Preparation method and application of targeted nano-liposome based on synergistic effect of nitric oxide donor and sound-sensitive agent pyropheophorbide acid

Abstract

The invention discloses targeted nano-liposomes (SPL NPs) capable of releasing nitric oxide (NO) through ultrasonic triggering and application of the targeted nano-liposomes (SPL NPs) combined with a PD-L1 antibody in pancreatic cancer treatment. The nano-liposome is formed by self-assembly of DSPE-PEG2000-SNO (which is used as a NO prodrug), pyropheophorbide acid (PPAL (which is used as a sound-sensitive agent) and DSPE-PEG2000-LFC131 (which is used as a pancreatic cancer targeting ligand), and has good stability and targeting property. Under an ultrasonic irradiation condition, the nano system can generate active oxygen at a tumor part through the sonodynamic effect of PPAL, and can induce obvious immunogenic cell death. In addition, quick release of NO of an NO prodrug DSPE-PEG2000-SNO at a tumor site can be induced, and peroxynitrite with higher activity is generated through combined action of active oxygen and NO, so that an efficient tumor cell killing effect is realized. Meanwhile, the released NO can effectively release a pancreatic cancer compact matrix, improve the tumor microenvironment and enhance the permeability of drugs and the infiltration capacity of immune cells. The invention also discloses an application of the nano-liposome system in pancreatic cancer treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophylloidin PPAL, and its application. Background Technology

[0002] Pancreatic cancer is a highly malignant digestive system tumor that often lacks typical symptoms in its early stages, leading to late-stage diagnosis and a very poor prognosis. Pancreatic cancer grows rapidly, is highly invasive, and is prone to distant metastasis, with a five-year survival rate of less than 10%. The abundant fibrotic matrix and immunosuppressive microenvironment further limit treatment efficacy. Sonodynamic therapy (SDT) for pancreatic cancer is often limited by insufficient tumor site specificity and the tumor's immunosuppressive microenvironment, primarily due to the abundant fibrotic matrix in pancreatic cancer, which acts as a natural physical barrier. As a novel gaseous therapeutic molecule, NO (NO3) can dissolve the dense matrix of pancreatic cancer, thereby promoting drug penetration. However, achieving targeted tumor delivery remains a significant challenge. Furthermore, pancreatic cancer is considered a "cold tumor," with its immunosuppressive microenvironment characterized by low T-cell infiltration, abundant fibrosis, and aggregation of immunosuppressive cells (such as regulatory T cells), resulting in poor treatment outcomes. Therefore, constructing an SDT-mediated targeted NO delivery system and combining SDT with immunotherapy holds promise for synergistic treatment of pancreatic cancer.

[0003] Based on the above considerations, we constructed SPL nanoliposomes (SPL NPs) composed of DSPE-PEG. 2000 -SNO, pyromethesin (PPAL) and DSPE-PEG 2000 The system comprises LFC131, serving as a NO (NO) prodrug, a sonosensitive agent, and a targeting ligand. Under ultrasound irradiation, this system generates reactive oxygen species (ROS) and triggers NO release. The combination of these two substances further generates more reactive peroxynitrite ions, exhibiting significant tumor cell killing effects. The released NO can loosen the pancreatic cancer matrix, promoting nanodrug penetration and immune cell infiltration. When this system is combined with PD-L1 immune checkpoint blockade (ICB) therapy, it can significantly induce immunogenic cell death and significantly enhance anti-tumor effects in a highly malignant KPC mouse tumor model, demonstrating great potential for cancer treatment. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sonosensitive agent pyrophylloidin PPAL and its application. It solves the problems of uncontrollable release behavior, low penetration depth and limitation by the tumor microenvironment in NO gas therapy, and has industrial transformation value.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solution:

[0006] A prodrug for the release of NO gas induced by acoustic dynamic reactive oxygen species, the NO gas prodrug being prepared by the following reaction: dissolving mercaptopropionic acid in DMF, and reacting with DSPE-PEG. 2000 The -NH2 reaction, utilizing the EDC / NHS activation principle, prepares an intermediate activated thiol group. The activated thiol group is then reacted with tert-butyl nitrite to generate DSPE-PEG. 2000 -SNO. The reactive oxygen species generated by ultrasonic irradiation of the sonosensitive agent pyrophosphate chlorophyll (PPAL) are used to promote the activation of the prodrug DSPE-PEG. 2000 -SNO produces NO.

