Intelligent medical coupling agent as well as preparation method and application thereof

Abstract

The invention discloses an intelligent medical coupling agent as well as a preparation method and application thereof, and belongs to the technical field of medical materials. Comprising the following steps: dissolving SiO2 nanoparticles in deionized water, adding an HPTS dye and gamma-aminopropyltriethoxysilane, purifying and concentrating to obtain a pH response nanoprobe; the preparation method comprises the following steps: dissolving dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidyl glycerol and a therapeutic drug in a chloroform-methanol mixed solvent, and evaporating and drying to obtain a lipid film; adding a PBS (Phosphate Buffer Solution) into the lipid film, introducing perfluoropropane, carrying out rotary shearing emulsification, and collecting micro-bubbles to obtain drug-loaded ultrasonic response micro-bubbles; the preparation method comprises the following steps: adding the carbomer powder into deionized water, and then adding glycerol, triethanolamine and methylparaben to obtain basic gel; adding a pH response nanoprobe into the basic gel, adding drug-loaded ultrasonic response microbubbles after the fluorescence intensity CV value is less than or equal to 5%, and performing multi-stage gradient vacuum treatment to obtain the intelligent medical coupling agent. The intelligent medical coupling agent provided by the invention realizes targeted delivery of drugs and physiological status feedback of tissues.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a smart medical coupling agent, its preparation method, and its application. Background Technology

[0002] Medical coupling agent is a functional medium used in ultrasound diagnosis and treatment. It is filled between the skin and the diagnostic probe to eliminate air, reduce acoustic impedance, and improve energy transmission efficiency. Its core function is to eliminate the air gap between the probe and the skin. Because the acoustic impedance difference between air and human tissue is huge, most of the ultrasound waves will be reflected, thus making it impossible to obtain effective diagnostic images or achieve efficient transmission of therapeutic energy.

[0003] Currently, the vast majority of medical coupling agents on the market and in published patents are basically composed of water-soluble polymers, water, humectants, and preservatives. For example, carbomer and sodium hyaluronate are used as gel matrices, supplemented with moisturizing ingredients such as glycerin to prevent excessive evaporation of water, and preservatives are added to maintain product stability. These traditional water-based gel coupling agents have been developed over decades and are technically very mature, basically meeting the basic requirements of routine ultrasound examinations for sound wave transmission.

[0004] However, with the deepening of the concept of precision medicine and the increasing complexity of clinical surgical scenarios, these traditional coupling agents have gradually exposed their inherent technical limitations and functional singularities, and can no longer meet the growing high-end clinical needs. Specifically, the existing technology has the following prominent problems and defects: 1. Limited functionality: Traditional coupling agents are designed merely as a conductive medium, unable to provide doctors with any physiological information about tissue conditions (such as pH levels) or to achieve targeted drug delivery during treatment. In scenarios such as ultrasound-guided interventional therapy or high-intensity focused ultrasound therapy, doctors need to use additional drugs or equipment, rendering the coupling agent itself ineffective in the treatment process, resulting in functional disconnect and waste.

[0005] 2. The lack of a state awareness and feedback mechanism affects the reliability and continuity of diagnostic and treatment procedures. During prolonged ultrasound examinations or surgeries, the coupling agent gradually dries out due to moisture evaporation, leading to a decrease in acoustic coupling effect and image quality degradation. In severe cases, the procedure must be interrupted and reapplied, which not only affects diagnostic and treatment efficiency but may also pose risks during critical surgical steps. Existing coupling agents themselves do not possess any ability to sense their own state and cannot provide warnings to the operator, relying entirely on the operator's subjective experience.

[0006] To overcome these shortcomings, some improvements have been attempted in existing technologies. For example, viscosity and moisturizing properties have been improved by adjusting the types and proportions of polymers; or sterile coupling agents have been developed for use during surgery or on broken skin. Other studies have attempted to add anti-inflammatory and analgesic drugs to the coupling agent, but these are mostly simple physical mixtures, resulting in uncontrollable drug release, failure to achieve targeted delivery, and potential impact on the acoustic properties of the coupling agent itself.

[0007] In summary, there is an urgent need in this field for a technological breakthrough aimed at developing a new generation of intelligent medical coupling agents that not only possess excellent acoustic conduction performance but also integrate multiple functions such as real-time status perception, physiological parameter monitoring, and on-demand treatment, in order to address the severe challenges posed by modern precision medicine. Summary of the Invention

[0008] To address the aforementioned technical problems, this invention provides an intelligent medical coupling agent, its preparation method, and its application.

