Lysosome-based nanomedicine particles and uses thereof

Abstract

The present application relates to a kind of lysosome-based nano drug-loaded particles and its application, belong to the field of biological medicine.The present application will be lysosome membrane as the source of biomimetic membrane to be coated on the surface of drug nano-particle, while keeping the stability of lysosome membrane structure Maximum degree retains its naturally occurring hydrolytic enzyme activity, so that the nano drug-loaded particle can be in situ degradation to tumor tissue extracellular matrix during in vivo delivery, thereby effectively weakening the physical barrier of tumor tissue, significantly enhance the permeability, distribution and cell uptake capacity of nano drug in tumor tissue, and on this basis, improve the conditions for immune cells to enter tumor tissue, ultimately improve the tumor treatment effect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a lysosome-based nanoparticle for drug delivery and its application. Background Technology

[0002] Nanomedicines, due to their tunable physicochemical properties and multifunctional integration advantages, have broad application prospects in the field of tumor treatment. However, existing nanomedicines generally face the problem of insufficient penetration into tumor tissue during in vivo delivery. The dense extracellular matrix in tumor tissue is composed of collagen, hyaluronic acid, and various proteoglycans. Its abnormal deposition and high cross-linking not only hinder the deep diffusion of nanomedicines in tumor tissue but also limit the infiltration of immune cells into the tumor core region, thereby significantly reducing the therapeutic effect.

[0003] To improve tumor tissue permeability, existing technologies have proposed various strategies, including physical stimulation, interference with extracellular matrix synthesis, and degradation by exogenous bioenzymes. However, physical methods typically rely on external equipment and carry the risk of tissue damage; interference with extracellular matrix synthesis involves multiple signaling pathways and is subject to complex regulation; exogenous enzymes have short half-lives in vivo, poor tissue specificity, and are prone to triggering immune responses, limiting their clinical application. Existing nanomedicine delivery systems still face multiple challenges in biomedical applications such as tumor therapy. On the one hand, traditional inorganic or organic nanocarriers are easily recognized and cleared by the immune system in vivo, resulting in limited biocompatibility and in vivo stability; on the other hand, the complex physiological barriers of tumor tissue limit the effective penetration and cellular uptake of nanomedicines in the lesion area, leading to limited therapeutic efficiency. Existing biomimetic carriers constructed using cell membranes or exosomes often struggle to simultaneously meet the requirements of immune escape, extracellular matrix degradation, and efficient tumor cell internalization.

[0004] In recent years, biomembrane-coated nanoparticles have attracted attention due to their excellent biocompatibility and immune escape capabilities. However, existing biomembrane systems struggle to simultaneously meet multiple requirements, including immune escape, extracellular matrix degradation, and efficient tumor cell internalization. Therefore, there is an urgent need to develop a novel nanodelivery system that combines good biocompatibility with tumor tissue penetration capabilities to address the aforementioned issues in existing technologies. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to overcome the problems of poor tumor tissue penetration and poor biocompatibility of existing nano-drug delivery systems.

[0006] To address the aforementioned technical problems, this invention provides lysosome-based drug-carrying nanoparticles and their applications. Lysosomes, as important subcellular organelles, are naturally rich in various membrane proteins and hydrolases, playing a crucial role in the degradation of biomacromolecules such as proteins, lipids, and polysaccharides, especially in the degradation of extracellular matrix components and the remodeling of the tissue microenvironment, where they possess potential biological functions. However, in existing technologies, lysosomes are mainly studied for their intracellular structure or function; their overall structure and the hydrolases they carry have not been effectively used to construct biomimetic nanomedicine delivery carriers, let alone to improve nanomedicine delivery efficiency by regulating the extracellular matrix of tumor tissue. This invention uses the entire lysosome as a biomimetic membrane source to coat the surface of nanoparticles. While maintaining the structural stability of the lysosomal membrane, it maximizes the preservation of its naturally occurring hydrolases, enabling the drug-carrying nanoparticles to degrade the extracellular matrix of tumor tissue in situ during in vivo delivery. This effectively weakens the physical barrier of tumor tissue, significantly enhances the penetration, distribution, and cellular uptake of nanomedicines in tumor tissue, and improves the conditions for immune cells to enter tumor tissue, ultimately enhancing the therapeutic effect of tumor treatment.