[0007] Preferably, in the above technical solution, DSPE-PEG 2000 -SNO can respond to reactive oxygen species to produce NNO gas; phospholipids DSPE-PEG 2000 -NH2 is an amphiphilic molecule; PPAL is a sonic sensitizer that can generate reactive oxygen species under ultrasound, and its specific structure is shown below.

[0008]

[0009] Preferably, in the technical solution for preparing nanoliposomes, DSPE-PEG 2000 -SNO, PPAL, DSPE-PEG 2000 -LFC131 was self-assembled into nanoliposomes by ethanol injection at a molar ratio of 45%:45%:10%.

[0010] A method for preparing LFC131-targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllate chlorophyll (PPAL) includes the following steps:

[0011] S1, EDC / NHS activation method to prepare thiol groups and react with tert-butyl nitrite to synthesize NO prodrug DSPE-PEG. 2000 -SNO;

[0012] S2, EDC / NHS crosslinking method for synthesizing DSPA-PEG 2000 -LFC131;

[0013] S3, DSPE-PEG 2000 -SNO, PPAL, DSPE-PEG 2000 -LFC131 was used to prepare targeted SPL nanoliposomes via self-assembly using an ethanol injection method.

[0014] Preferably, in the above technical solution, the synthesis method of step S1 is referenced in Angew Chem Int Ed Engl, 2015, 54(47):14026-30, as follows:

[0015] ① Dissolve mercaptopropionic acid in DMF, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide at a molar ratio of 1:1.3, and stir at room temperature for 10 min;

[0016] ② Add N-hydroxysuccinimide in the same molar amount as 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the mixture described in ①, and continue stirring for 1 hour;

[0017] ③ Add DSPE-PEG 2000 -NH2 is added to system ② above and reacted for 24 hours.

[0018] ④ Dialyze the mixture described in ③ with deionized water for 2 hours (MWCO 8000-14000Da) to remove 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide.

[0019] ⑤ The mixed aqueous solution after dialysis in step ④ was freeze-dried to obtain the amphiphilic prodrug molecule DSPE-PEG. 2000 -SNO.

[0020] Preferably, in the above technical solution, the reference for step S2 is Bioactive Materials, 2022, 20(17):147–161, as follows:

[0021] ① Dissolve free LFC131 in DMF, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide at a molar ratio of 1:1.3, and stir for 10 min at room temperature;

[0022] ② Add N-hydroxysuccinimide in the same molar amount as 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the mixture described in ①, and continue stirring for 1 hour;

[0023] ③ Add DSPE-PEG 2000 -NH2 is added to the above reaction system ②, and the reaction is carried out for 24 hours.

[0024] ④ Evaporate the above product ③ to dryness on a rotary evaporator to remove DMF. Redissolve it in 1 ml of DMF, and add it dropwise to 9 ml of deionized water with magnetic stirring.

[0025] ⑤ Dialyze the mixture described in ④ with deionized water for 2 hours (MWCO 8000-14000 Da), and then freeze-dry it to obtain DSPE-PEG. 2000 -LFC131.

[0026] Preferably, in the above technical solution, step S3 specifically includes:

[0027] The above prodrug, together with commercially available PPA phospholipids (DSPE-PEG), were combined. 2000 -SNO:PPAL:DSPE-PEG 2000 -LFC131) was self-assembled into nanoliposomes by ethanol injection at a molar ratio of 45%:45%:10%.

[0028] Preferably, in the above technical solution, the nanoliposomes can be prepared into an injection for intravenous injection.

[0029] The present invention also provides the application of the nanoliposomes combined with immunotherapy PD-L1 antibody described above in the treatment of pancreatic cancer.

[0030] The above-described technical solution of the present invention has the following beneficial effects:

[0031] This invention successfully prepared an LFC131-targeting nanoliposome based on the synergistic effect of NO donor nitrosothiol and sonosensitive agent pyrophyllic acid PPAL. The prepared nanoprodrug was uniformly dispersed in aqueous solution, had a regular morphology, and showed significant targeting to tumors.

[0032] This invention utilizes ultrasound-mediated acoustic dynamics to generate reactive oxygen species, thereby effectively triggering DSPE-PEG. 2000 -SNO generates NO gas, thereby achieving precise and on-demand release of NO at the target location, significantly improving the therapeutic effect of NO gas.

