Melphalan gel based on ultrasonic response as well as preparation method and application of melphalan gel

Abstract

The invention discloses melphalan gel based on ultrasonic response as well as a preparation method and application of the melphalan gel. According to the preparation method disclosed by the invention, the melphalan hydrogel with a cross-linked nanofiber structure is rapidly formed by the melphalan under the ultrasonic action through non-covalent interaction. Melphalan hydrogels can be used to improve traditional HAIC treatment methods for liver cancer, including expediting catheter removal, reducing systemic toxicity, increasing local drug concentration, and prolonging drug-cell interaction time. In a rat model, the Melphalan hydrogel-assisted HAIC shows better effectiveness than the traditional HAIC, can significantly inhibit the growth of liver cancer, activates effective anti-tumor immune response, and realizes complete cure in part of liver cancer. In addition, the drug delivery capability of the melphalan hydrogel is expected to improve the pharmacokinetic characteristics of various drugs and promote the synergistic treatment effect of various drugs. Therefore, the invention provides a choice capable of obviously optimizing the treatment process and improving the overall treatment result for liver cancer treatment.

Description

Technical Field

[0001] The invention relates to the technical field of biomaterials, and in particular to a melphalan gel based on ultrasound response, and a preparation method and application thereof. Background Art

[0002] Liver cancer is the sixth most common malignancy and the third leading cause of cancer-related death worldwide, which poses a huge challenge to people due to its hidden nature and difficulty in early diagnosis. Most patients are diagnosed at intermediate or advanced stages, making them unsuitable for radical treatments such as surgical resection, liver transplantation, or ablation. To address this issue, palliative interventions such as hepatic arterial infusion chemotherapy (HAIC) or hepatic artery embolization (HAE) are often used preferentially to reduce tumor size to create favorable conditions for surgical resection or radical ablation. Among these methods, HAIC is often used as an interventional treatment for liver cancer, which involves direct cannulation of the blood vessels supplying the tumor and continuous administration of high-dose chemotherapy drugs through the catheter to effectively eliminate tumor cells. Compared with systemic intravenous chemotherapy, HAIC provides higher local drug concentrations. However, HAIC usually requires the placement of an arterial catheter to continuously infuse chemotherapy drugs over 48-72 hours, which often increases the risk of catheter displacement and migration, as well as arterial spasm that may cause symptoms such as severe upper abdominal pain. In addition, repeated and continuous local infusion of high-dose chemotherapy drugs can lead to severe systemic toxic reactions, including anemia, gastrointestinal discomfort, liver and kidney function damage, and even organ failure.

[0003] Melphalan (Mel), also known as L-phenylalanine mustard, is a chemotherapy drug used to treat multiple myeloma, malignant lymphoma, lymphocytic and granulocytic leukemia, childhood neuroblastoma, ovarian cancer, breast cancer, and uveal melanoma. It can be taken orally or intravenously. Mel belongs to the class of nitrogen mustard alkylating agents, which work by interfering with the production of DNA and RNA. Specifically, Mel chemically changes the DNA nucleotide guanine by alkylation and creates links between DNA strands. This chemical change inhibits DNA synthesis and RNA synthesis, functions that are necessary for cell survival. These changes can cause cytotoxicity in both dividing and non-dividing tumor cells. Mel was approved for medical use in the United States in 1964. It is included in the World Health Organization's list of essential medicines and is available as a generic drug. In August 2023, the U.S. Food and Drug Administration approved Mel as a liver-directed therapy for the treatment of adult uveal melanoma patients with unresectable liver metastases affecting less than 50% of the liver, with no extrahepatic disease or with extrahepatic disease limited to the bones, lymph nodes, subcutaneous tissue, or lungs that are amenable to resection or radiation. Recent studies have further demonstrated that Mel has the potential to induce immunogenic cell death (ICD), during which certain molecules released during ICD, called damage-associated molecular patterns (DAMPs), are able to bind to pattern recognition receptors present on the surface of DC cells and initiate a cascade of cellular responses that ultimately activate innate and adaptive immune responses. Summary of the invention

[0004] To solve the above technical problems, the present invention provides a melphalan gel based on ultrasound response, as well as a preparation method and application. The present invention found that Mel can be assembled into many spherical aggregates within a few seconds of exposure to an ultrasound field, and then transformed into worm-like aggregates, and finally form a macroscopic hydrogel (Mel-gel) with a cross-linked nanofiber structure through non-covalent interactions. The hydrogel formed by Mel is not subject to any chemical synthesis or modification, and is expected to have enhanced bioavailability and drug sustained release properties. Considering the unique advantages of Mel-gel, the present invention innovatively applies it to HAIC. Specifically, after hepatic artery catheter intervention, a single injection of Mel is directly administered to the liver cancer site through the hepatic artery, and then ultrasound is used at the liver cancer site to induce hydrogel formation in its blood vessels. Subsequently, the Mel-gel will gradually degrade in the tumor supply artery, continuously releasing Mel for up to two weeks to effectively eradicate tumor cells.

