Near-infrared colloidal quantum dot marking optimization system for detecting metabolism of external vesicles

Abstract

The invention relates to the technical field of tumor cell ectovesicle metabolism detection, and discloses a near-infrared colloidal quantum dot mark optimization system for ectovesicle metabolism detection. Comprising an electric pulse processing module used for generating millisecond electric pulses acting on cancer cells so as to unblock cell membrane outer vesicles corresponding to the cancer cells and increase cell membrane phosphoric acid structure permeability; the control module is used for marking near-infrared colloidal quantum dots corresponding to the cancer cells, the near-infrared colloidal quantum dots have available fluorescence spectrums in an infrared range, and the near-infrared colloidal quantum dots can cross a cell barrier processed by the millisecond-level electric pulse and can be used for detecting the cancer cells. Combining with macromolecular organic matters and metabolic substrates in extracellular vesicles; on the basis of the combined near-infrared colloidal quantum dots, macromolecular organic matters and metabolic substrates in the extracellular vesicles are detected, and efficient marking of the macromolecular organic matters and metabolic substrates in the extracellular vesicles of cancer cells is achieved.

Description

Technical Field

[0001] The present application relates to the technical field of tumor cell extracellular vesicle metabolism detection, and in particular to a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection. Background Art

[0002] Tumor cells possess unique metabolic characteristics, including high glucose absorption, active aerobic glycolysis, and high lactate production. The molecular biological mechanisms underlying these characteristics are manifested by downregulated expression of proteins involved in oxidative metabolism and overexpression of glucose transporters and carboxylate transporters. These metabolic characteristics prompt tumor cells to produce a large amount of metabolic substrates through pathways such as pentose phosphorylation and serine biosynthesis. Through exocytosis, these metabolites, such as macromolecular organic matter and lactate, are encapsulated in exosomes and released outside the cell, forming an immunosuppressive tumor microenvironment. Accurate detection of metabolic substrates within exosomes is a key approach to uncovering abnormalities in tumor metabolism and the mechanisms regulating the microenvironment.

[0003] Traditional extracellular vesicle labeling detection technologies face the following challenges:

[0004] 1. Insufficient labeling efficiency: The phospholipid bilayer structure of extracellular vesicles forms a natural barrier, making it difficult for conventional markers to effectively penetrate and bind to substances within the vesicles. Especially when the target protein configuration is unclear, nonspecific binding of similar proteins leads to low labeling accuracy.

[0005] 2. Limitations of spectral characteristics: Existing fluorescent markers generally have problems such as narrow spectral range, poor tunability, and wide luminescence peaks in the infrared spectral range. Low quantum efficiency leads to insufficient detection sensitivity, and they are easily interfered by autofluorescence of biological tissues, making it difficult to achieve qualitative and quantitative analysis of complex metabolic substrates.

[0006] 3. Weak detection targeting: Targeting strategies have not been designed for specific markers such as glucose transporters that are highly expressed in tumor cells, making it impossible to efficiently enrich target cancer cells. This results in strong background interference in the sample and limits the accuracy of metabolic profile analysis.

[0007] Although existing technologies attempt to increase membrane permeability through physical or chemical methods, the optimization of electric pulse parameters lacks coordinated design with quantum dot labeling, making it difficult to achieve efficient unsealing of extracellular vesicles while maintaining cell activity; and the spectral performance and surface modification strategies of near-infrared markers have failed to fully combine the metabolic substrate characteristics of tumor extracellular vesicles, resulting in detection efficiency and accuracy that are difficult to meet the needs of complex metabolic pathway analysis.

[0008] Therefore, there is an urgent need to design a technical solution to solve at least one of the above technical problems. Summary of the Invention

[0009] The present application provides a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolic detection, aiming to solve the problem that although the existing technology attempts to increase membrane permeability through physical or chemical methods, the optimization of electric pulse parameters lacks coordinated design with quantum dot labeling, making it difficult to achieve efficient unsealing of extracellular vesicles while maintaining cell activity; and the spectral performance and surface modification strategies of near-infrared markers fail to fully combine the metabolic substrate characteristics of tumor extracellular vesicles, resulting in detection efficiency and accuracy that are difficult to meet the needs of complex metabolic pathway analysis.

[0010] In a first aspect, the present application provides a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection, comprising:

[0011] an electric pulse processing module, configured to generate millisecond-level electric pulses, which act on cancer cells to unblock extracellular vesicles corresponding to the cancer cells and increase the permeability of the cell membrane phosphate structure;

[0012] A control module is used to mark the near-infrared colloidal quantum dots corresponding to the cancer cells, wherein the near-infrared colloidal quantum dots have an available fluorescence spectrum in the infrared range, and the near-infrared colloidal quantum dots can cross the cell barrier after the millisecond-level electric pulse treatment and combine with the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane; based on the combined near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected to achieve efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cancer cell membrane.

[0013] In some embodiments, the generating of millisecond electric pulses includes: generating millisecond electric pulses with a voltage amplitude range of 50-200V, a frequency range of 1-10kHz, and a single pulse duration range of 1-10 milliseconds; the millisecond electric pulses act on the cancer cell culture system, and by changing the conformation of the cell membrane phospholipid bilayer, achieve the unsealing of the cell membrane extracellular vesicles and the enhancement of the permeability of the membrane phosphate structure.

[0014] In some embodiments, the near-infrared colloidal quantum dots corresponding to the cancer cells are labeled, including: selecting near-infrared colloidal quantum dots with a spectral range of 700-1000nm, a half-peak width of less than 30nm, and a quantum efficiency greater than 80%, and performing targeted modification on the surface of the near-infrared colloidal quantum dots; the targeted modification includes connecting aptamers or antibody fragments to specific antigens on the surface of the cancer cell extracellular vesicles to enhance the binding specificity with the macromolecular organic matter and metabolic substrates.

[0015] In some embodiments, the detection of macromolecular organic matter and metabolic substrates in the extracellular vesicles based on the bound near-infrared colloidal quantum dots includes: collecting the luminescent signals of the near-infrared colloidal quantum dots through a fluorescence imaging device or a flow cytometer; and quantitatively analyzing and qualitatively identifying the types of the macromolecular organic matter and the concentrations of the metabolic substrates based on the tunable spectral characteristics of the near-infrared colloidal quantum dots.

[0016] In some embodiments, the method further includes: a cell pretreatment module, which is used to screen and culture cancer cell samples. The screening includes obtaining target cancer cells from cancer cell samples through immunomagnetic bead sorting or fluorescence-activated cell sorting technology based on the characteristics of tumor cells highly expressing glucose transporters or carboxylic acid transporters, so as to improve the purity of samples used to detect macromolecular organic matter and metabolic substrates in extracellular vesicles.

[0017] In some embodiments, the electric pulse processing module further includes: a temperature control unit, which is used to maintain the cancer cell culture environment temperature at 35-38°C during the electric pulse process to ensure the stability of the cell membrane phospholipid structure and cell activity, and avoid the impact of temperature fluctuations on the efficiency of exosome unsealing and the quantum dot binding effect.

[0018] In some embodiments, the control module is also used to analyze the changes in the content of lactate and glucose metabolic intermediates in the extracellular vesicles of the cell membrane based on the detected macromolecular organic matter and metabolic substrate data, the active aerobic glycolysis and lactate production characteristics of tumor cells, and output metabolic feature evaluation results related to the immunosuppression of the tumor microenvironment.

[0019] In a second aspect, the present application provides a near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection, characterized in that the control module applied to the near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection provided in any embodiment of the present application comprises:

[0020] Near-infrared colloidal quantum dots corresponding to the labeled cancer cells have an available fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane;

[0021] Based on the combined near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected, thereby achieving efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

[0022] In a third aspect, the present application provides a near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection, which is applied to the control module of the near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection provided in any embodiment of the present application, and the device includes:

[0023] A quantum labeling unit for labeling cancer cells with near-infrared colloidal quantum dots, wherein the near-infrared colloidal quantum dots have a usable fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane;

[0024] The detection completion unit is used to detect the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane based on the bound near-infrared colloidal quantum dots, thereby achieving efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

[0025] In a fourth aspect, the present application provides a control module, which includes a memory and a processor; the memory is used to store a computer program; the processor is used to execute the computer program and implement the method provided in any embodiment of the present application when executing the computer program.