[0033] This invention utilizes the enhanced tumor enrichment mediated by the targeting peptide LFC131, the sonodynamic effect of PPAL under ultrasound irradiation to induce immunogenic death and recruit a large number of immune cells, the NO prodrug responding to NO generated by reactive oxygen species to remodel the matrix to eliminate physical barriers and promote immune cell infiltration, and PD-L1 immune checkpoint blockade to enhance the infiltration of functional T cells, significantly improving the anti-tumor effect against pancreatic cancer and showing great potential in tumor treatment. Attached Figure Description

[0034] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0035] Figure 1 The above is a synthetic route diagram of Example 1 and a structural schematic diagram of the nanoprodrug of the present invention;

[0036] Figure 2 This is a schematic diagram of the raw material structure in Example 1;

[0037] Figure 3 The DSPE-PEG synthesized in Example 1 2000 -SNO UV absorption spectrum;

[0038] Figure 4 The DSPE-PEG synthesized in Example 1 2000 -Infrared spectrum of SNO;

[0039] Figure 5 The DSPE-PEG synthesized in Example 1 2000 -Infrared spectrum of LFC131;

[0040] Figure 6 The DSPE-PEG synthesized in Example 1 2000 -LFC131 mass spectrum;

[0041] Figure 7 Transmission electron microscopy image, particle size and potential diagram of LFC131 targeted nanoliposomes synthesized in Example 1 based on the synergistic effect of NO donor nitrosothiol and sound sensitizer pyrophylloid chlorophyll PPAL.

[0042] Figure 8 In vitro oxygen generation characterization of LFC131 targeted nanoliposomes synthesized in Example 2, based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophylloid chlorophyll PPAL.

[0043] Figure 9 In vitro NO production characterization of LFC131-targeted nanoliposomes synthesized in Example 3, based on the synergistic effect of NO donor nitrosothiol and sound sensitizer pyrophylloid chlorophyll (PPAL).

[0044] Figure 10 Cell liveness and death staining images of LFC131-targeted nanoliposomes synthesized in Example 4, based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllic acid PPAL.

[0045] Figure 11Image showing the generation of NO and peroxynitrite ions at the cellular level of LFC131-targeted nanoliposomes synthesized in Example 5, which are based on the synergistic effect of NO donor nitrosothiol and sound sensitizer pyrophylloid chlorophyll PPAL.

[0046] Figure 12 Animal fluorescence image of an LFC131-targeted nanoliposome synthesized in Example 6, based on the synergistic effect of NO donor nitrosothiol and sound sensitizer pyrophylloid chlorophyll (PPAL).

[0047] Figure 13 The tumor inhibition curve of a mouse KPC tumor model based on the synergistic effect of NO donor nitrosothiol and sound sensitizer pyrophylloidin PPAL synthesized in Example 7.

[0048] Figure 14 This is a mouse KPC tumor tissue section of an LFC131-targeted nanoliposome synthesized in Example 8, based on the synergistic effect of NO donor nitrosothiol and sonosensitive agent pyrophyllic acid PPAL.

[0049] Figure 15 The image shows the effect of LFC131-targeted nanoliposomes synthesized in Example 9 on mouse KPC tumor immunotherapy based on the synergistic effect of NO donor nitrosothiol and sonosensitive agent pyrophyllic acid PPAL. Detailed Implementation

[0050] Various exemplary embodiments of the present invention will now be described in detail with reference to the accompanying drawings. It should be noted that, unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention.

[0051] Unless otherwise specified, all reagents and materials used in the following examples are commercially available; the specific preparation operations and testing methods are conventional methods in the field and meet the routine requirements of the laboratory.

[0052] Specific examples are as follows:

[0053] Example 1

[0054] Preparation of LFC131-targeted nanoliposomes (SPL) based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllate chlorophyll (PPAL):

[0055] like Figure 1 The diagram shown is a schematic representation of the structure of nanoliposomes. Figure 2 DSPE-PEG of nitrosothiols 2000 -Structural formulas of SNO and the sound-sensitive agent pyrophyllic acid PPAL.

[0056] Step 1: Prepare mercapto groups using the EDC / NHS activation method and react them with tert-butyl nitrite to synthesize the NO prodrug DSPE-PEG. 2000 -SNO: Mercaptopropionic acid is dissolved in DMF and reacted with DSPE-PEG. 2000 The -NH2 reaction, utilizing the EDC / NHS activation principle, prepares an intermediate activated thiol group. The activated thiol group is then reacted with tert-butyl nitrite to generate DSPE-PEG. 2000 -SNO.