[0005] The present invention is achieved through the following technical solutions: The first object of the present invention is to provide a melphalan gel based on ultrasound response, wherein the melphalan gel is formed by mixing melphalan powder with a buffer solution and treating the mixture under ultrasound conditions.

[0006] In one embodiment of the present invention, the mass ratio of the melphalan powder to the buffer solution is 7-60:1000, preferably 1:50.

[0007] In one embodiment of the present invention, the buffer is selected from one or more of phosphate buffer, citrate buffer, carbonate buffer and acetate buffer.

[0008] In one embodiment of the present invention, the concentration of the buffer solution is 0.01 M-0.1 M; the pH of the buffer solution is 2-10.

[0009] In one embodiment of the present invention, the power of the ultrasound is 40 Hz-100 Hz, preferably 50 Hz-70 Hz; and the treatment time is 5 s-20 s.

[0010] The second object of the present invention is to provide a method for preparing melphalan gel based on ultrasound response, comprising the following steps: mixing melphalan drug powder with a phosphate solution to obtain a melphalan suspension; The melphalan suspension is treated under ultrasonic conditions to obtain the melphalan gel.

[0011] In one embodiment of the present invention, the mass percentage of the melphalan suspension is 1 wt%-6 wt%.

[0012] The third object of the present invention is to provide the use of the melphalan gel in the preparation of anti-tumor drugs.

[0013] In one embodiment of the present invention, the tumor is a liver tumor.

[0014] The fourth object of the present invention is to provide a pharmaceutical composition comprising melphalan gel.

[0015] The beneficial effects of the present invention are: 1. The present invention provides a melphalan gel based on ultrasound response, as well as a preparation method and application. Compared with traditional HAIC, Mel-gel allows the catheter to be removed immediately after administration, significantly reducing the risks and complications caused by the catheter remaining in the patient's body for a long time.

[0016] 2. Ultrasound-induced gelation limits the local dispersion of Mel in the tumor, greatly reduces its systemic toxicity, and protects other parts of the body from adverse effects.

[0017] 3. Local and sustained-release drug delivery enhances the concentration of therapeutic agents in the target area and increases the rate at which drugs are absorbed by tumor cells, ultimately significantly improving treatment outcomes.

[0018] 4. Mel has the ability to stimulate ICD and trigger subsequent immune responses, thereby enhancing its effectiveness against metastatic tumors.

[0019] 5. In a rat orthotopic liver cancer model, HAIC using Mel under ultrasound showed significantly enhanced inhibition of liver cancer growth, and a complete therapeutic response was obtained in 60% of the subjects. The superior performance emphasizes the potential of the present invention as an effective intervention for liver cancer treatment.

