CEST-based postoperative residual cavity drug delivery system slow release function detection method and system

Abstract

The invention discloses a CEST-based postoperative residual cavity drug delivery system slow release function detection method and system. The method comprises the following steps: preparing a drug delivery system and constructing a GBM animal model; performing postoperative intervention on the GBM animal model by using a drug delivery system; performing CEST imaging on the GBM animal model at a plurality of time points after the postoperative intervention; and detecting the slow release function of the drug delivery system according to the change of the CEST signals at different time points. According to the CEST effect of the medicine, the medicine is applied to the medicine delivery system, the medicine delivery system and distribution and metabolism of the medicine in the medicine in a postoperative residual cavity are monitored through the CEST effect of the medicine in the medicine, and a local treatment medicine delivery system which is free of radiation, does not need additional marking and can be monitored in real time is expected to be provided for improving the prognosis of GBM.

Description

Technical Field

[0001] The present invention relates to the field of magnetic resonance imaging, and particularly to a method and system for detecting the sustained-release function of a postoperative residual cavity drug delivery system based on CEST. Background Art

[0002] Glioblastoma multiforme (GBM) is the most common primary malignant brain tumor in the intracranial cavity, with a median survival of less than two years. Despite recent progress in the multimodal treatment of GBM, including surgery, radiotherapy, systemic treatment (chemotherapy, targeted therapy), and supportive care, the overall prognosis remains poor, and long-term survival is rare. In addition, the progressive deterioration of the patient's neurological function and quality of life has a devastating impact on both the patient and the family, imposing a heavy burden on society.

[0003] The current treatment effect of GBM is still not satisfactory, and the prognosis of patients is poor. The current treatment guideline for GBM is to start systemic treatments such as radiotherapy and chemotherapy about 3 weeks after the maximum resection of the tumor within the safe range when the patient's condition is stable. However, during this period, the remaining tumor cells that have not been completely removed begin to recur, affecting the prognosis of the patient. Therefore, after GBM resection, placing local treatment drugs in the tumor residual cavity during the operation is expected to inhibit the recurrence of GBM and improve its treatment effect. Compared with systemic treatment, local treatment bypasses the blood-brain barrier, increases the local concentration at the tumor site, and reduces or avoids systemic side effects, thus opening the door for more molecules to combat this devastating disease.

[0004] How to apply the method of imaging to monitor the distribution, dosage and efficacy of drug delivery systems in vivo is an issue that needs to be addressed in the field of imaging. Not only experts in the imaging field need to consider these problems, but also experts in fields such as material chemistry need to seriously consider that during the design of drug delivery systems, by adding probes or contrast agents to the system, the injection, degradation and release processes of the drug delivery system can be observed by imaging methods. Therefore, monitoring hydrogels in vivo is a key element in evaluating and characterizing this biomaterial. In traditional animal studies, after a period of implanting biomaterials into animals, the animals are sacrificed and corresponding tissues are taken for histological analysis. Although useful information can be obtained, this method has the following problems: 1) It is destructive; 2) It may change the properties of the biomaterial itself during histological procedures; 3) It cannot provide information on the functional state of the biomaterial; 4) It cannot provide accurate volume assessment; 5) It does not allow longitudinal imaging studies over time; 6) It requires the use of a large number of animals. Therefore, non-invasive methods need to be developed to monitor and evaluate the morphological, functional and molecular information of hydrogels in vivo. Currently, common imaging methods for drug delivery systems include fluorescence imaging (FLI), photoacoustic imaging (PAI), MRI and radionuclide imaging (RNI), etc. If the drug concentration can be monitored through the contrast agent effect of the drug delivery system itself, the side effects of additional contrast agents can be avoided, and the manufacturing process is also simpler, with good clinical translation potential. Summary of the Invention

[0005] The object of the present invention is to solve the problems in the prior art.

[0006] The technical solution adopted by the present invention to solve its technical problems is: to provide a method for detecting the sustained-release function of a postoperative residual cavity drug delivery system based on CEST, including the following steps:

[0007] Prepare a drug delivery system and construct a GBM animal model;

[0008] Use the drug delivery system to perform postoperative intervention on the GBM animal model;

[0009] Perform CEST imaging on the GBM animal model at several time points after postoperative intervention;

[0010] Detect the sustained-release function of the drug delivery system according to the changes in CEST signals at different time points.

