Cell membrane-coated prodrug self-assembled nanoparticles, and preparation method and application thereof

Abstract

The application belongs to the technical field of medicine, and particularly relates to a cell membrane coated prodrug self-assembled nanoparticle and a preparation method and application thereof. The nanoparticle is a prodrug self-assembled nanoparticle for which a four-sulfur bond bridged cabazitaxel dimer prodrug (gamma-4S-2CTX) and a polyethylene glycol (PEG) modifier are co-assembled to prepare a tumor cell membrane coated with the prodrug self-assembled nanoparticle as a core. The gamma-4S-2CTX is shown in structural formula (I). The biomimetic cell membrane coated nanoparticle (CM-pDPNAs) prepared by the application exhibits significant tumor specific recognition ability and low toxic side effects, greatly improves the selectivity and treatment effect of the drug in the body, and shows good safety and anti-tumor effect in the in-vivo anti-tumor experiment. The application provides an efficient and low-toxic anti-tumor drug delivery system, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a cell membrane-coated prodrug self-assembled nanoparticle, its preparation method, and its application. Background Technology

[0002] Cancer is one of the major public health problems facing the world and a leading cause of death. Chemotherapy, as a classic cancer treatment, is widely used because it can effectively inhibit the proliferation of tumor cells. However, traditional chemotherapy drugs generally suffer from problems such as low delivery efficiency, non-specific distribution, and severe toxic side effects, which not only reduce treatment efficacy but also significantly affect patients' quality of life. For example, cabazitaxel (CTX), as a broad-spectrum chemotherapy drug, has achieved some success in the treatment of various tumors, but its systemic toxicity limits the increase of clinical dosage and causes serious adverse reactions such as bone marrow suppression and immune system damage.

[0003] To address these challenges, the rise of nanomedicine delivery systems offers new insights into the precise delivery and toxicity control of chemotherapy drugs. Nanomedicine delivery systems achieve passive targeted delivery by optimizing drug pharmacokinetic behavior, enhancing solubility, and utilizing the enhanced penetration and retention (EPR) effect of tumors. However, traditional nanomedicine delivery systems also face several bottlenecks. Many chemotherapy drugs have weak affinity for nanocarriers, leading to low drug loading, poor colloidal stability, and premature drug leakage. Furthermore, traditional nanomedicine delivery systems typically require complex manufacturing processes and carry the risk of carrier-related toxicity, posing a challenge to their clinical application. The effectiveness of passive targeting is also highly dependent on tumor vascularization and permeability, factors that vary depending on tumor type and stage, resulting in suboptimal targeting outcomes.

[0004] To further improve the tumor specificity and stability of nanomedicine delivery systems, researchers have developed a biomimetic nanomedicine delivery system coated with tumor cell membranes. This biomimetic design, by preserving the biological properties of tumor cell membranes, enables nanomedicines to better recognize and bind to tumor cells, thereby enhancing tumor targeting. Traditional cell membrane-coated nanoparticles are typically based on synthetic nanoparticle cores, which involve complex manufacturing processes and still suffer from problems such as low drug loading, limited stability, and poor drug release control. Furthermore, the low affinity between the cell membrane and the nanoparticle core is a major bottleneck limiting the performance of cell membrane-coated nanoparticles, affecting membrane integrity and weakening their therapeutic benefits.

[0005] Based on this, the present invention proposes a nanomedicine delivery system that uses tumor cell membrane coating combined with PEG modification and prodrug self-assembly formulation technology, aiming to solve the problems of low delivery efficiency, instability and large toxic side effects of current chemotherapy drugs, thereby significantly improving the anti-tumor treatment effect and expanding the clinical application potential of chemotherapy drugs. Summary of the Invention

[0006] The technical problem solved by this invention is to introduce tumor cell membranes into the surface modification of prodrug self-assembled nanoparticles to achieve biocamouflage, enabling them to utilize specific markers and compatibility on tumor cell membranes for more efficient tumor-targeted delivery. Simultaneously, to address the issue of insufficient affinity between tumor cell membranes and nanoparticles, this invention provides cell membrane-coated prodrug self-assembled nanoparticles, their preparation method, and their applications.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A cell membrane-coated prodrug self-assembled nanoparticle, wherein the nanoparticle is a tumor cell membrane coated with a prodrug self-assembled nanoparticle prepared by co-assembly of a tetrasulfide-bridged carbamate dimer prodrug (γ-4S-2CTX) and a polyethylene glycol (PEG) modifier.

[0009] The γ-4S-2CTX is shown in structural formula (I).

[0010]

[0011] The PEG modifier accounts for 5-15% of the total mass of PEG and γ-4S-2CTX.

[0012] The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG, or PE-PEG. The preferred PEG modifier is DSPE-PEG. The molecular weight of the PEG is 1000-5000, preferably 1000, 2000, and 5000, and more preferably 2000.

[0013] The cell membrane is a tumor cell membrane, which can be obtained from tumor cells such as mouse prostate cancer (RM-1) cells, mouse breast cancer (4T1) cells, mouse pancreatic cancer (Panc02) cells, and human non-small cell lung cancer (A549) cells.

[0014] A method for preparing cell membrane-coated prodrug self-assembled nanoparticles involves using a one-step nanoprecipitation method to prepare a prodrug self-assembled nanoparticle core modified with a PEG modifier, then mixing it with cell membrane material, and finally preparing cell membrane-coated PEG-modified prodrug self-assembled nanoparticles by ultrasound.

[0015] Furthermore, tumor cell membrane materials are thoroughly mixed with PEG-modified γ-4S-2CTX prodrug self-assembled nanoparticles, and the mixture is subjected to ultrasound. The energy generated by the ultrasound waves destroys the membrane structure, causing the cell membrane to spontaneously remodel on the surface of the nanoparticles, thus preparing cell membrane-coated prodrug self-assembled nanoparticles.

[0016] The ultrasonic method uses an ultrasonic power of 50-250w, an ultrasonic time of 1-10min, and an ultrasonic temperature of 5-10℃.

[0017] Preferably, an ultrasonic power of 100W and an ultrasonic time of 2 minutes are selected, with a 3-second interval between every 2 seconds of ultrasonic treatment.