[0020] 6. Mel-gel can serve as a multifunctional drug delivery platform, capable of prolonging the circulation of various other small molecule drugs or macromolecular protein therapeutics. By optimizing their distribution in tumors and normal organs, the Mel-gel system is expected to not only improve the pharmacokinetic characteristics of various therapeutic agents, but also achieve synergistic therapeutic effects with Mel, thereby improving the overall treatment efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein: Figure 1 The preparation process of Mel-gel of the present invention is shown in the figure; Figure 2 is a representative electron microscope image of the Mel-gel of the present invention; wherein a is a transmission electron microscope and b is a scanning electron microscope; Figure 3 is the rheological behavior of the Mel-gel of the present invention that changes with oscillation frequency, time and stress; wherein G' is the storage modulus; G" is the loss modulus; Figure 4 The representative infrared spectra, UV-visible absorption spectra and fluorescence emission spectra of Mel and Mel-gel of the present invention; Figure 5 are TEM images of the Mel-gel of the present invention after degradation on the 0th day, the 7th day and the 14th day; Figure 6 is the degradation curve of the Mel-gel of the present invention; Figure 7 df is a diagram of immunofluorescence staining of CRT, HSP70 and HMGB1 expression on mouse H22 tumor cells treated differently according to the present invention; wherein ac is a representative image of immunofluorescence staining, and df is a quantitative statistic of ac; Figure 8 These are representative fluorescence activated cell sorting (FACS) effect diagrams of the expression of CRT (a), HSP70 (b) and HMGB1 (c) on mouse H22 tumor cells treated differently according to the present invention; Fig. 9is a graph showing the concentration of extracellular HMGB1 and ATP in the culture medium after different treatments of the present invention; Fig.10 is a diagram showing the treatment of subcutaneous liver cancer in mice with Mel-gel; a is the statistics of tumor growth dynamics in different groups; b is the survival curve corresponding to the tumor size in different groups of mice; Fig.11 It is a T2 enhanced MRI scan of rats with N1S1 liver cancer in situ inoculated with different treatment schemes according to the present invention; Fig.12 is a graph showing the growth curve of rat orthotopic N1S1 liver cancer tumors treated with different methods of the present invention; Fig.13 These are the immunofluorescence staining images and statistical graphs of the expression levels of (a) CRT cells, (b) HSP70 cells and (c) HMGB1 cells after different treatments of mouse liver cancer of the present invention; Fig.14 is a graph showing the concentration of ATP in mouse liver cancer tissue after different treatments of the present invention; Fig.15 is a graph showing the changes in immune cells in the mouse liver cancer tissue after different treatments of the present invention; wherein a is mature DCs (CD11c + CD80 + CD86 + ) percentage; b is the percentage of infiltrating CTLs (CD3 + CD8 + ) in CD3 + The percentage of T cells in the chromatin matrix is ​​shown in Figure 2; c is the proliferative CTLs (CD8 + Ki67 + ) in CD8 + d is the percentage of activated CTLs (CD8 + G JZ + ) in CD8 + The percentage of T cells is statistically shown in e; NKs (CD3 - CD49b + ) in CD45 + Statistical graph of percentage in cells; Fig.16 Representative images of rabbit ears after central artery treatment with Mel or Mel-gel according to the present invention. DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0024] The first aspect of the present invention provides a hydrogel, wherein the hydrogel is prepared by mixing melphalan drug powder with a PBS solution and stirring to form a melphalan suspension, which is then placed under ultrasonic conditions to form a Mel-gel.

[0025] The present invention demonstrates a new method for rapidly forming melphalan gel under the influence of ultrasound, named Mel-gel. The present invention involves injecting Mel directly into the liver tumor site via the hepatic artery, and then using ultrasound to form a hydrogel within the tumor vasculature. This innovative method significantly shortens the intervention time of the hepatic artery catheter, optimizes the chemotherapy infusion treatment process, and provides a new perspective for the treatment of liver cancer. In both the mouse subcutaneous liver cancer model and the rat in situ liver cancer interventional treatment model, the method showed a significant effect in inhibiting tumor growth and achieved a therapeutic effect.

[0026] Furthermore, in the raw materials of the hydrogel, the mass ratio of melphalan to phosphate saline solution (PBS) is 7-60:1000, preferably 1:50.

[0027] Furthermore, in the ultrasonic condition of the hydrogel, the power range of the ultrasonic cleaning machine is 40 Hz-100 Hz, preferably 50 Hz-70 Hz.

[0028] The second aspect of the present invention provides a method for preparing the hydrogel according to the first aspect, comprising the following steps: First, a certain amount of melphalan drug powder was weighed, then mixed and stirred with PBS solution to prepare a 2 wt% melphalan suspension, and finally placed in an ultrasonic cleaner for 10 s to form a Mel-gel.

[0029] In the present invention, when preparing Mel-gel, Mel-gels of different concentrations can be obtained by controlling the amount of Melphalan hydrochloride added to PBS.

[0030] Furthermore, the molecular weight of the melphalan hydrochloride is 302.

[0031] Furthermore, the pH of the PBS is 7.4.

[0032] Example 1: Preparation of Mel-sol and Mel-gel.

[0033] Mel (Mel) was dispersed in PBS (pH 7.4, 0.01 M) or other buffers by gentle stirring to obtain a Mel suspension (Mel-sol), followed by ultrasonic irradiation (KQ-100KDB ultrasonic bath (40 kHz, 0.5 W / cm 2 ) or NSE-UPH-I handheld ultrasound device (50 kHz, 0.55 W / cm 2 )) to promote the dissolution of the suspension and quickly convert it into a stable Mel hydrogel (Mel-gel) within seconds. The preparation process of Mel-gel is as follows Figure 1 shown.