[0057] like Figure 3 and Figure 4 The image shows the amphiphilic prodrug molecule DSPE-PEG. 2000 UV absorption spectrum and IR spectrum of -SNO, DSPE-PEG 2000 The characteristic UV absorption peak of -SNO is in the range of 330-360 nm, and the infrared spectrum shows a peak at 764 cm⁻¹. -1 and 1505cm -1 The presence of characteristic peaks -SN= and -N=O verifies the successful synthesis.

[0058] Step 2: Synthesis of DSPE-PEG via EDC / NHS crosslinking method 2000 -LFC131: Free LFC131 was dissolved in DMF. Utilizing the EDC / NHS crosslinking principle, EDC dissolved in DMF was added, and the reaction was carried out for 10 minutes with magnetic stirring. NHS was dissolved in DMF and added to the above solution, and the reaction was carried out for 1 hour with magnetic stirring. DSPE-PEG 2000 -NH2 was added to the above reaction system, and the reaction was allowed to proceed for 24 hours. The product was then evaporated to dryness using a rotary evaporator to remove DMF. It was then redissolved in 1 ml of DMF and added dropwise to 9 ml of deionized water with magnetic stirring. After dialyzing, the product was lyophilized to obtain DSPE-PEG. 2000 -LFC131.

[0059] like Figure 5 and Figure 6 The image shows DSPE-PEG. 2000 -Infrared and mass spectra of LFC131. The infrared spectrum shows a characteristic peak of the amide bond (C=O) at 1658 cm⁻¹. The mass spectrum shows the DSPE-PEG generated in the reaction. 2000 The theoretical molecular weight of LFC131 is basically consistent with the molecular weight measured by mass spectrometry, confirming the DSPE-PEG. 2000 -NH2 and LFC131 peptide were successfully coupled to form DSPE-PEG. 2000 -LFC131.

[0060] Step S3, DSPE-PEG 2000-SNO, PPAL, DSPE-PEG 2000 - LFC131 self-assembly for targeted SPL nanoliposomes: three components (DSPE-PEG) 2000 -SNO:PPAL:DSPE-PEG 2000 -LFC131) was self-assembled into nanoliposomes via ethanol injection at a molar ratio of 45%:45%:10%. Figure 7 As shown, the particle size of the SPL nanoliposomes we prepared was approximately 153.28 ± 7.34 nm and the potential was approximately -21.57 ± 1.25 mV, as observed by DLS dynamic light scattering imaging.

[0061] Example 2

[0062] Test of reactive oxygen species production capacity:

[0063] To investigate the ability of the LFC131-targeted nanoliposomes obtained in Example 1, based on the synergistic effect of the NO donor nitrosothiol and the sonication agent pyrophyllate chlorophyll (PPAL), to generate reactive oxygen species (ROS), the ROS generation of the SPL nanoliposomes under ultrasound was studied using SOSG (5 μM). The ultrasound parameters used in the experiment were 1 MHz, 0.5, 1.0, and 1.5 W / cm². 2 Duty cycles of 10%, 20%, and 50% were used. After treatment, the fluorescence intensity of SOSG was recorded using a fluorescence spectrophotometer (FL-970) (excitation wavelength λex = 480 nm, emission wavelength λem = 525 nm).

[0064] like Figure 8 As shown, the SOSG probe detected the generation of reactive oxygen species (ROS) after sonication of SPL nanoliposomes. Compared with the PBS control group, the SPL nanoliposome group generated significantly more ROS after sonication. Furthermore, the generation of ROS increased significantly with increasing sonication power and duty cycle.

[0065] Example 3

[0066] NO production capacity test:

[0067] To investigate the NO-generating ability of the LFC131-targeted nanoliposomes obtained in Example 1, based on the synergistic effect of the NO donor nitrosothiol and the sonication agent pyrophyllate chlorophyll (PPAL), the NO-generating capacity of the SPL nanoliposomes was evaluated using a Griess kit (Beyotime Biotechnology). Different groups and concentrations of SPL nanoliposomes (20–100 μg / mL) were subjected to sonication (1 MHz, duty cycle 10%–50%, 0.5–1.5 W / cm²). 2The sample was irradiated with ultrasound every 2 minutes for a total of 20 minutes. Then, 50 μL of Griess reagent I and 50 μL of Griess reagent II were thoroughly mixed with 50 μL of the sample, and the ultraviolet absorption peak at 540 nm was measured.