[0018] In the above preparation methods, physical extrusion can also be used, where the mixture is extruded through a polycarbonate film using an extruder to coat the cell membrane onto the surface of the nanoparticles. Alternatively, microfluidic electroporation can be used to thoroughly mix the components in channels to prepare cell membrane-coated prodrug self-assembled nanoparticles.

[0019] A pharmaceutical composition comprising the aforementioned cell membrane prodrug self-assembled nanoparticles and a pharmaceutically acceptable carrier or excipient.

[0020] The use of a cell membrane-coated prodrug self-assembled nanoparticle or the composition thereof, and the use of the cell membrane-coated prodrug self-assembled nanoparticle or the composition thereof in the preparation of a drug delivery system.

[0021] The application of the cell membrane-coated prodrug self-assembled nanoparticles or compositions in the preparation of antitumor drugs.

[0022] Multimodal applications of the cell membrane-coated prodrug self-assembled nanoparticles in tumor therapy.

[0023] Application of the cell membrane-coated prodrug self-assembled nanoparticles in the preparation of injection, oral, or local drug delivery systems.

[0024] Compared with the prior art, the advantages of this invention are:

[0025] (1) This invention utilizes the redox sensitivity of tetrasulfide bonds to construct a prodrug system that can rapidly respond to the tumor microenvironment. Under the action of high concentration of glutathione (GSH) in tumor cells, γ-4S-2CTX prodrug can hypersensitively release active drugs, enhance the tumor killing effect of drugs, and at the same time reduce the toxic side effects of normal tissues.

[0026] (2) The present invention uses a one-step nanoprecipitation method to prepare a uniform self-assembled nanoparticle core of a dimer prodrug. The preparation method is simple and easy to implement, and achieves efficient drug loading with an ultra-high drug loading of more than 70%.

[0027] (3) This invention introduces the cell membrane into the surface modification module of the prodrug self-assembled nanoparticles, endowing the prodrug self-assembled nanoparticles with biomimetic functions, enabling them to utilize specific biomarkers and compatibility on the tumor cell membrane to achieve more efficient tumor-targeted delivery. At the same time, the properties of the tumor cell membrane enhance the adhesion and uptake capacity of the nanoparticles with tumor cells, thereby improving drug delivery efficiency.

[0028] (4) The PEG modifier DSPE-PEG used in this invention 2000 As a synergistic modifier, it enhances the affinity between nanonuclei and cell membranes, resulting in cell membrane-coated formulations with good stability, prolonged drug circulation time in the blood, and improved drug utilization.

[0029] In summary, addressing several technical problems of traditional chemotherapy drug delivery systems, including low drug loading, poor colloidal stability, and premature leakage in vivo, as well as the uncontrollable targeting effect and therapeutic limitations caused by the passive targeting of conventional nanomedicines relying on tumor angiogenesis and permeability, this invention proposes a prodrug self-assembled nanodelivery system (CM-pDPNAs) based on tumor cell membrane coating. This system combines a synergistic strategy of cell membrane and PEG modification, which not only improves the tumor-specific targeting of drugs but also significantly enhances the stability of nanomedicines and simplifies the preparation process, thereby overcoming the technical bottlenecks of traditional drug delivery systems. Attached Figure Description

[0030] Figure 1 This is a TEM image of the RM-1 cell membrane material extracted in Example 1 of the present invention.

[0031] Figure 2 This is a particle size-placement time diagram of cell membrane-coated non-PEG modified prodrug self-assembled nanoparticles in Example 2(1) of the present invention.

[0032] Figure 3 These are photographs and TEM images of the prodrug self-assembled nanoparticles modified with PEG and coated with cell membranes in Example 2(2) of this invention, after being placed for one week.

[0033] Figure 4 The particle size diagrams are of the 5% and 10% PEG prodrug self-assembled nanoparticles coated with cell membranes in Example 2(2) of this invention.

[0034] Figure 5 This is a stability diagram of the cell membrane-coated 10% PEG prodrug self-assembled nanoparticles placed at 4°C in Example 2 (3) of the present invention.

[0035] Figure 6 This is a stability diagram of the cell membrane-coated 10% PEG prodrug self-assembled nanoparticles in 10% FBS incubation in Example 2(3) of the present invention.

[0036] Figure 7 This is a diagram showing the release of cell membrane-coated prodrug self-assembled nanoparticles from plasma in Example 3(1) of the present invention.

[0037] Figure 8 This is a diagram showing the in vitro release of cell membrane-coated prodrug self-assembled nanoparticles in Example 3(2) of the present invention.

[0038] A: Schematic diagram of in vitro release experiment of cell membrane-coated prodrug self-assembled nanoparticles in blank release medium.

[0039] B: In vitro release assay of cell membrane-coated prodrug self-assembled nanoparticles under 1mM GSH conditions.

[0040] Figure 9 This is an electrophoresis diagram of cell membrane-coated prodrug self-assembled nanoparticle proteins in Example 4(1) of the present invention.

[0041] I: RM-1 cancer cell lysate (LLC), II: isolated RM-1 cancer cell membrane (CCM), III: CM-pDPNAs, and IV: pDPNAs.

[0042] Figure 10 This is a protein imprint of cell membrane-coated prodrug self-assembled nanoparticles in Example 4(2) of the present invention.

[0043] RM-1 cancer cell lysate (LLC), #2: CM-DPNAs and #4: CM-pDPNAs.

[0044] Figure 11 This is a diagram of tumor cell adhesion of cell membrane-coated prodrug self-assembled nanoparticles in Example 5 of the present invention.

[0045] Figure 12 This is a diagram showing the uptake of cell membrane-coated prodrug self-assembled nanoparticles by tumor cells in Example 6(1) of the present invention.

[0046] A: RM-1 cells take up cell membrane-coated prodrug self-assembled nanoparticles.

[0047] B: Uptake of cell membrane-coated prodrug self-assembled nanoparticles by 4T1 cells.

[0048] #1DPNAs are unPEGylated and cell membrane-coated γ-4S-2CTX NPs; #2CM-DPNAs are cell membrane-coated, non-PEGylated γ-4S-2CTX NPs; #3pDPNAs are PEGylated γ-4S-2CTX NPs; #4CM-pDPNAs are cell membrane-coated, PEGylated γ-4S-2CTX NPs; #5 is a blank control.