[0034] Example 2: Characterization of Mel-gel.

[0035] TEM images were obtained by using a FEI FT20 transmission electron microscope. SEM images were obtained using a Zeiss Gemini500 scanning electron microscope. Photos were taken using a smartphone in JPEG image format. Rheological behavior was measured using a Thermo Fisher HAAKE Mars40 with a P20 Ti parallel plate at 25 °C. Absorption curves were recorded using a JascoV-750 spectrophotometer. FTIR spectra were performed in KBr pellets using a Bruker V70 & Hyperion1000 spectrometer. Fluorescence spectra were recorded using a HORIBA FL3 fluorescence spectrometer. Results are shown in Figure 2-4 .

[0036] Figure 2 These are representative TEM and SEM images of Mel-gel. From the TEM and SEM images, it can be observed that the microscopic morphology of Mel-gel is nanofiber-like.

[0037] Figure 3 It is the rheological behavior of Mel-gel that changes with oscillation frequency, time and stress. Figure 3 As can be seen in b, the storage modulus and loss modulus of Mel-gel remain approximately 10 kPa and 1 kPa respectively over time. Figure 3 a and Figure 3 As shown in c, when the frequency is greater than 70 Hz or when the stress is greater than 30%, the curves of the storage modulus and loss modulus of Mel-gel suddenly change or cross, proving that the critical values ​​of its rheological properties are 70 Hz oscillation frequency and 30% shear stress, respectively.

[0038] Figure 4 These are representative infrared spectra, UV-visible absorption spectra, and fluorescence emission spectra of Mel and Mel-gel. Figure 4 As shown in a in the figure, the characteristic absorption peak of the carboxyl group in the Mel molecule changes from 1734 cm -1 Red shifted to 1720 cm -1 , indicating that the carboxyl group forms an intermolecular hydrogen bond. Figure 4 As shown in Figure b, compared with the spectrum of Mel molecules free in ethanol, the maximum absorption peak of Mel-gel has a color shift of 7 nm, which proves that the aggregation type of Mel molecules is J-type aggregation. Figure 4 As shown in Figure c, the fluorescence emission spectrum peak of Mel shifted from 350 nm to 368 nm, and the fluorescence intensity decreased with the formation of hydrogel. This is due to the aggregation of Mel molecules and the tendency of fluorescence quenching. The red shift is mainly attributed to the presence of benzene rings, which mainly participate in the self-assembly process through π-π interactions.

[0039] Example 3: Degradation and release behavior of Mel-gel.

[0040] The degradation and release behavior of Mel-gel was measured using a transwell system (Corning, catalog number 3422) in PBS at 37°C to simulate a physiological environment. Mel-gel (100 μL, 2 wt%) was loaded into the upper chamber, and the released Mel was collected from the lower chamber and collected at a series of time points and quantified using HPLC (BioLogical DuoFlowTM) at 261 nm. In addition, the remaining Mel-gel in the upper chamber was collected on days 0, 7, and 14 to prepare TEM samples for microscopic observation. Mel-gel (50 μL, 2 wt%) gelled in situ on the back of mice was used to study its degradation behavior in vivo. The mice were sacrificed on days 0 and 21, and the skin containing the hydrogel was dissected and photographed. The results are shown in Figure 5 and Figure 6 .

[0041] Figure 5 The following are TEM images of Mel-gel after degradation on day 0, day 7, and day 14. As shown in the TEM images of Mel-gel at different time points, the nanofiber structure of Mel-gel under the microscopic field of view gradually breaks down over time until it is completely degraded into small Mel molecules.

[0042] Figure 6 This is the degradation curve of Mel-gel. As shown in the figure, the concentration of Mel small molecules in the solution at different time points was measured by HPLC, and the degradation curve of Mel-gel was drawn. The results showed that Mel-gel was degraded by about 80% within two weeks.

[0043] Example 4: In vitro evaluation of CRT, HMGB1, HSP70 and ATP.