[0007] The first objective of this invention is to provide a lysosome-based drug-loaded nanoparticle, which is prepared by the following method:

[0008] S1. The lysosomes are first extruded through a polycarbonate membrane with a pore size of 450-350 nm to obtain primary lysosomal vesicles. The primary lysosomal vesicles are then extruded through a polycarbonate membrane with a pore size of 150-250 nm to obtain secondary lysosomal vesicles.

[0009] S2. The secondary lysosomal vesicles of S1 are mixed with the drug to be loaded to obtain a mixed system. The mixed system is co-extruded through a polycarbonate membrane with a pore size of 150-250 nm at 0-4℃ to obtain the drug-loaded nanoparticles.

[0010] Furthermore, this invention does not limit the specific type of drug to be loaded. Any nanoparticle that can form a stable coating structure with a lysosomal membrane through physical co-extrusion and is suitable for drug delivery or diagnostic applications can serve as the core structure of the drug-loaded nanoparticles of this invention.

[0011] Further, in step S2, the drug to be loaded is selected from one or more of the following: hollow mesoporous Prussian blue nanoparticles, iron oxide nanoparticles, manganese-based nanoparticles, copper-based nanoparticles, mesoporous silica nanoparticles, mesoporous carbon nanoparticles, polylactic acid-glycolic acid copolymer nanoparticles, polycaprolactone nanoparticles, protein self-assembled nanoparticles, and lipid-polymer hybrid nanoparticles.

[0012] Further, in step S2, the mass ratio of the secondary lysosomal vesicles to the drug to be loaded is 0.5:1-2:1.

[0013] Furthermore, the number of times the first extrusion process is performed in step S1 is 5-15 times, the number of times the second extrusion process is 5-15 times, and the total number of extrusion processes in step S2 is 5-20 times.

[0014] Furthermore, in step S1, the lysosomes are prepared by separating and purifying cell homogenates.

[0015] Furthermore, the lysosomes are prepared by separating and purifying cell homogenates by differential centrifugation at 0-4°C. The differential centrifugation is as follows: centrifuging at 800-1200g for 3-6 minutes and discarding the precipitate; then centrifuging at 2800-3200g for 8-12 minutes and discarding the precipitate; centrifuging at 4800-5200g for 8-12 minutes and collecting the supernatant; centrifuging at 15,000-25,000g for 15-25 minutes and collecting the precipitate; resuspending the precipitate and then centrifuging at 15,000-25,000g for 15-25 minutes and collecting the precipitate.

[0016] Furthermore, the cell homogenate is obtained by grinding the cells.

[0017] Furthermore, in step S1, the lysosomes are derived from mammalian hepatocytes, tumor cells, or immune cells.

[0018] Furthermore, the lysosome contains one or more of cathepsin B, cathepsin K, cathepsin L, and cathepsin S. The lysosome-based drug-carrying nanoparticles can be used to prepare pharmaceutical compositions that improve drug delivery efficiency by in-situ modulating the extracellular matrix structure of tumor tissue. Because this carrier retains the activity of lysosome-related hydrolases during delivery, it can degrade the tumor extracellular matrix, thereby weakening the dense physical barrier of tumor tissue and enhancing the penetration and distribution of nanomedicines in tumor tissue. In addition, by remodeling the tumor extracellular matrix structure, the lysosome-based drug-carrying nanoparticles of the present invention can also remove the physical restrictions on the infiltration of immune cells by tumor tissue, creating favorable conditions for the entry of immune cells such as dendritic cells and cytotoxic T cells into tumor tissue, thereby enabling the preparation of pharmaceutical compositions that improve the tumor immune microenvironment and enhance anti-tumor immune responses.

[0019] A second objective of this invention is to provide the application of the above-mentioned drug-loaded nanoparticles in the preparation of antitumor drugs, wherein the antitumor drugs degrade the extracellular matrix of tumor cells in situ.

[0020] A third objective of this invention is to provide an antitumor drug comprising the aforementioned drug-loaded nanoparticles, wherein the antitumor drug delivers the loaded drug to tumor cells by degrading the tumor cell extracellular matrix.

[0021] Furthermore, the antitumor drug is administered via intravenous injection.

