Drug detection method based on micron drug motor and DESI-MSI technology

Abstract

The invention discloses a drug detection method based on a micron drug motor and a DESI-MSI technology, and belongs to the technical field of drug analysis and mass spectrometric detection.The method comprises the steps that (1) the micron drug motor is prepared, the micron drug motor comprises active metal particles and a polylactic acid-glycolic acid copolymer layer deposited on part of the surfaces of the active metal particles, and the active metal particles are dispersed in the polylactic acid-glycolic acid copolymer layer; a medicine layer and a chitosan layer are sequentially arranged on the outer surface of the polylactic acid-glycolic acid copolymer layer from inside to outside; (2) after an experimental animal takes in the micron drug for a certain time, taking out animal tissues, putting the animal tissues into an embedding medium, freezing, slicing, performing DESI-MSI analysis, acquiring corresponding mass spectrum imaging data, and obtaining a distribution result of the drug in the animal tissues. The method is high in detection sensitivity and shows important application potential in precise treatment and curative effect evaluation of related diseases.

Description

Technical Field

[0001] This invention belongs to the field of drug analysis and mass spectrometry detection technology, specifically relating to a method for drug detection based on a micron-sized drug-pharmaceutical motor and DESI-MSI technology. Background Technology

[0002] Desorption electrospray ionization mass spectrometry imaging (DESI-MSI) is a novel analytical chemistry technique. With its unique advantages of being label-free, in-situ, and high-throughput, it can directly acquire spatial distribution information of chemical components on the sample surface under ambient pressure. This technique is applicable to both solid and liquid samples, requiring minimal or no sample pretreatment for direct analysis. DESI-MSI technology provides a powerful solution for drug distribution studies, analysis of metabolic heterogeneity in diseased tissues, and spatial localization of biomarkers.

[0003] Micromotors are a class of micrometer-scale (1~100 μm) drug-loaded materials with autonomous movement capabilities or the ability to respond to external stimuli. They can convert energy from the external environment into their own kinetic energy and can be driven by chemical fuels, ultrasound, light, and bioenzymes, showing broad application prospects in targeted drug delivery, precision medicine, biosensing, and environmental remediation. Micromotors can load drugs through physical embedding, covalent coupling, electrostatic adsorption, and pore filling. Unlike traditional passive diffusion-based drug delivery methods, these micromotors can actively navigate to the target site and release drugs on demand, thereby significantly improving therapeutic efficacy and reducing side effects.

[0004] DESI-MSI suffers from low detection sensitivity in drug analysis. Chinese patent document CN115753956A discloses a method for processing DESI mass spectrometry imaging of biological brain tissue, including the following steps: first, animal brain tissue is taken, frozen, and sectioned to obtain brain tissue slices; then, the brain tissue slices are adhered to a glass slide and frozen for preservation; then, the frozen slide with the adhered brain tissue slices is taken and subjected to DESI mass spectrometry imaging; the scan data is processed by data processing software to export ion imaging maps and mass spectra. However, the feasibility of this method needs to be proven if other DESI scanning equipment with lower precision than that used in this invention is employed; furthermore, this invention only targets DESI imaging enhancement of brain tissue, and its effect on DESI enhancement of other tissues and organs is not yet clear. Chinese patent document CN119985664A discloses a method for studying the specific distribution of multi-component complex systems in animal tissues based on mass spectrometry imaging and multivariate statistical analysis. The method includes: treating a multi-component complex system (a traditional Chinese medicine system) and administering it to animals for a certain period; euthanizing the animals and collecting their tissues; performing mass spectrometry imaging analysis and multivariate statistical analysis on the tissue sections to obtain the specific distribution results of the multi-component complex system in the animal tissues. This invention establishes a DESIMSI analysis method for whole mouse tissue sections based on frozen sectioning and mass spectrometry imaging techniques. However, this invention only targets the distribution in animal brain tissue and cannot fundamentally improve detection sensitivity. Summary of the Invention

[0005] To enhance the detection sensitivity of DESI-MSI technology, this invention provides a method for drug detection based on a micron-sized drug-pharmaceutical motor and DESI-MSI technology. This method can be used to analyze the uptake and distribution of drug molecules in biological tissues, has high detection sensitivity, and is beneficial for the detection of low-content drugs and in-situ drug molecule imaging.

