Systems and methods for ultrasound modulation of blood cells

Abstract

Embodiments of the present invention provide devices, systems, and methods for reducing inflammation. A method of reducing inflammation includes directing ultrasound towards a plurality of blood cells, wherein directing ultrasound towards the plurality of blood cells causes reduction of inflammation associated with the plurality of blood cells. A system configured to mitigate inflammation includes an ultrasound transducer configured to direct ultrasound to a plurality of blood cells to cause mitigation of inflammation caused by or associated with the plurality of blood cells.

Description

[0001] Cross-references to related applications

[0002] This application is based on and claims priority to U.S. Patent Application Serial No. 63 / 537,752, filed on September 11, 2023, the entire disclosure of which is incorporated herein by reference.

[0003] Statement on Federally Funded Research

[0004] This invention was made with government support under license number 140D0419C0092 granted by the Defense Advanced Research Projects Agency (DARPA). The government holds certain rights to this invention. Background Technology

[0005] Recent research has sought to suppress inflammatory responses by applying ultrasound stimulation to the nervous system, for example, by stimulating the vagus and splenic nerves. This stimulation is thought to drive neural activity in the spleen, thereby triggering a cascade of physiological processes that drive anti-inflammatory effects. However, the mechanisms and cellular targets remain not fully understood. Therefore, further research and understanding of this anti-inflammatory response are needed. Summary of the Invention

[0006] Embodiments of the present invention provide apparatus, systems, and methods for reducing inflammation. In one embodiment, the method for reducing inflammation includes directing ultrasound to a plurality of blood cells, wherein directing ultrasound to the plurality of blood cells causes a reduction in inflammation associated with said plurality of blood cells.

[0007] In another embodiment, a system for reducing inflammation is provided. The system includes an ultrasound transducer configured to direct ultrasound waves toward a plurality of blood cells to induce a reduction in inflammation associated with the plurality of blood cells. Attached Figure Description

[0008] The various objects, features, and advantages of the disclosed subject matter can be more fully understood when considered in conjunction with the accompanying drawings, and with reference to the following detailed description of the subject matter, in which the same reference numerals denote the same elements.

[0009] Figure 1A This is a flowchart illustrating an exemplary process of applying ultrasound stimulation to blood cells according to some aspects of this disclosure.

[0010] Figure 1B This is a flowchart illustrating an exemplary process of applying ultrasound stimulation to blood cells in an in vitro environment according to some aspects of this disclosure.

[0011] Figure 1CThis is a flowchart illustrating another exemplary process of applying ultrasound stimulation to blood cells in an in vitro environment according to some aspects of this disclosure.

[0012] Figure 2 This is an exemplary ultrasound stimulation system that can be used in accordance with the systems and methods described herein.

[0013] Figure 3 This is a block diagram of an ultrasound system that can be used in accordance with the systems and methods described herein.

[0014] Figure 4A This is a block diagram of a non-limiting example of an ultrasound device for providing stimulation.

[0015] Figure 4B This is a cross-sectional view of a non-limiting exemplary treatment module housing.

[0016] Figure 4C This is a cross-sectional view of a non-limiting exemplary treatment module base.

[0017] Figure 4D This is a cross-sectional view of a non-limiting exemplary assembled treatment module.

[0018] Figure 5 This is a block diagram of a non-limiting exemplary ultrasound system configuration.

[0019] Figure 6A Exemplary data from a first embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0020] Figure 6B Further exemplary data from a first embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0021] Figure 6C Further exemplary data from a first embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0022] Figure 7A Exemplary data from a second embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0023] Figure 7B Further exemplary data from a second embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0024] Figure 7C Further exemplary data from a second embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0025] Figure 8AExemplary data from a third embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0026] Figure 8B Further exemplary data from a third embodiment of experimental results obtained using the systems and methods of this disclosure are shown.

[0027] Figure 8C Further exemplary data from a third embodiment of experimental results obtained using the systems and methods of this disclosure are shown. Detailed Implementation

[0028] Before providing a detailed description of any embodiment of the invention, it should be understood that the invention is not limited in its application to the details of the construction and the arrangement of components set forth in the following description or illustrated in the accompanying drawings. The invention is capable of having other embodiments and can be practiced or performed in a variety of ways. Furthermore, it should be understood that the wording and terminology used herein are for descriptive purposes and should not be considered limiting. The use of “comprising,” “including,” or “having,” and variations thereof herein is intended to cover the items listed thereafter and their equivalents, as well as additional items. Unless otherwise specified or limited, the terms “mounting,” “connection,” “support,” and “coupled,” and variations thereof are used extensively and cover both direct and indirect mounting, connection, support, and coupling. Moreover, “connection” and “coupled” are not limited to physical or mechanical connections or couplings.

[0029] The following discussion is presented to enable those skilled in the art to prepare and use embodiments of the invention. Various modifications to the illustrated embodiments will be apparent to those skilled in the art, and the general principles herein can be applied to other embodiments and applications without departing from the embodiments of the invention. Therefore, embodiments of the invention are not intended to be limited to the illustrated embodiments, but should be given the widest scope consistent with the principles and features disclosed herein. The following detailed description will be read with reference to the accompanying drawings, wherein the same elements in the different drawings have the same reference numerals. The drawings are not necessarily to scale, depict selected embodiments, and are not intended to limit the scope of embodiments of the invention. Those skilled in the art will recognize that the examples provided herein have many available alternatives and fall within the scope of embodiments of the invention.

[0030] This disclosure provides systems and methods for targeting blood cells with ultrasound (US) energy to directly apply ultrasound stimulation to blood cells (e.g., immune cells). For example, the systems and methods provide direct immunomodulation of immune cells (which may be referred to as leukocytes). Directing low-intensity ultrasound stimulation to the blood cell group can induce a reduction in inflammation, measurable by inflammatory markers or other clinical symptoms such as swelling or redness, without requiring stimulation of nerves (e.g., the vagus or splenic nerve) or axonal terminals. Such anti-inflammatory responses can have a wide range of clinical, research, and consumer applications. For example, clinical applications may include treating one or more of the following: autoimmune diseases (e.g., rheumatoid arthritis, lupus, multiple sclerosis), inflammatory diseases (e.g., inflammatory bowel disease, Crohn's disease, acute SARS-CoV-2 infection, acute sequelae of SARS-CoV-2 infection, sepsis, viral infections), preoperative or postoperative suppression of inflammatory responses, protection of organs (i.e., kidneys, lungs, heart, intestines, brain) from ischemia-reperfusion injury, pancreatitis, cancer, joint or tissue swelling, healing of infected sites, bleeding or wound sites, neurodegenerative diseases, metabolic diseases, digestive diseases, cardiovascular diseases, lung or respiratory diseases, stroke, nerve or nerve injury problems such as spinal cord injury, etc. The method of directing therapeutic ultrasound to blood cells discussed is far simpler than previously implemented non-invasive methods of directing ultrasound to nerves in vivo. It eliminates the complexities of targeting areas as small as nerves, such as the necessary accuracy and precision required to target specific nerves. Targeting nerves with ultrasound can be complex due to shielding caused by the diversity of patient anatomy (e.g., bone or adipose tissue). Instead, ultrasound can be directed to larger, more blood-rich targets, such as lymph nodes, spleen, or large veins and arteries. By diversifying ultrasound targets, treatment is not limited to nerve accessibility but can be used in multiple areas of the body, potentially changing locations based on accessibility. The systems and methods can be applied in vivo to subjects (e.g., research volunteers, patients, or animals). They can also be applied ex vivo, i.e., after blood cells are removed, ultrasound stimulation is applied, and the blood cells are returned to the subject or another subject. The systems and methods can also be used in vitro for research or clinical applications. In this way, the time cost to the subject can be minimized to include only the time period during which sample collection occurs, while parameters can be optimized downstream in vitro. This in vitro method can also be used to alter natural or artificial cell and blood samples in petri dishes or experiments for other research purposes or to return them to animals.