[0022] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0023] (1) This invention introduces the whole lysosome as a biomimetic membrane source into the nanomedicine delivery system for the first time. During the preparation process, the activity of various hydrolytic enzymes naturally present in the lysosome is effectively preserved, so that the nanodrug-carrying particles can degrade the extracellular matrix of tumor tissue cells in situ during in vivo delivery, thereby significantly weakening the dense physical barrier of tumor tissue and providing favorable conditions for the deep penetration and uniform distribution of nanomedicine in tumor tissue. This is a technical effect that existing biomimetic carriers such as red blood cell membranes, tumor cell membranes or exosomes do not have.

[0024] (2) By degrading and remodeling the extracellular matrix of tumor cells, the lysosome-based nanoparticles of the present invention can significantly enhance the penetration ability and distribution uniformity of drugs in tumor tissues, thereby improving their enrichment level and tumor cell uptake efficiency in tumor tissues, and achieving an overall improvement in delivery efficiency.

[0025] (3) On the basis of the weakening of the extracellular matrix barrier, the nano-drug-carrying particles of the present invention can effectively remove the physical restriction of tumor tissue on the infiltration of immune cells, creating conditions for immune cells such as dendritic cells and cytotoxic T cells to enter the core area of ​​the tumor, thereby facilitating the occurrence and maintenance of anti-tumor immune response;

[0026] (4) The nano-drug-carrying particles of the present invention use lysosomal membranes as natural biological membrane sources, which have good biocompatibility and in vivo stability, which helps to reduce the risk of non-specific protein adsorption and immune system clearance, thereby prolonging blood circulation time and further improving the tumor tissue enrichment capacity.

[0027] (5) The preparation method of the nano-drug-loaded particles of the present invention is simple, mild and highly applicable, and can be adapted to various types of nanoparticle cores. It has good repeatability and industrial application prospects. Attached Figure Description

[0028] Figure 1 This is a transmission electron microscope image of the hollow mesoporous Prussian blue nanoparticles (HMPB) prepared in Example 1.

[0029] Figure 2This is a statistical result of the morphology and particle size of lysosomes extracted from mouse liver tissue in Example 2;

[0030] Figure 3 This is a transmission electron microscope image of the lysosome-based drug-carrying nanoparticles (L@H) in Example 3;

[0031] Figure 4 The diagram shows the hydration particle size distribution and zeta potential (ζ-potential) comparison results of lysosomes (LYSO), hollow mesoporous Prussian blue nanoparticles (HMPB), and lysosome-based drug-carrying nanoparticles (L@H) in Examples 1-3.

[0032] Figure 5 This is an electrophoresis result of lysosome-related membrane proteins and hydrolases in lysosomes (LYSO) and lysosome-based drug-carrying nanoparticles (L@H) in Example 4;

[0033] Figure 6 This is a comparison of the diffusion behavior of different samples in the extracellular matrix simulated gel in Example 5, where au represents the relative fluorescence intensity value;

[0034] Figure 7 This is a graph showing the uptake and lysosomal colocalization analysis results of the control group, lysosome-based drug-carrying nanoparticles (L@H), and erythrocyte membrane-based drug-carrying nanoparticles (R@H) in tumor cells in Example 6. "***" indicates a P value < 0.001.

[0035] Figure 8 This is a graph showing the results of lysosomal colocalization analysis of lysosome-based drug-carrying nanoparticles (L@H) and erythrocyte membrane-based drug-carrying nanoparticles (R@H) in tumor cells in Example 6, where Rr represents the Pearson correlation coefficient;

[0036] Figure 9 This is an imaging result of the tissue distribution and tumor enrichment of lysosome-based drug-carrying nanoparticles (L@H) and erythrocyte membrane-based drug-carrying nanoparticles (R@H) in tumor-bearing mice in Example 7.