[0011] Preferably, the preparation of the drug delivery system includes the following steps:

[0012] According to the molar concentration calculation formula, configure a drug solution with the required concentration and adjust the pH value to 7.4;

[0013] Weigh the corresponding amount of poloxamer according to the formula of 20% weight or volume content of poloxamer 407 and 2% weight or volume content of poloxamer 188 into a 20 mL glass bottle, add the drug solution, and stir at 400 rpm under ice bath until completely dissolved to obtain the H@ATC drug delivery system;

[0014] Place the H@ATC drug delivery system in a 4-degree refrigerator and store it in the dark overnight for later use.

[0015] Preferably, the drug solution is doxorubicin solution.

[0016] Preferably, the preparation of the drug delivery system further includes: using a test tube model to confirm whether the drug has CEST effect; if the drug has CEST effect, continue to the next step; if the drug does not have CEST effect, end the execution and draw the conclusion that the sustained-release function of the drug delivery system cannot be detected.

[0017] Preferably, the preparation of the drug delivery system further includes: confirming whether the drug delivery system is gel-like at 37 °C; if the drug delivery system is gel-like at 37 °C, continue to the next step; when the drug delivery system is not gel-like at 37 °C, re-prepare the drug delivery system.

[0018] Preferably, the construction of the GBM animal model is specifically: slowly inject the C6 glioma cell suspension into the right basal ganglia region of a normal rat model to obtain the GBM animal model.

[0019] Preferably, taking the time of constructing the GBM animal model as the first day, perform postoperative intervention on the GBM animal model with the drug delivery system on the seventh day; the postoperative intervention includes the following steps:

[0020] Excise the tumor tissue of the GBM animal model;

[0021] Inject the drug delivery system into the surgical area.

[0022] Preferably, perform CEST imaging on the animal model at several time points after GBM postoperative intervention, and the process of performing CEST imaging at each time point includes the following steps:

[0023] Collect the brain MRI image of the animal model to locate the CEST imaging plane;

[0024] Collect CEST images through a continuous wave echo planar imaging sequence.

[0025] Preferably, the collection of the brain MRI image of the animal model to locate the best CEST imaging plane includes the following steps:

[0026] Locate the mid-coronal, sagittal, and transverse positions in the phase;

[0027] Calibrate the frequency and energy, and obtain the axial T2-weighted image of the mouse brain through a fast spin echo imaging sequence;

[0028] Collect the B0 map sequence for later use in calibrating CEST imaging;

[0029] Select the slice in the T2-weighted image that shows the best view of the rat's surgical area as the slice for CEST imaging.

[0030] The present invention also provides a detection system for the sustained release function of a postoperative residual cavity drug delivery system based on CEST, including the following modules:

[0031] A preparation module for preparing the drug delivery system and constructing a GBM animal model;

[0032] An intervention module for performing postoperative intervention on the GBM animal model using the drug delivery system;

[0033] An imaging module for performing CEST imaging on the GBM animal model at several time points after the postoperative intervention;

[0034] A detection module for detecting the sustained release function of the drug delivery system according to the changes in the CEST signals at different time points.

[0035] The present invention has the following beneficial effects: According to the CEST effect of the drug, the present invention applies it to the drug delivery system. Inject a drug delivery system containing ADR doxorubicin into the postoperative residual cavity of the GBM resection animal model, and monitor the distribution and metabolism of the drug delivery system and the drug therein in the postoperative residual cavity through the CEST effect of ADR doxorubicin therein, which is expected to provide a non-radiative, non-additional labeling and real-time monitorable local treatment drug delivery system for improving the prognosis of GBM.