[0006] The specific technical solution adopted is as follows: A method for drug detection based on a micron-sized drug-motor and DESI-MSI technology includes the following steps: (1) Preparation of a micron-sized drug motor, wherein the micron-sized drug motor comprises active metal particles and a polylactic acid-glycolic acid copolymer layer deposited on a portion of the surface of the active metal particles, wherein a drug layer and a chitosan layer are sequentially disposed on the outer surface of the polylactic acid-glycolic acid copolymer layer from the inside to the outside; wherein the particle size of the micron-sized drug motor is 20~45 μm, and the active metal particles are partially exposed to form an opening with a diameter of 5~15 μm, wherein the active metal particles are selected from magnesium particles and zinc particles, which can generate hydrogen gas by reacting with gastric acid to drive the motor to move, thereby realizing the active penetration of the drug into the gastric mucosa, and having good biosafety; (2) After the experimental animal ingests the micron drug motor of step (1) for a certain period of time, the animal tissue is taken out, the animal tissue is placed in the embedding agent, frozen and sliced, and DESI-MSI analysis is performed to obtain the corresponding mass spectrometry imaging data and obtain the distribution results of the drug in the animal tissue. The animal tissue in question is animal kidney tissue.

[0007] The kidney possesses a highly heterogeneous micron-scale structure, comprising tightly packed functional units such as the cortex, medulla, renal corpuscles (glomeruli + Bowman's capsule), proximal / distal tubules, and loops of Henle. These structures are interwoven within a range of hundreds of micrometers, posing a significant challenge to the spatial resolution of DESI-MSI. Furthermore, as a metabolic and excretory organ, kidney tissue contains high concentrations of salts, urea, and drug metabolites, severely interfering with ionization efficiency and leading to a high degree of signal suppression. In other words, detecting drug distribution within kidney tissue is challenging. To address these technical problems, this invention develops a micron-sized drug delivery motor for efficient and precise drug delivery, enabling prolonged drug accumulation and retention at the target site. Combined with DESI-MSI technology, this allows for drug detection, revealing drug intensity and tissue distribution, which is of great significance for enhancing the sensitivity of mass spectrometry detection and imaging.

[0008] Furthermore, the drug in the drug layer is canagliflozin (CANA), which is used to treat diabetic nephropathy, and the loading of canagliflozin on the micron-sized drug delivery motor is 180~220 μg / mg.

[0009] Furthermore, the experimental animals were diabetic nephropathy model mice. After ingesting the micron drug motor from step (1) for 4 to 12 hours, the mice were sacrificed, and the kidney tissue was removed and stored at -75 to -85°C for later use.

[0010] Preferably, the micron-sized drug motor is prepared by the following method: active metal particles are placed on a carrier plate, and then coated sequentially with an ethyl acetate solution of polylactic acid-glycolic acid copolymer, a drug solution, and a chitosan solution. After each coating, the particles are dried, and the product is separated from the carrier plate to obtain the micron-sized drug motor.

[0011] Preferably, the encapsulating agent is a sodium carboxymethyl cellulose solution with a concentration of 0.5~5 wt%.

[0012] Specifically, before starting the slicing operation, the temperature of the microtome's freezing chamber is set to -15~-25 ℃ and the temperature of the blade is set to -25~-35 ℃ for pre-cooling. The slice thickness is 10 μm~20 μm, and the resulting slices are stored at -75~-85 ℃ for later use.

[0013] Preferably, during the DESI-MSI experiment, the spray capillary solvent is an acetonitrile and water system, and formic acid is used as a correction solution; more preferably, it is 70%~90% acetonitrile ACN + 10%~30% H2O (containing 0.1% formic acid FA), and most preferably, it is 80% ACN + 20% H2O (containing 0.1% formic acid).