[0037] Figure 10 This is a growth curve of tumor volume changes over time in mice of different treatment groups in Example 7;

[0038] Figure 11 These are histological analysis results of major organs and tumor tissues in mice from different treatment groups in Example 7;

[0039] Figure 12 This is a graph showing the experimental results of the effects of different treated samples on tumor cell viability in Example 8;

[0040] Figure 13The results show the morphology and particle size statistics of red blood cell membranes extracted from mouse blood in Comparative Example 1. Detailed Implementation

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0042] Example 1: Preparation of hollow mesoporous Prussian blue nanoparticles

[0043] (1) The specific preparation steps of hollow mesoporous Prussian blue nanoparticles are as follows:

[0044] 132 mg of potassium ferricyanide and 3 g of polyvinylpyrrolidone were weighed and dissolved in 40 mL of 0.01 M hydrochloric acid solution. The solution was magnetically stirred for 30 min at room temperature to form a homogeneous solution. The solution was then transferred to a constant temperature oven and reacted at 80 °C for 24 h. After the reaction was completed, the reaction solution was centrifuged at 13000 rpm for 20 min, and the supernatant was discarded. The resulting precipitate was washed five times with ultrapure water to remove residual reagents. The washed precipitate was then freeze-dried under vacuum to obtain Prussian blue nanoparticles.

[0045] 20 mg of the Prussian blue nanoparticles prepared above and 200 mg of polyvinylpyrrolidone were weighed and dispersed in 0.1 M hydrochloric acid solution and magnetically stirred for 3 h at room temperature. The suspension was then transferred to a polytetrafluoroethylene-lined high-pressure reactor and reacted at 140 °C for 3 h. After the reaction was completed and cooled to room temperature, the precipitate was collected by centrifugation at 13000 rpm for 20 min and washed five times with ultrapure water. Finally, hollow mesoporous Prussian blue nanoparticles (HMPB) were obtained by vacuum freeze-drying.

[0046] Transmission electron microscopy results as follows Figure 1 The results show that the obtained HMPB nanoparticles have a hollow mesoporous structure with an average particle size of 104.4±26.2 nm, making them suitable as the core structure for drug-carrying nanoparticles.

[0047] (2) In addition to the hollow mesoporous Prussian blue nanoparticles mentioned above, the following nanoparticles can also be selected as the nanoparticle core:

[0048] Metal or metal oxide nanoparticles, such as iron oxide nanoparticles, manganese-based nanoparticles, copper-based nanoparticles or composite nanoparticles thereof, can be prepared by co-precipitation, thermal decomposition or hydrothermal methods.

[0049] Porous inorganic nanoparticles, such as mesoporous silica nanoparticles and mesoporous carbon nanoparticles, can be prepared by template method or sol-gel method;

[0050] Polymer nanoparticles, such as polylactic acid-glycolic acid copolymer (PLGA) nanoparticles and polycaprolactone (PCL) nanoparticles, can be prepared by emulsification-solvent evaporation or nanoprecipitation.

[0051] Biomacromolecules or biomimetic nanoparticles, such as protein self-assembled nanoparticles and lipid-polymer hybrid nanoparticles.

[0052] All of the above-mentioned different types of nanoparticles can be coated with lysosomal membranes using the same physical co-extrusion method as in Example 3, thereby constructing lysosomal-based drug-loaded nanoparticles.

[0053] Example 2: Extraction and Characterization of Lysosomes

[0054] Using liver tissue from healthy female Balb / c mice as an example, the extraction and purification process of lysosomes is described in detail. A commercially available lysosome isolation kit (brand: Solarbio, catalog number: EX2670) was used for extraction, with appropriate adjustments made. The specific steps are as follows:

[0055] Liver tissue was minced and placed in pre-cooled lysosome separation buffer (lysosome extraction solution A) and incubated on ice for 10 min. Then, it was homogenized using a high-speed tissue homogenizer at 65 Hz for 30 s to obtain a homogeneous cell homogenate. The homogenate was then subjected to differential centrifugation at 4°C: 1000 g for 5 min (discarding the precipitate); 3000 g for 10 min (discarding the precipitate); and 5000 g for 10 min (collecting the supernatant).

[0056] The supernatant was centrifuged at 21400 g for 20 min at 4°C to obtain crude lysosome precipitate. The precipitate was resuspended in lysosome purification buffer (lysosome extraction buffer B) and centrifuged again at 21400 g for 20 min to further remove impurities. Finally, the precipitate was resuspended in PBS buffer, and the protein concentration was determined using a BCA (bisquinolinic acid method) quantitative kit. The obtained lysosomes (LYSO) were stored at 4°C for later use.

[0057] The morphology and particle size characterization results of the extracted lysosomes are as follows: Figure 2 As shown, the extracted lysosomes are spherical and have a size of 121.3 ± 39.8 nm.

