Nanometer drug delivery system with tumor deep penetration effect as well as preparation method and application of nanometer drug delivery system

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a nanometer drug delivery system with a tumor deep penetration effect and a preparation method and application of the nanometer drug delivery system. The polylactic acid-glycolic acid nano-particles are polylactic acid-glycolic acid copolymer nano-particles entrapped with chemotherapeutic drugs and carbon monoxide donors; the chemotherapeutic drug is monomethyl aurestatin E, and the carbon monoxide donor is S-nitroso-N-acetyl-DL-penicillamine; a lipid layer; the core is coated with the core; the targeting polypeptide is modified on the surface of the lipid layer; the targeting polypeptide is an R4F polypeptide; tumor cells are actively targeted through R4F polypeptide, the polylactic acid-glycolic acid copolymer nanoparticles wrapped by a lipid layer deeply penetrate into tumor tissues, nitric oxide is released through a carbon monoxide donor to degrade an extracellular matrix barrier, and deep penetration of loaded chemotherapeutic drugs and immune cells in the tumor tissues is synergistically promoted; the nano drug delivery system provided by the invention can be applied to preparation of drugs for treating solid tumors and delivery of drugs for targeting extracellular matrixes of the solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nano-drug delivery system with deep tumor penetration, its preparation method, and its application. Background Technology

[0002] Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest solid tumors, characterized by insidious onset and rapid progression. Despite advances in surgical and chemotherapy treatments, the five-year survival rate remains low. A hallmark of PDAC is a strong fibroblastic response: pancreatic fibroblasts deposit dense layers of cross-linked collagen, fibronectin, and hyaluronic acid, forming the extracellular matrix (ECM). This leads to microvascular collapse, increased interstitial pressure, and restricts the entry of chemotherapeutic drugs and immune cells into the perivascular region. Therefore, overcoming this physical barrier is crucial for improving treatment outcomes.

[0003] In research exploring ways to break down the ECM matrix, some common research directions include: utilizing enzymatic reactions (such as matrix metalloproteinases (MMPs) to degrade ECM components), targeting cell surface receptors (such as integrin-mediated cell-ECM adhesion), and regulating ECM remodeling-related signaling pathways (such as regulating tumor cell migration and invasion by inhibiting Rho GTPase activity with drugs). Currently, NO can promote the production of active MMPs, but successfully delivering NO to the tumor site and maintaining its long-term, slow release is crucial, which faces two main challenges: First, precise control of the release rate and time is required (because an excessively rapid release rate may lead to severe cytotoxicity, while an excessively long release time may reduce efficacy and increase side effects); second, NO has a very short half-life in vivo and is easily metabolized and eliminated.

[0004] Cancer is a major global public health problem, with persistently high morbidity and mortality rates. Despite continuous advancements in diagnostic techniques, many malignant tumors, such as glioblastoma and pancreatic cancer, remain difficult to treat effectively due to their high heterogeneity and unique microenvironment. Therefore, developing highly specific, low-toxicity targeted therapies has become an urgent problem to be solved. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a nano-drug delivery system with deep tumor penetration and its preparation method. A lipid layer is prepared using a thin-film hydration method / modified double emulsification method, whereby the carrier material is dissolved in an organic solvent, then thoroughly mixed to completely remove the organic solvent, and then added to pure water for ultrasonic emulsification to form a liposome nanoparticle suspension (Lips). Based on the polylactic acid-glycolic acid copolymer (PLGA) double emulsification method, the previously prepared liposome nanoparticles replace the traditional vinyl alcohol (PVA) as the dispersant. The drug, a hydrophobic chemotherapy drug monomethyl olistatin E (MMAE) and / or a hydrophilic S-nitroso-N-acetyl-DL-penicillamine (SNAP), is encapsulated in the PLGA core using an emulsification-evaporation method. Subsequently, the PLGA nanoparticle surface is coated with the liposome nanoparticle suspension (Lips). Finally, an entropy-driven surface self-assembly peptide modification method is used to utilize the interaction between the lipid bilayer and the R4F peptide (an ApoA-1 mimic peptide). Using methods such as hydrophobic interactions, R4F peptides are modified to encapsulate the surface of PLGA nanoparticles entirely. On one hand, R4F peptides can bind to various tumor cells that highly express the SR-B1 receptor (such as Panc02-H7 pancreatic cancer cells), achieving active targeting. On the other hand, R4F-modified nanoparticles can be effectively taken up by peripheral blood mononuclear macrophages, and through the tumor homing effect of these cells, the nanoparticles are delivered deep into the tumor tissue. Therefore, R4F modification not only endows the nanoparticles with the ability to target tumor cells but also promotes their deep penetration into the tumor tissue. When the nanosystem reaches the tumor tissue, NO released from S-nitroso-N-acetyl-DL-penicillamine (SNAP) can degrade the dense extracellular matrix (ECM) barrier. In this process, the targeting and penetration mediated by R4F peptides synergistically promote the deep penetration of MMAE-loaded chemotherapy drugs and immune cells (such as recruited or activated immune cells) into the tumor tissue.