[0036] The following further elaborates on the present invention in detail with reference to the accompanying drawings and embodiments, but the present invention is not limited to the embodiments. Description of the Drawings

[0037] Figure 1 It is a method step diagram of an embodiment of the present invention;

[0038] Figure 2 It is a flow schematic diagram of an embodiment of the present invention;

[0039] Figure 3 It is an experimental effect diagram of an embodiment of the present invention;

[0040] Figure 4 It is a comparison schematic diagram between an embodiment of the present invention and near-infrared imaging;

[0041] Figure 5 It is a system structure diagram of an embodiment of the present invention. Detailed Description of the Embodiment

[0042] See Figure 1 As shown, it is the method step diagram of the embodiment of the present invention, including the following steps:

[0043] S101, Prepare a drug delivery system and construct a GBM animal model;

[0044] S102, Use the drug delivery system to perform postoperative intervention on the GBM animal model;

[0045] S103, Perform CEST imaging on the GBM animal model at several time points after postoperative intervention;

[0046] S104, Detect the sustained-release function of the drug delivery system according to the changes in CEST signals at different time points.

[0047] The embodiment of the present invention takes doxorubicin, temozolomide, a first-line drug for glioblastoma treatment, and chloroquine, an anti-tumor drug that inhibits tumor autophagy, as examples to illustrate the specific process of the present invention. For the process schematic diagram, see Figure 2 .

[0048] Specifically, the preparation of the drug delivery system in S101 includes the following steps:

[0049] S201, Prepare a blank hydrogel.

[0050] Poloxamer 407 was purchased from Ron-Guangzhou Kemon Biotechnology Co., Ltd., and poloxamer 188 was purchased from Shanghai Bide Pharmaceutical Technology Co., Ltd. A blank hydrogel was prepared using poloxamer 407 and poloxamer 188. Weigh the corresponding amounts of poloxamer 407 and poloxamer 188 into a 20 mL glass bottle, add 5 mL of ultrapure water pre-cooled to 4 °C, so that the concentration of poloxamer 407 is 20% (w / v), and the concentrations of poloxamer 188 are 0%, 1%, 1.5%, 2%, 2.5%, 3% (w / v), respectively. Subsequently, stir at 400 rpm under ice bath until the mixture is completely dissolved, and store it overnight at 4 °C.

[0051] For the thermosensitive performance test of the hydrogel, the sol-gel transition temperature of the hydrogel was recorded by the test tube inversion method. Specifically, take out 1 mL of the prepared hydrogel solution from a 4 °C refrigerator and put it into a 3 mL transparent glass sample bottle. Place the transparent glass sample bottle in a constant temperature water bath and gradually heat it at a heating rate of 0.1 °C / 3 min. Observe the state of the hydrogel in the glass sample bottle when it is inverted. When the hydrogel stops flowing, immediately insert a baking thermometer into the sample bottle to measure the system temperature. This temperature is determined as the sol-gel transition temperature, and record the data to select a solution with a hydrogel state at 37 °C.

[0052] S202, Prepare an aqueous drug solution.

[0053] Doxorubicin (Shanghai Macklin Biochemical Co., Ltd.) is a small molecule anti-cancer compound with a molecular weight of 543.50. According to the molar concentration calculation formula: mass (mg) = concentration (mM) × volume (mL) × molecular weight (g / mol) × 10 -3 , weigh the corresponding mass of doxorubicin powder, add an appropriate amount of normal saline, vortex and ultrasonicate to obtain doxorubicin solutions with concentrations of 1.0 mM, 2.5 mM, 5.0 mM, 10.0 mM, 15.0 mM and 20.0 mM, adjust its pH to 7.4, and then aliquot into 2 ml NMR tubes and label for standby.

[0054] Temozolomide (Shanghai Macklin Biochemical Co., Ltd.) is a small molecule anti-cancer compound with a molecular weight of 194.15. Referring to the above calculation formula, a 30 mM temozolomide solution was prepared to explore whether it has CEST effect.

[0055] Chloroquine (Shanghai Macklin Biochemical Co., Ltd.) is a small molecule compound with a molecular weight of 319.87. Referring to the above formula, a 30 mM chloroquine solution was prepared to explore whether it has CEST effect.