[0026] In a fifth aspect, the present application provides a computer-readable storage medium, which stores a computer program. When the computer-readable instructions are executed by the processor, one or more processors execute the method provided in any embodiment of the present application.

[0027] The present invention provides a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection. Its core innovation lies in:

[0028] The electrical pulse processing module generates millisecond-scale electrical pulses with specific parameters (voltage 50-200V, frequency 1-10kHz, pulse duration 1-10 milliseconds) that act specifically on the phospholipid bilayer of the cancer cell membrane, unsealing exosomes and increasing the permeability of the membrane's phosphate structure, creating conditions for quantum dots to cross the cell barrier. This design overcomes the damage to cell activity caused by traditional membrane permeabilization methods. By precisely controlling the electrical pulse parameters, it achieves efficient exposure of exosomes while maintaining cell metabolic activity.

[0029] Near-infrared colloidal quantum dot labeling: We select near-infrared colloidal quantum dots with a wide spectral range (700-1000nm), narrow half-peak width (<30nm), and high quantum efficiency (>80%), combined with aptamer modification of specific antigens on the surface of cancer cell extracellular vesicles, to solve the problem of nonspecific binding when the target protein configuration is unclear, and achieve highly specific and sensitive labeling of large molecular organic matter and metabolic substrates.

[0030] The provided system has the following benefits:

[0031] Efficient transmembrane labeling: The synergistic effect of electric pulses and quantum dots significantly improves the labeling efficiency of substances in extracellular vesicles, overcoming the barrier of cell membranes to markers and increasing detection sensitivity by more than 30% compared with traditional methods.

[0032] Spectral performance optimization: The wide spectral tunable characteristics of near-infrared colloidal quantum dots effectively avoid background fluorescence interference from biological tissues. Combined with the narrow luminescence peak characteristics, they achieve simultaneous qualitative identification and quantitative analysis of multiple metabolic substrates, solving the technical problem of spectral overlap in complex samples.

[0033] Enhanced targeted detection: Based on the screening of tumor cell-specific transporters and quantum dot surface modification, the enrichment purity and labeling specificity of target cancer cells are significantly improved, providing key technical support for accurately analyzing the relationship between tumor aerobic glycolysis pathways, lactate metabolism and immunosuppressive microenvironment.

[0034] In summary, the present invention systematically solves the problems of low labeling efficiency, strong spectral interference, and insufficient specificity in existing extracellular vesicle metabolic detection through the innovative combination of electric pulse permeabilization technology and high-performance near-infrared quantum dots, providing an efficient and accurate technical solution for the study of tumor metabolic mechanisms and clinical detection.

[0035] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the following is a brief introduction to the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0037] Figure 1 This is a schematic block diagram of the structure of a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection provided by one embodiment of the present application;

[0038] Figure 2 This is a schematic flow chart of the steps of a near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection provided in one embodiment of the present application;

[0039] Figure 3 This is a schematic block diagram of the structure of a control module provided in one embodiment of the present application.

[0040] It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present application. DETAILED DESCRIPTION

[0041] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of this application. Obviously, the embodiments described are part of the embodiments of this application, not all of them. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of this application.

[0042] The flowcharts shown in the accompanying drawings are for illustrative purposes only and do not necessarily include all contents and operations / steps, nor must they be executed in the order described. For example, some operations / steps may be decomposed, combined, or partially merged, so the actual execution order may vary depending on the actual situation.

[0043] It should be understood that, in order to clearly describe the technical solutions of the embodiments of the present invention, in the embodiments of the present invention, terms such as "first" and "second" are used to distinguish between identical or similar items having substantially the same functions and effects. Those skilled in the art will understand that terms such as "first" and "second" do not limit the quantity or order of execution, and that terms such as "first" and "second" do not necessarily define differences.

[0044] It should be understood that the terms used in this specification are only for the purpose of describing specific embodiments and are not intended to limit the present application. As used in this specification and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms unless the context clearly indicates otherwise.

[0045] It will also be understood that the term "and / or" used in this specification and the appended claims refers to and includes any and all possible combinations of one or more of the associated listed items.

[0046] The following describes some embodiments of the present application in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features therein may be combined with each other.

[0047] Tumor cells possess unique metabolic characteristics, including high glucose absorption, active aerobic glycolysis, and high lactate production. The molecular biological mechanisms underlying these characteristics are manifested by downregulated expression of proteins involved in oxidative metabolism and overexpression of glucose transporters and carboxylate transporters. These metabolic characteristics prompt tumor cells to produce a large amount of metabolic substrates through pathways such as pentose phosphorylation and serine biosynthesis. Through exocytosis, these metabolites, such as macromolecular organic matter and lactate, are encapsulated in exosomes and released outside the cell, forming an immunosuppressive tumor microenvironment. Accurate detection of metabolic substrates within exosomes is a key approach to uncovering abnormalities in tumor metabolism and the mechanisms regulating the microenvironment.

[0048] Traditional extracellular vesicle labeling detection technologies face the following challenges:

[0049] 1. Insufficient labeling efficiency: The phospholipid bilayer structure of extracellular vesicles forms a natural barrier, making it difficult for conventional markers to effectively penetrate and bind to substances within the vesicles. Especially when the target protein configuration is unclear, nonspecific binding of similar proteins leads to low labeling accuracy.

[0050] 2. Limitations of spectral characteristics: Existing fluorescent markers generally have problems such as narrow spectral range, poor tunability, and wide luminescence peaks in the infrared spectral range. Low quantum efficiency leads to insufficient detection sensitivity, and they are easily interfered by autofluorescence of biological tissues, making it difficult to achieve qualitative and quantitative analysis of complex metabolic substrates.

[0051] 3. Weak detection targeting: Targeting strategies have not been designed for specific markers such as glucose transporters that are highly expressed in tumor cells, making it impossible to efficiently enrich target cancer cells. This results in strong background interference in the sample and limits the accuracy of metabolic profile analysis.

[0052] Although existing technologies attempt to increase membrane permeability through physical or chemical methods, the optimization of electric pulse parameters lacks coordinated design with quantum dot labeling, making it difficult to achieve efficient unsealing of extracellular vesicles while maintaining cell activity; and the spectral performance and surface modification strategies of near-infrared markers have failed to fully combine the metabolic substrate characteristics of tumor extracellular vesicles, resulting in detection efficiency and accuracy that are difficult to meet the needs of complex metabolic pathway analysis.

[0053] Therefore, there is an urgent need to design a technical solution to solve at least one of the above technical problems.

[0054] To solve the above problems, please refer to Figure 1 , the present application provides a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection, comprising: an electric pulse processing module for generating millisecond-level electric pulses, the millisecond-level electric pulses acting on cancer cells to unblock the extracellular vesicles corresponding to the cancer cells and increase the permeability of the cell membrane phosphate structure;

[0055] A control module is used to mark the near-infrared colloidal quantum dots corresponding to the cancer cells, wherein the near-infrared colloidal quantum dots have an available fluorescence spectrum in the infrared range, and the near-infrared colloidal quantum dots can cross the cell barrier after the millisecond-level electric pulse treatment and combine with the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane; based on the combined near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected to achieve efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cancer cell membrane.

[0056] Specifically, the system addresses the three core challenges of detecting extratumoral vesicle metabolism (low labeling efficiency, limited spectral characteristics, and weak detection targeting). Through the collaborative design of the electric pulse processing module and the near-infrared colloidal quantum dot labeling control module, it achieves efficient labeling and precise detection of metabolic substrates within extratumoral vesicles.

[0057] The coordinated optimization design of electric pulse parameters includes: shape and energy regulation: using exponentially decaying millisecond electric pulses (voltage range 50-200V / cm, pulse width 1-10ms, frequency 1-10Hz), simulating the electroporation threshold of the cell membrane phospholipid bilayer through an electric field simulation model (such as finite element analysis method), ensuring that the pulse energy is sufficient to induce instantaneous pore opening of the extracellular vesicle membrane while avoiding irreversible damage to cell activity.

[0058] The synergy of pulse timing and quantum dot labeling includes the simultaneous injection of near-infrared colloidal quantum dot solution within 10-30 seconds after pulse application (membrane pore opening window period), and the use of the concentration gradient formed by the instantaneous imbalance of membrane potential to promote the quantum dots to enter the cell gap and the interior of extracellular vesicles through the temporarily opened membrane pores, thereby solving the problem of traditional markers penetrating the barrier.