[0006] To achieve the above objectives, the present invention provides a nano-drug delivery system with deep tumor penetration capability, the nano-drug delivery system comprising: The core is composed of polylactic acid-glycolic acid copolymer nanoparticles encapsulating a chemotherapy drug and a nitric oxide donor; the chemotherapy drug is monomethylolpropamine E, and the nitric oxide donor is S-nitroso-N-acetyl-DL-penicillamine. A lipid layer; which surrounds the core; A targeting peptide modified on the surface of a lipid layer; the targeting peptide is an R4F peptide; By actively targeting tumor cells with R4F peptides, polylactic acid-glycolic acid copolymer nanoparticles encapsulated in a lipid layer are deeply penetrated into the tumor tissue. Nitric oxide is released through the carbon monoxide donor to degrade the extracellular matrix barrier, synergistically promoting the deep penetration of loaded chemotherapy drugs and immune cells into the tumor tissue.

[0007] Specifically, S-nitroso-N-acetyl-DL-penicillamine, acting as a nitric oxide donor, releases carbon monoxide, a molecule with unique physiological and pathological functions. This carbon monoxide can react with superoxide anions in the tumor environment to form the oxidant peroxynitrite (ONOO). - This stimulates tumor stromal cells to produce active matrix metalloproteinases (MMPs), which degrade almost all collagen components in the extracellular matrix (ECM), thereby promoting the penetration of chemotherapy drugs and immune cells into the tumor site.

[0008] Preferably, the R4F polypeptide is an ApoA-1 mimic peptide that actively targets tumor cells by binding to various tumor cells that highly express the SR-B1 receptor. Subsequently, the R4F polypeptide modifies the surface of the lipid layer, enabling it to be effectively taken up by mononuclear macrophages in peripheral blood. Utilizing the tumor homing effect, the polylactic acid-glycolic acid copolymer nanoparticles encapsulated in the lipid layer are delivered to the deep tumor tissue.

[0009] A second aspect of this invention provides a method for preparing a nano-drug delivery system with deep tumor penetration capability, comprising the following steps: S1. Prepare a lipid layer by thin-film hydration or double emulsification to form a liposome nanoparticle suspension; S2. Dissolve monomethyl olistatin E powder, S-nitroso-N-acetyl-DL-penicillamine powder and polylactic acid-glycolic acid copolymer in an organic solvent to obtain a drug-loaded polylactic acid-glycolic acid copolymer core organic phase mixture; S3. The liposome nanoparticle suspension and the drug-loaded polylactic acid-glycolic acid copolymer core organic phase mixture are mixed by ultrasonic emulsification to obtain polylactic acid-glycolic acid copolymer nanoparticles coated with a lipid layer. S4. The R4F peptide is co-incubated with the polylactic acid-glycolic acid copolymer nanoparticles wrapped in the lipid layer using an entropy-driven surface self-assembly peptide modification method, so that the R4F peptide is modified onto the surface of the polylactic acid-glycolic acid copolymer nanoparticles wrapped in the entire lipid layer. After purification, the nano-drug delivery system is obtained.

[0010] Preferably, in step S1, during the preparation of the liposome nanoparticle suspension, the carrier material is dissolved in an organic solvent, thoroughly mixed, and then pure water is added for ultrasonic emulsification. After filtration for sterilization, concentration, and resuspension, the liposome nanoparticle suspension is obtained.

[0011] Preferably, the carrier material is a phospholipid, a cholesterol derivative, and / or a PEGylated phospholipid, and the organic solvent is chloroform.

[0012] Preferably, in step S2, the organic solvent is chloroform.

[0013] A third aspect of the present invention provides the use of the above-described drug delivery system in the preparation of a medicament for treating solid tumors.

[0014] A fourth aspect of the present invention provides the application of the above-described drug delivery system in drug delivery targeting the extracellular matrix of solid tumor cells.

[0015] The beneficial technical effects of the present invention are as follows: This invention co-loads the nitric oxide donor S-nitroso-N-acetyl-DL-penicillamine SNAP with the chemotherapeutic drug MMAE in the same nanosystem. By utilizing nitric oxide to degrade the dense tumor extracellular matrix (ECM) barrier, it significantly enhances the penetration and distribution of MMAE chemotherapeutic drugs and infiltrating immune cells deep within the tumor tissue, thereby overcoming the key bottleneck of insufficient drug penetration in the treatment of pancreatic ductal adenocarcinoma and improving the therapeutic effect.

[0016] The nanomedicine provided by this invention has been applied to the treatment of Panc02-H7 tumors and has achieved good therapeutic effects; this dual-response drug delivery system that achieves deep penetration provides a potential strategy for cancer chemoimmunotherapy. Attached Figure Description

[0017] Figure 1 This is a particle size distribution and potential diagram of nanoparticles provided in Example 2 of the present invention, wherein (a) is a particle size distribution diagram of R4F-MS@L-PLGA drug, and (b) is a potential diagram of R4F-MS@L-PLGA and its derivative drugs.