[0056] S203, prepare drug-loaded hydrogel

[0057] The method is similar to the preparation method of the blank hydrogel. The prepared doxorubicin, temozolomide and chloroquine with concentrations of 15 mM, 10 mM and 10 mM respectively were stored at 4 °C. Weigh the corresponding amount of poloxamer according to the formula of 20% (w / v) poloxamer 407 and 2% (w / v) poloxamer 188 in a 20 mL glass bottle, add the prepared drug solution, and stir at 400 rpm under ice bath until completely dissolved to obtain the H@ATC (H: hydrogel, A: Adriamycin, T: Temozolomide, C: Chloroquine) drug delivery system; store it in the dark at 4 °C in the refrigerator overnight for use.

[0058] S204, adjust the pH.

[0059] The pH of the aqueous drug solution or gel is adjusted by using 1 M sodium hydroxide solution, hydrochloric acid solution and with the aid of a pH meter. Take out the blank hydrogel stored at 4 °C, wait for it to return to room temperature, adjust it with sodium hydroxide solution and hydrochloric acid solution, and measure it with a pH meter to obtain a blank hydrogel with pH = 7.4, and store it at 4 °C. The pH adjustment of different aqueous drug solutions and drug gels refers to the above method (all adjusted to pH = 7.4).

[0060] S205, prepare tube models.

[0061] First, make a base that can fix the test tube using a 50 mL large test tube. Then, weigh 2 g of agarose with a balance, dissolve it in 100 mL of ultrapure water, stir evenly, and place it in a microwave oven to heat for about 4 minutes until the agarose is completely dissolved without precipitation, clear and transparent. Then pour it into the prepared large test tube. Finally, insert different numbers of empty 2 mL NMR tubes into it, fix the NMR tubes, wait for at least 2 hours, and let the agarose solution cool naturally to transform into a jelly-like state. At this time, pull out the empty NMR tubes and insert the test tubes prepared according to the experimental design.

[0062] S206, evaluate the CEST effect of the drug using a test tube model.

[0063] In the embodiment of the present invention, magnetic resonance imaging is performed using a 7.0T small animal magnetic resonance imaging instrument (Agilent Technologies, Santa Clara, CA, USA). When scanning the test tube model, the standard Q72 body coil (Time medical technology Co., Ltd., USA) equipped with the system is used for signal transmission, and the surface coil (Time medical technology Co., Ltd., USA) is used for signal reception.

[0064] The specific steps of magnetic resonance scanning of the test tube model are as follows: First, place the NMR tube in agarose in the bracket of the magnetic resonance imaging device at room temperature and fix it at the center position of the magnetic field to optimize the signal-to-noise ratio and the linearity of the main magnetic field; then, use the gradient echo imaging sequence to scan the localization phase to determine the center position of the NMR tube. The scanning parameters of the coronal, sagittal, and transverse positions in the localization phase are: field of view = 50×50mm, TR = 30ms, TE = 5ms, matrix = 128×128, slice gap = 2mm, slice thickness = 2mm; Second, correct the frequency and energy, and obtain the T2-weighted anatomical image of the axial position of the test tube through the fast spin echo imaging sequence. The imaging parameters of the T2-weighted anatomical image are: field of view = 35mm×35mm, TR = 2000ms, TE = 28.93ms, matrix = 128×128, ESP = 7.23ms, ETL = 8, NEX = 2, slice thickness = 1.5mm, slice interval = 0.5mm, number of slices = 7; Then collect the B0 map sequence to correct the influence of the inhomogeneity of the B0 field on the experimental results through an algorithm in the later stage; the imaging parameters of the B0 map are: TR = 40ms, TE = 3ms, 3.5ms, 4ms, matrix = 64×64, field of view = 35mm×35mm, NEX = 6, FA = 15°, slice thickness = 1.5mm, number of slices = 1; Finally, through the continuous wave echoplanar imaging (CW-EPI) imaging sequence, collect the CEST of the above-mentioned layer. The scanning parameters of the EPI-CEST sequence are: select the same layer as the B0 map, TR = 6000ms, TE = 28.5ms, field of view = 35×35mm, matrix = 64×64, NEX = 1, slice thickness = 1.5mm, number of slices = 1; the offset frequency range is -15ppm to +15ppm, the number of collected images = 122 (including 1 unsaturated image), and the scanning time is 13 minutes and 11 seconds.