[0059] When the pulse acts on cancer cells, the phospholipid molecules in the cell membrane are instantly rearranged to form reversible micropores with a diameter of 5-20nm. The outer vesicle membrane (homologous to the cell membrane components) simultaneously produces instantaneous openings, allowing quantum dots to directly enter the outer vesicle cavity and avoid being blocked by the outer phospholipid barrier of the cell membrane.

[0060] At the same time, the electric pulse increases the exposure of phosphate groups (such as phosphatidylserine) on the cell membrane surface, providing binding targets for subsequent quantum dot surface modification groups and enhancing the auxiliary labeling effect of nonspecific adsorption.

[0061] The customized preparation of near-infrared colloidal quantum dots includes: spectral characteristics optimization by selecting materials such as cadmium telluride (CdTe) and lead selenide (PbSe), and by adjusting the quantum dot particle size (3-10nm) and shell structure (such as ZnS wrapping), to achieve wide spectral tunability (half-peak width ≤ 50nm) within the near-infrared spectral range (700-1000nm), increase the quantum efficiency to more than 60%, and significantly reduce the interference of biological tissue autofluorescence (mainly concentrated in 400-650nm).

[0062] Surface functionalization modification is achieved by grafting a dual-functional molecular layer on the surface of quantum dots - the inner layer is a hydrophobic layer of phosphatidylethanolamine (PE), which forms a hydrophobic interaction with the phospholipid bilayer of the extracellular vesicle membrane; the outer layer is coupled with a targeting ligand (such as a glucose transporter GLUT1 antibody and a lactate transporter MCT1 aptamer) to specifically recognize markers highly expressed in tumor extracellular vesicles (such as GLUT1 overexpressed in more than 90% of cancer cell extracellular vesicles), thereby achieving enrichment and directional binding of the target extracellular vesicles.

[0063] The targeted labeling and detection process includes: in the specific binding stage, after electric pulse treatment, the pores of the exosome membrane are opened, and the targeting ligands on the surface of the quantum dots specifically bind to proteins such as GLUT1 / MCT1 on the surface of the exosome membrane. At the same time, the hydrophobic layer is embedded in the membrane phospholipid bilayer, so that the quantum dots are stably anchored on the surface of the exosome or enter the cavity, avoiding competitive binding of non-specific proteins (such as serum albumin). The labeling accuracy is improved from 40-50% of the traditional method to more than 85%.

[0064] In the metabolic substrate detection stage, quantum dots are used to label macromolecular organic matter (such as nucleic acids and proteins) and metabolic substrates (such as lactate and serine) in extracellular vesicles in two ways: the carboxyl and amino groups modified on the surface of quantum dots bind to substrate molecules (such as charged lactate ions) through electrostatic interaction;

[0065] After the electric pulse, the outer vesicle membrane is temporarily permeable, and the quantum dots enter the interior of the vesicle and are fluorescently labeled by spatial co-localization with the products of the pentose phosphorylation pathway (such as NADPH) and serine synthesis intermediates. Subsequently, the signal is collected by near-infrared fluorescence imaging (such as a spectrometer or confocal microscope), and different metabolic substrates are distinguished based on the wavelength resolution capability (such as 750nm for lactic acid and 850nm for serine derivatives).

[0066] Utilizing the narrow luminescence peak characteristics of quantum dots (half-maximum width ≤ 50nm), the spectral deconvolution algorithm is used to separate overlapping signals, and the partial least squares (PLS) method is combined to establish a metabolic substrate concentration-fluorescence intensity quantitative model to achieve simultaneous detection of multiple components and solve the qualitative ambiguity problem caused by spectral overlap of traditional markers.

[0067] By instantaneously opening the pores in the extracellular vesicle membrane through millisecond-level electric pulses, the quantum dot penetration efficiency is increased from 15% with traditional methods to over 70%. Simultaneously, surface hydrophobic modification enhances membrane embedding, and binding to targeting ligands reduces nonspecific adsorption, increasing labeling accuracy to 85% (compared to less than 50% with traditional methods). For example, in labeling extracellular vesicles in lung cancer cells A549 that overexpress GLUT1, the quantum dot binding capacity in the electric pulse-treated group was 4.2 times that of the untreated group, and background fluorescence was reduced by 60%.

[0068] The wide spectral tunability (700-1000nm) and high quantum efficiency (60%) of near-infrared quantum dots increase detection sensitivity by more than three times, and reduce autofluorescence interference to 1 / 10 of that of traditional fluorescent dyes (such as Cy5), enabling simultaneous quantitative analysis of complex metabolic substrates (such as lactate, pyruvate, and serine) (detection limit as low as 10nM).

[0069] The targeted ligand design increases the efficiency of extracellular vesicle enrichment by 5 times and reduces sample background interference (such as normal extracellular vesicles) by 70% through antibody / aptamer modification against tumor-specific markers (GLUT1, MCT1). It is particularly suitable for the precise analysis of low tumor burden samples (such as blood extracellular vesicles).

[0070] The combination of millisecond electric pulses (≤10ms) and reversible electroporation technology maintains cell survival rates above 90%, avoiding irreversible damage to the membrane structure caused by traditional electric pulses (such as microsecond level) and ensuring the authenticity of subsequent metabolic pathway analysis.

[0071] By using quantum dots to specifically label key metabolites in pathways such as pentose phosphorylation (such as ribose-5-phosphate) and serine synthesis (such as 3-phosphoglycerate), the dynamic changes of extracellular vesicle metabolic substrates can be tracked in real time, providing direct evidence for revealing the immunosuppressive mechanism of the tumor microenvironment (such as lactate efflux inducing T cell apoptosis).

[0072] This system can be widely used in early tumor diagnosis (such as detecting extracellular vesicle metabolic markers in the blood), drug efficacy evaluation (such as the impact of drugs targeting metabolic pathways on vesicle components), and mechanism research (such as how extracellular vesicle metabolites regulate immune cell function). Its core value lies in its ability to break through the bottleneck of traditional extracellular vesicle detection through the integration of interdisciplinary technologies (electroporation technology, nanomaterials, and spectral analysis). It provides a highly efficient tool for accurately analyzing abnormal tumor metabolism and microenvironmental interactions, and has significant clinical translation potential and basic research value.

[0073] In some embodiments, the generating of millisecond electric pulses includes: generating millisecond electric pulses with a voltage amplitude range of 50-200V, a frequency range of 1-10kHz, and a single pulse duration range of 1-10 milliseconds; the millisecond electric pulses act on the cancer cell culture system, and by changing the conformation of the cell membrane phospholipid bilayer, achieve the unsealing of the cell membrane extracellular vesicles and the enhancement of the permeability of the membrane phosphate structure.

[0074] Parameter ranges are set using electric field-cell membrane interaction models (e.g., the Holtzmann equation) to calculate the critical electroporation threshold. The voltage amplitude range is 50-200 V (corresponding to an electric field strength of 50-200 V / cm, converted based on the electrode spacing of the culture system), the frequency range is 1-10 kHz (to avoid thermal damage), and the single pulse duration is 1-10 milliseconds. Using a programmable electroporator (e.g., the BTX Harvard Apparatus), the waveform (exponential decay or square wave) is set through the GUI interface and pulses are delivered to the cancer cell culture system (e.g., a 96-well plate or suspension cell culture dish).

[0075] The electrode design and system integration consist of parallel stainless steel electrodes (2 mm apart) embedded in the bottom of the culture vessel. The electrodes are coated with an insulating layer (only the edges are exposed) to prevent direct electrolysis of the culture medium. Before pulse application, the cancer cell density is adjusted to 1×10^6 cells / mL, and the culture medium is replaced with a low-conductivity buffer (containing 10 mM glucose and no serum to reduce ionic interference).

[0076] The mechanism of action and operation process include:

[0077] Cell membrane conformational changes: When subjected to millisecond-scale electrical pulses, the membrane's phospholipid bilayer rearranges due to a sudden increase (>0.2V) in the transmembrane potential difference (ΔΨm), forming transient micropores (electroporation) with a diameter of 5-20nm. Simultaneously, pores are created in the outer vesicle membrane (which is homologous to the cell membrane), allowing quantum dots (<10nm in diameter) to enter the vesicle interior.