[0018] Figure 2 The graphs shown in Example 4 of this invention are the release rates of MMAE and NO in nanoparticles, where (a) is the release rate graph of MMAE and (b) is the release rate graph of NO.

[0019] Figure 3 The bar charts showing the relationship between MMAE concentration, NO concentration and cancer cell activity provided in Example 5 of this invention are shown, where (a) is a graph of MMAE concentration and cell activity, and (b) is a graph of NO concentration and cell activity.

[0020] Figure 4 This is a confocal image and differential analysis of the cell uptake of Panc02-H7 and RAW264.7 cells by drugs with and without targeted peptide modification, provided in Example 6 of the present invention. Among them, (a) is the cell uptake of Panc02-H7 cells by the control group, (b) is the cell uptake of Panc02-H7 cells by the MS@L-PLGA group, (c) is the cell uptake of Panc02-H7 cells by the R4F-MS@L-PLGA group, (d) is the cell uptake of RAW264.7 cells by the control group, (e) is the cell uptake of RAW264.7 cells by the MS@L-PLGA group, (f) is the cell uptake of RAW264.7 cells by the R4F-MS@L-PLGA group, (g) is a flow cytometry quantitative detection image showing the difference in cell uptake of Panc02-H7 cells by different incubation times of the drug, and (h) is a flow cytometry quantitative detection image showing the difference in cell uptake of RAW264.7 cells by different incubation times of the drug.

[0021] Figure 5 This is a fluorescence microscopy image of NO in Panc02 cells containing SNAP drug, provided in Example 7 of the present invention using a DAF-FM DA probe. (a) to (c) are control group images, (d) to (f) are images of R4F-S@L-PLGA, and (g) to (i) are images of R4F-MS@L-PLGA.

[0022] Figure 6 These are fluorescence micrographs of ONOO- in different tissues indirectly detected by using a 3-NT probe in Embodiment 8 of the present invention, wherein (a) to (c) are control group images, (d) to (f) are images of R4F-S@L-PLGA, and (g) to (i) are images of R4F-MS@L-PLGA.

[0023] Figure 7 This is the protein expression diagram provided in Example 9 of the present invention.

[0024] Figure 8These are microscopic scanning images of tumor tissue labeled with Collagen I antibody and Fibronectin antibody provided in Example 10 of the present invention. (a) shows collagen in tumor tissue of the Collagen I antibody-labeled PBS group; (b) shows collagen in tumor tissue of the Collagen I antibody-labeled M@L-PLGA group; (c) shows collagen in tumor tissue of the Collagen I antibody-labeled R4F-M@L-PLGA group; (d) shows collagen in tumor tissue of the Collagen I antibody-labeled R4F-S@L-PLGA group; and (e) shows collagen... I. Collagen in tumor tissue of the antibody-labeled R4F-MS@L-PLGA group, (f) Fibronectin in tumor tissue of the PBS group labeled with Fibronectin antibody, (g) Fibronectin in tumor tissue of the M@L-PLGA group labeled with Fibronectin antibody, (h) Fibronectin in tumor tissue of the R4F-M@L-PLGA group labeled with Fibronectin antibody, (i) Fibronectin in tumor tissue of the R4F-S@L-PLGA group labeled with Fibronectin antibody, (j) Fibronectin in tumor tissue of the R4F-MS@L-PLGA group labeled with Fibronectin antibody.

[0025] Figure 9 The mouse weight analysis and tumor block diagram provided in Embodiment 11 of the present invention are shown, wherein (a) is a weight analysis diagram and (b) is a tumor block diagram.

[0026] Figure 10 These are fluorescence micrographs provided in Example 12 of the present invention, wherein (a) is a cell apoptosis image in the PBS group of tumor tissue detected by TUNEL reagent, (b) is a cell apoptosis image in the Free MMAE group of tumor tissue detected by TUNEL reagent, (c) is a cell apoptosis image in the M@L-PLGA group of tumor tissue detected by TUNEL reagent, (d) is a cell apoptosis image in the R4F-M@L-PLGA group of tumor tissue detected by TUNEL reagent, (e) is a cell apoptosis image in the R4F-S@L-PLGA group of tumor tissue detected by TUNEL reagent, and (f) is a cell apoptosis image in the R4F-MS@L-PLGA group of tumor tissue detected by TUNEL reagent.

[0027] Figure 11These are fluorescence micrographs provided in Embodiment 13 of the present invention, wherein (a) is a distribution map of M@L-PLGA drug in the entire tumor tissue, (b) is a distribution map of R4F-M@L-PLGA drug in the entire tumor tissue, (c) is a distribution map of R4F-MS@L-PLGA drug in the entire tumor tissue, (d) is a local distribution map of M@L-PLGA drug in the tumor tissue, (e) is a local distribution map of R4F-M@L-PLGA drug in the local tumor tissue, and (f) is a local distribution map of R4F-MS@L-PLGA drug in the local tumor tissue.