[0014] Preferably, the parameters of DESI-MSI also include: a spray capillary solvent flow rate of 0.5~2 μL / min, a gas pressure of 10~15 psi, a spray voltage of 0.5~1 kV, a capillary temperature of 100~120 ℃, a molecular weight detection range of 100-1500 Da; a sampling mode of negative ion mode, a pixel size of 50 μm×50 μm ~200 μm×200 μm, and a scanning rate of 100~200 μm / s.

[0015] For the drug canagliflozin (CANA), in negative ion mode, its ion signals are located around 443.1328, 479.1089, and 489.1379, which belong to [CANA-H]. - [CANA+Cl] - and [CANA+HCOO] - .

[0016] By setting characteristic mass spectrometry peaks for the target ions and conducting control experiments, the distribution of drugs in tissues and the impact of using micron-sized drug-pharmaceutical motors on drug detection sensitivity can be elucidated.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) The method for drug detection based on micron drug motor and DESI-MSI technology provided by the present invention is beneficial for low-content drug detection and in-situ drug molecule imaging, thereby reducing the toxicity of high-dose administration. It shows important application potential in the precision treatment and efficacy evaluation of related diseases. Moreover, the method is simple and efficient, and the results are reproducible.

[0018] (2) It is difficult to detect the distribution of drugs in kidney tissue. The method of the present invention utilizes micron drug motor coupled with DESI-MSI technology to actively transport drugs in the kidney and improve the local drug concentration. Combined with DESI-MSI technology, it can significantly improve imaging contrast and quantitative accuracy, and realize accurate detection of drug distribution in kidney tissue. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the structure and function of the micron-sized drug delivery motor Mg / PLGA / CANA / CHI.

[0020] Figure 2(a) shows the drug release mass spectrum of the micron-sized drug motor Mg / PLGA / CANA / CHI in 1% Tween PBS buffer, and (b) shows the mass spectrum of CANA in 1% Tween PBS buffer.

[0021] Figure 3 (a) is the chromatogram of drug release from the micron-sized drug-eluting motor Mg / PLGA / CANA / CHI in 1% Tween PBS buffer, and (b) is the chromatogram of CANA in 1% Tween PBS buffer.

[0022] Figure 4 DESI-MSI images of kidney sections from different groups of mice, where (a) represents [CANA-H]. - (b) is [CANA+Cl] - (c) is [CANA+HCOO] - . Detailed Implementation

[0023] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0024] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0025] Example 1: Micron-sized drug delivery motor Mg / PLGA / CANA / CHI Magnesium powder was washed three times with acetone to remove impurities, then vacuum dried. 2.0 mg of magnesium particles, each 20–40 μm in size, were then evenly sprinkled onto a glass slide (6.25 cm²). 2The mixture was then coated with 120 μL of a 20 mg / mL polylactic-co-glycolic acid copolymer (PLGA) ethyl acetate solution and dried overnight. Next, it was coated with 300 μL of a 10 mg / mL canagliflozin (CANA) ethanol solution and rapidly dried in a 75°C oven. Following this, it was coated with 100 μL of a 0.5 mg / mL chitosan (CHI) solution and dried at room temperature for 15 min. After scraping, a micronized drug-eluting motor Mg / PLGA / CANA / CHI was obtained. The particle size of this micronized drug-eluting motor was 20–45 μm, with locally exposed Mg cores forming openings with a diameter of 5–15 μm. Its structure is as follows. Figure 1 As shown.

[0026] The prepared micron-sized drug delivery motor Mg / PLGA / CANA / CHI was placed in a solution containing 1% Tween PBS and detected by UPLC-HRMS. The UPLC-HRMS detection conditions are as follows: The chromatographic column was a C18 column with a packing particle size of 1.7 μm, an inner diameter of 2.1 mm, a column length of 50 mm, a column temperature of 4 ℃, and an injection volume of 1 μL. The mobile phase A was water, and mobile phase B was acetonitrile, with a volume ratio of A to B of 95:5, and a flow rate of 0.4 L / min. Mass spectrometry detection conditions were as follows: molecular weight / mass-to-charge ratio detection range: 50-1200 Da; correction solution: sodium formate; real-time standard: leucine enkephalin; capillary voltage: 1.0 kV.