[0058] Example 3: Preparation and basic physicochemical characterization of lysosome-based drug-loaded nanoparticles

[0059] The film coating process is performed using a physical co-extrusion method, and the specific steps are as follows:

[0060] The lysosomal suspension obtained in Example 2 was placed in an ice bath for later use to prevent the lysosomal membrane structure and its internal components from being inactivated or damaged during processing. The lysosomal suspension was transferred to an Avanti mini-extruder liposome extruder and extruded sequentially through polycarbonate membranes with pore sizes of 400 nm and 200 nm. The number of extrusions was 5-15 times under each pore size condition, preferably 10 times, to obtain lysosomal-derived vesicles with relatively uniform particle size.

[0061] Subsequently, the lysosomal vesicles were mixed with the hollow mesoporous Prussian blue nanoparticles (HMPB) prepared in Example 1 at a certain mass ratio, which was adjusted within the range of 0.5:1 to 2:1, preferably about 1:1. While maintaining the system at a low temperature (0-4°C), the mixture was co-extruded through a polycarbonate membrane with a pore size of 200 nm. The number of extrusions was controlled within the range of 5-20 times, preferably not less than 10 times, thereby promoting the uniform coating of the lysosomal membrane onto the surface of the nanoparticles under external mechanical shear force.

[0062] After co-extrusion, the resulting sample was immediately placed on ice to prevent damage to the lysosomal membrane structure due to temperature rise. Following the above steps, lysosome-based drug-loaded nanoparticles L@H were obtained.

[0063] Experiments revealed that when the co-extrusion process was not carried out at low temperatures, or when the number of extrusions was less than 5, it was difficult to form a continuous and stable film structure on the surface of the resulting nanoparticles. Furthermore, when stepwise pore size extrusion was not employed (e.g., a single extrusion using only a 200 nm polycarbonate membrane) or when external extrusion power was insufficient (e.g., fewer than 3 extrusions or lack of continuous and stable mechanical shear force), it was also difficult to achieve effective coating of nanoparticles with lysosomal membranes. This indicates that physical co-extrusion with appropriate pore size and number of extrusions under low-temperature conditions is a key technical condition for achieving stable coating of nanoparticles with lysosomal membranes.

[0064] The transmission electron microscopy results of the prepared L@H are as follows: Figure 3 As shown, a continuous film structure can be observed on the surface of the nanoparticles, indicating that the lysosomal membrane was successfully coated on the nanoparticle surface. To further characterize the basic physicochemical properties of the prepared drug-loaded nanoparticles, the hydrated particle size and Zeta potential of all samples during the preparation process were measured using a dynamic light scattering instrument. The results show that the prepared L@H has a stable particle size distribution, and its hydrated particle size is increased compared to the uncoated nanoparticle core; at the same time, its surface Zeta potential also changes accordingly, reflecting that the lysosomal membrane was successfully coated on the nanoparticle surface. Figure 4The above results demonstrate that physical co-extrusion can effectively encapsulate nanoparticles with lysosomal membranes and yield structurally stable drug-loaded nanoparticles.

[0065] Example 4: Verification of lysosomal characteristics and biological function retention

[0066] To verify the structural characteristics of the lysosome-based drug-loaded nanoparticles and their retention of lysosomal biological properties, the prepared drug-loaded nanoparticles were characterized by protein composition analysis. The specific steps are as follows:

[0067] The protein composition of the samples was analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot, and lysosomal marker proteins and hydrolases were detected. The results are as follows: Figure 5 As shown, various lysosomal membrane proteins and hydrolases can still be detected in L@H, including lysosome-associated membrane protein 1 (LAMP1), cathepsin B, cathepsin K, cathepsin L, and cathepsin S, indicating that the biological characteristics of lysosomes are effectively preserved during the preparation process.