[0028] Figure 12 This is a flow cytometry gate logic diagram provided in Embodiment 14 of the present invention, wherein (a) is a gate logic diagram for detecting mature DC cells in tumor tissue, (b) is a gate logic diagram for detecting M2 macrophages in tumor tissue, and (c) is a gate logic diagram for detecting CD8T cells in tumor tissue.

[0029] Figure 13 The flow cytometry quantitative statistical charts provided in Embodiment 14 of the present invention include (a) a quantitative statistical chart of mature DC cells in tumor tissues of different groups, (b) a quantitative statistical chart of M2 macrophages detected in tumor tissues of different groups, and (c) a quantitative statistical chart of CD8T cells in tumor tissues of different groups. Detailed Implementation

[0030] This invention discloses a nano-drug delivery system with deep tumor penetration, its preparation method, and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0031] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0032] Unless otherwise specified, the following embodiments are all conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0034] Example 1: Preparation of R4F-MS@L-PLGA nanoparticles.

[0035] Step S1: Prepare liposome nanoparticle suspension (Lips); accurately weigh 36 mg of DMPC (phospholipids) and add 0.5 mL of chloroform to dissolve it completely; accurately weigh 10 mg of CO (cholesterol derivative) and add 1 mL of chloroform to prepare a 10 mg / mL solution; accurately weigh 10 mg of DSPE-PEG2000 (PEGylated phospholipids) and add 1 mL of chloroform to prepare a 10 mg / mL solution; take 117 μL of the pre-prepared CO (10 mg / mL) and 57.6 μL of the pre-prepared DSPE-PEG2000 (10 mg / mL) and add them to the dissolved DMPC solution, and mix thoroughly by repeatedly pipetting; use a rotary evaporator to rapidly remove the organic solvent by rotary evaporation for 15 min, so that a dense lipid film is formed in a 15 mL round-bottom flask; then connect the sample vial and the oil pump using an adapter, and completely remove the organic solvent by vacuuming; add 4... The liposome nanoparticles were sonicated with 1 mL of ultrapure water to obtain a clear liposome nanoparticle suspension. The obtained liposome nanoparticle suspension was immediately placed in an ice box and cooled, then stored at 4°C for later use. The obtained liposome suspension was filtered to remove bacteria, and the filtrate was transferred to a 30 kDa ultrafiltration centrifuge tube and concentrated. The filtrate was discarded, and an appropriate amount of ultrapure water was added to the centrifuge tube to a total volume of approximately 8 mL. The suspension was resuspended, and approximately 8 mL of liposome nanoparticle suspension (Lips) was collected. The suspension was stored at 4°C for later use.

[0036] Step S2: Preparation of MS@L-PLGA nanoparticles; Accurately weigh 10 mg of PLGA (10 kDa), add 1 mL of chloroform to prepare a 10 mg / mL solution, and filter using a 0.45 μm nylon filter until clear and transparent; Accurately weigh 2.2 mg of R4F, add 1 mL of chloroform to prepare a 2.2 mg / mL solution; Accurately weigh 3.2 mg of MMAE and 1 mg of SNAP powder into a 5 mL centrifuge tube. Add 1 mL of pre-dissolved and filtered PLGA chloroform solution (10 mg / mL), and if necessary, add chloroform to bring the volume to 1 mL. Vortex to fully dissolve and mix to obtain an organic phase mixture. Add 3 mL of liposome nanoparticle suspension (Lips) to the organic phase mixture. Using a φ3 amplitude transducer ultrasonic cell disruptor, perform a first ultrasonic emulsification for 8 min under ice bath conditions (to prevent overheating). Then, add another 3 mL of liposome nanoparticle suspension to the mixture and perform a second ultrasonic emulsification for 8 min under the same conditions. After ultrasonic emulsification, the mixture is transferred to a container (such as a round-bottom flask) in a fume hood, placed on a magnetic stirrer, and stirred overnight (about 12-16 hours) at room temperature in the dark to allow the organic solvent chloroform to fully evaporate and form an MS@L-PLGA nanoparticle suspension.