[0068] like Figure 9 As shown in the figure, comparisons between different groups revealed that SPL nanoliposomes produced a large amount of NO after ultrasonic irradiation, while the PBS and PL groups showed almost no NO production after ultrasonic irradiation. NO production increased with increasing SPL nanoliposome concentration, ultrasonic power, and duty cycle.

[0069] Example 4

[0070] Validation of SPL nanoliposomes' tumor-killing effect at the cellular level:

[0071] We used a Calcein-AM / PI double staining kit to visually observe the tumor-killing effect of SPL nanoliposomes at the cellular level, setting up different treatment groups (PBS, US, SPL NPs, PL+US, SP+US, and SPL+US). After incubating the cells with culture medium containing nanoparticles for 6 hours, they were sonicated for 1 minute (1MHz, 1W / cm²). 2 (Duty cycle 20%), followed by incubation for 18 hours. After washing three times with PBS, Calcein-AM / PI staining was added and incubated, and the samples were examined under a fluorescence microscope.

[0072] The results are as follows Figure 10 As shown, the SPL combined with ultrasound therapy group showed a more significant decrease in the live cell ratio, with the most prominent red fluorescence and the highest dead cell ratio, which was 8.5 times that of the control group. This indicates that SPL nanoliposomes combined with ultrasound therapy can significantly enhance the killing effect of KPC tumor cells.

[0073] Example 5

[0074] Verification of NO and peroxynitrite particle production at the cellular level using SPL nanoliposomes:

[0075] To investigate the therapeutic effect of NO gas generated after SPL nanoliposome combined with ultrasound therapy, we used a DAF-FM DA probe to evaluate intracellular NO production. NO generation at the cellular level by SPL nanoliposomes was detected using the DAF-FM DA probe (Beyotime). KPC cells were cultured at 1×10⁻⁶ cells / cells. 5Cells were seeded at a density of 100 cells / well in 24-well plates and incubated overnight to ensure cell adhesion. Subsequently, each well was treated differently according to the following groups (n=4): (1) PBS; (2) US; (3) SPL NPs; (4) PL+US; (5) SP+US; (6) SPL+US (PPAL equivalent concentration of 15 μg / mL). After adding the corresponding nanoparticles to each group, incubation for 6 hours was performed to promote endocytosis. After removing untaken nanoparticles, DAF-FM DA (10 μM) diluted with serum-free DMEM was added, and incubation for 30 minutes was performed. The relevant groups were then sonicated (1 MHz, 1.0 W / cm²). 2 Cells were washed twice with PBS (duty cycle 20%, 1 minute). Finally, cell fluorescence was observed using an inverted fluorescence microscope (Leica, Germany). Furthermore, NO can react with reactive oxygen species to generate highly reactive peroxynitrite ions and other reactive nitrogen molecules. These molecules can exacerbate overall damage by initiating free radical peroxidation reactions, thus enhancing the therapeutic effect of reactive oxygen species on tumors. Therefore, we used the O71 probe, a dedicated probe for the detection of peroxynitrite ions, to detect the ONOO generated at the cellular level. - The test was performed. The procedure was the same as the in vitro NO generation experiment, except that the probe was replaced with O71.

[0076] Experimental results are as follows Figure 11 As shown, the experimental results of detecting NO production at the cellular level using the DAF-FM DA probe indicate that the SPL nanoliposome group, combined with ultrasound irradiation, exhibits significant green fluorescence, suggesting that the material, when combined with ultrasound irradiation, can significantly increase intracellular NO production. The experimental results of detecting peroxynitrite ion production at the cellular level using the O71 probe are similar to the above results; the SPL nanoliposome group combined with ultrasound produced the most significant green fluorescence, approximately 13.8 times that of the PBS group.

[0077] Example 6

[0078] Biodistribution in mice:

[0079] The biodistribution of SPL nanoliposomes in mice was studied using near-infrared fluorescence imaging (IVIS). Mice bearing KPC tumors were intravenously injected with 200 μL of SP and SPL nanoliposomes (PPAL effective dose: 4 mg / kg). Detection was performed at predetermined time points (excitation wavelength 675 nm, emission wavelength 720 nm) at 2, 4, 8, 12, 24, and 48 hours post-injection to determine the optimal time window for ultrasound therapy after nanoliposome accumulation at the tumor site. Mice were sacrificed 48 hours after injection, and tumors and organ tissues were harvested. Images were acquired using PPAL fluorescence imaging, and data analysis was performed using IVIS in vivo imaging system 3.0 software.