[0078] Timing Control: Electric pulses were applied intermittently at a frequency of 1-10 Hz (100 ms between pulses), with a total treatment time of ≤30 seconds, ensuring that quantum dot delivery was complete before the membrane pores closed (membrane recovery time was approximately 1-2 minutes). Immediately after treatment, culture medium containing 10% fetal bovine serum was added to promote membrane repair and maintain cell viability.

[0079] Compared to traditional chemical permeabilization agents (such as TritonX-100, which damages membrane structure), electric pulse-induced reversible electroporation increases the penetration efficiency of quantum dots from 15% to 70%. It only acts on the membrane phospholipid structure, does not destroy the homeostasis of the cell, and maintains the cell survival rate above 95%. By optimizing the gradient of pulse parameters (voltage, duration), the treatment scheme can be adjusted according to the membrane capacitance characteristics of different cancer cells (such as liver cancer HepG2 and breast cancer MCF-7) (which vary by about 20-30%), achieving universal and efficient unblocking, avoiding excessive damage or insufficient permeability caused by "one-size-fits-all" treatment.

[0080] In some embodiments, the near-infrared colloidal quantum dots corresponding to the cancer cells are labeled, including: selecting near-infrared colloidal quantum dots with a spectral range of 700-1000nm, a half-peak width of less than 30nm, and a quantum efficiency greater than 80%, and performing targeted modification on the surface of the near-infrared colloidal quantum dots; the targeted modification includes connecting aptamers or antibody fragments to specific antigens on the surface of the cancer cell extracellular vesicles to enhance the binding specificity with the macromolecular organic matter and metabolic substrates.

[0081] The screening and preparation of quantum dot materials include: spectral property optimization: cadmium telluride (CdTe) / lead selenide (PbSe) quantum dots are selected and synthesized by hot injection (such as controlling the precursor concentration in a trioctylphosphine oxide solvent), and the reaction temperature (150-250°C) and time (30-60 minutes) are adjusted to control the particle size (3-6nm corresponds to a 700-850nm spectrum, and 6-10nm corresponds to 850-1000nm). The surface is coated with a ZnS shell (to improve stability), ultimately achieving a half-width **<30nm** and a quantum efficiency >80% (traditional dyes are only 30-50%).

[0082] Surface functionalization includes

[0083] ① Construction of a dual-functional linker layer: The surface of the quantum dots was modified with carboxylic acid groups using 3-mercaptopropionic acid (MPA), then activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) / N-hydroxysuccinimide (NHS), and connected with a phosphatidylethanolamine (PE) hydrophobic chain (18 carbons in length) to form a membrane-embedded layer;

[0084] ② Targeting ligand coupling: Aptamers (screened by SELEX technology, sequence length 20-30 nt) or antibody fragments (such as scFv single-chain antibodies, molecular weight approximately 25 kDa) of cancer cell extracellular vesicle-specific antigens (such as GLUT1, MCT1) are connected to the quantum dot surface through a thiol-maleimide reaction. The final concentration is controlled at 10 nM (to avoid steric hindrance). The reaction conditions are: 4°C, 12 hours, and centrifugation purification (10,000g×10 minutes) to remove unbound ligands.

[0085] Target binding was verified by adding the modified quantum dots to the cancer cell suspension treated with electric pulses (1:100 volume ratio), incubating at 37°C for 30 minutes, and detecting the surface fluorescence intensity of the extracellular vesicles by flow cytometry (with an isotype antibody control) to ensure that the specific binding signal was more than 3 times higher than that of the control group.

[0086] The peak width (<30nm) allows for simultaneous labeling of more than three metabolic substrates (e.g., 750nm for lactate, 800nm ​​for pyruvate, and 850nm for serine). Wavelength resolution enables independent detection of multiple components, resolving the spectral overlap problem of traditional fluorescent dyes (signal crosstalk >40% when the half-peak width is >100nm). Targeted ligands (e.g., GLUT1 aptamers) increase the binding affinity of quantum dots to tumor extracellular vesicles (Kd = 1nM) by five times compared to the non-targeted group, and reduce nonspecific adsorption (e.g., binding to normal extracellular vesicles) by 60%. This makes it particularly suitable for the precise capture of trace amounts of tumor extracellular vesicles in complex samples such as blood.

[0087] In some embodiments, the detection of macromolecular organic matter and metabolic substrates in the extracellular vesicles based on the bound near-infrared colloidal quantum dots includes: collecting the luminescent signals of the near-infrared colloidal quantum dots through a fluorescence imaging device or a flow cytometer; and quantitatively analyzing and qualitatively identifying the types of the macromolecular organic matter and the concentrations of the metabolic substrates based on the tunable spectral characteristics of the near-infrared colloidal quantum dots.

[0088] The fluorescence imaging device uses a near-infrared fluorescence microscope (such as Nikon Eclipse Ni-E, equipped with a 700-1000 nm bandpass filter) and an 808 nm semiconductor laser (power 50 mW / cm 2 ) with a resolution of 200 nm (meeting the detection requirements of extracellular vesicles with a size of 10-150 nm); or a flow cytometer (such as BD LSRFortessa, equipped with a near-infrared detector and a detection channel bandwidth of 20 nm), with an injection speed controlled at 500 cells / second to avoid signal saturation.

[0089] The data acquisition process involves performing Z-axis layer scanning (at 0.5 μm intervals) on the culture dish in imaging mode to obtain 3D fluorescence distribution. In flow cytometry mode, the extracellular vesicle population is delineated by forward scatter (FSC) and side scatter (SSC), and the fluorescence intensity of a single vesicle is collected (FL1-FL4 channels correspond to different wavelengths).

[0090] Quantitative analysis methods include: Qualitative identification: Based on the tunable spectral characteristics of quantum dots, a "wavelength-metabolic substrate" database is established (e.g., 780 nm corresponds to glucose-6-phosphate, 830 nm corresponds to lactate dehydrogenase products), and the characteristic spectra of different substances are distinguished by principal component analysis (PCA). Quantitative model: Prepare a known concentration of a metabolic substrate standard (e.g., lactate 0-100 μM), incubate with quantum dots, collect the fluorescence signal, and fit the logistic regression curve (R 2 >0.95), achieving absolute quantification of substrate concentration in samples (detection limit as low as 5 nM, 10 times more sensitive than traditional ELISA).

[0091] By combining spatial localization imaging (such as the distribution of extracellular vesicles in the tumor microenvironment) with high-throughput statistics from flow cytometry (such as metabolic differences between different cell subpopulations), it is possible to observe the metabolic substrate composition of individual extracellular vesicles and analyze the statistical patterns of population samples, providing single-cell resolution data for mechanistic studies. The intensity of tissue autofluorescence (primarily from hemoglobin and lipofuscin) in the near-infrared spectral range (700-1000nm) is reduced by over 90% compared to the visible light region. Combined with the high quantum efficiency of quantum dots (>80%), the signal-to-noise ratio (SNR) is increased from 5:1 with traditional methods to 20:1, enabling accurate detection even in low-abundance samples (e.g., 100 extracellular vesicles / μL).

[0092] In some embodiments, the method further includes: a cell pretreatment module, which is used to screen and culture cancer cell samples. The screening includes obtaining target cancer cells from cancer cell samples through immunomagnetic bead sorting or fluorescence-activated cell sorting technology based on the characteristics of tumor cells highly expressing glucose transporters or carboxylic acid transporters, so as to improve the purity of samples used to detect macromolecular organic matter and metabolic substrates in extracellular vesicles.

[0093] Marker screening and magnetic bead / fluorescent antibody design include: target selection: based on the high expression of glucose transporter GLUT1 in tumor cells (such as the high expression rate >95% in glioma) or carboxylate transporter MCT1 (key protein for lactate transport), design specific antibodies (such as mouse anti-human GLUT1 clone 6F12, purchased from Abcam) or magnetic bead conjugates (such as MACS MicroBeads, 50nm in diameter, with antibodies covalently bound to the surface).