[0031] In some configurations, it is desirable to target a pool of blood or a blood bank or reservoir. Such targets may include quiescent, nearly quiescent, or slowly moving blood cells, allowing for repeated stimulation of the blood cells during the treatment period. For example, target locations may include pools of blood or fluids located in: spleen, liver, kidney, pancreas, lymph nodes, lymphoid tissue, glymphatic tissue, heart, brain, intestine, blood vessels, veins, arteries, capillaries, swelling in joints, swelling in tissues, muscles, tumors, eyeballs, bruises or hematomas, ear regions or inner ear regions, gums, nasal cavity or regions, etc. In some configurations, the target may be in the brain, such as in the meninges of the subject's brain, which may contain a high density of immune cells. In some configurations, quiescent or nearly quiescent pools of blood or fluids may be targeted. In other aspects, the system and method can be applied to an ex vivo environment. For example, blood, bone marrow, lymph, etc., can be removed from a subject or patient, treated with ultrasound stimulation, and returned to the subject or another subject or even an animal. In other configurations, biological fluids can be extracted from animal objects, processed, and returned to human objects. In another instance, donated blood that has undergone ex vivo ultrasound treatment can be transfused into a blood-type-matched recipient, such as a human or animal object. In still other configurations, flowing blood can be targeted, which will be described in further detail below.

[0032] Figure 1A A flowchart illustrating an exemplary process 100 for applying ultrasound stimulation to immune cells is shown. Process 100 can be applied in vivo using an ultrasound device. For example, the ultrasound device can be a portable or wearable device that can intermittently apply ultrasound stimulation over a period of time (e.g., hours, days, weeks, etc.). Process 100 can also be applied in ex vivo environments, such as... Figure 1B As shown, this will be described in further detail below.

[0033] Process 100 begins at box 104, where patient or subject data can be acquired. Patient data may include imaging data that allows for the identification of target blood cells and guides the further identification of ultrasound stimulation targets in the process (e.g., box 106). For example, box 104 may include medical imaging such as ultrasound, magnetic resonance imaging (MRI), X-ray, computed tomography (CT), fluoroscopy, positron emission tomography (PET), single-photon emission computed tomography (SPECT), etc. In some configurations, imaging may preferably utilize ultrasound. When ultrasound imaging is used, the imaging ultrasound transducer may be integrated with the transducer used for ultrasound stimulation, or a dual-mode transducer may be used to simultaneously or alternately generate images and stimulation. Such images can guide the ultrasound stimulation target. For example, images can be used to identify blood pools containing a large number of immune cells. Imaging can also identify high-density vascular regions that can be targeted.

[0034] Acquiring imaging data in box 104 may also include dynamic imaging, which can indicate the velocity of blood movement in a target. For example, an imaging tracer can be used to track blood flow. As a supplement or alternative, MRI can be used in conjunction with flow imaging techniques, such as phase-contrast methods or gradient echo techniques, to generate dynamic angiographic images. Doppler ultrasound can also be used to estimate blood flow through vessels and can be enhanced by using microbubble contrast agents. Such dynamic blood flow data can provide information on target location or stimulation timing parameters in boxes 106 and 108, respectively. For example, such dynamic imaging can indicate areas where blood flow is stationary or nearly stationary, which can be targeted with a fixed ultrasound device. Alternatively, blood flow velocity can guide the use of a movable ultrasound device or a steerable ultrasound beam that can track blood flow through the body to target moving blood cells.

[0035] Patient data may also include clinical data, which can further guide the calibration of stimulation parameters in the workflow (e.g., box 108). Clinical patient data may include patient health history, demographic data, family history, historical biomarker data, current biomarker data, dynamic biomarker data, genetic expression data, metabolomics data, immune data, body size data, etc. Biomarkers may include measurements of cytokines, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin / d-dimer, etc. For example, cytokine biomarkers may include interleukin-1β (IL-1β), interferon-gamma (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-alpha (TNF-α), and nuclear factor kappa-light-chain enhancer of activated B cells (NF-κB). Other data about the patient's body, such as body mass index (BMI) or body dimensions of the target area, can be used to determine the optimal parameters and sites for ultrasound stimulation. Various types of patient data can be used alone or in combination to determine the patient's inflammatory status.

[0036] Patient data acquired in box 104 may include measurements of the patient's inflammatory state. In some configurations, such inflammatory state can be determined from blood draws used to measure cytokine information or other biomarkers. Blood samples can be collected, and results can be input into a data server. Such a server may be wirelessly or otherwise connected to the ultrasound stimulation device. The patient's inflammatory state may also be stored on a computer system based on previous clinical tests or patient history and accessed from computer memory. Patient data may alternatively be acquired using external sensors. For example, cytokine sensors may be implanted into the subject's vascular system, or implanted via a minimally invasive wearable patch or device using microneedles into the skin, oral region, or ear region (e.g., the ear canal, tympanic membrane, or other endocardial membrane near blood vessels close to the surface of the membrane).

[0037] An ultrasound stimulation target can be set in box 106. The stimulation target can be determined from the patient data acquired in box 104. For example, the target can be determined based on the patient's condition, health history, or cytokine levels. Additionally, imaging data can be used to target immune cells. For example, imaging data can be used to identify and locate blood pools containing high levels of immune cells or blood cells. Imaging data can also be used to identify areas of high vascular density.

[0038] Ultrasonic stimulation parameters can be set in box 108. These parameters may include stimulation time or duration, ultrasound frequency, peak pressure of ultrasound energy, pulse width, and repetition frequency. These parameters may be set to constants during the treatment period or may vary dynamically during the treatment. Treatment parameters may also include a duty cycle, where process 100 may be repeated once per duty cycle, separated by desired treatment interruptions.

[0039] Treatment parameters can be determined, at least in part, based on patient data acquired in box 104. For example, the patient's inflammatory status can guide treatment parameters. For instance, if the patient exhibits a low inflammatory response, the ultrasound energy used can be minimized. Conversely, if the patient is experiencing a higher-than-average inflammatory state, the amount of ultrasound therapy delivered can be increased by extending the amount of device use or increasing the frequency or load cycles of treatment. Furthermore, stimulation parameters can be adjusted to drive a greater inflammatory response, or even reduce anti-inflammatory effects, if the response falls below a certain threshold, based on the inflammatory response measured by various biomarkers or clinical symptoms (which will differ for each individual). Ultrasound stimulation parameters can also be determined in part by imaging data optionally acquired in box 104. For example, imaging data can determine the available time to process blood at a specific target location, which informs the duration of stimulation and influences the intensity parameters required to achieve the desired response within the limited available time. Ultrasound parameters can also be adjusted based on the patient's body metrics. For example, if there is a significant layer of adipose tissue between the target and the ultrasound transducer that could weaken and diminish treatment, the ultrasound intensity can be increased.

[0040] Some non-limiting exemplary stimulation parameters include stimulation frequencies of 50 kHz to 7 MHz, such as 500 kHz to 1.5 MHz, and pressures of 100 kPa to 1 MPa or 25 kPa to 10 MPa, depending on the stimulation frequency. The stimulation duration can occur within sessions of 1 minute to 1 hour, or more specifically, 5 minutes to 20 minutes; and the pulse width can be 1 μs to 5 ms, or more specifically, 100 μs to 500 μs. The pulse repetition frequency can be 100 Hz to 10 kHz, or more specifically, 1 or 2 kHz. The number of repetitions can be predetermined between a single repetition of these sessions and hundreds of repetitions, or more specifically, one or two sessions per day. Alternatively, these parameters can be adjusted based on an evaluation of the immune response, as shown in boxes 112 and 114.

[0041] The treatment parameters set in box 108 can also indicate whether ultrasound stimulation is required. For example, if the patient's immune response is within the expected range, as shown in box 104, the ultrasound stimulation parameters can indicate that ultrasound stimulation should not be applied to the patient. The immune response can be further re-evaluated in the procedure (e.g., boxes 112 and 114) or by repeating procedure 100.

[0042] Box 110 may include the application of ultrasound stimulation, if indicated in box 108. The target location of the ultrasound stimulation in box 110 and the stimulation parameters used may be set in boxes 106 and 108, as previously described. Such ultrasound stimulation may be applied directly to blood cells or other immune cells without explicitly requiring stimulation of the nervous system. Therefore, ultrasound stimulation may have a direct effect on immune cells, evoking an anti-inflammatory response. For example, ultrasound stimulation may be applied to peripheral blood mononuclear cells (PBMCs), which may include lymphocytes (e.g., T cells, B cells, NK cells), monocytes, and dendritic cells.