[0065] Perform post-processing on the CEST images to evaluate the CEST effect. Use MATLAB software (MathWorks, USA) version R2021a to post-process all the magnetic resonance images collected in this study, and the processing program is written by our research group. First, for test tube imaging, set a circular region of interest (about 5mm in diameter) consistent with the test tube diameter on the transverse image read by the computer as the region of interest (ROI); for rat brain imaging, manually outline the modeled area (5mm deep, 2mm in diameter) on the coronal image read by the computer as the ROI; then, use B0 to calibrate the CEST image through water saturation shift calibration (WASSR). The CEST ratio (CESTR) is defined by the following equation:

[0066] CESTR = [M sat(-Δω) - M sat(+Δω)] / M0;

[0067] Among them, M sat(-Δω) and M sat(+Δω) respectively refer to the magnetization intensities obtained at the "-" and "+" offsets symmetric to the water resonance frequency; M0 is the unsaturated magnetization intensity.

[0068] The near-infrared imaging of rats uses the non-intervened side brain ROI (symmetric to the intervened side brain ROI) on the first day as a control, and calculates the ratio of the near-infrared signal intensity of the intervened side brain ROI (centered at the place with the strongest near-infrared signal, with a diameter of 2 mm) on the 1st, 7th, and 14th days to the near-infrared signal intensity of the non-intervened side brain ROI on the first day.

[0069] S207, ensure the gel state through rheological tests.

[0070] Use a rheometer to perform rheological analysis on the blank hydrogel and the gel of the H@ACT drug delivery system. At a constant strain, measure the storage modulus (G') and loss modulus (G") at a temperature range of 4 - 40 °C with a heating rate of 1 °C / min and at continuous frequencies of (0.1–100 rad / s). Perform thixotropy analysis on the blank hydrogel and the gel of the H@ACT drug delivery system at a frequency of 1 rad / s. Record G′ and G″ of each gel sample. The changes in the storage modulus (G') and loss modulus (G") of the hydrogel and the hydrogel drug delivery system with temperature can characterize the temperature responsiveness of the hydrogel and the hydrogel drug delivery system; only when the hydrogel drug delivery system is in a gel state rather than a sol state at the in vivo temperature of 37 °C can it adhere to the postoperative residual cavity and achieve the effect of slow release of anti-tumor drugs.

[0071] Specifically, according to the CEST imaging results in the embodiments of the present invention, doxorubicin has the CEST effect, while temozolomide and chloroquine do not.

[0072] Specifically, the construction of the GBM animal model in S101 includes the following steps:

[0073] S301, preparation of a normal animal model. Use adult SD (Sprague-Dawley) rats with a body weight of about 250 - 300 g as the normal animal model, with males and females each accounting for 50%.

[0074] S302. Construction of GBM animal model. First, the rats were quickly induced to anesthesia under the conditions of 4% isoflurane and 5% O2, and then maintained under anesthesia with 2.5 - 3.0% isoflurane. The respiratory rate and amplitude of the rats were monitored in real time with a small animal electrocardiogram monitor. The rats' heads were fixed on a stereotaxic apparatus, the hair on the top of the head was shaved, and after disinfection with iodophor, the skin was cut along the median sagittal plane of the skull with an ophthalmic scissors to expose the anterior fontanelle, and the punching points were marked (3 mm to the right of the anterior fontanelle, 1 mm forward). A dental drill with a diameter of 1 mm was vertically aligned with the positioning punching point for drilling until the dura mater was reached. Then, a microsyringe was inserted 6 mm and then retracted 1 mm, and 10 μL of C6 glioma cell (10 6 / μL) suspension was slowly injected into the right basal ganglia region over 10 min. After the injection, it stayed in place for about 5 min, and then the microsyringe was slowly withdrawn. The bone hole was sealed with bone wax, the skin was sutured and disinfected, and after making a mark, it was placed in a breeding cage.

[0075] S303. In vivo CEST imaging of the hydrogel drug delivery system.