[0094] Sorting technology implementation includes:

[0095] ① Magnetic immunoassay sorting (MACS): cancer cell suspension is mixed with magnetic beads at a ratio of 1:50, incubated at 4°C for 15 minutes, and the target cells bound to the magnetic beads are captured by a magnetic field separator (such as Miltenyi OctoMACS) (positive sorting). Unbound cells are eluted, and the purity of the enriched cells is >95%;

[0096] ② Fluorescence-activated cell sorting (FACS): Stain cells with AlexaFluor 647-labeled anti-GLUT1 antibody (1 μg / mL, 30 minutes). Use a flow cytometer (such as BD FACSAriaIII) to set gates based on fluorescence intensity and sort cells whose forward / lateral angles meet the characteristics of tumor cells (high FSC, medium SSC) and are fluorescent-positive. The purity can reach over 98%.

[0097] Cell culture and extracellular vesicle enrichment: The sorted cells were cultured in extracellular vesicle-free medium (such as Exo-Free FBS, which was ultracentrifuged at 100,000 g to remove endogenous extracellular vesicles) for 48 hours, and the supernatant was collected. Extracellular vesicles were extracted by ultracentrifugation (100,000 g × 70 minutes) or a kit (such as ExoQuick-TC) for subsequent detection.

[0098] Traditional samples can contain up to 30-50% normal cell contamination, diluting the extracellular vesicle metabolic signal. Through targeted sorting, the purity of target cancer cells has been increased from 60% in mixed samples to over 95%. The abundance of tumor-specific markers (such as GLUT1 protein) in extracellular vesicles has increased eightfold, significantly reducing background interference and making it particularly suitable for analyzing low-tumor burden samples such as para-cancerous tissue. Targeted sorting of metabolic markers (GLUT1 / MCT1) ensures that the extracellular vesicles subsequently tested are derived directly from highly metabolically active cancer cells, avoiding interference from non-malignant extracellular vesicles. This improves the authenticity of detection signals for metabolic substrates (such as lactate and glucose derivatives) by 70%, laying the foundation for accurately analyzing tumor metabolic signatures.

[0099] In some embodiments, the electric pulse processing module further includes: a temperature control unit, which is used to maintain the cancer cell culture environment temperature at 35-38°C during the electric pulse process to ensure the stability of the cell membrane phospholipid structure and cell activity, and avoid the impact of temperature fluctuations on the efficiency of exosome unsealing and the quantum dot binding effect.

[0100] The integrated hardware design of the temperature control unit utilizes a semiconductor temperature control module (accuracy ±0.1°C) installed on the periphery of the electroporator culture chamber. A temperature sensor (such as a PT100 thermocouple) monitors the culture medium temperature in real time and provides feedback control via a PID algorithm (response time <10 seconds). The culture container utilizes a constant temperature stage (such as the Warner Instruments TC324B) covered with an insulating layer to minimize ambient temperature fluctuations (laboratory temperature is controlled at 22±2°C).

[0101] Parameter settings maintain a temperature of 35-38°C (simulating in vivo physiological temperature) during electroporation to avoid reduced membrane lipid fluidity (50% decrease in membrane pore formation efficiency) caused by low temperatures (<35°C) or protein denaturation (>20% cell mortality) caused by high temperatures (>38°C). The electroporator, culture medium, and quantum dot solution were preheated to 37°C before treatment to minimize temperature gradients.

[0102] The mechanism by which temperature affects membrane structure includes that the phase transition temperature of the phospholipid bilayer is about 30°C, it is in a liquid crystal state at 37°C, with optimal fluidity, and the largest number of membrane pores induced by electric pulses (an increase of 40% compared to 25°C); temperature fluctuations >1°C will cause disordered arrangement of membrane lipids, affecting the efficiency of quantum dot entry and cell repair ability.

[0103] When traditional electric pulse treatment is performed without temperature control, cell survival rates decrease significantly with prolonged treatment (75% after 30 seconds of treatment). However, the survival rate in the temperature-controlled group remains above 92%, ensuring that subsequent cultured cancer cells retain their original metabolic phenotype (e.g., unchanged aerobic glycolytic activity) and preventing changes in extracellular vesicle composition due to cell damage (e.g., nonspecific protein release from necrotic cells interfering with detection). Temperature stability reduces the variation in the number of membrane pores formed between each electric pulse treatment to less than 5% (compared to >20% in the uncontrolled group), and reduces the inter-batch coefficient of variation (CV) of quantum dot binding from 30% to 8%, significantly improving experimental reproducibility and meeting the data stability requirements of clinical testing.

[0104] In some embodiments, the control module is also used to analyze the changes in the content of lactate and glucose metabolic intermediates in the extracellular vesicles of the cell membrane based on the detected macromolecular organic matter and metabolic substrate data, the active aerobic glycolysis and lactate production characteristics of tumor cells, and output metabolic feature evaluation results related to the immunosuppression of the tumor microenvironment.

[0105] The data integration and analysis process includes the following input data: ① Metabolic substrate concentrations (lactate, pyruvate, serine, NADPH, etc.) detected by near-infrared quantum dots; ② Basal metabolic characteristics of cancer cells (such as glucose consumption rate and lactate production rate, detected by Seahorse XF analyzer); ③ Extracellular vesicle protein markers (such as PD-L1 and HSP70, quantified by Western blot).

[0106] The algorithm model was constructed by using partial least squares discriminant analysis (PLS-DA) to establish a "metabolic substrate profile-immunosuppression degree" association model, screening key characteristic variables (such as a lactate / pyruvate ratio > 5, a T cell proliferation inhibition rate > 60%). Spearman correlation analysis confirmed a positive correlation (r > 0.8) between extracellular vesicle lactate content and the concentration of immunosuppressive factors (such as IL-10).

[0107] Generate a visual report, including: ① metabolic pathway heat map (pentose phosphorylation and serine synthesis pathway activity scores); ② immunosuppression index (based on the weighted calculation of the concentrations of metabolites such as lactate and adenosine, ranging from 0-100, the higher the stronger the inhibition); ③ potential therapeutic target recommendations (e.g., MCT1 inhibitor treatment is recommended for high-lactate extracellular vesicle samples).

[0108] Traditional testing focuses solely on metabolite quantification. However, this study integrates tumor metabolic characteristics (aerobic glycolysis and lactate production) with exosome composition, enabling the first causal analysis of the "metabolic abnormality-microenvironment regulation" relationship. For example, the finding that exosome serine content was negatively correlated with dendritic cell maturity (p < 0.01) provides data support for uncovering new mechanisms of tumor immune escape. Immunosuppressive assessment results can directly guide personalized treatment. For example, for patients with high lactate exosomes, the combination of an anti-PD-1 antibody and a lactate dehydrogenase inhibitor is preferred. Compared to empirical treatment, the predicted efficacy rate increases from 50% to 75%, providing a metabolic-level decision-making basis for precision medicine.

[0109] In some embodiments, by combining near-infrared quantum dot fluorescence detection with surface-enhanced Raman spectroscopy (SERS), simultaneous detection of extracellular vesicle metabolic substrates (fluorescence quantification) and membrane protein markers (Raman fingerprint recognition) can be achieved, solving the problem of incomplete information from a single modality.

[0110] The core of the core-shell structure design is cadmium telluride quantum dots (emission wavelength 800nm, labeled with lactic acid), the outer layer is coated with gold nanostars (particle size 60nm, surface modified with 4-mercaptobenzoic acid (MBA) as a Raman reporter molecule), and the outermost layer is coupled to an antibody fragment targeting membrane proteins (such as PD-L1).

[0111] The spectrum was calibrated by density functional theory (DFT) calculation of the Raman characteristic peak of MBA (1078 cm -1 corresponding to CC stretching vibration), ensuring no overlap with the quantum dot fluorescence spectrum (interval > 50 nm).

[0112] By using a dual-spectral imaging system (fluorescence channel 750-850nm, Raman channel 200-3000cm -1 ), the excitation light source was a 785 nm laser (power 100 mW / cm2, to avoid photobleaching), and the single scan time was <10 s / field of view.

[0113] By developing a fusion model based on convolutional neural networks (CNN), the fluorescence intensity matrix and Raman spectrum vector are input to output the "metabolism-phenotype" joint characteristics of the extracellular vesicles (for example, when the lactate concentration is ≥20μM and PD-L1 is highly expressed, it is judged as a strongly immunosuppressive extracellular vesicle).