[0009] According to one aspect of the present invention, a method for preparing a smart medical coupling agent is provided, comprising: Step 1, Preparation of SiO2 nanoparticle suspension: Anhydrous ethanol and ammonia water are stirred and mixed, and ethyl silicate dissolved in anhydrous ethanol is added dropwise to react and obtain monodisperse SiO2 nanoparticle suspension. Step 2, Preparation of pH-responsive nanoprobes: The monodisperse SiO2 nanoparticle suspension was centrifuged, the precipitate was collected, washed with anhydrous ethanol, and then redispersed in deionized water. 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt dye was added, and physical adsorption was completed by stirring. γ-aminopropyltriethoxysilane was then added to continue the reaction. After the reaction was completed, the product was purified by dialysis and concentrated by evaporation to obtain pH-responsive nanoprobes, which were then stored in the dark under cold conditions. Step 3, preparation of lipid film: dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol and therapeutic drugs are dissolved in chloroform-methanol mixed solvent, and the organic solvent is removed by evaporation and vacuum drying to obtain lipid film. The therapeutic drugs include anti-inflammatory drugs, anti-tumor drugs, antibiotics and / or local anesthetics. Step 4, Preparation of drug-loaded ultrasound-responsive microbubbles: The lipid film obtained in step 3 is added to PBS buffer, hydrated, and then perfluoropropane gas is introduced to replace the air. Under ice bath conditions, the microbubbles are sheared and emulsified, and 1-3 micrometer microbubbles are collected by flotation to obtain drug-loaded ultrasound-responsive microbubbles, which are then refrigerated for later use. Step 5, Preparation of basic gel: Add carbomer powder to deionized water to ensure no clumping, then add glycerol and stir until homogeneous. Then add triethanolamine dropwise to adjust the pH to 7.2±0.1. Separately dissolve methylparaben in deionized water and add it to the system. Continue stirring to obtain a transparent and homogeneous basic gel. Step 6, preparation of intelligent medical coupling agent: The basic gel obtained in step 5 is added to the pH-responsive nanoprobe obtained in step 2 under the condition of rotation speed, and stirred until the color is uniform. After sampling and detecting the fluorescence intensity CV value ≤ 5%, the rotation speed is reduced and the drug-loaded ultrasonic-responsive microbubbles obtained in step 4 are added in layers. The mixture is stirred to obtain a mixed gel. The mixed gel is then placed in a vacuum environment for multi-stage gradient vacuum treatment. After the treatment is completed, the pressure is restored to normal to obtain the intelligent medical coupling agent.

[0010] Optionally, in step one, the concentration of ammonia is 15 mol / L, the volume ratio of anhydrous ethanol to ammonia is 20:1, and the volume of ethyl silicate in the ethyl silicate-ethanol solution is 1 / 10 of that of anhydrous ethanol.

[0011] Optionally, in step two, 2 g of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt dye is added and stirred at 600 rpm for 12 hours in a 40°C water bath to complete physical adsorption; 1.0 mL of γ-aminopropyltriethoxysilane is further added and reacted for 6 hours for covalent fixation, followed by dialysis purification for 72 hours, and then rotary evaporation is performed at 35-40°C and 80-100 rpm for 1.5-3 hours to concentrate to a solid content of 10% to obtain a pH-responsive nanoprobe, which is stored at 4°C in the dark.

[0012] Optionally, in step three, the mass ratio of added dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, and the therapeutic drug is 1:2:1; the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 2:1.

[0013] Optionally, in step three, the anti-inflammatory drug is dexamethasone; the antitumor drug is docetaxel or paclitaxel; the antibiotic is vancomycin; and the local anesthetic is ropivacaine.

[0014] Optionally, in step four, collecting 1-3 micron microbubbles by flotation includes: transferring the microbubble suspension obtained after shear emulsification to a jacketed glass graduated cylinder, keeping the graduated cylinder vertical, stationary, and vibration-free for 20-30 minutes, with the circulating water bath continuously running during the process to ensure that cooling water at 4°C continuously passes through the graduated cylinder jacket; after settling, removing and discarding the >5μm microbubbles on the surface of the graduated cylinder using a pipette, and then using a wide-mouth pipette to draw up the upper layer of 1-3 micron microbubbles from the top of the graduated cylinder.

[0015] Optionally, step five specifically includes: weighing 727.0g of deionized water and adding it to a 2L reactor. After forming a vortex at 800rpm, 10.0g of carbomer powder is sieved through a 100-mesh sieve. The feeding time is controlled to be no less than 15 minutes to ensure no clumping. After the feeding is completed, the speed is increased to 1000rpm and stirred continuously for 45 minutes. Then the speed is reduced to 400rpm, and 150.0g of glycerol is added along the reactor wall. Stirring is continued for 15 minutes until completely homogeneous. Further, while maintaining a speed of 400rpm, triethanolamine is added dropwise at a rate of 0.4g / min using a micro-injection pump. The pH change is monitored in real time, and a sample is taken for testing after every 1g is added. Finally, the pH is adjusted to 7.2±0.1, and stirring is continued for 15 minutes. Separately, 1.0g of methylparaben is dissolved in 10g of 60°C deionized water and added to the reactor. Stirring is continued for 15 minutes to obtain a transparent and homogeneous basic gel.

[0016] Optionally, step six specifically includes: adding 10.0g of the pH-responsive nanoprobe obtained in step two to the base gel obtained in step five at a speed of 150 rpm, stirring for 30 minutes until the color is uniform, taking a sample and using a fluorescence microplate reader to detect the fluorescence intensity CV value ≤5%, then further reducing the speed to 80 rpm, and adding 100.0g of the drug-loaded ultrasonic-responsive microbubbles obtained in step four in layers using a wide-mouth dropper, wherein the layered addition is as follows: first add 1 / 3, stir for 15 minutes; then add another 1 / 3, stir for 15 minutes; finally add all, stir for 30 minutes to obtain a mixed gel, then transfer the mixed gel to a 2L vacuum desiccator, maintain at -0.05MPa for 15 minutes, then at -0.08MPa for 15 minutes, and finally at -0.095MPa for 15 minutes, and then restore to normal pressure to obtain the intelligent medical coupling agent.