[0044] (1) Immunofluorescence staining. For the evaluation of CRT and HSP70, suspended H22 or N1S1 cells were cultured in 24-well plates (non-tissue culture treated, 1 mL of culture medium); the groups were untreated (PBS group), treated with ultrasound (0.55 W / cm², 10 seconds) (PBS+US group), Mel-sol (2.5 μL, 2 wt%) (Mel group), or Mel-gel (2.5 μL, 2 wt%) (Mel group) for 6 hours. For the evaluation of HMGB1, H22 or N1S1 cells were treated in the same manner, but the treatment time was 12 hours. Then, H22 or N1S1 cells were collected by centrifugation (1200 rpm, 3 minutes), dropped onto a slide, fixed with 0.25% formaldehyde for 10 minutes, and blocked with FBS for 30 minutes. Next, cells were incubated with primary antibodies anti-mouse / rat CRT (Abcam, catalog number ab92516), anti-mouse / rat HMGB1 (Abcam, catalog number ab79823), and anti-mouse / rat HSP70 (Abcam, catalog number ab181606) at a dilution of 1:800 at 4°C overnight (cell membranes were perforated with 0.1% Triton X-100 before staining with anti-mouse / rat HMGB1 or anti-mouse / rat HSP70). Subsequently, cells were washed with PBS and incubated with Alexa Fluor® 488-labeled goat anti-rabbit IgG H&L (Abcam, catalog number ab150077) secondary antibodies at a dilution of 1:400 for 1 hour at room temperature and stained with 4, 6-diamidino-2-phenylindole (DAPI) for 10 minutes. Finally, the slides were mounted and imaged by confocal microscopy (Zeiss LSM 800). Results are shown in Figure 7 .

[0045] Figure 7 Figure 2 is the immunofluorescence staining of CRT, HSP70 and HMGB1 expression on H22 tumor cells of mice treated with different treatments. As shown in Figures ac, US treatment did not cause any changes in DAMP levels in H22 and N1S1 tumor cells. In contrast, significant upregulation of CRT and HSP70 proteins was observed in H22 and N1S1 cells treated with Mel and Mel-gel, while HMGB1 was released into the extracellular medium (Figures df).

[0046] (2) Flow cytometry analysis. The ability of Mel and Mel-gel to induce ICD in H22 and N1S1 liver cancer cells was quantitatively determined by flow cytometry. For the evaluation of CRT and HSP70, suspended H22 or N1S1 cells were cultured in 12-well plates (non-tissue culture treated, 2 mL of culture medium); the groups were untreated (PBS group, G1), treated with ultrasound (0.55 W / cm², 10 seconds) (PBS+US group, G2), Mel-sol (2.5 μL, 2 wt%) (Mel group, G3), or Mel-gel (2.5 μL, 2 wt%) (Mel group, G4) for 6 hours. For the evaluation of HMGB1, H22 or N1S1 cells were treated in the same manner, but the treatment time was 12 hours. Then, cells were collected into Eppendorf tubes by centrifugation (1200 rpm, 3 minutes), fixed with 0.25% formaldehyde for 10 minutes, and blocked with anti-CD16 / 32 (BioLegend, 101302) antibody. Next, cells were incubated with anti-mouse / rat CRT (Abcam, catalog number ab92516), anti-mouse / rat HMGB1 (Abcam, catalog number ab79823), and anti-mouse / rat HSP70 (Abcam, catalog number ab181606) primary antibodies at a dilution of 1:500 for 1 hour at room temperature (before staining with anti-mouse / rat HMGB1 or anti-mouse / rat HSP70, cell membranes were perforated with 0.1% Triton X-100). Then, cells were washed with PBS and incubated with Alexa Fluor® 488-labeled goat anti-rabbit IgG H&L (Abcam, catalog number ab150077) secondary antibody at a dilution of 1:400 for 1 hour at room temperature. Finally, H22 or N1S1 cells of each sample were tested using a BD AccuriC6 plus flow cytometer and analyzed using FlowJo software (Ver. 10.0.7). Figure 8 .

[0047] Figure 8 The following are representative fluorescence activated cell sorting (FACS) results of CRT, HSP70, and HMGB1 expression on mouse H22 tumor cells treated with different methods. Figure 8 As shown in Figures ac, after treatment with Mel and Mel-gel, the fluorescence signals of CRT, HMGB1 and HSP70 proteins in H22 cells changed significantly. Figure 8 As shown in df, after treatment with Mel and Mel-gel, the fluorescence signals of CRT, HMGB1 and HSP70 proteins in N1S1 cells also changed significantly. These results prove that Mel can effectively induce immunogenic death of liver tumor cells before and after gelation.