[0068] Example 5: Evaluation of permeability in extracellular matrix simulated gels

[0069] To evaluate the diffusion and penetration performance of lysosome-based drug-loaded nanoparticles in a simulated tissue environment, an extracellular matrix-simulated gel system was constructed, and its penetration behavior was experimentally verified. The specific steps are as follows:

[0070] On ice, sequentially mix 48 μL of 10× phosphate-buffered saline (PBS), 12.8 μL of 1 M sodium hydroxide solution, 24.3 μL of ultrapure water, 300 μL of 5 mg / mL rat tail type I collagen solution, and 160 μL of 5 mg / mL hyaluronic acid solution dissolved in PBS. Gently vortex the mixture to ensure homogeneity and degas under vacuum on ice to eliminate air bubbles. Inject 50 μL of the pre-gel solution into a 1.2 mm inner diameter glass capillary tube, seal both ends, and incubate overnight at 37°C to complete gelation.

[0071] Far-red fluorescent (DiD) labeled erythrocyte membrane vesicles (DiD-RBCVs) were mixed with PBS, lysosomes (LYSO), and lysosome-based drug-carrying nanoparticles (L@H) to obtain the DiD-RBCVs+PBS group, the DiD-RBCVs+LYSO group, and the DiD-RBCVs+L@H group, respectively. These were then gently added to the gel surface, the capillary ends were resealed, and the mixtures were incubated at 37°C. At specified time points, diffusion was visualized and monitored using an IVIS Lumina XR in vivo imaging system, and the diffusion extent of the drug-carrying nanoparticles was quantitatively analyzed using ImageJ software.

[0072] The results are as follows Figure 6 As shown, the diffusion depth and diffusion area of ​​the DiD-RBCVs+L@H group in the gel were significantly better than those of the DiD-RBCVs+PBS group, indicating that it has better tissue penetration ability.

[0073] Example 6: Tumor cell uptake experiment and colocalization analysis

[0074] To evaluate the uptake behavior of lysosome-based drug-carrying nanoparticles in tumor cells and their targeting ability to the lysosomal pathway, their intracellular distribution characteristics were validated through tumor cell uptake experiments and co-localization analysis. Specific steps included:

[0075] First, L@H prepared in Example 3 and R@H prepared in Comparative Example 1 were respectively mixed with DiD fluorescent dye (the concentration of each was 100 μg / mL, calculated based on HMPB in L@H or R@H), and shaken on a shaker in the dark for 20-30 min. The reaction solution was centrifuged at 1000 g for 3 min, and the supernatant was collected; then the precipitate was collected by centrifugation at 13000 g for 10 min to obtain fluorescently labeled DiD-R@H and DiD-L@H.

[0076] 3×10 per dish 4 4T1 cells (mouse breast cancer cell line) were seeded into confocal culture dishes at a density of 1 / 2 cells and incubated at 37°C in a 5% CO2 incubator for 12 h. Then, DiD-R@H and DiD-L@H were added and co-incubated for 24 h (with medium without DiD-R@H or DiD-L@H serving as a control). After incubation, the cells were washed twice with PBS, and then stained sequentially with LysoTracker Green fluorescent probe (lysosomal green fluorescent probe) diluted in serum-free medium and Hoechst 33342 fluorescent dye (a blue fluorescent nuclear dye that can penetrate the cell membrane), for incubation times of 60 min and 20 min, respectively. Cell uptake images were then observed using a confocal microscope, and intracellular fluorescence intensity was quantitatively analyzed using ImageJ software. Results are as follows: Figure 7 As shown, the fluorescence signal of L@H in tumor cells is stronger than that of R@H, indicating that lysosome-based nanoparticles can effectively promote cellular uptake of nanomedicines.

[0077] Further colocalization analysis was performed on the confocal images using ImageJ software. The Pearson correlation coefficient was calculated using the ColocalizationFinder plugin to assess the overlap between the two fluorescence signals (red fluorescence and lysosomal green fluorescence). Simultaneously, regions of interest were selected, and the Plot Profile method (an image processing tool in ImageJ) was used to compare and analyze the fluorescence signal curves of the two channels to evaluate the targeting ability of DiD-L@H to intracellular lysosomes. The colocalization analysis results are as follows: Figure 8 As shown, DiD-L@H has a higher colocalization coefficient compared to DiD-R@H, confirming that the lysosome loading strategy can endow the vector with stronger lysosome pathway targeting specificity.