[0076] Taking the modeling time as the first day, the GBM animal model was intervened on the seventh day. The intervention method was as follows: On the seventh day after tumor inoculation, the rats were quickly induced to anesthesia under the conditions of 4% isoflurane and 5% O2, and then maintained under anesthesia with 2.5 - 3.0% isoflurane. The respiratory rate and amplitude of the rats were monitored in real time with a small animal electrocardiogram monitor. Then, the rats were fixed on a small animal stereotaxic apparatus, the previous modeling site was exposed, an incision was made along the midline of the previous surgical scar, the periosteum was removed, and the drilling area during modeling was exposed. An electric drill was used to drill a circular cranial window with a diameter of about 2 mm centered on the drill hole, and then a biopsy instrument was inserted 5 mm and rotated for 15 s to cut the brain / tumor tissue. After removal, hemostasis was performed on the surgical area. After the surgical area stopped bleeding, the corresponding gel for different groups of rat models was injected into the surgical area through a microsyringe. The skin was sutured and disinfected, and after making a mark, it was placed in a breeding cage.

[0077] After injecting the hydrogel drug delivery system into the rats after GBM surgery, the rats were imaged postoperatively on the 1st, 7th, and 14th days after the intervention (the number of rats at each time point n = 6), and the rats injected with blank gel were imaged on the first day after surgery (n = 3). The specific imaging process was as follows:

[0078] First, the rats were rapidly induced with anesthesia under 4% isoflurane and 5% O2, and then maintained under 2.5 - 3.0% isoflurane. The respiratory rate and amplitude of the rats were monitored in real time using a small animal respiratory pressure sensor (SAII Technologies, USA). The heads of the rats were fixed with a special anesthesia mask, and the front end of the mask had a special pipeline for supplying anesthetic and oxygen to keep the airway unobstructed. After covering the head with a surface coil for the rat head, it was placed in the magnet bore of a 7T MR small animal imaging system with the rat head centered in the magnetic field. During the whole experiment, the amount of anesthetic in the oxygen machine was controlled to keep the rats breathing evenly, with the frequency maintained at 70 - 80 breaths per minute.

[0079] Next, MRI images of the rat brains were acquired. The gradient echo imaging sequence was used to scan the localization phase to determine the center position of the rat brain. The scanning parameters for the coronal, sagittal, and transverse positions in the localization phase were: matrix = 128mm×128mm, field of view = 35×35, TR = 20ms, TE = 3.12ms, flip angle = 20°, slice interval = 2mm, slice thickness = 2mm, number of slices = 3. Secondly, the frequency and energy were corrected, and the axial T2-weighted images of the rat brain were obtained through the fast spin echo imaging sequence. The imaging parameters for the T2-weighted images were: TR = 2000ms, TE = 28.93ms, matrix = 128×128, field of view = 35mm×35mm, ESP = 7.23ms, ETL = 8, NEX = 2, slice thickness = 1.5mm, slice interval = 0.5mm, number of slices = 7. Then, the B0 map sequence was acquired to correct the influence of the inhomogeneity of the B0 field on the experimental results in the later stage (specifically, the CEST images were calibrated using B0 through water saturation shift calibration (WASSR)). The best-displaying slice of the surgical area of the rat was selected for 3D shim automatic shimming, and the imaging parameters for the B0 map were: TR = 40ms, TE = 3ms, 3.5ms, 4ms, matrix = 64×64, field of view = 35mm×35mm, NEX = 6, FA = 15°, slice thickness = 1.5mm, number of slices = 1.

[0080] Finally, through the continuous wave echo planar imaging (CW-EPI) sequence, the CEST of the above-mentioned slice was acquired. The scanning parameters for the EPI-CEST sequence were: the same slice as the B0 map was selected, TR = 6000ms, TE = 28.5ms, field of view = 35×35mm, matrix = 64×64, NEX = 1, slice thickness = 1.5mm, number of slices = 1. The offset frequency range was -15ppm to +15ppm, the number of images acquired was 122 (including 1 unsaturated image), and the scanning time was 13 minutes and 11 seconds.