[0017] According to another aspect of the present invention, a smart medical coupling agent is provided, which is prepared based on the preparation method of the smart medical coupling agent as described above. The smart medical coupling agent integrates a pH-responsive nanoprobe and a drug-loaded ultrasonic-responsive microbubble. The pH-responsive nanoprobe converts the biochemical information pH value into a detectable fluorescence signal through fluorescence imaging, and triggers the drug release function of the drug-loaded ultrasonic-responsive microbubble through ultrasound.

[0018] According to another aspect of the present invention, the application of the smart medical coupling agent as described above in the preparation of medical coupling agent materials is provided.

[0019] The beneficial effects of this invention are: This invention's intelligent medical coupling agent integrates a pH-responsive nanoprobe with drug-loaded ultrasound-responsive microbubbles (wherein the pH-responsive nanoprobe converts biochemical information pH into a detectable fluorescent signal, achieving diagnostic enhancement, and the drug-loaded ultrasound-responsive microbubbles release drugs at specific points under external ultrasound commands, achieving therapeutic functions), making it an active platform integrating diagnosis and treatment. In diagnosis, it can generate a real-time pH distribution map reflecting the biochemical state of tissues based on conventional ultrasound imaging, providing doctors with crucial information beyond anatomical imaging for locating lesions such as tumors and inflammation (whose microenvironments are often acidic). In treatment, it can release drugs at specific points and in a controlled manner in a specific area via external ultrasound pulse commands, achieving a closed-loop integrated diagnosis and treatment system of "detection during diagnosis and immediate treatment after detection," greatly improving diagnostic efficiency and accuracy.

[0020] The core dye of the pH-responsive nanoprobe, HPTS, is a ratiometric pH-sensitive fluorescent dye whose molecular structure contains protonation (H2O). + (Bound) and dissociated groups, when the ambient pH decreases (H) + When the concentration increases, its molecular structure changes, leading to a significant decrease in its fluorescence emission intensity at 510 nm. This change exhibits a highly reproducible linear relationship within the pH range of 6.0–8.0. Therefore, by emitting excitation light of a specific wavelength (e.g., 450 nm) towards the target area through optical components, nanoprobes distributed at the tissue-coupler interface and superficial tissues can be excited to emit fluorescence signals (e.g., at 510 nm) corresponding to the local pH. This allows for the sensing and feedback of the pH value of the tissue microenvironment, and visualization of the lesion area.

[0021] Drug-loaded ultrasound-responsive microbubbles are a highly flexible drug delivery platform with a wide range of encapsulated drugs, including anti-inflammatory drugs, anti-tumor drugs, antibiotics, and local anesthetics. By changing different drugs, the smart coupling agent of the same technology platform can be applied to multiple medical fields such as oncology, infectious diseases, pain management, rehabilitation, and plastic surgery.

[0022] Furthermore, the drug-loaded ultrasound-responsive microbubbles are controlled to a size of 1–3 μm. This size range makes them difficult to pass through normal vascular endothelial spaces. However, in tumor or inflammatory areas, the intercellular spaces of vascular endothelial cells are often widened and structurally disordered, making it easier for microbubbles to leak from blood vessels and remain in the lesion tissue spaces. The phospholipids that make up the microbubble shell themselves carry a certain negative charge. In an acidic environment, their surface charge and membrane stability may undergo subtle changes, which is conducive to their longer retention in acidic areas.

[0023] The coupling agent of this invention is applied directly, in large quantities, and uniformly to the skin surface corresponding to the lesion. The drug-loaded microbubbles (i.e., the drug-loaded ultrasound-responsive microbubbles of this invention) directly contact the subcutaneous tissue through the coupling agent matrix, or enhance penetration through ultrasound microfluidics. This allows high-concentration microbubbles to form extremely high local initial concentrations in the area corresponding to the lesion without going through systemic circulation, achieving the most direct "local accumulation," which is an advantage that traditional intravenous injection microbubbles cannot match.

[0024] The therapeutic drug is securely encapsulated within the phospholipid bilayer membrane of ultrasound-responsive microbubbles. When not triggered, the drug is virtually leak-free, preventing exposure to non-target tissues. The release mechanism is controlled by the operating physician. After confirming the target location using pH-responsive nanoprobe imaging, the physician manually triggers the targeted drug release mechanism via ultrasound. This mechanism converts externally applied ultrasound energy into mechanical forces that disrupt the microbubble structure and release the drug encapsulated within. These forces destroy the phospholipid bilayer that forms the microbubble shell, causing the microbubble to disintegrate. The drug encapsulated within the phospholipid layer or its internal water core is then instantly released into the surrounding microenvironment. This allows for efficient drug concentration at the lesion site, avoiding the side effects of systemic administration and achieving minimally invasive or even non-invasive precision treatment, thus improving efficacy and safety. Attached Figure Description

[0025] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are used to explain the invention, but do not constitute an undue limitation of the invention. In the drawings: Figure 1 TEM image of a pH-responsive nanoprobe; Figure 2 TEM image of drug-loaded ultrasound-responsive microbubbles; Figure 3 TEM image of drug-loaded ultrasonically responsive microbubbles after ultrasonic fragmentation; Figure 4 Bright-field image of a smart medical coupling agent; Figure 5 A bar chart showing the fluorescence intensity for performance testing; Figure 6 A bar chart showing the drug release rate for performance testing. Detailed Implementation

[0026] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present application, and not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort should fall within the scope of protection of the present application. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present application can be combined with each other.