[0048] (3) Determination of extracellular HMGB1 and ATP. The levels of HMGB1 and ATP in the supernatant of H22 and N1S1 liver cancer cells after treatment with Mel and Mel-gel were investigated using HMGB1 and ATP detection kits. The culture medium of H22 or N1S1 cells after different treatments was collected and measured using a mouse / rat HMGB1 ELISA kit (Beyotime, catalog number PH406) and an ATP assay kit (Beyotime, catalog number S0027) according to the manufacturer's instructions. The results are shown in Fig. 9 .

[0049] Fig. 9 Figure 2 is a graph showing the concentration of extracellular HMGB1 and ATP in the culture medium after different treatments. Fig. 9 a and Fig. 9 As shown in b, after treatment with Mel and Mel-gel, the levels of HMGB1 and ATP in the supernatant of H22 cells and N1S1 cells increased significantly, indicating that H22 cells and N1S1 cells released a large amount of HMGB1 and ATP into the extracellular matrix. This experimental result further proves that Mel-gel can effectively induce immunogenic death of liver tumor cells.

[0050] Example 5: In vivo treatment of mouse H22 liver cancer.

[0051] To establish a subcutaneous H22 mouse liver cancer model, each Balb / c mouse was subcutaneously inoculated with 1.0 × 10 6 H22 cells. After 7 days, mice with tumors (volume about 100 mm 3 ) were randomly divided into four groups (six in each group) for in vivo experiments, including no treatment group, ultrasound treatment group (0.55 W / cm 2 , 10 seconds), Mel-sol treatment group (20 μL, 2 wt%) and Mel-gel treatment group (20 μL, 2 wt%). For distant tumor inoculation, each Balb / c mouse was injected subcutaneously in the left flank 3 days after primary tumor inoculation with 1.0×10 6 H22 cells. Mice in the Mel-sol group received Mel solution treatment via intratumoral injection, while mice in the Mel-gel group received Mel suspension injection into the tumor followed by ultrasound-triggered in situ gelation. The length (L) and width (W) of the tumors were measured with a vernier caliper every other day. Tumor growth curves were monitored by calculating tumor volume (V) using the following formula: V = W 2 × L × 0.5. When mice showed signs of being unhealthy or the tumor volume exceeded 1000mm 3 The mice were killed when Fig.10 .

[0052] Fig.10 Figure 2 is a diagram of the treatment of subcutaneous liver cancer in mice with Mel-gel. As shown in Figure a, ultrasound alone did not affect tumor progression. In the Mel group, rapid systemic elimination of freely diffusing drugs from the tumor site resulted in low drug retention and modest tumor growth inhibition. In contrast, the Mel-gel group injected with Mel suspension and irradiated with US showed significantly enhanced tumor inhibition and improved survival (Figure b), which should be attributed to the sustained release of the drug and prolonged interaction with tumor cells.

[0053] Example 6: Hepatic artery infusion chemotherapy for the treatment of rat orthotopic liver cancer.

[0054] The anesthetized rat was placed on the operating table and its limbs were fixed. A midline abdominal incision was made starting from the xiphoid process. The fascia, muscles, and peritoneum were carefully dissected to expose the liver tissue, and then 5.0×10 6N1S1 cells were inoculated into the liver. One week later, the rats were randomly divided into four groups: PBS (1 mL), PBS (100 μL) + US, Mel-Sol (1 mL, 0.2 wt%), and Mel-gel (100 μL, 2 wt%) for hepatic artery perfusion. The specific steps of the operation were as follows: the anesthetized rats were fixed on a sterile operating table, and a midline abdominal incision was performed to separate all tissue layers. Subsequently, the gastroduodenal artery, common hepatic artery, and proper hepatic artery were exposed to form a "Y"-shaped structure. The common hepatic artery was temporarily ligated to temporarily stop the blood flow. Subsequently, one-third of the gastroduodenal artery was transected with microsurgical scissors and retrogradely cannulated to the proper hepatic artery. Then, superselective cannulation of the blood supply artery of the liver lobe where the tumor was located was performed, and blood flow was restored by reopening the occlusion of the common hepatic artery. The catheter placement was confirmed by DSA imaging to ensure accurate positioning after suture fixation. The liquid was slowly injected (about 1 hour) along the catheter to the liver tumor site using a syringe pump to simulate the long catheterization process required for traditional HAIC, during which it was crucial to observe the anesthesia state of the rats and prevent the catheter from falling off. The procedure was performed on the PBS group and the Mel-SOL group. Rats in the PBS+US and Mel-gel groups completed perfusion administration within 1 minute, and the tumor was ultrasounded at the same time. In the Mel-gel group, hydrogel formation occurred in all blood supply arteries within the rat tumor. After administration, the incision was closed and disinfected. Abdominal scans were performed using 3.0 T MRI (General Electric Company, USA) before treatment and on days 3, 7, and 14 after treatment to evaluate tumor growth. The largest cross-section of the tumor was selected to measure the length (L) and width (W), while the longitudinal diameter (Lo) was measured based on the scanning layer thickness and the observable layer of the tumor. The volume (V) of the rat orthotopic liver tumor was calculated as follows: V = (1 / 2L × 1 / 2W × 1 / 2L O ) × 4 / 3π. The result is shown in Figure 11-12 .