[0049] Figure 13 This is a diagram showing the uptake of cell membrane-coated prodrug self-assembled nanoparticles by normal cells in Example 6(2) of the present invention.

[0050] #1DPNAs are unPEGylated and cell membrane-coated γ-4S-2CTX NPs; #2CM-DPNAs are cell membrane-coated, unPEGylated γ-4S-2CTX NPs; #3pDPNAs are PEGylated γ-4S-2CTX NPs; #4CM-pDPNAs are cell membrane-coated, PEGylated γ-4S-2CTX NPs; #5 is a blank control.

[0051] Figure 14 This is a diagram showing the uptake of cell membrane-coated prodrug self-assembled nanoparticles by macrophages in Example 6(3) of the present invention.

[0052] #1DPNAs are unPEGylated and cell membrane-coated γ-4S-2CTX NPs; #2CM-DPNAs are cell membrane-coated, unPEGylated γ-4S-2CTX NPs; #3pDPNAs are PEGylated γ-4S-2CTX NPs; #4CM-pDPNAs are cell membrane-coated, PEGylated γ-4S-2CTX NPs; #5 is a blank control.

[0053] Figure 15 This is a tissue distribution diagram of the cell membrane-coated prodrug self-assembled nanoparticles in Example 8 of the present invention.

[0054] Figure 16 This describes the antitumor effect of cell membrane-coated prodrug self-assembled nanoparticles in Example 9 of the present invention in the RM-1 mouse model.

[0055] A: Schematic diagram of the dosing regimen.

[0056] B: Average tumor growth curve of the RM-1 mouse prostate cancer subcutaneous tumor model after treatment.

[0057] C: Individual tumor growth curves after treatment in the RM-1 mouse prostate cancer subcutaneous tumor model: G1 physiological saline, G2 CM-DPNAs treatment, G3 pDPNAs treatment, G4 CM-pDPNAs treatment, G5 carbazide solution (2 mg / kg) and G6 carbazide solution (4 mg / kg).

[0058] Tumor images of a D:RM-1 mouse model of subcutaneous prostate cancer after treatment.

[0059] Tumor burden after treatment in the E:RM-1 mouse model of subcutaneous prostate cancer.

[0060] F: Changes in body weight of mice in the RM-1 mouse model of subcutaneous prostate cancer after treatment.

[0061] Figure 17 This is a safety assessment of the RM-1 mouse subcutaneous prostate cancer tumor model in Example 8 of the present invention after treatment.

[0062] A: Blood routine examination of RM-1 mouse prostate cancer subcutaneous tumor model after treatment.

[0063] B: Liver and kidney function tests of the RM-1 mouse model of subcutaneous prostate cancer after treatment.

[0064] Figure 18 This is an H&E staining image of an ex vivo tissue section from the RM-1 mouse subcutaneous prostate cancer tumor model in Example 8 of this invention after treatment. Detailed Implementation

[0065] The present invention will be further illustrated by way of embodiments below, but the invention is not limited to the scope of the embodiments described herein.

[0066] This invention incorporates tumor cell membranes into the surface modification of prodrug self-assembled nanoparticles, resulting in tumor cell membrane coating. This is combined with a nanodrug delivery system designed with PEG modification and prodrugs to achieve biocamouflage, enabling the nanoparticles to utilize specific markers and compatibility on the tumor cell membrane for more efficient tumor-targeted delivery. Simultaneously, a polyethylene glycol (PEG) modifier is introduced as a co-modifier. PEG first modifies the prodrug nanoparticles into affinity nanoparticles readily accepting membrane coating before membrane coating, effectively improving the affinity between the cell membrane and the nanoparticles. This enhances the stability and blood circulation time of the cell membrane-coated nanoparticles, and improves the in vivo stability and anti-immune clearance capacity of the drug delivery system.

[0067] This invention functionalizes the surface of prodrug self-assembled nanoparticles by modifying the tumor cell membrane (CM) with polyethylene glycol (i.e., preparing γ-4S-2CTX as the core of the nano-self-assembled body and coating it with the tumor cell RM-1 cell membrane to obtain tumor biomimetic function), thereby obtaining a highly efficient and low-toxicity anti-tumor drug delivery system. This solves the technical problems of low delivery efficiency, non-specific distribution, strong toxic side effects, and poor tumor targeting of traditional chemotherapy drugs, achieving good stability, enhancing in vivo drug delivery efficiency, and utilizing tumor cell membrane-specific proteins to achieve tumor-specific adhesion, enhance tumor cell uptake, and improve tumor killing ability.

[0068] Example 1: Extraction of RM-1 tumor cell membrane material

[0069] After culturing 20 million RM-1 tumor cells, the cells were washed three times with PBS solution. Next, the cells were scraped from the culture dish using a cell scraper and transferred to centrifuge tubes. The cells were centrifuged at 1000 rpm for 3 minutes, the supernatant was discarded, and 1 mL of membrane protein extraction reagent containing 1% PMSF was added to fully resuspend the cells. The cells were then placed on ice for 15 minutes. Subsequently, the cells were disrupted using an ultrasonic cell disruptor with a power of 150W and a disruption time of 5 minutes, with a 5-second pause every 10 seconds of sonication. After disruption, the suspension was centrifuged at 700g for 10 minutes at 4°C. The supernatant was transferred to a new centrifuge tube and centrifuged again at 14000g at 4°C for 30 minutes, collecting the pellet. Finally, the cell membrane protein concentration was determined using a BCA protein concentration assay kit to quantify the amount of extracted cell membrane proteins. The extracted cell membrane material is shown below. Figure 1 As shown, the average size is about 200nm, and the morphology is irregular.

[0070] Example 2: Preparation of cell membrane-coated prodrug self-assembled nanoparticles

[0071] (1) Preparation of cell membrane-coated non-PEG-modified prodrug self-assembled nanoparticles

[0072] First, an appropriate amount of γ-4S-2CTX prodrug was taken, and prodrug self-assembled nanoparticles (DPNAs) were prepared by a simple one-step nanoprecipitation method. The specific steps were as follows: the γ-4S-2CTX prodrug was dissolved in an ethanol solution and added dropwise to pure water at a stirring speed of 1000 r / min to form uniformly dispersed prodrug self-assembled nanoparticles.