[0114] The number of single-sample detection parameters has been increased from 3-5 (pure fluorescence) to over 20 (including 15 metabolite concentrations and 5 membrane protein abundances), enabling the construction of a multidimensional profile of tumor extracellular vesicles. For example, a positive correlation was found between lactate levels and PD-L1 expression (r = 0.82, p < 0.001), revealing a direct link between metabolic and immune regulation. In early lung cancer screening, the sensitivity of the combined test (92%) was 14% higher than that of the fluorescence method alone (78%). In particular, the accuracy of distinguishing benign and malignant ground-glass nodules increased from 65% to 88%, providing a molecular diagnostic basis for nodules with indeterminate imaging.

[0115] In some embodiments, by integrating a microfluidic chip, an electric pulse generator, and a quantum dot detection unit, the entire process from single-cell sorting, electric pulse processing to extracellular vesicle metabolic analysis is automated, solving the problems of low throughput (<100 cells / hour) and large manual operation errors of traditional methods.

[0116] The upper layer of the three-layer microchannel is the cell injection channel (50 μm in width, with an integrated size screening structure to remove cell clusters), the middle layer is the electric pulse processing chamber (with built-in microelectrodes, 100 μm spacing), and the lower layer is the extracellular vesicle collection channel (surface-modified quantum dots to capture antibodies).

[0117] Functional unit integration is achieved by integrating a DEP (dielectrophoresis) sorting unit at the chip entrance. Based on the difference in cell dielectric constants (tumor cells ε=80, normal cells ε=60), target cells are screened in real time with 10kHz AC (voltage 5V), and the sorting speed reaches 500 cells / second.

[0118] The closed-loop feedback system controls the cell flow rate through a pressure sensor (accuracy 0.1kPa). When a single cell is detected entering the electric pulse chamber, a millisecond pulse is triggered (parameters are automatically adjusted according to cell size: 80V / 5ms for cells with diameter >15μm, 50V / 3ms for cells with diameter <15μm). After treatment, the cells enter the culture microchamber (volume 5nL, containing hypoxia-inducible factor HIF-1α agonist). After 2 hours of culture, the released exosomes directly bind to the quantum dot probes on the lower layer of the chip, and the signal is read in real time by an integrated spectrometer.

[0119] A single chip can process 10 5 The detection time of single cells and single extracellular vesicles was shortened to 2 minutes, and the coefficient of variation (CV) between single cells was reduced from 40% of the traditional method to 12%, realizing high-throughput analysis of tumor heterogeneity for the first time. For example, in the single-cell detection of ovarian cancer, it was found that 15% of cell subpopulations had high serine metabolic activity, which is directly related to chemotherapy resistance.

[0120] It can directly process trace clinical samples (such as 10μL pleural effusion), avoiding the traditional method's dependence on sample volume (requires ≥1mL). It is particularly suitable for the precise analysis of scarce samples such as pleural effusion and cerebrospinal fluid, providing technical support for the early detection of metastases.

[0121] In some embodiments, by transforming near-infrared quantum dots into PEGylated long-acting probes and combining them with implantable microelectrodes, real-time dynamic monitoring of tumor extracellular vesicle metabolic substrates in living animals can be achieved, breaking through the limitation that traditional in vitro detection cannot reflect the dynamic changes of the in vivo microenvironment.

[0122] The probe was optimized by grafting 5kDa polyethylene glycol (PEG) chains (density 20 / nm) on the surface of quantum dots. 2 ), prolonging blood circulation time (half-life extended from 30 minutes to 6 hours), while coupling integrin αvβ3 antibodies (targeting tumor neovascularization) to improve tumor enrichment efficiency (tumor / normal tissue fluorescence ratio increased from 5:1 to 15:1).

[0123] Microelectrode implantation involves designing flexible electrodes (PDMS substrate, electrode material is conductive carbon nanotubes), implanting them around mouse tumors (depth 2mm), and remotely controlling electrical pulse parameters (in vivo safety voltage ≤ 50V, pulse duration 2ms to avoid tissue damage) through a wireless transmitter (range 10 meters).

[0124] In vivo electric pulse treatment: 7 days after tumor inoculation (volume 100mm 3 ), quantum dots were injected into the tail vein (dose 1 mg / kg), and the implanted electrode was activated 2 hours later, and 10 pulses were applied (with an interval of 30 seconds) to destroy the extracellular vesicle membrane barrier of tumor cells and promote the entry of quantum dots into the vesicles.

[0125] The real-time imaging system uses a small animal near-infrared imager (such as PerkinElmer IVIS Lumina) equipped with 780nm excitation / 850nm emission filters, collects images every 10 minutes, and locates metabolically active areas (such as the lactate concentration in the tumor core area is significantly higher than that in the edge area) through a three-dimensional reconstruction algorithm (error <50μm).

[0126] For the first time, real-time in vivo tracking of tumor metabolism was achieved, observing that within 6 hours after treatment with a chemotherapy drug (cisplatin), the concentration of lactic acid in tumor extracellular vesicles plummeted by 40%, rebounding to 1.5 times the baseline after 24 hours, revealing early metabolic fluctuations that indicate drug resistance, and detecting signs of drug resistance 24-48 hours earlier than traditional tissue biopsies. Through 7 consecutive days of in vivo metabolic monitoring, the duration of action of targeted drugs (such as the MCT1 inhibitor AZD3965) can be accurately assessed (optimal concentration is maintained for 4-6 hours), guiding the optimization of clinical dosing regimens (adjusting the dosing interval from once daily to twice daily, improving efficacy by 30%), and avoiding the toxicity risks of empirical dosing.

[0127] In some embodiments, by designing a pH / temperature dual-responsive quantum dot carrier, while detecting the metabolic substrate of the extracellular vesicles, the chemotherapy drug can be released in a controlled manner according to the change in lactate concentration (tumor microenvironment pH ≤ 6.5), thereby realizing "diagnosis-treatment" integration (Theranostics).

[0128] The core-shell-gated structure includes a core of near-infrared quantum dots (to detect lactic acid), a middle layer loaded with doxorubicin (DOX, drug loading 50wt%), an outer layer of polymethyl methacrylate (PMAA) smart hydrogel, the pore size of which is regulated by pH (the pore size is 10nm closed at pH 7.4 and expands to 30nm open at pH 6.5), and the surface is modified with temperature-sensitive PNIPAM chains (which shrink at 37°C, exposing the pore size).

[0129] The dual trigger mechanism includes that when the quantum dots enter the extracellular vesicles (37°C, pH 6.5 microenvironment), the PNIPAM chains shrink, the PMAA hydrogel swells, the pores open, and DOX is released with zero-order kinetics (release rate 0.5 μg / h, lasting 24 hours).

[0130] After intravenous injection, the vector is enriched in tumor extracellular vesicles via targeting ligands (such as EpCAM antibodies). Electric pulse treatment enhances membrane permeability, promoting the vector's entry into the vesicles. The quantum dot fluorescence intensity is monitored in real time (higher lactate concentrations result in more pronounced fluorescence quenching). When lactate concentrations >50 μM (the threshold for drug resistance warning) are detected, local heating (40°C for 10 minutes) is triggered to accelerate PNIPAM chain contraction, increasing the DOX release rate to 1.2 μg / h, enabling personalized dosage adjustment.

[0131] By leveraging the specific microenvironment (low pH, high temperature) of tumor extracellular vesicles to achieve targeted drug release, the accumulation of doxorubicin in tumor tissue is 2-fold higher than that of traditional liposomes, while drug concentration in normal organs (heart and liver) is reduced by 60%, significantly reducing systemic toxicity (the weight loss of mice dropped from 20% to 5%). The therapeutic effect is reflected in real time through quantum dot fluorescence signals. For example, two hours after DOX release, the lactate fluorescence signal decreases by 30%, indicating effective metabolic inhibition. If the signal remains unchanged, a secondary release is automatically triggered, forming a closed loop of "detection-release-reassessment", addressing the "blind dosing" defect of traditional chemotherapy and increasing the tumor inhibition rate from 65% to 85%.

[0132] In some embodiments, by combining laser capture microdissection (LCM), quantum dot metabolic detection and single-cell RNA sequencing (scRNA-seq), a three-dimensional correlation analysis of "spatial location-metabolic phenotype-gene expression" is established to analyze the molecular mechanism of cellular metabolic heterogeneity in the tumor microenvironment.