[0043] In some configurations, ultrasound stimulation applied in box 110 can target blood cells or immune cells without stimulating the nervous system, such as nerves, axons, terminals, and other neural tissues. Therefore, stimulation can be directed to blood cells instead of the target's neural tissue. In this way, anti-inflammatory responses can be directly targeted by bypassing possible intermediate steps in regulating complex neuroimmune signaling pathways. Direct targeting of immune cells reduces the required stimulation frequency, repetition, intensity, etc., improving treatment safety and reducing costs and burdens on healthcare systems and patients. Direct targeting of immune cells also expands the target sites in vivo that are susceptible to ultrasound-mediated immunomodulation and eliminates the need to target small and difficult-to-dissect neural structures. Furthermore, the target patient population can be expanded to include individuals lacking end organs and / or relevant target nerves. By directly targeting immune cells, rather than altering nerve and organ function through ultrasound stimulation and avoiding such alterations, the risk of unintended side effects from activation of downstream neural circuits is reduced.

[0044] In some configurations, applying ultrasound stimulation in box 110 may include directing the stimulation to two or more locations. For example, ultrasound stimulation may be applied to blood cells at different locations as blood flows through a blood vessel. Several methods can be used to apply ultrasound at multiple points along the flow path. For example, an ultrasound array can dynamically guide ultrasound waves along the blood path. As a complement or alternative, several ultrasound devices may be used, each positioned at various points along the blood flow path. Each device may be triggered based on blood flow velocity. In some configurations, the ultrasound stimulation device may be dynamically moved along the path of the blood vessel at a speed similar to or equal to the blood flow velocity. In any case, the target location and triggering timing may be determined based on blood flow velocity. For example, the blood vessel path and flow velocity may be determined in box 104. Blood tracers may also be used to verify the location and flow velocity of target blood cells, which may be imaged simultaneously or nearly simultaneously with ultrasound stimulation. Ultrasound may also be applied to multiple blood pools throughout the body, such as different organs or vascular clusters.

[0045] In box 110, applying ultrasound stimulation to blood cells can elicit an anti-inflammatory immune response. For example, immune cells may reduce cytokine production or decrease signal transduction, thereby reducing cytokines in the blood. Therefore, the immune response can be measured or monitored in box 112. For example, inflammatory markers in treated or nearby blood can be monitored to indicate an anti-inflammatory effect. This effect can be measured using standard or other known clinical or experimental methods for measuring inflammatory markers or biomarkers. For example, the presence of multiple cytokines can be measured. As a non-limiting example, cytokines may include interleukin-1β (IL-1β), interferon-γ (IFN-γ), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10), tumor necrosis factor-α (TNF-α), and nuclear factor κ-light chain enhancer (NF-κB) for activated B cells, etc. For example, cytokines can be measured by a cytokine sensor implanted in a patient and / or by blood draws from which direct protein or RNA transcript quantification is performed. In one non-limiting example, reverse transcription quantitative real-time PCR (RT-qPCR) can be used to measure cytokines in the blood. Other techniques, such as ELISA, can be used to measure cytokine levels. Other biomarkers may include erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin / d-dimer, etc. Other biomarkers may be determined based on gene expression data or other quantitative methods from samples collected from the user. Such biomarkers can be used alone or in combination with other biomarkers or other patient data (e.g., age, health status, metabolomics, etc.). The ratio of multiple biomarkers or the change of biomarkers over time can also be used as a measure of inflammatory status.

[0046] After measuring the immune response in box 112, the measured inflammatory status can be assessed in box 114. For example, thresholds can be set for biomarkers or combinations of biomarkers of the target inflammation (e.g., IL-1β, IFN-γ, MCP-1, IL-8, TNF-α, NF-κB, ESR, CRP, IL-6, IL-10, MAPK14, etc.). Other thresholds can be set for other clinically relevant inflammatory biomarkers (e.g., erythrocyte sedimentation rate (ESR) and ferritin / d-dimer). An adequate immune response to ultrasound stimulation can be considered if the measured biomarker cytokine levels are modulated to a clinically significant target range. Alternatively or complementary to this approach, the immune response can be evaluated based on changes in inflammatory biomarkers between those measured in box 112 and box 104. The procedure can be terminated if a reduction in inflammatory biomarkers is considered sufficient based on the desired threshold or change. However, if the anti-inflammatory response is insufficient, the procedure can be repeated. The ultrasound stimulation parameters can be optionally adjusted in box 116 based on the measured immune response. For example, the intensity, repetition rate, or load cycle rate of ultrasound stimulation can be increased. Target location can also be adjusted based on the measured inflammatory status. A delay time can also be set in box 116, where repeated treatment is paused to provide treatment interruption and maintain a safe limit of stimulation, or to accommodate target movement. Ultrasound stimulation can be repeatedly applied in box 110 with the desired load cycles. This process can continue repeatedly until the desired response is obtained in box 114.

[0047] Procedure 100 can be repeated at a desired frequency, including all or some of the boxes in Procedure 100. For example, Procedure 100 can be repeated once daily to assess the patient's immune status. If immune modulation is deemed necessary in box 108, the procedure can continue with ultrasound stimulation in box 110. However, if patient data obtained in box 104 indicates that the immune response is adequately suppressed, the procedure can continue without modulation (i.e., by setting the ultrasound stimulation parameters to no ultrasound stimulation). In this way, the patient's biomarkers can be monitored periodically, and ultrasound stimulation can be applied as needed based on biomarker measurements.

[0048] In some configurations, it may be desirable to apply ultrasound stimulation relative to an object in an ex vivo environment, such as in an in vitro setting or in an in vivo animal model. Figures 1B to 1C Non-limiting exemplary procedures 120 and 130 are shown that can be used to apply ultrasound stimulation to immune cells in an in vitro environment. For example, Figure 1B A general process 120 is provided for processing biological fluids for a variety of applications. Figure 1C A more detailed exemplary process 130 is provided for removing a biological fluid or blood from an object, processing the biological fluid outside the body, and returning the biological fluid of the object to the object's body.

[0049] refer to Figure 1B An exemplary process 120 for processing a biological fluid via ultrasound stimulation is provided. Process 120 can be used for a variety of clinical or research applications, some of which will be described. The biological fluid can be obtained in box 122. For example, the biological fluid may include blood, plasma, serum, lymph, bone marrow, intestinal fluid, urine, etc. As a non-limiting example, obtaining a biological fluid may include removing it from a subject or donor, obtaining it from an animal (e.g., a non-human primate, a mouse), obtaining an artificially prepared biological fluid, obtaining blood from a blood bank, or otherwise obtaining the desired biological fluid. Box 122 may also include processing or preparing the biological fluid. For example, the biological fluid may be separated (e.g., by centrifugation) into individual components such as plasma, red blood cells, platelets, cryoprecipitate, etc. For example, blood cells may be separated from blood and suspended in cell cultures, agar, or otherwise stored for processing. The biological fluid may be cleaned, tested, or otherwise processed (e.g., to reduce white blood cells, stain, cool, etc.) to prepare it for ultrasound processing, storage, or transplantation. In some configurations, the preparation of biofluids may include transplanting blood into animal models for ultrasound treatment or experimental purposes.

[0050] Once prepared, the target biological fluid or cells can be treated with ultrasound stimulation in box 124. For example, ultrasound stimulation can be applied in an in vitro environment (e.g., a petri dish). As another non-limiting example, ultrasound stimulation can be applied in an in vivo animal model. Treating the biological fluid or cells in box 124 may include process 100 (… Figure 1A The process involves all or some steps of the treatment, such as applying the treatment to a suitable environment for a desired application. Once treated, the biofluid or cells in box 126 are available for the desired application. For example, box 126 may include replacing the treated biofluid back into a donor. As another example, box 126 may include transplanting the treated blood into another recipient, such as a patient recovering from trauma or suffering from an infection. As another non-limiting example, box 126 may include using the treated blood for research (e.g., testing for inflammatory markers over time, transplanting blood into diseased animal models, etc.). Box 126 may also include replacing or transplanting the treated biofluid into a donor or recipient animal. Box 126 may also include storing the treated biofluid for future use. In this way, anti-inflammatory blood or other biofluids can be prepared and stored for use in clinical or hospital settings, such as for trauma patients in an emergency room.

[0051] refer to Figure 1CAnother non-limiting exemplary process 130 is provided for processing biological fluids in an ex vivo setting. In box 132, the biological fluid can be removed from an object. For example, the biological fluid may include blood, plasma, serum, lymph, bone marrow, intestinal fluid, urine, etc. The biological fluid can be removed, for example, by IV, blood draw, surgery, bone marrow aspiration, or other known methods. In box 124, the fluid can be processed by an ex vivo ultrasound stimulation process that induces an anti-inflammatory response. For example, box 124 may include process 100 applied in an ex vivo setting (…). Figure 1A The steps described in box 136 are as follows: After the cells have been modulated with ultrasound energy in an ex vivo environment, the biofluid can be returned to the object body in box 136. Alternatively, the biofluid can be transferred to another object in box 136.