[0027] The detection results showed that the CANA released by the micron-sized drug delivery motor exhibited a single peak at 3.69 min in negative ion mode, and CANA fragment ions appeared at 479.1089 and 489.1378, which were attributed to [CANA+Cl], respectively. - and [CANA+HCOO] - This is consistent with the test results of pure CANA in the same solution, proving that the micron-sized drug delivery motor successfully loaded CANA without significantly affecting the ionic signal and intensity of CANA. The detection results are as follows: Figure 2 (a) and (b) in the middle, and Figure 3 As shown in (a) and (b) in the figure.

[0028] The above demonstrates the successful preparation of the micron-sized drug-drive motor Mg / PLGA / CANA / CHI, which can generate hydrogen gas by reacting with gastric acid to drive the motor, thereby enabling the active penetration of drugs into the gastric mucosa.

[0029] Example 2 Drug release behavior The drug-eluting micron-sized motors Mg / PLGA / CANA / CHI and Mg / PLGA / CHI unloaded particles (their preparation method differs from that of the drug-eluting micron-sized motor in Example 1 only in that the CANA coating step is omitted, while the other steps and parameters are the same) were tested in simulated gastric fluid containing 1% Tween to observe the release behavior of CANA from the carrier.

[0030] The aforementioned micron-sized drug-eluting motors were dispersed in simulated gastric fluid containing 1% Tween, with three groups set up. The mixture was incubated in a shaker at 37°C and 100 rpm for 12 h. After 12 h, 100 μL of supernatant was taken from the simulated gastric fluid and diluted to 1 mL with simulated gastric fluid containing 1% Tween. The absorption peak of CANA at 295 nm was measured using a UV-Vis spectrophotometer to calculate the loading capacity and loading efficiency of the CANA micron-sized drug-eluting motor.

[0031] Calculations show that the loading capacity of CANA on the drug-loaded micromotor is 180~220 μg / mg, and the loading rate is 20~27%.

[0032] Example 3: Preparation of tissue sections Five groups of mice were set up: healthy mice, diabetic nephropathy mice (DN), diabetic nephropathy mice orally administered CANA (CANA), diabetic nephropathy mice orally administered unloaded motor carrier (MG), and diabetic nephropathy mice micron-loaded motor (MOTOR). The MOTOR and CANA groups were administered CANA by gavage at a standard dose of 7.5 mg / kg, while the healthy mice and DN groups were administered an equal volume of PBS buffer by gavage.

[0033] All mice were fasted for 12 hours before the experiment. After being gavaged for 4 hours, the mice were euthanized by cervical dislocation. The kidneys were surgically removed, rinsed in sterile PBS, blotted dry with clean filter paper, and stored at -80 °C until sectioning.

[0034] Before starting the sectioning operation, the cryostat was pre-cooled by setting the freezing chamber temperature to -20 ℃ and the blade temperature to -31 ℃. The kidney tissue was removed and placed in a container with an appropriate amount of 4 wt% CMC-Na embedding medium and then rapidly frozen in dry ice. The section thickness was adjusted to 15 μm. After trimming the section to the largest surface of the kidney tissue, the sectioning began. After transferring the kidney sections to glass slides, they were stored in a -80 ℃ freezer until the subsequent DESI-MSI experiment.

[0035] Example 4 DESI-MSI Analysis The DESI-MSI equipment parameters were set as follows: spray capillary solvent was 80% ACN + 20% H2O (containing 0.1% FA), flow rate was 2 μL / min, gas pressure was 13 psi, spray voltage was 0.8 kV, capillary temperature was 120 ℃, and molecular weight detection range was 100-1200 Da. The sampling mode was negative ion mode, pixel size was 100 μm × 100 μm, scan rate was 200 μm / s, and the imaging data was processed using HDI 1.7 software to obtain the final DESI image, as shown below. Figure 4 As shown.

[0036] In the healthy mouse group and the DN group that did not use CANA, the mass spectrometry signals generated were all background signals.