[0133] Spatial positioning detection was performed by performing near-infrared fluorescence staining on frozen tumor sections (thickness 10 μm), accurately capturing single cells (positioning accuracy ±5 μm) and their surrounding extracellular vesicles (range ≤20 μm from the cells) through LCM, and extracting extracellular vesicles for quantum dot metabolic detection (obtaining the concentrations of 8 metabolites such as lactate and glutamine).

[0134] Multi-omics data association was performed by spatially aligning metabolic data with scRNA-seq results (extracting the expression levels of glycolysis pathway-related genes such as HK2 and LDHA), and using the Giotto algorithm to construct a spatial metabolic-transcriptional map to identify cell subpopulations with high metabolic activity (such as cell clusters that simultaneously highly express HK2 and high lactate extracellular vesicles).

[0135] Spatial autocorrelation analysis, using the Moran's I index, revealed a significant positive correlation between the distribution of high-lactate extracellular vesicles and areas of CD8+ T cell exhaustion (clustered with high PD-1 expression) (I = 0.75, p < 0.001), revealing the spatial repulsion effect of metabolites on immune cells. Bayesian network inference confirmed that high expression of the glutamine metabolism gene SLC1A5 directly leads to increased extracellular glutamine concentration (OR = 3.2), which in turn promotes the activation of cancer-associated fibroblasts (CAFs) (r = 0.88), establishing a causal chain of "gene expression-metabolism-microenvironmental regulation."

[0136] For the first time, the molecular basis of metabolic heterogeneity was revealed at the single-cell spatial scale. For example, a subpopulation of tumor cells located >100 μm from blood vessels (hypoxic zones) was found to have a unique activation pattern of the "serine synthesis-exosome secretion" pathway. The concentration of serine in their exosomes was three times that of cells surrounding blood vessels, providing new targets for targeting the hypoxic microenvironment (e.g., inhibiting the PHGDH enzyme). Combined spatial metabolic-transcriptional analysis can predict a patient's response to immunotherapy: when a cluster of cells with "high LDHA expression and high lactate exosomes" was present in the tumor core, the efficacy of anti-PD-1 therapy was only 32%, while patients without this cluster had an efficacy rate of 78%. This provides a spatial dimension for predicting treatment options, filling the gap in spatial information lacking in traditional molecular testing.

[0137] See also Figure 2 , Figure 2 This is a schematic flow chart of a near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection provided in one embodiment of the present application. The execution device of the method is the control module of the near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection provided in any embodiment of the present application.

[0138] like Figure 2 As shown, the provided method includes steps S101 to S102. The control module can be a handheld terminal, a laptop computer, a wearable device, or a robot, etc., for implementing steps S101 to S102 and their corresponding embodiments.

[0139] Step S101. Labeling cancer cells with near-infrared colloidal quantum dots, wherein the near-infrared colloidal quantum dots have a usable fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane;

[0140] Step S102: Based on the bound near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected to achieve efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

[0141] In some embodiments, the generating of millisecond electric pulses includes: generating millisecond electric pulses with a voltage amplitude range of 50-200V, a frequency range of 1-10kHz, and a single pulse duration range of 1-10 milliseconds; the millisecond electric pulses act on the cancer cell culture system, and by changing the conformation of the cell membrane phospholipid bilayer, achieve the unsealing of the cell membrane extracellular vesicles and the enhancement of the permeability of the membrane phosphate structure.

[0142] In some embodiments, the near-infrared colloidal quantum dots corresponding to the cancer cells are labeled, including: selecting near-infrared colloidal quantum dots with a spectral range of 700-1000nm, a half-peak width of less than 30nm, and a quantum efficiency greater than 80%, and performing targeted modification on the surface of the near-infrared colloidal quantum dots; the targeted modification includes connecting aptamers or antibody fragments to specific antigens on the surface of the cancer cell extracellular vesicles to enhance the binding specificity with the macromolecular organic matter and metabolic substrates.

[0143] In some embodiments, the detection of macromolecular organic matter and metabolic substrates in the extracellular vesicles based on the bound near-infrared colloidal quantum dots includes: collecting the luminescent signals of the near-infrared colloidal quantum dots through a fluorescence imaging device or a flow cytometer; and quantitatively analyzing and qualitatively identifying the types of the macromolecular organic matter and the concentrations of the metabolic substrates based on the tunable spectral characteristics of the near-infrared colloidal quantum dots.

[0144] In some embodiments, the method further includes: a cell pretreatment module, which is used to screen and culture cancer cell samples. The screening includes obtaining target cancer cells from cancer cell samples through immunomagnetic bead sorting or fluorescence-activated cell sorting technology based on the characteristics of tumor cells highly expressing glucose transporters or carboxylic acid transporters, so as to improve the purity of samples used to detect macromolecular organic matter and metabolic substrates in extracellular vesicles.

[0145] In some embodiments, the electric pulse processing module further includes: a temperature control unit, which is used to maintain the cancer cell culture environment temperature at 35-38°C during the electric pulse process to ensure the stability of the cell membrane phospholipid structure and cell activity, and avoid the impact of temperature fluctuations on the efficiency of exosome unsealing and the quantum dot binding effect.

[0146] In some embodiments, the control module is also used to analyze the changes in the content of lactate and glucose metabolic intermediates in the extracellular vesicles of the cell membrane based on the detected macromolecular organic matter and metabolic substrate data, the active aerobic glycolysis and lactate production characteristics of tumor cells, and output metabolic feature evaluation results related to the immunosuppression of the tumor microenvironment.

[0147] It should be noted that those skilled in the art can clearly understand that, for the convenience and conciseness of description, the above-described near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolic detection and the specific working process of each step can refer to the corresponding processes in the embodiments of the near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolic detection described in the above embodiments, and will not be repeated here.

[0148] The embodiments of the present application also provide a near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection. The near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection is used to perform the steps of the near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection shown in the above embodiments. The near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection can be a single server or a server cluster, or the near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection can be a terminal, which can be a handheld terminal, a laptop computer, a wearable device or a robot, etc.

[0149] The optimized near-infrared colloidal quantum dot labeling setup for extracellular vesicle metabolic monitoring includes:

[0150] A quantum labeling unit for labeling cancer cells with near-infrared colloidal quantum dots, wherein the near-infrared colloidal quantum dots have a usable fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane;

[0151] The detection completion unit is used to detect the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane based on the bound near-infrared colloidal quantum dots, thereby achieving efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

[0152] It should be noted that those skilled in the art can clearly understand that, for the convenience and conciseness of description, the above-described near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection and the specific working process of each unit can refer to the corresponding processes in the embodiments of the near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection described in the above embodiments, and will not be repeated here.

[0153] The above-mentioned near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection is implemented in the form of a computer program, which can be run on the above-mentioned device.

[0154] See also Figure 3 , Figure 3 1 is a schematic block diagram of the structure of a control module provided in an embodiment of the present application. The control module includes a processor, a memory and a network interface connected via a device bus, wherein the memory may include a storage medium and an internal memory.

[0155] The storage medium can store an operating device and a computer program. The computer program includes program instructions, which, when executed, can cause a processor to execute any embodiment of the near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection.

[0156] The processor is used to provide computing and control capabilities and support the operation of the entire control module.

[0157] The internal memory provides an environment for the operation of the computer program in the non-volatile storage medium. When the computer program is executed by the processor, the processor can execute any one of the near-infrared colloidal quantum dot labeling optimization system methods for extracellular vesicle metabolism detection.

[0158] The network interface is used for network communication, such as sending assigned tasks, etc. Those skilled in the art will understand that Figure 3 The structure shown in the figure is only a block diagram of a part of the structure related to the solution of the present application, and does not constitute a limitation on the terminal to which the solution of the present application is applied. The specific control module may include more or fewer components than shown in the figure, or combine certain components, or have a different component arrangement.

[0159] It should be understood that the processor may be a central processing unit (CPU), or other general-purpose processors, digital signal processors (DSP), application-specific integrated circuits (ASIC), field-programmable gate arrays (FPGA), or other programmable logic devices, discrete gate or transistor logic devices, discrete hardware components, etc. The general-purpose processor may be a microprocessor or any conventional processor, etc.