[0078] Example 7: Application of lysosome-based drug-carrying nanoparticles in tumor therapy

[0079] To evaluate the efficacy of lysosome-based drug-carrying nanoparticles in tumor therapy, a mouse subcutaneous tumor-bearing model was constructed for in vivo experimental validation. The specific steps are as follows:

[0080] Healthy female Balb / c mice (6-8 weeks old) were selected, and 4T1 breast cancer cells were introduced into each mouse at a rate of 3 × 10⁻⁶ cells under sterile conditions. 6 A subcutaneous tumor-bearing model was established by subcutaneous inoculation of mice with 100 cells / 100 μL PBS in the right hind limb. Tumor growth was monitored daily after inoculation, and the tumor was monitored when it reached approximately 100-200 mm in size. 3 At that time, the drug administration experiment began.

[0081] Experimental animals were randomly divided into four groups: a control group (PBS group), a control group without nanoparticle coating (HMPB group), a group treated with erythrocyte membrane-based drug-loaded nanoparticles (R@H group), and a group treated with lysosome-based drug-loaded nanoparticles (L@H group). Each treatment group was administered the drug via tail vein injection at a volume of 200 μL per animal (HMPB, R@H, and L@H groups were treated as 1 mg / mL of HMPB). The control group received an equal volume of PBS solution.

[0082] At different time points (8 h, 24 h, and 72 h) after drug administration, some experimental animals were sacrificed, and major organs (including heart, liver, spleen, lung, and kidney) and tumor tissue were collected. The distribution of nanomedicine carriers in each tissue was analyzed using in vitro fluorescence imaging. The results are as follows: Figure 9As shown in the image, the imaging results indicate that the L@H group had a higher enrichment level in tumor tissue compared to the R@H group.

[0083] During the efficacy evaluation phase, tumor volume changes in each group of mice were continuously monitored, and the long and short diameters of the tumors were measured every two days. Tumor volume was calculated using the formula V = (long diameter × short diameter). 2 The result was calculated as () / 2, and a tumor growth curve was plotted. The experimental results are as follows: Figure 10 As shown, the tumor growth rate in the L@H group was significantly slowed down, and the tumor volume was significantly reduced.

[0084] After the experiment, all experimental animals were euthanized, and major organs and tumor tissues were collected for histological analysis to further evaluate the treatment efficacy and systemic toxicity. Results are as follows: Figure 11 As shown, lysosome-based drug-carrying nanoparticles significantly inhibited tumor growth without causing tissue damage to major organs, indicating good biocompatibility.

[0085] The experimental results above indicate that lysosome-based drug-carrying nanoparticles can effectively improve the distribution and utilization efficiency of nanomedicines in tumor tissues, thus demonstrating good application effects and prospects in tumor treatment.

[0086] Example 8: Cytotoxicity Evaluation of Lysosome-Based Drug-Loaded Nanoparticles

[0087] To rule out the possibility of lysosomes themselves acting as a therapeutic component and exerting direct cytotoxic effects on tumor cells, in vitro cytotoxicity evaluations were conducted on lysosomes alone (LYSO group), nanoparticles (HMPB group), and lysosome-based drug-loaded nanoparticles (L@H group). The specific experimental methods were as follows:

[0088] 4T1 cells (mouse breast cancer cell line) and HepG2 cells (human liver cancer cell line) were seeded into 96-well plates. After cell attachment, sample suspensions of different concentrations (equivalent nanoparticle concentrations: 0, 12.5, 25, 50, and 100 μg / mL) were added for treatment (as shown in Table 1) for 24 h. Cell viability was assessed using the CCK-8 assay.

[0089] Table 1 Sample processing details

[0090]

[0091] The results are as follows Figure 12As shown, in both tumor cell models, no significant decrease in cell viability was observed in the LYSO group, indicating that lysosomes themselves do not exhibit significant cytotoxicity. In contrast, the L@H group showed a significantly stronger cytotoxic effect than HMPB, exhibiting a certain concentration-dependent characteristic. Under the same treatment conditions, the cell survival rate of the L@H group was significantly lower than that of the HMPB-treated group, indicating that lysosome loading can effectively enhance the killing effect of HMPB in tumor cells.

[0092] The above results indicate that the antitumor effect of lysosome-based drug-carrying nanoparticles does not originate from the direct cytotoxic effect of lysosomes as therapeutic components, but is mainly attributed to their enhancing role as biomimetic carriers in improving the intracellular delivery efficiency and action conditions of nanoparticles.