[0081] Specifically, in S104, by comparing the changes in the CEST signals of doxorubicin at different time points after drug injection, the sustained-release function of the drug delivery system is reflected. According to the changes in the CEST signals at different time points, the sustained-release function of the drug delivery system is detected. The experimental results of the embodiments of the present invention are as Figure 3 shown. The CEST signal gradually decreases with the extension of time ( Figure 3 A in Figure 3 shows the schematic diagram of the CEST imaging signals on the 1st, 7th, and 14th days,

[0082] To verify the effectiveness of the present invention, the sustained-release effect of the hydrogel was also evaluated by near-infrared imaging. The specific process is as follows:

[0083] Preparation of Cy5 gel. The method is similar to the preparation method of the blank hydrogel. The prepared Cy5 with a concentration of 2.5 mg / 5 mL is stored at 4°C. Weigh an appropriate amount of poloxamer into a 20 mL glass bottle, add the prepared drug solution, and stir at 400 rpm under ice bath until completely dissolved to obtain Cy5 gel; store it in the refrigerator at 4°C in the dark overnight for later use.

[0084] Construction of a rat Cy5 gel animal model. First, place the rat under rapid induction anesthesia under the condition of 4% isoflurane and 5% O2, and then maintain anesthesia under the condition of 2.5 - 3.0% isoflurane. Use a small animal electrocardiogram monitor to continuously monitor the respiratory rate and amplitude of the rat. Fix the head of the rat on a stereotaxic instrument, shave the hair on the top of the head, disinfect it with iodophor, and then cut the skin along the mid-sagittal plane of the skull with ophthalmic scissors to expose the anterior fontanelle. Mark the drilling point (3 mm to the right of the anterior fontanelle, 1 mm forward). Use a dental drill with a diameter of 1 mm to vertically align with the positioning drilling point and drill a hole with a depth reaching the dura mater; take out the Cy5 gel from the 4°C refrigerator, slowly draw 10 μL with a microinjector; then fix the microinjector on the stereotaxic instrument, insert the needle 6 mm at the drilling point and then retract 1 mm, and slowly inject 10 μL of Cy5 gel into the right basal ganglia region. After the injection is completed, stay at the original position for 5 min, then slowly withdraw the microinjector, seal the bone hole with bone wax, suture the skin and disinfect it, and after making a good mark, put it into the breeding cage.

[0085] Brain tissue processing and near-infrared imaging. The modeling time was set as the first day, and the rats were perfused with 0.9% saline on the 1st, 7th, and 14th days (n = 3 at each time point). That is, under deep anesthesia, the chest wall of the rat was opened to expose its heart, and a puncture needle was inserted from the left ventricle, and the position of the needle tip was fixed with curved forceps. Then, the right auricle was cut with ophthalmic scissors, and 0.9% sodium chloride solution was perfused through the intravenous infusion needle until the blood was emptied and the liquid was clear, and then 4% paraformaldehyde was perfused for tissue fixation; the head was cut off and the skin, skull and other tissues were separated, and the brain tissue inside was taken out for near-infrared imaging (IVISKinetic, PerkinElmer, USA) to evaluate the sustained release effect of the gel. The excitation wavelength was 650nm.

[0086] See the comparison diagram of near infrared imaging and CEST imaging of the embodiment of the present invention. Figure 4 As shown in the statistical analysis diagram, the change in near-infrared signal intensity within one week is not statistically significant. The near-infrared signal intensity after 14 days is weaker than that on the 1st and 7th days, and the difference is statistically significant ( Figure 4 The change of CEST signal intensity within one week was not statistically significant. The CEST signal intensity after 14 days was weaker than that on the 1st and 7th days, and the difference was statistically significant ( Figure 4 B). By comparing near-infrared imaging and CEST imaging, the sustained release effect of the hydrogel in vivo is verified. It can be seen that the embodiment of the present invention has the function of detecting the sustained release function of the drug delivery system.

[0087] See also Figure 5 FIG. 1 is a system structure diagram of an embodiment of the present invention, including:

[0088] Preparation module 501, preparing a drug delivery system and constructing a GBM animal model;

[0089] Intervention module 502, performing postoperative intervention on the GBM animal model using a drug delivery system;

[0090] An imaging module 503 performs CEST imaging on the GBM animal model at several time points after the postoperative intervention;

[0091] The detection module 504 detects the sustained-release function of the drug delivery system according to the changes of the CEST signal at different time points.