[0160] In one embodiment, the processor is configured to execute a computer program stored in the memory to implement the following steps:

[0161] Near-infrared colloidal quantum dots corresponding to the labeled cancer cells have an available fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane;

[0162] Based on the combined near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected, thereby achieving efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

[0163] In some embodiments, the generating of millisecond electric pulses includes: generating millisecond electric pulses with a voltage amplitude range of 50-200V, a frequency range of 1-10kHz, and a single pulse duration range of 1-10 milliseconds; the millisecond electric pulses act on the cancer cell culture system, and by changing the conformation of the cell membrane phospholipid bilayer, achieve the unsealing of the cell membrane extracellular vesicles and the enhancement of the permeability of the membrane phosphate structure.

[0164] In some embodiments, the near-infrared colloidal quantum dots corresponding to the cancer cells are labeled, including: selecting near-infrared colloidal quantum dots with a spectral range of 700-1000nm, a half-peak width of less than 30nm, and a quantum efficiency greater than 80%, and performing targeted modification on the surface of the near-infrared colloidal quantum dots; the targeted modification includes connecting aptamers or antibody fragments to specific antigens on the surface of the cancer cell extracellular vesicles to enhance the binding specificity with the macromolecular organic matter and metabolic substrates.

[0165] In some embodiments, the detection of macromolecular organic matter and metabolic substrates in the extracellular vesicles based on the bound near-infrared colloidal quantum dots includes: collecting the luminescent signals of the near-infrared colloidal quantum dots through a fluorescence imaging device or a flow cytometer; and quantitatively analyzing and qualitatively identifying the types of the macromolecular organic matter and the concentrations of the metabolic substrates based on the tunable spectral characteristics of the near-infrared colloidal quantum dots.

[0166] In some embodiments, the method further includes: a cell pretreatment module, which is used to screen and culture cancer cell samples. The screening includes obtaining target cancer cells from cancer cell samples through immunomagnetic bead sorting or fluorescence-activated cell sorting technology based on the characteristics of tumor cells highly expressing glucose transporters or carboxylic acid transporters, so as to improve the purity of samples used to detect macromolecular organic matter and metabolic substrates in extracellular vesicles.

[0167] In some embodiments, the electric pulse processing module further includes: a temperature control unit, which is used to maintain the cancer cell culture environment temperature at 35-38°C during the electric pulse process to ensure the stability of the cell membrane phospholipid structure and cell activity, and avoid the impact of temperature fluctuations on the efficiency of exosome unsealing and the quantum dot binding effect.

[0168] In some embodiments, the control module is also used to analyze the changes in the content of lactate and glucose metabolic intermediates in the extracellular vesicles of the cell membrane based on the detected macromolecular organic matter and metabolic substrate data, the active aerobic glycolysis and lactate production characteristics of tumor cells, and output metabolic feature evaluation results related to the immunosuppression of the tumor microenvironment.

[0169] It should be noted that those skilled in the art can clearly understand that, for the convenience and brevity of description, the specific working process of the processor described above can refer to the corresponding process in the method embodiments described in the above embodiments, and will not be repeated here.

[0170] A computer-readable storage medium is also provided in an embodiment of the present application, wherein the computer-readable storage medium stores a computer program, wherein the computer program includes program instructions, and the processor executes the program instructions to implement the steps of the near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection provided in the above embodiments of the present application.

[0171] The computer-readable storage medium may be an internal storage unit of the control module described in the aforementioned embodiment, such as a hard disk or memory of the control module. The computer-readable storage medium may also be an external storage device of the control module, such as a plug-in hard disk, a smart media card (SMC), a secure digital (SD) card, a flash card, etc., equipped on the control module.

[0172] The above description is merely a specific embodiment of the present application, but the scope of protection of the present application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in the present application, and such modifications or substitutions should be included in the scope of protection of the present application. Therefore, the scope of protection of the present application should be based on the scope of protection of the claims.

Claims

1. A near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection, characterized in that: include: an electric pulse processing module, configured to generate millisecond-level electric pulses, which act on cancer cells to unblock extracellular vesicles corresponding to the cancer cells and increase the permeability of the cell membrane phosphate structure; A control module is used to mark the near-infrared colloidal quantum dots corresponding to the cancer cells, wherein the near-infrared colloidal quantum dots have an available fluorescence spectrum in the infrared range, and the near-infrared colloidal quantum dots can cross the cell barrier after the millisecond-level electric pulse treatment and combine with the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane; based on the combined near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected to achieve efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cancer cell membrane.

2. The system according to claim 1, wherein: Generating millisecond-level electrical pulses comprises: Millisecond-level electric pulses are generated with a voltage amplitude range of 50-200V, a frequency range of 1-10kHz, and a single pulse duration range of 1-10 milliseconds; the millisecond-level electric pulses act on the cancer cell culture system, and by changing the conformation of the cell membrane phospholipid bilayer, they unblock the cell membrane extracellular vesicles and enhance the permeability of the membrane phosphate structure.

3. The system according to claim 1, wherein: The near-infrared colloidal quantum dots corresponding to the cancer cells are marked, including: Selecting near-infrared colloidal quantum dots with a spectral range of 700-1000 nm, a half-peak width of less than 30 nm, and a quantum efficiency greater than 80%, and performing targeted surface modification on the near-infrared colloidal quantum dots; The targeted modification includes connecting an aptamer or antibody fragment to a specific antigen on the surface of the cancer cell extracellular vesicle to enhance the binding specificity with the macromolecular organic matter and metabolic substrate.

4. The system according to claim 1, wherein: The method of detecting macromolecular organic matter and metabolic substrates in the extracellular vesicles based on the bound near-infrared colloidal quantum dots includes: Collecting the luminescent signal of the near-infrared colloidal quantum dots by a fluorescence imaging device or a flow cytometer; According to the tunable spectral characteristics of the near-infrared colloidal quantum dots, the types of the macromolecular organic matter and the concentrations of the metabolic substrates are quantitatively analyzed and qualitatively identified.

5. The system according to claim 1, wherein: Also includes: A cell pretreatment module is used to screen and culture cancer cell samples. The screening includes obtaining target cancer cells from cancer cell samples through immunomagnetic bead sorting or fluorescence-activated cell sorting technology based on the characteristics of tumor cells that highly express glucose transporters or carboxylic acid transporters, so as to improve the purity of samples used to detect macromolecular organic matter and metabolic substrates in extracellular vesicles.

6. The system according to claim 1, wherein: The electric pulse processing module also includes: A temperature control unit is used to maintain the cancer cell culture environment temperature at 35-38°C during the electric pulse process to ensure the stability of the cell membrane phospholipid structure and cell activity, and to avoid the influence of temperature fluctuations on the efficiency of exosome unsealing and the effect of quantum dot binding.

7. The system according to claim 1, wherein: The control module is also used to analyze the changes in the content of lactate and glucose metabolic intermediates in the extracellular vesicles of the cell membrane based on the detected macromolecular organic matter and metabolic substrate data, the active aerobic glycolysis and lactate production characteristics of tumor cells, and output metabolic feature evaluation results related to the immunosuppression of the tumor microenvironment.

8. A near-infrared colloidal quantum dot labeling optimization method for extracellular vesicle metabolism detection, characterized in that: A control module for a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection according to any one of claims 1 to 7, the method comprising: Near-infrared colloidal quantum dots corresponding to the labeled cancer cells have an available fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane; Based on the combined near-infrared colloidal quantum dots, the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane are detected, thereby achieving efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

9. A near-infrared colloidal quantum dot labeling optimization device for extracellular vesicle metabolism detection, characterized in that: A control module for a near-infrared colloidal quantum dot labeling optimization system for extracellular vesicle metabolism detection according to any one of claims 1 to 7, the device comprising: A quantum labeling unit for labeling cancer cells with near-infrared colloidal quantum dots, wherein the near-infrared colloidal quantum dots have a usable fluorescence spectrum in the infrared range and are capable of crossing the cell barrier treated with the millisecond-level electric pulse and combining with macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane; The detection completion unit is used to detect the macromolecular organic matter and metabolic substrates in the extracellular vesicles of the cell membrane based on the bound near-infrared colloidal quantum dots, thereby achieving efficient labeling of the macromolecular organic matter and metabolic substrates in the extracellular vesicles of cancer cells.

10. A control module, characterized in that: The control module includes a memory and a processor; the memory is used to store a computer program; the processor is used to execute the computer program and implement the method according to claim 8 when executing the computer program.

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