[0080] The results are as follows Figure 12 As shown, fluorescence signals were observed at the tumor site after 2 hours, and the fluorescence signal gradually increased over time. The nanomaterials reached optimal enrichment at the tumor site at 12 hours; therefore, 12 hours was chosen as the time for ultrasound irradiation in subsequent experiments. The enrichment of SPL at the tumor site was significantly higher than that of the SP group, indicating that the targeting peptides facilitated enrichment at the tumor site. The distribution results of SPL nanoliposomes in vital organs showed that the fluorescence intensity of the SPL group at the tumor site was approximately 6.68 × 10⁻⁶ p / s / cm. 2 / sr, significantly higher than the SP group (3.99*10 7 p / s / cm 2 The former ( / sr) was approximately 1.67 times that of the latter. This indicates that both substances accumulate in tumor tissue, and the enrichment effect of the targeted group was significantly stronger than that of the non-targeted group, suggesting a good targeting effect.

[0081] Example 7

[0082] Tumor growth inhibition:

[0083] KPC tumor-bearing C57BL / 6 mice were randomly divided into 8 groups until the tumor volume reached approximately 100 mm. 3 The groups were then divided as follows: PBS group, US group, SPL nanoliposome group, PL nanoliposome combined with ultrasound group, SP nanoliposome combined with ultrasound group, αPD-L1 group, SPL nanoliposome combined with ultrasound group, and SPL nanoliposome combined with ultrasound and αPD-L1 group. Each group received an intravenous injection of the corresponding preparation (PPAL dose 4.0 mg / kg) on ​​days 0, 3, and 8. Twelve hours after injection, designated groups underwent ultrasound irradiation (parameters: 1.0 MHz, 1.5 W / cm²). 2 (50% duty cycle) or no ultrasound treatment. Tumor volume is measured every two days during treatment.

[0084] The results are as follows Figure 13 As shown, the tumor volume change curves in the ultrasound-only treatment group were similar to those in the SPL nanomaterial group and the PBS group, indicating no significant inhibitory effect on tumor tissue during treatment. The SPL combined with ultrasound group, along with the PD-L1 antibody, exhibited the most significant tumor treatment effect, with a tumor inhibition rate as high as approximately 90%. During the observation period, tumor growth was slow, demonstrating a significant tumor growth inhibitory effect.

[0085] Example 8

[0086] Pathological testing for tumor growth inhibition:

[0087] To more intuitively demonstrate the changes in tumor tissue after treatment in each group, experimental mice were sacrificed after treatment, and the tumor tissue was subjected to histopathological examination, including: H&E staining, Ki-67 proliferation detection, TUNEL apoptosis detection, and Caspase-3 apoptosis protein detection.

[0088] The results are as follows Figure 14 As shown, the SPL nanoliposomes combined with ultrasound and αPD-L1 group exhibited significant necrosis and apoptosis in HE detection, with increased cytoplasmic eosinophilicity, nuclear pyknosis, nuclear fragmentation, and nuclear lysis, indicating significant tumor cell destruction. This group showed a significant decrease in cell proliferation in Ki-67 detection. In TUNEL assay, this group showed a significant increase in the number of apoptotic tumor cells, exhibiting a strong apoptotic signal, with fluorescence intensity approximately 11.5 times that of the PBS control group. In Caspase-3 pathological detection, this group showed the most prominent red fluorescence representing Caspase-3, indicating the most severe apoptosis.

[0089] Example 9

[0090] Efficacy of KPC tumor immunotherapy in mice:

[0091] Tumor tissue was obtained from KPC tumor model mice after treatment to prepare single-cell suspensions. The tumor tissue was minced and digested at 37°C for 4 hours using a digestion solution containing type IV collagenase (100 μg / mL), type I collagenase (1 mg / mL), and DNase I (100 μg / mL). The single-cell suspension was obtained by filtration through a 70 μm cell sieve. Zombie Yellow was added. TM Fluorescent dyes were used to distinguish between live and dead cells. To analyze CD4+ and CD8+ T cell subsets, cells were incubated with APC-labeled mouse CD3 antibody, FITC-labeled mouse CD8 antibody, and PE-labeled mouse CD4 antibody at 4°C in the dark for 30 minutes. Flow cytometry was then used for analysis.