[0037] Figure 4 (a) indicates that there is no... m / z [CANA-H] was observed at position 443.1328. - The signal.

[0038] Figure 4 (b) indicates that almost no [CANA+Cl] was detected in the CANA and MG groups. - Ion signals. Compared to the CANA and MG groups, a strong CANA signal was detected in the MOTOR group, and CANA was mainly distributed in the renal cortex, [CANA+Cl]. - The strength of the ions depends in part on the concentration of chloride ions in the kidney tissue.

[0039] Figure 4 (c) indicates that a mass-to-charge ratio of 489.1379 was detected only in the MOTOR group, determined to be [CANA+HCOO]. - The signal, and Figure 4 The results are similar to those in (b). These results indicate that the micron-sized drug-injection motor Mg / PLGA / CANA / CHI significantly improves the detection sensitivity of CANA. Conventional oral administration combined with DESI-MSI technology cannot reveal the drug distribution of CANA, while the micron-sized drug-injection motor combined with DESI-MSI technology provides high imaging quality, significantly superior to conventional methods. CANA is mainly distributed in the renal cortex.

[0040] The embodiments described above provide a detailed explanation of the technical solutions of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, additions, or similar substitutions made within the scope of the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology, characterized in that, Includes the following steps: (1) A micron-sized drug motor is prepared, wherein the micron-sized drug motor includes active metal particles and a polylactic acid-glycolic acid copolymer layer deposited on a portion of the surface of the active metal particles. A drug layer and a chitosan layer are sequentially disposed on the outer surface of the polylactic acid-glycolic acid copolymer layer from the inside to the outside. The particle size of the micron-sized drug motor is 20~45 μm, and the active metal particles are partially exposed to form an opening with a diameter of 5~15 μm. The active metal particles are selected from magnesium particles or zinc particles. (2) After the experimental animal ingests the micron drug motor of step (1) for a certain period of time, the animal tissue is taken out, the animal tissue is placed in the embedding agent, frozen and sliced, and DESI-MSI analysis is performed to obtain the corresponding mass spectrometry imaging data and obtain the distribution results of the drug in the animal tissue. The animal tissue in question is animal kidney tissue.

2. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 1, characterized in that, The drug in the drug layer is canagliflozin.

3. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 2, characterized in that, The loading capacity of canagliflozin on the micron-sized drug delivery motor is 180~220 μg / mg.

4. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 2, characterized in that, The experimental animals were diabetic nephropathy model mice. After ingesting the micron drug motor in step (1) for 4 to 12 hours, the mice were sacrificed and the kidney tissue was removed and stored at -75 to -85°C for later use.

5. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 1, characterized in that, The micron-sized drug motor is prepared by the following method: active metal particles are placed on a carrier plate, and then coated sequentially with an ethyl acetate solution of polylactic acid-glycolic acid copolymer, a drug solution, and a chitosan solution. After each coating, the particles are dried, and the product is separated from the carrier plate to obtain the micron-sized drug motor.

6. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 1, characterized in that, The embedding agent is a sodium carboxymethyl cellulose solution with a concentration of 0.5~5 wt%.

7. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 1, characterized in that, Before starting the slicing operation, set the temperature of the microtome's freezing chamber to -15~-25 ℃ and the temperature of the blade to -25~-35 ℃ for pre-cooling. The slice thickness should be 10 μm~20 μm. The resulting slices should be stored at -75~-85 ℃ for later use.

8. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 1, characterized in that, During the DESI-MSI experiment, the solvent in the spray capillary was a system of acetonitrile and water, and formic acid was used as a correction solution.

9. The method for drug detection based on a micron-sized drug-eluting motor and DESI-MSI technology according to claim 1, characterized in that, The parameters of DESI-MSI also include: spray capillary solvent flow rate of 0.5~2 μL / min, gas pressure of 10~15 psi, spray voltage of 0.5~1 kV, capillary temperature of 100~120 ℃, molecular weight detection range of 100-1500 Da; sampling mode of negative ion mode, pixel size of 50 μm×50 μm~200 μm×200 μm, and scanning rate of 100~200 μm / s.

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