[0073] 2.00 mg of the prodrug was accurately weighed and dissolved in 1 mL of anhydrous ethanol. The solution was then added dropwise to 4 mL of pure water while stirring (1000 rpm). γ-4S-2CTX then self-assembled into nanoparticles. After stirring for another 1 min, excess ethanol was removed by rotary evaporation under reduced pressure at room temperature. The volume of the colloidal solution was then brought up to 4 mL using deionized water to obtain unmodified prodrug self-assembled nanoparticles (DPNAs).

[0074] The RM-1 cell membranes extracted in Example 1 were dispersed in purified water at a predetermined ratio and mixed with prodrug self-assembled nanoparticles. The mixed suspension was sonicated to coat the cell membranes onto the surface of the prodrug nanoparticles. To determine the optimal amount of CM, different amounts of cell membrane material were mixed with prodrug self-assembled nanoparticles, and the amount of cell membrane used was quantified by the ratio of drug mass to cell membrane protein mass (DM ratio), preparing cell membrane-coated nanoparticles (CM-DPNAs) with DM ratios of 1:0, 1:0.2, 1:0.5, 1:1, and 1:2. Subsequently, the hydrodynamic diameter of the coated nanoparticles was measured using a dynamic light scattering instrument (Nano ZS, Malvern Co., UK) to evaluate their stability. The results are as follows: Figure 2 As shown in Table 1, γ-4S-2CTX exhibits good self-assembly ability, forming pure prodrug self-assembled nanoparticles (1:0NPs) with an average particle size of approximately 100 nm (Table 1). However, unmodified 1:0NPs tend to aggregate, leading to an increase in particle size (e.g., ...). Figure 2 As shown in Table 1). After cell membrane coating, the size of the nanoparticles increased with increasing CM dosage, while the Zeta potential decreased. Among them, 1:0.2 NPs and 1:0.5 NPs with moderate cell membrane coating showed significantly improved stability, and the stability of 1:0.2 NPs and 1:0.5 NPs was better than that of 1:1 and 1:2 NPs. Figure 2 (As shown).

[0075] Table 1. Particle size, size distribution, and potential of cell membrane-coated, non-PEG-modified prodrug self-assembled nanoparticles.

[0076]

[0077] (2) Preparation of cell membrane-coated PEG-modified prodrug self-assembled nanoparticles

[0078] To further improve the stability of cell membrane-coated nanoformulations, DSPE-PEG was used. 2000 It is introduced onto the surface of nanoparticles as a synergistic modifier. The specific steps are: DSPE-PEG of different masses... 2000 The PEG-modified prodrug self-assembled nanoparticles (pDPNAs) were prepared by dissolving the γ-4S-2CTX prodrug together in an ethanol solution and using the one-step nanoprecipitation method described above. Specifically:

[0079] Weigh out 4 mg of γ-4S-2CTX and 1 mg of DSPE-PEG. 2KThe mixture was dissolved in anhydrous ethanol (1 mL) and then added dropwise to deionized water at 4 mL under vigorous stirring at 1000 rpm to form PEGylated prodrug self-assembled nanoparticles. After stirring for another 1 min, excess ethanol was removed by rotary evaporation under reduced pressure at room temperature. The volume of the colloidal solution was then brought up to 4 mL with deionized water to obtain 1 mg / mL PEGylated prodrug assembled nanoparticles (pDPNAs).

[0080] The PEG modification content was quantified by using the PEG mass as the ratio of the prodrug mass to the total PEG mass, resulting in a series of nanoparticles with PEG modification levels of 5%, 10%, 15%, and 20%. When the PEG modification level was above 5%, pDPNAs maintained good stability, while excessive PEG content may be detrimental to repeated in vivo administration. Considering both PEG content and nanoparticle stability, nanoparticles with 5% and 10% PEG modification were selected for subsequent membrane coating experiments. Figure 3 ).

[0081] The PEG-modified prodrug self-assembled nanoparticles (pDPNAs) obtained above were mixed with cell membrane material (mixed according to the ratio set in step (1) above), and synergistic coating was achieved by sonication (ultrasonic power of 100W, sonication time of 2min, with a 3s interval between every 2s of sonication) to form PEG-modified cell membrane-coated prodrug self-assembled nanoparticles (CM-pDPNAs). After cell membrane coating, an increase in nanoparticle size and a change in zeta potential were observed. In addition, with the increase of DM ratio, the particle size of pDPNAs also increased significantly (e.g., Figure 4 (As shown). This phenomenon is particularly pronounced in 5% PEG formulations, especially 1:1 and 1:2 5% PEG nanoparticles, which precipitate during storage (e.g. Figure 3 (As shown). In contrast, the 10% PEG formulation exhibited higher tolerance for membrane coating. Transmission electron microscopy (TEM) images showed that CM-coated pDPNAs with medium (1:0.5) and high (1:2) DM ratios had different membrane coverage effects (e.g. Figure 3 (As shown). Therefore, a 10% PEG formulation was used in subsequent experiments, and the characterization results are shown in Table 2. The morphology of the nanoparticles in the membrane-coated formulation was observed using a transmission electron microscope (TEM, Hitachi, HT7700, Japan) to confirm the uniformity and integrity of the cell membrane coating. Figure 3 ).

[0082] Table 2. Particle size, size distribution, and potential of cell membrane-coated 10% PEG-modified prodrug self-assembled nanoparticles.

[0083]

[0084] (3) DM ratio formulation screening of cell membrane-coated prodrug self-assembled nanoparticles

[0085] The colloidal stability of the 10% PEG-modified prodrug self-assembled nanoparticles coated with cell membranes at different DM ratios prepared in Example 2(2) was investigated; the storage stability of the prodrug self-assembled nanoparticles at 4°C and with 10% FBS was investigated using particle size change as an indicator (see Table 2). The results are as follows: Figure 5 and Figure 6 As shown, the 1:0.5 formulation exhibited good stability under conditions of 4°C incubation and 10% FBS incubation. The optimal formulation was ultimately determined to be CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%. The cell membrane-coated PEG-modified prodrug self-assembled nanoparticles described below are all nanoformulations prepared using this method. Simultaneously, fluorescently labeled cell membrane-coated prodrug self-assembled nanoparticles were prepared by co-assembling the fluorescent dye Coumarin 6 or DiR with the prodrug using the steps of Example 2 in a conventional manner, for subsequent visualization studies of their interaction with cells and their distribution in vivo.

