wall.

[0116] Figure 4This figure illustrates the changes in gene expression and signaling pathways in RAW264.7 cells (a macrophage cell line) under different stimuli. The left side of the figure shows cellular metabolic pathways, including oxidative phosphorylation in mitochondria and glycolysis in the cytoplasm. The right side shows the effects of nanomaterials on cellular signaling pathways.

[0117] Figure 4 The diagram illustrates the conversion of pyruvate to acetyl-CoA and the subsequent metabolic processes within the tricarboxylic acid cycle (citric acid cycle). These processes produce ATP, carbon dioxide, and water, and are central to cellular energy metabolism. Mitochondria play a crucial role in energy production and oxidative stress responses; the diagram shows the generation of oxygen, hydrogen peroxide, and water. The right-hand illustration shows how nanomaterials influence the PPAR signaling pathway, thereby regulating gene expression and function in macrophages. PPARγ is a key transcription factor that affects lipid metabolism and inflammatory responses. The diagram also shows the activation of the m-TOR signaling pathway, a vital regulator of cell growth, protein synthesis, and cellular metabolism. By influencing these signaling pathways, nanomaterials can modulate the polarization state of macrophages, thereby affecting their anti-inflammatory or pro-inflammatory functions. The diagram shows how nanomaterials promote the expression of anti-inflammatory genes (such as Prdm1) by modulating signaling pathways, which helps alleviate inflammatory responses. By inhibiting the PPARγ protein nuclear switch and restricting lipid synthesis, nanomaterials may further enhance their anti-inflammatory effects. Therefore... Figure 4 This study demonstrates that nanomaterials may play a role in anti-inflammatory therapy by modulating metabolic pathways and signaling pathways in RAW264.7 cells, thereby affecting their gene expression and function.

[0118] Figure 5 This study illustrates the changes in gene expression and phenotypic effects of signaling pathway inhibitors in RAW264.7 cells under different stimuli. ROS, m-TOR, and PPAR-δ regulate macrophage activation by promoting metabolism. Figure 5(a) qPCR analysis of mRNA expression levels of the M2 marker gene after stimulation of M0 cells for 6 h under different culture conditions with PPIX-L and PP242 m-TOR inhibitors (a), norathyriol PPAR-δ inhibitors (c), and GKT831 ROS inhibitors (e). (b), (d), and (f) Changes in macrophage morphological polarization. ns: no significant difference; *P<0.05, **P<0.01, unpaired, two-tailed Student's test. Data represent the cumulative results of 3 independent experiments with 4 samples per group (a, c, e) or 3 independent experiments (b, d, f). (g) Immunostaining of M0 (g1), M1 (g2), and M2 (g3) macrophages with phalloidin and DAPI. Scale bar: 20 µm. (h) qPCR analysis of m-TOR and PPAR-δ mRNA expression levels with and without PPIX-L, 1 nM ROS inhibitor GKT931, and 1 nM PPAR-δ norathyriol inhibitor, respectively. (i) Heatmaps show that DEGs are enriched in biological processes involving immune and inflammatory regulation, and they all play important roles in macrophage differentiation. Data represent either 3 independent experiments or 2 independent experiments, with 3 samples per group.

[0119] Figure 5 This study illustrates the changes in gene expression and phenotypic effects of signaling pathway inhibitors in RAW264.7 cells (a macrophage line) under different stimuli. The figures demonstrate that ROS, m-TOR, and PPAR-δ regulate macrophage activation by promoting metabolism. Figure 5 Images (a), (c), and (e) show the qPCR analysis results of the mRNA expression levels of M2 marker genes after stimulating M0 macrophages with PPIX-L, PP242 (m-TOR inhibitor), norathyriol (PPAR-δ inhibitor), and GKT831 (ROS inhibitor) for 6 hours under different culture conditions. These results indicate that these inhibitors can significantly affect gene expression in M2 macrophages. Figure 5 (b), (d), and (f) in the figure show the changes in the degree of macrophage morphological polarization, reflecting the effects of different inhibitors on macrophage polarization. Figure 5 Immunostaining of macrophages M0 (g1), M1 (g2), and M2 (g3) with phalloidin and DAPI showed the morphological changes of macrophages under different polarization states. The scale bar is 20 µm. Figure 5qPCR analysis of m-TOR and PPAR-δ mRNA expression levels with or without PPIX-L, 1 nM ROS inhibitor GKT931, and 1 nM PPAR-δ norathyriol inhibitor (h) further illustrates the effects of these inhibitors on specific signaling pathways. Figure 5 (i) shows the enrichment of differentially expressed genes (DEGs) in biological processes involving immune and inflammatory regulation. These genes play important roles in macrophage differentiation. The heatmap uses color changes (from blue to red) to represent changes in gene expression, with red indicating upregulation and blue indicating downregulation.