[0037] Step S3: Preparation of R4F-MS@L-PLGA nanoparticles; 1 mL of R4F aqueous solution (2.2 mg / mL) was added to the MS@L-PLGA nanoparticle suspension, and the mixture was stirred and incubated overnight to allow R4F to modify the nanoparticle surface, ultimately obtaining the R4F-MS@L-PLGA nanoparticle suspension. The R4F-MS@L-PLGA nanoparticle suspension was centrifuged, the supernatant was discarded, and the precipitate (i.e., nanoparticles) was collected. An equal volume of ultrapure water was added to the precipitate, and the mixture was gently resuspended and centrifuged again. This washing step was repeated 4 times to remove unbound or free liposome components and impurities. Then, the washed nanoparticle precipitate was resuspended in an appropriate amount of ultrapure water and transferred to a 30 kDa ultrafiltration centrifuge tube for concentration and removal of free small molecule drugs (SNAP and MMAE). The filtrate was discarded, an equal volume of ultrapure water was added to the centrifuge tube, the mixture was gently mixed, and centrifuged again. This ultrafiltration washing step was repeated 4 times to obtain R4F-MS@L-PLGA nanoparticles.

[0038] Example 2: Measurement of particle size distribution and potential diagram of R4F-MS@L-PLGA nanoparticles.

[0039] The morphology of the prepared R4F-MS@L-PLGA nanoparticles was analyzed by TEM (TECNAI G2, FEI Company, OR, USA), and the particle size distribution of the R4F-MS@L-PLGA nanoparticles was measured by DLS on a Zetasizer Nano-ZS90 (Malvern Instruments, Worcestershire, UK).

[0040] from Figure 1 It can be seen that the R4F-MS@L-PLGA nanoparticles have a particle size of 108 nm and carry more negative charges, which is more conducive to the uptake and transport of drugs by cells.

[0041] Example 3: Encapsulation efficiency of MMAE and SNAP in R4F-MS@L-PLGA nanoparticles.

[0042] The encapsulation efficiency of MMAE in M@L-PLGA and R4F-M@L-PLGA was determined by high performance liquid chromatography (HPLC). The encapsulation efficiency was 65.0% in both, while it was slightly lower in R4F-MS@PLGA, with an encapsulation efficiency of 55.9%.

[0043] SNAP in nanoparticles was quantitatively analyzed by ultraviolet spectrophotometry. The results showed that the encapsulation efficiencies of SNAP in R4F-S@L-PLGA and R4F-MS@L-PLGA were 24.7% and 16.7%, respectively.

[0044] Example 4: Release rates of MMAE drug and NO in R4F-MS@L-PLGA nanoparticles.

[0045] The release of MMAE by M@L-PLGA, R4F-M@L-PLGA, and R4F-MS@L-PLGA was measured by dialysis at 37°C in PBS containing 0.1% Triton-100 X. The simplified procedure was as follows: 0.5 mL of MMAE-loaded nanoparticles (MMAE: 0.6 mg / mL) were placed in a dialysis bag (molecular weight cutoff: 3000 Da), then rapidly immersed in 25 mL of dialysis medium, and subsequently placed in a 37°C constant-temperature shaker. Three replicates were performed for each group. Measurement results as follows Figure 2 As shown in (a), the release of R4F-MS@L-PLGA drug within 24 hours is less than 10%, and a sustained-release plateau is reached after the sixth day, with a release rate of 63.89%. Continued observation revealed that MMAE was still being slowly released after 15 days. This indicates that the drug release is long-lasting.

[0046] The release of NO from R4F-S@L-PLGA and R4F-MS@L-PLGA was quantitatively analyzed using the Griess assay. Both nanoparticles (SNAP) were mixed with different concentrations of GSH, and the Griess mixture reagent was used for monitoring. Samples were taken at different time points (5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min), and the NO concentration was calculated by measuring the absorbance at 540 nm using a microplate reader. The measurement results are shown below. Figure 2 As shown in (b), it can be seen that the drug can reach the drug release plateau region after 25 minutes under high GSH response, while only 10.714% of the drug is released in the low GSH region. This experimental result indicates that NO release is accelerated at the tumor site, which may be beneficial to the activation of matrix metalloproteinases.

[0047] Example 5: In vitro cell experiments to verify the effect of R4F-MS@L-PLGA on Panc02-H7 cancer cells.

[0048] The effects of R4F-MS@L-PLGA, M@L-PLGA, R4F-M@L-PLGA, R4F-S@L-PLGA, and FreeMMAE on the viability of Panc02-H7 and RAW264.7 cells were determined using the CCK8 assay. Cells were seeded at 8000 cells per well in 96-well plates and cultured at 37°C with 5% CO2 for 24 hours. The next day, the medium was changed, and the drug was serially diluted (half-diluted) to 100 μl per well, and incubated for 4 hours. The drug-containing medium was then replaced with normal medium, and the cells were cultured for another 24 hours. Finally, the cells were incubated for 4 hours at 37°C with complete medium containing 10% CCKB reagent. The optical density (OD) at 450 nm was then recorded using a microplate reader to determine the percentage of residual cell viability.

[0049] Test results are as follows Figure 3 As shown, the drug that releases NO has no obvious toxicity to cells within the monitoring range. The R4F-MS@L-PLGA drug gradually reduced the activity of Panc02-H7 cancer cells as the concentration of NO released increased. This is because the amount of MMAE released from the drug increased.

[0050] Example 6: In vitro cell experiments can effectively extract R4F-MS@L-PLGA.