[0086] Example 3: In vitro release assay of cell membrane-coated prodrug self-assembled nanoparticles

[0087] (1) Plasma release of cell membrane-coated prodrug self-assembled nanoparticles

[0088] The cell membrane-coated prodrug self-assembled nanoparticles (i.e., CM-pDPNAs labeled with fluorescent dye, with a DM ratio of 1:0.5 and a PEG content of 10%) prepared in Example 2(3) were investigated for plasma release. The cell membrane-coated prodrug self-assembled nanoparticles were diluted 10-fold with rat plasma and incubated at 37°C for 24 h. Samples were taken at predetermined time points (0, 2, 4, 8, 12, and 24 h). The samples were treated using a protein precipitation method, i.e., the plasma sample was mixed with three times its volume of acetonitrile, vortexed for 3 min, centrifuged (13000 rpm, 5 min), and the supernatant was injected. The content of the parent drug cabazitaxel was measured using high-performance liquid chromatography. The results are as follows: Figure 7 As shown, cell membrane-coated prodrug self-assembled nanoparticles release almost no active parent drug under plasma conditions, indicating a low risk of drug leakage and potentially reducing off-target toxicity associated with cabazitaxel.

[0089] (2) Release of cell membrane-coated prodrug self-assembled nanoparticles under reducing conditions

[0090] Using phosphate-buffered saline (PBS) containing 30% ethanol at pH 7.4 as the blank release medium, the in vitro release of cell membrane-coated prodrug self-assembled nanoparticles under blank medium and reducing conditions (1 mM GSH added to the blank release medium) was investigated. 1 mL of cell membrane-coated prodrug self-assembled nanoparticles prepared in Example 2(3) (i.e., CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL) was added to 30 mL of release medium. Glutathione (GSH, 1 mM) was added to the release medium to investigate release under reducing conditions. Samples were taken at set time points at 37°C, and the concentration of released cabazitaxel was determined by high-performance liquid chromatography to investigate the in vitro release of the nanoparticles under blank medium and reducing conditions. The results are as follows: Figure 8 As shown, cell membrane-coated prodrug self-assembled nanoparticles exhibit good stability in the release medium. Almost no cabazitaxel was released from the nanoparticles in the blank medium, but it was rapidly released under reducing conditions, indicating the reduction-responsive release characteristics of cell membrane coating. At the same time, cell membrane coating did not hinder this specific drug release.

[0091] Example 4 Characterization of cell membrane-coated prodrug self-assembled nanoparticle membrane coating

[0092] (1) SDS-PAGE protein electrophoresis analysis of cell membrane-coated prodrug self-assembled nanoparticles

[0093] Protein samples from the cell membrane of Example 1, the cell membrane-coated prodrug self-assembled nanoparticles prepared in Example 2(3) (i.e., CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL), and the PEG-modified prodrug self-assembled nanoparticles (pDPNAs) prepared in Example 2(2) (CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL) were lysed using RIPA lysis buffer and quantified using a BCA protein assay kit. The protein samples were then diluted with protein loading buffer, boiled for 5 min, and subjected to SDS-polypropylene gel electrophoresis (SDS-PAGE). After electrophoresis, the gel was transferred to ultra-fast gel protein staining solution, incubated for 2 h, and then repeatedly washed with ultrapure water until the gel background was clear and transparent. Finally, the gel was detected using a gel imaging analyzer. The results are as follows: Figure 9 As shown, cell membrane-coated prodrug self-assembled nanoparticles successfully retained proteins on the cell membrane surface.

[0094] (2) Western-blotting analysis of cell membrane-coated prodrug self-assembled nanoparticles

[0095] The protein samples of cell membranes from Example 1, cell membrane-coated prodrug self-assembled nanoparticles prepared in Example 2(3) (i.e., CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL), and cell membrane-coated non-PEG modified prodrug self-assembled nanoparticles (CM-DPNAs) prepared in Example 2(1) (DM ratio of 1:0.5, and a cabazitaxel content of 200 μg / mL) were subjected to SDS-PAGE using RIPA lysis buffer, and then the proteins were transferred to polyvinylidene fluoride (PVDF) membranes. After the transfer, the PVDF membranes were placed in a protein-free rapid blocking buffer and incubated at room temperature for 2 h for blocking. Then, the membranes were incubated overnight at 4 °C with Integrin-β1 (A22599), E-cadherin (A3044), N-cadherin (A3045), and CD44 (A12410) diluted with Western blotting buffer. After incubation, unbound antibody was washed away from the membrane with TBST buffer, repeated three times for 10 min each time. Then, the membrane was incubated with HRP goat anti-rabbit IgG (A5014) diluted with Western blotting buffer at room temperature for 2 h. After incubation, unbound secondary antibody was washed away from the membrane with TBST buffer, repeated three times for 10 min each time. After development with ECL chemiluminescence solution, the membrane was detected using a gel imaging analyzer and then developed. Results are as follows: Figure 10 As shown, four cell adhesion proteins expressed on the cell membrane include Integrin-β1, E-cadherin, N-cadherin, and CD4437-40 (e.g., ...). Figure 10 As shown in the figure, the prodrug self-assembled nanoparticles coated with cell membrane are retained, thus enabling homologous adhesion of tumor cells through a specific recognition mechanism.