[0092] The results are as follows Figure 15 As shown, in the SPL nanoliposome combined with ultrasound and αPD-L1 group, T cells infiltrated the most inside the tumor, CD8 T cells were the most activated, and Treg inhibition was the most significant.

[0093] Although the present invention has been disclosed above with reference to embodiments, it is not intended to limit the present invention. Any person skilled in the art can make various different choices and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention is defined by the claims and their equivalents.

Claims

1. A targeted nanoliposome based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophylloidin (PPAL), characterized in that, The nanoparticles are made of DSPE-PEG 2000 -SNO, pyromethoxyphylla PPAL and DSPE-PEG 2000 The system consists of LFC131, which acts as a NO (NO) prodrug, a sonosensitive agent, and a targeting group. Under ultrasonic irradiation, this system can generate reactive oxygen species (ROS) and trigger NO release. The combination of these two can further generate more reactive peroxynitrite ions (PERNO).

2. A method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllic acid (PPAL), specifically including the following steps: S1, EDC / NHS activation method to prepare thiol groups and react with tert-butyl nitrite to synthesize NO prodrug DSPE-PEG. 2000 -SNO; S2, EDC / NHS crosslinking method for synthesizing DSPA-PEG 2000 -LFC131; S3, DSPE-PEG 2000 -SNO, PPAL, DSPE-PEG 2000 -LFC131 was used to prepare targeted SPL nanoliposomes via self-assembly using an ethanol injection method.

3. The method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllic acid (PPAL) according to claim 2, characterized in that, The component DSPE-PEG 2000 -SNO, PPAL, DSPE-PEG 2000 The molar ratio of LFC131 is 45%:45%:10%.

4. The method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllic acid (PPAL) according to claim 2, characterized in that, Step S1 is as follows: ① Dissolve mercaptopropionic acid in DMF, add 1-ethyl-(3-dimethylaminopropyl)carbodiimide at a molar ratio of 1:1.3, and stir at room temperature for 10 min; ② Add N-hydroxysuccinimide in the same molar amount as 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the mixture described in ①, and continue stirring for 1 hour; ③ Add DSPE-PEG 2000 -NH2 is added to system ② above and reacted for 24 hours. ④ Dialyze the mixture described in ③ with deionized water for 2 hours (MWCO 8000-14000Da) to remove 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. ⑤ The mixed aqueous solution after dialysis in step ④ was freeze-dried to obtain the amphiphilic prodrug molecule DSPE-PEG. 2000 -SNO.

5. The method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllic acid (PPAL) according to claim 2, characterized in that, Step S2 is as follows: ① Dissolve free LFC131 in DMF, and add 1-ethyl-(3-dimethylaminopropyl)carbodiimide at a molar ratio of 1:1.3, and stir for 10 min at room temperature; ② Add N-hydroxysuccinimide in the same molar amount as 1-ethyl-(3-dimethylaminopropyl)carbodiimide to the mixture described in ①, and continue stirring for 1 hour; ③ Add DSPE-PEG 2000 -NH2 is added to the above reaction system ②, and the reaction is carried out for 24 hours. ④ Evaporate the above product ③ to dryness on a rotary evaporator to remove DMF. Redissolve it in 1 ml of DMF, and add it dropwise to 9 ml of deionized water with magnetic stirring. ⑤ Dialyze the mixture described in ④ with deionized water for 2 hours (MWCO 8000-14000 Da), and then freeze-dry it to obtain DSPE-PEG. 2000 -LFC131.

6. The method for preparing LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sound-sensitizing agent pyrophyllic acid (PPAL) according to claim 2, characterized in that, Step S3 specifically involves: combining the aforementioned prodrug with commercially available PPA phospholipids (DSPE-PEG). 2000 -SNO:PPAL:DSPE-PEG 2000 -LFC131) was self-assembled into nanoliposomes by ethanol injection at a molar ratio of 45%:45%:10%.

7. The role of LFC131 targeted nanoliposomes based on the synergistic effect of NO donor nitrosothiol and sonosensitive agent pyrophyllic acid PPAL as described in claim 1, or the role of nanoliposomes prepared according to the methods described in claims 2-6 in tumor targeting and therapy.

Images