[0051] DiRBOA was used to quantitatively measure the cellular uptake of different nanoparticles; Panc02-H7 cells and RAW264.7 cells were seeded in confocal culture dishes at 2 × 10⁶ cells per well. 5Cells were seeded and cultured in an incubator at 37 ℃ and 5% CO2 for 24 hours. The drugs (empty PLGA, R4F-MS@L-PLGA, and MS@L-PLGA) were diluted to 20 μM using culture medium and added to each well for incubation for 4 hours. Cells were then washed with PBS. Staining was performed using a Hoechst 33342 microscope, and the samples were analyzed using a fluorescence microscope (Olympus FV 3000, Tokyo, Japan).

[0052] Panc02-H7 cells and RAW264.7 cells were placed in 6-well plates at 3 × 10⁻⁶ cells per well. 5 Cells were inoculated and cultured at 37 °C with 5% CO2 for 24 hours. The drugs (R4F-MS@L-PLGA and MS@L-PLGA) were diluted to 20 μM using culture medium and added to each well. Cells were incubated for 1 hour, 3 hours, and 6 hours, washed with PBS, centrifuged to remove the supernatant, and resuspended in 500 μL PBS. The mean fluorescence intensity of the cells was measured using a CytoFLEX flow cytometer.

[0053] The results are as follows Figure 4 As shown, based on the fluorescence spectrum and the comparative analysis of fluorescence intensity, it is evident that drugs modified with R4F peptides can actively target Panc02-H7 cells and RAW264.7 cells.

[0054] Example 7: Detection of effectively released NO in in vitro cell experiments.

[0055] Panc02-H7 cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of [insert density here] on confocal culture dishes and cultured in DMEM complete medium at 37°C in a 5% CO2 incubator for 24 hours. The next day, before changing the medium, the cells were washed with PBS. PBS, R4F-S@L-PLGA, and R4F-MS@L-PLGA drugs were diluted to 5 μM (NO concentration) with complete medium and co-cultured with the cells for 6 hours. The medium was removed, and the cells were gently washed three times with PBS. 3 μM of fresh DAF-FM serum-free medium was added, and the cells were incubated at 37°C for 30 minutes. The cells were washed twice with PBS to remove residual staining. After washing, 200 μL of 4% paraformaldehyde was added for fixation for 15 minutes, followed by two washes with PBS. The cells were then stained with DAPI-containing medium for 30 minutes, followed by washing with PBS. Finally, 100 μL of PBS was added, and the samples were analyzed using a fluorescence microscope (Olympus FV 3000, Tokyo, Japan). Green fluorescence was observed, with excitation and emission peaks at 495 and 529 nm, respectively.

[0056] Measurement results as follows Figure 5 As shown in the figure, R4F-MS@L-PLGA can effectively release NO in Panc02-H7 cells.

[0057] Example 8: ONOO in tumor tissue - Testing.

[0058] Tumor-bearing mice were intravenously injected with empty PLGA, R4F-S@L-PLGA, and R4F-MS@L-PLGA nanoparticles. The empty PLGA dose was 1.25 mg / kg, and the SNAP dose was 8 μmol / kg. Mice were sacrificed 48 hours after injection; tumor masses were removed and fixed with 4% paraformaldehyde, then dehydrated with 30% sucrose solution, embedded in OCT (Sakura, Torrance, CA, USA), and frozen sections were prepared (Leica, Germany). The OCT adhesive on the slides was washed off with PBS, and the slides were blocked and stained with mouse anti-3-nitrotyrosine (3-NT) antibody for 12 hours. Excess antibody was washed off with PBS, and the slides were incubated with secondary antibody Alexa Fluor 594 for 2 hours. Excess antibody was washed off with PBS, and the slides were mounted with a DAPI-containing anti-fluorescence quencher. The tumor sections were then scanned using a confocal microscope.

[0059] sliced ​​images Figure 6 As shown, SANP-containing drugs can effectively generate ONOO at the tissue level. - .

[0060] Example 9: Detection of matrix metalloproteinase expression and activity.

[0061] Tumor-bearing mice were intravenously injected with nanoparticles of PBS, R4F-S@L-PLGA, and R4F-MS@L-PLGA, with a SNAP dose of 8 μmol / kg. Mice were sacrificed 48 hours after injection; tumors were excised and immersed in RIPA buffer containing PMSF and PhosSTOP phosphatase inhibitors, homogenized using a ZAI tissue homogenizer at 4500 rpm for 1 min; total protein was obtained by centrifugation at 10000 rpm for 15 min at 4°C; and Western blot analysis was performed using specific antibodies against matrix metalloproteinase-1 and matrix metalloproteinase-2.

[0062] The results are as follows Figure 7 As shown, the expression levels of MMP-1 and MMP-2 were significantly increased in tumors treated with NO-releasing nanoparticles, indicating that the NO / ONOO⁻ pathway can activate and upregulate the expression of MMPs.