[0120] According to one or more embodiments, an in vitro photon-induced macrophage system includes an in vitro photon modulation device, a nanomaterial input and cell culture device, and further includes:

[0121] A light source module for emitting photons, including a tunable laser and beam control components;

[0122] The optical path adjustment module is used to control the photons emitted by the light source and to process the beam, and to adjust the incident light illumination range.

[0123] Cell and sample input module, used to culture and acclimatize macrophages using the photon acclimatization system;

[0124] The cell domestication and culture module is used to domesticate macrophages under photon irradiation after regulation and to test the polarization of macrophages.

[0125] Furthermore, the aforementioned in vitro photon acclimatization system includes: a laser, a broadband beam splitter, a polarizer, a quarter-wave plate, an energy regulator, a beam expander, a temperature and CO2 controller, a system chamber stage (culture dish and conveyor belt), a microsyringe, and a sample input pipeline (plastic or stainless steel).

[0126] The photon modulation components of the aforementioned in vitro photon domestication system include photon energy modulation, photon polarization modulation, photon frequency modulation, and photon phase modulation. The components are arranged and assembled in the aforementioned order.

[0127] The wavelength range of the light source module is 300-1400nm, the photon energy modulation range is 1%-99%, the photon frequency modulation range is the visible light band, and the quarter-wave plate modulates the photon phase.

[0128] The cell and sample input module includes a rapid and precise micro-injector with an injection rate in the range of nl / s-ml / s. The syringe tip is connected to a microchannel and directly enters the system chamber, vertically injecting into the system culture dish. The injection rate is controllable and the position is adjustable, ensuring that the number of injected samples and cells is within a controllable range.

[0129] The cell domestication and culture module consists of a culture dish in the sample injection system chamber, which is then transported by a conveyor belt to be irradiated by an expanded beam of light.

[0130] According to one or more embodiments, a macrophage screening and polarization method based on an in vitro photon-induced macrophage system includes the following steps:

[0131] Extraction and differentiation of mouse bone marrow macrophages;

[0132] Stimulation of macrophages by drug administration;

[0133] The extraction and differentiation of mouse bone marrow macrophages includes:

[0134] 1. Isolation of the tibia and femur of mice: First, 4-8 week old C57BL / 6 mice are euthanized by cervical dislocation and thoroughly disinfected with 75% alcohol. Under aseptic conditions, the tibia and femur of the mice are isolated and removed, taking care not to break the bones, and then placed in a cell culture dish containing 75% alcohol;

[0135] 2. Bone marrow rinsing: Transfer the separated tibia and femur to a biosafety cabinet for further separation and removal of surrounding tissues. Then, wash the tibia and femur 2-3 times in a cell culture dish containing PBS. Finally, transfer the bone marrow cells to a cell culture dish containing complete culture medium (1% penicillin-streptomycin + 10% fetal bovine serum + DMEM basal medium). Cut open both ends of the tibia and femur with ophthalmic scissors, then use a 1ml syringe to draw up the complete culture medium and rinse the bone marrow cells from one end of the bone into a 50ml sterile centrifuge tube. Repeat this process several times until the bone turns white.

[0136] 3. Removal of red blood cells and other impurities: Add 5 times the volume of red blood cell lysis buffer to a 50ml centrifuge tube, repeatedly pipette, and let stand for 15 minutes. After standing, centrifuge at 1000 rpm for 10 minutes and discard the supernatant. Resuspend the cells in an appropriate amount of DMEM cell culture medium, and then filter the cells through a 200-mesh filter. Centrifuge again at 1000 rpm for 10 minutes, discard the supernatant, and repeat twice to wash the cells.