[0027] The terms “comprising” and “having”, and any variations thereof, in the specification and claims of this application are intended to cover non-exclusive inclusion, for example, a process, method, product, or apparatus that includes a series of steps or units is not necessarily limited to those steps or units that are explicitly listed, but may include other steps or units that are not explicitly listed or that are inherent to such process, method, product, or apparatus.

[0028] Example 1: This example provides a method for preparing a smart medical coupling agent, including the following steps: Step 1: Prepare a suspension of SiO2 nanoparticles; Add 400 mL of anhydrous ethanol and 20 mL of 15 mol / L ammonia to a 1 L three-necked flask, place it in a water bath and mechanically stir at 25°C and 300 rpm for 30 min. Then, dissolve 8 mL of tetraethyl orthosilicate in 80 mL of anhydrous ethanol and add it dropwise to the three-necked flask at a rate of 2 mL / min using a constant pressure dropping funnel. After the addition is complete, react for 6 hours to obtain a monodisperse SiO2 (silicon dioxide) nanoparticle suspension (the particle size of SiO2 nanoparticles is 50~80 nm).

[0029] Step 2: Prepare pH-responsive nanoprobes; The monodisperse SiO2 nanoparticle suspension obtained in step one was centrifuged at 12,000 rpm for 30 min, the precipitate was collected, washed three times with anhydrous ethanol, and redispersed in 200 mL of deionized water. 2.0 g of 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt (HPTS) dye was added, and the mixture was stirred at 600 rpm for 12 hours in a 40°C water bath to complete physical adsorption. 1.0 mL of γ-aminopropyltriethoxysilane was then added, and the reaction was continued for 6 hours for covalent fixation. Subsequently, the mixture was purified by dialysis for 72 hours, and then concentrated to a solid content of 10% using a rotary evaporator at 35-40°C and 80-100 rpm for 1.5-3 hours. The pH-responsive nanoprobe was then stored at 4°C in the dark.

[0030] TEM (Transmission Electron Microscope) images of pH-responsive nanoprobes are shown below. Figure 1 As shown, Figure 1 In the study, the probes exhibited regular morphology and uniform dispersion of spherical shapes, with an average particle size between 50 and 80 nm.

[0031] The purpose of dialysis purification in this step is to remove free 8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt dye molecules that are not covalently or physically adsorbed onto the SiO2 nanoparticles. Dialysis purification is achieved using a standard regenerated cellulose dialysis bag with a molecular weight cutoff of 8,000-14,000 Da. This size is much smaller than silica nanoparticles but much larger than HPTS dye molecules (molecular weight approximately 524 Da). Therefore, the silica nanoparticles are trapped inside the bag, while the free dye molecules can diffuse freely outside the bag. The specific dialysis purification procedure is as follows: Approximately 200 mL of silica nanoparticle suspension, after completing the dye loading reaction of trisodium 8-hydroxypyrene-1,3,6-trisulfonic acid, is carefully transferred to a dialysis bag using a glass funnel. The dialysis bag is then securely sealed at both ends using a dedicated dialysis clamp or by tightly tying it. The sealed dialysis bag is placed in a 5L beaker, and 4L of external dialysis buffer (0.1 mM PBS buffer, pH 7.4) is added. The beaker is placed on a magnetic stirrer and slowly stirred at 150-200 rpm in a 4°C refrigerator. The dialysis buffer is changed every 6 hours. After 72 hours of continuous dialysis, the dialysis buffer should be almost colorless and transparent, completing the dialysis purification process.

[0032] Step 3: Prepare the lipid film; 8g dipalmitoylphosphatidylcholine, 1.6g dipalmitoylphosphatidylglycerol, and 0.8g therapeutic drug were dissolved in 200mL of chloroform-methanol (volume ratio 2:1) mixed solvent. The mixture was rotary evaporated at 40°C and 60rpm until a uniform lipid film was formed. The film was then vacuum dried for 24 hours to completely remove the organic solvent and obtain the lipid film. The therapeutic drug is selected from the following group: anti-inflammatory drugs, antitumor drugs, antibiotics, and local anesthetics. Further, the anti-inflammatory drug may be dexamethasone; the antitumor drug may be docetaxel or paclitaxel; the antibiotic may be vancomycin; and the local anesthetic may be ropivacaine.

[0033] Step 4: Prepare drug-loaded ultrasonic-responsive microbubbles; The lipid film obtained in step 3 was added to 200 mL of PBS buffer (pH=7.4) and hydrated at 55°C for 1 hour. Then, perfluoropropane gas was introduced to replace the air, and the film was sheared and emulsified at 10,000 rpm for 3 minutes under ice bath conditions. 1-3 micron microbubbles were collected by flotation to obtain drug-loaded ultrasonically responsive microbubbles, which were stored at 4°C for later use.