[0055] Figure 11-12 The following are T2 enhanced MRI scans and tumor growth curves of rat orthotopic N1S1 liver cancer treated with different treatments. As shown in the figure, on day 14, conventional HAIC using Mel showed a certain inhibitory effect on rat orthotopic liver cancer, but failed to achieve complete tumor remission. In contrast, in the Mel-gel group, complete tumor eradication was observed in 60% of rats, and the remaining 40% of rats showed significant tumor shrinkage.

[0056] Example 7: Evaluation of CRT, HMGB1, HSP70 and ATP in tumor tissues.

[0057] Given the impressive therapeutic effects of Mel-gel, subsequent studies aimed to determine whether the “sustained release effect” of Mel-gel could trigger more potent immunogenic cell death in vivo compared with free drugs, thereby potentially eliciting a stronger antitumor immune response. Immunofluorescence staining of CRT, HMGB1, and HSP70 was therefore performed. Mice were sacrificed on day 2 after treatment, and their tumors were collected and frozen in optimal cutting temperature (OCT) compound at -80 °C. After 12 h, the tumors were cut into slices (thickness = 10 μm) at -25 °C using a cryostat (Lecia CM1860), mounted on slides and fixed with 4% paraformaldehyde. Then, the same staining procedure as in the cell experiment was performed. For the determination of ATP, the collected tumors were thoroughly ground in RIPA lysis buffer at 4 °C and centrifuged (12800 rpm, 5 min) to remove the precipitate. Then, ATP levels in the supernatant were measured by an ATP assay kit (Beyotime, catalog number S0027) according to the manufacturer's instructions. Results are shown in Figure 13-14 .

[0058] Fig.13 These are immunofluorescence staining images and statistical graphs of the expression of CRT cells, HSP70 cells, and HMGB1 cells after different treatments of mouse liver cancer. Immunofluorescence staining of tumor tissues showed that Mel-gel treatment resulted in increased expression levels of CRT, HSP70, and HMGB1 proteins in tumor tissues relative to Mel, due to a prolonged effective interaction time with tumor cells. It is worth noting that compared with in vitro cell experiments, in vivo experiments showed that the drug stimulated the significant release of HMGB1 protein synthesized by tumor cells into tumor tissues. Therefore, immunofluorescence showed enhanced HMGB1 signals after treatment.

[0059] Fig.14 The figure shows the concentration of ATP in mouse liver cancer tissue after different treatments. A higher level of ATP was observed in the supernatant of the ground tumor tissue samples treated with Mel-gel, further proving that Mel-gel can also effectively induce immunogenic death of liver tumor cells in vivo.

[0060] Example 8: Immune cell analysis by flow cytometry.

[0061] The presence of immune cells in tumor lymph nodes and tumor tissues was further investigated using flow cytometry. For the subcutaneous H22 murine hepatocellular carcinoma model, tumors and tumor-draining lymph nodes harvested from mice were collected on day 5 after treatment and homogenized into a single-cell suspension in cold FACS buffer solution (1% FBS in PBS). Then, the Fc receptors of the cells were blocked with anti-CD16 / 32 (BioLegend, catalog number 101302) antibodies to avoid nonspecific adsorption and stained with the following antibodies according to the manufacturer's instructions: FITC anti-mouse CD45 (BioLegend, catalog number 147710), PerCP anti-mouse CD11c (BioLegend, catalog number 117326), APC anti-mouse CD80 (BioLegend, catalog number 104714), PE anti-mouse CD86 (BioLegend, catalog number 1051 06), PerCP anti-mouse CD3 (BioLegend, catalog number 100326), APC anti-mouse CD4 (BioLegend, catalog number 100412), PE anti-mouse CD8 (BioLegend, catalog number 100708), FITC anti-mouse Ki67 (BioLegend, catalog number 151212), PE / Cy7 anti-mouse GzmB (BioLegend, catalog number 396410), APC anti-mouse CD49b (BioLegend, catalog number 103516). A total of 2.0 × 10 cells / mL were collected for each sample using a C6 plus flow cytometer. 5 targets and were analyzed by FlowJo software (version 10.0.7). For orthotopic N1S1 rat liver cancer, tumors from rats were collected on day 7 and the same experimental procedures as above were performed. The antibodies used were as follows: APC anti-rat CD45 (BioLegend, catalog number 202314), FITC anti-rat CD3 (BioLegend, catalog number 201403), PE / Cy7 anti-rat CD4 (BioLegend, catalog number 201516), PE anti-rat CD8 (BioLegend, catalog number 200607). Results are shown in Fig.15 .