[0137] 4. Cell Seeding: After the final centrifugation, discard the supernatant and resuspend the cells in DMEM complete medium containing 10 ng / ml M-CSF to induce bone marrow cells to differentiate into macrophages. After cell counting, adjust the cell density to 1 x 10^6 cells / ml and seed the cells in culture dishes. Incubate at 37°C with 5% CO2 for further research.

[0138] The macrophage administration stimulation includes:

[0139] 1. After LPS stimulates cell adhesion, the prepared 1 mg / ml LPS solution is taken out of the -20℃ freezer to thaw, diluted with culture medium, and used to culture cells. The cells are then incubated in an incubator for 12-24 hours.

[0140] 2. After il-4 stimulation of cell adhesion, dilute the prepared 0.1 mg / ml il-4 solution with culture medium and use it to culture cells. Incubate in an incubator for 12-24 hours. 3. After nanomaterial + laser stimulation of cell adhesion, add an appropriate amount of carbon quantum dots to the cell culture medium. After reacting for 8 hours, irradiate the cells with a laser for an appropriate time and incubate in an in vitro photon acclimatization system.

[0141] It should be understood that in the embodiments of the present invention, the term "and / or" is merely a description of the relationship between associated objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Furthermore, the character " / " in this document generally indicates that the preceding and following associated objects have an "or" relationship.

[0142] It is worth noting that although the spirit and principles of this invention have been described with reference to several specific embodiments, it should be understood that this invention is not limited to the disclosed specific embodiments, and the division of aspects does not imply that the features in these aspects cannot be combined; such division is merely for the convenience of description. This invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

Claims

1. An in vitro photon-induced macrophage training system, characterized in that, The system includes: An external photon modulation unit is used for the emission and modulation of photons. The external photon modulation unit, arranged sequentially along the optical path, includes: a laser, an optical path adjustment component, an energy regulator, and a beam expander. The optical path adjustment component, arranged sequentially along the optical path, includes a broadband beam splitter, a polarizer, and a quarter-wave plate. The photon frequency modulation range is in the visible light band, and the quarter-wave plate modulates the photon phase. An input unit for inputting macrophage samples into the system, the input unit comprising a microsyringe and a sample input channel connected to the microsyringe; An acclimatization and culture unit, connected to the input unit, is used to acclimatize the macrophage sample under controlled photon irradiation. The acclimatization and culture unit has a culture chamber with a conveyor belt on a stage within the chamber, and culture dishes placed on the conveyor belt. Initially, these culture dishes are connected to the sample input channel of the input unit. A laser emits photons of a specific wavelength, which are then split by a broadband beam splitter. The beams are then polarized and phase-controlled by polarizers and quarter-wave plates, and finally, the photon energy is adjusted by an energy control device. The regulated light beam is expanded by a beam expander to uniformly illuminate the macrophage sample in the culture dish. After obtaining the samples to be acclimatized and cultured, the culture dishes containing the macrophage samples are transported by conveyor belt to be irradiated and acclimatized under a beam of expanded light.

2. The system according to claim 1, characterized in that, This system uses micro-nano photonic materials to couple with photons onto macrophage samples. It controls macrophage metabolism by coordinating photon energy, frequency, polarization, and phase, thereby inducing macrophages to M2 polarization.

3. The system according to claim 2, characterized in that, The micro / nano photonic material is PPIX-L, which is composed of carbon dots and protoporphyrin IX to form a PpIX@CDs composite material.

4. The system according to claim 1, characterized in that, The system reprograms macrophage genes through photon domestication.

5. The system according to claim 1, characterized in that, The culture chamber is equipped with temperature and CO2 controllers.

6. A method for in vitro photon-induced macrophage training, characterized in that, Based on the system of claim 1, the system includes the following steps: Extraction and differentiation of mouse bone marrow macrophages; Input the macrophage sample into the system culture dish; By coupling the micro-nano photonic material PPIX-L with an optical field, macrophage metabolism is controlled through the coordinated control of photon energy, frequency, polarization, and phase, inducing macrophages to polarize towards the M2 type and enhancing their ability to clear OX-LDL and apoptotic erythrocytes.

7. The method according to claim 6, characterized in that, Mouse bone marrow macrophage extraction and differentiation include, (1) Separate the tibia and femur of the mouse; (2) Flushing the bone marrow; (3) Remove impurities, including red blood cells; (4) Perform cell seeding plate operation.

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