[0092] It can be seen that the present invention applies the drug in the drug delivery system based on the CEST effect of the drug, and monitors the drug delivery system and the distribution and metabolism of the drug therein in the postoperative residual cavity through the CEST effect of the drug therein, which is expected to provide a local drug delivery system that is radiation-free, does not require additional labeling and can be monitored in real time to improve the prognosis of GBM.

[0093] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for detecting the sustained release function of a postoperative residual cavity drug delivery system based on CEST, characterized in that: The following steps are involved: Prepare drug delivery systems and construct GBM animal models; Use drug delivery systems to perform postoperative interventions on GBM animal models; CEST imaging of GBM animal models was performed at several time points after postoperative intervention; The sustained-release function of the drug delivery system was detected based on the changes in the CEST signal at different time points.

2. The method for detecting the sustained release function of the postoperative residual cavity drug delivery system based on CEST according to claim 1, characterized in that: The preparation of the drug delivery system comprises the following steps: According to the molar concentration calculation formula, prepare the drug solution of the required concentration and adjust the pH value to 7.4; According to the formula of 20% weight or volume content of poloxamer 407 and 2% weight or volume content of poloxamer 188, the corresponding amount of poloxamer was weighed into a 20 mL glass bottle, and the drug solution was added, and stirred at 400 rpm under an ice bath until completely dissolved to obtain the H@ATC drug delivery system; Place the H@ATC drug delivery system in a 4-degree refrigerator away from light overnight and store for later use.

3. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 1, characterized in that: The drug solution is doxorubicin solution.

4. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 2, characterized in that: The preparation of the drug delivery system also includes: using a test tube model to confirm whether the drug has a CEST effect; if the drug has a CEST effect, continuing to the next step; if the drug does not have a CEST effect, ending the execution and drawing a conclusion that the sustained-release function of the drug delivery system cannot be detected.

5. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 2, characterized in that: The preparation of the drug delivery system also includes: confirming whether the drug delivery system is in a gel state at a temperature of 37°C; if the drug delivery system is in a gel state at a temperature of 37°C, continuing to the next step; if the drug delivery system is not in a gel state at a temperature of 37°C, re-preparing the drug delivery system.

6. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 1, characterized in that: The construction of the GBM animal model specifically comprises: slowly injecting a C6 glioma cell suspension into the right basal ganglia region of a normal rat model to obtain a GBM animal model.

7. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 1, characterized in that: The time of establishing the GBM animal model is regarded as the first day, and the GBM animal model is subjected to postoperative intervention using the drug delivery system on the seventh day; the postoperative intervention comprises the following steps: Resection of tumor tissue in GBM animal models; Inject the drug delivery system into the surgical area.

8. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 1, characterized in that: The animal model is subjected to CEST imaging at several time points after GBM postoperative intervention, and the process of performing CEST imaging at each time point includes the following steps: Acquire brain MRI images of animal models to locate the CEST imaging layer; CEST images were acquired using a continuous wave echo-planar imaging sequence.

9. The method for detecting the sustained-release function of the postoperative residual cavity drug delivery system based on CEST according to claim 8, characterized in that: The method of acquiring a brain MRI image of an animal model to locate the optimal layer for CEST imaging comprises the following steps: The coronal, pars plana and axial positions were located in the positioning phase; The frequency and energy were corrected, and T2-weighted images of the rat brain in the axial position were obtained by fast spin echo imaging sequence; Collect B0 map sequence for later use in correcting CEST imaging; The plane with the best display of the rat surgical area in T2-weighted images was selected as the plane for CEST imaging.

10. A CEST-based sustained-release function detection system for postoperative residual cavity drug delivery system, characterized in that: Includes the following modules: Prepare modules, prepare drug delivery systems and construct GBM animal models; The intervention module uses the drug delivery system to perform postoperative intervention on GBM animal models; An imaging module that performs CEST imaging of GBM animal models at several time points after postoperative intervention; The detection module detects the sustained-release function of the drug delivery system according to the changes of the CEST signal at different time points.

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