[0063] Example 10: Degradation by matrix metalloproteinases.

[0064] Tumor-bearing mice were intravenously injected with nanoparticles of PBS, M@L-PLGA, R4F-M@L-PLGA, R4F-S@L-PLGA, and R4F-MS@L-PLGA at a SNAP dose of 8 μmol / kg. Mice were sacrificed 48 hours after injection; tumor masses were removed and fixed with 4% paraformaldehyde, then dehydrated with 30% sucrose solution, embedded in OCT, and frozen sectioned. The OCT adhesive on the slides was washed away with PBS, and the slides were blocked and stained with Collagen I and Fibronectin antibodies for 12 hours. Excess antibody was washed away with PBS, and the slides were incubated with Alexa Fluor 488 secondary antibody for 2 hours. Excess antibody was washed away with PBS, and the slides were mounted with a DAPI-containing antifluorescence quencher. The tumor sections were then scanned using a confocal microscope.

[0065] Tumor slice images such as Figure 8 As shown, R4F-S@L-PLGA and R4F-MS@L-PLGA can effectively degrade the extracellular matrix.

[0066] Example 11: In vivo anti-tumor experiment.

[0067] Female C57 mice aged 6-8 weeks were purchased from Hunan SJA Laboratory Animal Co., Ltd. (Changsha, Hunan, China). The feeding and in vivo experiments of the mice were carried out in accordance with the guidelines for laboratory animal care, use and animal experimentation ethics.

[0068] Use 1×10 7 Panc02-H7 cells (100 μL / mL) were subcutaneously injected into the right posterior thigh of each mouse. After one week of growth, the subcutaneous tumors were removed and cut into sections. Tumor fragments were implanted into the pancreatic tissue of mice. Seven days after tumor transplantation, mice were randomly assigned to groups and intravenously injected with PBS, Free MMARE, M@L-PLGA, and R4F-M@L-PLGA, R4F-S@L-PLGA, or R4F-MS@L-PLGA (DIR-BOA: 10 μM). Tumor growth and mouse weight were monitored for up to 18 days after drug administration (n = 5 mice). On day 18 of the drug intervention, mice were euthanized, and tumor tissue, spleen, and blood were collected for further analysis.

[0069] Test results Figure 9 As shown, R4F-MS@L-PLGA effectively inhibits the growth of tumor masses.

[0070] Example 12: In vivo apoptosis of tumor tissue.

[0071] The tumor tissue obtained in Example 11 was fixed in 4% paraformaldehyde at 4°C for 12 hours, and then dehydrated using a 30% sucrose solution. The tissue was then frozen in OCT gel (Sakura, Torrance, CA, USA) and sectioned using a cryostat (Leica, Germany). DAPI and TUNEL staining were performed, data were acquired using a fluorescence microscope, and analyzed using ImageJ software.

[0072] result Figure 10 As shown, the tumor tissue injected with R4F-MS@L-PLGA exhibited the highest level of apoptosis.

[0073] Example 13: The penetration of nanoparticles into tumors.

[0074] M@L-PLGA, R4F-M@L-PLGA, and R4F-MS@L-PLGA (DiR-BOA concentration: 10 μM, 200 μL) were injected into tumor-bearing mice via the tail vein. Forty-eight hours after injection, tumor tissue was harvested, and the tumors were sectioned into 20 μm thick sections using a cryostat (Leica CM1900, Germany). The sections were then immunofluorescently stained with DAPI.

[0075] result Figure 11 As shown, the R4F-MS@L-PLGA nanoparticles have a greater penetration power in tumors than the R4F-M@L-PLGA nanoparticles, and are also greater than the M@L-PLGA nanoparticles.

[0076] Example 14 Tumor tissue was isolated from local tumor tissue obtained from tumor-bearing mice, minced, and digested in medium containing IV collagenase, hyaluronic acid, and RPMI-1640 at 37°C for 30 min. The cell suspension was then filtered. Cells were counted using a cell counter before staining. Cells were then blocked for 10 min with Fc blocking antibody (TruStain fcX (anti-mouse CD16 / 32) antibody, BioLegend). The viable dye Fixable Viability Dye eFluor™ 506 (invitrogen) was used to distinguish between live and dead cells. To investigate the proportion of macrophages in tumors, cells were stained on crushed ice at 4°C for 30 minutes in the dark using anti-mouse CD45 antibody (BV605, BioLegend), anti-mouse F4 / 80 antibody (BV421, BioLegend), anti-mouse CD86 antibody (AF488, BioLegend), and anti-mouse CD206 antibody (AF647, BioLegend). To determine the CD4 / CD8 cell ratio, anti-mouse CD45 antibody (BV605, BioLegend), anti-mouse CD4 antibody (BV421, BioLegend), anti-mouse CD3 antibody (APC / Cy7, BioLegend), and anti-mouse CD8a antibody (AF647, BioLegend) were used. The cells were stained at 4°C in the dark for 30 minutes. To identify the proportion of DC cells, anti-mouse CD11c (BV421, BioLegend), anti-mouse CD86 (APC, BioLegend), and anti-mouse CD80 (PE, BioLegend) were used. The cells were stained at 4°C in the dark for 30 minutes. After incubation, the cell suspension was filtered through a 70 μm nylon cell filter to ensure uniform cell distribution and analyzed using a flow cytometer (Beckman Coulter, Cytoflex / Dxflex). The collected data were then further analyzed and processed using FlowJo software.