[0052] Figure 2 A block diagram of an ultrasound stimulation system 200 is shown, providing a non-limiting example of a system that can be used to apply ultrasound stimulation to blood cells or immune cells and receive external signals. As a non-limiting example, system 200 can perform process 100. The system includes a controller 202, which typically includes an ultrasound stimulation mode module 210, an ultrasound target module 212, a control module 216, a processor 220, a power supply 230, a memory 232, a clock module 234, and a communication module 236. The system may also include an ultrasound device 246 capable of applying ultrasound stimulation with a specified waveform to a target. The ultrasound device 246 may also be capable of performing ultrasound imaging. The ultrasound device 246 will be described in further detail below.

[0053] System 200 may also include an external device 242. For example, external device 242 may include a server capable of storing patient data, system commands, imaging data, etc. External device 242 may also include a user interface capable of displaying system parameters or receiving user input or operational commands. Specifically, input may include various user interface elements, such as a mouse, keyboard, touchpad, touchscreen, buttons, etc. Input may also include various drivers and receivers, such as flash drives, USB drives, CD / DVD drives, and other computer-readable media receivers, for receiving various data and information. For this purpose, input may also include various communication ports and modules, such as Ethernet, Bluetooth, WiFi, etc., for exchanging data and information with various external computers, systems, devices, machines, mainframes, servers, or networks. External device 242 may also include various other sensors, such as external imaging devices, cytokine sensors, positioning sensors, etc. Ultrasound device 246 and external device 242 may be connected to controller 202 via appropriate connections (e.g., data cables, Wi-Fi, Bluetooth, etc.).

[0054] The external device 242 may also be controlled by a processor 220, which can determine instructions and provide them to the external device 242 via a control module 216. For example, the external device 242 may include a cytokine sensor, and the control module 216 may trigger cytokine measurements at different times throughout the stimulation process.

[0055] The controller 202 may also include various connectivity, terminal, or wireless communication connections for transmitting signals generated by the ultrasound stimulation mode module 210, the ultrasound target module 212, or the control module 216, or signals measured by the external device 242. Any or all components may be housed in one or more wearable devices, in one or more portable devices carried externally to the body, or on an external server, and have suitable wired or wireless connectivity between the components.

[0056] Processor 220 may be configured or programmed to perform various functions for operating controller 202 using instructions stored in memory 232 in the form of non-transitory computer-readable media or by inputting received instructions. In some embodiments, processor 220 may control the sending and receiving of instructions and operating parameters (e.g., via a wireless transcutaneous link in communication module 236), the storage of operating or stimulation parameters and instructions in memory 232, the transmission of operating parameters and selective triggering to ultrasound device 246, and the synchronization of various functions using clock module 234. For example, processor 220 may communicate with clock module 234 to determine the timing and synchronization of different stimuli. Processor 220 may also communicate with clock module 234, ultrasound stimulation mode module 210, and other hardware and digital logic circuitry to accurately store activation times and parameters in memory 232. For example, processor 220 may be a programmable microprocessor or microcomputer.

[0057] The ultrasound stimulation mode module 210 can determine ultrasound stimulation parameters and provide instructions to the ultrasound device 246. For example, the ultrasound stimulation mode module 210 can provide waveform parameters to the ultrasound device, such as ultrasound intensity, ultrasound frequency, pulse width, pulse timing, load cycles, etc. The ultrasound stimulation mode module 210 can determine such ultrasound stimulation parameters based on input data from the external device 242. For example, ultrasound parameters can be determined from measured cytokine levels or other patient data.

[0058] The ultrasound target module 212 can determine the target location for ultrasound stimulation. For example, the ultrasound target module 212 can use data (such as imaging data or a user interface) provided by the external device 242 to determine the target location for ultrasound stimulation. For example, the ultrasound target module 212 can automatically identify the target location based on imaging data. For example, the ultrasound target module 212 can automatically analyze imaging data to identify areas of high vascular density or stagnant blood pools. Alternatively, the user can input the target location using a user interface. The ultrasound target module 212 can also locate the ultrasound device based on imaging data acquired solely by the ultrasound device or using other imaging data provided by an external imaging device or server.

[0059] The ultrasound target module 212 can also provide positioning instructions to the ultrasound device 246 or the user. For example, the ultrasound target module 212 can determine the settings for the phased array, thereby stimulating the target position with high positioning accuracy. For example, a phased array can achieve a spatial accuracy of 1 mm × 1 mm × 1 mm. The ultrasound target module 212 can also provide instructions to the device user via an external device 242 (e.g., a user interface or display). Such instructions can guide the user or patient on how to position the device on the patient to target the desired location.

[0060] In some embodiments, controller 202, together with ultrasound device 246, may be part of a standalone stimulation system. Alternatively, controller 202 may be a portable, wearable, or implantable unit that can be programmed or configured using an external device, computer, or system. For this purpose, communication module 236 may be configured to transmit and receive various signals, as well as receive power. Specifically, communication module 236 may include an antenna, or input-output coils, receivers and transmitters, data converters, and other hardware components. As a non-limiting example, receivers and transmitters may be configured to receive and transmit radio-frequency (RF) signals. In some embodiments, antenna may be configured for transcutaneous wireless bidirectional communication with an external wearable device, transmitting and receiving signals when the external wearable device is placed in close proximity. Communication signals may be transmitted via magnetic induction and include information for operating and / or programming processor 220. For example, communication signals may include trigger or command signals for generating stimulation or for transmitting patient data. In some configurations, the transmitted signals may also be configured to power or charge a battery assembly that provides power to controller 202. The antenna can be connected to a receiver and a transmitter, which in turn can be connected to serial-to-parallel and parallel-to-serial data converters, respectively. Then, as described, any information transmitted or received can be processed by processor 220.

[0061] The controller 202 may be powered by an internal and / or external power source 230. For example, the internal source may include a standard rechargeable battery, similar to those used in implantable devices such as pacemakers. As a complement or alternative, the internal power source may include a capacitor (e.g., a single-ended primary inductor converter or a DC-DC converter) combined with a regulator, which together can generate a constant current or voltage output for short periods. In some embodiments, the capacitor may be charged by an external wearable device. Therefore, the controller 202 may include an induction coil or a thin, tightly wound coil that allows for RF telemetry and / or battery recharging via an external wearable or portable device configured as part of the communication module 236 or as separate hardware. Other charging methods may also be used.

[0062] Figure 3 An exemplary ultrasound system is shown that can be used to apply ultrasound modulation to blood cells according to this disclosure. As a non-limiting example, ultrasound system 300 may be part of stimulation system 200, such as... Figure 2 The ultrasonic device 246 is included. The ultrasonic system 300 includes a transducer. The transducer may be a transducer array 302 comprising a plurality of independently driven transducer elements 304. The transducer array 302 may include any suitable ultrasonic transducer array, including linear arrays, curved arrays, phased arrays, etc. Similarly, the transducer array 302 may include 1D transducers, 1.5D transducers, 1.75D transducers, 2D transducers, 3D transducers, etc.

[0063] When excited by transmitter 306, a given transducer element 304 generates an ultrasonic energy pulse. The ultrasonic energy (e.g., echo) reflected back to transducer array 302 from the object under study is converted into an electrical signal (e.g., echo signal) by each transducer element 304 and can be individually applied to receiver 308 via a set of switches 310. Transmitter 306, receiver 308, and switches 310 operate under the control of controller 312, which may include one or more processors. As an example, controller 312 may include a computer system.

[0064] Transmitter 306 can be programmed to emit unfocused or focused ultrasonic waves. In some configurations, transmitter 306 can also be programmed to emit diverged waves, spherical waves, cylindrical waves, plane waves, or combinations thereof. Furthermore, transmitter 306 can be programmed to emit spatially or temporally encoded pulses.

[0065] Receiver 308 can be programmed to implement a suitable detection sequence for an existing imaging task. In some embodiments, the detection sequence may include one or more of progressive scan, compound plane wave imaging, synthetic aperture imaging, and compounding diverging beam imaging.