[0093] Comparative Example 1: Preparation of drug-carrying nanoparticles based on erythrocyte membranes

[0094] To illustrate the impact of different biomembrane sources on the performance of drug-loaded nanoparticles, we constructed drug-loaded nanoparticles (R@H) based on erythrocyte membranes using erythrocyte membranes as the biomimetic membrane source. The specific preparation process is as follows:

[0095] First, erythrocyte membranes were extracted: Blood was collected from the orbital region of healthy female Balb / c mice and placed in centrifuge tubes containing heparin anticoagulant. The blood sample was centrifuged at 1000 g for 5 min at 4°C, and the supernatant plasma was discarded. The precipitate was resuspended in PBS buffer and centrifuged and washed three times to thoroughly remove plasma and leukocyte components. The washed erythrocyte precipitate was then added to ice-cold ultrapure water and lysed under hypotonic conditions for 1 h to release the cell contents. Subsequently, the erythrocyte membrane precipitate was collected by centrifugation at 10000 g for 5 min. The washing steps were repeated until the supernatant was clear and colorless. Finally, the erythrocyte membrane was resuspended in PBS for later use.

[0096] Red blood cell membrane suspension was sonicated for 5 min under ice bath conditions, followed by extrusion through 400 nm and 200 nm polycarbonate membranes 10 times each, using the same extrusion process as in Example 3, to prepare homogeneous vesicles derived from red blood cell membranes. The obtained red blood cell membrane vesicles were mixed with HMPB and co-extruded through a 200 nm membrane at least 10 times to obtain red blood cell membrane-based drug-loaded nanoparticles R@H. Their morphology and coating structure were verified by transmission electron microscopy. Figure 13 ).

[0097] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. The application of lysosome-based drug-carrying nanoparticles in the preparation of antitumor drugs, characterized in that, The antitumor drug degrades the tumor cell extracellular matrix in situ, and the drug-loaded nanoparticles are prepared by the following method: S1. The lysosomes are first extruded through a polycarbonate membrane with a pore size of 450-350 nm to obtain primary lysosomal vesicles. The primary lysosomal vesicles are then extruded through a polycarbonate membrane with a pore size of 150-250 nm to obtain secondary lysosomal vesicles. S2. The secondary lysosomal vesicles of S1 are mixed with the drug to be loaded to obtain a mixed system. The mixed system is co-extruded through a polycarbonate membrane with a pore size of 150-250 nm at 0-4℃ to obtain the drug-loaded nanoparticles. The drug to be loaded is selected from one or more of hollow mesoporous Prussian blue nanoparticles, iron oxide nanoparticles, manganese-based nanoparticles, and copper-based nanoparticles.

2. The application according to claim 1, characterized in that, In step S2, the mass ratio of the secondary lysosomal vesicles to the drug to be loaded is 0.5:1 to 2:

1.

3. The application according to claim 1, characterized in that, The number of extrusion processes in step S1 is 5-15 times for the first extrusion process, the number of extrusion processes in the second extrusion process is 5-15 times for the second extrusion process, and the total number of extrusion processes in step S2 is 5-20 times.

4. The application according to claim 1, characterized in that, In step S1, the lysosomes are prepared by separating and purifying cell homogenates.

5. The application according to claim 4, characterized in that, The lysosomes are prepared by separating and purifying cell homogenates by differential centrifugation at 0-4°C. The differential centrifugation is as follows: centrifuge at 800-1200g for 3-6 minutes, discard the precipitate, then centrifuge at 2800-3200g for 8-12 minutes, discard the precipitate, centrifuge at 4800-5200g for 8-12 minutes, collect the supernatant, centrifuge at 15000-25000g for 15-25 minutes, collect the precipitate, resuspend, and then centrifuge at 15000-25000g for 15-25 minutes to collect the precipitate.

6. The application according to claim 1, characterized in that, In step S1, the lysosomes are derived from mammalian hepatocytes, tumor cells, or immune cells.

7. An antitumor drug, characterized in that, The antitumor drug includes the drug-loaded nanoparticles as described in any one of claims 1-6, wherein the antitumor drug delivers the drug to be loaded to tumor cells by degrading the tumor cell extracellular matrix.

8. The antitumor drug according to claim 7, characterized in that, The antitumor drug is administered via intravenous injection.

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