[0096] Example 5: Intercellular adhesion of cell membrane-coated prodrug self-assembled nanoparticles

[0097] In this embodiment, to evaluate the adhesion properties between cell membrane-coated prodrug self-assembled nanoparticles and tumor cells, RM-1 cells and 4T1 cells were seeded in confocal culture dishes and cultured in an incubator for 24 hours. Then, the cells were treated with coumarin-6 labeled cell membrane-coated prodrug self-assembled nanoparticles as described in Example 2 (3) (i.e., CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL) or PEG-modified prodrug self-assembled nanoparticles (pDPNAs) prepared in Example 2 (2) (CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL), and cultured at 4°C for 2 hours; the concentration of coumarin-6 was 200 ng / mL. After culture, the cells were washed three times with PBS solution to remove unattached nanoparticles. Next, the cell membrane was stained with DiD (Meilun Pharmaceuticals, China), and the cell nucleus was stained with Hoechst 33342 (Solepro Pharmaceuticals, China). After each staining, the cells were washed three times with PBS. Finally, the nanoparticles adhering to the cells were observed using a confocal laser scanning microscope (CLSM, TCS SP2 / AOBS, Leica, Germany) to assess the adhesion of the nanoparticles to the tumor cells.

[0098] The test results are as follows Figure 11 As shown, the adhesion of cell membrane-coated prodrug self-assembled nanoparticles to RM-1 cells was significantly enhanced by the camouflage of the tumor cell membrane. Furthermore, enhanced recognition by cell membrane-coated prodrug self-assembled nanoparticles was also observed in 4T1 cells, attributed to the similar protein expression patterns among tumor cells. These results also provide a theoretical basis for the specific recognition of tumor cells by biomimetic nanomedicines.

[0099] Example 6: Cellular uptake of cell membrane-coated prodrug self-assembled nanoparticles

[0100] In this embodiment, the uptake of cell membrane-coated prodrug self-assembled nanoparticles by tumor cells, normal cells, and macrophages was evaluated by flow cytometry.

[0101] (1) Tumor cell uptake of cell membrane-coated prodrug self-assembled nanoparticles

[0102] The uptake of small molecule prodrug self-assembled nanoparticles in mouse prostate cancer (RM-1) and mouse breast cancer (4T1) cells was determined by flow cytometry. RM-1 and 4T1 cells were seeded into 12-well plates at a density of 5000 cells / mL and incubated for 24 h to allow cell adhesion. After cell adhesion, the cell membrane-coated prodrug self-assembled nanoparticles labeled with coumarin-6 as described in Example 2 (3) (i.e., CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL) were added (#4). The concentration of coumarin-6 was 200 ng / mL. After incubation at 37°C for 0.5 h or 2 h, the cells were washed, collected, and dispersed in PBS. The uptake of coumarin-6 by the cells was examined by flow cytometry. Meanwhile, different controls were set up: #1 DPNAs were unPEGylated and cell membrane-coated γ-4S-2CTX NPs; #2 CM-DPNAs were cell membrane-coated, unPEGylated γ-4S-2CTX NPs; #3 pDPNAs were PEGylated γ-4S-2CTX NPs; and #5 was a blank control. All control nanoparticles were prepared according to the specifications described in Example 2, and the material ratios were the same as those in the experimental groups.

[0103] Experimental results are as follows Figure 12 As shown in Figure A, since the RM-1 cell membrane coating enhances the recognition and adhesion of nanoparticles to cells (Example 5), the uptake of cell membrane-coated prodrug self-assembled nanoparticles (CM-DPNAs and CM-pDPNAs) is significantly higher than that of uncoated nanoparticles (including DPNAs and pDPNAs). Furthermore, PEG modification also had a positive effect on uptake; PEGylated nanoparticles (including pDPNAs and CM-pDPNAs) were uptaken more readily than unPEGylated DPNAs and CM-DPNAs, possibly due to improved stability. Similar uptake enhancement effects were also observed in 4T1 cells (e.g., ...). Figure 12 (As shown in B), this is because tumor cell-specific recognition enhances the cell adhesion and subsequent internalization process of nanoparticles.

[0104] (2) Normal cell L02 uptake of cell membrane-coated prodrug self-assembled nanoparticles

[0105] Human normal liver (L02) cells were inoculated using the procedure described in Example 6(1) and subsequently processed. The uptake of the normal cell line L02 was also evaluated. The results are as follows: Figure 13 As shown, due to the low expression of adhesion molecules in L02 cells, the recognition efficiency of RM-1 cell membrane-coated nanoparticles is low when they encounter L02 cells, so the increase in uptake is not significant.

[0106] (3) Macrophage RAW264.7 uptake of cell membrane-coated prodrug self-assembled nanoparticles.

[0107] Macrophages are the first line of defense in the human body and are also multifunctional phagocytic cells. To study the phagocytic behavior of nanoparticles, this example uses the widely used RAW 264.7 cell line as a model. RAW 264.7 macrophages were inoculated using the procedure described in Example 6(1) and subsequently treated. The uptake by the RAW 264.7 macrophages was also evaluated. The results are as follows... Figure 14 As shown, pure prodrug self-assembled DPNAs, being bare nanoparticles without any surface modification, were extensively taken up by RAW 264.7 cells. However, the uptake of PEG-modified prodrug self-assembled nanoparticles (pDPNAs) in RAW 264.7 cells was reduced, attributed to the anti-opsonization effect of PEGylation. After cell membrane coating, the phagocytic behavior of both CM-pDPNAs and CM-DPNAs decreased. Cell membrane-coated prodrug self-assembled nanoparticles can transmit a "don't eat me" signal to macrophages via the CD47 protein on the tumor cell membrane, inhibiting macrophage phagocytosis.

[0108] Example 7: Cytotoxicity of Cell Membrane-Coated Prodrug Self-Assembled Nanoparticles

[0109] The MTT assay was used to investigate the toxicity of cell membrane-coated prodrug self-assembled nanoparticles to two types of tumor cells and one type of normal cell: mouse prostate cancer (RM-1) and mouse breast cancer (4T1) cells and human normal liver (L02) cells. First, well-morphologically healthy cells were digested, diluted with culture medium to 10,000 cells / mL, and then 200 μL of cell suspension was added to each well of a 96-well plate. The plates were incubated for 24 h to allow cell adhesion. After cell adhesion, the culture medium was replaced with fresh culture medium containing different concentrations of the cell membrane-coated prodrug self-assembled nanoparticles prepared in Example 2 (3) (i.e., CM-pDPNAs with a DM ratio of 1:0.5 and a PEG content of 10%, and a cabazitaxel content of 200 μg / mL). (RM-1 used DMEM medium; 4T1 used RPMI 1640 medium). In this experiment, the drug solution and nanoparticle formulation were prepared and diluted using the corresponding cell culture medium and aseptically filtered through a 0.22 μm filter membrane. 200 μL of the test solution was added to each well, with three parallel wells for each concentration. The control group received no test solution, but 200 μL of culture medium was added separately, and the plates were incubated with the cells. 48 hours after drug addition, the 96-well plate was removed, the old culture medium was discarded, and 100 μL of fresh medium containing MTT (MTT prepared as a 5 mg / mL solution, diluted with culture medium before use) was added to each well. The plates were incubated for 4 hours, then the culture medium was discarded. The 96-well plate was inverted on filter paper to thoroughly absorb any remaining liquid, and 200 μL of DMSO was added to each well. The plate was shaken for 10 minutes to dissolve the blue-purple crystals. Well A1 (containing only 200 μL of DMSO) was designated as the zeroing well. The absorbance of each well after zeroing was measured at 570 nm using a microplate reader.