[0066] In some configurations, transmitter 306 and receiver 308 can be programmed to implement a high frame rate. For example, a frame rate associated with a pulse repetition frequency (“PRF”) of at least 100 Hz can be implemented. In some configurations, ultrasound system 300 can sample and store a set of at least one hundred echo signals in the time direction.

[0067] The controller 312 can be programmed to implement imaging or stimulation sequences using the techniques described in this disclosure or other methods known in the art. In some embodiments, the controller 312 receives user input defining multiple factors used in the design of the imaging or stimulation sequence.

[0068] Scanning or stimulation can be performed by setting switch 310 to its transmission position, thereby guiding transmitter 306 to momentarily open during a single transmission event to excite transducer element 304 according to a designed imaging or stimulation sequence. Switch 310 can then be set to its receiving position, and subsequent echo signals generated by transducer element 304 in response to one or more detected echoes can be measured and applied to receiver 308, for example, during an imaging sequence. Individual echo signals from transducer element 304 can be combined in receiver 308 to generate a single echo signal.

[0069] In some configurations, the ultrasound system 300 may include more than one ultrasound array 302. At least one of the arrays may be used to emit ultrasound for therapeutic treatment, and at least one other array may be used to emit and / or receive ultrasound for imaging. In a non-limiting example, the frequency used for treatment may be lower than the frequency used to produce high-quality images. In this way, the ultrasound system 300 can provide an integrated imaging and stimulation system, such that imaging can provide information for the localization of the stimulation target. Additional ultrasound arrays 302 may be used to provide stimulation at multiple targets within the object.

[0070] The echo signal is transmitted to a processing unit (not shown), which can be implemented by a hardware processor and memory, to process the echo signal or an image generated from the echo signal. As a non-limiting example, the processing unit may use the methods described in this disclosure to target blood cells for ultrasound stimulation to treat inflammation. The image generated from the echo signal by the processing unit can be displayed on display system 314.

[0071] In one non-limiting example, a wearable / miniaturized phased array ultrasound device can be used with ultrasound system 300, but other types of ultrasound stimulation devices (e.g., conventional / cart-based ultrasound systems, portable systems, etc.) can also be used. The wearable device can be placed on the subject and worn for extended periods. In some configurations, a reprogrammed smartphone or tablet device can be used to control and / or monitor the stimulation. In one non-limiting example, the reprogrammed smartphone or tablet device can allow for Bluetooth wireless control of the stimulation device. For example, the smartphone or tablet device can provide a user interface for inputting patient data, desired stimulation parameters, or target location. In some configurations, the control and / or monitoring system can be an application mounted on a smartphone, tablet, etc. In another non-limiting example, multiple devices distributed around the subject can be used to provide ultrasound stimulation. When using miniaturized ultrasound array technology, the overall size of the wearable device can be similar to that of other consumer electronics wearable devices, such as smartwatches. In one non-limiting example, the total footprint of the device can be less than 1.2 cm × 1.2 cm. In another non-limiting example, the device may occupy less than 40 mm × 40 mm. In some non-limiting examples, the total space occupied by the device may be larger, for example, to cover a larger area of ​​the spleen, liver, kidney, intestine, target blood vessels, lymphatic tissue, or other organs. In one non-limiting example, the space occupied by the device is the same as the size of the spleen (which may be 8 to 16 cm) or another target organ.

[0072] In some configurations, the ultrasound transducer can be movable. For example, the ultrasound transducer can be moved manually or automatically by a motorized system. In this way, ultrasound stimulation can track blood flow.

[0073] In some configurations, the ultrasonic transducer elements 304 can be activated simultaneously to form a planar wavefront. In other configurations, the ultrasonic transducer elements 304 can be activated sequentially to form an angular or focused wavefront.

[0074] In some configurations, beam steering can be used to target selected locations, such as blood pools or areas of high vascular density, using ultrasound stimulation. Locations can be selected to optimize therapeutic effects, for example, by targeting immune cells.

[0075] In some configurations, wearable power delivery devices will have the ability to image a designated target and deliver ultrasound energy to it. Imaging and / or beam steering can also be used to compensate for blood movement (e.g., blood flow) or target movement, such as changes in depth due to object motion. Target organs or blood vessels can move naturally. For example, the spleen moves within the abdominal cavity during heavy breathing and changes in body position. Using imaging feedback, the ultrasound beam focus can be re-aligned as the distance to the target surface changes relative to the transducer array. This offers the potential for closed-loop devices to operate with automated reorientation algorithms. The ultrasound beam focus can also track blood flow guided by imaging data.

[0076] To complete the beam-direction scanning, the transmitter 306 imparts a time delay T to the corresponding pulse 316 applied to the continuous transducer element 304. i If the time delay is zero (T... i If the time delay increment T is 0, all transducer elements 304 will be excited simultaneously, and the resulting ultrasonic beam will be guided along the axis 318 perpendicular to the face of transducer 302 and originating from the center of transducer array 302. i In addition, the ultrasonic beam is guided away from the central axis 318 at an angle θ. The time delay increment T is sequentially added from one end i = 1 to the other end i = n of the transducer array 302 to each i-th signal. i The relationship between them is given by the following relation:

[0077] ;

[0078] Where S is the equal spacing between the centers of adjacent transducer elements 304; c is the speed of sound in the object under study; R is the range or depth at which the transmitted beam will be focused; and T0 is the delay offset, which ensures that all calculated time delay increments Tᵢ are positive.

[0079] The second term in equation (1) tunes the beam to the desired angle θ, and the third term is used when the transmitted beam is focused within a fixed range R. This is achieved by gradually changing the time delay T during continuous excitation. i A sector scan is performed. In this way, the angle θ changes incrementally to adjust the transmitted beam in a continuous direction. When the beam direction is above the central axis 318, the pulse timing is reversed; however, equation (1) still applies in this case.

[0080] The echo signals generated by each ultrasonic energy pulse are emitted from a reflecting object located at a continuous range or depth R along the ultrasonic beam. These are sensed individually by each transducer element 304 in the transducer array 302, and a sample of the amplitude of each echo signal at a specific time point represents the amount of reflection occurring at the specific range R. However, due to the difference in the propagation path between the focus P and each transducer element 304, these echo signals do not occur simultaneously, and their amplitudes are not equal. The function of the receiver 308 is to amplify and demodulate these individual echo signals, assign an appropriate time delay to each echo signal, and sum them together to provide a single echo signal that accurately indicates the total ultrasonic energy reflected from each focus P located at a continuous range R along the ultrasonic beam oriented at angle θ.

[0081] Guided by the digital controller 312, the receiver 308 provides a delay during scanning, enabling it to be tuned to track the beam direction tuned by the transmitter 306, and to sample the echo signal over a continuous range R, providing an appropriate delay to dynamically focus along the beam at point P. Thus, each transmission of an ultrasonic pulse results in the acquisition of a series of data points representing the amount of sound reflected from a corresponding series of points P located along the ultrasonic beam.

[0082] In some configurations, physical devices (such as cones) may be used to direct the ultrasound beam toward the target location. Those skilled in the art will understand that other options for directing ultrasound to the target location may be used in conjunction with this disclosure.

[0083] refer to Figure 4A The diagram illustrates a block diagram of a non-limiting exemplary device for providing stimulation. Other ultrasound or stimulation devices may also be used. A treatment module 400 may be included and is capable of providing ultrasound stimulation to a subject. A control module 410 may be used to control the treatment module 400. The control module 410 may include stored instructions for controlling the treatment module 400. A biomarker feedback sensor 440 may provide feedback data, such as measurements of cytokine levels, and may contact the subject, for example, through the skin layer, through contact with blood via an IV or implanted sensor, or through additional contact with the subject. The biomarker feedback sensor 440 may wirelessly communicate 460 with the control module 410. Power for the system may be provided by a power supply / adapter 420. The power supply / adapter 420 may be an AC power adapter that plugs into a wall socket for standard power input. The treatment module 400 may be connected to the control module 410 using a cable 430. Alternatively, the treatment module 400 may wirelessly communicate with the control module 410.