[0034] In this step, the flotation method is used to collect 1-3 micrometer microbubbles. Specifically, the microbubble suspension obtained after high-speed shear emulsification is immediately transferred to a jacketed glass graduated cylinder. A circulating water bath is turned on, allowing 4°C cooling water to continuously pass through the graduated cylinder jacket. The entire process must be carried out at low temperature to maintain the stability of the phospholipid film and the solubility of perfluoropropane gas. The graduated cylinder is allowed to stand vertically, still, and without vibration for 20-30 minutes. During this period, due to the huge density difference, large-sized microbubbles (>5μm) and microbubble clusters will rise the fastest due to the greatest buoyancy, forming a thick, white "foam layer" or "cream layer" on the liquid surface. Microbubbles that are too small (<1μm) rise very slowly due to insufficient buoyancy, and most of them remain dispersed in the solution. Microbubbles of the target size (1-3μm) are in an intermediate state, with some rising to the upper part of the graduated cylinder and some still slowly rising. After settling, carefully remove and discard the thick "cream layer" on top of the liquid surface using a pipette (this layer mainly consists of excessively large and unstable microbubbles, which can affect the homogeneity and stability of the final product). After primary flotation, the liquid in the graduated cylinder will exhibit a concentration gradient from top to bottom: the supernatant, the liquid at the top of the graduated cylinder, contains the floated, highly concentrated 1-3 μm microbubbles; the middle layer is clear with a lower microbubble concentration; and the bottom precipitate may contain unencapsulated free drug, lipid aggregates, or a very small number of heavy impurities. Using a wide-mouth pipette, starting from the top of the graduated cylinder, slowly aspirate the upper layer of liquid, collecting 20%-30% of the total volume from the liquid surface downwards, completing the collection of 1-3 μm microbubbles.

[0035] TEM images of drug-loaded ultrasound-responsive microbubbles as follows Figure 2 As shown, the intact spherical microbubble structure can be observed, and the TEM morphology after ultrasonic disruption is as follows. Figure 3 , Figure 3 The microbubble membrane structure underwent significant rupture, clearly demonstrating the structural basis for drug-loaded ultrasound-responsive microbubbles triggering drug release under ultrasound.

[0036] Step 5: Prepare the basic gel; Accurately weigh 727.0g of deionized water and add it to a 2L reactor. After a moderate vortex is formed at 800rpm, slowly sift 10.0g of carbomer powder (such as carbomer 934, carbomer 941, carbomer 980, or carbomer 974P) through a 100-mesh sieve, controlling the feeding time to be no less than 15 minutes to ensure no clumping. After the feeding is complete, increase the speed to 1000rpm and stir continuously for 45 minutes. Then reduce the speed to 400rpm and slowly add 150.0g of glycerol, letting it flow down the reactor wall, and continue stirring for 15 minutes until completely homogeneous. Further, while maintaining a rotation speed of 400 rpm, triethanolamine was added dropwise at a rate of 0.4 g / min using a micro-injection pump, with real-time monitoring of pH changes (samples were taken for testing after every 1 g was added). The pH was finally adjusted to 7.2 ± 0.1, and stirring was continued for 15 minutes. 1.0 g of methylparaben was dissolved in 10 g of 60°C deionized water and slowly added to the reaction vessel. Stirring was continued for 15 minutes to obtain a transparent and homogeneous base gel.

[0037] Step six: Prepare the intelligent medical coupling agent; The base gel obtained in step five was slowly added to 10.0g of the pH-responsive nanoprobe obtained in step two at 150rpm. The mixture was stirred for 30 minutes until the color was uniform. After the fluorescence intensity CV value was measured using a fluorescence microplate reader and found to be ≤5%, the stirring speed was further reduced to 80rpm. 100.0g of the drug-loaded ultrasonic-responsive microbubbles obtained in step four were slowly added using a wide-mouth dropper. A layered addition technique was used: first, 1 / 3 was added and stirred for 15 minutes; then another 1 / 3 was added and stirred for 15 minutes; finally, the remaining 1 / 3 was added and stirred for 30 minutes to obtain a mixed gel. The mixed gel was then transferred to a 2L vacuum desiccator and kept at -0.05MPa for 15 minutes, then at -0.08MPa for 15 minutes, and finally at -0.095MPa for 15 minutes before returning to normal pressure to obtain the intelligent medical coupling agent.

[0038] The obtained bright-field image of the smart medical coupling agent is as follows Figure 4 As shown, the intelligent medical coupling agent is a transparent and uniform gel with no obvious particle aggregation or stratification. The functional components (pH-responsive nanoprobes and drug-loaded ultrasonic-responsive microbubbles) are well compatible with the basic gel system and do not damage the physical morphology of the gel.

[0039] Example 2: In this example, except that the pH-responsive nanoprobe added in step six is ​​5g and the drug-loaded ultrasonic-responsive microbubbles are 50g, the rest of the steps are the same as in Example 1.

[0040] Example 3: In this example, except that the pH-responsive nanoprobe added in step six is ​​20g, the other steps are the same as in Example 1.

[0041] Example 4: In this example, except that the amount of drug-loaded ultrasonic response microbubbles added in step six is ​​200g, the other steps are the same as in Example 1.

[0042] Comparative Example 1: This comparative example does not include the addition of pH-responsive nanoprobes and drug-loaded ultrasonic-responsive microbubbles in step six; the remaining steps are the same as in Example 1.

[0043] The performance tests of the intelligent medical coupling agent in this invention are as follows: Fluorescence intensity was measured using a fluorescence spectrophotometer with an excitation wavelength of 450 nm and an emission wavelength of 510 nm. The samples prepared in the above examples and comparative examples were diluted 20 times before measurement.

[0044] Drug release rate was measured using a Franz diffusion cell under 37°C with a 1.2 MI ultrasonic pulse (frequency 1 MHz, duration 60 seconds). The calculation formula was: (released drug amount / total encapsulated drug amount) × 100%.

[0045] The performance results of Examples 1-4 and Comparative Example 1 (the prepared sample) are shown in Table 1 below (fluorescence intensity bar chart as shown). Figure 5 Release rate histogram as shown Figure 6 ).