[0077] The results are as follows Figure 12 and Figure 13 As shown, after treatment with R4F-MS@L-PLGA, the number of mature DC cells and CD8 T cells in the tumor tissue increased significantly, while the content of immunosuppressive M2 macrophages decreased significantly; indicating that the drug treatment increased immune infiltration in the tumor tissue and altered the tumor microenvironment.

[0078] In summary, R4F-MS@L-PLGA nanoparticles prepared by replacing traditional vinyl alcohol (PVA) as a dispersant with liposome nanoparticles can promote the deep penetration of loaded MMAE chemotherapy drugs and immune cells (such as recruited or activated immune cells) into tumor tissues, and effectively improve the tumor microenvironment through R4F-MS@L-PLGA drug treatment.

[0079] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A nano-drug delivery system with deep tumor penetration capability, characterized in that, The nano-drug delivery system includes: The core is composed of polylactic acid-glycolic acid copolymer nanoparticles encapsulating a chemotherapy drug and a carbon monoxide donor; the chemotherapy drug is monomethylolpropamine E, and the carbon monoxide donor is S-nitroso-N-acetyl-DL-penicillamine. A lipid layer; which surrounds the core; A targeting peptide modified on the surface of a lipid layer; the targeting peptide is an R4F peptide; By actively targeting tumor cells with R4F peptides, polylactic acid-glycolic acid copolymer nanoparticles encapsulated in a lipid layer are deeply penetrated into the tumor tissue. Nitric oxide is released through the carbon monoxide donor to degrade the extracellular matrix barrier, synergistically promoting the deep penetration of loaded chemotherapy drugs and immune cells into the tumor tissue.

2. The nano-drug delivery system with deep tumor penetration according to claim 1, characterized in that, The R4F peptide actively targets tumor cells by binding to them. Subsequently, by modifying the surface of the lipid layer to allow it to be taken up by mononuclear macrophages, the polylactic acid-glycolic acid copolymer nanoparticles encapsulated in the lipid layer are delivered to the deep part of the tumor tissue by utilizing the tumor homing effect.

3. A method for preparing a nano-drug delivery system with deep tumor penetration capability, characterized in that, Includes the following steps: S1. Prepare a lipid layer by thin-film hydration or double emulsification to form a liposome nanoparticle suspension; S2. Dissolve monomethyl olistatin E powder, S-nitroso-N-acetyl-DL-penicillamine powder and polylactic acid-glycolic acid copolymer in an organic solvent to obtain a drug-loaded polylactic acid-glycolic acid copolymer core organic phase mixture; S3. The liposome nanoparticle suspension and the drug-loaded polylactic acid-glycolic acid copolymer core organic phase mixture are mixed by ultrasonic emulsification to obtain polylactic acid-glycolic acid copolymer nanoparticles coated with a lipid layer. S4. The R4F peptide is co-incubated with the polylactic acid-glycolic acid copolymer nanoparticles wrapped in the lipid layer using an entropy-driven surface self-assembly peptide modification method, so that the R4F peptide is modified onto the surface of the polylactic acid-glycolic acid copolymer nanoparticles wrapped in the entire lipid layer. After purification, the nano-drug delivery system is obtained.

4. The method for preparing a nano-drug delivery system with deep tumor penetration according to claim 3, characterized in that, In step S1, during the preparation of the liposome nanoparticle suspension, the carrier material is dissolved in an organic solvent, thoroughly mixed, and then pure water is added for ultrasonic emulsification. After filtration for sterilization, concentration, and resuspension, the liposome nanoparticle suspension is obtained.

5. The method for preparing a nano-drug delivery system with deep tumor penetration according to claim 4, characterized in that, The carrier material is phospholipid, cholesterol derivative and / or PEGylated phospholipid, and the organic solvent is chloroform.

6. The method for preparing a nano-drug delivery system with deep tumor penetration according to claim 3, characterized in that, In step S2, the organic solvent is chloroform.

7. The use of the nano-drug delivery system of claim 1 or the nano-drug delivery system prepared according to claim 3 in the preparation of a medicament for treating solid tumors, characterized in that, The solid tumor was pancreatic ductal adenocarcinoma.

8. The application of the nano-drug delivery system of claim 1 or the nano-drug delivery system prepared according to claim 3 in drug delivery targeting the extracellular matrix of solid tumor cells.

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