[0084] The control module can be used for device operation and power conversion of the transmitting transducer. In a non-limiting example, the control module 410 can generate + / - 90 V and 20 V DC outputs. These DC outputs are provided to the treatment module 400 to power the beamforming IC, MCU, and other secondary components. Based on pre-programmed parameters, the beamforming IC can generate a tone-burst pulse with a maximum peak-to-peak voltage of approximately 180 V (+ / - 90 V). Using these tone-burst pulses, the phased array transducer can activate piezoelectric elements (e.g., 128 piezoelectric elements) in a predefined manner and generate ultrasonic pressure waves. For each device, the ultrasonic pressure output can be measured and adjusted to ensure an appropriate intensity below a safety threshold.

[0085] refer to Figures 4B to 4D A non-limiting exemplary treatment module 400 is shown in more detail below. (See reference) Figure 4B The treatment module 400 may include a housing 470 and a transmitter array 472. (Reference) Figure 4C The base 474 may include a cut-out portion 476 for accommodating the transmission array 472. (See reference) Figure 4D A cross-sectional view of a treatment module 400 assembled with a housing 470 and a base 474 is shown. In some configurations, the treatment module 400 may be a small, lightweight, wearable component. In a non-limiting example, the treatment module 400 houses a beamforming integrated circuit (IC), a phased array ultrasonic transducer, and other auxiliary circuitry (Bluetooth, memory, etc.).

[0086] The treatment module 400 can be attached to the body of an object using a disposable adhesive coupling pad. In a non-limiting exemplary arrangement, the treatment module 400 can be placed on the torso or head of the object, or otherwise close to the target blood cells.

[0087] refer to Figure 5 The diagram illustrates a non-limiting exemplary ultrasound system configuration 500. A power generator 510 (which may be a DC power supply in some configurations) provides power to a processor and a beamforming integrated circuit 520, which controls the stimulation delivered by a phased array transducer 530. A clock generator 540 provides a signal for triggering the beamforming integrated circuit 520. A wireless communication module 550 may be coupled to the beamforming integrated circuit 520 and provides diagnostic access, data input / output, user interface access, etc.

[0088] The system interface allows authorized users (e.g., designated technicians, professionals, clinicians, or nurses) to switch selected features for management and control via a customized tablet app. During treatment procedures, device use can be limited to on / off operations with an automatic shut-off feature, eliminating the need for a separate tablet unit. Internally, phasing algorithms are provided for individual channel control, allowing for the focusing and orientation of the ultrasound beam. This allows the ultrasound beam to be optimized for high intensity and pressure at the target site of blood cells. Additional parameters, including pulse duration and repetition frequency, can be optimized to achieve the desired therapeutic effect.

[0089] In one configuration, the system can be configured with 128 or 256 channels of beamforming capability for high-resolution directional and focusing. The peak frequency can be from 400 to 600 kHz, and the channel spacing is 1.5 mm, equivalent to half the length (λ / 2) of ultrasound waves in water at frequencies below 500 kHz. This optimizes the directional and focusing capabilities of the ultrasound beam by avoiding energy loss due to sidelobe generation. The peak frequency can also be from 600 kHz to 1 MHz or from 1 MHz to 2 MHz. An ultra-compact design is possible, where the wearable system is smaller than a standard business card.

[0090] In one configuration, the transducers used for the system may include 128 transducer elements. Each element can be electrically and physically isolated, allowing for individual phasing and improving efficiency by virtually eliminating all acoustic crosstalk between channels. The array of 128 elements can be divided into four identical sections, each with 32 elements. Each of the 32 elements in a given section can be controlled in series with three equivalent elements from the other sections. This allows the system to operate efficiently in a manner equivalent to a 128-channel system for center beam focusing for blood cell stimulation.

[0091] In one configuration, the transducer may include 32 channels connected to 32 elements for full orientation and phasing along both the X and Y axes. The transducer system can generate peak negative sound pressure levels exceeding 1 MPa and deliver energy to multiple depths depending on the target location. For example, the target depth can range from 1 mm to 30 cm. In some configurations, transducers with up to 512 elements and channels are available for higher pressures and a wider acoustic range.

[0092] For blood cell targeting, the system has the ability to directional and focus the beam as needed to achieve the desired therapeutic effect. In one configuration, the beam can be effectively directional to the desired location by phasing 64 channels of the system and transducer. For wearable devices, a large number of programs can be stored in internal memory for very rapid rasterizing between directional and focused states. Phaging conditions can be pre-programmed based on achievable targets of the ultrasound device and the desired targets for treatment. For example, in cases of blood stimulation in the spleen, kidneys, liver, heart, or intestines, the device can be configured to target a wide range of people with different organ or tissue depths and other bodily disturbances, such as the ribs. For some treatments, this can include hundreds or thousands of phasing algorithms that can be used very rapidly in raster pattern (e.g., switching), potentially performing hundreds of programs in one second or a few seconds.

[0093] Phased array transducers can be designed to produce custom ultrasonic beam spectra with optimal energy or pressure spectra (i.e., peak pressure, depth, beam area, etc.).

[0094] The electrical system may include a multi-channel beamforming microprocessor chip for controlling 32 individual channels for phasing and focusing. The wearable device can be wired to a power adapter that accommodates standard power inputs (i.e., 100 to 240 V, 50 / 60 Hz).

[0095] In some configurations, the system incorporates a small wearable form factor, low cost, and adaptability for scalable fabrication. Existing instruments for focused ultrasound medical applications are significantly more expensive; standard multichannel systems typically cost $50,000 to $200,000 per unit and have large vehicle-mounted or benchtop-based form factors. Conventional wearable ultrasound energy emitters are designed for other purposes, such as wound healing, and operate at inappropriate ultrasound frequencies, or have only one or a few channels operating, which cannot perform sufficient beamforming for focusing. Handheld medical ultrasound devices are designed for imaging purposes and are also insufficient to meet the energy delivery requirements for stimulating specific targets or blood cells in the body, due to linear array designs that can only perform beamforming in one axis or tend to operate at high frequencies (e.g., above 3 to 10 MHz).

[0096] In some configurations, a hybrid system and transducer can be provided for ultrasound imaging feedback. When targeting blood, immune cells, organs, and obstacles, beam steering mechanisms can be used in the process to avoid artifacts in the resulting images.

[0097] The systems and methods according to this disclosure can be configured to use feedback provided by biomarker sensors, some of which provide immediate or recent information about cytokine levels that may be correlated with treatments delivered by the system.

[0098] One non-limiting exemplary biomarker sensor includes a portable biosensor system capable of detecting cytokines (e.g., IL-1β, IFN-γ, MCP-1, IL-8, TNF-α, NF-κB, IL-6, IL-10, MAPK14, MCP-1) in untreated whole blood using a filter paper-based immunosensor and smartphone imaging. This biosensor can detect minute changes in cytokine levels within 20 minutes. The paper-based immunosensor generates a colorimetric signal, and the pixel intensity of the colorimetric signal can be evaluated using real-time density determination implemented by a user-guided smartphone app. The biosensor can also be an electrochemical aptamer-based sensing platform that measures the concentration of specific molecules directly in blood and even in vivo, or it can be a label-free electrochemical impedance spectroscopy platform with multiple types of electrodes. The ultrasound stimulation system can be controlled via Bluetooth using a smartphone app, which can also receive information from the cytokine biosensor app installed on the same smartphone device; in some embodiments, the biosensor app can be integrated into the ultrasound stimulation system app. By incorporating data from cytokine monitoring over the past few hours or days, and based on predetermined algorithms and thresholds, the level of ultrasound stimulation can be modulated in response to increases or decreases in detected cytokines.

[0099] One non-limiting exemplary biomarker sensor includes a miniaturized sensor that uses a fluid (such as saliva or sweat) to measure cytokines or other biomarkers associated with inflammation levels. These devices can utilize a variety of technologies, such as light waves (i.e., ultraviolet or infrared).

[0100] Communication with the system can be provided in real-time or with delay using communication protocols such as Bluetooth, RFID, near-field communication, or WiFi. In some configurations, the biomarker sensor can be physically separated from the rest of the system while still communicating wirelessly. Direct integration of the biomarker sensor into the system is also available. In this configuration, the biomarker sensor is directly integrated into the device.

[0101] It can also provide communication with the system via an intermediate system (such as a networked server). The biomarker sensor can be internet-enabled and provide data to a management server, which then pushes the data back to the system via the internet, and the system can also be networked accordingly. The data can be processed by the intermediate server or transmitted directly to the system.