[0046] Table 1 Performance Test Results Group pH-responsive nanoprobe content (g) Drug-loaded ultrasound-responsive microbubble content (g) Fluorescence intensity (RFU) Drug release rate (%) Example 1 10 100 29,800±920 68.7±2.5 Example 2 5 50 15,250±850 42.3±3.1 Example 3 20 100 58,500±1,250 69.1±2.3 Example 4 10 200 30,100±950 85.4±1.8 Comparative Example 1 0 0 0 0

[0047] As shown in Table 1: Comparative Example 1: No pH fluorescence response and drug release function.

[0048] Example 1: With appropriate amounts of pH-responsive nanoprobes and drug-loaded ultrasonic-responsive microbubbles (10g, 100g), high fluorescence signals (~29,800 RFU) and drug release rates (~68.7%) were provided, indicating that the formulation has reliable performance in both diagnostic sensitivity and therapeutic efficacy and can be used as a basic standard formulation.

[0049] Example 2: By halving the dosage of pH-responsive nanoprobes and drug-loaded ultrasound-responsive microbubble components (5g, 50g), a proportionally reduced fluorescence intensity and drug release rate were achieved. This formulation is suitable for scenarios where high diagnostic signal and therapeutic intensity are not required, or where cost control is necessary.

[0050] Example 3: By doubling the amount of pH-responsive nanoprobe to 20g, a significantly enhanced fluorescence signal (~58,500 RFU) was obtained, while the drug release rate was comparable to that of Example 1. This indicates that its core advantage lies in greatly improving the contrast and signal-to-noise ratio of pH imaging, making it particularly suitable for scenarios requiring precise localization of lesion boundaries (such as invasive tumors) or quantitative studies.

[0051] Example 4: By doubling the dosage of drug-loaded ultrasound-responsive microbubbles to 200g, the drug release rate was significantly increased to ~85.4% while maintaining a good diagnostic signal. This demonstrates that the formulation can carry and release higher doses of therapeutic drugs, making it suitable for refractory lesions or large lesion areas requiring enhanced treatment effects.

[0052] In practical applications, different ratios of intelligent medical coupling agents can be used according to different clinical needs.

[0053] Furthermore, to clarify the drug release behavior of this invention at non-target sites, a comparative experiment was conducted comparing the release rates under ultrasound-triggered and non-ultrasound-triggered conditions, such as... Figure 6 The results showed that the cumulative release rate of Examples 1-4 and Comparative Example 1 was less than 5% within 24 hours without ultrasound application. However, after ultrasound (frequency 1.0 MHz, mechanical index 1.2) triggering, the drug release rate significantly increased, reaching 68.7±2.5% in Example 1, 69.1±2.3% in Example 3, and 85.4±1.8% in Example 4. This comparative data clearly demonstrates that the drug is efficiently released only in the ultrasound-targeted area, with almost no drug leakage at non-targeted sites, fully meeting the technical requirements of "targeted and controllable release" and exhibiting significant ultrasound response characteristics.

[0054] The pH-responsive nanoprobes in this invention's intelligent medical coupling agent can sense and provide real-time feedback on the pH value of the tissue microenvironment, visualizing the lesion area and solving the problem of not being able to see biochemical information (pH). Drug-loaded ultrasound-responsive microbubbles release drugs through ultrasound triggering, solving the problem of being able to observe but not treat.

[0055] Furthermore, the intelligent medical coupling agent prepared in this invention has a storage temperature of 15~25°C, should be kept away from freezing, and has a shelf life of 24 months. Its specific usage method is as follows: Apply an appropriate amount of coupling agent evenly to the examination site, with a thickness of approximately 2~3 mm. Using a fusion ultrasound / fluorescence imaging system, first perform a conventional B-ultrasound scan, then switch to fluorescence imaging mode, with an excitation wavelength of 450 nm and a receiving fluorescence wavelength of 510 nm. The system generates a pH distribution pseudo-color image, which is superimposed on the B-ultrasound image. After locating the pH abnormal area, switch to treatment mode and set the ultrasound parameters: frequency 1.0 MHz, mechanical index 1.2, duty cycle 50%. The treatment time is set according to the size of the lesion, typically 60~120 seconds. The ultrasound can trigger the release of drug-loaded ultrasound-responsive microbubbles. After treatment, pH imaging can be performed again to assess the effect.

[0056] The principle behind this invention, which uses intelligent medical coupling agent fluorescence imaging to locate pH abnormal regions, is as follows: Solid tumor tissues, due to the prevalent aerobic glycolysis, produce and accumulate large amounts of acidic metabolites such as lactic acid during their metabolic processes. This, coupled with increased proton pump activity, leads to a characteristic decrease in extracellular pH, creating an acidic microenvironment (typically pH 6.2–6.9), significantly different from normal physiological tissues (pH approximately 7.4). Active inflammatory lesions, due to ischemia, hypoxia, and activated immune cell metabolism, also produce excessive lactic acid through anaerobic glycolysis, resulting in local tissue acidification. This tissue acidification is an early-appearing and universal pathochemical marker independent of morphological changes. The pH-responsive nanoprobe core dye HPTS in this invention is a ratiometric pH-sensitive fluorescent dye with protonation (H2O) in its molecular structure. + (Bound) and dissociated groups, when the ambient pH decreases (H) + When the concentration increases, its molecular structure changes, leading to a significant decrease in fluorescence emission intensity at 510 nm. This change exhibits a highly reproducible linear relationship within the pH range of 6.0–8.0. This is achieved using an ultrasound imaging device with an integrated optical module. During scanning, the device's optical components emit excitation light of a specific wavelength (e.g., 450 nm) towards the target area, exciting nanoprobes distributed at the tissue-coupler interface and superficial tissues, causing them to emit fluorescence signals (e.g., at 510 nm) corresponding to the local pH. This process encodes and associates spatial location (determined by the ultrasound probe scanning path) with local biochemical information (fluorescence intensity determined by pH). The system's built-in or external signal processing unit, based on a pre-established "fluorescence intensity-pH value" standard calibration curve, converts the fluorescence intensity collected at each spatially encoded point into a specific pH value in real time. The resulting digital matrix containing spatial coordinates and pH values ​​is then mapped, through image processing algorithms, into a functional image that characterizes pH levels using pseudo-color tones (e.g., warm tones representing acidic regions). Finally, the pH functional distribution map was pixel-level registered and fused with the concurrently acquired ultrasound grayscale image reflecting the anatomical structure to generate a composite image that combines anatomical location and biochemical information, thus realizing real-time sensing and feedback of the pH value of the tissue microenvironment.