[0062] Fig.15This is a graph showing the changes in immune cells in mouse liver cancer tissue after different treatments. As shown in Figure a, Mel-gel treatment induced a higher degree of DC maturation in the tumor-draining lymph nodes compared with Mel, indicating that the enhanced immunogenic cell death triggered by Mel-gel not only increased the presence of tumor-associated antigens, but also meant that these DC cells may be more effective in processing antigens and presenting them to cytotoxic T lymphocytes (CTLs). In turn, this enhanced the infiltration (Figure b), proliferation (Figure c) and activation (Figure d) of CTLS in the tumor site. Interestingly, Mel-gel also promoted the infiltration of natural killer (NK) cells into the tumor microenvironment (Figure e). These observations demonstrate that Mel-gel can induce a more effective anti-tumor immune response by affecting the post-hydrogel formation through its sustained release.

[0063] Example 9: Verification of Mel gelation in blood vessels.

[0064] In order to better observe the formation of Mel-gel in the blood vessels in the complex and dynamic in vivo environment and evaluate the sustained efficacy of Mel in blocking blood flow, Mel was injected into the middle ear artery of rabbits, and ultrasound was applied at the junction where the central artery bifurcated into branch arteries. First, the rabbit was fixed in a fixator and the ears were shaved for easy observation. The lower end of the central ear artery was disinfected with iodine. Then, the Mel suspension (50 μL, 2 wt%) was slowly injected into the central ear artery through the catheter. At the same time, the terminal branches of the central ear artery were ultrasounded with a handheld ultrasound device (0.55 W / cm 2 ). Finally, the morphological changes of rabbit ears at different time points were recorded by taking photos. Fig.16 .

[0065] Fig.16 Representative images of rabbit ears after central artery treatment with Mel or Mel-gel. Monitoring over several days showed that the ears treated with ultrasound (Mel-gel group) turned dark purple from the first to third postoperative day, with progressive swelling and cyanosis, indicating tissue hypoxia caused by arterial embolism. By day 5, necrosis began to appear in the ears treated with ultrasound, and by day 7, the necrotic area gradually expanded. In contrast, rabbit ears that did not receive ultrasound treatment only showed surgery-related congestion on the first postoperative day, which completely resolved by day 7. These results confirm that once Mel-gel forms within the vasculature, it effectively obstructs blood supply for an extended period of time. This leads to tissue necrosis - a key basis for subsequent therapeutic interventions against orthotopic liver tumors in rats.

[0066] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A melphalan gel based on ultrasound response, characterized in that: The melphalan gel is formed by mixing melphalan powder and buffer and treating the mixture under ultrasonic conditions.

2. The melphalan gel according to claim 1, characterized in that The mass ratio of the melphalan powder to the buffer solution is 7-60:1000.

3. The melphalan gel according to claim 1, characterized in that The buffer is selected from one or more of a phosphate buffer, a citrate buffer, a carbonate buffer and an acetate buffer.

4. The melphalan gel according to claim 1, characterized in that The concentration of the buffer solution is 0.01 M-0.1 M; the pH of the buffer solution is 2-10.

5. The melphalan gel according to claim 1, characterized in that The power of the ultrasound is 40 Hz-100 Hz; the treatment time is 5 s-20 s.

6. A method for preparing melphalan gel based on ultrasound response, characterized in that: The following steps are involved: mixing melphalan drug powder with a phosphate solution to obtain a melphalan suspension; The melphalan suspension is treated under ultrasonic conditions to obtain the melphalan gel.

7. The preparation method according to claim 6, characterized in that: The mass percentage of the melphalan suspension is 1 wt%-6 wt%.

8. Use of the melphalan gel according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

9. The use according to claim 8, characterized in that: The tumor is a liver tumor.

10. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises melphalan gel.

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