[0102] Example

[0103] EDTA tubes containing whole blood were collected from healthy volunteers from commercial blood banks. The blood was mixed 1:1 with phosphate-buffered solution (PBS) containing 2% fetal bovine serum (FBS). Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation, which was performed by floating the blood + PBS mixture on top of a lymphoprep layer in a 50 mL conical tube, followed by centrifugation. The PBMCs were removed from the resulting layered mixture, washed in another PBS + 2% FBS solution, and then centrifuged to form a pellet. The pellet was separated and resuspended in RPMI-1640 medium. At this point, the cell suspension was divided into three conditions: a control without sonication (US) or lipopolysaccharide (LPS) treatment, an LPS control, and experimental LPS treated with sonication (US) (LPS + US). The LPS + US condition was activated using different levels of sonication, as described below, in 3D-printed tubes placed in a water bath.

[0104] The 3D-printed tubes were designed to be compatible with our ultrasonic transducer focusing cone, reducing temperature variations and minimizing ultrasonic reflections at the tube interface. After sonication, all cells were positioned at approximately 0.5 × 10⁻⁶. 6 Cells / mL were diluted and placed in RPMI-1640 cells / mL in 12-well plates. LPS was added to appropriate wells at different concentrations, and the plates were incubated at 37°C and 5% CO2 for 4 hours. Next, the cell suspension was transferred to tubes and pelleted. The pellet was lysed with buffer from the Qiagen RNeasy Mini Kit, and RNA was extracted using the kit. RNA concentration was quantified using a qubit fluorescence assay. The RNA was then reverse transcribed into cDNA at an appropriate concentration and tested using real-time PCR (RT-qPCR).

[0105] The provided qPCR data were replicated using the technique. Primers were purchased from Integrated DNA Technologies. The primers were mixed with the Sybr Green master mix. The RPLP0 gene was used as the housekeeping gene. The cycle threshold (C) of each target gene was measured. t This indicates the number of amplification cycles required for detection. Therefore, a higher C... t The value indicates a lower number of copies of the gene transcript present. (The C value is used to represent this value.) t The value is averaged across technical replicates. This is achieved by averaging the C values ​​from the target gene. t Subtract the average C of the housekeeper t To calculate ΔC tThis corrects for the C value relative to the gene (RPLP0) that is not altered by experimental intervention. t Value. Next, from the LPS condition and the LPS + US condition, ΔC t Subtract ΔC from the control condition (no LPS and US) t This provides ΔΔC under two conditions. t Value. The relative fold change in gene expression under two conditions can be defined as: This fold change represents the percentage change from the control condition to the target condition. For example, a fold change of 2 for the LPS condition means that the LPS sample produced 200% more RNA transcripts of the target gene than the control. Therefore, if ultrasound therapy is intended to reduce inflammation, the fold change in inflammatory cytokines under the LPS + US condition should be lower than that under the LPS condition. The fold changes of LPS + US compared to the control under the three experimental conditions are plotted below. Figure 6A , 7A And 8A.

[0106] To visualize the data in another way, we also calculated the multiple change of the LPS + US condition compared to the LPS condition, which means the change from ΔC under the LPS + US condition. t Subtracting ΔC from the LPS condition t Then take This fold change shows the change in expression from LPS to LPS+US. In this case, a fold change of less than 1 indicates that the LPS+US condition produces less transcript than the LPS condition. As described below, the fold changes of the LPS+US condition compared to the LPS condition for the three experimental conditions are plotted on [the graph / plot]. Figure 6B , 7B And 8B.

[0107] To further visualize the data, we plotted the percentage change from LPS to LPS + US. This percentage change was calculated by taking (1 - multiple of change). The reciprocal of 100% is calculated as a percentage of production. For example, a -50% result means that the amount of RNA transcripts produced under LPS + US conditions is 50% less than that produced under LPS conditions. The percentage change from LPS to LPS + US under the three experimental conditions is plotted below. Figure 6C , 7C And in 8C.

[0108] Example 1

[0109] For the US experimental conditions, stimulation was applied for 10 minutes using a 1 MHz transducer at a negative peak pressure of approximately 550 kPa. The pulse width was set to 136 μs, and the trigger frequency was 2 kHz. For the LPS control and US + LPS experimental conditions, inflammation was induced in the culture with a concentration of 10 μg / mL of LPS. The transcripts of cytokines IL-1β, IFN-γ, IL-8, TNF-α, and NF-κB were measured under all three experimental conditions.

[0110] Figure 6A The fold changes of five inflammatory cytokines (IL-1β, IFN-γ, IL-8, TNF-α, and NF-κB) relative to the negative control are shown in both LPS and LPS+US conditions using RPLP0 as the housekeeping gene. Ultrasound stimulation reduced the upregulation of all five inflammatory cytokines following LPS injury. Figure 6B The fold change of each cytokine relative to the LPS-only condition is shown. When ultrasound stimulation was applied, it reduced the upregulation of each inflammatory cytokine following LPS injury compared to the LPS-only condition. Similarly, Figure 6C The results show the percentage changes in cytokine transcripts after LPS + US stimulation relative to LPS alone. All five inflammatory cytokines showed a decrease after ultrasound stimulation.

[0111] Example 2

[0112] For the US experimental conditions, ultrasound stimulation was applied for 20 minutes using a 1 MHz transducer at a negative peak pressure of approximately 780 kPa. The pulse width was set to 136 μs, and the trigger frequency was 1 kHz. For the LPS control and US + LPS experimental conditions, inflammation was induced in the culture with a concentration of 10 μg / mL LPS. The transcripts of cytokines IL-1β, IFN-γ, IL-8, and TNF-α were measured under all three experimental conditions.

[0113] Figure 7A The fold changes in four inflammatory cytokines (IL-1β, IFN-γ, IL-8, and TNF-α) relative to the negative control are shown in both LPS and LPS+US conditions using RPLP0 as the housekeeping gene. Ultrasound stimulation reduced the upregulation of all four inflammatory cytokines following LPS injury. Figure 7B The fold change of each cytokine relative to the LPS-only condition is shown. When ultrasound stimulation was applied, it reduced the upregulation of each inflammatory cytokine following LPS injury compared to the LPS-only condition. Similarly, Figure 7C The results show the percentage changes in cytokine transcripts after LPS + US stimulation relative to LPS alone. All four inflammatory cytokines showed a decrease after ultrasound stimulation.

[0114] Example 3

[0115] For the US experimental conditions, ultrasound stimulation was applied for 10 minutes using a 1 MHz transducer at a negative peak pressure of approximately 550 kPa. The pulse width was set to 136 μs, and the trigger frequency was 2 kHz. For the LPS control and US + LPS experimental conditions, inflammation was induced in the culture with a concentration of 0.01 μg / mL LPS. The transcripts of cytokines IL-1β, IFN-γ, MCP-1, IL-8, TNF-α, and NF-κB were measured under all three experimental conditions.

[0116] Figure 8A The fold changes of six inflammatory cytokines (IL-1β, IFN-γ, MCP-1, IL-8, TNF-α, and NF-κB) relative to the negative control are shown in both LPS and LPS+US conditions using RPLP0 as the housekeeping gene. Ultrasound stimulation reduced the upregulation of all six inflammatory cytokines following LPS injury. Figure 6B The fold change of each cytokine relative to the LPS-only condition is shown. When ultrasound stimulation was applied, it reduced the upregulation of each inflammatory cytokine following LPS injury compared to the LPS-only condition. Similarly, Figure 6C The results show the percentage changes in cytokine transcripts after LPS + US stimulation relative to LPS alone. All six inflammatory cytokines showed a decrease after ultrasound stimulation.

[0117] Those skilled in the art will understand that although the invention has been described above in conjunction with specific embodiments and examples, the invention is not limited thereto, and many other embodiments, examples, uses, modifications, and variations thereof are intended to be covered by the appended claims. The full disclosure of each patent and publication cited herein is incorporated by reference as if each such patent or publication were individually incorporated herein by reference. Many features and advantages of the invention are set forth in the appended claims.

Claims

1. Methods to reduce inflammation include: Directing ultrasound to multiple blood cells The ultrasound is directed to the plurality of blood cells to reduce inflammation caused by or related to the plurality of blood cells.

2. The method of claim 1, further comprising: Monitor inflammatory markers in at least a portion of the multiple blood cells.

3. The method of claim 1, wherein guiding ultrasound to a plurality of blood cells further comprises: The ultrasound is directed to multiple blood cells in the target.

4. The method of claim 1, wherein guiding ultrasound to a plurality of blood cells further comprises: The ultrasound is directed to multiple blood cells in the target. The multiple blood cells are arranged in a pooled or slowly moving blood reserve area.