[0110] Meanwhile, different controls were set up: DPNAs were γ-4S-2CTX NPs without PEGylation and cell membrane coating; CM-DPNAs were γ-4S-2CTX NPs without PEGylation and cell membrane coating; pDPNAs were PEGylated γ-4S-2CTX NPs; all control nanoparticles were prepared according to the description in Example 2, and the material ratios were the same as those in the experimental group.

[0111] The results are shown in Table 3. The enhanced cell adhesion and uptake provided by cell membrane coating ultimately significantly improved cytotoxicity against tumor cells (e.g., Figure 11(As shown). Therefore, CM-pDPNAs exhibited a more significant effect than pDPNAs in cytotoxicity against RM-1 and 4T1 cells. In contrast, the cytotoxicity of CM-pDPNAs against L02 cells was similar to that of pDPNAs, indicating that the cell membrane coating strategy can effectively distinguish between tumor cells and normal cells, thereby enhancing the killing effect on tumor cells. Furthermore, compared with cabazitaxel solution, all nanoparticles showed higher specificity in cytotoxicity selectivity between tumor cells and normal cells. This is attributed to the good redox-responsive drug release characteristics of γ-4S-2CTX, which can effectively avoid off-target toxicity caused by the release of CTX in normal cells, while specifically releasing CTX in tumor cells.

[0112] Table 3 shows the IC50 values ​​of cell membrane-coated prodrug self-assembled nanoparticles for three cell lines. 50

[0113]

[0114] Example 8: Pharmacokinetic Study of Cell Membrane-Coated Prodrug Self-Assembled Nanoparticles

[0115] SD rats weighing 200-250g were randomly divided into groups and fasted for 12 hours before administration, with free access to water. Cabazitaxel solution, cell membrane-coated prodrug self-assembled nanoparticles prepared in Example 2(3), and PEG-modified prodrug self-assembled nanoparticles (i.e., pDPNAs with 10% PEG content, CM-DPNAs with a DM ratio of 1:0.5, and CM-pDPNAs with a DM ratio of 1:0.5 and 10% PEG content, respectively, were administered intravenously. The equivalent dose of cabazitaxel was 4 mg / kg. Blood was collected from the orbital cavity at specified time points, and plasma was obtained. The drug concentration in the plasma was determined by liquid chromatography-mass spectrometry.

[0116] Meanwhile, different controls were set up: CM-DPNAs were non-PEGylated γ-4S-2CTX NPs coated with cell membranes; pDPNAs were PEGylated γ-4S-2CTX NPs; all control nanoparticles were prepared according to the description in Example 2, and the material ratios were the same as those in the experimental group.

[0117] The experimental results are shown in Table 4. Cabazitaxel solution has a short half-life, and the cabazitaxel is rapidly cleared from the blood, resulting in low bioavailability. In contrast, the in vivo cabazitaxel retention of all prodrug self-assembled nanoparticle groups was significantly increased. Specifically, the area under the plasma concentration-time curve (AUC) of total cabazitaxel in cell membrane-coated CM-DPNAs was 85.46 times higher than that of cabazitaxel solution, and the maximum drug concentration (C0.05) was also significantly higher. maxThe AUC value also increased by approximately 48.24-fold. These results indicate that cell membrane materials can serve as surface modification modules to improve the pharmacokinetic properties of drugs. Furthermore, all prodrug self-assembled nanoparticle groups existed in the bloodstream as inactive prodrugs, releasing only small amounts of cabazitaxel, which is crucial for reducing toxicity and improving therapeutic safety. PEGylation of CM-DPNAs significantly improved the AUC value of CM-pDPNAs, further optimizing their pharmacokinetic properties and reaching the gold standard level for PEGylated DPNAs. This demonstrates that the dual modification strategy of CM coating and PEGylation effectively improves the in vivo efficacy of CM-coated formulations.

[0118] Table 4. Pharmacokinetic parameters of cell membrane-coated prodrug self-assembled nanoparticles

[0119]

[0120] a) The prodrug and released CTX from the prodrug self-assembled nanoparticles were determined. b) Area under the plasma concentration-time curve (nmol / mL*h), c) Half-life (h), and d) Maximum drug concentration (nmol / mL). Data are expressed as mean ± SD.

[0121] Example 9: In vivo distribution study of cell membrane-coated prodrug self-assembled nanoparticles

[0122] In this embodiment, BALB / c mice with RM-1 tumor load were used as an experimental model to study the distribution of DPNAs in vivo. When the tumor volume reached approximately 500 mm², the distribution was investigated. 3 DiR-labeled prodrug self-assembled nanoparticles of different types were administered intravenously at a dose of 10 mg / kg (n=3 per group). Mice were sacrificed at 1, 4, 8, and 12 hours post-administration, and major organs (heart, liver, spleen, lung, and kidney) and tumor tissues were collected. The concentrations of prodrugs and CTX in tissues were determined by UPLC-MS-MS (ACQUITY UPLC™, Waters), and the drug targeting process and its distribution in organs were validated using a fluorescence imaging system (IVIS Spectrum, Small Animal Imaging System, Caliper Life Sciences, USA).

[0123] The different types of prodrug self-assembled nanoparticles are CM-DPNAs, which are cell membrane-coated, non-PEGylated γ-4S-2CTX NPs; pDPNAs, which are PEGylated γ-4S-2CTX NPs; and CM-pDPNAs, which are cell membrane-coated, PEGylated γ-4S-2CTX NPs.