[0057] The principle of ultrasound-responsive microbubble targeted drug release is as follows: Therapeutic drugs (anti-inflammatory drugs, anti-tumor drugs, antibiotics, and / or local anesthetics) are securely encapsulated within the phospholipid bilayer membrane of ultrasound-responsive microbubbles. When not triggered, the drugs exhibit minimal leakage, preventing exposure to non-target tissues. The release is controlled by the operating physician. After confirming the target location using pH-responsive nanoprobe imaging, the physician manually triggers drug release via ultrasound. The externally applied ultrasound energy is converted into mechanical forces that disrupt the microbubble structure and release the encapsulated drug. These forces destroy the phospholipid bilayer that forms the microbubble shell, causing the microbubble to disintegrate. The drug, encapsulated within the phospholipid layer or its internal water core, is then instantly released into the surrounding microenvironment. This allows for efficient drug concentration at the lesion site, avoiding the side effects of systemic administration and achieving minimally invasive or even non-invasive precision treatment.

[0058] In summary, the intelligent medical coupling agent of this invention integrates pH-responsive nanoprobes and drug-loaded ultrasound-responsive microbubbles, achieving tissue physiological state feedback and targeted drug delivery, making it an active platform integrating diagnosis and treatment, which greatly improves diagnostic efficiency and accuracy.

[0059] It should be further noted that those skilled in the art will understand that the purpose of this invention is to provide a relatively universal overall preparation process or technical principle, and to cover as many different applicable scenarios and conditions as possible (such as different raw material characteristics, production scale, product demand, etc.). Therefore, some specific operations can be flexibly adjusted according to the actual implementation, as long as the expected or similar technical effects as in the embodiments of this invention can be achieved. For example, in step two, "rotary evaporation for 1.5 to 3 hours, concentrating to a solid content of 10%", the evaporation time can be extended or shortened according to the actual preparation scale or sample conditions (large preparation scale, large quantity, extend evaporation time; low sample water content, dry, shorten evaporation time), as long as the sample is dried (solid content reaches 10%). Other operations are similar and will not be elaborated further.

[0060] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0061] The steps in the method of this invention can be adjusted, combined, or deleted according to actual needs. The technical features can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the embodiments are described. However, as long as the combinations of these technical features do not contradict each other, they should all be considered within the scope of this invention.

[0062] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for preparing an intelligent medical couplant, characterized in that, The application relates to a preparation method of an intelligent medical coupling agent. Step one, preparation of SiO2 nanoparticle suspension: Anhydrous ethanol and ammonia water are stirred and mixed, a reaction is carried out by dropwise adding a tetraethyl orthosilicate-ethanol solution of tetraethyl orthosilicate dissolved in anhydrous ethanol, and a monodisperse SiO2 nanoparticle suspension is obtained; Step two, preparation of pH response nanoprobe: The monodisperse SiO2 nanoparticle suspension is centrifuged, the precipitate is collected, washed with anhydrous ethanol, and then dispersed in deionized water again, 8-hydroxy pyrene-1, 3, 6-trisulfonic acid trisodium salt dye is added, physical adsorption is completed by stirring, further gamma-aminopropyl triethoxysilane is added for continuous reaction, and the obtained product is purified by dialysis, concentrated by evaporation, and then the pH response nanoprobe is obtained, which is stored in a cold environment and protected from light; Step three, preparation of lipid film: Dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol and therapeutic drugs are dissolved in a chloroform-methanol mixed solvent, and the organic solvent is removed by evaporation and vacuum drying to obtain a lipid film, wherein the therapeutic drugs include anti-inflammatory drugs, antitumor drugs, antibiotics and / or local anesthetics; Step four, preparation of drug-loaded ultrasound response microbubbles: The lipid film obtained in step three is added to a PBS buffer solution, and after hydration, the air is replaced by perfluoropropane gas, and the shearing emulsification is carried out under ice bath conditions; the 1-3 micron microbubbles are collected by using the flotation method, and the drug-loaded ultrasound response microbubbles are obtained and stored in a cold environment for standby use; Step five, preparation of base gel: Carbomer powder is added to deionized water to ensure that there is no lumping, then glycerol is added, and stirring is carried out until the mixture is uniform; then triethanolamine is added dropwise, the pH is adjusted to 7.2+ / -0.1, and then nipagin methyl is dissolved in deionized water and added to the system; the stirring is continued, and the transparent and uniform base gel is obtained; Step six, preparation of intelligent medical coupling agent: The base gel obtained in step five is added to the pH response nanoprobe obtained in step two under the condition of rotating speed, and stirring is carried out until the color is uniform; after the sample detection fluorescence intensity CV value is less than or equal to 5%, the rotating speed is reduced, the drug-loaded ultrasound response microbubbles obtained in step four are layered and added, and stirring is carried out to obtain a mixed gel; the mixed gel is placed in a vacuum environment for multi-stage gradient vacuum treatment, and after the treatment is completed, the normal pressure is restored, and the intelligent medical coupling agent is obtained.