5. The method of claim 3, wherein the plurality of blood cells in the ultrasound-guided object further comprises: The ultrasound is directed at multiple blood cells in the object. The plurality of blood cells are disposed in at least one of the following organs of the subject: spleen, liver, kidney, pancreas, lymph nodes, lymphoid tissue, lymphoid tissue, heart, brain, intestine, blood vessels, veins, arteries, capillaries, joint swelling, tissue swelling, muscle, tumor, eyeball, bruise or hematoma, external ear region, inner ear region, gums, and nasal cavity region.

6. The method of claim 3, wherein the plurality of blood cells in the ultrasound-guided object further comprises: The ultrasound is directed at multiple blood cells in the object. The multiple blood cells are arranged within the meninges of the subject's brain.

7. The method of claim 3, wherein the plurality of blood cells in the ultrasound-guided object further comprises: The ultrasound is directed to multiple blood cells in the object, rather than to the nerve tissue in the object.

8. The method of any one of the preceding claims, wherein the plurality of blood cells comprises immune cells.

9. The method of any one of the preceding claims, wherein the plurality of blood cells comprises peripheral blood mononuclear cells (PBMCs).

10. The method of claim 9, wherein the PBMC comprises at least one of T cells, B cells, NK cells, monocytes, or dendritic cells.

11. The method of claim 2, wherein the inflammatory marker comprises at least one of ESR, CRP, ferritin / d-dimer, IL-1β, IFN-γ, MCP-1, IL-8, TNF-α, NF-κB, IL-6, IL-10, MAPK14 and MCP-1.

12. The method of any one of the preceding claims, wherein guiding ultrasound to a plurality of blood cells further comprises: The ultrasound was directed to the multiple blood cells using a 1 MHz transducer.

13. The method of claim 12, wherein directing ultrasound to a plurality of blood cells further comprises: The ultrasound was directed to the plurality of blood cells for 20 minutes using the 1 MHz transducer at a pressure of 780 kPa, a pulse width of 136 μs, and a repetition frequency of 1 or 2 kHz.

14. The method of claim 12, wherein directing ultrasound to a plurality of blood cells further comprises: The ultrasound was directed to the plurality of blood cells for 10 minutes using the 1 MHz transducer at a pressure of 550 kPa, a pulse width of 136 μs, and a repetition frequency of 1 or 2 kHz.

15. The method of any one of the preceding claims, wherein guiding ultrasound to a plurality of blood cells further comprises: Ultrasound is directed to the multiple blood cells using a wearable transducer.

16. The method of any one of the preceding claims, wherein before directing the ultrasound to a plurality of blood cells, the method comprises: The object is imaged to identify the plurality of blood cells.

17. The method of claim 16, wherein imaging the object to identify the plurality of blood cells further comprises: The object is imaged using ultrasound.

18. The method of any one of the preceding claims, wherein guiding ultrasound to a plurality of blood cells further comprises: The ultrasound is directed to multiple blood cells in the target. The plurality of blood cells are arranged in at least two different locations within the object.

19. The method of claim 18, wherein directing ultrasound to a plurality of blood cells further comprises: In at least two different locations within a blood vessel, ultrasound is directed to multiple blood cells within the target. The ultrasound is directed to at least two different locations within the blood vessel based on the blood flow rate through the vessel.

20. The method of claim 19, wherein guiding the ultrasound-guided plurality of blood cells in the target at at least two different locations within the blood vessel further comprises: By adding a tracer substance to the blood, ultrasound is directed to multiple blood cells in the subject at at least two different locations in the blood vessel.

21. The method of claim 1, wherein guiding ultrasound to a plurality of blood cells further comprises: Remove the plurality of blood cells from the object. Ultrasound is directed to the plurality of blood cells outside the object, and Return the plurality of blood cells to the object.

22. The method of claim 1, wherein directing ultrasound to a plurality of blood cells further comprises: The plurality of blood cells were removed from the first object. Ultrasound is directed to the plurality of blood cells outside the first object to produce a plurality of processed blood cells, and The treated blood cells are then applied to a second object.

23. The method of claim 22, wherein the first object is one of a first human object or a first animal object, and wherein the second object is one of a second human object or a second animal object.

24. The method of claim 1, wherein directing ultrasound to a plurality of blood cells further comprises: Ultrasound is directed to the multiple blood cells in an in vitro environment.

25. The method of claim 1, wherein directing ultrasound to a plurality of blood cells further comprises: In an in vivo animal model, ultrasound was directed to the aforementioned multiple blood cells.

26. A system configured to reduce inflammation, the system comprising: An ultrasound transducer is configured to direct ultrasound to a plurality of blood cells to induce a reduction in inflammation caused by or associated with the plurality of blood cells.

27. The system of claim 26, further comprising: The sensor is configured to monitor inflammatory markers in at least a portion of the plurality of blood cells.

28. The system of claim 26, wherein the transducer is further configured to: The ultrasound is directed to multiple blood cells in the target.

29. The system of claim 26, wherein the transducer is further configured to: The ultrasound is directed to multiple blood cells in the target. The multiple blood cells are arranged in a pooled or slowly moving blood reserve area.

30. The system of claim 28, wherein the plurality of blood cells are disposed in at least one of the following of the subject: spleen, liver, kidney, pancreas, lymph nodes, lymphoid tissue, lymphoid tissue, heart, brain, intestine, blood vessels, veins, arteries, capillaries, swelling in joints, swelling in tissues, muscles, tumors, eyeballs, bruises or hematomas, external ear region, inner ear region, gums, and nasal cavity region.

31. The system of claim 28, wherein the plurality of blood cells are arranged within the meninges of the brain of the subject.

32. The system of claim 28, wherein the transducer is further configured to: The ultrasound is directed to multiple blood cells in the object, rather than to the nerve tissue in the object.

33. The system of any one of the preceding claims, wherein the plurality of blood cells comprises immune cells.

34. The system of any one of the preceding claims, wherein the plurality of blood cells comprises peripheral blood mononuclear cells (PBMCs).

35. The system of claim 34, wherein the PBMC comprises at least one of T cells, B cells, NK cells, monocytes, or dendritic cells.

36. The system of claim 27, wherein the inflammatory marker comprises at least one of ESR, CRP, ferritin / d-dimer, IL-1β, IFN-γ, MCP-1, IL-8, TNF-α, NF-κB, IL-6, IL-10, MAPK14 and MCP-1.

37. The system of any one of the preceding claims, wherein the ultrasonic transducer comprises: 1 MHz transducer.

38. The system of claim 37, wherein the transducer is configured to use the 1 MHz transducer for 20 minutes at a pressure of 780 kPa, a pulse width of 136 μs, and a repetition frequency of 1 or 2 kHz when directing ultrasound to a plurality of blood cells.

39. The system of claim 37, wherein the transducer is configured to use the 1 MHz transducer for 10 minutes at a pressure of 550 kPa, a pulse width of 136 μs, and a repetition frequency of 1 or 2 kHz when directing ultrasound to a plurality of blood cells.

40. The system of any one of the preceding claims, wherein the ultrasonic transducer comprises a wearable transducer.

41. The system of any one of the preceding claims, further comprising an imaging system, said imaging system being configured to The object is imaged to identify the plurality of blood cells.

42. The system of claim 41, wherein the imaging system comprises an ultrasound imaging system.

43. The system of any one of the preceding claims, wherein the plurality of blood cells are arranged in at least two different locations within the object.

44. The system of claim 43, wherein the transducer is further configured to: In at least two different locations within a blood vessel, ultrasound is directed to multiple blood cells within the target. The ultrasound is directed to at least two different locations within the blood vessel based on the blood flow rate through the vessel.

45. The system of claim 44, wherein the transducer is located at at least two different positions in the blood vessel, and is further configured to: By adding a tracer substance to the blood, ultrasound is directed to multiple blood cells in the subject at at least two different locations in the blood vessel.

46. ​​The system of claim 26, wherein the transducer is further configured to: After the plurality of blood cells are removed from the object, ultrasound is directed onto the plurality of blood cells outside the object.

47. The system of claim 26, wherein the transducer is further configured to: Ultrasound is directed to the multiple blood cells in an in vitro environment.

48. The system of claim 26, wherein the transducer is further configured to: In an in vivo animal model, ultrasound was directed to the aforementioned multiple blood cells.

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