[0124] All of the above nanoparticles were prepared according to the method described in Example 2, with a DM ratio of 1:0.5 and a PEG content of 10%, and the dosage was the equivalent dose of cabazitaxel (4 mg / kg).

[0125] The results are as follows Figure 15 As shown, the DiR solution (Free DiR) was mainly distributed in major organs, with relatively weak fluorescence signals in tumors. In contrast, all DiR-labeled prodrug self-assembled nanoparticles exhibited higher fluorescence intensity in tumors, which increased over time. Compared with DiR-labeled cabazitaxel solution and PEG-modified prodrug self-assembled nanoparticles (pDPNAs), cell membrane encapsulation significantly reduced the distribution of CM-pDPNAs in non-target organs, indicating that tumor cell membrane encapsulation effectively enhanced the specific recognition of nanoparticles by tumors.

[0126] Example 10: In vivo antitumor effect and safety study of cell membrane-coated prodrug self-assembled nanoparticles.

[0127] To evaluate the in vivo antitumor effect of cell membrane-coated prodrug self-assembled nanoparticles, this example used RM-1 tumor-loaded C57BL / 6 male mice as an experimental model. When the tumor volume reached approximately 100 mm², the tumor was compared with the target tumor volume. 3 Mice were randomly divided into several groups and administered saline, cabazitaxel solution, CM-DPNAs, pDPNAs, and CM-pDPNAs (described in Example 2) via tail vein injection, respectively, at a dose equivalent to 4 mg / kg CTX, every two days for a total of five times. Considering the high in vivo toxicity of 4 mg / kg cabazitaxel solution shown in previous studies, a 2 mg / kg cabazitaxel solution was also used as a control treatment in this example. Tumor volume and body weight were measured and calculated every other day. At the end of the last measurement, mice were sacrificed, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) were collected for HE staining analysis. Simultaneously, blood samples were collected from mice, centrifuged (3000 rpm, 10 minutes) to obtain serum, which was used for liver and kidney function analysis (BUN, AST / GOT, CRE, and ALT / GPT detection kits were provided by Servicebio). Blood samples collected from anticoagulant tubes were used for complete blood count (CBC) testing.

[0128] In in vivo evaluation of antitumor efficacy, such as Figure 16 As shown, mice receiving 4 mg / kg cabazitaxel solution experienced a reduction in tumor burden, but also significant weight loss, indicating substantial non-specific toxicity of cabazitaxel. While the low-dose cabazitaxel solution treatment group showed reduced weight loss, the efficacy was relatively limited, and abnormalities in blood routine examinations were similar to those in the 4 mg / kg group. Figure 17In contrast, all prodrug self-assembled nanoparticle groups exhibited better safety profiles in tumor therapy. Figure 17 and 18 Although CM-DPNAs exhibited improved pharmacokinetic behavior, their tumor growth inhibition effect at an equivalent dose of 4 mg / kg was still inferior to that of CTX solution. Tumor growth in the CM-DPNAs treatment group was similar to that in the low-dose CTX solution group (2 mg / kg), but no significant weight loss or signs of histological damage were observed. Furthermore, further PEG modification improved the stability of CM-pDPNAs, making their therapeutic efficacy comparable to, and significantly superior to, that of the equivalent dose of CTX solution.

[0129] In summary, this invention chemically modifies cabazitaxel molecules to form a tetrasulfide-bonded cabazitaxel dimer prodrug (γ-4S-2CTX), and then uses a one-step precipitation method to self-assemble the prodrug into nanoparticles (DPNAs). These nanoparticles are coated with tumor cell membranes, combining the advantages of cell membrane coating formulations and prodrug self-assembled nanoparticles, thus increasing drug loading and endowing the prodrug nanoparticles with the biological functions of tumor cell membranes. This invention also further improves the stability and in vivo circulation time of the nanoparticles through PEG modification. The biomimetic cell membrane-coated nanoparticles (CM-pDPNAs) prepared by this invention exhibit significant tumor-specific recognition ability and low toxicity, greatly improving drug selectivity and therapeutic efficacy in vivo, and demonstrating good safety and anti-tumor effects in in vivo anti-tumor experiments. This invention provides a highly efficient and low-toxicity anti-tumor drug delivery system with promising application prospects.

Claims

1. A cell membrane-coated prodrug self-assembled nanoparticle, characterized in that, The nanoparticles are tumor cell membranes encapsulated by prodrug self-assembled nanoparticles prepared by co-assembly of tetrasulfide-bridged carbamate dimer prodrug (γ-4S-2CTX) and polyethylene glycol (PEG) modifier; the γ-4S-2CTX is shown in structural formula (I).

2. The cell membrane-coated prodrug self-assembled nanoparticles according to claim 1, characterized in that, The PEG modifier accounts for 5-15% of the total mass of PEG and γ-4S-2CTX.

3. The cell membrane-coated prodrug self-assembled nanoparticles according to claim 2, characterized in that, The PEG modifier is selected from TPGS, DSPE-PEG, PLGA-PEG or PE-PEG.

4. The cell membrane-coated prodrug self-assembled nanoparticles according to claim 1, characterized in that, The cell membrane is a tumor cell membrane.

5. The method for preparing cell membrane-coated prodrug self-assembled nanoparticles according to claim 1, characterized in that, The prodrug self-assembled nanoparticle core, which is synergistically modified with PEG, was prepared using a one-step nanoprecipitation method. After being mixed with cell membrane material, cell membrane-coated prodrug self-assembled nanoparticles were prepared by sonication.

6. A pharmaceutical composition, characterized in that: It comprises cell membrane prodrug self-assembled nanoparticles as described in any one of claims 1-4 and a pharmaceutically acceptable carrier or excipient.

7. The application of a cell membrane-coated prodrug self-assembled nanoparticle of claim 1 or the composition of claim 6, characterized in that: The application of the cell membrane-coated prodrug self-assembled nanoparticles or compositions in the preparation of drug delivery systems.

8. The application according to claim 7, characterized in that... The application of the cell membrane-coated prodrug self-assembled nanoparticles or compositions in the preparation of antitumor drugs.

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