2. The method of claim 1, wherein the medical couplant is prepared by the steps of: In step one, the concentration of ammonia water is 15 mol / L, and the volume ratio of anhydrous ethanol to ammonia water is 20:1; the volume of tetraethyl orthosilicate in the tetraethyl orthosilicate-ethanol solution is 1 / 10 of that of anhydrous ethanol.

3. The method for preparing the intelligent medical coupling agent according to claim 1, characterized in that, In step two, 2g of 8-hydroxy pyrene-1, 3, 6-trisulfonic acid trisodium salt dye is added, stirring is carried out at 600 rpm under a 40°C water bath for 12 hours, and the physical adsorption is completed; 1.0 mL of gamma-aminopropyl triethoxysilane is further added, covalent fixation is carried out by reaction for 6 hours, and then dialysis purification is carried out for 72 hours; the rotary evaporator is used for rotary evaporation at 80-100 rpm under the condition of 35-40°C for 1.5-3 hours, and the obtained pH response nanoprobe is concentrated to a solid content of 10%, and stored in a cold environment and protected from light.

4. The preparation method of the intelligent medical coupling agent according to claim 1, characterized in that, In step three, the mass ratio of dipalmitoyl phosphatidylcholine, dipalmitoyl phosphatidylglycerol and therapeutic drugs added is 1:2:1; the volume ratio of chloroform to methanol in the chloroform-methanol mixed solvent is 2:

1.

5. The method for preparing the intelligent medical coupling agent according to claim 1, characterized in that, In step three, the anti-inflammatory drug is dexamethasone; the antitumor drug is docetaxel or paclitaxel; the antibiotic is vancomycin; and the local anesthetic is ropivacaine.

6. The method for preparing the intelligent medical coupling agent according to claim 1, characterized in that, In step four, the 1-3 micron microbubbles are collected by using a flotation method, which includes: The microbubble suspension obtained after shear emulsification is transferred to a jacketed glass measuring cylinder, the measuring cylinder is kept vertical, stationary and vibration-free for 20-30 minutes, and the circulating water bath is turned on throughout the process to make the jacket of the measuring cylinder continuously pass through 4°C cooling water. After the standing is completed, the >5 micron microbubbles on the liquid surface of the measuring cylinder are removed and discarded by using a pipette, and then the 1-3 micron microbubbles on the top of the measuring cylinder are taken from the top of the measuring cylinder by using a wide-mouth pipette.

7. The method for preparing the intelligent medical coupling agent according to claim 1, characterized in that, Step five specifically includes: After the addition is completed, the stirring speed is increased to 1000 rpm, and the stirring is continued for 45 minutes. Then the stirring speed is reduced to 400 rpm, 150.0 g of glycerol is added along the wall of the reactor, and the stirring is continued for 15 minutes until it is completely uniform. Further, under the condition of maintaining the stirring speed at 400 rpm, triethanolamine is added dropwise at a speed of 0.4 g / min using a micro-injection pump, and the pH change is monitored in real time. Every 1 g is sampled and detected, and finally the pH is adjusted to 7.2±0.

1. The stirring is continued for 15 minutes, and then 1.0 g of nipagin methyl ester is dissolved in 10 g of 60°C deionized water and added to the reactor. The stirring is continued for 15 minutes to obtain a transparent and uniform base gel.

8. The method for preparing the intelligent medical coupling agent according to claim 1, characterized in that, Step six specifically includes: The base gel obtained in step five is added to 10.0 g of pH-responsive nanoprobes obtained in step two under the condition of a stirring speed of 150 rpm, and the stirring is continued for 30 minutes until the color is uniform. After the sample is detected by using a fluorescence microplate reader and the fluorescence intensity CV value is ≤5%, the stirring speed is further reduced to 80 rpm, and 100.0 g of drug-loaded ultrasound-responsive microbubbles obtained in step four are added in layers using a wide-mouth dropper. The addition in layers is as follows: first add 1 / 3, stir for 15 minutes; then add 1 / 3, stir for 15 minutes; and finally add the rest, stir for 30 minutes to obtain a mixed gel. The mixed gel is then transferred to a 2L vacuum dryer, and the pressure is maintained at -0.05 MPa for 15 minutes, then at -0.08 MPa for 15 minutes, and finally at -0.095 MPa for 15 minutes before returning to normal pressure to obtain an intelligent medical coupling agent.

9. An intelligent medical couplant, characterized by, The preparation method of the intelligent medical coupling agent according to any one of claims 1 to 8 is prepared, the intelligent medical coupling agent integrates pH-responsive nanoprobes and drug-loaded ultrasound-responsive microbubbles, converts the biochemical information pH value into a detectable fluorescence signal through pH-responsive nanoprobes fluorescence imaging, and triggers the drug release function of the drug-loaded ultrasound-responsive microbubbles through ultrasound.

10. The use of the intelligent medical coupling agent according to claim 9 in the preparation of a medical coupling agent material.

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